Establishment of an in vitro transcription system for Peste des petits ruminant virus.

PubMed

Yunus, Mohammad; Shaila, Melkote S

2012-12-05

Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.

Three-dimensional culture system can induce expression of casein in immortalized bovine mammary epithelial cells.

PubMed

Zhan, Kang; Lin, Miao; Liu, MingMei; Sui, YangNan; Babekir, Haitham Mohammed; Zhao, GuoQi

2017-05-01

Primary bovine mammary epithelial cells (BMECs) are not ideal models for long-term studies of lactation mechanisms because these cells in a monolayer culture system cannot be polarized to simulate the physiological functions in vitro. We investigate the effects of different culture models and karyotypes on casein expression in a three-dimensional (3D) culture system. The immortalized cells' karyotypes were analyzed at passages 10, 20, 30 and 40 to detect the effects of chromosome stability. Western blotting examined that whether or not the immortalized cells at passages 5, 10, 20, 30, 40 and 50 could induce expression of casein in a 3D culture system. The proper polarization of the acinar structures was monitored. BMECs were successfully immortalized. The cell karyotype at passage 30 remained at 60 chromosomes and the average value was 57.1 ± 0.40 after passage 40. The polarized protein's levels were up-regulated in 3D culture compared to 2D culture. Expression of αs1, β and κ-casein could be detectable in a passage range in 3D culture. Expression of αs2-casein was undetectable in all experimental groups. However, all casein expressions were barely detectable in traditional 2D culture system. Therefore, 3D culture system is an important tool for the long-term study of lactation mechanisms in vitro. © 2016 Japanese Society of Animal Science.

In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shivachandra, Sathish B.; Rao, Mangala; Janosi, Laszlo

2006-02-05

An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc {sup -} phage particles. Binding was specific, stable, and of high affinity. Thismore » defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.« less

Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.

PubMed

Urrego, R; Bernal-Ulloa, S M; Chavarría, N A; Herrera-Puerta, E; Lucas-Hahn, A; Herrmann, D; Winkler, S; Pache, D; Niemann, H; Rodriguez-Osorio, N

2017-04-01

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

Development of an in vitro test system measuring transcriptional downregulatory activities on IL-13.

PubMed

Choi, Jeong June; Park, Bo-Kyung; Park, Sunyoung; Yun, Chi-Young; Kim, Dong Hee; Kim, Jin Sook; Hwang, Eun Sook; Jin, Mirim

2009-03-01

Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a wellknown antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.

Heterogeneous Nuclear Ribonucleoprotein F Suppresses Angiotensinogen Gene Expression and Attenuates Hypertension and Kidney Injury in Diabetic Mice

PubMed Central

Lo, Chao-Sheng; Chang, Shiao-Ying; Chenier, Isabelle; Filep, Janos G.; Ingelfinger, Julie R.; Zhang, Shao Ling; Chan, John S.D.

2012-01-01

We investigated the impact of heterogeneous nuclear ribonucleoprotein F (hnRNP F) overexpression on angiotensinogen (Agt) gene expression, hypertension, and renal proximal tubular cell (RPTC) injury in high-glucose milieu both in vivo and in vitro. Diabetic Akita transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs were created, and the effects on systemic hypertension, Agt gene expression, renal hypertrophy, and interstitial fibrosis were studied. We also examined immortalized rat RPTCs stably transfected with control plasmid or plasmid containing hnRNP F cDNA in vitro. The results showed that hnRNP F overexpression attenuated systemic hypertension, suppressed Agt and transforming growth factor-β1 (TGF-β1) gene expression, and reduced urinary Agt and angiotensin II levels, renal hypertrophy, and glomerulotubular fibrosis in Akita hnRNP F-Tg mice. In vitro, hnRNP F overexpression prevented the high-glucose stimulation of Agt and TGF-β1 mRNA expression and cellular hypertrophy in RPTCs. These data suggest that hnRNP F plays a modulatory role and can ameliorate hypertension, renal hypertrophy, and interstitial fibrosis in diabetes. The underlying mechanism is mediated, at least in part, via the suppression of intrarenal Agt gene expression in vivo. hnRNP F may be a potential target in the treatment of hypertension and kidney injury in diabetes. PMID:22664958

Expression stability of two housekeeping genes (18S rRNA and G3PDH) during in vitro maturation of follicular oocytes in buffalo (Bubalus bubalis).

PubMed

Aswal, Ajay Pal Singh; Raghav, Sarvesh; De, Sachinandan; Thakur, Manish; Goswami, Surender Lal; Datta, Tirtha Kumar

2008-01-15

The present study was undertaken to evaluate the expression stability of two housekeeping genes (HKGs), 18S rRNA and G3PDH during in vitro maturation (IVM) of oocytes in buffalo, which qualifies their use as internal controls for valid qRT-PCR estimation of other oocyte transcripts. A semi quantitative RT-PCR system was used with optimised qRT-PCR parameters at exponential PCR cycle for evaluation of temporal expression pattern of these genes over 24 h of IVM. 18S rRNA was found more stable in its expression pattern than G3PDH.

Peptide-functionalized quantum dots for potential applications in the imaging and treatment of obesity.

PubMed

Thovhogi, Ntevheleni; Sibuyi, Nicole Remaliah Samantha; Onani, Martin Opiyo; Meyer, Mervin; Madiehe, Abram Madimabe

2018-01-01

Obesity is a worldwide epidemic affecting millions of people. The current pharmacological treatment of obesity remains limited and ineffective due to drugs' undesirable side effects. Hence, there is a need for novel or improved strategies for long-term therapies that will help prevent the disease progression into other chronic diseases. Nanotechnology holds the future for the treatment of obesity because of its versatility, as shown by improved drug efficiency and safety in cancer clinical trials. Nano-based drug delivery systems could potentially do the same for obesity through targeted drug delivery. This study investigated the use of peptide-functionalized quantum dots (QDs) for the imaging of prohibitin (PHB)-expressing cells in vitro and in diet-induced obese rats, which could potentially be used as nanocarriers of antiobesity drugs. Cadmium (Cd)-based QDs were functionalized with an adipose homing peptide (AHP) and injected intravenously into lean and obese Wistar rats. Biodistribution of the QDs was analyzed by an IVIS ® Lumina XR imaging system and inductively coupled plasma optical emission spectroscopy (ICP-OES). For in vitro studies, PHB-expressing (Caco-2 and MCF-7) and non-PHB-expressing (KMST-6 and CHO) cells were exposed to either unfunctionalized QDs (QD625) or AHP-functionalized QDs (AHP-QD625) and analyzed by fluorescence microscopy. AHP-QD625 accumulated significantly in PHB-expressing cells in vitro when compared with non-PHB-expressing cells. In vivo data indicated that QD625 accumulated mainly in the reticuloendothelial system (RES) organs, while the AHP-QD625 accumulated mostly in the white adipose tissues (WATs). AHP-functionalized QDs were successfully and selectively delivered to the PHB-expressing cells in vitro (Caco-2 and MCF-7 cells) and in the WAT vasculature in vivo. This nanotechnology-based approach could potentially be used for dual targeted drug delivery and molecular imaging of adipose tissues in obese patients in real time.

Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

PubMed Central

Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Sun, Guochang

2012-01-01

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium. PMID:23166741

Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.

PubMed

Ferreira, A R; Machado, G M; Diesel, T O; Carvalho, J O; Rumpf, R; Melo, E O; Dode, M A N; Franco, M M

2010-07-01

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8- to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. (c) 2010 Wiley-Liss, Inc.

Chronic wasting disease prion infection of differentiated neurospheres.

PubMed

Iwamaru, Yoshifumi; Mathiason, Candace K; Telling, Glenn C; Hoover, Edward A

2017-07-04

A possible strategy to develop more diverse cell culture systems permissive to infection with naturally occurring prions is to exploit culture of neurospheres from transgenic mice expressing the normal prion protein (PrP) of the native host species. Accordingly, we developed differentiated neurosphere cultures from the cervid PrP-expressing mice to investigate whether this in vitro system would support replication of non-adapted cervid-origin chronic wasting disease (CWD) prions. Here we report the successful amplification of disease-associated PrP in differentiated neurosphere cultures within 3 weeks after exposure to CWD prions from both white-tailed deer or elk. This neurosphere culture system provides a new in vitro tool that can be used to assess non-adapted CWD prion propagation and transmission.

Comparison of L1000 and Affymetrix Microarray for In Vitro Concentration-Response Gene Expression Profiling (SOT)

EPA Science Inventory

Advances in high-throughput screening technologies and in vitro systems have opened doors for cost-efficient evaluation of chemical effects on a diversity of biological endpoints. However, toxicogenomics platforms remain too costly to evaluate large libraries of chemicals in conc...

N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

PubMed

Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

2010-07-01

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

PubMed Central

Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

2011-01-01

Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor.

PubMed

Kawakami, Takashi; Ogawa, Koji; Hatta, Tomohisa; Goshima, Naoki; Natsume, Tohru

2016-06-17

N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liquid chromatography-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.

Pathway-Based Concentration Response Profiles from Toxicogenomics Data

EPA Science Inventory

Microarray analysis of gene expression of in vitro systems could be a powerful tool for assessing chemical hazard. Differentially expressed genes specific to cells, chemicals, and concentrations can be organized into molecular pathways that inform mode of action. An important par...

Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

PubMed Central

Laitala, T; Väänänen, H K

1994-01-01

The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption. Images PMID:8200964

Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Aihua; Cheng Guangli; Zhu Genghui

Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increasedmore » in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling.« less

Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism

PubMed Central

Jansen, Felix; Yang, Xiaoyan; Baumann, Katharina; Przybilla, David; Schmitz, Theresa; Flender, Anna; Paul, Kathrin; Alhusseiny, Adil; Nickenig, Georg; Werner, Nikos

2015-01-01

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE−/− mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced. PMID:26081516

Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism.

PubMed

Jansen, Felix; Yang, Xiaoyan; Baumann, Katharina; Przybilla, David; Schmitz, Theresa; Flender, Anna; Paul, Kathrin; Alhusseiny, Adil; Nickenig, Georg; Werner, Nikos

2015-09-01

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE-/- mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate.

PubMed

Park, Soohyun; Hong, Soohye; Pack, Seung Pil; Lee, Jinwon

2014-02-01

Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5-30 °C) and weakly basic conditions (pH 8.5-10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.

The in vitro GcMAF effects on endocannabinoid system transcriptionomics, receptor formation, and cell activity of autism-derived macrophages.

PubMed

Siniscalco, Dario; Bradstreet, James Jeffrey; Cirillo, Alessandra; Antonucci, Nicola

2014-04-17

Immune system dysregulation is well-recognized in autism and thought to be part of the etiology of this disorder. The endocannabinoid system is a key regulator of the immune system via the cannabinoid receptor type 2 (CB2R) which is highly expressed on macrophages and microglial cells. We have previously published significant differences in peripheral blood mononuclear cell CB2R gene expression in the autism population. The use of the Gc protein-derived Macrophage Activating Factor (GcMAF), an endogenous glycosylated vitamin D binding protein responsible for macrophage cell activation has demonstrated positive effects in the treatment of autistic children. In this current study, we investigated the in vitro effects of GcMAF treatment on the endocannabinoid system gene expression, as well as cellular activation in blood monocyte-derived macrophages (BMDMs) from autistic patients compared to age-matched healthy developing controls. To achieve these goals, we used biomolecular, biochemical and immunocytochemical methods. GcMAF treatment was able to normalize the observed differences in dysregulated gene expression of the endocannabinoid system of the autism group. GcMAF also down-regulated the over-activation of BMDMs from autistic children. This study presents the first observations of GcMAF effects on the transcriptionomics of the endocannabinoid system and expression of CB2R protein. These data point to a potential nexus between endocannabinoids, vitamin D and its transporter proteins, and the immune dysregulations observed with autism.

The in vitro GcMAF effects on endocannabinoid system transcriptionomics, receptor formation, and cell activity of autism-derived macrophages

PubMed Central

2014-01-01

Background Immune system dysregulation is well-recognized in autism and thought to be part of the etiology of this disorder. The endocannabinoid system is a key regulator of the immune system via the cannabinoid receptor type 2 (CB2R) which is highly expressed on macrophages and microglial cells. We have previously published significant differences in peripheral blood mononuclear cell CB2R gene expression in the autism population. The use of the Gc protein-derived Macrophage Activating Factor (GcMAF), an endogenous glycosylated vitamin D binding protein responsible for macrophage cell activation has demonstrated positive effects in the treatment of autistic children. In this current study, we investigated the in vitro effects of GcMAF treatment on the endocannabinoid system gene expression, as well as cellular activation in blood monocyte-derived macrophages (BMDMs) from autistic patients compared to age-matched healthy developing controls. Methods To achieve these goals, we used biomolecular, biochemical and immunocytochemical methods. Results GcMAF treatment was able to normalize the observed differences in dysregulated gene expression of the endocannabinoid system of the autism group. GcMAF also down-regulated the over-activation of BMDMs from autistic children. Conclusions This study presents the first observations of GcMAF effects on the transcriptionomics of the endocannabinoid system and expression of CB2R protein. These data point to a potential nexus between endocannabinoids, vitamin D and its transporter proteins, and the immune dysregulations observed with autism. PMID:24739187

Tadalafil modulates aromatase activity and androgen receptor expression in a human osteoblastic cell in vitro model.

PubMed

Aversa, A; Fittipaldi, S; Bimonte, V M; Wannenes, F; Papa, V; Francomano, D; Greco, E A; Lenzi, A; Migliaccio, S

2016-02-01

Phosphodiesterase type-5 inhibitor (PDE5i) tadalafil administration in men with erectile dysfunction is associated with increased testosterone/estradiol ratio, leading to hypothesize a potential increased effect of androgen action on target tissues. We aimed to characterize, in a cellular model system in vitro, the potential modulation of aromatase and sex steroid hormone receptors upon exposure to tadalafil (TAD). Human osteoblast-like cells SAOS-2 were chosen as an in vitro model system since osteoblasts are target of steroid hormones. Cells were tested for viability upon TAD exposure, which increased cell proliferation. Then, cells were treated with/without TAD for several times to evaluate potential modulation in PDE5, aromatase (ARO), androgen (AR) and estrogen (ER) receptor expression. Osteoblasts express significant levels of both PDE5 mRNA and protein. Exposure of cells to increasing concentrations of TAD (10(-8)-10(-7) M) decreased PDE5 mRNA and protein expression. Also, TAD inhibited ARO mRNA and protein expression leading to an increase in testosterone levels in the supernatants. Interestingly, TAD increased total AR mRNA and protein expression and decreased ERα, with an increased ratio of AR/ER, suggesting preferential androgenic vs estrogenic pathway activation. Our results demonstrate for the first time that TAD decreases ARO expression and increases AR protein expression in human SAOS-2, strongly suggesting a new control of steroid hormones pathway by PDE5i. These findings might represent the first evidence of translational actions of PDE5i on AR, which leads to hypothesize a growing relevance of this molecule in men with prostate cancer long-term treated with TAD for sexual rehabilitation.

[The role of the serotonin system in the stress response of various cells

NASA Technical Reports Server (NTRS)

Belzhelarskaia, S. N.; Satton, F. F.; Sutton, F. (Principal Investigator)

2003-01-01

The recombinant mouse brain serotonin receptor (5HT1c) was used to study the response of plant cells and oocytes to a stress signal activated by the serotonin-serotonin receptor interaction and associated Ca2+ flow. Based on plant expression vectors, recombinant constructs were obtained to direct production of 5HT1c fused with the green fluorescent protein in plant cells. The mRNAs for hybrid proteins were synthesized in an in vitro transcription system. The expression and function of the hybrid protein and the function of the associated ion channels were electrophysiologically studied in Xenopus laevis oocytes injected with the hybrid mRNA. The hybrid protein was functional and changed the operation of the Ca2+ channel in oocytes. To study the expression of the hybrid constructs in plant cells, the in vitro transcription product was inoculated in tobacco leaves, which then fluoresced.

Expression of TNF-α system members in bovine ovarian follicles and the effects of TNF-α or dexamethasone on preantral follicle survival, development and ultrastructure in vitro.

PubMed

Silva, A W B; Ribeiro, R P; Menezes, V G; Barberino, R S; Passos, J R S; Dau, A M P; Costa, J J N; Melo, L R F; Bezerra, F T G; Donato, M A M; Peixoto, C A; Matos, M H T; Gonçalves, P B D; van den Hurk, R; Silva, J R V

2017-07-01

This study was conducted to detect the protein expression of TNF-α system members (TNF-α/TNFR1/TNFR2) in bovine ovarian follicles and to evaluate the effects of TNF-α or dexamethasone on the survival and growth of primordial follicles in vitro, as well as on gene expression in cultured ovarian tissue. It was hypothesized that TNF-α induces follicular atresia in ovarian tissues cultured in vitro, and that dexamethasone suppresses the production of endogenous TNF-α, which can improve follicle viability in vitro. Ovarian fragments were cultured for 6days in α-MEM + supplemented with TNF-α (0, 1, 10, 100 or 200ng/ml) or dexamethasone (0, 1, 10, 100 or 200ng/ml). After culture, the expression of mRNAs for BCL-2, BAX, P53, TNF-α, and CASP3 and CASP6 were evaluated. Immunohistochemical results showed that the TNF-α system members, were detected in bovine preantral and antral follicles. After 6days, the TNF-α (10ng/ml) treatment reduced the percentage of normal preantral follicles and increased the number of TUNEL-positive cells in cultured tissue. Dexamethasone (10ng/ml) during 6days of culture did maintain the percentage of normal follicles and the ultrastructure of follicles, while the presence of TNF-α or dexamethasone did not influence primordial follicle activation. However, TNF-α or dexamethasone had no effect on the levels of mRNA for P53, BCL-2, BAX and CASP6, in cultured tissues, but the presence of dexamethasone reduced the levels of CASP3 compared to ovarian slices cultured in control medium (α-MEM + ). In conclusion, proteins of the TNF-α system are expressed at different bovine follicle stages. The addition of TNF-α in culture reduces follicle survival and increases the number of apoptotic cells in ovarian tissue, while the presence of dexamethasone maintains follicle ultrastructure in cultured tissue. Copyright © 2017. Published by Elsevier B.V.

Evidence for a dopamine intrinsic direct role in the regulation of the ovary reproductive function: in vitro study on rabbit corpora lutea.

PubMed

Parillo, Francesco; Maranesi, Margherita; Mignini, Fiorenzo; Marinelli, Lisa; Di Stefano, Antonio; Boiti, Cristiano; Zerani, Massimo

2014-01-01

Dopamine (DA) receptor (DR) type 1 (D1R) has been found to be expressed in luteal cells of various species, but the intrinsic role of the DA/DRs system on corpora lutea (CL) function is still unclear. Experiments were devised to characterize the expression of DR types and the presence of DA, as well as the in vitro effects of DA on hormone productions by CL in pseudopregnant rabbits. Immunoreactivity and gene expression for D1R decreased while that for D3R increased in luteal and blood vessel cells from early to late pseudopregnant stages. DA immunopositivity was evidenced only in luteal cells. The DA and D1R agonist increased in vitro release of progesterone and prostaglandin E2 (PGE2) by early CL, whereas the DA and D3R agonist decreased progesterone and increased PGF2α in vitro release by mid- and late CL. These results provide evidence that the DA/DR system exerts a dual modulatory function in the lifespan of CL: the DA/D1R is luteotropic while the DA/D3R is luteolytic. The present data shed new light on the physiological mechanisms regulating luteal activity that might improve our ability to optimize reproductive efficiency in mammal species, including humans.

Distinct Properties of Human M-CSF and GM-CSF Monocyte-Derived Macrophages to Simulate Pathological Lung Conditions In Vitro: Application to Systemic and Inflammatory Disorders with Pulmonary Involvement.

PubMed

Lescoat, Alain; Ballerie, Alice; Augagneur, Yu; Morzadec, Claudie; Vernhet, Laurent; Fardel, Olivier; Jégo, Patrick; Jouneau, Stéphane; Lecureur, Valérie

2018-03-17

Macrophages play a central role in the pathogenesis of inflammatory and fibrotic lung diseases. However, alveolar macrophages (AM) are poorly available in humans to perform in vitro studies due to a limited access to broncho-alveolar lavage (BAL). In this study, to identify the best alternative in vitro model for human AM, we compared the phenotype of AM obtained from BAL of patients suffering from three lung diseases (lung cancers, sarcoidosis and Systemic Sclerosis (SSc)-associated interstitial lung disease) to human blood monocyte-derived macrophages (MDMs) differentiated with M-CSF or GM-CSF. The expression of eight membrane markers was evaluated by flow cytometry. Globally, AM phenotype was closer to GM-CSF MDMs. However, the expression levels of CD163, CD169, CD204, CD64 and CD36 were significantly higher in SSc-ILD than in lung cancers. Considering the expression of CD204 and CD36, the phenotype of SSc-AM was closer to MDMs, from healthy donors or SSc patients, differentiated by M-CSF rather than GM-CSF. The comparative secretion of IL-6 by SSc-MDMs and SSc-AM is concordant with these phenotypic considerations. Altogether, these results support the M-CSF MDM model as a relevant in vitro alternative to simulate AM in fibrotic disorders such as SSc.

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

PubMed Central

Guo, Zhong; Johnston, Wayne; Kovtun, Oleksiy; Mureev, Sergey; Bröcker, Cornelia; Ungermann, Christian; Alexandrov, Kirill

2013-01-01

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data. PMID:24312556

RAPID COMMUNICATION: Nerve growth factor influences cleavage rate and embryo development in sheep.

PubMed

Crispo, M; Dos Santos-Neto, P C; Vilariño, M; Mulet, A P; de León, A; Barbeito, L; Menchaca, A

2016-10-01

Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.

In vitro protein expression: an emerging alternative to cell-based approaches.

PubMed

He, Mingyue

2011-04-30

Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

Different Subcellular Localization of ALCAM Molecules in Neuroblastoma: Association with Relapse

PubMed Central

Corrias, Maria Valeria; Gambini, Claudio; Gregorio, Andrea; Croce, Michela; Barisione, Gaia; Cossu, Claudia; Rossello, Armando; Ferrini, Silvano; Fabbi, Marina

2010-01-01

Background: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system. Methods: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively. Results: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P = 0.044 and P < 0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype. Conclusions: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up. PMID:20208136

Effect of timing of development on total cell number and expression profile of HSP-70.1 and GLUT-1 in buffalo (Bubalus bubalis) oocytes and preimplantation embryos produced in vitro.

PubMed

Rajhans, Rajib; Kumar, G Sai; Dubey, Pawan K; Sharma, G Taru

2010-03-29

The present study was designed to compare the expression profile of two developmentally important genes (HSP-70.1 and GLUT-1) and TCN (total cell number) count in fast (group A) and slow (group B) cleaved buffalo embryos to access their in vitro developmental competence. Buffalo COCs (cumulus oocyte complexes) were collected from local abattoir ovaries and subjected to in vitro maturation in: TCM-199 supplemented with 10% FBS (fetal bovine serum), BSA (3 mg/ml), sodium pyruvate (0.25 mM) and 20 ng/ml EGF (epidermal growth factor) at 38.5 degrees C under 5% CO2. In vitro derived embryos were collected at 4-8, 8-16 cell, morula and blastocyst stages at specific time points for gene expression analysis and total cell count. A semiquantitative RT-PCR (reverse transcriptase-PCR) assay was used to determine the HSP-70.1 and GLUT-1 transcripts. Results showed that developmental competence and TCN count in fast (group A)-cleaving embryos was significantly (P<0.05) higher than in the slow group (group B). The gene transcript of HSP-70.1 and GLUT-1 was expressed in oocytes (immature and mature) and throughout the embryonic developmental stages in the fast group (group A), while in the slow (group B) cleaving embryos, the expression of HSP-70.1 was absent in all the embryonic developmental stages, and expression of GLUT-1 was absent after 8-16 cell stage. In conclusion, TCN count and expression profile of HSP-70.1 and GLUT-1 genes in buffalo embryos are different taking into account the cleavage rate. Quality of such embryos for research purposes, TCN and expression profiling of developmentally important genes should be employed to optimize the in vitro culture system to produce superior quality of embryos.

Regulation of axonal and dendritic growth by the extracellular calcium-sensing receptor (CaSR)

PubMed Central

Vizard, Thomas N.; O'Keeffe, Gerard W.; Gutierrez, Humberto; Kos, Claudine H.; Riccardi, Daniela; Davies, Alun M.

2009-01-01

The extracellular calcium-sensing receptor (CaSR) monitors the systemic extracellular free ionized calcium level ([Ca2+]o) in organs involved in systemic [Ca2+]o homeostasis. However, the CaSR is also expressed in the nervous system where its role is unknown. Here we find high levels of the CaSR in perinatal mouse sympathetic neurons when their axons are innervating and branching extensively in their targets. Manipulating CaSR function in these neurons by varying [Ca2+]o, using CaSR agonists and antagonists or expressing a dominant-negative CaSR markedly affects neurite growth in vitro Sympathetic neurons lacking the CaSR have smaller neurite arbors in vitro, and sympathetic innervation density is reduced in CaSR-deficient mice in vivo. Hippocampal pyramidal neurons, which also express the CaSR, have smaller dendrites when transfected with dominant-negative CaSR in postnatal organotypic cultures. Our findings reveal a crucial role for the CaSR in regulating the growth of neural processes in the peripheral and central nervous systems. PMID:18223649

Alteration of gene expression by zinc oxide nanoparticles or zinc sulfate in vivo and comparison with in vitro data: A harmonious case.

PubMed

Zhang, Wei-Dong; Zhao, Yong; Zhang, Hong-Fu; Wang, Shu-Kun; Hao, Zhi-Hui; Liu, Jing; Yuan, Yu-Qing; Zhang, Peng-Fei; Yang, Hong-Di; Shen, Wei; Li, Lan

2016-08-01

Granulosa cells (GCs) are those somatic cells closest to the female germ cell. GCs play a vital role in oocyte growth and development, and the oocyte is necessary for multiplication of a species. Zinc oxide (ZnO) nanoparticles (NPs) readily cross biologic barriers to be absorbed into biologic systems that make them promising candidates as food additives. The objective of the present investigation was to explore the impact of intact NPs on gene expression and the functional classification of altered genes in hen GCs in vivo, to compare the data from in vivo and in vitro studies, and finally to point out the adverse effects of ZnO NPs on the reproductive system. After a 24-week treatment, hen GCs were isolated and gene expression was quantified. Intact NPs were found in the ovary and other organs. Zn levels were similar in ZnO-NP-100 mg/kg- and ZnSO4-100 mg/kg-treated hen ovaries. ZnO-NP-100 mg/kg and ZnSO4-100 mg/kg regulated the expression of the same sets of genes, and they also altered the expression of different sets of genes individually. The number of genes altered by the ZnO-NP-100 mg/kg and ZnSO4-100 mg/kg treatments was different. Gene Ontology (GO) functional analysis reported that different results for the two treatments and, in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, 12 pathways (out of the top 20 pathways) in each treatment were different. These results suggested that intact NPs and Zn(2+) had different effects on gene expression in GCs in vivo. In our recent publication, we noted that intact NPs and Zn(2+) differentially altered gene expression in GCs in vitro. However, GO functional classification and KEGG pathway enrichment analyses revealed close similarities for the changed genes in vivo and in vitro after ZnO NP treatment. Furthermore, close similarities were observed for the changed genes after ZnSO4 treatments in vivo and in vitro by GO functional classification and KEGG pathway enrichment analyses. Therefore, the effects of ZnO NPs on gene expression in vitro might represent their effects on gene expression in vivo. The results from this study and our earlier studies support previous findings indicating ZnO NPs promote adverse effects on organisms. Therefore, precautions should be taken when ZnO NPs are used as diet additives for hens because they might cause reproductive issues. Copyright © 2016 Elsevier Inc. All rights reserved.

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

PubMed

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

Persistent Alterations of Gene Expression Profiling of Human Peripheral Blood Mononuclear Cells From Smokers

PubMed Central

Weng, Daniel Y.; Chen, Jinguo; Taslim, Cenny; Hsu, Ping-Ching; Marian, Catalin; David, Sean P.; Loffredo, Christopher A.; Shields, Peter G.

2016-01-01

The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco-related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross-sectional study. We profiled global transcriptional changes in cultured smokers’ peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well-characterized smokers’ blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5-fold-change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5-fold-change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty-one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non-small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect. PMID:26294040

Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system.

PubMed

Zichel, R; Mimran, A; Keren, A; Barnea, A; Steinberger-Levy, I; Marcus, D; Turgeman, A; Reuveny, S

2010-05-01

Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

Gene expression analysis of a porcine hepatocyte/bile duct in vitro differentiaion model

USDA-ARS?s Scientific Manuscript database

A serum-free, feeder-cell-dependent, inductive differentiation culture system of porcine hepatocytes and bile ductules was analyzed for differential gene expression on a porcine genome microarray. Primary cultures of baby pig hepatocytes (BPH) were matured in culture as a monolayer of hepatocytes w...

Identification and expression of the tig gene coding for trigger factor from psychrophilic bacteria with no information of genome sequence available.

PubMed

Lee, Kyunghee; Choi, Hyojung; Im, Hana

2009-08-01

Trigger factor (TF) plays a key role as a molecular chaperone with a peptidyl-prolyl cis-trans isomerase (PPIase) activity by which cells promote folding of newly synthesized proteins coming out of ribosomes. Since psychrophilic bacteria grow at a quite low temperature, between 4 and 15 degrees C, TF from such bacteria was investigated and compared with that of mesophilic bacteria E. coli in order to offer an explanation of cold-adaptation at a molecular level. Using a combination of gradient PCRs with homologous primers and LA PCR in vitro cloning technology, the tig gene was fully identified from Psychromonas arctica, whose genome sequence is not yet available. The resulting amino acid sequence of the TF was compared with other homologous TFs using sequence alignments to search for common domains. In addition, we have developed a protein expression system, by which TF proteins from P. arctica (PaTF) were produced by IPTG induction upon cloning the tig gene on expression vectors, such as pAED4. We have further examined the role of expressed psychrophilic PaTF on survival against cold treatment at 4 degrees C. Finally, we have attempted the in vitro biochemical characterization of TF proteins with His-tags expressed in a pET system, such as the PPIase activity of PaTF protein. Our results demonstrate that the expressed PaTF proteins helped cells survive against cold environments in vivo and the purified PaTF in vitro display the functional PPIase activity in a concentration dependent manner.

Expression and in vitro functional analyses of recombinant Gam1 protein

PubMed Central

Avila, Gustavo A.; Ramirez, Daniel H.; Hildenbrand, Zacariah L.; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2014-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1’s roles in viral replication. PMID:25450237

Expression and in vitro functional analyses of recombinant Gam1 protein.

PubMed

Avila, Gustavo A; Ramirez, Daniel H; Hildenbrand, Zacariah L; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2015-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Toll-Like Receptor 4 Mediates Methamphetamine-Induced Neuroinflammation through Caspase-11 Signaling Pathway in Astrocytes

PubMed Central

Du, Si-Hao; Qiao, Dong-Fang; Chen, Chuan-Xiang; Chen, Si; Liu, Chao; Lin, Zhoumeng; Wang, Huijun; Xie, Wei-Bing

2017-01-01

Methamphetamine (METH) is an amphetamine-typed stimulant drug that is increasingly being abused worldwide. Previous studies have shown that METH toxicity is systemic, especially targeting dopaminergic neurons in the central nervous system (CNS). However, the role of neuroinflammation in METH neurotoxicity remains unclear. We hypothesized that Toll-like receptor 4 (TLR4) and Caspase-11 are involved in METH-induced astrocyte-related neuroinflammation. We tested our hypothesis by examining the changes of TLR4 and Caspase-11 protein expression in primary cultured C57BL/6 mouse astrocytes and in the midbrain and striatum of mice exposed to METH with western blot and double immunofluorescence labeling. We also determined the effects of blocking Caspase-11 expression with wedelolactone (a specific inhibitor of Caspase-11) or siRNA on METH-induced neuroinflammation in astrocytes. Furthermore, we determined the effects of blocking TLR4 expression with TAK-242 (a specific inhibitor of TLR4) or siRNA on METH-induced neuroinflammation in astrocytes. METH exposure increased Caspase-11 and TLR4 expression both in vitro and in vivo, with the effects in vitro being dose-dependent. Inhibition of Caspase-11 expression with either wedelolactone or siRNAs reduced the expression of inflammasome NLRP3 and pro-inflammatory cytokines. In addition, blocking TLR4 expression inhibited METH-induced activation of NF-κB and Caspase-11 in vitro and in vivo, suggesting that TLR4-Caspase-11 pathway is involved in METH-induced neuroinflammation. These results indicate that Caspase-11 and TLR4 play an important role in METH-induced neuroinflammation and may be potential gene targets for therapeutics in METH-caused neurotoxicity. PMID:29311802

A quantitative framework to evaluate modeling of cortical development by neural stem cells

PubMed Central

Stein, Jason L.; de la Torre-Ubieta, Luis; Tian, Yuan; Parikshak, Neelroop N.; Hernandez, Israel A.; Marchetto, Maria C.; Baker, Dylan K.; Lu, Daning; Hinman, Cassidy R.; Lowe, Jennifer K.; Wexler, Eric M.; Muotri, Alysson R.; Gage, Fred H.; Kosik, Kenneth S.; Geschwind, Daniel H.

2014-01-01

Summary Neural stem cells have been adopted to model a wide range of neuropsychiatric conditions in vitro. However, how well such models correspond to in vivo brain has not been evaluated in an unbiased, comprehensive manner. We used transcriptomic analyses to compare in vitro systems to developing human fetal brain and observed strong conservation of in vivo gene expression and network architecture in differentiating primary human neural progenitor cells (phNPCs). Conserved modules are enriched in genes associated with ASD, supporting the utility of phNPCs for studying neuropsychiatric disease. We also developed and validated a machine learning approach called CoNTExT that identifies the developmental maturity and regional identity of in vitro models. We observed strong differences between in vitro models, including hiPSC-derived neural progenitors from multiple laboratories. This work provides a systems biology framework for evaluating in vitro systems and supports their value in studying the molecular mechanisms of human neurodevelopmental disease. PMID:24991955

FOXO3a is broadly neuroprotective in vitro and in vivo against insults implicated in motor neuron diseases

PubMed Central

Mojsilovic-Petrovic, Jelena; Nedelsky, Natalia; Boccitto, Marco; Mano, Itzhak; Georgiades, Savvas N.; Zhou, Weiguo; Liu, Yuhong; Neve, Rachael L.; Taylor, J. Paul; Driscoll, Monica; Clardy, Jon; Merry, Diane; Kalb, Robert G.

2009-01-01

Aging is a risk factor for the development of adult-onset neuro-degenerative diseases. While some of the molecular pathways regulating longevity and stress resistance in lower organisms are defined (i.e., those activating the transcriptional regulators DAF-16 and HSF-1 in C. elegans), their relevance to mammals and disease susceptibility are unknown. We studied the signaling controlled by the mammalian homolog of DAF-16, FOXO3a, in model systems of motor neuron disease. Neuron death elicited in vitro by excitotoxic insult or the expression of mutant SOD1, mutant p150glued or polyQ expanded androgen receptor was abrogated by expression of nuclear-targeted FOXO3a. We identify a compound (Psammaplysene A, PA) that increases nuclear localization of FOXO3a in vitro and in vivo and show that PA also protects against these insults in vitro. Administration of PA to invertebrate model systems of neurodegeneration similarly blocked neuron death in a DAF-16/FOXO3a-dependent manner. These results indicate that activation of the DAF-16/FOXO3a pathway, genetically or pharmacologically, confers protection against the known causes of motor neuron diseases. PMID:19553463

Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver. Up-regulation during in vitro activation and in hepatic tissue repair.

PubMed

Knittel, T; Aurisch, S; Neubauer, K; Eichhorst, S; Ramadori, G

1996-08-01

Ito cells (lipocytes, stellate cells) are regarded as the principle matrix-producing cell of the liver and have been shown recently to express glial fibrillary acidic protein, an intermediate filament typically found in glia cells of the nervous system. The present study examines 1) whether Ito cells of rat liver express central nervous system typical adhesion molecules, namely, neural cell adhesion molecule (N-CAM), in a cell-type-specific manner and 2) whether N-CAM expression is affected by activation of Ito cells in vitro and during rat liver injury in vivo. As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, and immunocytochemistry of freshly isolated and cultivated hepatic cells, N-CAM expression was restricted to Ito cells and was absent in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Ito cells expressed predominantly N-CAM-coding transcripts of 6.1 and 4.8 kb in size and 140-kd isoforms of the N-CAM protein, which was localized on the cell surface membrane of Ito cells. In parallel to glial fibrillary acidic protein down-regulation and smooth muscle alpha-actin up-regulation, N-CAM expression was increased during in vitro transformation of Ito cells from resting to activated (myofibroblast-like) cells and by the fibrogenic mediator transforming growth factor-beta 1. By immunohistochemistry, N-CAM was detected in normal rat liver in the portal field as densely packed material and in a spot as well as fiber-like pattern probably representing nerve structures. However, after liver injury, N-CAM expression became detectable in mesenchymal cells within and around the necrotic area and within fibrotic septae. In serially cut tissue sections, N-CAM-positive cells were predominantly co-distributed with smooth muscle alpha-actin-positive cells rather than glial fibrillary acidic protein-positive cells, especially in fibrotic livers. The experimental results illustrate that N-CAM positivity in the liver cannot be solely ascribed to nerve endings as, among the different types of resident liver cells, Ito cells specifically express N-CAM in vitro and presumably in vivo. In addition to its role as potential cell-type-specific marker protein for activated Ito cells, the induction of N-CAM expression might illustrate a mechanism by which mesenchymal cell proliferation might be inhibited when tissue repair is concluded.

EXPRESSION AND CHARACTERIZATION OF THE RECOMBINANT JUVENILE HORMONE EPOXIDE HYDROLASE (JHEH) FROM MANDUCA SEXTA. (R825433)

EPA Science Inventory

The cDNA of the microsomal Juvenile Hormone Epoxide Hydrolase (JHEH) from Manduca sexta was expressed in vitro in the baculovirus system. In insect cell culture, the recombinant enzyme (Ms-JHEH) was produced at a high level (100 fold over background EH catalytic activit...

E2-EPF UCP regulates stability and functions of missense mutant pVHL via ubiquitin mediated proteolysis.

PubMed

Park, Kyeong-Su; Kim, Ju Hee; Shin, Hee Won; Chung, Kyung-Sook; Im, Dong-Soo; Lim, Jung Hwa; Jung, Cho-Rok

2015-10-26

Missense mutation of VHL gene is frequently detected in type 2 VHL diseases and linked to a wide range of pVHL functions and stability. Certain mutant pVHLs retain ability to regulate HIFs but lose their function by instability. In this case, regulating of degradation of mutant pVHLs, can be postulated as therapeutic method. The stability and cellular function of missense mutant pVHLs were determine in HEK293T transient expressing cell and 786-O stable cell line. Ubiquitination assay of mutant VHL proteins was performed in vitro system. Anticancer effect of adenovirus mediated shUCP expressing was evaluated using ex vivo mouse xenograft assay. Three VHL missense mutants (V155A, L158Q, and Q164R) are directly ubiquitinated by E2-EPF UCP (UCP) in vitro. Mutant pVHLs are more unstable than wild type in cell. Missense mutant pVHLs interact with UCP directly in both in vitro and cellular systems. Lacking all of lysine residues of pVHL result in resistance to ubiquitination thereby increase its stability. Missense mutant pVHLs maintained the function of E3 ligase to ubiquitinate HIF-1α in vitro. In cells expressing mutant pVHLs, Glut-1 and VEGF were relatively upregulated compared to their levels in cells expressing wild-type. Depletion of UCP restored missense mutant pVHLs levels and inhibited cell growth. Adenovirus-mediated shUCP RNA delivery inhibited tumor growth in ex vivo mouse xenograft model. These data suggest that targeting of UCP can be one of therapeutic method in type 2 VHL disease caused by unstable but functional missense mutant pVHL.

Methylome reorganization during in vitro dedifferentiation and regeneration of Populus trichocarpa

PubMed Central

2013-01-01

Background Cytosine DNA methylation (5mC) is an epigenetic modification that is important to genome stability and regulation of gene expression. Perturbations of 5mC have been implicated as a cause of phenotypic variation among plants regenerated through in vitro culture systems. However, the pattern of change in 5mC and its functional role with respect to gene expression, are poorly understood at the genome scale. A fuller understanding of how 5mC changes during in vitro manipulation may aid the development of methods for reducing or amplifying the mutagenic and epigenetic effects of in vitro culture and plant transformation. Results We investigated the in vitro methylome of the model tree species Populus trichocarpa in a system that mimics routine methods for regeneration and plant transformation in the genus Populus (poplar). Using methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq), we compared the methylomes of internode stem segments from micropropagated explants, dedifferentiated calli, and internodes from regenerated plants. We found that more than half (56%) of the methylated portion of the genome appeared to be differentially methylated among the three tissue types. Surprisingly, gene promoter methylation varied little among tissues, however, the percentage of body-methylated genes increased from 9% to 14% between explants and callus tissue, then decreased to 8% in regenerated internodes. Forty-five percent of differentially-methylated genes underwent transient methylation, becoming methylated in calli, and demethylated in regenerants. These genes were more frequent in chromosomal regions with higher gene density. Comparisons with an expression microarray dataset showed that genes methylated at both promoters and gene bodies had lower expression than genes that were unmethylated or only promoter-methylated in all three tissues. Four types of abundant transposable elements showed their highest levels of 5mC in regenerated internodes. Conclusions DNA methylation varies in a highly gene- and chromosome-differential manner during in vitro differentiation and regeneration. 5mC in redifferentiated tissues was not reset to that in original explants during the study period. Hypermethylation of gene bodies in dedifferentiated cells did not interfere with transcription, and may serve a protective role against activation of abundant transposable elements. PMID:23799904

Global gene expression and Ingenuity biological functions analysis on PCBs 153 and 138 induced human PBMC in vitro reveals differential mode(s) of action in developing toxicities.

PubMed

Ghosh, Somiranjan; Zang, Shizhu; Mitra, Partha S; Ghimbovschi, Svetlana; Hoffman, Eric P; Dutta, Sisir K

2011-07-01

Several reports have indicated that low level of polychlorinated biphenyl (PCB) exposure can adversely affect a multitude of physiological disorders and diseases in in vitro, in vivo, and as reported in epidemiological studies. This investigation is focused on the possible contribution of two most prevalent PCB congeners in vitro in developing toxicities. We used PCBs 138 and 153 at the human equivalence level as model agents to test their specificity in developing toxicities. We chose a global approach using oligonucleotide microarray technology to investigate modulated gene expression for biological effects, upon exposure of PCBs, followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We performed in vitro studies with human peripheral blood mononuclear cells (PBMC), where PBMC cells were exposed to respective PCBs for 48 h. Overall, our observation on gene expression indicated that PCB produces a unique signature affecting different pathways, specific for each congener. While analyzing these data through IPA, the prominent and interesting disease and disorders were neurological disease, cancer, cardiovascular disease, respiratory disease, as well as endocrine system disorders, genetic disorders, and reproductive system disease. They showed strong resemblances with in vitro, in vivo, and in the epidemiological studies. A distinct difference was observed in renal and urological diseases, organisimal injury and abnormalities, dental disease, ophthalmic disease, and psychological disorders, which are only revealed by PCB 138 exposure, but not in PCB 153. The present study emphasizes the challenges of global gene expression in vitro and was correlated with the results of exposed human population. The microarray results give a molecular mechanistic insight and functional effects, following PCB exposure. The extent of changes in genes related to several possible mode(s) of action highlights the changes in cellular functions and signaling pathways that play major roles. In addition to understanding the pathways related to mode of action for chemicals, these data could lead to the identification of genomic signatures that could be used for screening of chemicals for their potential to cause disease and developmental disorders. Copyright © 2011 Elsevier Ltd. All rights reserved.

Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine.

PubMed

Rozov, F N; Grinenko, T S; Levit, G L; Krasnov, V P; Belyavsky, A V

2010-09-15

Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders. 2010 Elsevier Inc. All rights reserved.

Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.

PubMed

Hribal, R; Jewgenow, K; Braun, B C; Comizzoli, P

2013-04-01

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. © 2012 Blackwell Verlag GmbH.

Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

PubMed Central

Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

2015-01-01

Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

Comparison of NMDA and AMPA Channel Expression and Function between Embryonic and Adult Neurons Utilizing Microelectrode Array Systems.

PubMed

Edwards, Darin; Sommerhage, Frank; Berry, Bonnie; Nummer, Hanna; Raquet, Martina; Clymer, Brad; Stancescu, Maria; Hickman, James J

2017-12-11

Microelectrode arrays (MEAs) are innovative tools used to perform electrophysiological experiments for the study of electrical activity and connectivity in populations of neurons from dissociated cultures. Reliance upon neurons derived from embryonic tissue is a common limitation of neuronal/MEA hybrid systems and perhaps of neuroscience research in general, and the use of adult neurons could model fully functional in vivo parameters more closely. Spontaneous network activity was concurrently recorded from both embryonic and adult rat neurons cultured on MEAs for up to 10 weeks in vitro to characterize the synaptic connections between cell types. The cultures were exposed to synaptic transmission antagonists against NMDA and AMPA channels, which revealed significantly different receptor profiles of adult and embryonic networks in vitro. In addition, both embryonic and adult neurons were evaluated for NMDA and AMPA channel subunit expression over five weeks in vitro. The results established that neurons derived from embryonic tissue did not express mature synaptic channels for several weeks in vitro under defined conditions. Consequently, the embryonic response to synaptic antagonists was significantly different than that of neurons derived from adult tissue sources. These results are especially significant because most studies reported with embryonic hippocampal neurons do not begin at two to four weeks in culture. In addition, the utilization of MEAs in lieu of patch-clamp electrophysiology avoided a large-scale, labor-intensive study. These results establish the utility of this unique hybrid system derived from adult hippocampal tissue in combination with MEAs and offer a more appropriate representation of in vivo function for drug discovery. It has application for neuronal development and regeneration as well as for investigations into neurodegenerative disease, traumatic brain injury, and stroke.

In vitro CpG methylation increases the transformation efficiency of Borrelia burgdorferi strains harboring the endogenous linear plasmid lp56.

PubMed

Chen, Qiang; Fischer, Joshua R; Benoit, Vivian M; Dufour, Nicholas P; Youderian, Philip; Leong, John M

2008-12-01

Borrelia burgdorferi is the causative agent of Lyme disease, the most common vector-borne illness in the Northern hemisphere. Low-passage-number infectious strains of B. burgdorferi exhibit extremely low transformation efficiencies-so low, in fact, as to hinder the genetic study of putative virulence factors. Two putative restriction-modification (R-M) systems, BBE02 contained on linear plasmid 25 (lp25) and BBQ67 contained on lp56, have been postulated to contribute to this poor transformability. Restriction barriers posed by other bacteria have been overcome by the in vitro methylation of DNA prior to transformation. To test whether a methylation-sensitive restriction system contributes to poor B. burgdorferi transformability, shuttle plasmids were treated with the CpG methylase M.SssI prior to the electroporation of a variety of strains harboring different putative R-M systems. We found that for B. burgdorferi strains that harbor lp56, in vitro methylation increased transformation by at least 1 order of magnitude. These results suggest that in vitro CpG methylation protects exogenous DNA from degradation by an lp56-contained R-M system, presumably BBQ67. The utility of in vitro methylation for the genetic manipulation of B. burgdorferi was exemplified by the ease of plasmid complementation of a B. burgdorferi B31 A3 BBK32 kanamycin-resistant (B31 A3 BBK32::Kan(r)) mutant, deficient in the expression of the fibronectin- and glycosaminoglycan (GAG)-binding adhesin BBK32. Consistent with the observation that several surface proteins may promote GAG binding, the B. burgdorferi B31 A3 BBK32::Kan(r) mutant demonstrated no defect in the ability to bind purified GAGs or GAGs expressed on the surfaces of cultured cells.

Establishment of an in vitro cell line experimental system for the study of inhalational anesthetic mechanisms.

PubMed

Nagamoto, Seiji; Iijima, Norio; Ishii, Hirotaka; Takumi, Ken; Higo, Shimpei; Aikawa, Satoko; Anzai, Megumi; Matsuo, Izumi; Nakagawa, Shinji; Takashima, Naoyuki; Shigeyoshi, Yasufumi; Sakamoto, Atsuhiro; Ozawa, Hitoshi

2016-05-04

General anesthesia affects the expression of clock genes in various organs. Expression of Per2, a core component of the circadian clock, is markedly and reversibly suppressed by sevoflurane in the suprachiasmatic nucleus (SCN), and is considered to be a biochemical marker of anesthetic effect in the brain. The SCN contains various types of neurons, and this complexity makes it difficult to investigate the molecular mechanisms of anesthesia. Here, we established an in vitro experimental system using a cell line to investigate the mechanisms underlying anesthetic action. Development of the system comprised two steps: first, we developed a system for application of inhalational anesthetics and incubation; next, we established cultures of anesthetic-responsive cells expressing mPer2 promoter-dLuc. GT1-7 cells, derived from the mouse hypothalamus, responded to sevoflurane by reversibly decreasing mPer2-promoter-driven bioluminescence. Interestingly, the suppression of bioluminescence was found only in the serum-starved GT1-7 cells, which showed neuron-like morphology, but not in growing cells, suggesting that neuron-like characteristics are required for anesthetic effects in GT1-7 cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs.

PubMed

Romanienko, Peter J; Giacalone, Joseph; Ingenito, Joanne; Wang, Yijie; Isaka, Mayumi; Johnson, Thomas; You, Yun; Mark, Willie H

2016-01-01

The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA) and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6). However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

PubMed

Torelli, A; Soragni, E; Bolchi, A; Petrucco, S; Ottonello, S; Branca, C

1996-12-01

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

In vitro Catecholamine Exposure Produces Variable Effects on the B7 Costimulatory Pathway in Human Monocytic Cells

NASA Technical Reports Server (NTRS)

Salicru, A. N.; Crucian, B.; Sams, Clarence; Actor, J. K.; Marshall, G. D., Jr.

2006-01-01

Catecholamines have been associated with immunomodulation of the adaptive immune system towards a Th2 response in vitro. We therefore examined the role of in vitro epinephrine (EPI) and norepinephrine (NE) exposure on the B7 costimulatory expression of antigen presenting cells (APC) from human monocytic cell lines and human peripheral blood mononuclear cells (PBMC). THP1 monocytic cells and CD14+ cells from normal human PBMC were stimulated with lipopolysaccharide (LPS) and incubated with physiologic stress levels (10(exp -6) - 10(exp -8)M) of EPI or NE for 24 hours. Cells were subsequently stained with CD80 FITC, CD86 PE, and CD14 PC5 antibodies and analyzed by flow cytometry for changes in fluorescence and mean fluorescence intensity (MFI). Exposure of THP1 to EPI in vitro at concentrations of 10(exp -6), 10(exp -7) and 10(exp -8)M significantly decreased mean CD80 from 42 plus or minus 0.7% to 11 plus or minus 0.44%, 19.1 plus or minus 2.0%, and 30.7 plus or minus 2.1% expression, respectively (p less than 0.01). In addition, CD86 expression increased with EPI at 10(exp -6), 10(exp -7) and 10(exp -8) M from 9.2 plus or minus 0.52% to 41 plus or minus 3.8%, 26.4 plus or minus 1.9%, and 15.74 plus or minus 1.8% expression, respectively (p less than 0.01). Similar results for mean CD80 and CD86 percent expression were observed for CD14+ cells from PBMC with a sample size of N = 6 and for NE when substituted for EPI. The data show that in vitro exposure to catecholamines significantly decreases %CD86 expression and significantly increases %CD86 expression in THP1 cells and human CD14+ APC. Previous studies have suggested an association between increased CD86 expression and TH2 activity. Thus, these data suggest that immunomodulation by catecholamines results in part by the variable effects of the B7 costimulatory pathway in APC.

A Metaproteomics Approach to Elucidate Host and Pathogen Protein Expression during Catheter-Associated Urinary Tract Infections (CAUTIs)

PubMed Central

Lassek, Christian; Burghartz, Melanie; Chaves-Moreno, Diego; Otto, Andreas; Hentschker, Christian; Fuchs, Stephan; Bernhardt, Jörg; Jauregui, Ruy; Neubauer, Rüdiger; Becher, Dörte; Pieper, Dietmar H.; Jahn, Martina; Jahn, Dieter; Riedel, Katharina

2015-01-01

Long-term catheterization inevitably leads to a catheter-associated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro- and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosa seems to express secreted and surface-exposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system. PMID:25673765

A metaproteomics approach to elucidate host and pathogen protein expression during catheter-associated urinary tract infections (CAUTIs).

PubMed

Lassek, Christian; Burghartz, Melanie; Chaves-Moreno, Diego; Otto, Andreas; Hentschker, Christian; Fuchs, Stephan; Bernhardt, Jörg; Jauregui, Ruy; Neubauer, Rüdiger; Becher, Dörte; Pieper, Dietmar H; Jahn, Martina; Jahn, Dieter; Riedel, Katharina

2015-04-01

Long-term catheterization inevitably leads to a catheter-associated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (1) to unravel bacterial community structure and function of such a uropathogenic biofilm and (2) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro- and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, that is, Pseudomonas aeruginosa, Morganella morganii, and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Although P. aeruginosa seems to express secreted and surface-exposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages, and the complement system in the patient's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of catheter-associated urinary tract infections might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Self-renewal and multilineage differentiation of mouse dental epithelial stem cells.

PubMed

Chang, Julia Yu Fong; Wang, Cong; Jin, Chengliu; Yang, Chaofeng; Huang, Yanqing; Liu, Junchen; McKeehan, Wallace L; D'Souza, Rena N; Wang, Fen

2013-11-01

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies. © 2013.

Self-renewal and Multilineage Differentiation of Mouse Dental Epithelial Stem Cells

PubMed Central

Chang, Julia Yu Fong; Wang, Cong; Jin, Chengliu; Yang, Chaofeng; Huang, Yanqing; Liu, Junchen; McKeehan, Wallace L.; D’Souza, Rena N.; Wang, Fen

2013-01-01

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49fBright population was enriched in sphere-forming cells. In addition, the CD49fBright population includes both slow-cycling and Lgr5+ DESCs. The in vitro sphere culture system and identification of CD49fBright as a DESC marker provide a novel plateform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies. PMID:23906788

RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures.

PubMed

Morabito, Caterina; Steimberg, Nathalie; Mazzoleni, Giovanna; Guarnieri, Simone; Fanò-Illic, Giorgio; Mariggiò, Maria A

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

RCCS Bioreactor-Based Modelled Microgravity Induces Significant Changes on In Vitro 3D Neuroglial Cell Cultures

PubMed Central

Mazzoleni, Giovanna; Fanò-Illic, Giorgio; Mariggiò, Maria A.

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions. PMID:25654124

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of themore » TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.« less

Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

PubMed Central

Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

2013-01-01

Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

PubMed Central

Wang, Yarong; Du, Dewei; Li, Zhanting; Wei, Junxia; Yang, Angang

2012-01-01

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats. PMID:22990115

In Vivo and In Vitro Characterization of a Plasmodium Liver Stage-Specific Promoter

PubMed Central

Horstmann, Sebastian; Annoura, Takeshi; del Portillo, Hernando A.; Khan, Shahid M.; Heussler, Volker T.

2015-01-01

Little is known about stage-specific gene regulation in Plasmodium parasites, in particular the liver stage of development. We have previously described in the Plasmodium berghei rodent model, a liver stage-specific (lisp2) gene promoter region, in vitro. Using a dual luminescence system, we now confirm the stage specificity of this promoter region also in vivo. Furthermore, by substitution and deletion analyses we have extended our in vitro characterization of important elements within the promoter region. Importantly, the dual luminescence system allows analyzing promoter constructs avoiding mouse-consuming cloning procedures of transgenic parasites. This makes extensive mutation and deletion studies a reasonable approach also in the malaria mouse model. Stage-specific expression constructs and parasite lines are extremely valuable tools for research on Plasmodium liver stage biology. Such reporter lines offer a promising opportunity for assessment of liver stage drugs, characterization of genetically attenuated parasites and liver stage-specific vaccines both in vivo and in vitro, and may be key for the generation of inducible systems. PMID:25874388

GCaMP expression in retinal ganglion cells characterized using a low-cost fundus imaging system

NASA Astrophysics Data System (ADS)

Chang, Yao-Chuan; Walston, Steven T.; Chow, Robert H.; Weiland, James D.

2017-10-01

Objective. Virus-transduced, intracellular-calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. Due to the existence of an optimal time window for the expression of calcium indicators, a suitable tool for tracking GECI expression in vivo following transduction is highly desirable. Approach. We developed a noninvasive imaging approach based on a custom-modified, low-cost fundus viewing system that allowed us to monitor and characterize in vivo bright-field and fluorescence images of the mouse retina. AAV2-CAG-GCaMP6f was injected into a mouse eye. The fundus imaging system was used to measure fluorescence at several time points post injection. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array and used calcium imaging to evaluate the responsiveness of retinal ganglion cells (RGCs) to external electrical stimulation. Main results. The noninvasive fundus imaging system clearly resolves individual (RGCs and axons. RGC fluorescence intensity and the number of observable fluorescent cells show a similar rising trend from week 1 to week 3 after viral injection, indicating a consistent increase of GCaMP6f expression. Analysis of the in vivo fluorescence intensity trend and in vitro neurophysiological responsiveness shows that the slope of intensity versus days post injection can be used to estimate the optimal time for calcium imaging of RGCs in response to external electrical stimulation. Significance. The proposed fundus imaging system enables high-resolution digital fundus imaging in the mouse eye, based on off-the-shelf components. The long-term tracking experiment with in vitro calcium imaging validation demonstrates the system can serve as a powerful tool monitoring the level of genetically-encoded calcium indicator expression, further determining the optimal time window for following experiment.

Functional characterization of an unusual phytochelatin synthase, LjPCS3, of Lotus japonicus.

PubMed

Ramos, Javier; Naya, Loreto; Gay, Marina; Abián, Joaquín; Becana, Manuel

2008-09-01

In plants and many other organisms, phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins from glutathione in the presence of certain metals and metalloids. We have used budding yeast (Saccharomyces cerevisiae) as a heterologous system to characterize two PCS proteins, LjPCS1 and LjPCS3, of the model legume Lotus japonicus. Initial experiments revealed that the metal tolerance of yeast cells in vivo depends on the concentrations of divalent cations in the growth medium. Detailed in vivo (intact cells) and in vitro (broken cells) assays of PCS activity were performed with yeast expressing the plant enzymes, and values of phytochelatin production for each metal tested were normalized with respect to those of cadmium to correct for the lower expression level of LjPCS3. Our results showed that lead was the best activator of LjPCS1 in the in vitro assay, whereas, for both assays, arsenic, iron, and aluminum were better activators of LjPCS3 and mercury was similarly active with the two enzymes. Most interestingly, zinc was a powerful activator, especially of LjPCS3, when assayed in vivo, whereas copper and silver were the strongest activators in the in vitro assay. We conclude that the in vivo and in vitro assays are useful and complementary to assess the response of LjPCS1 and LjPCS3 to a wide range of metals and that the differences in the C-terminal domains of the two proteins are responsible for their distinct expression levels or stabilities in heterologous systems and patterns of metal activation.

Toxicology studies of primycin-sulphate using a three-dimensional (3D) in vitro human liver aggregate model.

PubMed

Pénzes, Ágota; Mahmud Abdelwahab, Elhusseiny Mohamed; Rapp, Judit; Péteri, Zsanett A; Bovári-Biri, Judit; Fekete, Csaba; Miskei, György; Kvell, Krisztián; Pongrácz, Judit E

2017-11-05

Primycin-sulphate is a highly effective compound against Gram (G) positive bacteria. It has a potentially synergistic effect with vancomycin and statins which makes primycin-sulphate a potentially very effective preparation. Primycin-sulphate is currently used exclusively in topical preparations. In vitro animal hepatocyte and neuromuscular junction studies (in mice, rats, snakes, frogs) as well as in in vitro human red blood cell experiments were used to test toxicity. During these studies, the use of primycin-sulphate resulted in reduced cellular membrane integrity and modified ion channel activity. Additionally, parenteral administration of primycin-sulphate to mice, dogs, cats, rabbits and guinea pigs indicated high level of acute toxicity. The objective of this study was to reveal the cytotoxic and gene expression modifying effects of primycin-sulphate in a human system using an in vitro, three dimensional (3D) human hepatic model system. Within the 3D model, primycin-sulphate presented no acute cytotoxicity at concentrations 1μg/ml and below. However, even at low concentrations, primycin-sulphate affected gene expressions by up-regulating inflammatory cytokines (e.g., IL6), chemokines (e.g., CXCL5) and by down-regulating molecules of the lipid metabolism (e.g., peroxisome proliferator receptor (PPAR) alpha, gamma, etc). Down-regulation of PPAR alpha cannot just disrupt lipid production but can also affect cytochrome P450 metabolic enzyme (CYP) 3A4 expression, highlighting the need for extensive drug-drug interaction (DDI) studies before human oral or parenteral preparations can be developed. Copyright © 2017 Elsevier B.V. All rights reserved.

Transgene Expression and Host Cell Responses to Replication-Defective, Single-Cycle, and Replication-Competent Adenovirus Vectors.

PubMed

Crosby, Catherine M; Barry, Michael A

2017-02-18

Most adenovirus (Ad) vectors are E1 gene deleted replication defective (RD-Ad) vectors that deliver one transgene to the cell and all expression is based on that one gene. In contrast, E1-intact replication-competent Ad (RC-Ad) vectors replicate their DNA and their transgenes up to 10,000-fold, amplifying transgene expression markedly higher than RD-Ad vectors. While RC-Ad are more potent, they run the real risk of causing adenovirus infections in vector recipients and those that administer them. To gain the benefits of transgene amplification, but avoid the risk of Ad infections, we developed "single cycle" Ad (SC-Ad) vectors. SC-Ads amplify transgene expression and generated markedly stronger and more persistent immune responses than RD-Ad as expected. However, they also unexpectedly generated stronger immune responses than RC-Ad vectors. To explore the basis of this potency here, we compared gene expression and the cellular responses to infection to these vectors in vitro and in vivo. In vitro, in primary human lung epithelial cells, SC- and RC-Ad amplified their genomes more than 400-fold relative to RD-Ad with higher replication by SC-Ad. This replication translated into higher green fluorescent protein (GFP) expression for 48 h by SC- and RC-Ad than by RD-Ad. In vitro, in the absence of an immune system, RD-Ad expression became higher by 72 h coincident with cell death mediated by SC- and RC-Ad and release of transgene product from the dying cells. When the vectors were compared in human THP-1 Lucia- interferon-stimulated gene (ISG) cells, which are a human monocyte cell line that have been modified to quantify ISG activity, RC-Ad6 provoked significantly stronger ISG responses than RD- or SC-Ad. In mice, intravenous or intranasal injection produced up to 100-fold genome replication. Under these in vivo conditions in the presence of the immune system, luciferase expression by RC and SC-Ad was markedly higher than that by RD-Ad. In immunodeficient mice, SC-Ad drove stronger luciferase expression than RC- or RD-Ad. These data demonstrate better transgene expression by SC- and RC-Ad in vitro and in vivo than RD-Ad. This higher expression by the replicating vectors results in a peak of expression within 1 to 2 days followed by cell death of infected cells and release of transgene products. While SC- and RC-Ad expression were similar in mice and in Syrian hamsters, RC-Ad provoked much stronger ISG induction which may explain in part SC-Ad's ability to generate stronger and more persistent immune responses than RC-Ad in Ad permissive hamsters.

The Transcription Factor p53 Influences Microglial Activation Phenotype

PubMed Central

Jayadev, Suman; Nesser, Nicole K.; Hopkins, Stephanie; Myers, Scott J.; Case, Amanda; Lee, Rona J.; Seaburg, Luke A.; Uo, Takuma; Murphy, Sean P.; Morrison, Richard S.; Garden, Gwenn A.

2011-01-01

Several neurodegenerative diseases are influenced by the innate immune response in the central nervous system (CNS). Microglia, have pro-inflammatory and subsequently neurotoxic actions as well as anti-inflammatory functions that promote recovery and repair. Very little is known about the transcriptional control of these specific microglial behaviors. We have previously shown that in HIV associated neurocognitive disorders (HAND), the transcription factor p53 accumulates in microglia and that microglial p53 expression is required for the in vitro neurotoxicity of the HIV coat glycoprotein gp120. These findings suggested a novel function for p53 in regulating microglial activation. Here we report that in the absence of p53, microglia demonstrate a blunted response to interferon-γ, failing to increase expression of genes associated with classical macrophage activation or secrete pro-inflammatory cytokines. Microarray analysis of global gene expression profiles revealed increased expression of genes associated with anti-inflammatory functions, phagocytosis and tissue repair in p53 knockout (p53−/−) microglia compared with those cultured from strain matched p53 expressing (p53+/+) mice. We further observed that p53−/− microglia demonstrate increased phagocytic activity in vitro and expression of markers for alternative macrophage activation both in vitro and in vivo. In HAND brain tissue, the alternative activation marker CD163 was expressed in a separate subset of microglia than those demonstrating p53 accumulation. These data suggest that p53 influences microglial behavior, supporting the adoption of a pro-inflammatory phenotype, while p53 deficiency promotes phagocytosis and gene expression associated with alternative activation and anti-inflammatory functions. PMID:21598312

[Regulating human interferon-gamma gene expression in marrow stromal cells in mice by Tet-off system].

PubMed

Qin, Xin-Tian; Lu, Yue; Tan, Yin-Duo; Chen, Xiao-Qin; Gen, Qi-Rong

2008-01-01

We have constructed plasmid "pTre-IFN-gamma" and proved that the Tet-off system could regulate the expression of human interferon-gamma (IFN-gamma) gene in murine marrow stromal cells in vitro. This study was to investigate the regulatory reversibility of Tet-off system and its effect on the expression of human IFN-gamma gene in murine marrow stromal cells in mice. Plasmids pTet-off and pTre-IFN-gamma were co-transfected into murine marrow stromal cells. The expression of IFN-gamma in marrow stromal cells was detected with ELISA. The marrow stromal cells were transfused into BABL/c naked mice after co-transfection. The expression of IFN-gamma mRNA in the spleen was detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR). IFN-gamma protein was detected in the culture solution of marrow stromal cells after co-transfection. The secretion peak appeared within the first 72 h. The protein level of IFN-gamma was significantly lower in 300 ng/ml tetracycline hydrochloride-treated marrow stroma cells than in untreated cells [(67.11+/-22.14) pg/1 x 10(7) cells vs. (319.96+/-29.04) pg/1 x 10(7) cells, P<0.001]; its expression was increased when removed tetracycline hydrochloride (P=0.032). The expression of human IFN-gamma mRNA was detected in the spleen. The mRNA level of IFN-gamma was significantly higher in untreated group than in continuous tetracycline hydrochloride-treated group [(1.5+/-0.7)x10(5) copies . (100 mg)(-1) vs. (6.9+/-5.3)x10(2) copies . (100 mg)(-1), P<0.001]; its expression in the mice received tetracycline hydrochloride for one single time lay between the above two groups with significant difference. In mice, Tet-off system could rapidly, efficiently and reversibly regulate the expression of human IFN-gamma gene in marrow stromal cells in vitro and in vivo.

Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

EPA Science Inventory

Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

Downregulation of SS18-SSX1 expression in synovial sarcoma by small interfering RNA enhances the focal adhesion pathway and inhibits anchorage-independent growth in vitro and tumor growth in vivo.

PubMed

Takenaka, Satoshi; Naka, Norifumi; Araki, Nobuhito; Hashimoto, Nobuyuki; Ueda, Takafumi; Yoshioka, Kiyoko; Yoshikawa, Hideki; Itoh, Kazuyuki

2010-04-01

Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by a unique t(X;18) translocation resulting in expression of SS18-SSX fusion protein. In order to investigate the biological function of this fusion protein and to develop a novel therapeutic option, we examined downregulation of SS18-SSX1 expression by small interfering RNA targeting SS18-SSX1 in three human SS cell lines. Microarray analysis comparing SS18-SSX1-silenced cells with control cells in three SS cell lines showed that SS18-SSX1 mainly affected the focal adhesion pathway. In accord with the array data, silencing of SS18-SSX1 enhances adhesion to the extracellular matrix through the induction of expression of myosin light-chain kinase. Furthermore, the silencing of SS18-SSX1 inhibits anchorage-independent growth in vitro and systemic delivery of siRNA against SS18-SSX1 using a nanoparticle system inhibited tumor growth in a nude mouse xenograft model. Our results demonstrate that siRNA targeting of SS18-SSX1 has therapeutic potential for the treatment of SS.

Long-term Neuroglial Cocultures as a Brain Aging Model: Hallmarks of Senescence, MicroRNA Expression Profiles, and Comparison With In Vivo Models.

PubMed

Bigagli, Elisabetta; Luceri, Cristina; Scartabelli, Tania; Dolara, Piero; Casamenti, Fiorella; Pellegrini-Giampietro, Domenico E; Giovannelli, Lisa

2016-01-01

Our purpose was to evaluate long-term neuroglial cocultures as a model for investigating senescence in the nervous system and to assess its similarities with in vivo models. To this aim, we maintained the cultures from 15 days in vitro (mature cultures) up to 27 days in vitro (senescent cultures), measuring senescence-associated, neuronal, dendritic, and astrocytic markers. Whole microRNA expression profiles were compared with those measured in the cortex of 18- and 24-month-old C57Bl/6J aged mice and of transgenic TgCRND8 mice, a model of amyloid-β deposition. Neuroglial cocultures displayed features of cellular senescence (increased senescence-associated-β-galactosidase activity, oxidative stress, γ-H2AX expression, IL-6 production, astrogliosis) that were concentration dependently counteracted by the antiaging compound resveratrol (1-5 µM). Among the 1,080 microRNAs analyzed, 335 were downregulated or absent in 27 compared with 15 days in vitro and resveratrol reversed this effect. A substantial overlapping was found between age-associated changes in microRNA expression profiles in vitro and in TgCRND8 mice but not in physiologically aged mice, indicating that this culture model displays more similarities with pathological than physiological brain aging. Our results demonstrate that neuroglial cocultures aged in vitro can be useful for investigating the cellular and molecular mechanisms of brain aging and for preliminary testing of protective compounds. © The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription.

PubMed Central

Brady, J; Radonovich, M; Thoren, M; Das, G; Salzman, N P

1984-01-01

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294. Images PMID:6321950

Human breast cancer histoid: an in vitro 3-dimensional co-culture model that mimics breast cancer tissue.

PubMed

Kaur, Pavinder; Ward, Brenda; Saha, Baisakhi; Young, Lillian; Groshen, Susan; Techy, Geza; Lu, Yani; Atkinson, Roscoe; Taylor, Clive R; Ingram, Marylou; Imam, S Ashraf

2011-12-01

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 µm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.

Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation

PubMed Central

2012-01-01

Background The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (HBG) to replace abnormal adult sickle βS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to β-globin (γ/β) switch observed throughout in vitro erythroid differentiation. Results We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified. The same approach was used to generate profile-2 genes where expression was up-regulated over 28 days in culture. IPA for the 2,437 genes with > 1.5-fold induction identified the mitotic roles of polo-like kinase, aryl hydrocarbon receptor, cell cycle control, and ATM (Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included KLF1, GATA1 and NFE2 among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the γ/β-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways. Conclusions The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is an excellent model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation. PMID:22537182

Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9.

PubMed

Kurihara, Takeshi; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Uemura, Kentaro; Okamoto, Toru; Sugiyama, Masaya; Motooka, Daisuke; Nakamura, Shota; Ikawa, Masato; Mizokami, Masashi; Maehara, Yoshihiko; Matsuura, Yoshiharu

2017-07-21

Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.

Inhibition of inflammatory gene expression in keratinocytes using a composition containing carnitine, thioctic Acid and saw palmetto extract.

PubMed

Chittur, Sridar; Parr, Brian; Marcovici, Geno

2011-01-01

Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA). Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr) and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS) provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4) associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maneckjee, R.; Minna, J.D.

Using specific radioactively-labeled ligands, the authors find that lung cancer cell lines of diverse histologic types express multiple, high-affinity membrane receptors for {mu}, {delta}, and {kappa} opioid agonists and for nicotine and {alpha}-bungarotoxin. These receptors are biologically active because cAMP levels decreased in lung cancer cells after opioid and nicotine application. Nicotine at concentrations found in the blood of smokers had no effect on in vitro lung cancer cell growth, whereas {mu}, {delta}, and {kappa} opioid agonists at low concentrations inhibited lung cancer growth in vitro. They also found that lung cancer cells expressed various combinations of immunoreactive opioid peptidesmore » ({beta}-endorphin, enkephalin, or dynorphin), suggesting the participation of opioids in a negative autocrine loop or tumor-suppressing system. Due to the almost universal exposure of patients with lung cancer to nicotine, they tested whether nicotine affected the response of lung cancer cell growth to opioids and found that nicotine at concentrations of 100-200 nM partially or totally reversed opioid-induced growth inhibition in 9/14 lung cancer cell lines. These in vitro results for lung cancer cells suggest that opioids could function as part of a tumor suppressor system and that nicotine can function to circumvent this system in the pathogenesis of lung cancer.« less

Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey.

PubMed

Thoma, Eva C; Heckel, Tobias; Keller, David; Giroud, Nicolas; Leonard, Brian; Christensen, Klaus; Roth, Adrian; Bertinetti-Lapatki, Cristina; Graf, Martin; Patsch, Christoph

2016-10-25

Due to their broad differentiation potential, pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced, similar systems for non-human primates (NHPs) are still lacking. However, as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals, the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here, we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs, we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover, we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions, such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species.

Wnt1a maintains characteristics of dermal papilla cells that induce mouse hair regeneration in a 3D preculture system.

PubMed

Dong, Liang; Hao, Haojie; Liu, Jiejie; Tong, Chuan; Ti, Dongdong; Chen, Deyun; Chen, Li; Li, Meirong; Liu, Huiling; Fu, Xiaobing; Han, Weidong

2017-05-01

Hair follicle morphogenesis and regeneration depend on intensive but well-orchestrated interactions between epithelial and mesenchymal components. Therefore, an alternative strategy to reproduce the process of epithelial-mesenchymal interaction in vitro could use a 3D system containing appropriate cell populations. The 3D air-liquid culture system for reproducibly generating hair follicles from dissociated epithelial and dermal papilla (DP) cells combined with a collagen-chitosan scaffold is described in this study. Wnt-CM was prepared from the supernatant of Wnt1a-expressing bone marrow mesenchymal stem cells (BM-MSCs) that maintain the hair-inducing gene expression of DP cells. The collagen-chitosan scaffold cells (CCS cells) were constructed using a two-step method by inoculating the Wnt-CM-treated DP cells and epidermal (EP) cells into the CCS. The cells in the air-liquid culture formed dermal condensates and a proliferative cell layer in vitro. The CCS cells were able to induce hair regeneration in nude mice. The results demonstrate that Wnt-CM can maintain the hair induction ability of DP cells in expansion cultures, and this approach can be used for large-scale preparation of CCS cells in vitro to treat hair loss. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

In vitro culture of stress erythroid progenitors identifies distinct progenitor populations and analogous human progenitors.

PubMed

Xiang, Jie; Wu, Dai-Chen; Chen, Yuanting; Paulson, Robert F

2015-03-12

Tissue hypoxia induces a systemic response designed to increase oxygen delivery to tissues. One component of this response is increased erythropoiesis. Steady-state erythropoiesis is primarily homeostatic, producing new erythrocytes to replace old erythrocytes removed from circulation by the spleen. In response to anemia, the situation is different. New erythrocytes must be rapidly made to increase hemoglobin levels. At these times, stress erythropoiesis predominates. Stress erythropoiesis is best characterized in the mouse, where it is extramedullary and utilizes progenitors and signals that are distinct from steady-state erythropoiesis. In this report, we use an in vitro culture system that recapitulates the in vivo development of stress erythroid progenitors. We identify cell-surface markers that delineate a series of stress erythroid progenitors with increasing maturity. In addition, we use this in vitro culture system to expand human stress erythroid progenitor cells that express analogous cell-surface markers. Consistent with previous suggestions that human stress erythropoiesis is similar to fetal erythropoiesis, we demonstrate that human stress erythroid progenitors express fetal hemoglobin upon differentiation. These data demonstrate that similar to murine bone marrow, human bone marrow contains cells that can generate BMP4-dependent stress erythroid burst-forming units when cultured under stress erythropoiesis conditions. © 2015 by The American Society of Hematology.

Assessment of anti-oxidant activity of plant extracts using microbial test systems.

PubMed

Oktyabrsky, O; Vysochina, G; Muzyka, N; Samoilova, Z; Kukushkina, T; Smirnova, G

2009-04-01

To evaluate the anti-oxidant properties of extracts from 20 medicinal herbs growing in western Siberia using microbial test systems and different in vitro methods. In vivo anti-oxidant activity of extracts was evaluated for their capacity to protect bacteria, Escherichia coli, against bacteriostatic and bactericidal effects of H(2)O(2) and menadione, and action on anti-oxidant gene expression. In vitro anti-oxidant activity has been examined by a number of methods including: the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH(*))-scavenging assay, chelating activity and capacity to protect plasmid DNA against oxidative damage. In addition, total polyphenol content was determined. The extracts of Fragaria vesca, Rosa majalis, Pentaphylloides fruticosa, Alchemilla vulgaris and Pulmonaria mollis possessed the highest levels of anti-oxidant activity in vivo and in vitro. The protective properties were more closely related to the DPPH(*) radical-scavenging activity, tannin content and action on anti-oxidant gene expression than to other parameters. The extracts of medicinal plants may have anti-oxidant effects on bacteria simultaneously through several different pathways, including direct inhibition of reactive oxygen species, iron chelation and anti-oxidant genes induction. Using microbial test systems, we revealed herbs that may be used as potential sources of natural anti-oxidants.

Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte‐derived dendritic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sebastian, Katrin, E-mail: ksebastian@ukaachen.de; Ott, Hagen; Zwadlo-Klarwasser, Gabriele

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides – which are the most frequent cause of adverse drug reactions – were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIRmore » and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines. -- Highlights: ► We tested the sensitizing potential of small molecular weight drugs in vitro. ► In vitro assays were performed with moDCs and THP-1 cells. ► Beta-lactam antibiotics can be recognized as sensitizing compounds. ► They affect the expression of metabolic enzymes, cytokines and transcription factors. ► Sulfamethoxazole has no measurable effect on THP-1 cells and moDCs.« less

Renin knockout rat: control of adrenal aldosterone and corticosterone synthesis in vitro and adrenal gene expression

PubMed Central

Gehrand, Ashley; Bruder, Eric D.; Hoffman, Matthew J.; Engeland, William C.; Moreno, Carol

2014-01-01

The classic renin-angiotensin system is partly responsible for controlling aldosterone secretion from the adrenal cortex via the peptide angiotensin II (ANG II). In addition, there is a local adrenocortical renin-angiotensin system that may be involved in the control of aldosterone synthesis in the zona glomerulosa (ZG). To characterize the long-term control of adrenal steroidogenesis, we utilized adrenal glands from renin knockout (KO) rats and compared steroidogenesis in vitro and steroidogenic enzyme expression to wild-type (WT) controls (Dahl S rat). Adrenal capsules (ZG; aldosterone production) and subcapsules [zona reticularis/fasciculata (ZFR); corticosterone production] were separately dispersed and studied in vitro. Plasma renin activity and ANG II concentrations were extremely low in the KO rats. Basal and cAMP-stimulated aldosterone production was significantly reduced in renin KO ZG cells, whereas corticosterone production was not different between WT and KO ZFR cells. As expected, adrenal renin mRNA expression was lower in the renin KO compared with the WT rat. Real-time PCR and immunohistochemical analysis showed a significant decrease in P450aldo (Cyp11b2) mRNA and protein expression in the ZG from the renin KO rat. The reduction in aldosterone synthesis in the ZG of the renin KO adrenal seems to be accounted for by a specific decrease in P450aldo and may be due to the absence of chronic stimulation of the ZG by circulating ANG II or to a reduction in locally released ANG II within the adrenal gland. PMID:25394830

Pseudomonas aeruginosa overexpression system of nitric oxide reductase for in vivo and in vitro mutational analyses.

PubMed

Yamagiwa, Raika; Kurahashi, Takuya; Takeda, Mariko; Adachi, Mayuho; Nakamura, Hiro; Arai, Hiroyuki; Shiro, Yoshitsugu; Sawai, Hitomi; Tosha, Takehiko

2018-05-01

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N 2 O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO 2 - ) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies. Copyright © 2018 Elsevier B.V. All rights reserved.

In vitro Multi-Species Biofilms of Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa and Their Host Interaction during In vivo Colonization of an Otitis Media Rat Model

PubMed Central

Yadav, Mukesh K.; Chae, Sung-Won; Go, Yoon Young; Im, Gi Jung; Song, Jae-Jun

2017-01-01

Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are known to cause biofilm-related infections. MRSA and PA have been frequently isolated from chronically infected wounds, cystic fibrosis, chronic suppurative otitis media (CSOM), and from indwelling medical devices, and these bacteria co-exist; however, their interaction with each-other or with the host is not well known. In this study, we investigated MRSA and PA multi-species biofilm communities in vitro and their interaction with the host during in vivo colonization using an OM rat-model. In-vitro biofilm formation and in-vivo colonization were studied using CV-microtiter plate assay and OM rat-model respectively. The biofilms were viewed under scanning electron microscope and bacteria were enumerated using cfu counts. The differential gene expressions of rat mucosa colonized with single or multi-species of MRSA or PA were studied using RNA-sequencing of total transcriptome. In multi-species in-vitro biofilms PA partially inhibited SA growth. However, no significant inhibition of MRSA was detected during in-vivo colonization of multi-species in rat bullae. A total of 1,797 genes were significantly (p < 0.05) differentially expressed in MRSA or PA or MRSA + PA colonized rat middle ear mucosa with respect to the control. The poly-microbial colonization of MRSA and PA induced the differential expression of a significant number of genes that are involved in immune response, inflammation, signaling, development, and defense; these were not expressed with single species colonization by either MRSA or PA. Genes involved in defense, immune response, inflammatory response, and developmental process were exclusively up-regulated, and genes that are involved in nervous system signaling, development and transmission, regulation of cell growth and development, anatomical and system development, and cell differentiation were down-regulated after multi-species inoculation. These results indicate that poly-microbial colonization induces a host response that is different from that induced by single species infection. PMID:28459043

Thyroid Hormone Receptor β (TRβ) and Liver X Receptor (LXR) Regulate Carbohydrate-response Element-binding Protein (ChREBP) Expression in a Tissue-selective Manner*

PubMed Central

Gauthier, Karine; Billon, Cyrielle; Bissler, Marie; Beylot, Michel; Lobaccaro, Jean-Marc; Vanacker, Jean-Marc; Samarut, Jacques

2010-01-01

Thyroid hormone (TR) and liver X (LXR) receptors are transcription factors involved in lipogenesis. Both receptors recognize the same consensus DNA-response element in vitro. It was previously shown that their signaling pathways interact in the control of cholesterol elimination in the liver. In the present study, carbohydrate-response element-binding protein (ChREBP), a major transcription factor controlling the activation of glucose-induced lipogenesis in liver, is characterized as a direct target of thyroid hormones (TH) in liver and white adipose tissue (WAT), the two main lipogenic tissues in mice. Using genetic and molecular approaches, ChREBP is shown to be specifically regulated by TRβ but not by TRα in vivo, even in WAT where both TR isoforms are expressed. However, this isotype specificity is not found in vitro. This TRβ specific regulation correlates with the loss of TH-induced lipogenesis in TRβ−/− mice. Fasting/refeeding experiments show that TRβ is not required for the activation of ChREBP expression particularly marked in WAT following refeeding. However, TH can stimulate ChREBP expression in WAT even under fasting conditions, suggesting completely independent pathways. Because ChREBP has been described as an LXR target, the interaction of LXR and TRβ in ChREBP regulation was assayed both in vitro and in vivo. Each receptor recognizes a different response element on the ChREBP promoter, located only 8 bp apart. There is a cross-talk between LXR and TRβ signaling on the ChREBP promoter in liver but not in WAT where LXR does not regulate ChREBP expression. The molecular basis for this cross-talk has been determined in in vitro systems. PMID:20615868

Comparative expression analysis reveals lineage relationships between human and murine gliomas and a dominance of glial signatures during tumor propagation in vitro.

PubMed

Henriquez, Nico V; Forshew, Tim; Tatevossian, Ruth; Ellis, Matthew; Richard-Loendt, Angela; Rogers, Hazel; Jacques, Thomas S; Reitboeck, Pablo Garcia; Pearce, Kerra; Sheer, Denise; Grundy, Richard G; Brandner, Sebastian

2013-09-15

Brain tumors are thought to originate from stem/progenitor cell populations that acquire specific genetic mutations. Although current preclinical models have relevance to human pathogenesis, most do not recapitulate the histogenesis of the human disease. Recently, a large series of human gliomas and medulloblastomas were analyzed for genetic signatures of prognosis and therapeutic response. Using a mouse model system that generates three distinct types of intrinsic brain tumors, we correlated RNA and protein expression levels with human brain tumors. A combination of genetic mutations and cellular environment during tumor propagation defined the incidence and phenotype of intrinsic murine tumors. Importantly, in vitro passage of cancer stem cells uniformly promoted a glial expression profile in culture and in brain tumors. Gene expression profiling revealed that experimental gliomas corresponded to distinct subclasses of human glioblastoma, whereas experimental supratentorial primitive neuroectodermal tumors (sPNET) correspond to atypical teratoid/rhabdoid tumor (AT/RT), a rare childhood tumor. ©2013 AACR.

The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo

PubMed Central

Meerbrey, Kristen L.; Hu, Guang; Kessler, Jessica D.; Roarty, Kevin; Fang, Justin E.; Herschkowitz, Jason I.; Burrows, Anna E.; Ciccia, Alberto; Sun, Tingting; Schmitt, Earlene M.; Bernardi, Ronald J.; Fu, Xiaoyong; Bland, Christopher S.; Cooper, Thomas A.; Schiff, Rachel; Rosen, Jeffrey M.; Westbrook, Thomas F.; Elledge, Stephen J.

2011-01-01

The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets. PMID:21307310

Adipogenesis of bovine perimuscular preadipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taniguchi, Masaaki; Le Luo Guan; Zhang Bing

2008-02-01

In this study, non-transformed progeny adipofibroblasts, derived from mature adipocyte dedifferentiation, was used as a novel in vitro model to study adipogenic gene expression in cattle. Adipofibroblasts from dedifferentiated mature perimuscular fat (PMF) tissue were cultured with differentiation stimulants until the cells exhibited morphological differentiation. Treated cells were harvested from day 2 to 16 for RNA extraction, whereas control cells were cultured without addition of stimulants. Results from time course gene expression assays by quantitative real-time PCR revealed that peroxisome proliferator-activated receptor gamma (PPAR-{gamma}), sterol regulatory element binding protein 1 (SREBP-1) and their six down-stream genes were co-expressed at daymore » 2 post-differentiation induction. When compared to other adipogenesis culture systems, the adipogenic gene expression of bovine PMF adipofibroblasts culture was different, especially to the rodent model. Collectively, these results demonstrated PPAR-{gamma} and SREBP-1 cooperatively play a key role to regulate the re-differentiation of bovine adipofibroblasts, during early conversion stages in vitro.« less

Optimizing Culture Medium Composition to Improve Oligodendrocyte Progenitor Cell Yields In Vitro from Subventricular Zone-Derived Neural Progenitor Cell Neurospheres

PubMed Central

Franco, Paula G.; Pasquini, Juana M.; Silvestroff, Lucas

2015-01-01

Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC. PMID:25837625

Thyroid endocrine system disruption by pentachlorophenol: an in vitro and in vivo assay.

PubMed

Guo, Yongyong; Zhou, Bingsheng

2013-10-15

The present study aimed to evaluate the disruption caused to the thyroid endocrine system by pentachlorophenol (PCP) using in vitro and in vivo assays. In the in vitro assay, rat pituitary GH3 cells were exposed to 0, 0.1, 0.3, and 1.0 μM PCP. PCP exposure significantly downregulated basal and triiodothyronine (T3)-induced Dio 1 transcription, indicating the antagonistic activity of PCP in vitro. In the in vivo assay, zebrafish embryos were exposed to 0, 1, 3, and 10 μg/L of PCP until 14 days post-fertilization. PCP exposure resulted in decreased thyroxine (T4) levels, but elevated contents of whole-body T3. PCP exposure significantly upregulated the mRNA expression of genes along hypothalamic-pituitary-thyroid (HPT) axis, including those encoding thyroid-stimulating hormone, sodium/iodide symporter, thyroglobulin, Dio 1 and Dio 2, alpha and beta thyroid hormone receptor, and uridinediphosphate-glucuronosyl-transferase. PCP exposure did not influence the transcription of the transthyretin (TTR) gene. The results indicate that PCP potentially disrupts the thyroid endocrine system both in vitro and in vivo. Copyright © 2013 Elsevier B.V. All rights reserved.

Phenotypic Alterations Involved in CD8+ Treg Impairment in Systemic Sclerosis

PubMed Central

Negrini, Simone; Fenoglio, Daniela; Parodi, Alessia; Kalli, Francesca; Battaglia, Florinda; Nasi, Giorgia; Curto, Monica; Tardito, Samuele; Ferrera, Francesca; Filaci, Gilberto

2017-01-01

Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis, vasculopathy, and autoimmunity. Although the exact pathogenetic mechanisms behind SSc remain to be fully elucidated, a great deal of evidence suggests the existence of an unbalanced ratio between the effector and regulatory arms of the immune system. With regard to the T regulatory (Treg) compartment, we observed that CD8+ Treg subsets display functional defects in SSc-affected patients. Since CD127 down-modulation and CD39 upregulation have been observed on Treg subsets, the phenotypic expression of these molecules was analyzed on the CD8+CD28− Treg precursors and on CD8+ Treg cells generated in vitro through interleukin-10 commitment. Immunophenotypic data from SSc patients were compared to those obtained from healthy subjects. The analyses performed on ex vivo-isolated CD8+CD28− Treg precursors did not show any significant differences in CD39 or CD127 expression as compared to values obtained from healthy donors. On the contrary, in vitro-generated CD8+ Tregs obtained from SSc patients displayed reduced expression of the CD39 molecule as compared to controls. Moreover, the percentage of CD127+ cells was significantly higher in in vitro-generated CD8+ Tregs from SSc patients compared to CD8+ Tregs obtained from healthy donors. Taken together, these findings may indicate an impairment of maturation processes affecting CD8+ Treg cells in SSc patients. This impairment of maturation involves phenotypic alterations that are mainly characterized by a deficient CD39 upregulation and a lack of down-modulation of the CD127 molecule. PMID:28154567

Aseptic Culture Systems of Radopholus similis for In Vitro Assays on Musa spp. and Arabidopsis thaliana

PubMed Central

Elsen, A.; Lens, K.; Nguyet, D. T. M.; Broos, S.; Stoffelen, R.; De Waele, D.

2001-01-01

Radopholus similis is one of the most damaging nematodes in bananas. Chemical control is currently the most-used method, but nematode control through genetic improvement is widely encouraged. The objective of this study was to establish an aseptic culture system for R. similis and determine whether R. similis can infect and reproduce on in vitro banana plantlets and in vitro Arabidopsis thaliana. In the study's first part, a suitable aseptic culture system was developed using alfalfa callus. Radopholus similis could penetrate and reproduce in the callus. Six weeks after inoculation with 25 females, the reproduction ratio was 26.3 and all vermiform stages were present. The reproduction ratio increased to 223.2 after 12 weeks. Results of a greenhouse test showed that R. similis did not lose its pathogenicity after culturing on alfalfa callus. In the study's second part, the infection and reproduction of the nematodes cultured on the callus were studied on both in vitro banana plantlets and A. thaliana. Radopholus similis infected and reproduced on both banana and A. thaliana. Furthermore, nematode damage was observed in the root systems of both hosts. These successful infections open new perspectives for rapid in vitro screening for resistance in banana cultivars and anti-nematode proteins expressed in A. thaliana. PMID:19266012

Implantable wireless powered light emitting diode (LED) for near-infrared photoimmunotherapy: device development and experimental assessment in vitro and in vivo.

PubMed

Nakajima, Kohei; Kimura, Toshihiro; Takakura, Hideo; Yoshikawa, Yasuo; Kameda, Atsushi; Shindo, Takayuki; Sato, Kazuhide; Kobayashi, Hisataka; Ogawa, Mikako

2018-04-13

The aim of this study was to develop and assess a novel implantable, wireless-powered, light-emitting diode (LED) for near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT is a recently developed cancer therapy that uses NIR light and antibody-photosensitizer conjugates and is able to induce cancer-specific cell death. Due to limited light penetration depth it is currently unable to treat tumors in deep tissues. Use of implanted LED might potentially overcome this limitation. The wireless LED system was able to emit NIR light up to a distance of 20 cm from the transmitter coil by using low magnetic fields as compliant with limits for use in humans. Results indicated that the LED system was able to kill tumor cells in vitro and to suppress tumor growth in implanted tumor-bearing mice. Results indicated that the proposed implantable wireless LED system was able to suppress tumor growth in vivo . These results are encouraging as wireless LED systems such as the one here developed might be a possible solution to treat tumors in deep regions in humans. Further research in this area would be important. An implantable LED system was developed. It consisted of a LED capsule including two LED sources and a receiver coil coupled with an external coil and power source. Wireless power transmission was guaranteed by using electromagnetic induction. The system was tested in vitro by using EGFR-expressing cells and HER2-expressing cells. The system was also tested in vivo in tumor-bearing mice.

Tumor-specific expression of shVEGF and suicide gene as a novel strategy for esophageal cancer therapy.

PubMed

Liu, Ting; Wu, Hai-Jun; Liang, Yu; Liang, Xu-Jun; Huang, Hui-Chao; Zhao, Yan-Zhong; Liao, Qing-Chuan; Chen, Ya-Qi; Leng, Ai-Min; Yuan, Wei-Jian; Zhang, Gui-Ying; Peng, Jie; Chen, Yong-Heng

2016-06-21

To develop a potent and safe gene therapy for esophageal cancer. An expression vector carrying fusion suicide gene (yCDglyTK) and shRNA against vascular endothelial growth factor (VEGF) was constructed and delivered into EC9706 esophageal cancer cells by calcium phosphate nanoparticles (CPNP). To achieve tumor selectivity, expression of the fusion suicide gene was driven by a tumor-specific human telomerase reverse transcriptase (hTERT) promoter. The biologic properties and therapeutic efficiency of the vector, in the presence of prodrug 5-fluorocytosine (5-FC), were evaluated in vitro and in vivo. Both in vitro and in vivo testing showed that the expression vector was efficiently introduced by CPNP into tumor cells, leading to cellular expression of yCDglyTK and decreased VEGF level. With exposure to 5-FC, it exhibited strong anti-tumor effects against esophageal cancer. Combination of VEGF shRNA with the fusion suicide gene demonstrated strong anti-tumor activity. The shVEGF-hTERT-yCDglyTK/5-FC system provided a novel approach for esophageal cancer-targeted gene therapy.

Regulation of carotenoid accumulation and the expression of carotenoid metabolic genes in citrus juice sacs in vitro.

PubMed

Zhang, Lancui; Ma, Gang; Kato, Masaya; Yamawaki, Kazuki; Takagi, Toshihiko; Kiriiwa, Yoshikazu; Ikoma, Yoshinori; Matsumoto, Hikaru; Yoshioka, Terutaka; Nesumi, Hirohisa

2012-01-01

In the present study, to investigate the mechanisms regulating carotenoid accumulation in citrus, a culture system was set up in vitro with juice sacs of three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). The juice sacs of all the three varieties enlarged gradually with carotenoid accumulation. The changing patterns of carotenoid content and the expression of carotenoid metabolic genes in juice sacs in vitro were similar to those ripening on trees in the three varieties. Using this system, the changes in the carotenoid content and the expression of carotenoid metabolic genes in response to environmental stimuli were investigated. The results showed that carotenoid accumulation was induced by blue light treatment, but was not affected by red light treatment in the three varieties. Different regulation of CitPSY expression, which was up-regulated by blue light while unaffected by red light, led to different changes in carotenoid content in response to these two treatments in Satsuma mandarin and Valencia orange. In all three varieties, increases in carotenoid content were observed with sucrose and mannitol treatments. However, the accumulation of carotenoid in the two treatments was regulated by distinct mechanisms at the transcriptional level. With abscisic acid (ABA) treatment, the expression of the genes investigated in this study was up-regulated in Satsuma mandarin and Lisbon lemon, indicating that ABA induced its own biosynthesis at the transcriptional level. This feedback regulation of ABA led to decreases in carotenoid content. With gibberellin (GA) treatment, carotenoid content was significantly decreased in the three varieties. Changes in the expression of genes related to carotenoid metabolism varied among the three varieties in response to GA treatment. These results provided insights into improving carotenoid content and composition in citrus during fruit maturation.

Regulation of carotenoid accumulation and the expression of carotenoid metabolic genes in citrus juice sacs in vitro

PubMed Central

Zhang, Lancui; Ma, Gang; Kato, Masaya; Yamawaki, Kazuki; Takagi, Toshihiko; Kiriiwa, Yoshikazu; Ikoma, Yoshinori; Matsumoto, Hikaru; Yoshioka, Terutaka; Nesumi, Hirohisa

2012-01-01

In the present study, to investigate the mechanisms regulating carotenoid accumulation in citrus, a culture system was set up in vitro with juice sacs of three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). The juice sacs of all the three varieties enlarged gradually with carotenoid accumulation. The changing patterns of carotenoid content and the expression of carotenoid metabolic genes in juice sacs in vitro were similar to those ripening on trees in the three varieties. Using this system, the changes in the carotenoid content and the expression of carotenoid metabolic genes in response to environmental stimuli were investigated. The results showed that carotenoid accumulation was induced by blue light treatment, but was not affected by red light treatment in the three varieties. Different regulation of CitPSY expression, which was up-regulated by blue light while unaffected by red light, led to different changes in carotenoid content in response to these two treatments in Satsuma mandarin and Valencia orange. In all three varieties, increases in carotenoid content were observed with sucrose and mannitol treatments. However, the accumulation of carotenoid in the two treatments was regulated by distinct mechanisms at the transcriptional level. With abscisic acid (ABA) treatment, the expression of the genes investigated in this study was up-regulated in Satsuma mandarin and Lisbon lemon, indicating that ABA induced its own biosynthesis at the transcriptional level. This feedback regulation of ABA led to decreases in carotenoid content. With gibberellin (GA) treatment, carotenoid content was significantly decreased in the three varieties. Changes in the expression of genes related to carotenoid metabolism varied among the three varieties in response to GA treatment. These results provided insights into improving carotenoid content and composition in citrus during fruit maturation. PMID:21994171

In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes.

PubMed

Quinlan, Jonathan M; Yu, Wei-Yuan; Hornsey, Mark A; Tosh, David; Slack, Jonathan M W

2006-05-25

Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable.

Novel Nano-Therapeutic Approach Actively Targets Human Ovarian Cancer Stem Cells after Xenograft into Nude Mice.

PubMed

Abou-ElNaga, Amoura; Mutawa, Ghada; El-Sherbiny, Ibrahim M; Abd-ElGhaffar, Hassan; Allam, Ahmed A; Ajarem, Jamaan; Mousa, Shaker A

2017-04-12

The power of tumorigenesis, chemo-resistance and metastasis in malignant ovarian tumors resides in a tiny population of cancer cells known as ovarian cancer stem cells (OCSCs). Developing nano-therapeutic targeting of OCSCs is considered a great challenge. The potential use of poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) was investigated as a drug delivery system for paclitaxel (PTX) against OCSCs in vitro and in vivo. PTX-loaded PLGA NPs were prepared by an emulsion solvent evaporation method, supported by incorporation of folic acid (FA) as the ligand. NPs were characterized for size, surface morphology, drug loading, and encapsulation efficiency. In vitro cytotoxicity of PTX-loaded FA/PLGA NPs was tested against OCSCs with MTT assay. In vivo anti-tumoral efficiency and active targeting potential of prepared NPs against tumors in nude mice were investigated. In vitro results revealed that IC 50 of PTX was significantly reduced after loading on PLGA NPs. On the other hand, in vivo results showed that PLGA NPs enhanced the tumor suppression efficiency of PTX. Investigation with real time quantitative PCR analysis revealed the limiting expression of chemo-resistant genes ( ABCG2 and MDR1 ) after applying PLGA NPs as a drug delivery system for PTX. Histopathological examination of tumors showed the effective biological influence of PTX-loaded FA/PLGA NPs through the appearance of reactive lymphoid follicles. Targeting potential of PTX was activated by FA/PLGA NPs through significant preservation of body weight ( p < 0.0001) and minimizing the systemic toxicity in healthy tissues. Immunohistochemical investigation revealed a high expression of apoptotic markers in tumor tissue, supporting the targeting effect of FA/PLGA NPs. A drug delivery system based on FA/PLGA NPs can enhance PTX's in vitro cytotoxicity and in vivo targeting potential against OCSCs.

Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells

PubMed Central

Takayama, Naoya; Nishimura, Satoshi; Nakamura, Sou; Shimizu, Takafumi; Ohnishi, Ryoko; Endo, Hiroshi; Yamaguchi, Tomoyuki; Otsu, Makoto; Nishimura, Ken; Nakanishi, Mahito; Sawaguchi, Akira; Nagai, Ryozo; Takahashi, Kazutoshi; Yamanaka, Shinya; Nakauchi, Hiromitsu

2010-01-01

Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a+CD42b+ platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b+ platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones. PMID:21098095

Characterization of surface interleukin-2 receptor expression on gated populations of peripheral blood mononuclear cells from manatees, Trichechus manatus latirostris.

PubMed

Sweat, J M; Johnson, C M; Marikar, Y; Gibbs, E P

2005-12-15

An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin.

Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.

PubMed Central

Kass-Eisler, A; Falck-Pedersen, E; Alvira, M; Rivera, J; Buttrick, P M; Wittenberg, B A; Cipriani, L; Leinwand, L A

1993-01-01

To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system. Images Fig. 1 Fig. 5 PMID:8265580

Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression

PubMed Central

QI, Xiaonan; LI, Huatao; CONG, Xia; WANG, Xin; JIANG, Zhongling; CAO, Rongfeng; TIAN, Wenru

2016-01-01

Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation. PMID:27478062

Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

PubMed

Leon, P M M; Campos, V F; Kaefer, C; Begnini, K R; McBride, A J A; Dellagostin, O A; Seixas, F K; Deschamps, J C; Collares, T

2013-08-01

The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

Regulation of the gut-specific carboxypeptidase: a study using the binary Gal4/UAS system in the mosquito Aedes aegypti

PubMed Central

Zhao, Bo; Kokoza, Vladimir A.; Saha, Tusar T.; Wang, Stephanie; Roy, Sourav; Raikhel, Alexander S.

2015-01-01

Pathogen transmission by mosquitoes is tightly linked to blood feeding which, in turn, is required for egg development. Studies of these processes would greatly benefit from genetic methods, such as the binary Gal4/UAS system. The latter has been well established for model organisms, but its availability is limited for mosquitoes. The objective of this study was to develop the blood-meal-activated, gut-specific Gal4/UAS system for the yellow-fever mosquito Aedes aegypti and utilize it to investigate the regulation of gut-specific gene expression. A 1.1-kb, 5' upstream region of the carboxypeptidase A (CP) gene was used to genetically engineer the CP-Gal4 driver mosquito line. The CP-Gal4 specifically activated the Enhanced Green Fluorescent Protein (EGFP) reporter only after blood feeding in the gut of the CP-Gal4>UAS-EGFP female Ae. aegypti. We used this system to study the regulation of CP gene expression. In vitro treatments with either amino acids (AAs) or insulin stimulated expression of the CP-Gal4>UAS-EGFP transgene; no effect was observed with 20-hydroxyecdysone (20E) treatments. The transgene activation by AAs and insulin was blocked by rapamycin, the inhibitor of the Target-of-Rapamycin kinase (TOR). RNA interference (RNAi) silence of the insulin receptor (IR) reduced the expression of the CP-Gal4>UAS-EGFP transgene. Thus, in vitro and in vivo experiments have revealed that insulin and TOR pathways control expression of the digestive enzyme CP. In contrast, 20E, the major regulator of post-blood-meal vitellogenic events in female mosquitoes, has no role in regulating the expression of this gene. This novel CP-Gal4/UAS system permits functional testing of midgut-specific genes that are involved in blood digestion and interaction with pathogens in Ae. aegypti mosquitoes. PMID:25152428

Astrocytes from adult Wistar rats aged in vitro show changes in glial functions.

PubMed

Souza, Débora Guerini; Bellaver, Bruna; Raupp, Gustavo Santos; Souza, Diogo Onofre; Quincozes-Santos, André

2015-11-01

Astrocytes, the most versatile cells of the central nervous system, play an important role in the regulation of neurotransmitter homeostasis, energy metabolism, antioxidant defenses and the anti-inflammatory response. Recently, our group characterized cortical astrocyte cultures from adult Wistar rats. In line with that work, we studied glial function using an experimental in vitro model of aging astrocytes (30 days in vitro after reaching confluence) from newborn (NB), adult (AD) and aged (AG) Wistar rats. We evaluated metabolic parameters, such as the glucose uptake, glutamine synthetase (GS) activity, and glutathione (GSH) content, as well as the GFAP, GLUT-1 and xCT expression. AD and AG astrocytes take up less glucose than NB astrocytes and had decreased GLUT1 expression levels. Furthermore, AD and AG astrocytes exhibited decreased GS activity compared to NB cells. Simultaneously, AD and AG astrocytes showed an increase in GSH levels, along with an increase in xCT expression. NB, AD and AG astrocytes presented similar morphology; however, differences in GFAP levels were observed. Taken together, these results improve the knowledge of cerebral senescence and represent an innovative tool for brain studies of aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Efficacy of HSV-tk/GCV system on human laryngeal squamous cell cancer in vitro].

PubMed

Ding, Xiu-yong; Qin, Yong; Li, Fu-ying; Cong, Tie-chuan

2006-05-01

Efficacy of HSV-tk/GCV system antitumor effects was assessed on human laryngeal cancer cell line Hep-2 in vitro. To assess the HSV-tk/CGV system whether has an antitumour effect on human laryngeal squamous cell cancer Hep-2 in vitro. The mechanisms of cytotoxity were also assessed. Hep-2 cells were transfected with HSV-tk gene by lipofection. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the HSV-tk gene expression. MTT was utilized to test for the cytotoxicity of this system. The cell-circle arrest and apoptosis were analyzed by flowcytometry assay. HSV-tk gene transfected cells demonstrated obvious cytoreductivity followed by ganciclovir (GCV) administration and this cytoreductivity showed partial GCV dose-independent. HSV-tk gene transfected cells demonstrated obvious s-phase arrest, no apoptosis and necrosis occurred. The HSV-tk/GCV system can inhabit the growth of Hep-2 cells effectively. S-phase arrest perhaps is the main reason that leads to the cell inhibition in our study. HSV-tk/GCV system has potential antitumor effects for the future clinical practice.

Surfactant protein A is expressed in the central nervous system of rats with experimental autoimmune encephalomyelitis, and suppresses inflammation in human astrocytes and microglia

PubMed Central

Yang, Xue; Yan, Jun; Feng, Juan

2017-01-01

The collectin surfactant protein-A (SP-A), a potent host defense molecule, is well recognized for its role in the maintenance of pulmonary homeostasis and the modulation of inflammatory responses. While previous studies have detected SP-A in numerous extrapulmonary tissues, there is still a lack of information regarding its expression in central nervous system (CNS) and potential effects in neuroinflammatory diseases, such as multiple sclerosis (MS). The present study used experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS, to investigate the expression of SP-A in the CNS at different stages of disease progression. In addition, in vitro experiments with lipopolysaccharide (LPS)-stimulated human astrocytes and microglia were performed to investigate the potential role of SP-A in the modulation of CNS inflammatory responses. The results of the present study demonstrated widespread distribution of SP-A in the rat CNS, and also identified specific expression patterns of SP-A at different stages of EAE. In vitro, the current study revealed that treatment of human astrocytes and microglia with LPS promoted SP-A expression in a dose-dependent manner. Furthermore, exogenous SP-A protein significantly decreased Toll-like receptor 4 and nuclear factor-κB expression, and reduced interleukin-1β and tumor necrosis factor-α levels. The results of the current study indicate a potential role for SP-A in the modulation of CNS inflammatory responses. PMID:28393255

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin.

PubMed

Figueira, Cláudio Pereira; Croda, Julio; Choy, Henry A; Haake, David A; Reis, Mitermayer G; Ko, Albert I; Picardeau, Mathieu

2011-06-09

In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis.

Pluripotent stem cells reveal the developmental biology of human megakaryocytes and provide a source of platelets for clinical application.

PubMed

Takayama, Naoya; Eto, Koji

2012-10-01

Human pluripotent stem cells [PSCs; including human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] can infinitely proliferate in vitro and are easily accessible for gene manipulation. Megakaryocytes (MKs) and platelets can be created from human ESCs and iPSCs in vitro and represent a potential source of blood cells for transfusion and a promising tool for studying the human thrombopoiesis. Moreover, disease-specific iPSCs are a powerful tool for elucidating the pathogenesis of hematological diseases and for drug screening. In that context, we and other groups have developed in vitro MK and platelet differentiation systems from human pluripotent stem cells (PSCs). Combining this co-culture system with a drug-inducible gene expression system enabled us to clarify the novel role played by c-MYC during human thrombopoiesis. In the next decade, technical advances (e.g., high-throughput genomic sequencing) will likely enable the identification of numerous gene mutations associated with abnormal thrombopoiesis. Combined with such technology, an in vitro system for differentiating human PSCs into MKs and platelets could provide a novel platform for studying human gene function associated with thrombopoiesis.

Development of a Synthetic Oxytetracycline-Inducible Expression System for Streptomycetes Using de Novo Characterized Genetic Parts.

PubMed

Wang, Weishan; Yang, Tongjian; Li, Yihong; Li, Shanshan; Yin, Shouliang; Styles, Kathryn; Corre, Christophe; Yang, Keqian

2016-07-15

Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (KD = 1.7 × 10(-10) M) and oxytetracycline induced dissociation of the OtrR/DNA complex in a concentration-dependent manner. On the basis of these genetic parts, a synthetic inducible expression system Potr* was optimized. Induction of Potr* with 0.01-4 μM of oxytetracycline triggered a wide-range expression level of gfp reporter gene in different Streptomyces species. Benchmarking Potr* against the widely used constitutive promoters ermE* and kasOp* revealed greatly enhanced levels of expression when Potr* was fully induced. Finally, Potr* was used as a tool to activate and optimize the expression of the silent jadomycin biosynthetic gene cluster in Streptomyces venezuelae. Altogether, the synthetic Potr* presents a new versatile tool for fine-tuning gene expression in streptomycetes.

Macrophage-Specific Expression of IL-37 in Hyperlipidemic Mice Attenuates Atherosclerosis.

PubMed

McCurdy, Sara; Baumer, Yvonne; Toulmin, Emma; Lee, Bog-Hieu; Boisvert, William A

2017-11-15

Atherosclerosis, the progressive buildup of plaque within arterial blood vessels, can lead to fatal downstream events, such as heart attack or stroke. A key event contributing to the development of atherosclerosis is the infiltration of monocytes and its associated inflammation, as well as the formation of lipid-laden macrophage foam cells within the vessel wall. IL-37 is recognized as an important anti-inflammatory cytokine expressed especially by immune cells. This study was undertaken to elucidate the role of macrophage-expressed IL-37 in reducing the production and effects of proinflammatory cytokines, preventing foam cell formation, and reducing the development of atherosclerosis. Expression of human IL-37 was achieved with a macrophage-specific overexpression system, using the CD68 promoter in mouse primary bone marrow-derived macrophages via retroviral transduction. Macrophage IL-37 expression in vitro resulted in decreased mRNA (e.g., IL-1B, IL-6, and IL-12) and secreted protein production (e.g., IL-6, M-CSF, and ICAM-1) of key inflammatory mediators. IL-37 expression also inhibited macrophage proliferation, apoptosis, and transmigration, as well as reduced lipid uptake, compared with controls in vitro. The in vivo effects of macrophage-expressed IL-37 were investigated through bone marrow transplantation of transduced hematopoietic stem cells into irradiated atherosclerosis-prone Ldlr -/- mice. After 10 wk on a high-fat/high-cholesterol diet, mice with IL-37-expressing macrophages showed reduced disease pathogenesis, which was demonstrated by significantly less arterial plaque development and systemic inflammation compared with control mice. The athero-protective effect of macrophage-expressed IL-37 has implications for development of future therapies to treat atherosclerosis, as well as other chronic inflammatory diseases. Copyright © 2017 by The American Association of Immunologists, Inc.

Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds.

PubMed

Jarrahy, Reza; Huang, Weibiao; Rudkin, George H; Lee, Jane M; Ishida, Kenji; Berry, Micah D; Sukkarieh, Modar; Wu, Benjamin M; Yamaguchi, Dean T; Miller, Timothy A

2005-08-01

Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(l-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions.

CXCL14 is a candidate biomarker for Hedgehog signalling in idiopathic pulmonary fibrosis.

PubMed

Jia, Guiquan; Chandriani, Sanjay; Abbas, Alexander R; DePianto, Daryle J; N'Diaye, Elsa N; Yaylaoglu, Murat B; Moore, Heather M; Peng, Ivan; DeVoss, Jason; Collard, Harold R; Wolters, Paul J; Egen, Jackson G; Arron, Joseph R

2017-09-01

Idiopathic pulmonary fibrosis (IPF) is associated with aberrant expression of developmental pathways, including Hedgehog (Hh). As Hh signalling contributes to multiple pro-fibrotic processes, Hh inhibition may represent a therapeutic option for IPF. However, no non-invasive biomarkers are available to monitor lung Hh activity. We assessed gene and protein expression in IPF and control lung biopsies, mouse lung, fibroblasts stimulated in vitro with sonic hedgehog (SHh), and plasma in IPF patients versus controls, and cancer patients before and after treatment with vismodegib, a Hh inhibitor. Lung tissue from IPF patients exhibited significantly greater expression of Hh-related genes versus controls. The gene most significantly upregulated in both IPF lung biopsies and fibroblasts stimulated in vitro with SHh was CXCL14 , which encodes a soluble secreted chemokine whose expression is inhibited in vitro by the addition of vismodegib. CXCL14 expression was induced by SHh overexpression in mouse lung. Circulating CXCL14 protein levels were significantly higher in plasma from IPF patients than controls. In cancer patients, circulating CXCL14 levels were significantly reduced upon vismodegib treatment. CXCL14 is a systemic biomarker that could be used to identify IPF patients with increased Hh pathway activity and monitor the pharmacodynamic effects of Hh antagonist therapy in IPF. Post-results, NCT00968981. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

PAMAM dendrimer-baculovirus nanocomplex for microencapsulated adipose stem cell-gene therapy: in vitro and in vivo functional assessment.

PubMed

Paul, Arghya; Shao, Wei; Abbasi, Sana; Shum-Tim, Dominique; Prakash, Satya

2012-09-04

The present study aims to develop a new stem cell based gene delivery system consisting of human adipose tissue derived stem cells (hASCs) genetically modified with self-assembled nanocomplex of recombinant baculovirus and PAMAM dendrimer (Bac-PAMAM) to overexpress the vascular endothelial growth factor (VEGF). Cells were enveloped into branched PEG surface functionalized polymeric microcapsules for efficient transplantation. In vitro analysis confirmed efficient transduction of hASCs expressing 7.65 ± 0.86 ng functionally active VEGF per 10(6) microencapsulated hASCs (ASC-VEGF). To determine the potential of the developed system, chronically infarcted rat hearts were treated with either empty microcapsules (MC), microencapsulated hASCs expressing MGFP reporter protein (MC+ASC-MGFP), or MC+ASC-VEGF, and analyzed for 10 weeks. Post-transplantation data confirmed higher myocardial VEGF expressions with significantly enhanced neovasculature in the MC+ASC-VEGF group. In addition, the cardiac performance, as measured by percentage ejection fraction, also improved significantly in the MC+ASC-VEGF group (48.6 ± 6.1%) compared to that in MC+ASC-MGFP (38.8 ± 5.3%) and MC groups (31.5 ± 3.3%). Collectively, these data demonstrate the feasibility of this system for improved stem cell therapy applications.

Use of in vivo Expression Technology for the Identification of Putative Host Adaptation Factors of the Lyme Disease Spirochete.

PubMed

Casselli, Timothy; Bankhead, Troy

2015-01-01

The causative agent of Lyme disease, Borrelia burgdorferi, is an obligate parasite that requires either a tick vector or a mammalian host for survival. Identification of the bacterial genes that are specifically expressed during infection of the mammalian host could provide targets for novel therapeutics and vaccines. In vivo expression technology (IVET) is a reporter-based promoter trap system that utilizes selectable markers to identify promoters of bacterial host-specific genes. Using previously characterized genes for in vivo and in vitro selection, this study utilized an IVET system that allows for selection of B. burgdorferi sequences that act as active promoters only during murine infection. This promoter trap system was able to successfully distinguish active promoter sequences both in vivo and in vitro from control sequences and a library of cloned B. burgdorferi genomic fragments. However, a bottleneck effect during the experimental mouse infection limited the utility for genome-wide promoter screening. Overall, IVET was demonstrated as a tool for the identification of in vivo-induced promoter elements of B. burgdorferi, and the observed infection bottleneck apparent using a polyclonal infection pool provides insight into the dynamics of experimental infection with B. burgdorferi. © 2015 S. Karger AG, Basel.

The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro

PubMed Central

Manczinger, Máté; Bocsik, Alexandra; Kocsis, Gabriella F.; Vörös, Andrea; Hegedűs, Zoltán; Marton, Annamária; Vízler, Csaba; Tubak, Vilmos; Deli, Mária; Kemény, Lajos; Nagy, István; Lakatos, Lóránt

2015-01-01

To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis. PMID:26366412

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin*♦

PubMed Central

Samuel, Premila P.; Smith, Lucian P.; Phillips, George N.; Olson, John S.

2015-01-01

Expression levels in animal muscle tissues and in Escherichia coli vary widely for naturally occurring mammalian myoglobins (Mb). To explore this variation, we developed an in vitro transcription and wheat germ extract-based translation assay to examine quantitatively the factors that govern expression of holoMb. We constructed a library of naturally occurring Mbs from two terrestrial and four deep-diving aquatic mammals and three distal histidine mutants designed to enhance apoglobin stability but decrease hemin affinity. A strong linear correlation is observed between cell-free expression levels of holo-metMb variants and their corresponding apoglobin stabilities, which were measured independently by guanidine HCl-induced unfolding titrations using purified proteins. In contrast, there is little dependence of expression on hemin affinity. Our results confirm quantitatively that deep diving mammals have highly stable Mbs that express to higher levels in animal myocytes, E. coli, and the wheat germ cell-free system than Mbs from terrestrial mammals. Our theoretical analyses show that the rate of aggregation of unfolded apoMb is very large, and as a result, the key factor for high level expression of holoMb, and presumably other heme proteins, is an ultra high fraction of folded, native apoglobin that is capable of rapidly binding hemin. This fraction is determined by the overall equilibrium folding constant and not hemin affinity. These results also demonstrate that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability without the need to generate large amounts of protein for in vitro unfolding measurements. PMID:26205820

Adhesion mechanisms in embryogenesis and in cancer invasion and metastasis.

PubMed

Thiery, J P; Boyer, B; Tucker, G; Gavrilovic, J; Valles, A M

1988-01-01

Cell-substratum and cell-cell adhesion mechanisms contribute to the development of animal form. The adhesive status of embryonic cells has been analysed during epithelial-mesenchymal cell interconversion and in cell migrations. Clear-cut examples of the modulation of cell adhesion molecules (CAMs) have been described at critical periods of morphogenesis. In chick embryos the three primary CAMs (N-CAM. L-CAM and N-cadherin) present early in embryogenesis are expressed later in a defined pattern during morphogenesis and histogenesis. The axial mesoderm derived from gastrulating cells expresses increasing amounts of N-cadherin and N-CAM. During metamerization these two adhesion molecules become abundant at somitic cell surfaces. Both CAMs are functional in an in vitro aggregation assay; however, the calcium-dependent adhesion molecule N-cadherin is more sensitive to perturbation by specific antibodies. Neural crest cells which separate from the neural epithelium lose their primary CAMs in a defined time-sequence. Adhesion to fibronectins via specific surface receptors becomes a predominant interaction during the migratory process, while some primary and secondary CAMs are expressed de novo during the ontogeny of the peripheral nervous system. In vitro, different fibronectin functional domains have been identified in the attachment, spreading and migration of neural crest cells. The fibronectin receptors which transduce the adhesive signals play a key role in the control of cell movement. All these results have prompted us to examine whether similar mechanisms operate in carcinoma cell invasion and metastasis. In vitro, rat bladder transitional carcinoma cells convert reversibly into invasive mesenchymal cells. A rapid modulation of adhesive properties is found during the epithelial-mesenchymal carcinoma cell interconversion. The different model systems analysed demonstrate that a limited repertoire of adhesion molecules, expressed in a well-defined spatiotemporal pattern, is involved in tissue formation and in key processes of tumour spread.

Effects of environmental lead contamination on cattle in a lead/zinc mining area: changes in cattle immune systems on exposure to lead in vivo and in vitro.

PubMed

Ikenaka, Yoshinori; Nakayama, Shouta M M; Muroya, Taro; Yabe, John; Konnai, Satoru; Darwish, Wageh Sobhy; Muzandu, Kaampwe; Choongo, Kennedy; Mainda, Geoffrey; Teraoka, Hiroki; Umemura, Takashi; Ishizuka, Mayumi

2012-10-01

The Republic of Zambia is rich in mineral resources, such as zinc (Zn) and lead (Pb), and mining is a key industry in Zambia. A previous study of Pb pollution in Kabwe, one of the main mining areas, found that soil was contaminated with high levels of toxic metals over a substantial area. In the present study, the authors focus on toxic metal pollution in cattle, one of the most important domestic animals in Zambia. Blood samples from cattle in Kabwe and a control area (Lusaka) were tested for toxic metal content. They also measured mRNA expression of metal-responsive proteins and cytokines in white blood cells using real-time reverse transcriptase polymerase chain reaction. In the present in vitro study, The authors cultured peripheral blood mononuclear cells (PBMCs) from cattle, exposing them to Pb acetate for 24 h and analyzing mRNA expression of metal-responsive proteins and selected cytokines. Lead concentrations in cattle blood from Kabwe were significantly greater than those from Lusaka, as were the mRNA expressions of metallothionein-2 (MT-2), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1β (IL-1β), IL-6, and inducible nitric oxide synthase (iNOS). The present in vitro study demonstrated that Pb exposure led to an increase in the expressions of MT-2, TNF-α, IL-1β, and iNOS, similar to those found in vivo. These results indicate the possibility of immune system modulations in cattle from the Kabwe area. Copyright © 2012 SETAC.

N-octanoylated ghrelin peptide inhibits bovine oocyte meiotic resumption.

PubMed

Xu, X L; Bai, J H; Feng, T; Xiao, L L; Song, Y Q; Xiao, Y X; Liu, Y

2018-07-01

Studies have shown that ghrelin plays an important role in the mammalian reproductive system, including the central, gonadal levels, and also during in vitro maturation of oocytes; however, the functions of ghrelin in bovine oocyte meiosis require further investigation. We aimed to evaluate the effects of an n-octanoylated ghrelin peptide on oocyte meiotic resumption and the developmental competence of mature oocytes in vitro. design: The expression of GHRL (encoding ghrelin) mRNA and its receptor (the growth hormone secretagogue receptor, GHSR) in the cumulus-oocyte complex (COCs), denuded oocytes (DOs), and cumulus cells (CCs) was assessed using quantitative real-time reverse transcription PCR (qRT-PCR), and the effects of the n-octanoylated ghrelin peptide on meiotic resumption were studied at four different doses (0, 10, 50, and 100 ng/mL) in a 6 h culture system. qRT-PCR analysis showed that GHRL and GHSR mRNAs were expressed in all tested samples; however, GHRL was predominantly expressed in DOs, and GHSR was predominantly expressed in CCs. Germinal vesicle breakdown was inhibited significantly by 50 ng/mL ghrelin compared with that in the negative control (P < 0.05). Further studies showed that n-octanoylated ghrelin increased the levels of cAMP and cGMP in the CCs and DOs, which inhibited the meiotic resumption of bovine oocytes. And the inhibitory role in the developmental competence of mature oocytes were also included, ghrelin could significantly improve the cleavage rate (P < 0.05) and blastocyst rate (P < 0.05). N-octanoylated ghrelin maintained bovine oocytes meiotic arrest and further improved their developmental competence; therefore, n-octanoylated ghrelin could be considered as a potential pharmaceutical inhibitor of meiosis for the in vitro maturation of bovine oocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

Transient Expression of Lumbrokinase (PI239) in Tobacco (Nicotiana tabacum) Using a Geminivirus-Based Single Replicon System Dissolves Fibrin and Blood Clots.

PubMed

Dickey, Alexia; Wang, Nan; Cooper, Edwin; Tull, Lauren; Breedlove, Drew; Mason, Hugh; Liu, Dehu; Wang, Kevin Yueju

2017-01-01

Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild-type (WT) and plant codon-optimized (OP) PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum) leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.

A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

PubMed

Lu, M; Farrell, P J; Johnson, R; Iatrou, K

1997-12-05

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant proteins and the capability of expression of proteins from genomic as well as cDNA sequences.

Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis, Daniel D.; Department of Biomedical Engineering, University of California Davis, Davis, CA; Villarreal, Fernando D.

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with amore » special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.« less

Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

PubMed Central

Lewis, Daniel D.; Villarreal, Fernando D.; Wu, Fan; Tan, Cheemeng

2014-01-01

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems. PMID:25538941

Synthetic biology outside the cell: linking computational tools to cell-free systems.

PubMed

Lewis, Daniel D; Villarreal, Fernando D; Wu, Fan; Tan, Cheemeng

2014-01-01

As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.

Expression analysis of MDR1, BCRP and MRP3 transporter proteins in different in vitro and ex vivo cornea models for drug absorption studies.

PubMed

Verstraelen, Jessica; Reichl, Stephan

2013-01-30

Ocular drug absorption studies are required for the development of new drugs or drug delivery systems for eye treatment. Such preclinical investigations on transcorneal drug absorption are performed ex vivo with the excised corneas of experimental animals or in vitro using corneal cell culture models. The data currently available on the expression of ABC transporter proteins in corneal tissue is limited or contradictory. This study describes, for the first time, the comparison of the expression of ABC transporters, in particular, MDR1, BCRP and MRP3, between human cornea cell culture models and the most commonly used ex vivo models, namely, rabbit and porcine corneas, conducted in the same laboratory. The expression levels and functionality were determined by means of PCR, western blot, immunohistochemistry and bidirectional permeation studies using specific substrates and inhibitors. The results clearly indicate species-dependent expression of the studied efflux transporters. In the rabbit cornea, the expression and activity of MDR1 transporter was confirmed, whereas human cell culture models and porcine corneas did not show MDR1 expression. However, human cornea models possessed MRP3 and BCRP expression, whereas no functional expression was found in rabbit and porcine corneas. Therefore, the translation of transcorneal permeation data from animal experiments to humans should be performed with caution. Copyright © 2012 Elsevier B.V. All rights reserved.

[EFFECT OF RECOMBINANT ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 12 TRANSFECTION ON DIFFERENTIATION OF PERIPHERAL BLOOD MESENCHYMAL STEM CELLS INTO TENDON/LIGAMENT CELLS].

PubMed

Fu, Weili; Chen, Gang; Tang, Xin; Li, Qi; Ll, Jian

2015-04-01

To research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. Peripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. GFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type I gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061+/- 0.013 vs. 0.004 +/- 0.002, t = -7.700, P=0.031; 0.029 +/- 0.008 vs. 0.003 +/- 0.001, t = -5.741, P=0.020; 0.679 +/- 0.067 vs. 0.142 +/- 0.024, t = -12.998, P=0.000). Ad-BMP-12 can significantly promote differentiation of peripheral blood MSCs into tendon/ligament fibroblasts and enhance the expressions of tendon/ligament-specific phenotypic differentiation, which would provide the evidence for peripheral blood MSCs applied for tendon/ligament regeneration.

Targeted expression of miR-34a using the T-VISA system suppresses breast cancer cell growth and invasion.

PubMed

Li, Laisheng; Xie, Xinhua; Luo, Jinmei; Liu, Min; Xi, Shaoyan; Guo, Jiaoli; Kong, Yanan; Wu, Minqing; Gao, Jie; Xie, Zeming; Tang, Jun; Wang, Xi; Wei, Weidong; Yang, Mingtian; Hung, Mien-Chie; Xie, Xiaoming

2012-12-01

Recurrence and metastasis result in a poor prognosis for breast cancer patients. Recent studies have demonstrated that microRNAs (miRNAs) play vital roles in the development and metastasis of breast cancer. In this study, we investigated the therapeutic potential of miR-34a in breast cancer. We found that miR-34a is downregulated in breast cancer cell lines and tissues, compared with normal cell lines and the adjacent nontumor tissues, respectively. To explore the therapeutic potential of miR-34a, we designed a targeted miR-34a expression plasmid (T-VISA-miR-34a) using the T-VISA system, and evaluated its antitumor effects, efficacy, mechanism of action, and systemic toxicity. T-VISA-miR-34a induced robust, persistent expression of miR-34a, and dramatically suppressed breast cancer cell growth, migration, and invasion in vitro by downregulating the protein expression levels of the miR-34a target genes E2F3, CD44, and SIRT1. In an orthotopic mouse model of breast cancer, intravenous injection of T-VISA-miR-34a:liposomal complex nanoparticles significantly inhibited tumor growth, prolonged survival, and did not induce systemic toxicity. In conclusion, T-VISA-miR-34a lead to robust, specific overexpression of miR-34a in breast cancer cells and induced potent antitumor effects in vitro and in vivo. T-VISA-miR-34a may provide a potentially useful, specific, and safe-targeted therapeutic approach for breast cancer.

Targeted Expression of miR-34a Using the T-VISA System Suppresses Breast Cancer Cell Growth and Invasion

PubMed Central

Li, Laisheng; Xie, Xinhua; Luo, Jinmei; Liu, Min; Xi, Shaoyan; Guo, Jiaoli; Kong, Yanan; Wu, Minqing; Gao, Jie; Xie, Zeming; Tang, Jun; Wang, Xi; Wei, Weidong; Yang, Mingtian; Hung, Mien-Chie; Xie, Xiaoming

2012-01-01

Recurrence and metastasis result in a poor prognosis for breast cancer patients. Recent studies have demonstrated that microRNAs (miRNAs) play vital roles in the development and metastasis of breast cancer. In this study, we investigated the therapeutic potential of miR-34a in breast cancer. We found that miR-34a is downregulated in breast cancer cell lines and tissues, compared with normal cell lines and the adjacent nontumor tissues, respectively. To explore the therapeutic potential of miR-34a, we designed a targeted miR-34a expression plasmid (T-VISA-miR-34a) using the T-VISA system, and evaluated its antitumor effects, efficacy, mechanism of action, and systemic toxicity. T-VISA-miR-34a induced robust, persistent expression of miR-34a, and dramatically suppressed breast cancer cell growth, migration, and invasion in vitro by downregulating the protein expression levels of the miR-34a target genes E2F3, CD44, and SIRT1. In an orthotopic mouse model of breast cancer, intravenous injection of T-VISA-miR-34a:liposomal complex nanoparticles significantly inhibited tumor growth, prolonged survival, and did not induce systemic toxicity. In conclusion, T-VISA-miR-34a lead to robust, specific overexpression of miR-34a in breast cancer cells and induced potent antitumor effects in vitro and in vivo. T-VISA-miR-34a may provide a potentially useful, specific, and safe-targeted therapeutic approach for breast cancer. PMID:23032974

The urokinase plasminogen activator system components are regulated by vascular endothelial growth factor D in bovine oviduct.

PubMed

García, Daniela C; Russo-Maenza, Agostina; Miceli, Dora C; Valdecantos, Pablo A; Roldán-Olarte, Mariela

2018-06-08

SummaryThe mammalian oviduct plays a pivotal role in the success of early reproductive events. The urokinase plasminogen activator system (uPAS) is present in the bovine oviduct and is involved in extracellular matrix remodelling through plasmin generation. This system can be regulated by several members of the vascular endothelial growth factors (VEGF) and their receptors. In this study, the VEGF-D effect on the regulation of uPAS was evaluated. First, RT-polymerase chain reaction (PCR) analyses were used to evidence the expression of VEGF-D and its receptors in oviductal epithelial cells (BOEC). VEGF-D, VEGFR2 and VEGFR3 transcripts were found in ex vivo and in vitro BOEC, while only VEGFR2 mRNA was present after in vitro conditions. VEGF-D showed a regulatory effect on uPAS gene expression in a dose-dependent manner, inducing an increase in the expression of both uPA and its receptor (uPAR) at 24 h post-induction and decreases in the expression of its inhibitor (PAI-1). In addition, the regulation of cell migration induced by VEGF-D and uPA in BOEC monolayer cultures was analyzed. The wound areas of monolayer cultures incubated with VEGF-D 10 ng/ml or uPA 10 nM were modified and significant differences were found at 24 h for both stimulations. These results indicated that uPAS and VEGF-D systems can modify the arrangement of the bovine oviductal epithelium and contribute to the correct maintenance of the oviductal microenvironment.

Melatonin Scavenger Properties against Oxidative and Nitrosative Stress: Impact on Gamete Handling and In Vitro Embryo Production in Humans and Other Mammals

PubMed Central

Loren, Pía; Sánchez, Raúl; Arias, María-Elena; Felmer, Ricardo; Risopatrón, Jennie; Cheuquemán, Carolina

2017-01-01

Oxidative and nitrosative stress are common problems when handling gametes in vitro. In vitro development in mammalian embryos is highly affected by culture conditions, especially by reactive oxygen species (ROS) and reactive nitrogen species (RNS), because their absence or overproduction causes embryo arrest and changes in gene expression. Melatonin in gamete co-incubation during in vitro fertilization (IVF) has deleterious or positive effects, depending on the concentration used in the culture medium, demonstrating the delicate balance between antioxidant and pro-oxidant activity. Further research is needed to better understand the possible impact of melatonin on the different IVP steps in humans and other mammals, especially in seasonal breeds where this neuro-hormone system highly regulates its reproduction physiology. PMID:28613231

Advances in recombinant protein expression for use in pharmaceutical research.

PubMed

Assenberg, Rene; Wan, Paul T; Geisse, Sabine; Mayr, Lorenz M

2013-06-01

Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells. Copyright © 2013. Published by Elsevier Ltd.

In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells.

PubMed

Jung, Kwang Bo; Lee, Hana; Son, Ye Seul; Lee, Ji Hye; Cho, Hyun-Soo; Lee, Mi-Ok; Oh, Jung-Hwa; Lee, Jaemin; Kim, Seokho; Jung, Cho-Rok; Kim, Janghwan; Son, Mi-Young

2018-01-01

Human intestinal organoids (hIOs) derived from human pluripotent stem cells (hPSCs) have immense potential as a source of intestines. Therefore, an efficient system is needed for visualizing the stage of intestinal differentiation and further identifying hIOs derived from hPSCs. Here, 2 fluorescent biosensors were developed based on human induced pluripotent stem cell (hiPSC) lines that stably expressed fluorescent reporters driven by intestine-specific gene promoters Krüppel-like factor 5 monomeric Cherry (KLF5 mCherry ) and intestine-specific homeobox enhanced green fluorescence protein (ISX eGFP ). Then hIOs were efficiently induced from those transgenic hiPSC lines in which mCherry- or eGFP-expressing cells, which appeared during differentiation, could be identified in intact living cells in real time. Reporter gene expression had no adverse effects on differentiation into hIOs and proliferation. Using our reporter system to screen for hIO differentiation factors, we identified DMH1 as an efficient substitute for Noggin. Transplanted hIOs under the kidney capsule were tracked with fluorescence imaging (FLI) and confirmed histologically. After orthotopic transplantation, the localization of the hIOs in the small intestine could be accurately visualized using FLI. Our study establishes a selective system for monitoring the in vitro differentiation and for tracking the in vivo localization of hIOs and contributes to further improvement of cell-based therapies and preclinical screenings in the intestinal field.-Jung, K. B., Lee, H., Son, Y. S., Lee, J. H., Cho, H.-S., Lee, M.-O., Oh, J.-H., Lee, J., Kim, S., Jung, C.-R., Kim, J., Son, M.-Y. In vitro and in vivo imaging and tracking of intestinal organoids from human induced pluripotent stem cells. © FASEB.

Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors: assessment of immunophenotype and differentiation capacity in vitro.

PubMed

van Vollenstee, Fiona A; Jackson, Carlo; Hoffmann, Danie; Potgieter, Marnie; Durandt, Chrisna; Pepper, Michael S

2016-10-01

Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies.

Combinatorial Approaches for the Identification of Brain Drug Delivery Targets

PubMed Central

Stutz, Charles C.; Zhang, Xiaobin; Shusta, Eric V.

2018-01-01

The blood-brain barrier (BBB) represents a large obstacle for the treatment of central nervous system diseases. Targeting endogenous nutrient transporters that transcytose the BBB is one promising approach to selectively and noninvasively deliver a drug payload to the brain. The main limitations of the currently employed transcytosing receptors are their ubiquitous expression in the peripheral vasculature and the inherent low levels of transcytosis mediated by such systems. In this review, approaches designed to increase the repertoire of transcytosing receptors which can be targeted for the purpose of drug delivery are discussed. In particular, combinatorial protein libraries can be screened on BBB cells in vitro or in vivo to isolate targeting peptides or antibodies that can trigger transcytosis. Once these targeting reagents are discovered, the cognate BBB transcytosis system can be identified using techniques such as expression cloning or immunoprecipitation coupled with mass spectrometry. Continued technological advances in BBB genomics and proteomics, membrane protein manipulation, and in vitro BBB technology promise to further advance the capability to identify and optimize peptides and antibodies capable of mediating drug transport across the BBB. PMID:23789958

Hapten-derivatized nanoparticle targeting and imaging of gene expression by multimodality imaging systems.

PubMed

Cheng, C-M; Chu, P-Y; Chuang, K-H; Roffler, S R; Kao, C-H; Tseng, W-L; Shiea, J; Chang, W-D; Su, Y-C; Chen, B-M; Wang, Y-M; Cheng, T-L

2009-01-01

Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39+/-0.5 nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100 microM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

In-vitro Cell Exposure Studies for the Assessment of Nanoparticle Toxicity in the Lung - A Dialogue between Aerosol Science and Biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanns-Rudolf, Paur; Cassee, Flemming R.; Teeguarden, Justin G.

The rapid introduction of engineered nanostructured materials into numerous industrial and consumer products will result in enhanced exposure to engineered nanoparticles. Workplace exposure has been identified as the most likely source of uncontrolled inhalation of engineered aerosolized nanoparticles, but release of engineered nanoparticles may occur at any stage of the lifecycle of consumer products. The dynamic development of new nanomaterials with possibly unknown toxicological effects poses a challenge for the assessment of nanoparticle induced toxicity and safety. In this consensus document from a workshop on in-vitro cell systems for nanotoxicity testing an overview is given of the main issues concerningmore » inhalation exposure to nanoparticles, lung physiology, nanoparticle-related biological mechanisms, in-vitro cell exposure systems for nanoparticles and social aspects of nanotechnology. The workshop participants recognized the large potential of in-vitro cell exposure systems for reliable, high-throughput screening of nanotoxicity. For the investigation of pulmonary nanotoxicity, a strong preference was expressed for air-liquid interface (ALI) cell exposure systems (rather than submerged cell exposure systems) as they closely resemble in-vivo conditions in the lungs and they allow for unaltered and dosimetrically accurate delivery of aerosolized nanoparticles to the cells. The members of the workshop believe that further advances in in-vitro cell exposure studies would be greatly facilitated by a more active role of the aerosol scientists. The technical know-how for developing and running ALI in-vitro exposure systems is available in the aerosol community and at the same time biologists/toxicologists are required for proper assessment of the biological impact of nanoparticles.« less

A Modular Lentiviral and Retroviral Construction System to Rapidly Generate Vectors for Gene Expression and Gene Knockdown In Vitro and In Vivo

PubMed Central

Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R.; de Bruyns, Angeline; Dutchak, Kendall L.; Garnett, Samantha; Dankort, David

2013-01-01

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems. PMID:24146852

Characterization of in vitro transcriptional responses of dorsal root ganglia cultured in the presence and absence of blastema cells from regenerating salamander limbs

PubMed Central

Athippozhy, Antony; Lehrberg, Jeffrey; Monaghan, James R.; Gardiner, David M.

2014-01-01

Abstract During salamander limb regeneration, nerves provide signals that induce the formation of a mass of proliferative cells called the blastema. To better understand these signals, we developed a blastema−dorsal root ganglia (DRG) co‐culture model system to test the hypothesis that nerves differentially express genes in response to cues provided by the blastema. DRG with proximal and distal nerve trunks were isolated from axolotls (Ambystoma mexicanum), cultured for 5 days, and subjected to microarray analysis. Relative to freshly isolated DRG, 1541 Affymetrix probe sets were identified as differentially expressed and many of the predicted genes are known to function in injury and neurodevelopmental responses observed for mammalian DRG. We then cultured 5‐day DRG explants for an additional 5 days with or without co‐cultured blastema cells. On day 10, we identified 27 genes whose expression in cultured DRG was significantly affected by the presence or absence of blastema cells. Overall, our study established a DRG−blastema in vitro culture system and identified candidate genes for future investigations of axon regrowth, nerve−blastema signaling, and neural regulation of limb regeneration. PMID:25750744

Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis.

PubMed

Werner, Sean R; Morgan, John A

2009-07-15

Glycosyltransferases are promising biocatalysts for the synthesis of small molecule glycosides. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase (GT) from Dianthus caryophyllus (carnation) was investigated as a whole-cell biocatalyst. Two yeast expression systems were compared using the flavonoid naringenin as a model substrate. Under in vitro conditions, naringenin-7-O-glucoside was formed and a higher specific glucosyl transfer activity was found using a galactose inducible expression system compared to a constitutive expression system. However, S. cerevisiae expressing the GT constitutively was significantly more productive than the galactose inducible system under in vivo conditions. Interestingly, the glycosides were recovered directly from the culture broth and did not accumulate intracellularly. A previously uncharacterized naringenin glycoside formed using the D. caryophyllus GT was identified as naringenin-4'-O-glucoside. It was found that S. cerevisiae cells hydrolyze naringenin-7-O-glucoside during whole-cell biocatalysis, resulting in a low final glycoside titer. When phloretin was added as a substrate to the yeast strain expressing the GT constitutively, the natural product phlorizin was formed. This study demonstrates S. cerevisiae is a promising whole-cell biocatalyst host for the production of valuable glycosides.

Nanocapsule-mediated cytosolic siRNA delivery for anti-inflammatory treatment.

PubMed

Jiang, Ying; Hardie, Joseph; Liu, Yuanchang; Ray, Moumita; Luo, Xiang; Das, Riddha; Landis, Ryan F; Farkas, Michelle E; Rotello, Vincent M

2018-06-05

The use of nanoparticle-stabilized nanocapsules for cytosolic siRNA delivery for immunomodulation in vitro and in vivo is reported. These NPSCs deliver siRNA directly to the cytosol of macrophages in vitro with concomitant knockdown of gene expression. In vivo studies showed directed delivery of NPSCs to the spleen, enabling gene silencing of macrophages, with preliminary studies showing 70% gene knockdown at a siRNA dose of 0.28 mg/kg. Significantly, the delivery of siRNA targeting tumor necrosis factor-α efficiently silenced TNF-α expression in LPS-challenged mice, demonstrating efficacy in modulating immune response in an organ-selective manner. This research highlights the potential of the NPSC platform for targeted immunotherapy and further manipulation of the immune system. Copyright © 2018 Elsevier B.V. All rights reserved.

Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

NASA Technical Reports Server (NTRS)

Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1994-01-01

A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

Mechanisms of Chemical Modulation and Toxicity of the Immune System.

DTIC Science & Technology

1988-05-15

expressing the L3T4 surface antigens and suppressor/cytotoxic cells bearing Lyt-2 surface antigens. Autoimmune or immunodeficient diseases have been...Ill. Written Publications (cumulative list) A. Suppression of mitogen-induced blastogenesis of feline lymphocytes by in vitro incubation with

A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Webb, Carol F., E-mail: carol-webb@omrf.org; Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK; Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK

Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights:more » • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.« less

Transient and Big Are Key Features of an Invertebrate T-type Channel (LCav3) from the Central Nervous System of Lymnaea stagnalis*

PubMed Central

Senatore, Adriano; Spafford, J. David

2010-01-01

Here we describe features of the first non-mammalian T-type calcium channel (LCav3) expressed in vitro. This molluscan channel possesses combined biophysical properties that are reminiscent of all mammalian T-type channels. It exhibits T-type features such as “transient” kinetics, but the “tiny” label, usually associated with Ba2+ conductance, is hard to reconcile with the “bigness” of this channel in many respects. LCav3 is 25% larger than any voltage-gated ion channel expressed to date. It codes for a massive, 322-kDa protein that conducts large macroscopic currents in vitro. LCav3 is also the most abundant Ca2+ channel transcript in the snail nervous system. A window current at typical resting potentials appears to be at least as large as that reported for mammalian channels. This distant gene provides a unique perspective to analyze the structural, functional, drug binding, and evolutionary aspects of T-type channels. PMID:20056611

Hypoxia reduces testosterone synthesis in mouse Leydig cells by inhibiting NRF1-activated StAR expression

PubMed Central

Zou, Zhiran; Wang, Dan; Lu, Yapeng; Dong, Zhangji; Zhu, Li

2017-01-01

Male fertility disorders play a key role in half of all infertility cases. Reduction in testosterone induced by hypoxia might cause diseases in reproductive system and other organs. Hypoxic exposure caused a significant decrease of NRF1. Software analysis reported that the promoter region of steroidogenic acute regulatory protein (StAR) contained NRF1 binding sites, indicating NRF1 promoted testicular steroidogenesis. The purpose of this study is to determine NRF1 is involved in testosterone synthesis; and under hypoxia, the decrease of testosterone synthesis is caused by lower expression of NRF1. We designed both in vivo and in vitro experiments. Under hypoxia, the expressions of NRF1 in Leydig cells and testosterone level were significantly decreased both in vivo and in vitro. Overexpression and interference NRF1 could induced StAR and testosterone increased and decreased respectively. ChIP results confirmed the binding of NRF1 to StAR promoter region. In conclusion, decline of NRF1 expression downregulated the level of StAR, which ultimately resulted in a reduction in testosterone synthesis. PMID:28146428

Age-related alterations in basal expression and in vitro, tumour necrosis factor alpha mediated, upregulation of CD11b.

PubMed

Armstrong, M E; Alexander, H D; Ritchie, J L; McMillan, S A; Rea, I M

2001-01-01

The beta(2-)integrin CD11b (Mac-1) plays a crucial role in the firm attachment of leucocytes to the endothelium during the inflammatory response. This study aimed to determine whether the increased incidence of infections witnessed in elderly individuals compared to their younger counterparts was associated with deficiencies in basal expression and/or upregulation of CD11b. Flow cytometry was used to measure CD11b expression, before and after in vitro tumour necrosis factor alpha (TNF-alpha) stimulation, on neutrophils, monocytes and lymphocytes from healthy volunteers aged less than 36 years and Senieur-approximated 70-85 and over 85 year olds. The TNF-alpha levels in serum were measured using a commercially available enzyme-linked immunoassay technique. The basal expression of CD11b on monocytes and lymphocytes was highest in the 70-85-year-olds and lowest in the > 85-year-olds. Following in vitro stimulation using low (10 IU) and high (100 IU) TNF-alpha concentrations, subjects > 85 years consistently showed significantly lower increases in CD11b expression on each of the three cell types. The maximal increase in CD11b expression was in the 70-85-year age group for neutrophils and monocytes and in < 36-year-olds for lymphocytes. Serum TNF-alpha was significantly higher in the elderly groups. Regression analysis showed a significant association between TNF-alpha and expression of CD11b on lymphocytes before and after TNF-alpha stimulation and for neutrophils before stimulation. The results of this study suggest that CD11b expression on leucocytes may not be consistent throughout life. Such age-related changes could compromise the inflammatory response, rendering individuals > 85 years old more susceptible to infections. Alternatively, the lower levels of CD11b expression in this group may represent downregulation and protection against excess leucocyte activation within the vascular system and may, therefore, provide a mechanism for successful ageing. Copyright 2001 S. Karger AG, Basel

Verification of γ-Amino-Butyric Acid (GABA) Signaling System Components in Periodontal Ligament Cells In Vivo and In Vitro.

PubMed

Konermann, Anna; Kantarci, Alpdogan; Wilbert, Steven; Van Dyke, Thomas; Jäger, Andreas

2016-11-01

CNS key neurotransmitter γ-amino-butyric acid (GABA) and its signaling components are likewise detectable in non-neuronal tissues displaying inter alia immunomodulatory functions. This study aimed at identifying potential glutamate decarboxylase (GAD)65 and GABA receptor expression in periodontal ligament (PDL) cells in vivo and in vitro, with particular regard to inflammation and mechanical loading. Gene expression was analyzed in human PDL cells at rest or in response to IL-1ß (5 ng/ml) or TNFα (5 ng/ml) challenge via qRT-PCR. Western blot determined constitutive receptor expression, and confocal laser scanning fluorescence microscopy visualized expression changes induced by inflammation. ELISA quantified GAD65 release. Immunocytochemistry was performed for GABA component detection in vitro on mechanically loaded PDL cells, and in vivo on rat upper jaw biopsies with mechanically induced root resorptions. Statistical significance was set at p < 0.05. GABA B1 , GABA B2 , GABA A1 , and GABA A3 were ubiquitously expressed both on gene and protein level. GABA A2 and GAD65 were undetectable in resting cells, but induced by inflammation. GABA B1 exhibited the highest basal gene expression (6.97 % ± 0.16). IL-1ß markedly increased GABA B2 on a transcriptional (57.28-fold ± 12.40) and protein level seen via fluorescence microscopy. TNFα-stimulated PDL cells released GAD65 (3.68 pg/ml ± 0.17 after 24 h, 5.77 pg/ml ± 0.65 after 48 h). Immunocytochemistry revealed GAD65 expression in mechanically loaded PDL cells. In vivo, GABA components were varyingly expressed in an inflammatory periodontal environment. PDL cells differentially express GABA signaling components and secrete GAD65. Inflammation and mechanical loading regulate these neurotransmitter molecules, which are also detectable in vivo and are potentially involved in periodontal pathophysiology.

A three-dimensional cell culture system as an in vitro canine mammary carcinoma model for the expression of connective tissue modulators.

PubMed

Cardoso, T C; Sakamoto, S S; Stockmann, D; Souza, T F B; Ferreira, H L; Gameiro, R; Vieira, F V; Louzada, M J Q; Andrade, A L; Flores, E F

2017-06-01

In this study, derived complex carcinoma (CC) and simple carcinoma (SC) cell lines were established and cultured under two-dimensional (2D) and three-dimensional (3D) conditions. The 3D was performed in six-well AlgiMatrix™ (LifeTechnologies®, Carlsbad, CA, USA) scaffolds, resulting in spheroids sized 50-125 µm for CC and 175-200 µm for SC. Cell viability was demonstrated up to 14 days for both models. Epidermal growth factor receptor (EGFR) was expressed in CC and SC in both systems. However, higher mRNA and protein levels were observed in SC 2D and 3D systems when compared with CC (P < 0.005). The connective tissue modulators, metalloproteinases-1, -2, -9 and -13 (MMPs), relaxin receptors 1 and 2 (RXR1 and RXR2) and E-cadherin (CDH1) were quantitated. All were upregulated similarly when canine mammary tumour (CMT)-derived cell lines were cultured under 3D AlgiMatrix, except CDH1 that was downregulated (P < 0.005). These results are promising towards the used of 3D system to increase a high throughput in vitro canine tumour model. © 2016 John Wiley & Sons Ltd.

MicroRNA-133 controls vascular smooth muscle cell phenotypic switch in vitro and vascular remodeling in vivo.

PubMed

Torella, Daniele; Iaconetti, Claudio; Catalucci, Daniele; Ellison, Georgina M; Leone, Angelo; Waring, Cheryl D; Bochicchio, Angela; Vicinanza, Carla; Aquila, Iolanda; Curcio, Antonio; Condorelli, Gianluigi; Indolfi, Ciro

2011-09-30

MicroRNA (miR)-1 and -133 play a crucial role in skeletal and cardiac muscle biology and pathophysiology. However, their expression and regulation in vascular cell physiology and disease is currently unknown. The aim of the present study was to evaluate the role, if any, of miR-1 and miR-133 in vascular smooth muscle cell (VSMC) phenotypic switch in vitro and in vivo. We demonstrate here that miR-133 is robustly expressed in vascular smooth muscle cells (VSMCs) in vitro and in vivo, whereas miR-1 vascular levels are negligible. miR-133 has a potent inhibitory role on VSMC phenotypic switch in vitro and in vivo, whereas miR-1 does not have any relevant effect per se. miR-133 expression is regulated by extracellular signal-regulated kinase 1/2 activation and is inversely correlated with VSMC growth. Indeed, miR-133 decreases when VSMCs are primed to proliferate in vitro and following vascular injury in vivo, whereas it increases when VSMCs are coaxed back to quiescence in vitro and in vivo. miR-133 loss- and gain-of-function experiments show that miR-133 plays a mechanistic role in VSMC growth. Accordingly, adeno-miR-133 reduces but anti-miR-133 exacerbates VSMC proliferation and migration in vitro and in vivo. miR-133 specifically suppresses the transcription factor Sp-1 expression in vitro and in vivo and through Sp-1 repression regulates smooth muscle gene expression. Our data show that miR-133 is a key regulator of vascular smooth muscle cell phenotypic switch in vitro and in vivo, suggesting its potential therapeutic application for vascular diseases.

Transcriptome Profiling of Human FoxP3+ Regulatory T Cells

PubMed Central

Bhairavabhotla, Ravikiran; Kim, Yong C.; Glass, Deborah D.; Escobar, Thelma M.; Patel, Mira C.; Zahr, Rami; Nguyen, Cuong K.; Kilaru, Gokhul K.; Muljo, Stefan A.; Shevach, Ethan M.

2015-01-01

The major goal of this study was to perform an in depth characterization of the “gene signature” of human FoxP3+ T regulatory cells (Tregs). Highly purified Tregs and T conventional cells (Tconvs) from multiple healthy donors (HD), either freshly explanted or activated in vitro, were analyzed via RNA sequencing (RNA-seq) and gene expression changes validated using the nCounter system. Additionally, we analyzed microRNA (miRNA) expression using TaqMan low-density arrays. Our results confirm previous studies demonstrating selective gene expression of FoxP3, IKZF2, and CTLA4 in Tregs. Notably, a number of yet uncharacterized genes (RTKN2, LAYN, UTS2, CSF2RB, TRIB1, F5, CECAM4, CD70, ENC1 and NKG7) were identified and validated as being differentially expressed in human Tregs. We further characterize the functional roles of RTKN2 and LAYN by analyzing their roles in vitro human Treg suppression assays by knocking them down in Tregs and overexpressing them in Tconvs. In order to facilitate a better understanding of the human Treg gene expression signature, we have generated from our results a hypothetical interactome of genes and miRNAs in Tregs and Tconvs, PMID:26686412

Regulation of period 1 expression in cultured rat pineal

NASA Technical Reports Server (NTRS)

Fukuhara, Chiaki; Dirden, James C.; Tosini, Gianluca

2002-01-01

The aim of the present study was to investigate the in vitro expression of Period 1 (Per1), Period 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) genes in the rat pineal gland to understand the mechanism(s) regulating the expression of these genes in this organ. Pineals, when maintained in vitro for 5 days, did not show circadian rhythmicity in the expression of any of the three genes monitored. Norepinephrine (NE) induced AA-NAT and Per1, whereas its effect on Per2 was negligible. Contrary to what was observed in other systems, NE stimulation did not induce circadian expression of Per1. The effect of NE on Per1 level was dose- and receptor subtype-dependent, and both cAMP and cGMP induced Per1. Per1 was not induced by repeated NE - or forskolin - stimulation. Protein synthesis was not necessary for NE-induced Per1, but it was for reduction of Per1 following NE stimulation. Per1 transcription in pinealocytes was activated by BMAL1/CLOCK. Our results indicate that important differences are present in the regulation of these genes in the mammalian pineal. Copyright 2002 S. Karger AG, Basel.

Cellular respiration: replicating in vivo systems biology for in ...

EPA Pesticide Factsheets

This editorial develops a philosophy for expanding the scope of Journal of Breath Research (JBR) into the realm of cellular level study, and links certain topics back to more traditional systemic research for understanding human health based on exhaled breath constituents. The express purpose is to provide a publication outlet for novel breath related research that includes in vitro studies, especially those that explore the biological origin and expression of compounds that may ultimately influence the constituents of exhaled breath. The new topics include all manner of methods and instrumentations for making in vivo and in vitro measurements, the use of different biological media (blood, urine saliva, swabs) including human and microbial cell-lines, in vitro kinetic studies of metabolism, and advances in ex vivo methods for maintaining metabolic competency and viability of biological samples. Traditionally, JBR has published articles on human breath analysis for diagnosing disease, tracking health state, assessing the dose and effect of exogenous chemicals, and contributions of malodorous compounds from the oral/nasal cavity. These have also included research describing novel sampling and analytical technologies, most notably those implementing mass spectrometry, chemical sensors and optical measurement instrumentation (Amann and Smith 2013). The journal’s original scope has also embraced animal models as surrogates for human sampling, new mathematical and

Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.

PubMed

Cottet, Sandra; Corthésy-Theulaz, Irène; Spertini, François; Corthésy, Blaise

2002-09-13

Molecular dissection of the mechanisms underlying Helicobacter pylori infection suffers from the lack of in vitro systems mimicking in vivo observations. A system was developed whereby human epithelial cells (Caco-2) grown as polarized monolayers and bacteria can communicate with each other under culture conditions optimal for each partner. Caco-2 cells grown on filter supports were inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO(2) at their basolateral surface (aerophilic conditions) and 5% CO(2), 5% O(2), 90% N(2) (microaerophilic conditions) in the apical compartment. Remarkably, the epithelial polarized layer was stable under these asymmetric culture conditions for at least 24 h, and the presence of Caco-2 cells was necessary to maintain H. pylori growth. In contrast to previous studies conducted with non-polarized Caco-2 cells and other cell lines kept under aerophilic conditions, we found H. pylori-dependent stimulation of cytokine secretion (MCP-1 (monocyte chemoattractant protein-1), GRO-alpha (growth-regulated oncogene-alpha), RANTES (regulated on activation normal T cell expressed and secreted)). This correlated with nuclear translocation of NF-kappaB p50 and p65 subunits. Tyrosine phosphorylation of nine cellular proteins was induced or enhanced; we identified p120(RasGAP), p190(RhoGAP), p62dok (downstream of tyrosine kinases), and cortactin as H. pylori-inducible targets. Moreover, reduction of H. pylori urease expression was observed in adherent bacteria as compared with bacteria in suspension. In addition to mimicking several observations seen in the inflamed gastric mucosa, the novel in vitro system was allowed to underscore complex cellular events not seen in classical in vitro analyses of microaerophilic bacteria-epithelial cell cross-talk.

Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit.

PubMed

Rettig, J; Heinemann, S H; Wunder, F; Lorra, C; Parcej, D N; Dolly, J O; Pongs, O

1994-05-26

Structural and functional diversity of voltage-gated Kv1-type potassium channels in rat brain is enhanced by the association of two different types of subunits, the membrane-bound, poreforming alpha-subunits and a peripheral beta-subunit. We have cloned a beta-subunit (Kv beta 1) that is specifically expressed in the rat nervous system. Association of Kv beta 1 with alpha-subunits confers rapid A-type inactivation on non-inactivating Kv1 channels (delayed rectifiers) in expression systems in vitro. This effect is mediated by an inactivating ball domain in the Kv beta 1 amino terminus.

Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification☆

PubMed Central

Wilson, K.; Mole, D.J.; Binnie, M.; Homer, N.Z.M.; Zheng, X.; Yard, B.A.; Iredale, J.P.; Auer, M.; Webster, S.P.

2014-01-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington’s disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

PubMed

Qiu, Ji; LaBaer, Joshua

2011-01-01

Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

Strong Correlation of Genome-Wide Expression after Traumatic Brain Injury In Vitro and In Vivo Implicates a Role for SORLA

PubMed Central

Lamprecht, Michael R.; Elkin, Benjamin S.; Kesavabhotla, Kartik; Crary, John F.; Hammers, Jennifer L.; Huh, Jimmy W.; Raghupathi, Ramesh

2017-01-01

Abstract The utility of in vitro models of traumatic brain injury (TBI) depends on their ability to recapitulate the in vivo TBI cascade. In this study, we used a genome-wide approach to compare changes in gene expression at several time points post-injury in both an in vitro model and an in vivo model of TBI. We found a total of 2073 differentially expressed genes in our in vitro model and 877 differentially expressed genes in our in vivo model when compared to noninjured controls. We found a strong correlation in gene expression changes between the two models (r = 0.69), providing confidence that the in vitro model represented at least part of the in vivo injury cascade. From these data, we searched for genes with significant changes in expression over time (analysis of covariance) and identified sorting protein-related receptor with A-type repeats (SORLA). SORLA directs amyloid precursor protein to the recycling pathway by direct binding and away from amyloid-beta producing enzymes. Mutations of SORLA have been linked to Alzheimer's disease (AD). We confirmed downregulation of SORLA expression in organotypic hippocampal slice cultures by immunohistochemistry and Western blotting and present preliminary data from human tissue that is consistent with these experimental results. Together, these data suggest that the in vitro model of TBI used in this study strongly recapitulates the in vivo TBI pathobiology and is well suited for future mechanistic or therapeutic studies. The data also suggest the possible involvement of SORLA in the post-traumatic cascade linking TBI to AD. PMID:26919808

Biofilms promote survival and virulence of Salmonella enterica sv. Tennessee during prolonged dry storage and after passage through an in vitro digestion system.

PubMed

Aviles, Bryan; Klotz, Courtney; Eifert, Joseph; Williams, Robert; Ponder, Monica

2013-04-01

Salmonella enterica serotypes have been linked to outbreaks associated with low water activity foods. While the biofilm-forming abilities of Salmonella improve its survival during thermal processing and sanitation it is unclear whether biofilms enhance survival to desiccation and gastric stresses. The purpose of this study was to quantify the effect of physiological state (planktonic versus biofilm) and prior exposure to desiccation and storage in dry milk powder on Salmonella survival and gene expression after passage through an in vitro digestion model. Planktonic cells of Salmonella enterica serotype Tennessee were deposited onto membranes while biofilms were formed on glass beads. The cells were subsequently dried at room temperature and stored in dried milk powder (a(w)=0.3) for up to 30 days. Salmonella survival was quantified by serial dilution onto Brilliant Green Agar before desiccation, after desiccation, after 1-day storage and after 30-day storage. At each sampling period both physiological states were tested for survival through a simulated gastrointestinal system. RNA was extracted at the identical time points and Quantitative Real-Time PCR was used to determine relative expression for genes associated with stress response (rpoS, otsB), virulence (hilA, invA, sipC) and a housekeeping gene 16S rRNA. The physiological state and length of storage affected the survival and gene expression of Salmonella within the desiccated milk powder environment and after passage through an in vitro digestion system (p<0.05). Larger numbers of S. Tennessee were recovered by plate counts for biofilms compared to planktonic, however, the numbers of Salmonella genomes detected by qPCR were not significantly different suggesting entry of the planktonic cells of S. Tennessee into a viable but non-culturable state. The increased expression of stress response genes rpoS and otsB correlated with survival, indicating cross-protection to low water activity and acid stress. Increased expression of virulence-associated genes was seen in cells exposed to dry storage for short periods, however the largest amount of expression occurred in biofilm cells stored for 30 days at aw 0.3, suggesting increased virulence potential. Copyright © 2013 Elsevier B.V. All rights reserved.

The Differentiation of Human Adipose-Derived Stem Cells towards a Urothelium-Like Phenotype In Vitro and the Dynamic Temporal Changes of Related Cytokines by Both Paracrine and Autocrine Signal Regulation

PubMed Central

Zhou, Zhe; Zhang, Ke; Zhou, Juan; Zhao, Yang; Wang, Zhong; Lu, Mu-jun

2014-01-01

Purpose To investigate the differentiation ability of human adipose-derived stem cells (ASCs) towards urothelium-like cells in vitro and the dynamic changes of related cytokines and cytokine receptors in the culture medium. Materials and Methods The ASCs were induced using both conditioned media (CM) and the transwell co-culture system with an immortalized urothelium cell line (SV-HUC-1,HUC) for 21 days. Protein and mRNA expression of the mature urothelium specific markers uroplakin-IA (UP-1A) and uroplakin-II (UP-II) were detected by immunofluorescence and quantitative real-time PCR, respectively. Array detection was used to screen 41 cytokines and receptors in the upper medium of urothelium, non-induced ASCs and urothelium-induced ASCs at three time points, early (12 hours), intermediate (7 days) and late (21 days). Results After induction for 7 days, the ASCs grown in both CM and transwell co-culture system expressed uroplakin-IA (13.54±2.00%; 17.28±1.84%) and uroplakin-II (19.49±1.73%; 13.98±1.47%). After induction for 21 days, ASCs grown in co-culture had significantly increased expression of uroplakin-IA (48.03±1.25%; 49.57±2.85%) and uroplakin-II (45.38±2.50%; 46.58±1.95%). In the upper medium of urothelium, 28 cytokines and 8 cytokine receptors had significantly higher expression than the counterpart of non-induced ASCs. After 7 days induction, the expression of 22 cytokines and 8 cytokine receptors was significantly elevated in the upper medium of induced ASCs compared to non-induced ASCs. At the early and intermediate time points, ASCs secreted high levels of relative cytokines and soluble receptors, but their expressions decreased significantly at the late time point. Conclusion The adipose-derived stem cells have the potential to be differentiated into urothelium-like cells in vitro by both CM and transwell co-culture system with mature urothelium. Numerous cytokines and receptors were involved in the differentiation process with dynamic temporal changes by both paracrine and autocrine signal regulation. Further studies should be carried out to determine the detailed mechanism of cytokines and receptors and to enhance the urothelium differentiation efficiency of ASCs. PMID:24752317

The differentiation of human adipose-derived stem cells towards a urothelium-like phenotype in vitro and the dynamic temporal changes of related cytokines by both paracrine and autocrine signal regulation.

PubMed

Zhang, Ming; Xu, Ming-Xi; Zhou, Zhe; Zhang, Ke; Zhou, Juan; Zhao, Yang; Wang, Zhong; Lu, Mu-Jun

2014-01-01

To investigate the differentiation ability of human adipose-derived stem cells (ASCs) towards urothelium-like cells in vitro and the dynamic changes of related cytokines and cytokine receptors in the culture medium. The ASCs were induced using both conditioned media (CM) and the transwell co-culture system with an immortalized urothelium cell line (SV-HUC-1,HUC) for 21 days. Protein and mRNA expression of the mature urothelium specific markers uroplakin-IA (UP-1A) and uroplakin-II (UP-II) were detected by immunofluorescence and quantitative real-time PCR, respectively. Array detection was used to screen 41 cytokines and receptors in the upper medium of urothelium, non-induced ASCs and urothelium-induced ASCs at three time points, early (12 hours), intermediate (7 days) and late (21 days). After induction for 7 days, the ASCs grown in both CM and transwell co-culture system expressed uroplakin-IA (13.54±2.00%; 17.28±1.84%) and uroplakin-II (19.49±1.73%; 13.98±1.47%). After induction for 21 days, ASCs grown in co-culture had significantly increased expression of uroplakin-IA (48.03±1.25%; 49.57±2.85%) and uroplakin-II (45.38±2.50%; 46.58±1.95%). In the upper medium of urothelium, 28 cytokines and 8 cytokine receptors had significantly higher expression than the counterpart of non-induced ASCs. After 7 days induction, the expression of 22 cytokines and 8 cytokine receptors was significantly elevated in the upper medium of induced ASCs compared to non-induced ASCs. At the early and intermediate time points, ASCs secreted high levels of relative cytokines and soluble receptors, but their expressions decreased significantly at the late time point. The adipose-derived stem cells have the potential to be differentiated into urothelium-like cells in vitro by both CM and transwell co-culture system with mature urothelium. Numerous cytokines and receptors were involved in the differentiation process with dynamic temporal changes by both paracrine and autocrine signal regulation. Further studies should be carried out to determine the detailed mechanism of cytokines and receptors and to enhance the urothelium differentiation efficiency of ASCs.

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin

PubMed Central

2011-01-01

Background In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. Results The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. Conclusions This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis. PMID:21658265

Effects of intense magnetic fields on sedimentation pattern and gene expression profile in budding yeast

NASA Astrophysics Data System (ADS)

Ikehata, Masateru; Iwasaka, Masakazu; Miyakoshi, Junji; Ueno, Shoogo; Koana, Takao

2003-05-01

Effects of magnetic fields (MFs) on biological systems are usually investigated using biological indices such as gene expression profiles. However, to precisely evaluate the biological effects of MF, the effects of intense MFs on systematic material transport processes including experimental environment must be seriously taken into consideration. In this study, a culture of the budding yeast, Saccharomyces cerevisiae, was used as a model for an in vitro biological test system. After exposure to 5 T static vertical MF, we found a difference in the sedimentation pattern of cells depending on the location of the dish in the magnet bore. Sedimented cells were localized in the center of the dish when they were placed in the lower part of the magnet bore while the sedimentation of the cells was uniform in dishes placed in the upper part of the bore because of the diamagnetic force. Genome wide gene expression profile of the yeast cells after exposure to 5 T static MF for 2 h suggested that the MF did not affect the expression level of any gene in yeast cells although the sedimentation pattern was altered. In addition, exposure to 10 T for 1 h and 5 T for 24 h also did not affect the gene expression. On the other hand, a slight change in expressions of several genes which are related to respiration was observed by exposure to a 14 T static MF for 24 h. The necessity of estimating the indirect effects of MFs on a study of its biological effect of MF in vitro will be discussed.

Non-invasive neural stem cells become invasive in vitro by combined FGF2 and BMP4 signaling.

PubMed

Sailer, Martin H M; Gerber, Alexandra; Tostado, Cristóbal; Hutter, Gregor; Cordier, Dominik; Mariani, Luigi; Ritz, Marie-Françoise

2013-08-15

Neural stem cells (NSCs) typically show efficient self-renewal and selective differentiation. Their invasion potential, however, is not well studied. In this study, Sox2-positive NSCs from the E14.5 rat cortex were found to be non-invasive and showed only limited migration in vitro. By contrast, FGF2-expanded NSCs showed a strong migratory and invasive phenotype in response to the combination of FGF2 and BMP4. Invasive NSCs expressed Podoplanin (PDPN) and p75NGFR (Ngfr) at the plasma membrane after exposure to FGF2 and BMP4. FGF2 and BMP4 together upregulated the expression of Msx1, Snail1, Snail2, Ngfr, which are all found in neural crest (NC) cells during or after epithelial-mesenchymal transition (EMT), but not in forebrain stem cells. Invasive cells downregulated the expression of Olig2, Sox10, Egfr, Pdgfra, Gsh1/Gsx1 and Gsh2/Gsx2. Migrating and invasive NSCs had elevated expression of mRNA encoding Pax6, Tenascin C (TNC), PDPN, Hey1, SPARC, p75NGFR and Gli3. On the basis of the strongest upregulation in invasion-induced NSCs, we defined a group of five key invasion-related genes: Ngfr, Sparc, Snail1, Pdpn and Tnc. These genes were co-expressed and upregulated in seven samples of glioblastoma multiforme (GBM) compared with normal human brain controls. Induction of invasion and migration led to low expression of differentiation markers and repressed proliferation in NSCs. Our results indicate that normal forebrain stem cells have the inherent ability to adopt a glioma-like invasiveness. The results provide a novel in vitro system to study stem cell invasion and a novel glioma invasion model: tumoral abuse of the developmental dorsoventral identity regulation.

The SGBS cell strain as a model for the in vitro study of obesity and cancer.

PubMed

Allott, Emma H; Oliver, Elizabeth; Lysaght, Joanne; Gray, Steven G; Reynolds, John V; Roche, Helen M; Pidgeon, Graham P

2012-10-01

The murine adipocyte cell line 3T3-L1 is well characterised and used widely, while the human pre-adipocyte cell strain, Simpson-Golabi-Behmel Syndrome (SGBS), requires validation for use in human studies. Obesity is currently estimated to account for up to 41 % of the worldwide cancer burden. A human in vitro model system is required to elucidate the molecular mechanisms for this poorly understood association. This work investigates the relevance of the SGBS cell strain for obesity and cancer research in humans. Pre-adipocyte 3T3-L1 and SGBS were differentiated according to standard protocols. Morphology was assessed by Oil Red O staining. Adipocyte-specific gene expression was measured by qPCR and biochemical function was assessed by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity. Differential gene expression in oesophageal adenocarcinoma cell line OE33 following co-culture with SGBS or primary omental human adipocytes was investigated using Human Cancer Profiler qPCR arrays. During the process of differentiation, SGBS expressed higher levels of adipocyte-specific transcripts and fully differentiated SGBS expressed more similar morphology, transcript levels and biochemical function to primary omental adipocytes, relative to 3T3-L1. Co-culture with SGBS or primary omental adipocytes induced differential expression of genes involved in adhesion (ITGB3), angiogenesis (IGF1, TEK, TNF, VEGFA), apoptosis (GZMA, TERT) and invasion and metastasis (MMP9, TIMP3) in OE33 tumour cells. Comparable adipocyte-specific gene expression, biochemical function and a shared induced gene signature in co-cultured OE33 cells indicate that SGBS is a relevant in vitro model for obesity and cancer research in humans.

FERRITIN EXPRESSION AFTER IN VITRO EXPOSURES OF HUMAN ALVEOLAR MACROPHAGES TO SILICA IS IRON-DEPENDENT

EPA Science Inventory

The increased availability of catalytically active iron after silica exposure can present an oxidative injury to a living system. Sequestration of reactive iron would, therefore, confer a protective effect. The intracellular storage of iron by ferritin within macrophages can limi...

In vivo and in vitro invasion in relation to phenotypic characteristics of human colorectal carcinoma cells.

PubMed Central

de Vries, J. E.; Dinjens, W. N.; De Bruyne, G. K.; Verspaget, H. W.; van der Linden, E. P.; de Bruïne, A. P.; Mareel, M. M.; Bosman, F. T.; ten Kate, J.

1995-01-01

In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7841040

Discovery of a stem-like multipotent cell fate.

PubMed

Paffhausen, Emily S; Alowais, Yasir; Chao, Cara W; Callihan, Evan C; Creswell, Karen; Bracht, John R

2018-01-01

Adipose derived stem cells (ASCs) can be obtained from lipoaspirates and induced in vitro to differentiate into bone, cartilage, and fat. Using this powerful model system we show that after in vitro adipose differentiation a population of cells retain stem-like qualities including multipotency. They are lipid (-), retain the ability to propagate, express two known stem cell markers, and maintain the capacity for trilineage differentiation into chondrocytes, adipocytes, and osteoblasts. However, these cells are not traditional stem cells because gene expression analysis showed an overall expression profile similar to that of adipocytes. In addition to broadening our understanding of cellular multipotency, our work may be particularly relevant to obesity-associated metabolic disorders. The adipose expandability hypothesis proposes that inability to differentiate new adipocytes is a primary cause of metabolic syndrome in obesity, including diabetes and cardiovascular disease. Here we have defined a differentiation-resistant stem-like multipotent cell population that may be involved in regulation of adipose expandability in vivo and may therefore play key roles in the comorbidities of obesity.

Suppression of inflammation-associated factors by indole-3-carbinol in mice fed high-fat diets and in isolated, co-cultured macrophages and adipocytes.

PubMed

Chang, H-P; Wang, M-L; Hsu, C-Y; Liu, M-E; Chan, M-H; Chen, Y-H

2011-12-01

This study investigated the effects of indole-3-carbinol (I3C), a compound from cruciferous vegetables, on various parameters related to obesity, in particular, the parameters of infiltration by macrophages and of inflammatory cytokines expressed during the co-culture of adipocytes and macrophages. Male C57BL/6 mice were fed with a control diet (C group), high-fat diet (HF group) and HF+5 mg kg(-1) I3C (HFI group). The I3C was intraperitoneally injected (HFI group) for 12 weeks. Epididymal adipose tissue (AT) was collected and stained for F4/80, a marker of macrophages. The immunohistochemical staining for F4/80 indicated a greater presence of macrophages in the HF group than in AT from the control and HFI groups. Furthermore, I3C treatment, in an in vitro cell culture system, decreased expression of inducible nitric oxide synthase (iNOS), decreased nitrite content and enhanced expression of peroxisome proliferator-activated receptor (PPAR-γ). Moreover, in vitro cell culture studies revealed that I3C inhibited intracellular lipid accumulation in hypertrophied adipocytes. In macrophage and primary adipocyte co-culture, I3C inhibited expression of interleukin-6 (IL-6). In vivo treatment with I3C reduced the infiltration of macrophages in AT, and in vitro addition of I3C to co-cultured macrophages and adipocytes reduced nitrite production and IL-6 expression. With cultures of adipocytes only, I3C inhibited accumulation of intracellular lipid, either by disrupting differentiation, or by directly inhibiting triglyceride synthesis.

Cell-Free Optogenetic Gene Expression System.

PubMed

Jayaraman, Premkumar; Yeoh, Jing Wui; Jayaraman, Sudhaghar; Teh, Ai Ying; Zhang, Jingyun; Poh, Chueh Loo

2018-04-20

Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, their vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modeling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of the target gene to generate oscillatory response. The deterministic model developed was able to recapitulate the system behavior and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.

Mifepristone-inducible transgene expression in neural progenitor cells in vitro and in vivo

PubMed Central

Hjelm, BE; Grunseich, C; Gowing, G; Avalos, P; Tian, J; Shelley, BC; Mooney, M; Narwani, K; Shi, Y; Svendsen, CN; Wolfe, JH; Fischbeck, KH; Pierson, TM

2016-01-01

Numerous gene and cell therapy strategies are being developed for the treatment of neurodegenerative disorders. Many of these strategies use constitutive expression of therapeutic transgenic proteins, and although functional in animal models of disease, this method is less likely to provide adequate flexibility for delivering therapy to humans. Ligand-inducible gene expression systems may be more appropriate for these conditions, especially within the central nervous system (CNS). Mifepristone’s ability to cross the blood–brain barrier makes it an especially attractive ligand for this purpose. We describe the production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure that the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for use in the development of safe and efficient gene therapy for neurological disorders. PMID:26863047

Multipotent mesenchymal stromal cells decrease transforming growth factor β1 expression in microglia/macrophages and down-regulate plasminogen activator inhibitor 1 expression in astrocytes after stroke.

PubMed

Xin, Hongqi; Chopp, Michael; Shen, Li Hong; Zhang, Rui Lan; Zhang, Li; Zhang, Zheng Gang; Li, Yi

2013-05-10

Multipotent mesenchymal stromal cells (MSCs) decrease the expression of transforming growth factor β1 (TGFβ1) in astrocytes and subsequently decrease astrocytic plasminogen activator inhibitor 1 (PAI-1) level in an autocrine manner. Since activated microglia/macrophages are also a source of TGFβ1 after stroke, we therefore tested whether MSCs regulate TGFβ1 expression in microglia/macrophages and subsequently alters PAI-1 expression after ischemia. TGFβ1 and its downstream effector phosphorylated SMAD 2/3 (p-SMAD 2/3) were measured in mice subjected to middle cerebral artery occlusion (MCAo). MSC treatment significantly decreased TGFβ1 protein expression in both astrocytes and microglia/macrophages in the ischemic boundary zone (IBZ) at day 14 after stroke. However, the p-SMAD 2/3 was only detected in astrocytes and decreased after MSC treatment. In vitro, RT-PCR results showed that the TGFβ1 mRNA level was increased in both astrocytes and microglia/macrophages in an astrocyte-microglia/macrophage co-culture system after oxygen-glucose deprived (OGD) treatment. MSCs treatment significantly decreased the above TGFβ1 mRNA level under OGD conditions, respectively. OGD increased the PAI-1 mRNA in astrocytes in the astrocyte-microglia/macrophage co-culture system, and MSC administration significantly decreased this level. PAI-1 mRNA was very low in microglia/macrophages compared with that in astrocytes under different conditions. Western blot results also verified that MSC administration significantly decreased p-SMAD 2/3 and PAI-1 level in astrocytes in astrocyte-microglia/macrophage co-culture system under OGD conditions. Our in vivo and in vitro data, in concert, suggest that MSCs decrease TGFβ1 expression in microglia/macrophages in the IBZ which contribute to the down-regulation of PAI-1 level in astrocytes. Published by Elsevier Ireland Ltd.

Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire.

PubMed

Makeyev, E V; Kolb, V A; Spirin, A S

1999-02-12

A novel cloning-independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single-chain antibody repertoire and express it directly in a cell-free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.

Functional Analyses of the Crohn's Disease Risk Gene LACC1.

PubMed

Assadi, Ghazaleh; Vesterlund, Liselotte; Bonfiglio, Ferdinando; Mazzurana, Luca; Cordeddu, Lina; Schepis, Danika; Mjösberg, Jenny; Ruhrmann, Sabrina; Fabbri, Alessia; Vukojevic, Vladana; Percipalle, Piergiorgio; Salomons, Florian A; Laurencikiene, Jurga; Törkvist, Leif; Halfvarson, Jonas; D'Amato, Mauro

2016-01-01

Genetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn's disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression. We implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function. FAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems. FAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated.

Functional characterization of CXCR4 in mediating the expression of protein C system in experimental ulcerative colitis

PubMed Central

Lin, Xuhong; Wang, Huichao; Li, Yuxia; Yang, Jingnan; Yang, Ruilin; Wei, Dandan; Zhang, Junjie; Yang, Desheng; Wang, Bin; Ren, Xuequn; Cheng, Guanchang

2017-01-01

The present study aimed to explore the role of CXCR4 and protein C system (PCS) in the experimental ulcerative colitis (UC). The expression of CXCR3, CCR10, and CXCR4 in dextran sulfate sodium (DSS)-induced colitis mouse model was measured by immunohistochemistry and western blot analysis. In vitro studies with microvascular endothelial cells (MVECs) were performed. The expression of endothelial protein C receptor (EPCR) and thrombomodulin (TM) were detected by RT-PCR and western blot analysis. Activities of protein C (PC), protein S (PS), activated PC (APC) were evaluated in cells pre-treated with JNK inhibitor SP600125 and c-Jun silencing. DSS mice showed up-regulated expression of CXCR4, higher macroscopic score and histological score (P<0.05), as well as elevated levels of SDF-1α (P<0.05) compared with wild type, CXCR4-/-, or CXCR4-/- +DSS mice. In DSS mice, EPCR expression was down-regulated (P<0.05), accompanied by decreased activity of PC and PS (P<0.05 or P<0.01) with an up-regulated expression of pJNK MAPK and pc-Jun (P<0.05). Moreover, the macroscopic score and histological score index, SDF-1α levels, EPCR expression, PC activity, pJNK, and pc-Jun were reversed in CXCR4-/- +DSS mice (P<0.05). In vitro, SDF-1α-induced inhibition of the PCS was blunted by SP600125 (P<0.05). Meanwhile, down-regulation of c-Jun rescued the inhibition of PCS (P<0.05). MVECs with retrovirus-mediated transfection of c-Jun demonstrated a strong trans-inactivation effect on the EPCR promoter (P<0.05). These findings suggest that CXCR4 is involved in UC pathogenesis and could be a promising therapeutic target for UC treatment. PMID:29218082

A novel platform for biologically active recombinant human interleukin-13 production.

PubMed

Wang, David J; Brandsma, Martin; Yin, Ziqin; Wang, Aiming; Jevnikar, Anthony M; Ma, Shengwu

2008-06-01

Interleukin-13 (IL-13) is a pleiotropic regulatory cytokine with the potential for treating several human diseases, including type-1 diabetes. Thus far, conventional expression systems for recombinant IL-13 production have proven difficult and are limited by efficiency. In this study, transgenic plants were used as a novel expression platform for the production of human IL-13 (hIL-13). DNA constructs containing hIL-13 cDNA were introduced into tobacco plants. Transcriptional expression of the hIL-13 gene in transgenic plants was confirmed by reverse transcriptase-polymerase chain reaction and Northern blotting. Western blot analysis showed that the hIL-13 protein was efficiently accumulated in transgenic plants and present in multiple molecular forms, with an expression level as high as 0.15% of total soluble protein in leaves. The multiple forms of plant-derived recombinant hIL-13 (rhIL-13) are a result of differential N-linked glycosylation, as revealed by enzymatic and chemical deglycosylation, but not of disulphide-linked oligomerization. In vitro trypsin digestion indicated that plant rhIL-13 was more resistant than unglycosylated control rhIL-13 to proteolysis. The stability of plant rhIL-13 to digestion was further supported with simulated gastric and intestinal fluid digestion. In vitro bioassays using a factor-dependent human erythroleukaemic cell line (TF-1 cells) showed that plant rhIL-13 retained the biological functions of the authentic hIL-13 protein. These results demonstrate that transgenic plants are superior to conventional cell-based expression systems for the production of rhIL-13. Moreover, transgenic plants synthesizing high levels of rhIL-13 may prove to be an attractive delivery system for direct oral administration of IL-13 in the treatment of clinical diseases such as type-1 diabetes.

Failure of hepatocyte marker-expressing hematopoietic progenitor cells to efficiently convert into hepatocytes in vitro.

PubMed

Lian, Gewei; Wang, Chengyan; Teng, Chunbo; Zhang, Cong; Du, Liying; Zhong, Qian; Miao, Chenglin; Ding, Mingxiao; Deng, Hongkui

2006-03-01

Whether bone marrow (BM) hematopoietic stem/progenitor cells can directly differentiate into nonhematopoietic cells remains controversial. The aim of this study is to further investigate the potentiality of BM hematopoietic progenitor cells to convert into hepatocytes in vitro. Different subsets of BM cells from C57/BL6 mice were isolated using markers of hematopoietic stem cells by magnetic cell sorting and by flow cytometry. These cells were induced to transdifferentiate to hepatocytes in vitro in the presence of various cytokines or of hepatocytes (or tissue) from damaged liver, which have been reported to stimulate the conversion. Hepatic gene markers in freshly isolated or cultured BM cells were determined by reverse transcriptase polymerase chain reaction and immunofluorescence. Freshly isolated hematopoietic progenitor cells (HPC) expressed a low level of messenger RNAs of hepatic cell-specific markers including albumin and alpha-fetoprotein (AFP), but did not significantly upregulate expression of these markers, even in the presence of cytokines or cocultured hepatocytes (or tissue). HPCs induced in vitro did not express the message of alpha-anti-trypsin-a mature hepatocyte marker. At protein level, the specific staining of AFP was not detected in the HPCs, either freshly isolated or in vitro induced. Albumin protein was detected in freshly isolated albumin mRNA-positive and -negative BM cell subpopulations. Albumin-stained BM cells disappeared after being induced for 5 days, but restained if mouse serum was supplemented in medium for a 24-hour extended culture, suggesting that albumin was absorbed by BM cells instead of de novo expression. HPCs expressed mRNAs of hepatic cell markers, but could not efficiently convert into hepatocytes in vitro under our experimental conditions. Our observation raises a cautionary note in determining whether in vitro transdifferentiation of BM cells to hepatocytes can actually take place.

A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

PubMed

Zhao, Shuan; Liu, Zhen-Xing; Gao, Hui; Wu, Yi; Fang, Yuan; Wu, Shuai-Shuai; Li, Ming-Jie; Bai, Jia-Hua; Liu, Yan; Evans, Alexander; Zeng, Shen-Ming

2015-07-15

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation. Copyright © 2015 Elsevier Inc. All rights reserved.

Whey peptide Isoleucine-Tryptophan inhibits expression and activity of matrix metalloproteinase-2 in rat aorta.

PubMed

Kopaliani, Irakli; Martin, Melanie; Zatschler, Birgit; Müller, Bianca; Deussen, Andreas

2016-08-01

Aortic stiffness is an independent risk factor for development of cardiovascular diseases. Activation of renin-angiotensin-aldosterone system (RAAS) including angiotensin converting enzyme (ACE) activity leads to overproduction of angiotensin II (ANGII) from its precursor angiotensin I (ANGI). ANGII leads to overexpression and activation of matrix metalloproteinase-2 (MMP2), which is critically associated with pathophysiology of aortic stiffness. We previously reported that the whey peptide Isoleucine-Tryptophan (IW) acts as a potent ACE inhibitor. Herein, we critically elucidate the mechanism of action by which IW causes inhibition of expression and activity of MMP2 in aortic tissue. Effects of IW on expression and activity of MMP2 were assessed on endothelial and smooth muscle cells (ECs and SMCs) in vitro and ex vivo (isolated rat aorta). As controls we used the pharmaceutical ACE inhibitor - captopril and the ANGII type 1 receptor blocker - losartan. In vitro, both ANGII and ANGI stimulation significantly (P<0.01) increased expression of MMP2 assessed with western blot. Similarly, to captopril IW significantly (P<0.05) inhibited ANGI, but not ANGII mediated increase in expression of MMP2, while losartan also blocked effects of ANGII. Signaling pathways regulating MMP2 expression in ECs and SMCs were similarly inhibited after treatment with IW or captopril. In ECs IW significantly (P<0.05) inhibited JNK pathway, whereas in SMCs JAK2/STAT3 pathway, assessed with western blot. In vitro findings were fully consistent with results in isolated rat aorta ex vivo. Moreover, IW not only inhibited the MMP2 expression, but also its activation assessed with gelatin zymography. Our findings demonstrate that IW effectively inhibits expression and activation of MMP2 in rat aorta by decreasing local conversion of ANGI to ANGII. Thus, similar to pharmaceutical ACE inhibitor captopril the dipeptide IW may effectively inhibit ACE activity and prevent the age and hypertension associated rise of aortic stiffness. Copyright © 2016 Elsevier Inc. All rights reserved.

Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha6-integrin subunit.

PubMed

Kashimata, M; Gresik, E W

1997-02-01

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) regulate branching morphogenesis of fetal mouse submandibular gland (SMG) rudiments in vitro. The EGF system (EGF, TGF-alpha, and their shared receptor, EGFR) also regulates expression of integrins and their ligands in the extracellular matrix. We show here that inhibition of EGFR tyrosine-kinase activity by a tyrphostin retards in vitro development of SMGs. Using total RNA isolated from pooled SMGs taken from intact mouse fetuses, mRNA transcripts for EGF, TGF-alpha, and EGFR were detected by reverse transcription-polymerase chain reaction (RT-PCR), and age-dependent variations in the levels of these mRNA were quantitatively determined by nuclease protection assays. These findings suggest that the EGF system is operative in the in vivo development of this gland. alpha6-Integrin subunit was localized by immunofluorescence at the basal surface of epithelial cells. Branching morphogenesis of cultured SMG rudiments was inhibited by anti-alpha6 antibodies. Synthesis of alpha6-subunit in cultured SMGs, detected by metabolic labeling and immunoprecipitation, was increased by EGF and drastically reduced by tyrphostin. RT-PCR revealed that mRNAs for alpha6- and beta1- and beta4-integrin subunits are expressed at all ages between embryonic day 13 and postnatal day 7. These findings suggest that 1) the EGF system is a physiologic regulator of development of fetal mouse SMG, and 2) one mechanism by which it acts may be by regulating expression of integrins, which in turn control interaction of epithelial cells with the extracellular matrix.

NIR and MR imaging supported hydrogel based delivery system for anti-TNF alpha probiotic therapy of IBD

NASA Astrophysics Data System (ADS)

Janjic, Jelena M.; Berlec, Ales; Bagia, Christina; Liu, Lu S.; Jeric, Irenej; Gach, Michael; Janjic, Bratislav M.; Strukelj, Borut

2016-03-01

Current treatment of inflammatory bowel disease (IBD) is largely symptomatic and consists of anti-inflammatory agents, immune-suppressives or antibiotics, whereby local luminal action is preferred to minimize systemic side-effects. Recently, anti-TNFα therapy has shown considerable success and is now being routinely used. Here we present a novel approach of using perfluorocarbon (PFC) nanoemulsion containing hydrogels (nanoemulgels) as imaging supported delivery systems for anti-TNF alpha probiotic delivery in IBD. To further facilitate image-guided therapy a food-grade lactic acid bacterium Lactococcus lactis capable of TNFα-binding was engineered to incorporate infrared fluorescent protein (IRFP). This modified bacteria was then incorporated into novel PFC nanoemulgels. The nanoemulgels presented here are designed to deliver locally anti-TNFα probiotic in the lower colon and rectum and provide dual imaging signature of gel delivery (MRI) across the rectum and lower colon and bacteria release (NIR). NIR imaging data in vitro demonstrates high IRFP expressing and TNFα-binding bacteria loading in the hydrogel and complete release in 3 hours. Stability tests indicate that gels remain stable for at least 14 days showing no significant change in droplet size, zeta potential and pH. Flow cytometry analyses demonstrate the NIRF expressing bacteria L. lactis binds TNFα in vitro upon release from the gels. Magnetic resonance and near-infrared imaging in vitro demonstrates homogeneity of hydrogels and the imaging capacity of the overall formulation.

In vitro biomimetic platforms featuring a perfusion system and 3D spheroid culture promote the construction of tissue-engineered corneal endothelial layers.

PubMed

Li, Shanyi; Han, Yuting; Lei, Hao; Zeng, Yingxin; Cui, Zekai; Zeng, Qiaolang; Zhu, Deliang; Lian, Ruiling; Zhang, Jun; Chen, Zhe; Chen, Jiansu

2017-04-10

Corneal endothelial cells (CECs) are very important for the maintenance of corneal transparency. However, in vitro, CECs display limited proliferation and loss of phenotype via endothelial to mesenchymal transformation (EMT) and cellular senescence. In this study, we demonstrate that continuous supplementary nutrition using a perfusion culture bioreactor and three-dimensional (3D) spheroid culture can be used to improve CEC expansion in culture and to construct a tissue-engineered CEC layer. Compared with static culture, perfusion-derived CECs exhibited an increased proliferative ability as well as formed close cell-cell contact junctions and numerous surface microvilli. We also demonstrated that the CEC spheroid culture significantly down-regulated gene expression of the proliferation marker Ki67 and EMT-related markers Vimentin and α-SMA, whereas the gene expression level of the CEC marker ATP1A1 was significantly up-regulated. Furthermore, use of the perfusion system in conjunction with a spheroid culture on decellularized corneal scaffolds and collagen sheets promoted the generation of CEC monolayers as well as neo-synthesized ECM formation. This study also confirmed that a CEC spheroid culture on a curved collagen sheet with controlled physiological intraocular pressure could generate a CEC monolayer. Thus, our results show that the use of a perfusion system and 3D spheroid culture can promote CEC expansion and the construction of tissue-engineered corneal endothelial layers in vitro.

A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3′ untranslated region-mediated gene regulation

PubMed Central

Chaudhury, Arindam; Kongchan, Natee; Gengler, Jon P.; Mohanty, Vakul; Christiansen, Audrey E.; Fachini, Joseph M.; Martin, James F.; Neilson, Joel R.

2014-01-01

Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3′ untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3′ UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3′ UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome—a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3′ UTR of interest. The pBUTR (piggyBac-based 3′ UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting. PMID:24753411

In vitro study of stem cell communication via gap junctions for fibrocartilage regeneration at entheses.

PubMed

Nayak, Bibhukalyan Prasad; Goh, James Cho Hong; Toh, Siew Lok; Satpathy, Gyan Ranjan

2010-03-01

Entheses are fibrocartilaginous organs that bridge ligament with bone at their interface and add significant insertional strength. To replace a severely damaged ligament, a tissue-engineered graft preinstalled with interfacial fibrocartilage, which is being regenerated from stem cells, appears to be more promising than ligament-alone graft. Such a concept can be realized by a biomimetic approach of establishing a dynamic communication of stem cells with bone cells and/or ligament fibroblasts in vitro. The current study has two objectives. The first objective is to demonstrate functional coculture of bone marrow-derived stem cells (BMSCs) with mature bone cells/ligament fibroblasts as evidenced by gap-junctional communication in vitro. The second objective is to investigate the role of BMSCs in the regeneration of fibrocartilage within the coculture. Rabbit bone/ligament fibroblasts were dual-stained with DiI-Red and calcein (gap-junction permeable dye), and cocultured with unlabeled BMSCs at fixed ratio (1:10). The functional gap junction was demonstrated by the transfer of calcein from donor to recipient cells that was confirmed and quantified by flow cytometry. Type 2 collagen (cartilage extracellular matrix-specific protein) expressed by the mixed cell lines in the cocultures were estimated by real-time reverse transcription PCR and compared with that of the ligament-bone coculture (control). Significant transfer of calcein into BMSCs was observed and flow cytometry analyses showed a gradual increase in the percentage of BMSCs acquiring calcein with time. Cocultures that included BMSCs expressed significantly more type 2 collagen compared with the control. The current study, for the first time, reported the expression of gap-junctional communication of BMSCs with two adherent cell lines of musculoskeletal system in vitro and also confirmed that incorporation of stem cells augments fibrocartilage regeneration. The results open up a path to envisage a composite graft preinstalled with enthesial fibrocartilage using a stem cell-based coculture system.

Low-Intensity Pulsed Ultrasound Protects Retinal Ganglion Cell From Optic Nerve Injury Induced Apoptosis via Yes Associated Protein

PubMed Central

Zhou, Jia-Xing; Liu, Yun-Jia; Chen, Xi; Zhang, Xi; Xu, Jie; Yang, Ke; Wang, Dong; Lin, Sen; Ye, Jian

2018-01-01

Background: Low-intensity pulsed ultrasound (LIPUS) has been used in clinical studies. But little is known about its effects on the central nervous system (CNS), or its mechanism of action. Retinal ganglion cells (RGCs) are CNS neuronal cells that can be utilized as a classic model system to evaluate outcomes of LIPUS protection from external trauma-induced retinal injury. In this study, we aim to: (1) determine the pulse energy and the capability of LIPUS in RGC viability, (2) ascertain the protective role of LIPUS in optic nerve (ON) crush-induced retinal injury, and 3) explore the cellular mechanisms of RGC apoptosis prevention by LIPUS. Methods: An ON crush model was set up to induce RGC death. LIPUS was used to treat mice eyes daily, and the retina samples were dissected for immunostaining and Western blot. The expression of yes-associated protein (YAP) and apoptosis-related proteins was detected by immunostaining and Western blot in vitro and in vivo. Apoptosis of RGCs was evaluated by TUNEL staining, the survival of RGCs and retained axons were labeled by Fluoro-gold and Tuj1 antibody, respectively. Rotenone was used to set up an in vitro cellular degenerative model and siYAP was used to interfering the expression of YAP to detect the LIPUS protective function. Results: LIPUS protected RGC from loss and apoptosis in vivo and in vitro. The ratio of cleaved/pro-caspase3 also decreased significantly under LIPUS treatment. As a cellular mechanical sensor, YAP expression increased and YAP translocated to nucleus in LIPUS stimulation group, however, phospho-YAP was found to be decreased. When YAP was inhibited, the LIPUS could not protect RGC from caspase3-dependent apoptosis. Conclusion: LIPUS prevented RGCs from apoptosis in an ON crush model and in vitro cellular degenerative model, which indicates a potential treatment for further traumatic ON injury. The mechanism of protection is dependent on YAP activation and correlated with caspase-3 signaling.

Organotypic liver culture models: Meeting current challenges in toxicity testing

PubMed Central

LeCluyse, Edward L.; Witek, Rafal P.; Andersen, Melvin E.; Powers, Mark J.

2012-01-01

Prediction of chemical-induced hepatotoxicity in humans from in vitro data continues to be a significant challenge for the pharmaceutical and chemical industries. Generally, conventional in vitro hepatic model systems (i.e. 2-D static monocultures of primary or immortalized hepatocytes) are limited by their inability to maintain histotypic and phenotypic characteristics over time in culture, including stable expression of clearance and bioactivation pathways, as well as complex adaptive responses to chemical exposure. These systems are less than ideal for longer-term toxicity evaluations and elucidation of key cellular and molecular events involved in primary and secondary adaptation to chemical exposure, or for identification of important mediators of inflammation, proliferation and apoptosis. Progress in implementing a more effective strategy for in vitro-in vivo extrapolation and human risk assessment depends on significant advances in tissue culture technology and increasing their level of biological complexity. This article describes the current and ongoing need for more relevant, organotypic in vitro surrogate systems of human liver and recent efforts to recreate the multicellular architecture and hemodynamic properties of the liver using novel culture platforms. As these systems become more widely used for chemical and drug toxicity testing, there will be a corresponding need to establish standardized testing conditions, endpoint analyses and acceptance criteria. In the future, a balanced approach between sample throughput and biological relevance should provide better in vitro tools that are complementary with animal testing and assist in conducting more predictive human risk assessment. PMID:22582993

Recombinant Expression and Purification of the Shigella Translocator IpaB.

PubMed

Barta, Michael L; Adam, Philip R; Dickenson, Nicholas E

2017-01-01

Type III secretion systems (T3SS) are highly conserved virulence factors employed by a large number of pathogenic gram-negative bacteria. Like many T3SS translocators, recombinant expression of the hydrophobic Shigella protein IpaB requires the presence of its cognate chaperone IpgC. Chaperone-bound IpaB is maintained in a nonfunctional state, which has hampered in vitro studies aimed at understanding molecular structure and function of this important class of T3SS proteins. Herein, we describe an expression and purification protocol that utilizes mild detergents to produce highly purified, homogeneous IpaB of defined oligomeric states.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Zhijie; Jiang, Hequn; Liu, Ying

MicroRNAs (miRNAs) are a class of small non-coding RNAs that function as critical gene regulators by targeting mRNAs for translational repression or degradation. In this study, we showed that the expression level of miR-133b was decreased, while Sirt1 mRNA expression levels were increased in hepatocellular carcinoma (HCC) and cell lines, and we identified Sirt1 as a novel direct target of miR-133b. The over-expression of miR-133b suppressed Sirt1 expression. In addition, miR-133b over-expression resulted in attenuating HCC cell proliferation and invasion together with apoptosis increase in vitro. HepG2 cell transplantation revealed that up-regulation of miR-133b could inhibit HCC tumor genesis inmore » vivo. Forced expression of Sirt1 partly rescued the effect of miR-133b in vitro. Furthermore, our study showed that miR-133b over-expression or Sirt1 down-regulation elevated E-cadherin expression, and repressed glypican-3 (GPC3) and the anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1) expression. The inhibition of GPC3 expression repressed Bcl-2, Bcl-xL, and Mcl-1 expression, and elevated E-cadherin expression. Moreover, the Sirt1 up-regulation resulted in increases in HCC cell proliferation and invasion together with decreases apoptosis, and increases in the cytosolic accumulation and nuclear translocation of the transcription factor β-catenin in vitro. But the effect of Sirt1 up-regulation was partly reversed by GPC3 down-regulation in vitro. Taken together, these findings provide insight into the role and mechanism of miR-133b in regulating HCC cell proliferation, invasion and apoptosis via the miR-133b/Sirt1/GPC3/Wnt β-catenin axis, and miR-133b may serve as a potential therapeutic target in HCC in the future. - Highlights: • Sirt1 is a direct target of miR-133b in HCC. • miR-133b over-expression suppresses HCC progression in vitro and in vivo. • Sirt1 restoration reverses the effect of miR-133b over-expression on HCC cells. • GPC3 down-regulation reverses the effect of Sirt1 up-regulation on HCC cells. • Sirt1 activates Wnt β-catenin signaling by GPC3 in vitro.« less

Proteomic analysis of human follicular fluid in poor ovarian responders during in vitro fertilization.

PubMed

Oh, Jae Won; Kim, Seul Ki; Cho, Kyung-Cho; Kim, Min-Sik; Suh, Chang Suk; Lee, Jung Ryeol; Kim, Kwang Pyo

2017-03-01

Poor ovarian response (POR) in controlled ovarian stimulation is often observed during in vitro fertilization and embryo transfer cycles and it is a major problem. A POR has been found to be related to several factors, including advanced age, high body mass index, and history of ovarian or pelvic surgery. However, it is difficult to predict POR, as there are no specific biomarkers known. In this study, we used quantitative proteomic analyses to investigate potential biomarkers that can predict poor response during in vitro fertilization based on follicular fluid samples. A total of 1079 proteins were identified using a high-resolution orbitrap mass spectrometer coupled online to a nanoflow-LC system. It is notable that 65 upregulated and 66 downregulated proteins were found to be functionally enriched in poor responders. We also validated these differentially expressed proteins using a triple-quadrupole mass spectrometer for quantification of targeted proteins. Of the differentially expressed proteins, three proteins (pregnancy zone protein, renin, and sushi repeat-containing protein SRPX) were regarded as statistically significant (p < 0.05). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hydrogen-driven asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol by Ralstonia eutropha transformant expressing alcohol dehydrogenase from Kluyveromyces lactis.

PubMed

Oda, Takahiro; Oda, Koji; Yamamoto, Hiroaki; Matsuyama, Akinobu; Ishii, Masaharu; Igarashi, Yasuo; Nishihara, Hirofumi

2013-01-10

Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in a hydrogen-oxidizing bacterium: Ralstonia eutropha. This is the first report of hydrogen-driven in vivo coupling reaction of the alcohol dehydrogenase and indigenous soluble NAD-reducing hydrogenase. Asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol, which is a commercial building block for antibacterial agents, was performed using the transformant as the microbial cell catalyst. The two enzymes coupled in vitro in vials without a marked decrease of reactivity during the 20 hr reaction because of the hydrogenase reaction, which generates no by-product that affects enzymes. Alcohol dehydrogenase was expressed functionally in R. eutropha in an activity level equivalent to that of indigenous NAD-reducing hydrogenase under the hydrogenase promoter. The hydrogen-driven in vivo coupling reaction proceeded only by the transformant cell without exogenous addition of a cofactor. The decrease of reaction velocity at higher concentration of hydroxyacetone was markedly reduced by application of an in vivo coupling system. Production of (R)-1,2-propanediol (99.8% e.e.) reached 67.7 g/l in 76 hr with almost a constant rate using a jar fermenter. The reaction velocity under 10% PH2 was almost equivalent to that under 100% hydrogen, indicating the availability of crude hydrogen gas from various sources. The in vivo coupling system enabled cell-recycling as catalysts. Asymmetric reduction of hydroxyacetone by a coupling reaction of the two enzymes continued in both in vitro and in vivo systems in the presence of hydrogen. The in vivo reaction system using R. eutropha transformant expressing heterologous alcohol dehydrogenase showed advantages for practical usage relative to the in vitro coupling system. The results suggest a hopeful perspective of the hydrogen-driven bioprocess as an environmentally outstanding method to achieve industrial green innovation. Hydrogen-oxidizing bacteria can be useful hosts for the development of hydrogen-driven microbial cell factories.

Dual systemic tumor targeting with ligand-directed phage and Grp78 promoter induces tumor regression.

PubMed

Kia, Azadeh; Przystal, Justyna M; Nianiaris, Nastasia; Mazarakis, Nicholas D; Mintz, Paul J; Hajitou, Amin

2012-12-01

The tumor-specific Grp78 promoter is overexpressed in aggressive tumors. Cancer patients would benefit greatly from application of this promoter in gene therapy and molecular imaging; however, clinical benefit is limited by lack of strategies to target the systemic delivery of Grp78-driven transgenes to tumors. This study aims to assess the systemic efficacy of Grp78-guided expression of therapeutic and imaging transgenes relative to the standard cytomegalovirus (CMV) promoter. Combination of ligand and Grp78 transcriptional targeting into a single vector would facilitate systemic applications of the Grp78 promoter. We generated a dual tumor-targeted phage containing the arginine-glycine-aspartic acid tumor homing ligand and Grp78 promoter. Next, we combined flow cytometry, Western blot analysis, bioluminescence imaging of luciferase, and HSVtk/ganciclovir gene therapy and compared efficacy to conventional phage carrying the CMV promoter in vitro and in vivo in subcutaneous models of rat and human glioblastoma. We show that double-targeted phage provides persistent transgene expression in vitro and in tumors in vivo after systemic administration compared with conventional phage. Next, we showed significant tumor killing in vivo using the HSVtk/ganciclovir gene therapy and found a systemic antitumor effect of Grp78-driven HSVtk against therapy-resistant tumors. Finally, we uncovered a novel mechanism of Grp78 promoter activation whereby HSVtk/ganciclovir therapy upregulates Grp78 and transgene expression via the conserved unfolded protein response signaling cascade. These data validate the potential of Grp78 promoter in systemic cancer gene therapy and report the efficacy of a dual tumor targeting phage that may prove useful for translation into gene therapy and molecular imaging applications.

Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues.

PubMed

Lundquist, P; Artursson, P

2016-11-15

In this contribution, we review the molecular and physiological barriers to oral delivery of peptides and nanoparticles. We discuss the opportunities and predictivity of various in vitro systems with special emphasis on human intestine in Ussing chambers. First, the molecular constraints to peptide absorption are discussed. Then the physiological barriers to peptide delivery are examined. These include the gastric and intestinal environment, the mucus barrier, tight junctions between epithelial cells, the enterocytes of the intestinal epithelium, and the subepithelial tissue. Recent data from human proteome studies are used to provide information about the protein expression profiles of the different physiological barriers to peptide and nanoparticle absorption. Strategies that have been employed to increase peptide absorption across each of the barriers are discussed. Special consideration is given to attempts at utilizing endogenous transcytotic pathways. To reliably translate in vitro data on peptide or nanoparticle permeability to the in vivo situation in a human subject, the in vitro experimental system needs to realistically capture the central aspects of the mentioned barriers. Therefore, characteristics of common in vitro cell culture systems are discussed and compared to those of human intestinal tissues. Attempts to use the cell and tissue models for in vitro-in vivo extrapolation are reviewed. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Mammalian cell display technology coupling with AID induced SHM in vitro: an ideal approach to the production of therapeutic antibodies.

PubMed

Qin, Chang-Fei; Li, Guan-Cheng

2014-12-01

Traditional antibody production technology within non-mammalian cell expression systems has shown many unsatisfactory properties for the development of therapeutic antibodies. Nevertheless, mammalian cell display technology reaps the benefits of producing full-length all human antibodies. Together with the developed cytidine deaminase induced in vitro somatic hypermutation technology, mammalian cell display technology provides the opportunity to produce high affinity antibodies that might be ideal for therapeutic application. This review was concentrated on the development of the mammalian cell display technology as well as the activation-induced cytidine deaminase induced in vitro somatic hypermutation technology and their applications for the production of therapeutic antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo.

PubMed

Venalis, Paulius; Maurer, Britta; Akhmetshina, Alfiya; Busch, Nicole; Dees, Clara; Stürzl, Michael; Zwerina, Jochen; Jüngel, Astrid; Gay, Steffen; Schett, Georg; Distler, Oliver; Distler, Jörg H W

2009-10-01

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc.

Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo

PubMed Central

Venalis, Paulius; Maurer, Britta; Akhmetshina, Alfiya; Busch, Nicole; Dees, Clara; Stürzl, Michael; Zwerina, Jochen; Jüngel, Astrid; Gay, Steffen; Schett, Georg; Distler, Oliver; Distler, Jörg HW

2009-01-01

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc. PMID:18774958

Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models.

PubMed

Choi, Yoon Pyo; Lee, Joo Hyun; Gao, Ming-Qing; Kim, Baek Gil; Kang, Suki; Kim, Se Hoon; Cho, Nam Hoon

2014-11-01

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5β1 and αvβ3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis. © 2014 UICC.

Systemic and Gene Modified Mesenchymal Stem Cell Therapy for Metastatic Prostate Cancer

DTIC Science & Technology

2009-05-01

downregulate the expression of growth factors , receptor tyrosine kinases, proteases, and AR both in vitro and in vivo only when the cells express high... growth , and apoptosis as shown in Figure 1A. Downregulation of growth factors , FGF, Mdk, PDGF, Ptn, TGFb1 and TGFb3, tyrosine kinase receptors Ffgr3 and... growth factors and receptor tyrosine kinases mainly in andro- gen-sensitive and AR-overexpressing cells. The effect was more pronounced in Erk, Ras, and

Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

PubMed

Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

2014-03-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

PubMed

Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

2015-01-01

In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively. © 2014 Wiley Periodicals, Inc.

Corydalis bungeana Turcz. attenuates LPS-induced inflammatory responses via the suppression of NF-κB signaling pathway in vitro and in vivo.

PubMed

Zhai, Xiao-Ting; Chen, Jia-Quan; Jiang, Cui-Hua; Song, Jie; Li, Dong-Yu; Zhang, Hao; Jia, Xiao-Bin; Tan, Wei; Wang, Shu-Xia; Yang, Yi; Zhu, Fen-Xia

2016-12-24

Corydalis bungeana Turcz. (C. bungeana) is one of traditionally used medicines in China and possesses various biological effects, such as anti-inflammatory, antibacterial activity and inhibition of the immune function of the host. we studied the anti-inflammatory effect and molecular mechanism involved of C. bungeana both in vitro and in vivo model system in which the inflammatory response was induced by LPS treatment. Anti-inflammatory activity of C. bungeana was investigated by LPS-induced RAW 264.7 macrophages and BALB/c mice. The production and expression of pro-inflammatory cytokines were evaluated by Griess reagent, ELISA kits and RT-qPCR, respectively. Phosphorylation status of IκBα and p65 was illustrated by western blot assay. C. bungeana reduced the secretion of NO, TNF-α, IL-6 and IL-1β through inhibiting the protein expression of iNOS, TNF-α, IL-6 and IL-1β in vitro and in vivo. Western blot analysis suggested that C. bungeana supressed NF-κB activation via regulating the phosphorylation of IκBα and p65. Immunohistochemical assay also demostrated the histological inflammatory change in liver tissue. The results indicate that C. bungeana supresses the activation of NF-κB signaling pathway through inhibiting phosphorylation of IκBα and p65, which results in good anti-inflammatory effect. In addition, C. bungeana attenuates inflammatory reaction by supressing the expression of various inflammatory cytokines both in vivo and in vitro. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Human Breast Cancer Histoid

PubMed Central

Kaur, Pavinder; Ward, Brenda; Saha, Baisakhi; Young, Lillian; Groshen, Susan; Techy, Geza; Lu, Yani; Atkinson, Roscoe; Taylor, Clive R.; Ingram, Marylou

2011-01-01

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 µm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue. PMID:22034518

Expression of three isoforms of Na-K-2Cl cotransporter (NKCC2) in the kidney and regulation by dehydration.

PubMed

Itoh, Kazuko; Izumi, Yuichiro; Inoue, Takeaki; Inoue, Hideki; Nakayama, Yushi; Uematsu, Takayuki; Fukuyama, Takashi; Yamazaki, Taiga; Yasuoka, Yukiko; Makino, Takeshi; Nagaba, Yasushi; Tomita, Kimio; Kobayashi, Noritada; Kawahara, Katsumasa; Mukoyama, Masashi; Nonoguchi, Hiroshi

2014-10-24

Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro. Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro, NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo. These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b. Copyright © 2014 Elsevier Inc. All rights reserved.

Simple in vitro generation of human leukocyte antigen-G-expressing T-regulatory cells through pharmacological hypomethylation for adoptive cellular immunotherapy against graft-versus-host disease.

PubMed

Stamou, Panagiota; Marioli, Dimitra; Patmanidi, Alexandra L; Sgourou, Argyro; Vittoraki, Angeliki; Theofani, Efthymia; Pierides, Chryso; Taraviras, Stavros; Costeas, Paul A; Spyridonidis, Alexandros

2017-04-01

Major barriers in using classical FOXP3+ regulatory T cells (Tregs) in clinical practice are their low numbers in the circulation, the lack of specific cell surface markers for efficient purification and the loss of expression of Treg signature molecules and suppressive function after in vitro expansion or in a pro-inflammatory microenviroment. A surface molecule with potent immunosuppressive function is the human leukocyte antigen-G (HLA-G), which is normally expressed in placenta protecting the "semi-allogeneic" fetus from maternal immune attack. Because HLA-G expression is strongly regulated by methylation, we asked whether hypomethylating agents (HA) may be used in vitro to induce HLA-G expression on conventional T cells and convert them to Tregs. Human peripheral blood T cells were exposed to azacytidine/decitabine and analyzed for HLA-G expression and their in vitro suppressor properties. HA treatment induces de novo expression of HLA-G on T cells through hypomethylation of the HLA-G proximal promoter. The HA-induced CD4 + HLA-G pos T cells are FOXP3 negative and have potent in vitro suppression function, which is dependent to a large extent, but not exclusively, on the HLA-G molecule. Converted HLA-G pos suppressors retain their suppressor function in the presence of tumor necrosis factor (TNF) and preserve hypomethylated the HLA-G promoter for at least 2 days after azacytidine exposure. Decitabine-treated T cells suppressed ex vivo the proliferation of T cells isolated from patients suffering from graft-versus-host disease (GVHD). We propose, in vitro generation of HLA-G-expressing T cells through pharmacological hypomethylation as a simple, Good Manufacturing Practice (GMP)-compatible and efficient strategy to produce a stable Treg subset of a defined phenotype that can be easily purified for adoptive immunotherapy. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Octyl Methoxycinnamate Modulates Gene Expression and Prevents Cyclobutane Pyrimidine Dimer Formation but not Oxidative DNA Damage in UV-Exposed Human Cell Lines

PubMed Central

Duale, Nur; Olsen, Ann-Karin; Christensen, Terje; Butt, Shamas T.; Brunborg, Gunnar

2010-01-01

Octyl methoxycinnamate (OMC) is one of the most widely used sunscreen ingredients. To analyze biological effects of OMC, an in vitro approach was used implying ultraviolet (UV) exposure of two human cell lines, a primary skin fibroblast (GM00498) and a breast cancer (MCF-7) cell lines. End points include cell viability assessment, assay of cyclobutane pyrimidine dimers (CPDs) and oxidated DNA lesions using alkaline elution and lesion-specific enzymes, and gene expression analysis of a panel of 17 DNA damage–responsive genes. We observed that OMC provided protection against CPDs, and the degree of protection correlated with the OMC-mediated reduction in UV dose. No such protection was found with respect to oxidative DNA lesions. Upon UV exposure in the presence of OMC, the gene expression studies showed significant differential changes in some of the genes studied and the expression of p53 protein was also changed. For some genes, the change in expression seemed to be delayed in time by OMC. The experimental approach applied in this study, using a panel of 17 genes in an in vitro cellular system together with genotoxicity assays, may be useful in the initial screening of active ingredients in sunscreens. PMID:20071424

Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia

PubMed Central

Alves, Cecília J.; Leitão, Luís; Sousa, Daniela M.; Alencastre, Inês S.; Conceição, Francisco; Lamghari, Meriem

2017-01-01

Limited knowledge from mechanistic studies on adult sensory neuronal activity was generated, to some extent, in recapitulated adult in vivo 3D microenvironment. To fill this gap there is a real need to better characterize the adult dorsal root ganglia (aDRG) organotypic cultures to make these in vitro systems exploitable for different approaches, ranging from basic neurobiology to regenerative therapies, to address the sensory nervous system in adult stage. We conducted a direct head-to-head comparison of aDRG and embryonic DRG (eDRG) organotypic culture focusing on axonal growth, neuropeptides expression and receptors tyrosine kinase (RTK) activation associated with neuronal survival, proliferation and differentiation. To identify alterations related to culture conditions, these parameters were also addressed in retrieved aDRG and eDRG and compared with organotypic cultures. Under similar neurotrophic stimulation, aDRG organotypic cultures displayed lower axonal outgrowth rate supported by reduced expression of growth associated protein-43 and high levels of RhoA and glycogen synthase kinase 3 beta mRNA transcripts. In addition, differential alteration in sensory neuropeptides expression, namely calcitonin gene-related peptide and substance P, was detected and was mainly pronounced at gene expression levels. Among 39 different RTK, five receptors from three RTK families were emphasized: tropomyosin receptor kinase A (TrkA), epidermal growth factor receptors (EGFR, ErbB2 and ErbB3) and platelet-derived growth factor receptor (PDGFR). Of note, except for EGFR, the phosphorylation of these receptors was dependent on DRG developmental stage and/or culture condition. In addition, EGFR and PDGFR displayed alterations in their cellular expression pattern in cultured DRG. Overall we provided valuable information particularly important when addressing in vitro the molecular mechanisms associated with development, maturation and regeneration of the sensory nervous system. PMID:28742111

Epidermal growth factor-containing fibulin-like extracellular matrix protein 1 expression and regulation in uterine leiomyoma.

PubMed

Marsh, Erica E; Chibber, Shani; Wu, Ju; Siegersma, Kendra; Kim, Julie; Bulun, Serdar

2016-04-01

To determine the presence, differential expression, and regulation of epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) in uterine leiomyomas. Laboratory in vivo and in vitro study with the use of human leiomyoma and myometrial tissue and primary cells. Academic medical center. Leiomyoma and myometrial tissue samples and cultured cells. 5-Aza-2'-deoxycytidine (5-aza-dC) treatment. Fold-change difference between EFEMP1 and fibulin-3 expression in leiomyoma tissue and cells compared with matched myometrial samples, and fold-change difference in EFEMP1 expression with 5-Aza-dC treatment. In vivo, EFEMP1 expression was 3.19-fold higher in myometrial tissue than in leiomyoma tissue. EFEMP1 expression in vitro was 5.03-fold higher in myometrial cells than in leiomyoma cells. Western blot and immunohistochemistry staining of tissue and cells confirmed similar findings in protein expression. Treatment of leiomyoma cells with 5-Aza-dC resulted in increased expression of EFEMP1 in vitro. The EFEMP1 gene and its protein product, fibulin-3, are both significantly down-regulated in leiomyoma compared with myometrium when studied both in vivo and in vitro. The increase in EFEMP1 expression in leiomyoma cells with 5-Aza-dC treatment suggest that differential methylation is responsible, in part, for the differences seen in gene expression. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Specific immunotherapy of experimental myasthenia gravis in vitro and in vivo: the Guided Missile strategy.

PubMed

Sun, W; Adams, R N; Miagkov, A; Lu, Y; Juon, H-S; Drachman, D B

2012-10-15

Current immunotherapy of myasthenia gravis (MG) is often effective, but entails risks of infection and neoplasia. The "Guided Missile" strategy described here is designed to target and eliminate the individual's unique AChR-specific T cell repertoire, without otherwise interfering with the immune system. We genetically engineered dendritic cells to present AChR epitopes and simultaneously express Fas ligand in an ongoing EAMG model. In both in vitro and in vivo experiments, these engineered cells specifically killed AChR-responsive T cells without otherwise damaging the immune system. AChR antibodies were markedly reduced in the treated mice. Translation of this method to treat human MG is possible. Copyright © 2012 Elsevier B.V. All rights reserved.

Distinct Strains of Toxoplasma gondii Feature Divergent Transcriptomes Regardless of Developmental Stage

DOE PAGES

Croken, Matthew; Ma, Yan Fen; Markillie, Lye Meng; ...

2014-11-13

Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of Toxoplasma gondii representing three common genotypes under both in vitro tachyzoite and in vitro bradyzoite-inducing alkaline stress culture conditions. Strikingly, the differences in transcriptional profiles between the strains, RH, PLK, and CTG, is much greater than differences between tachyzoites and alkaline stressed in vitro bradyzoites. With an FDR of 10%, we identify 241 genes differentially expressed between CTG tachyzoites and in vitro bradyzoites, including 5 putative AP2 transcription factors. We also observe close association between cell cycle regulated genes and differentiation. By Gene Set Enrichment Analysismore » (GSEA), there are a number of KEGG pathways associated with the in vitro bradyzoite transcriptomes of PLK and CTG, including pyrimidine metabolism and DNA replication. These functions are likely associated with cell-cycle arrest. When comparing mRNA levels between strains, we identify 1,526 genes that are differentially expressed regardless of culture-condition as well as 846 differentially expressed only in bradyzoites and 542 differentially expressed only in tachyzoites between at least two strains. Using GSEA, we identify ribosomal proteins as being expressed at significantly higher levels in the CTG strain than in either the RH or PLK strains. This association holds true regardless of life cycle stage.« less

Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

PubMed

Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

2018-03-01

During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Homeopathic medicines do not alter growth and gene expression in prostate and breast cancer cells in vitro.

PubMed

Thangapazham, Rajesh L; Gaddipati, Jaya P; Rajeshkumar, N V; Sharma, Anuj; Singh, Anoop K; Ives, John A; Maheshwari, Radha K; Jonas, Wayne B

2006-12-01

Homeopathy is an alternative medical system practiced in all parts of the world. Although several theories are proposed to explain the mechanisms of action, none are scientifically verified. In this study, the authors investigate the effect of selected homeopathic remedies often used to treat prostate and breast cancer. The authors investigated the effect of the homeopathic medicines Conium maculatum, Sabal serrulata, Thuja occidentalis, Asterias, Phytolacca, and Carcinosin on prostate and breast cancer cell (DU-145, LNCaP, MAT-LyLu, MDA-MB-231) growth and on gene expression that regulates apoptosis, using MTT and multiprobe ribonuclease protection assay. None of the homeopathic remedies tested in different potencies produced significant inhibitory or growth-promoting activity in either prostate or breast cancer cells. Also, gene expression studies by ribonuclease protection assay produced no significant changes in mRNA levels of bax, bcl-2, bcl-x, caspase-1, caspase-2, caspase-3, Fas, or FasL after treatment with homeopathic medicines. The results demonstrate that the highly diluted homeopathic remedies used by homeopathic practitioners for cancer show no measurable effects on cell growth or gene expression in vitro using currently available methodologies.

Agrin Signalling Contributes to Cell Activation and Is Overexpressed in T Lymphocytes from Lupus Patients1

PubMed Central

Jury, Elizabeth C.; Eldridge, Jillian; Isenberg, David A.; Kabouridis, Panagiotis S.

2008-01-01

It is shown in this study that the heparan sulfate proteoglycan agrin is overexpressed in T cells isolated from patients with the autoimmune disease systemic lupus erythematosus (SLE). Freshly isolated CD4+ and CD8+ subpopulations both exhibited higher expression over healthy controls, which however, gradually declined when cells were cultured in vitro. Agrin expression was induced following in vitro activation of cells via their Ag receptor, or after treatment with IFN-α, a cytokine shown to be pathogenic in lupus. Furthermore, serum from SLE patients with active disease was able to induce agrin expression when added to T cells from healthy donors, an increase that was partially blocked by neutralizing anti-IFN-α Abs. Cross-linking agrin with mAbs resulted in rapid reorganization of the actin cytoskeleton, activation of the ERK MAPK cascade, and augmentation of anti-CD3-induced proliferation and IL-10 production, indicating that agrin is a functional receptor in T cells. These results demonstrate that agrin expression in human T cells is regulated by cell activation and IFN-α, and may have an important function during cell activation with potential implications for autoimmunity. PMID:18025246

The role of the megagametophyte in maintaining loblolly pine (Pinus taeda L.) seedling arginase gene expression in vitro.

PubMed

Todd, Christopher D; Gifford, David J

2002-05-01

Following loblolly pine (Pinus taeda L.) seed germination, storage-protein breakdown in the megagametophyte and in the seedling results in a large increase in the seedling's free amino acid pool. A substantial portion of both the storage proteins and the amino acid pool is arginine, a very efficient nitrogen-storage compound. Free arginine is hydrolyzed in the seedling by the enzyme arginase (EC 3.5.3.1), which is under strong developmental control. At present, regulation of arginase in conifers is not well understood. Here we report the utilization of an in vitro culture system to address the separate impacts of the seedling and megagametophyte tissues on arginase enzyme activity, protein levels and patterns of gene expression. We also describe the generation of an anti-arginase antibody prepared from a histidine-tagged loblolly pine arginase fusion protein expressed in Escherichia coli. Our results indicate that arginase gene expression in the seedling is initiated by the seedling itself and then maintained or up-regulated by the megagametophyte. The contribution of storage-protein breakdown and the free amino acid pool, particularly arginine, in this regulation is also addressed.

Establishment and culture optimization of a new type of pituitary immortalized cell line.

PubMed

Kokubu, Yuko; Asashima, Makoto; Kurisaki, Akira

2015-08-07

The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells under sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. Copyright © 2015 Elsevier Inc. All rights reserved.

In Vivo magnetic resonance imaging of xenografted tumors using FTH1 reporter gene expression controlled by a tet-on switch.

PubMed

He, Xiaoya; Cai, Jinhua; Li, Hao; Liu, Bo; Qin, Yong; Zhong, Yi; Wang, Longlun; Liao, Yifan

2016-11-29

As a promising magnetic resonance imaging (MRI) reporter, ferritin has been used to track cells in vivo; however, its continuous overexpression can be cytotoxic, which restricts its application. In this study, we aimed to develop a switch to turn this genetic reporter "on" or "off" while monitoring cell grafts via MRI. To accomplish this, we genetically modified the ferritin heavy chain (FTH1) with a Tet-On switch and assessed the expression of FTH1 in transduced neuroblastoma cells (SK-N-SH) in vitro and in xenografted tumors in vivo. We found that FTH1 expression induced by doxycycline (Dox) in SK-N-SH-FTH1 cells depended on treatment dose and duration. We successfully detected T2-weighted MRI contrast in cell grafts after switching "on" the reporter gene using Dox, and this contrast disappeared when we switched it "off". The genetic reporter FTH1 can thus be switched "on" or "off" throughout longitudinal monitoring of cell grafts, limiting expression to when MRI contrast is needed. The controllable imaging system we have developed minimizes risks from constitutive reporter gene overexpression and facilitates tumor cell monitoring in vitro and in vivo.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peres, Elodie A.; Valable, Samuel; Guillamo, Jean-Sebastien

Hypoxia has been shown to be one of the major events involved in EPO expression. Accordingly, EPO might be expressed by cerebral neoplastic cells, especially in glioblastoma, known to be highly hypoxic tumours. The expression of EPOR has been described in glioma cells. However, data from the literature remain descriptive and controversial. On the basis of an endogenous source of EPO in the brain, we have focused on a potential role of EPOR in brain tumour growth. In the present study, with complementary approaches to target EPO/EPOR signalling, we demonstrate the presence of a functional EPO/EPOR system on glioma cellsmore » leading to the activation of the ERK pathway. This EPO/EPOR system is involved in glioma cell proliferation in vitro. In vivo, we show that the down-regulation of EPOR expression on glioma cells reduces tumour growth and enhances animal survival. Our results support the hypothesis that EPOR signalling in tumour cells is involved in the control of glioma growth.« less

Safflomide increases the expression of adiponectin in vitro and in vivo: Implication for hypoadiponectemia, visceral obesity, and insulin resistance

USDA-ARS?s Scientific Manuscript database

Safflomide (N-caffeoyltryptamine) is a phenolic amide with serotonin-receptor antagonist and anti-oxidant activities. We investigated the effects of safflomide on the expression of adipokines in vitro and in vivo. Safflomide did not affect the expressions of TNF-a, IL-6, and MCP-1/CCL2 in hypertrop...

Characterisation of the cancer-associated glucocorticoid system: key role of 11β-hydroxysteroid dehydrogenase type 2.

PubMed

Cirillo, Nicola; Morgan, David J; Pedicillo, Maria Carmela; Celentano, Antonio; Lo Muzio, Lorenzo; McCullough, Michael J; Prime, Stephen S

2017-09-26

Recent studies have shown that production of cortisol not only takes place in several non-adrenal peripheral tissues such as epithelial cells but, also, the local inter-conversion between cortisone and cortisol is regulated by the 11β-hydroxysteroid dehydrogenases (11β-HSDs). However, little is known about the activity of this non-adrenal glucocorticoid system in cancers. The presence of a functioning glucocorticoid system was assessed in human skin squamous cell carcinoma (SCC) and melanoma and further, in 16 epithelial cell lines from 8 different tissue types using ELISA, western blotting and immunofluorescence. 11β-HSD2 was inhibited both pharmacologically and by siRNA technology. Naïve CD8 + T cells were used to test the paracrine effects of cancer-derived cortisol on the immune system in vitro. Functional assays included cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical data of 11β-HSD expression were generated using tissue microarrays of 40 cases of human SCCs as well as a database featuring 315 cancer cases from 15 different tissues. We show that cortisol production is a common feature of malignant cells and has paracrine functions. Cortisol production correlated with the magnitude of glucocorticoid receptor (GR)-dependent inhibition of tumour-specific CD8 + T cells in vitro. 11β-HSDs were detectable in human skin SCCs and melanoma. Analyses of publicly available protein expression data of 11β-HSDs demonstrated that 11β-HSD1 and -HSD2 were dysregulated in the majority (73%) of malignancies. Pharmacological manipulation of 11β-HSD2 activity by 18β-glycyrrhetinic acid (GA) and silencing by specific siRNAs modulated the bioavailability of cortisol. Cortisol also acted in an autocrine manner and promoted cell invasion in vitro and cell-cell adhesion and cohesion in two- and three-dimensional models. Immunohistochemical analyses using tissue microarrays showed that expression of 11β-HSD2 was significantly reduced in human SCCs of the skin. The results demonstrate evidence of a cancer-associated glucocorticoid system and show for the first time, the functional significance of cancer-derived cortisol in tumour progression.

Engineering artificial cells by combining HeLa-based cell-free expression and ultra-thin double emulsion template

PubMed Central

Ho, Kwun Yin; Murray, Victoria L.; Liu, Allen P.

2015-01-01

Generation of artificial cells provides the bridge needed to cover the gap between studying the complexity of biological processes in whole cells and studying these same processes in an in vitro reconstituted system. Artificial cells are defined as the encapsulation of biologically active material in a biological or synthetic membrane. Here, we describe a robust and general method to produce artificial cells for the purpose of mimicking one or more behaviors of a cell. A microfluidic double emulsion system is used to encapsulate a mammalian cell free expression system that is able to express membrane proteins into the bilayer or soluble proteins inside the vesicles. The development of a robust platform that allows the assembly of artificial cells is valuable in understanding subcellular functions and emergent behaviors in a more cell-like environment as well as for creating novel signaling pathways to achieve specific cellular behaviors. PMID:25997354

Synthetic miR-34a mimics as a novel therapeutic agent for Multiple Myeloma: in vitro and in vivo evidence

PubMed Central

Di Martino, Maria T.; Leone, Emanuela; Amodio, Nicola; Foresta, Umberto; Lionetti, Marta; Pitari, Maria R.; Gallo Cantafio, Maria E.; Gullà, Annamaria; Conforti, Francesco; Morelli, Eugenio; Tomaino, Vera; Rossi, Marco; Negrini, Massimo; Ferrarini, Manlio; Caraglia, Michele; Shammas, Masood A.; Munshi, Nikhil C.; Anderson, Kenneth C.; Neri, Antonino; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

2015-01-01

Purpose Deregulated expression of microRNAs (miRNAs) has been demonstrated in multiple myeloma (MM). A promising strategy to achieve a therapeutic effect by targeting the miRNA regulatory network is to enforce the expression of miRNAs that act as tumor suppressor genes, such as miR-34a. Experimental Design Here, we investigated the therapeutic potential of synthetic miR-34a against human MM cells in vitro and in vivo. Results Either transient expression of miR-34a synthetic mimics or lentivirus-based miR-34a-stable enforced expression triggered growth inhibition and apoptosis in MM cells in vitro. Synthetic miR-34a downregulated canonic targets BCL2, CDK6 and NOTCH1 at both the mRNA and protein level. Lentiviral vector-transduced MM xenografts with constitutive miR-34a expression showed high growth inhibition in SCID mice. The anti-MM activity of lipidic-formulated miR-34a was further demonstrated in vivo in two different experimental settings: i) SCID mice bearing non-transduced MM xenografts; and ii) SCID-synth-hu mice implanted with synthetic 3D scaffolds reconstituted with human bone marrow stromal cells and then engrafted with human MM cells. Relevant tumor growth inhibition and survival improvement were observed in mice bearing TP53-mutated MM xenografts treated with miR-34a mimics in the absence of systemic toxicity. Conclusions Our findings provide a proof-of-principle that formulated synthetic miR-34a has therapeutic activity in preclinical models and support a framework for development of miR-34a-based treatment strategies in MM patients. PMID:23035210

Synthetic miR-34a mimics as a novel therapeutic agent for multiple myeloma: in vitro and in vivo evidence.

PubMed

Di Martino, Maria T; Leone, Emanuela; Amodio, Nicola; Foresta, Umberto; Lionetti, Marta; Pitari, Maria R; Cantafio, Maria E Gallo; Gullà, Annamaria; Conforti, Francesco; Morelli, Eugenio; Tomaino, Vera; Rossi, Marco; Negrini, Massimo; Ferrarini, Manlio; Caraglia, Michele; Shammas, Masood A; Munshi, Nikhil C; Anderson, Kenneth C; Neri, Antonino; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

2012-11-15

Deregulated expression of miRNAs has been shown in multiple myeloma (MM). A promising strategy to achieve a therapeutic effect by targeting the miRNA regulatory network is to enforce the expression of miRNAs that act as tumor suppressor genes, such as miR-34a. Here, we investigated the therapeutic potential of synthetic miR-34a against human MM cells in vitro and in vivo. Either transient expression of miR-34a synthetic mimics or lentivirus-based miR-34a-stable enforced expression triggered growth inhibition and apoptosis in MM cells in vitro. Synthetic miR-34a downregulated canonic targets BCL2, CDK6, and NOTCH1 at both the mRNA and protein level. Lentiviral vector-transduced MM xenografts with constitutive miR-34a expression showed high growth inhibition in severe combined immunodeficient (SCID) mice. The anti-MM activity of lipidic-formulated miR-34a was further shown in vivo in two different experimental settings: (i) SCID mice bearing nontransduced MM xenografts; and (ii) SCID-synth-hu mice implanted with synthetic 3-dimensional scaffolds reconstituted with human bone marrow stromal cells and then engrafted with human MM cells. Relevant tumor growth inhibition and survival improvement were observed in mice bearing TP53-mutated MM xenografts treated with miR-34a mimics in the absence of systemic toxicity. Our findings provide a proof-of-principle that formulated synthetic miR-34a has therapeutic activity in preclinical models and support a framework for development of miR-34a-based treatment strategies in MM patients. ©2012 AACR.

Intra-articular administration of xenogeneic neonatal Mesenchymal Stromal Cells early after meniscal injury down-regulates metalloproteinase gene expression in synovium and prevents cartilage degradation in a rabbit model of osteoarthritis.

PubMed

Saulnier, N; Viguier, E; Perrier-Groult, E; Chenu, C; Pillet, E; Roger, T; Maddens, S; Boulocher, C

2015-01-01

The anti-inflammatory and anti-catabolic effects of neonatal Mesenchymal Stromal Cell (MSC) were investigated in a xenogeneic model of mild osteoarthritis (OA). The paracrine properties of MSC on synoviocytes were further investigated in vitro. OA was induced by medial meniscal release (MMR) in 30 rabbit knees. A single early (day 3) or delayed (day 15) intra-articular (IA) injection of MSC isolated from equine Umbilical Cord Wharton's jelly (UC-MSC) was performed. Rabbits were euthanized on days 15 or 56. OA grading was performed and gene expression of inflammatory cytokines and metalloproteinases was measured in synovial tissue. Paracrine effects of UC-MSC were investigated using UC-conditioned vs control medium on rabbit primary synoviocytes stimulated with interleukin 1 beta in vitro. No adverse local or systemic responses were observed clinically after xenogeneic UC-MSC injection. At study end point, cartilage fibrillation was lower in early treatment than in delayed treatment group. Cellular infiltrate was observed in the synovium of both UC-MSC groups. OA synovium exhibited a reduced expression of metalloproteinases-1, -3, -13 in the early cell-treated group at d56. In vitro, UC-conditioned medium exerted anti-inflammatory and anti-catabolic effects on synoviocytes exposed to pro-inflammatory stimulus. Early IA injection of equine UC-MSC was effective in preventing OA signs in rabbit knees following MMR. UC-MSC target the synovium and modulate the gene expression pattern of synoviocytes to promote an anti-catabolic environment. This confirms the synovium is a major target and mediator of MSC therapy, modulating the expression of matrix-degrading enzymes. Copyright © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

NASA Astrophysics Data System (ADS)

Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

2013-03-01

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

A dual system formed by the ARC and NR molybdoenzymes mediates nitrite-dependent NO production in Chlamydomonas.

PubMed

Chamizo-Ampudia, Alejandro; Sanz-Luque, Emanuel; Llamas, Ángel; Ocaña-Calahorro, Francisco; Mariscal, Vicente; Carreras, Alfonso; Barroso, Juan B; Galván, Aurora; Fernández, Emilio

2016-10-01

Nitric oxide (NO) is a relevant signal molecule involved in many plant processes. However, the mechanisms and proteins responsible for its synthesis are scarcely known. In most photosynthetic organisms NO synthases have not been identified, and Nitrate Reductase (NR) has been proposed as the main enzymatic NO source, a process that in vitro is also catalysed by other molybdoenzymes. By studying transcriptional regulation, enzyme approaches, activity assays with in vitro purified proteins and in vivo and in vitro NO determinations, we have addressed the role of NR and Amidoxime Reducing Component (ARC) in the NO synthesis process. N\\R and ARC were intimately related both at transcriptional and activity level. Thus, arc mutants showed high NIA1 (NR gene) expression and NR activity. Conversely, mutants without active NR displayed an increased ARC expression in nitrite medium. Our results with nia1 and arc mutants and with purified enzymes support that ARC catalyses the NO production from nitrite taking electrons from NR and not from Cytb5-1/Cytb5-Reductase, the component partners previously described for ARC (proposed as NOFNiR, Nitric Oxide-Forming Nitrite Reductase). This NR-ARC dual system would be able to produce NO in the presence of nitrate, condition under which NR is unable to do it. © 2016 John Wiley & Sons Ltd.

In Vitro Assembly of Alphavirus Cores by Using Nucleocapsid Protein Expressed in Escherichia coli

PubMed Central

Tellinghuisen, Timothy L.; Hamburger, Agnes E.; Fisher, Bonnie R.; Ostendorp, Ralf; Kuhn, Richard J.

1999-01-01

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19–264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19–264) or could inhibit assembly if its molar ratio relative to CP(19–264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly. PMID:10364277

The relationship between in vitro cellular aging and in vivo human age.

PubMed Central

Schneider, E L; Mitsui, Y

1976-01-01

Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging. PMID:1068470

Magnolol affects expression of IGF-1 and associated binding proteins in human prostate cancer cells in vitro.

PubMed

McKeown, Brendan T; Hurta, Robert A R

2014-11-01

This study investigated the effects of magnolol, a compound from Magnolia officinalis, on the behavior of LNCaP and PC3 human prostate cancer cells in vitro. In vitro cell culture approach with biochemical tests and Western blot analyses was used. Magnolol, (80 μM, 6 hour exposure) was found to affect the expression of insulin-like growth factor-1 (IGF-1) and associated proteins. In both cell lines, protein expression of IGF-1 and insulin-like growth factor binding protein-5 (IGFBP-5) were significantly decreased, while protein expression of IGFBP-3 was significantly increased. Additionally, protein expression of insulin-like growth factor-1 receptor (IGF-1R) was significantly increased and the phosphorylated form of IGF-1 (p-IGF-1R) was significantly decreased in PC3 cells, while IGFBP-4 protein expression was significantly increased in LNCaP cells. This study has demonstrated for the first time that magnolol can alter the expression of IGF-1 and associated proteins in human prostate cancer cells in vitro and suggests that magnolol may have a potential role as a novel anti-prostate cancer agent. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

PubMed Central

Nakagawa, Yusuke; Muneta, Takeshi; Otabe, Koji; Ozeki, Nobutake; Mizuno, Mitsuru; Udo, Mio; Saito, Ryusuke; Yanagisawa, Katsuaki; Ichinose, Shizuko; Koga, Hideyuki; Tsuji, Kunikazu; Sekiya, Ichiro

2016-01-01

Objective Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats. Methods For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages. Results In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage. Conclusion Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future. PMID:26867127

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo.

PubMed

Nakagawa, Yusuke; Muneta, Takeshi; Otabe, Koji; Ozeki, Nobutake; Mizuno, Mitsuru; Udo, Mio; Saito, Ryusuke; Yanagisawa, Katsuaki; Ichinose, Shizuko; Koga, Hideyuki; Tsuji, Kunikazu; Sekiya, Ichiro

2016-01-01

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats. For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages. In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage. Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.

Use of In Vitro Transcription System for Analysis of Corynebacterium glutamicum Promoters Recognized by Two Sigma Factors.

PubMed

Šilar, Radoslav; Holátko, Jiří; Rucká, Lenka; Rapoport, Andrey; Dostálová, Hana; Kadeřábková, Pavla; Nešvera, Jan; Pátek, Miroslav

2016-09-01

Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum.

Transformation of Solanum tuberosum plastids allows high expression levels of β-glucuronidase both in leaves and microtubers developed in vitro.

PubMed

Segretin, María Eugenia; Lentz, Ezequiel Matías; Wirth, Sonia Alejandra; Morgenfeld, Mauro Miguel; Bravo-Almonacid, Fernando Félix

2012-04-01

Plastid genome transformation offers an attractive methodology for transgene expression in plants, but for potato, only expression of gfp transgene (besides the selective gene aadA) has been published. We report here successful expression of β-glucuronidase in transplastomic Solanum tuberosum (var. Desiree) plants, with accumulation levels for the recombinant protein of up to 41% of total soluble protein in mature leaves. To our knowledge, this is the highest expression level reported for a heterologous protein in S. tuberosum. Accumulation of the recombinant protein in soil-grown minitubers was very low, as described in previous reports. Interestingly, microtubers developed in vitro showed higher accumulation of β-glucuronidase. As light exposure during their development could be the trigger for this high accumulation, we analyzed the effect of light on β-glucuronidase accumulation in transplastomic tubers. Exposure to light for 8 days increased β-glucuronidase accumulation in soil-grown tubers, acting as a light-inducible expression system for recombinant protein accumulation in tuber plastids. In this paper we show that plastid transformation in potato allows the highest recombinant protein accumulation in foliar tissue described so far for this food crop. We also demonstrate that in tubers high accumulation is possible and depends on light exposure. Because tubers have many advantages as protein storage organs, these results could lead to new recombinant protein production schemes based on potato.

A reporter model to visualize imprinting stability at the Dlk1 locus during mouse development and in pluripotent cells.

PubMed

Swanzey, Emily; Stadtfeld, Matthias

2016-11-15

Genomic imprinting results in the monoallelic expression of genes that encode important regulators of growth and proliferation. Dysregulation of imprinted genes, such as those within the Dlk1-Dio3 locus, is associated with developmental syndromes and specific diseases. Our ability to interrogate causes of imprinting instability has been hindered by the absence of suitable model systems. Here, we describe a Dlk1 knock-in reporter mouse that enables single-cell visualization of allele-specific expression and prospective isolation of cells, simultaneously. We show that this 'imprinting reporter mouse' can be used to detect tissue-specific Dlk1 expression patterns in developing embryos. We also apply this system to pluripotent cell culture and demonstrate that it faithfully indicates DNA methylation changes induced upon cellular reprogramming. Finally, the reporter system reveals the role of elevated oxygen levels in eroding imprinted Dlk1 expression during prolonged culture and in vitro differentiation. The possibility to study allele-specific expression in different contexts makes our reporter system a useful tool to dissect the regulation of genomic imprinting in normal development and disease. © 2016. Published by The Company of Biologists Ltd.

Retinoic acid, hemin and hexamethylen bisacetamide interference with "in vitro" differentiation of chick embryo chondrocytes.

PubMed

Manduca, P; Abelmoschi, M L

1992-01-01

We have investigated the effect of all-trans Retinoic acid, and of substances (Hemine and Hexamethylene bisacetamide) which interfere with "in vitro" differentiation of mesenchyme derived cell lineages on the expression of specific markers of hyperthrophy in "in vitro" differentiating chick embryo chondrocytes. (Castagnola P., et al., 1986). Continuous treatment of chondrogenic cells in conditions allowing differentiation "in vitro" with Retinoic acid resulted in persistence of type I collagen synthesis and in lack of type X collagen and Ch 21 protein expression. Hemin treated cells secreted a reduced amount of type X collagen. HMBA treatment inhibited type X collagen expression and caused reduction of the ratio between type II collagen and Ch 21 synthesized. The data indicate an independent regulation of these markers during chondrocyte differentiation.

Regulated Expression of Adenoviral Vectors-Based Gene Therapies

PubMed Central

Curtin, James F.; Candolfi, Marianela; Puntel, Mariana; Xiong, Weidong; Muhammad, A. K. M.; Kroeger, Kurt; Mondkar, Sonali; Liu, Chunyan; Bondale, Niyati; Lowenstein, Pedro R.; Castro, Maria G.

2008-01-01

Summary Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the “OFF” state, and expression should quickly reach therapeutic levels in the “ON” state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA2S-M2 inducer and tTSKid silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo. PMID:18470649

A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens.

PubMed

Visser, M; Stephan, D; Jaynes, J M; Burger, J T

2012-06-01

Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species. D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Systematic Analysis of Two-Component Systems in Citrobacter rodentium Reveals Positive and Negative Roles in Virulence.

PubMed

Thomassin, Jenny-Lee; Leclerc, Jean-Mathieu; Giannakopoulou, Natalia; Zhu, Lei; Salmon, Kristiana; Portt, Andrea; Daigle, France; Le Moual, Hervé; Gruenheid, Samantha

2017-02-01

Citrobacter rodentium is a murine pathogen used to model intestinal infections caused by the human diarrheal pathogens enterohemorrhagic and enteropathogenic Escherichia coli During infection, bacteria use two-component systems (TCSs) to detect changing environmental cues within the host, allowing for rapid adaptation by altering the expression of specific genes. In this study, 26 TCSs were identified in C. rodentium, and quantitative PCR (qPCR) analysis showed that they are all expressed during murine infection. These TCSs were individually deleted, and the in vitro and in vivo effects were analyzed to determine the functional consequences. In vitro analyses only revealed minor differences, and surprisingly, type III secretion (T3S) was only affected in the ΔarcA strain. Murine infections identified 7 mutants with either attenuated or increased virulence. In agreement with the in vitro T3S assay, the ΔarcA strain was attenuated and defective in colonization and cell adherence. The ΔrcsB strain was among the most highly attenuated strains. The decrease in virulence of this strain may be associated with changes to the cell surface, as Congo red binding was altered, and qPCR revealed that expression of the wcaA gene, which has been implicated in colanic acid production in other bacteria, was drastically downregulated. The ΔuvrY strain exhibited increased virulence compared to the wild type, which was associated with a significant increase in bacterial burden within the mesenteric lymph nodes. The systematic analysis of virulence-associated TCSs and investigation of their functions during infection may open new avenues for drug development. Copyright © 2017 American Society for Microbiology.

Role of YAP activation in nuclear receptor CAR-mediated proliferation of mouse hepatocytes.

PubMed

Abe, Taiki; Amaike, Yuto; Shizu, Ryota; Takahashi, Miki; Kano, Makoto; Hosaka, Takuomi; Sasaki, Takamitsu; Kodama, Susumu; Matsuzawa, Atsushi; Yoshinari, Kouichi

2018-06-08

Constitutive androstane receptor (CAR) is a xenobiotic-responsive nuclear receptor that is highly expressed in the liver. CAR activation induces hepatocyte proliferation and hepatocarcinogenesis in rodents, but the mechanisms remain unclear. In this study, we investigated the association of CAR-dependent cell proliferation with Yes-associated protein (YAP), which is a transcriptional cofactor controlling organ size and cell growth through the interaction with various transcriptional factors including TEAD. In mouse livers, TCPOBOP (a mouse CAR activator) treatment increased the nuclear YAP accumulation and mRNA levels of YAP target genes as well as cell-cycle related genes along with liver hypertrophy and verteporfin (an inhibitor of YAP/TEAD interaction) cotreatment tended to attenuate them. Furthermore, in cell-based reporter gene assays, CAR activation enhanced the YAP/TEAD-dependent transcription. To investigate the role of YAP/TEAD activation in the CAR-dependent hepatocyte proliferation, we sought to establish an in vitro system completely reproducing CAR-dependent cell proliferation. Since CAR was only slightly expressed in cultured mouse primary hepatocytes compared to mouse livers and no proliferation was observed after treatment with TCPOBOP, we overexpressed CAR using mouse CAR expressing adenovirus (Ad-mCAR-V5) in mouse primary hepatocytes. Ad-mCAR-V5 infection and TCPOBOP treatment induced hepatocyte proliferation. Similar results were obtained with immortalized normal mouse hepatocytes as well. In the established in vitro system, CAR-dependent proliferation was strongly inhibited by Yap knockdown and completely abolished by verteporfin treatment. Our present results obtained in in vivo and in vitro experiments suggest that YAP/TEAD activation plays key roles in CAR-dependent proliferation of murine hepatocytes.

Virulence of Erwinia amylovora, a prevalent apple pathogen: Outer membrane proteins and type III secreted effectors increase fitness and compromise plant defenses.

PubMed

Holtappels, Michelle; Noben, Jean-Paul; Valcke, Roland

2016-09-01

Until now, no data are available on the outer membrane (OM) proteome of Erwinia amylovora, a Gram-negative plant pathogen, causing fire blight in most of the members of the Rosaceae family. Since the OM forms the interface between the bacterial cell and its environment it is in direct contact with the host. Additionally, the type III secretion system, embedded in the OM, is a pathogenicity factor of E. amylovora. To assess the influence of the OM composition and the secretion behavior on virulence, a 2D-DIGE analysis and gene expression profiling were performed on a high and lower virulent strain, both in vitro and in planta. Proteome data showed an increase in flagellin for the lower virulent strain in vitro, whereas, in planta several interesting proteins were identified as being differently expressed between both the strains. Further, gene expression of nearly all type III secreted effectors was elevated for the higher virulent strain, both in vitro and in planta. As a first, we report that several characteristics of virulence can be assigned to the OM proteome. Moreover, we demonstrate that secreted proteins prove to be the important factors determining differences in virulence between the strains, otherwise regarded as homogeneous on a genome level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasmodium falciparum malaria skews globin gene expression balance in in-vitro haematopoietic stem cell culture system: Its implications in malaria associated anemia.

PubMed

Pathak, Vrushali; Colah, Roshan; Ghosh, Kanjaksha

2018-02-01

Understanding the pathophysiology and associated host parasite interactions of the malaria infection is the prerequisite for developing effective prevention and treatment strategies. The exact mechanism underlying malaria associated ineffective and dyserythropoiesis is not yet fully understood. Being an important protein, haemoglobin serves as the main amino acid reservoir available to the intra-erythrocytic plasmodium. It is important to check the expression profiling of globin genes which may help us to understand host parasite interactions and its potential contribution to both infection and disease. Here, an in-vitro culture system was used to study the effect of different doses of Plasmodium falciparum on haematopoietic stem cell expansion, differentiation and expression of globin genes. Upon exposure to the different doses of P. falciparum parasites of strains 3D7, Dd2 and RKL9 (intact and lysed form) at different stages of erythroid development, cells demonstrated suppression in growth and differentiation. At almost all stages of erythroid development upon parasite exposure, the γ globin gene was found to be downregulated and the α/β as well as α/non- α globin mRNA ratios in late stage erythroid cells were found to be reduced (p < .01) compared to the untreated controls. The imbalance in globin chain expression might be considered as one of the factors involved in malaria associated inappropriate erythropoietic responses. Copyright © 2018 Elsevier Inc. All rights reserved.

Tumor Targeting and Pharmacokinetics of a Near-Infrared Fluorescent-Labeled δ-Opioid Receptor Antagonist Agent, Dmt-Tic-Cy5.

PubMed

Huynh, Amanda Shanks; Estrella, Veronica; Stark, Valerie E; Cohen, Allison S; Chen, Tingan; Casagni, Todd J; Josan, Jatinder S; Lloyd, Mark C; Johnson, Joseph; Kim, Jongphil; Hruby, Victor J; Vagner, Josef; Morse, David L

2016-02-01

Fluorescence molecular imaging can be employed for the development of novel cancer targeting agents. Herein, we investigated the pharmacokinetics (PK) and cellular uptake of Dmt-Tic-Cy5, a delta-opioid receptor (δOR) antagonist-fluorescent dye conjugate, as a tumor-targeting molecular imaging agent. δOR expression is observed normally in the CNS, and pathologically in some tumors, including lung liver and breast cancers. In vitro, in vivo, and ex vivo experiments were conducted to image and quantify the fluorescence signal associated with Dmt-Tic-Cy5 over time using in vitro and intravital fluorescence microscopy and small animal fluorescence imaging of tumor-bearing mice. We observed specific retention of Dmt-Tic-Cy5 in tumors with maximum uptake in δOR-expressing positive tumors at 3 h and observable persistence for >96 h; clearance from δOR nonexpressing negative tumors by 6 h; and systemic clearance from normal organs by 24 h. Live-cell and intravital fluorescence microscopy demonstrated that Dmt-Tic-Cy5 had sustained cell-surface binding lasting at least 24 h with gradual internalization over the initial 6 h following administration. Dmt-Tic-Cy5 is a δOR-targeted agent that exhibits long-lasting and specific signal in δOR-expressing tumors, is rapidly cleared from systemic circulation, and is not retained in non-δOR-expressing tissues. Hence, Dmt-Tic-Cy5 has potential as a fluorescent tumor imaging agent.

Nicotine Inhibits Memory CTL Programming

PubMed Central

Sun, Zhifeng; Smyth, Kendra; Garcia, Karla; Mattson, Elliot; Li, Lei; Xiao, Zhengguo

2013-01-01

Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion in vitro, but impairs in vivo expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation in vitro. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development. PMID:23844169

Global gene expression and systems biology analysis of bovine monocyte-derived macrophages in response to in vitro challenge with Mycobacterium bovis.

PubMed

Magee, David A; Taraktsoglou, Maria; Killick, Kate E; Nalpas, Nicolas C; Browne, John A; Park, Stephen D E; Conlon, Kevin M; Lynn, David J; Hokamp, Karsten; Gordon, Stephen V; Gormley, Eamonn; MacHugh, David E

2012-01-01

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cell types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2:1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array. Comparison of M. bovis-challenged MDM gene expression profiles with those from the non-challenged MDM controls at each time point identified 3,064 differentially expressed genes 2 hours post-challenge, with 4,451 and 5,267 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤ 0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly higher than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors; and (3) apoptosis. The increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support roles for MyD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis.

A 5′ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

PubMed Central

Lu, Jiamiao; Williams, James A.; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A.

2017-01-01

We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo. PMID:27903072

Molecular changes associated with heat-shock treatment in avian mononuclear and lymphoid lineage cells.

PubMed

Miller, L; Qureshi, M A

1992-03-01

The induction of heat-shock protein (HSP) synthesis in avian cells of the mononuclear phagocytic system (MPS) and lymphoid system (LS) lineage was investigated by exposure to in vitro heat-shock conditions. In addition, the kinetics of HSP90 mRNA expression was examined in chicken peritoneal macrophages (PM) as well as heat-shock-induced HSP synthesis in PM from chickens, turkeys, quail, and ducks. Each MPS and LS cell type expressed three major (23, 70, and 90 kDa) HSP following a 1-h heat shock at 45 C. However, a unique heat-induced 32-kDa protein (P32) was expressed only by cells of MPS lineage. The expression of HSP90 mRNA in chicken PM was temperature- and time-dependent. These findings imply that avian PM undergo molecular changes in response to elevated environmental temperatures and that the pattern of HSP expression appears to be distinct for cells of the MPS and LS lineages in chickens.

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

PubMed

Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

2001-04-01

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

Expression patterns of poliovirus receptor, erythrocyte protein band 4.1-like 3, regulator of g-protein signaling 11, and oxytocin receptor in mouse ovarian cells during follicle growth and early luteinization in vitro and in vivo.

PubMed

Segers, Ingrid; Adriaenssens, Tom; Smitz, Johan

2012-01-01

Poliovirus receptor (Pvr), erythrocyte protein band 4.1-like 3 (Epb4.1l3), regulator of G-protein signaling 11 (Rgs11), and oxytocin receptor (Oxtr) expression were quantified in in vitro- and in vivo-grown mouse follicles. The expression of all genes was increased during antral growth in in vitro-grown cumulus cells, whereas only Rgs11 and Oxtr were increased and Pvr and Epb4.1l3 were decreased in in vivo grown cumulus cells. In vivo mural granulosa cells showed the highest expression of Pvr, Rgs11, and Oxtr. The in vitro granulosa + theca compartment responded to human chorionic gonadotropinduring early luteinization by either an upregulation (Pvr, Oxtr) or downregulation (Epb41l3, Rgs11). Oocytes expressed Epb4.1l3, not Rgs11, and Pvr only in in vitro-grown oocytes. Translation into protein was confirmed for Epb4.1l3 in in vitro-grown follicles and in vivo-grown cumulus-oocyte complexes. Protein 4.1B was present during antral growth in cumulus, granulosa cells, and oocytes. Hypothetical functions of Epb4.1l3 and Pvr involve cell adhesion regulation and Rgs11 could be involved in cAMP production in the follicle. Oxtr is known to be important during and after the ovulatory stimulus, but, as in bovine, was also regulated during folliculogenesis. High expression of Pvr and Epb4.1l3 with culture duration in cumulus cells might mark inappropriate differentiation into a mural granulosa-like cell type and function as negative follicle development marker. Rgs11 and Oxtr are both in vivo and in vitro upregulated in cumulus cells during antral follicle growth and might be considered positive markers for follicle development.

Gene expression of commensal Lactobacillus johnsonii strain NCC533 during in vitro growth and in the murine gut.

PubMed

Denou, Emmanuel; Berger, Bernard; Barretto, Caroline; Panoff, Jean-Michel; Arigoni, Fabrizio; Brüssow, Harald

2007-11-01

Work with pathogens like Vibrio cholerae has shown major differences between genes expressed in bacteria grown in vitro and in vivo. To explore this subject for commensals, we investigated the transcription of the Lactobacillus johnsonii NCC533 genome during in vitro and in vivo growth using the microarray technology. During broth growth, 537, 626, and 277 of the 1,756 tested genes were expressed during exponential phase, "adaptation" (early stationary phase), and stationary phase, respectively. One hundred one, 150, and 33 genes, respectively, were specifically transcribed in these three phases. To explore the in vivo transcription program, we fed L. johnsonii containing a resistance plasmid to antibiotic-treated mice. After a 2-day washout phase, we determined the viable-cell counts of lactobacilli that were in the lumina and associated with the mucosae of different gut segments. While the cell counts showed a rather uniform distribution along the gut, we observed marked differences with respect to the expression of the Lactobacillus genome. The largest number of transcribed genes was in the stomach (n = 786); the next-largest numbers occurred in the cecum (n = 391) and the jejunum (n = 296), while only 26 Lactobacillus genes were transcribed in the colon. In vitro and in vivo transcription programs overlapped only partially. One hundred ninety-one of the transcripts from the lactobacilli in the stomach were not detected during in vitro growth; 202 and 213 genes, respectively, were transcribed under all in vitro and in vivo conditions; but the core transcriptome for all growth conditions comprised only 103 genes. Forty-four percent of the NCC533 genes were not detectably transcribed under any of the investigated conditions. Nontranscribed genes were clustered on the genome and enriched in the variable-genome part. Our data revealed not only major differences between in vitro- and in vivo-expressed genes in a Lactobacillus gut commensal organism but also marked changes in the expression of genes along the digestive tract.

3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aly, Hussein Hassan; Shimotohno, Kunitada; Hijikata, Makoto

2009-02-06

Due to the high polymorphism of natural hepatitis C virus (HCV) variants, existing recombinant HCV replication models have failed to be effective in developing effective anti-HCV agents. In the current study, we describe an in vitro system that supports the infection and replication of natural HCV from patient blood using an immortalized primary human hepatocyte cell line cultured in a three-dimensional (3D) culture system. Comparison of the gene expression profile of cells cultured in the 3D system to those cultured in the existing 2D system demonstrated an up-regulation of several genes activated by peroxisome proliferator-activated receptor alpha (PPAR{alpha}) signaling. Furthermore,more » using PPAR{alpha} agonists and antagonists, we also analyzed the effect of PPAR{alpha} signaling on the modulation of HCV replication using this system. The 3D in vitro system described in this study provides significant insight into the search for novel anti-HCV strategies that are specific to various strains of HCV.« less

A Dual Luciferase Reporter System for B. burgdorferi Measures Transcriptional Activity during Tick-Pathogen Interactions

PubMed Central

Adams, Philip P.; Flores Avile, Carlos; Jewett, Mollie W.

2017-01-01

Knowledge of the transcriptional responses of vector-borne pathogens at the vector-pathogen interface is critical for understanding disease transmission. Borrelia (Borreliella) burgdorferi, the causative agent of Lyme disease in the United States, is transmitted by the bite of infected Ixodes sp. ticks. It is known that B. burgdorferi has altered patterns of gene expression during tick acquisition, persistence and transmission. Recently, we and others have discovered in vitro expression of RNAs found internal, overlapping, and antisense to annotated open reading frames in the B. burgdorferi genome. However, there is a lack of molecular genetic tools for B. burgdorferi for quantitative, strand-specific, comparative analysis of these transcripts in distinct environments such as the arthropod vector. To address this need, we have developed a dual luciferase reporter system to quantify B. burgdorferi promoter activities in a strand-specific manner. We demonstrate that constitutive expression of a B. burgdorferi codon-optimized Renilla reniformis luciferase gene (rlucBb) allows normalization of the activity of a promoter of interest when fused to the B. burgdorferi codon-optimized Photinus pyralis luciferase gene (flucBb) on the same plasmid. Using the well characterized, differentially regulated, promoters for flagellin (flaBp), outer surface protein A (ospAp) and outer surface protein C (ospCp), we document the efficacy of the dual luciferase system for quantitation of promoter activities during in vitro growth and in infected ticks. Cumulatively, the dual luciferase method outlined herein is the first dual reporter system for B. burgdorferi, providing a novel and highly versatile approach for strand-specific molecular genetic analyses. PMID:28620587

A Dual Luciferase Reporter System for B. burgdorferi Measures Transcriptional Activity during Tick-Pathogen Interactions.

PubMed

Adams, Philip P; Flores Avile, Carlos; Jewett, Mollie W

2017-01-01

Knowledge of the transcriptional responses of vector-borne pathogens at the vector-pathogen interface is critical for understanding disease transmission. Borrelia ( Borreliella ) burgdorferi , the causative agent of Lyme disease in the United States, is transmitted by the bite of infected Ixodes sp . ticks. It is known that B. burgdorferi has altered patterns of gene expression during tick acquisition, persistence and transmission. Recently, we and others have discovered in vitro expression of RNAs found internal, overlapping, and antisense to annotated open reading frames in the B. burgdorferi genome. However, there is a lack of molecular genetic tools for B. burgdorferi for quantitative, strand-specific, comparative analysis of these transcripts in distinct environments such as the arthropod vector. To address this need, we have developed a dual luciferase reporter system to quantify B. burgdorferi promoter activities in a strand-specific manner. We demonstrate that constitutive expression of a B. burgdorferi codon-optimized Renilla reniformis luciferase gene ( rluc Bb ) allows normalization of the activity of a promoter of interest when fused to the B. burgdorferi codon-optimized Photinus pyralis luciferase gene ( fluc Bb ) on the same plasmid. Using the well characterized, differentially regulated, promoters for flagellin ( flaBp ), outer surface protein A ( ospAp ) and outer surface protein C ( ospCp ), we document the efficacy of the dual luciferase system for quantitation of promoter activities during in vitro growth and in infected ticks. Cumulatively, the dual luciferase method outlined herein is the first dual reporter system for B. burgdorferi , providing a novel and highly versatile approach for strand-specific molecular genetic analyses.

Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

ERIC Educational Resources Information Center

Mokin, Maxim; Keifer, Joyce

2005-01-01

Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

Steroid and cytokine regulation of matrix metalloproteinase expression in endometriosis and the establishment of experimental endometriosis in nude mice.

PubMed

Bruner-Tran, Kaylon L; Eisenberg, Esther; Yeaman, Grant R; Anderson, Ted A; McBean, Judith; Osteen, Kevin G

2002-10-01

The cyclic expression of matrix metalloproteinases (MMPs) by human endometrium has been suggested to play a role in the invasive process necessary to establish endometriosis. The ability of progesterone exposure to inhibit endometrial MMP-3 and MMP-7 expression requires the local action of TGF beta and may also be linked to the local production of retinoic acid by stromal cells. A continuous expression of several MMPs in endometriotic lesions has been reported, indicating a failure of progesterone or locally produced factors to suppress these enzymes. To address cell-specific MMP regulation associated with endometriosis, we examined expression of MMP-3 and MMP-7 mRNA in eutopic endometrium and endometriotic lesions acquired during the secretory phase of the menstrual cycle. We examined the in vitro regulation of MMP-3 and MMP-7 protein in similar tissues. We also examined the in vitro regulation of MMP secretion by progesterone, retinoic acid, and TGF beta in endometriosis tissues relative to the establishment of experimental disease. Our studies indicate that either eutopic or ectopic tissue from women with endometriosis exhibit patterns of altered MMP regulation in vivo. A lack of responsiveness to progesterone was demonstrated in vitro, associated with a failure to suppress MMP expression and an enhanced ability of the tissue to establish experimental endometriosis. However, in vitro treatments with retinoic acid and TGF beta restored the ability of progesterone to suppress MMPs in vitro and prevented the establishment of experimental disease.

Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

NASA Astrophysics Data System (ADS)

Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

1990-09-01

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

Human immunodeficiency virus coinfection with hepatitis B virus leads to a decrease in extracellular and intracellular hepatitis B antigen.

PubMed

Pan, Wei; Wu, Zuoqiao; Wu, Shuwen; Guo, Deyin; Gong, Xiaoyan; Po, Tien

2015-04-01

Chronic hepatitis B virus (HBV) infection could cause severe liver disease including cirrhosis, hepatocellular carcinoma, and end-stage liver failure in HIV-positive individuals. The available data from clinical studies suggest that HIV infection modulates the HBV-specific T cell response. However, the virological and molecular aspects of HIV-HBV coinfection are currently poorly understood due to the lack of appropriate model systems. In this study, the effect of HIV infection on the life cycle of HBV was explored using an in vitro model system. The present data show that the extracellular and intracellular hepatitis B surface antigen (HBsAg) and e antigen (HBeAg) decrease significantly in HepG2 cells cotransfected with HIV NL4-3 and pHBV1.3 as compared to those cells transfected only with pHBV1.3. Moreover, a significant decrease in HBV DNA and mRNA expression was also observed in the cotransfected cells. HIV Rev protein, an RNA-bound regulatory protein, could significantly decrease the expression levels of extracellular and intracellular HBsAg and HBeAg by mediating the expression of HBV mRNA in cells cotransfected with plasmids containing HIV-1 Rev and pHBV1.3. Further experiments demonstrate that HIV Rev manipulated neither the promoters of HBV nor the nuclear export of HBV mRNA. These results from the in vitro model system might provide clues to further understand the rapid progression of liver disease in HIV-HBV-coinfected patients.

Isolation and identification of gene-specific microRNAs.

PubMed

Lin, Shi-Lung; Chang, Donald C; Ying, Shao-Yao

2006-01-01

Prediction of microRNA (miRNA) candidates using computer programming has identified hundreds and hundreds of genomic hairpin sequences, of which, the functions remain to be determined. Because direct transfection of hairpin-like miRNA precursors (pre)-miRNAs in mammalian cells is not always sufficient to trigger effective RNA-induced gene-silencing complex (RISC) assembly, a key step for RNA interference (RNAi)-related gene silencing, we developed an intronic miRNA-expressing system to overcome this problem, and successfully increased the efficiency and effectiveness of miRNA-associated RNAi induction in vitro and in vivo. By insertion of a hairpin-like pre-miRNA structure into the intron region of a gene, this intronic miRNA biogenesis system has been found to depend on a coupled interaction of nascent precursor messenger RNA transcription and intron excision within a specific nuclear region proximal to genomic perichromatin fibrils. The intronic miRNA was transcribed by RNA type II polymerases, coexpressed with a primary gene transcript, and excised out of its encoding gene transcript by intracellular RNA splicing and processing mechanisms. Currently, some ribonuclease III endonucleases have been found to be involved in the processing of spliced introns and probably facilitating the intronic miRNA maturation. Using this miRNA-expressing system, we have shown for the first time that the intron-derived miRNAs were able to induce strong RNAi effects in not only human and mouse cells but also zebrafish, chicken embryos, and adult mice. Based on the strand complementarity between the designed miRNA and its target gene sequence, we have also developed a miRNA isolation protocol to purify and identify the mature miRNAs generated by the intronic miRNA-expressing system. Several intronic miRNA identities and structures are currently confirmed to be active in vitro and in vivo. According to this proof- of-principle method, we now have the knowledge to design pre-miRNA inserts that are more efficient and effective for the intronic miRNA-expressing system.

Comparative transcriptome analysis by RNAseq of necrotic enteritis Clostridium perfringens during in vivo colonization and in vitro conditions.

PubMed

Parreira, Valeria R; Russell, Kay; Athanasiadou, Spiridoula; Prescott, John F

2016-08-12

Necrotic enteritis (NE) caused by netB-positive type A Clostridium perfringens is an important bacterial disease of poultry. Through its complex regulatory system, C. perfringens orchestrates the expression of a collection of toxins and extracellular enzymes that are crucial for the development of the disease; environmental conditions play an important role in their regulation. In this study, and for the first time, global transcriptomic analysis was performed on ligated intestinal loops in chickens colonized with a netB-positive C. perfringens strain, as well as the same strain propagated in vitro under various nutritional and environmental conditions. Analysis of the respective pathogen transcriptomes revealed up to 673 genes that were significantly expressed in vivo. Gene expression profiles in vivo were most similar to those of C. perfringens grown in nutritionally-deprived conditions. Taken together, our results suggest a bacterial transcriptome responses to the early stages of adaptation, and colonization of, the chicken intestine. Our work also reveals how netB-positive C. perfringens reacts to different environmental conditions including those in the chicken intestine.

Biomechanical forces promote embryonic haematopoiesis

PubMed Central

Adamo, Luigi; Naveiras, Olaia; Wenzel, Pamela L.; McKinney-Freeman, Shannon; Mack, Peter J.; Gracia-Sancho, Jorge; Suchy-Dicey, Astrid; Yoshimoto, Momoko; Lensch, M. William; Yoder, Mervin C.; García-Cardeña, Guillermo; Daley, George Q.

2009-01-01

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system1,2. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3–5), a master regulator of haematopoiesis, and give rise to haematopoietic cells4. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential6. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41+c-Kit+ haematopoietic progenitor cells7,concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the paraaortic splanchnopleura/aorta–gonads–mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling8, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development. PMID:19440194

Bisphenol A disrupts gene expression in human placental trophoblast cells.

PubMed

Rajakumar, Chandrew; Guan, Haiyan; Langlois, David; Cernea, Maria; Yang, Kaiping

2015-06-01

This study examined the effect of bisphenol A (BPA) on human placental gene expression using primary trophoblast cells as an in vitro model system. Trophoblast cells were isolated from human placentas at term, cultured and then exposed to environmentally relevant concentrations of BPA (0.1-2 μg/ml) for up to 24h, after which levels of 11β-HSD2 mRNA, protein and activity were determined by standard radiometric conversion assay, western blotting, and qRT-PCR, respectively. The mRNA levels of several other prominent placental hormones/factors were also assessed by qRT-PCR. BPA dramatically increased levels of 11β-HSD2 activity, protein and mRNA in a time- and concentration-dependent manner (> 4-fold). BPA also augmented aromatase, glucose transporter-1, CRH, and hCG mRNA levels while reducing the level of leptin mRNA. These findings demonstrate that BPA severely disrupts human placental gene expression in vitro, which suggests that exposure to BPA may contribute to altered placental function and consequent pregnancy complications. Copyright © 2015 Elsevier Inc. All rights reserved.

Noninvasive diode laser activation of transient receptor potential proteins and nociceptors

NASA Astrophysics Data System (ADS)

Jiang, Nan; Cooper, Brian Y.; Nemenov, Michael I.

2007-02-01

We investigated diode laser (980 nm) evoked activation of transient receptor potential proteins (TRPV1 and TRPV2). C and A-delta (Aδ) nociceptor families are primarily responsible for pain mediation in the peripheral nervous system. TRPV1 proteins have been associated with heat evoked pain in C fibers while Aδ fibers have been associated with TRPV2. Diode laser stimulation allows a margin of safety between non-invasive activation and damage 19, 22, 34. Laser pulses (20-50 ms, 0.1-10 W, 980 nm) were used to stimulate: A) in vitro: excised patches from HEK293 cells expressing TRPV1; B) in vitro: rat DRG nociceptors expressing either TRPV1 or TRPV2; and C) in vivo: C-fibers of the rat saphenous nerve (SN) trunk. Cell currents were recorded using standard patch clamp methods. The SN was also stimulated electrically with bipolar electrodes. Stimulation (20-50 ms) of HEK and DRG cells expressing TRPV1 was highly reproducible. Activation and peak currents were achieved at estimated peak temperatures of 55°C and 70°C. Threshold activation was also observed in DRG neurons expressing TRPV2. The conduction velocity for laser-activated saphenous nerve afferents was in the C fiber range (0.5-1 m/s). Electrically stimulated nerve contained stimulation artifacts and complex neural components with conduction velocities ranging from 0.3-30 m/s. Diode laser activation of TRPV1 protein is a reproducible and effective means to probe TRP activity in both in vivo and in vitro preparations

Large-scale production of foot-and-mouth disease virus (serotype Asia1) VLP vaccine in Escherichia coli and protection potency evaluation in cattle.

PubMed

Xiao, Yan; Chen, Hong-Ying; Wang, Yuzhou; Yin, Bo; Lv, Chaochao; Mo, Xiaobing; Yan, He; Xuan, Yajie; Huang, Yuxin; Pang, Wenqiang; Li, Xiangdong; Yuan, Y Adam; Tian, Kegong

2016-07-02

Foot-and-mouth disease (FMD) is an acute, highly contagious disease that infects cloven-hoofed animals. Vaccination is an effective means of preventing and controlling FMD. Compared to conventional inactivated FMDV vaccines, the format of FMDV virus-like particles (VLPs) as a non-replicating particulate vaccine candidate is a promising alternative. In this study, we have developed a co-expression system in E. coli, which drove the expression of FMDV capsid proteins (VP0, VP1, and VP3) in tandem by a single plasmid. The co-expressed FMDV capsid proteins (VP0, VP1, and VP3) were produced in large scale by fermentation at 10 L scale and the chromatographic purified capsid proteins were auto-assembled as VLPs in vitro. Cattle vaccinated with a single dose of the subunit vaccine, comprising in vitro assembled FMDV VLP and adjuvant, developed FMDV-specific antibody response (ELISA antibodies and neutralizing antibodies) with the persistent period of 6 months. Moreover, cattle vaccinated with the subunit vaccine showed the high protection potency with the 50 % bovine protective dose (PD50) reaching 11.75 PD50 per dose. Our data strongly suggest that in vitro assembled recombinant FMDV VLPs produced from E. coli could function as a potent FMDV vaccine candidate against FMDV Asia1 infection. Furthermore, the robust protein expression and purification approaches described here could lead to the development of industrial level large-scale production of E. coli-based VLPs against FMDV infections with different serotypes.

Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

PubMed

Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

2018-02-20

Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

In vitro cytotoxicity analysis of doxorubicin-loaded/superparamagnetic iron oxide colloidal nanoassemblies on MCF7 and NIH3T3 cell lines

PubMed Central

Tomankova, Katerina; Polakova, Katerina; Pizova, Klara; Binder, Svatopluk; Havrdova, Marketa; Kolarova, Mary; Kriegova, Eva; Zapletalova, Jana; Malina, Lukas; Horakova, Jana; Malohlava, Jakub; Kolokithas-Ntoukas, Argiris; Bakandritsos, Aristides; Kolarova, Hana; Zboril, Radek

2015-01-01

One of the promising strategies for improvement of cancer treatment is based on magnetic drug delivery systems, thus avoiding side effects of standard chemotherapies. Superparamagnetic iron oxide (SPIO) nanoparticles have ideal properties to become a targeted magnetic drug delivery contrast probes, named theranostics. We worked with SPIO condensed colloidal nanocrystal clusters (MagAlg) prepared through a new soft biomineralization route in the presence of alginate as the polymeric shell and loaded with doxorubicin (DOX). The aim of this work was to study the in vitro cytotoxicity of these new MagAlg–DOX systems on mouse fibroblast and breast carcinoma cell lines. For proper analysis and understanding of cell behavior after administration of MagAlg–DOX compared with free DOX, a complex set of in vitro tests, including production of reactive oxygen species, comet assay, cell cycle determination, gene expression, and cellular uptake, were utilized. It was found that the cytotoxic effect of MagAlg–DOX system is delayed compared to free DOX in both cell lines. This was attributed to the different mechanism of internalization of DOX and MagAlg–DOX into the cells, together with the fact that the drug is strongly bound on the drug nanocarriers. We discovered that nanoparticles can attenuate or even inhibit the effect of DOX, particularly in the tumor MCF7 cell line. This is a first comprehensive study on the cytotoxic effect of DOX-loaded SPIO compared with free DOX on healthy and cancer cell lines, as well as on the induced changes in gene expression. PMID:25673990

An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

PubMed Central

Janebodin, Kajohnkiart; Buranaphatthana, Worakanya; Ieronimakis, Nicholas; Hays, Aislinn L.; Reyes, Morayma

2013-01-01

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors. PMID:23841093

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Xiaozhong; Hong, Sung Woo; Moreira, Estefania G.

Gonocytes exist in the neonatal testis and represent a transient population of male germ-line stem cells. It has been shown that stem cell self-renewal and progeny production is probably controlled by the neighboring differentiated cells and extracellular matrix (ECM) in vivo known as niches. Recently, we developed an in vitro three-dimensional (3D) Sertoli cell/gonocyte co-culture (SGC) model with ECM overlay, which creates an in vivo-like niche and supports germ-line stem cell functioning within a 3D environment. In this study, we applied morphological and cytotoxicity evaluations, as well as microarray-based gene expression to examine the effects of different phthalate esters (PE)more » on this model. Known in vivo male developmentally toxic PEs (DTPE) and developmentally non-toxic PEs (DNTPE) were evaluated. We observed that DTPE induced significantly greater dose-dependent morphological changes, a decrease in cell viability and an increase in cytotoxicity compared to those treated with DNTPE. Moreover, the gene expression was more greatly altered by DTPE than by DNTPE and non-supervised cluster analysis allowed the discrimination of DTPE from the DNTPE. Our systems-based GO-Quant analysis showed significant alterations in the gene pathways involved in cell cycle, phosphate transport and apoptosis regulation with DTPE but not with DNTPE treatment. Disruptions of steroidogenesis related-gene expression such as Star, Cyp19a1, Hsd17b8, and Nr4a3 were observed in the DTPE group, but not in the DNTPE group. In summary, our observation on cell viability, cytotoxicity, and microarray-based gene expression analysis induced by PEs demonstrate that our in vitro 3D-SGC system mimicked in vivo responses for PEs and suggests that the 3D-SGC system might be useful in identifying developmental reproductive toxicants.« less

Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Noninternalizing Receptors.

PubMed

Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J

2017-03-28

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ∼3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or noninternalizing receptor (CD45). When lymphocytes were preloaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell antitumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors.

Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Non-Internalizing Receptors

PubMed Central

Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J.

2017-01-01

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ~3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or non-internalizing receptor (CD45). When lymphocytes were pre-loaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell anti-tumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors. PMID:28231431

AAV-CRISPR/Cas9-Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro.

PubMed

Wu, Wenyi; Duan, Yajian; Ma, Gaoen; Zhou, Guohong; Park-Windhol, Cindy; D'Amore, Patricia A; Lei, Hetian

2017-12-01

Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)-mediated CRISPR (clustered regularly interspaced short palindromic repeats)-associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. AAV-CRISRP/Cas9-mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.

In-vitro formation of the blood-testis barrier during long-term organotypic culture of human prepubertal tissue: comparison with a large cohort of pre/peripubertal boys.

PubMed

de Michele, F; Poels, J; Giudice, M G; De Smedt, F; Ambroise, J; Vermeulen, M; Gruson, D; Wyns, C

2018-03-12

How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in-vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical files. A longitudinal analysis of connexin 43, claudin 11 and AMH expressions on immunohistological sections of organotypic cultured testicular tissue from three prepubertal boys who underwent a biopsy for fertility preservation was performed. Immunostaining was evaluated at culture days 0, 1, 3, 10, 16, 27, 32, 53, 64 and 139 for two different types of culture media. Immunohistochemical control sections showed progressive maturation of Sertoli cells, as shown by the decrease in AMH expression, with increasing age (p ≤ 0.01) and the AMH expression was negatively correlated with the expression of connexin 43 and claudin 11 (p ≤ 0.01 for both proteins). AR (Androgen receptor) expression increased with age (p ≤ 0.01) and was significantly correlated with the expression of connexin 43 (p = 0.002) and claudin 11 (p = 0.03). A statistical correlation was also found between the reduction of AMH expression and both the advancement of Tanner stages (p ≤ 0.01) and the differentiation of germ cells (p ≤ 0.01). Furthermore, positive correlations between BTB formation (using connexin 43 and claudin 11 expression) and age (p ≤ 0.01 for both the proteins), higher Tanner stages (p ≤ 0.001 and p ≤ 0.01 for connexin 43 and claudin 11, respectively), and presence of more advanced germ cells (p ≤ 0.001 for both proteins) were observed. In the subanalysis on organotypic cultured ITT, where a significant decrease in AMH expression as a marker of SC maturation was already reported, we showed the onset of expression of connexin 43 at day 16 (p ≤ 0.001) and a constant expression of claudin 11 from day 0 to day 139, for all three patients, without differences between the two types of culture media. N/A. Accessibility of prepubertal human testicular tissue is a major limiting factor to the analysis of cultured tissue samples from a wide number of patients, as would be needed to assess the in-vitro development of the BTB according to the age. The impossibility of performing longitudinal studies on in-vivo BTB formation in the same patient prevents a comparison of the time needed to achieve effective BTB formation and protein expression patterns in vivo and in vitro. To the best of our knowledge, this is the first report describing the expression of two BTB proteins in samples from a cohort of prepubertal and peripubertal boys, for the in-vivo pattern, and in cultured ITT from a few prepubertal boys, for the in-vitro evaluation. Since the formation of this barrier is essential for spermatogenesis and because little is known about its protein expression patterns and development in humans, a deeper understanding of the testicular microenvironment is essential to improve ITT in-vitro culture conditions. The final aim is to restore fertility by acheiving in-vitro differentiation of spermatogonial stem cells, using cryopreserved ITT collected before gonadotoxic therapies. Funding was received from Fonds National de la Recherche Scientifique de Belgique (grant Télevie No. 7.4554.14F and No. 7.6511.16) and Fondation Salus Sanguinis. No conflict of interest has to be disclosed.

[Expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro].

PubMed

Yu, Sheng-ji; Qiu, Gui-xing; Burton, Yang; Sandra, Roth; Cari, Whyne; Albert, Yee

2005-12-15

To investigate the expression of integrin alpha5 and actin in the cells of intervertebral disc under cyclic hydrostatic pressure in vitro. The porcine lumbar intervertebral disc cells were isolated and cultured in vitro, and the cells underwent cyclic hydrostatic loading. After that, the expression of integrin alpha5 and actin in intervertebral disc cells were studied by means of morphology observing, Western blot and immunohistochemistry staining. The morphology of intervertebral disc cells were changed into smaller and flatten shape, and the expression of integrin alpha5 and actin were decreased after loading. The expression of integrin alpha5 decreases under cyclic hydrostatic pressure, and the actin is affected at the same time when signals are transferred into the cells by integrin alpha5. That may be one of the important mechanisms of the mechanotransduction in the cells of intervertebral disc.

Expression of pathogenicity-related genes of Xylella fastidiosa in vitro and in planta.

PubMed

de Souza, Alessandra A; Takita, Marco A; Pereira, Eridan O; Coletta-Filho, Helvécio D; Machado, Marcos A

2005-04-01

Xylella fastidiosa is responsible for several economically important plant diseases. It is currently assumed that the symptoms are caused by vascular occlusion due to biofilm formation. Microarray technology was previously used to examine the global gene expression profile of X. fastidiosa freshly isolated from symptomatic plants or after several passages by axenic culture medium, and different pathogenicity profiles have been obtained. In the present study the expression of some pathogenicity-related genes was evaluated in vitro and in planta by RT-PCR. The results suggest that adhesion is important at the beginning of biofilm formation, while the genes related to adaptation are essential for the organism's maintenance in planta. Similar results were observed in vitro mainly for the adhesion genes. The pattern of expression observed suggests that adhesion modulates biofilm formation whereas the expression of some adaptation genes may be related to the environment in which the organism is living.

Indian hedgehog contributes to human cartilage endplate degeneration.

PubMed

Wang, Shaowei; Yang, Kun; Chen, Shuai; Wang, Jiying; Du, Guoqing; Fan, Shunwu; Wei, Lei

2015-08-01

To determine the role of Indian hedgehog (Ihh) signaling in human cartilage endplate (CEP) degeneration. CEP-degenerated tissues from patients with Modic I or II changes (n = 9 and 45, respectively) and normal tissues from vertebral burst fracture patients (n = 17) were collected. Specimens were either cut into slices for organ culture ex vivo or digested to isolate chondrocytes for cell culture in vitro. Ihh expression and the effect of Ihh on cartilage degeneration were determined by investigating degeneration markers in this study. Ihh expression and cartilage degeneration markers significantly increased in the Modic I and II groups. The expression of cartilage degeneration markers was positively correlated with degeneration severity. Gain-of-function for Ihh promoted expression of cartilage degeneration markers in vitro, while loss-of-function for Ihh inhibited their expression both in vitro and ex vivo. These findings demonstrated that Ihh promotes CEP degeneration. Blocking Ihh pathway has potential clinical usage for attenuating CEP degeneration.

Comparison of MeHg-induced toxicogenomic responses across in vivo and in vitro models used in developmental toxicology.

PubMed

Robinson, Joshua F; Theunissen, Peter T; van Dartel, Dorien A M; Pennings, Jeroen L; Faustman, Elaine M; Piersma, Aldert H

2011-09-01

Toxicogenomic evaluations may improve toxicity prediction of in vitro-based developmental models, such as whole embryo culture (WEC) and embryonic stem cells (ESC), by providing a robust mechanistic marker which can be linked with responses associated with developmental toxicity in vivo. While promising in theory, toxicogenomic comparisons between in vivo and in vitro models are complex due to inherent differences in model characteristics and experimental design. Determining factors which influence these global comparisons are critical in the identification of reliable mechanistic-based markers of developmental toxicity. In this study, we compared available toxicogenomic data assessing the impact of the known teratogen, methylmercury (MeHg) across a diverse set of in vitro and in vivo models to investigate the impact of experimental variables (i.e. model, dose, time) on our comparative assessments. We evaluated common and unique aspects at both the functional (Gene Ontology) and gene level of MeHg-induced response. At the functional level, we observed stronger similarity in MeHg-response between mouse embryos exposed in utero (2 studies), ESC, and WEC as compared to liver, brain and mouse embryonic fibroblast MeHg studies. These findings were strongly correlated to the presence of a MeHg-induced developmentally related gene signature. In addition, we identified specific MeHg-induced gene expression alterations associated with developmental signaling and heart development across WEC, ESC and in vivo systems. However, the significance of overlap between studies was highly dependent on traditional experimental variables (i.e. dose, time). In summary, we identify promising examples of unique gene expression responses which show in vitro-in vivo similarities supporting the relevance of in vitro developmental models for predicting in vivo developmental toxicity. Copyright © 2011 Elsevier Inc. All rights reserved.

A 3-D Cardiac Muscle Construct for Exploring Adult Marrow Stem Cell Based Myocardial Regeneration

PubMed Central

Valarmathi, Mani T.; Goodwin, Richard L.; Fuseler, John W.; Davis, Jeffrey M.; Yost, Michael J.; Potts, Jay D.

2010-01-01

Adult bone marrow stromal cells (BMSCs) are capable of differentiating into cardiomyocyte-like cells in vitro and contribute to myocardial regeneration in vivo. Consequently, BMSCs may potentially play a vital role in cardiac repair and regeneration. However, this concept has been limited by inadequate and inconsistent differentiation of BMSCs into cardiomyocytes along with poor survival and integration of neo-cardiomyocytes after implantation into ischemic myocardium. In order to overcome these barriers and to explore adult stem cell based myocardial regeneration, we have developed an in vitro model of three-dimensional (3-D) cardiac muscle using rat ventricular embryonic cardiomyocytes (ECMs) and BMSCs. When ECMs and BMSCs were seeded sequentially onto a 3-D tubular scaffold engineered from topographically aligned type I collagen fibers and cultured in basal medium for 7, 14, 21, or 28 days, the maturation and co-differentiation into a cardiomyocyte lineage was observed. Phenotypic induction was characterized at morphological, immunological, biochemical and molecular levels. The observed expression of transcripts coding for cardiomyocyte phenotypic markers and the immunolocalization of cardiomyogenic lineage-associated proteins revealed typical expression patterns of neo-cardiomyogenesis. At the biochemical level differentiating cells exhibited appropriate metabolic activity and at the ultrastructural level myofibrillar and sarcomeric organization were indicative of an immature phenotype. Our 3-D co-culture system sustains the ECMs in vitro continuum of differentiation process and simultaneously induces the maturation and differentiation of BMSCs into cardiomyocyte-like cells. Thus, this novel 3-D co-culture system provides a useful in vitro model to investigate the functional role and interplay of developing ECMs and BMSCs during cardiomyogenic differentiation. PMID:20129663

Divergent Annexin A1 expression in periphery and gut is associated with systemic immune activation and impaired gut immune response during SIV infection

PubMed Central

Sena, Angela A. S.; Glavan, Tiffany; Jiang, Guochun; Sankaran-Walters, Sumathi; Grishina, Irina; Dandekar, Satya; Goulart, Luiz R.

2016-01-01

HIV-1 disease progression is paradoxically characterized by systemic chronic immune activation and gut mucosal immune dysfunction, which is not fully defined. Annexin A1 (ANXA1), an inflammation modulator, is a potential link between systemic inflammation and gut immune dysfunction during the simian immunodeficiency virus (SIV) infection. Gene expression of ANXA1 and cytokines were assessed in therapy-naïve rhesus macaques during early and chronic stages of SIV infection and compared with SIV-negative controls. ANXA1 expression was suppressed in the gut but systemically increased during early infection. Conversely, ANXA1 expression increased in both compartments during chronic infection. ANXA1 expression in peripheral blood was positively correlated with HLA-DR+CD4+ and CD8+ T-cell frequencies, and negatively associated with the expression of pro-inflammatory cytokines and CCR5. In contrast, the gut mucosa presented an anergic cytokine profile in relation to ANXA1 expression. In vitro stimulations with ANXA1 peptide resulted in decreased inflammatory response in PBMC but increased activation of gut lymphocytes. Our findings suggest that ANXA1 signaling is dysfunctional in SIV infection, and may contribute to chronic inflammation in periphery and with immune dysfunction in the gut mucosa. Thus, ANXA1 signaling may be a novel therapeutic target for the resolution of immune dysfunction in HIV infection. PMID:27484833

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

PubMed Central

Hirsh, J; Morgan, B A; Scholnick, S B

1986-01-01

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity. Images PMID:3099170

[Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

PubMed

Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

2007-09-01

To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

The GDNF System Is Altered in Diverticular Disease – Implications for Pathogenesis

PubMed Central

Böttner, Martina; Barrenschee, Martina; Hellwig, Ines; Harde, Jonas; Egberts, Jan-Hendrik; Becker, Thomas; Zorenkov, Dimitri; Schäfer, Karl-Herbert; Wedel, Thilo

2013-01-01

Background & Aims Absence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons. Methods Colonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system (GDNF, GDNF receptors GFRα1 and RET). To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons (receptor expression, neuronal differentiation and plasticity) were monitored. Results mRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression. Conclusions Our results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system. PMID:23805210

“Do-it-yourself in vitro vasculature that recapitulates in vivo geometries for investigating endothelial-blood cell interactions”

PubMed Central

Mannino, Robert G.; Myers, David R.; Ahn, Byungwook; Wang, Yichen; Margo Rollins; Gole, Hope; Lin, Angela S.; Guldberg, Robert E.; Giddens, Don P.; Timmins, Lucas H.; Lam, Wilbur A.

2015-01-01

Investigating biophysical cellular interactions in the circulation currently requires choosing between in vivo models, which are difficult to interpret due in part to the hemodynamic and geometric complexities of the vasculature; or in vitro systems, which suffer from non-physiologic assumptions and/or require specialized microfabrication facilities and expertise. To bridge that gap, we developed an in vitro “do-it-yourself” perfusable vasculature model that recapitulates in vivo geometries, such as aneurysms, stenoses, and bifurcations, and supports endothelial cell culture. These inexpensive, disposable devices can be created rapidly (<2 hours) with high precision and repeatability, using standard off-the-shelf laboratory supplies. Using these “endothelialized” systems, we demonstrate that spatial variation in vascular cell adhesion molecule (VCAM-1) expression correlates with the wall shear stress patterns of vascular geometries. We further observe that the presence of endothelial cells in stenoses reduces platelet adhesion but increases sickle cell disease (SCD) red blood cell (RBC) adhesion in bifurcations. Overall, our method enables researchers from all disciplines to study cellular interactions in physiologically relevant, yet simple-to-make, in vitro vasculature models. PMID:26202603

Analysis of surface protein expression in human bone marrow stromal cells: new aspects of culture-induced changes, inter-donor differences and intracellular expression.

PubMed

Schäck, Luisa Marilena; Noack, Sandra; Weist, Ramona; Jagodzinski, Michael; Krettek, Christian; Buettner, Manuela; Hoffmann, Andrea

2013-12-15

The most widely used technique for isolation of human bone marrow stromal cells (hBMSCs) from bone marrow includes density gradient centrifugation, recovery of the mononuclear cell population, and subsequent isolation of hBMSCs by virtue of their plastic adherence. During subsequent in vitro cultivation, they may lose their original characteristics since in vitro the stem cell niche cannot yet be properly mimicked. To further characterize these culture-induced changes in regard to mRNA and extra- and intracellular protein expression, as well as potential differences between hBMSCs from different donors, we investigated a panel of CD antigens for their presence on in vitro cultured hBMSCs. Interestingly, after culture-induced downregulation of their extracellular expression, both CD146 and CD271 persist intracellularly, which hints at the possibility that culture-induced changes may be reversed by appropriate stimuli. Further, CD34-a protein whose expression on hBMSCs is still controversial-is expressed at the intracellular level in hBMSCs of all donors independently of passage number. CD34 mRNA levels are significantly higher in female than in male donors. In summary, we further elucidate phenotypical changes induced by in vitro culture of hBMSCs, highlight interindividual differences in the phenotype of these cells and for the first time show the intracellular expression of CD34.

Olive Oil-Based Lipid Emulsions Do Not Influence Platelet Receptor Expression in Comparison to Medium and Long Chain Triglycerides In vitro.

PubMed

Stoetzer, Carsten; Nickel, Katja; Weißig, Annette; Großheim, Marieke; Scheinichen, Dirk; Doll, Thorben; Jüttner, Björn

2016-11-01

Lipid emulsions influence platelet aggregation and receptor expression. However, the effect on platelet function is not fully explained. Therefore, the aim of this study was to examine the influence of the lipids Lipofundin ® , Lipidem ® and ClinOleic ® on surface expressions of P-selectin, GPIb and GPIIb/IIIa on platelets in vitro. Whole blood was incubated in two different concentrations (0.06 and 0.6 mg/ml) of LCT/MCT, n-3/LCT/MCT and LCT-MUFA for 30 min, followed by activation with TRAP-6 or ADP for flow-cytometric assay. Rates of P-selectin, GPIb and GPIIb/IIIa expression were analyzed. There was a significant increase in GPIIb/IIIa- and P-selectin-expression after incubation with LCT/MCT and n-3/LCT/MCT at the concentration of 0.6 mg/ml, without and after stimulation with TRAP-6 and ADP. GPIb was significantly decreased. Accordingly, LCT-MUFA had no effect on receptor expression of platelets in vitro. We demonstrated that LCT-MUFA did not activate receptor expression of platelets whereas LCT/MCT significantly increased platelet aggregation in vitro. This finding should be noted for parenteral nutrition of intensive care patients and, in the future, might provide further insight into the pathogenic pathways of acute thromboembolic events. However, prospectively designed clinical studies are needed to support our results.

A Novel Source of Cultured Podocytes

PubMed Central

Da Sacco, Stefano; Lemley, Kevin V.; Sedrakyan, Sargis; Zanusso, Ilenia; Petrosyan, Astgik; Peti-Peterdi, Janos; Burford, James; De Filippo, Roger E.; Perin, Laura

2013-01-01

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies. PMID:24349133

Using the 2A Protein Coexpression System: Multicistronic 2A Vectors Expressing Gene(s) of Interest and Reporter Proteins.

PubMed

Luke, Garry A; Ryan, Martin D

2018-01-01

To date, a huge range of different proteins-many with cotranslational and posttranslational subcellular localization signals-have been coexpressed together with various reporter proteins in vitro and in vivo using 2A peptides. The pros and cons of 2A co-expression technology are considered below, followed by a simple example of a "how to" protocol to concatenate multiple genes of interest, together with a reporter gene, into a single gene linked via 2As for easy identification or selection of transduced cells.

Expression and regulation of the chemokine CXCL16 in Crohn’s disease and models of intestinal inflammation

PubMed Central

Diegelmann, Julia; Seiderer, Julia; Niess, Jan-Hendrik; Haller, Dirk; Göke, Burkhard; Reinecker, Hans-Christian; Brand, Stephan

2010-01-01

Background/Aims CXCL16 mediates adhesion and phagocytosis of both Gram-negative and Gram-positive bacteria and is a strong chemoattractant for CXCR6+ T cells. In this study, we determined the so far unknown expression and signal transduction of the novel CXCL16-CXCR6 chemokine-ligand receptor system in intestinal inflammation in vivo and in vitro. Methods CXCL16 mRNA was measured by quantitative PCR in human colonic biopsies of patients with Crohn’s disease (CD) as well as in the TNFΔARE mouse model of ileitis and in murine cytomegalovirus (MCMV)-induced colitis. CXCL16 serum levels were analyzed by ELISA. CXCL16-induced signal transduction was analyzed in IEC with phospho-specific antibodies for MAP kinases and Akt. Results We found an inverse expression pattern of CXCL16 and CXCR6 with highest CXCL16 mRNA levels in the proximal murine small intestine and highest CXCR6 mRNA expression in the distal colon. CXCL16 and CXCR6 mRNA were expressed in colorectal cancer (CRC)-derived IEC lines. CRC-expressed CXCR6 was functional as demonstrated by CXCL16-induced MAP kinase and Akt activation. Intestinal CXCL16 expression was elevated in the TNFΔARE mouse model of ileitis and in MCMV-induced colitis (p<0.05) and in the sera and colons of patients with CD (p<0.05), where its expression correlated highly with CXCR6 and IL-8 levels (r=0.85 and 0.89, respectively). Conclusion CRC-derived IEC express the functional CXCL16 receptor CXCR6. CXCL16 mRNA and protein expression is up-regulated in intestinal inflammation in vitro and in CD patients, suggesting an important role for this chemokine in intestinal inflammation. PMID:20848509

Keratinocyte growth factor expression in human gingival fibroblasts and stimulation of in vitro gene expression by retinoic acid.

PubMed

Mackenzie, I C; Gao, Z

2001-04-01

Keratinocyte growth factor (KGF) is a stromally derived growth factor of the fibroblast growth factor (FGF) family with paracrine effects targeted to influence the growth and differentiation of epithelia. Regional and temporal changes in KGF expression play important roles in the development and maintenance of epithelial structures and in epithelial wound healing. Differing patterns of expression of KGF by fibroblasts in the gingival region could therefore be related to the observed regional variation in the differentiation and behavior of gingival epithelia. The in vitro and in vivo patterns of expression of KGF mRNA in human gingival and periodontal fibroblasts were examined using reverse transcription polymerase chain reactions (RT-PCR) and in situ hybridization with digoxigenin-labeled riboprobes. The patterns observed for human gingiva were compared with those for human skin and for murine tissues. Gingival and periodontal fibroblasts showed expression of KGF transcripts in vitro, and the degree of expression was markedly influenced by the presence of retinoic acid, an agent known to influence patterns of epithelial differentiation. Sections of human and murine gingiva and skin showed regionally variable expression of transcripts with the cells expressing KGF in the subepithelial, rather than the deeper, connective tissues and periodontium. The results point to a role of KGF in the maintenance of normal growth and differentiation of gingival epithelia. A lack of KGF expression by periodontal fibroblasts in vivo is expected to hinder apical epithelial migration and thus stabilize the epithelial attachment. The effects of retinoic acid (RA) on KGF expression in vitro provide an indirect mechanism by which RA may regulate the growth and differentiation of gingival epithelia.

Defining the Genomic Signature of Totipotency and Pluripotency during Early Human Development

PubMed Central

Galan, Amparo; Diaz-Gimeno, Patricia; Poo, Maria Eugenia; Valbuena, Diana; Sanchez, Eva; Ruiz, Veronica; Dopazo, Joaquin; Montaner, David; Conesa, Ana; Simon, Carlos

2013-01-01

The genetic mechanisms governing human pre-implantation embryo development and the in vitro counterparts, human embryonic stem cells (hESCs), still remain incomplete. Previous global genome studies demonstrated that totipotent blastomeres from day-3 human embryos and pluripotent inner cell masses (ICMs) from blastocysts, display unique and differing transcriptomes. Nevertheless, comparative gene expression analysis has revealed that no significant differences exist between hESCs derived from blastomeres versus those obtained from ICMs, suggesting that pluripotent hESCs involve a new developmental progression. To understand early human stages evolution, we developed an undifferentiation network signature (UNS) and applied it to a differential gene expression profile between single blastomeres from day-3 embryos, ICMs and hESCs. This allowed us to establish a unique signature composed of highly interconnected genes characteristic of totipotency (61 genes), in vivo pluripotency (20 genes), and in vitro pluripotency (107 genes), and which are also proprietary according to functional analysis. This systems biology approach has led to an improved understanding of the molecular and signaling processes governing human pre-implantation embryo development, as well as enabling us to comprehend how hESCs might adapt to in vitro culture conditions. PMID:23614026

High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase.

PubMed

Kim, Sung Bae; Ozawa, Takeaki; Watanabe, Shigeaki; Umezawa, Yoshio

2004-08-10

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.

[Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells, and teratocarcinoma cells by TGFb family signaling factors].

PubMed

Gordeeva, O F; Nikonova, T M; Lifantseva, N V

2009-01-01

The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

Global analysis of gene expression in mineralizing fish vertebra-derived cell lines: new insights into anti-mineralogenic effect of vanadate

PubMed Central

2011-01-01

Background Fish has been deemed suitable to study the complex mechanisms of vertebrate skeletogenesis and gilthead seabream (Sparus aurata), a marine teleost with acellular bone, has been successfully used in recent years to study the function and regulation of bone and cartilage related genes during development and in adult animals. Tools recently developed for gilthead seabream, e.g. mineralogenic cell lines and a 4 × 44K Agilent oligo-array, were used to identify molecular determinants of in vitro mineralization and genes involved in anti-mineralogenic action of vanadate. Results Global analysis of gene expression identified 4,223 and 4,147 genes differentially expressed (fold change - FC > 1.5) during in vitro mineralization of VSa13 (pre-chondrocyte) and VSa16 (pre-osteoblast) cells, respectively. Comparative analysis indicated that nearly 45% of these genes are common to both cell lines and gene ontology (GO) classification is also similar for both cell types. Up-regulated genes (FC > 10) were mainly associated with transport, matrix/membrane, metabolism and signaling, while down-regulated genes were mainly associated with metabolism, calcium binding, transport and signaling. Analysis of gene expression in proliferative and mineralizing cells exposed to vanadate revealed 1,779 and 1,136 differentially expressed genes, respectively. Of these genes, 67 exhibited reverse patterns of expression upon vanadate treatment during proliferation or mineralization. Conclusions Comparative analysis of expression data from fish and data available in the literature for mammalian cell systems (bone-derived cells undergoing differentiation) indicate that the same type of genes, and in some cases the same orthologs, are involved in mechanisms of in vitro mineralization, suggesting their conservation throughout vertebrate evolution and across cell types. Array technology also allowed identification of genes differentially expressed upon exposure of fish cell lines to vanadate and likely involved in its anti-mineralogenic activity. Many were found to be unknown or they were never associated to bone homeostasis previously, thus providing a set of potential candidates whose study will likely bring insights into the complex mechanisms of tissue mineralization and bone formation. PMID:21668972

Optical imaging of reporter gene expression using a positron-emission-tomography probe

NASA Astrophysics Data System (ADS)

Liu, Hongguang; Ren, Gang; Liu, Shuanglong; Zhang, Xiaofen; Chen, Luxi; Han, Peizhen; Cheng, Zhen

2010-11-01

Reporter gene/reporter probe technology is one of the most important techniques in molecular imaging. Lately, many reporter gene/reporter probe systems have been coupled to different imaging modalities such as positron emission tomography (PET) and optical imaging (OI). It has been recently found that OI techniques could be used to monitor radioactive tracers in vitro and in living subjects. In this study, we further demonstrate that a reporter gene/nuclear reporter probe system [herpes simplex virus type-1 thymidine kinase (HSV1-tk) and 9-(4-18F-fluoro-3-[hydroxymethyl] butyl) guanine ([18F]FHBG)] could be successfully imaged by OI in vitro and in vivo. OI with radioactive reporter probes will facilitate and broaden the applications of reporter gene/reporter probe techniques in medical research.

Generation of Human Adult Mesenchymal Stromal/Stem Cells Expressing Defined Xenogenic Vascular Endothelial Growth Factor Levels by Optimized Transduction and Flow Cytometry Purification

PubMed Central

Helmrich, Uta; Marsano, Anna; Melly, Ludovic; Wolff, Thomas; Christ, Liliane; Heberer, Michael; Scherberich, Arnaud; Martin, Ivan

2012-01-01

Adult mesenchymal stromal/stem cells (MSCs) are a valuable source of multipotent progenitors for tissue engineering and regenerative medicine, but may require to be genetically modified to widen their efficacy in therapeutic applications. For example, overexpression of the angiogenic factor vascular endothelial growth factor (VEGF) at controlled levels is an attractive strategy to overcome the crucial bottleneck of graft vascularization and to avoid aberrant vascular growth. Since the regenerative potential of MSCs is rapidly lost during in vitro expansion, we sought to develop an optimized technique to achieve high-efficiency retroviral vector transduction of MSCs derived from both adipose tissue (adipose stromal cells, ASCs) or bone marrow (BMSCs) and rapidly select cells expressing desired levels of VEGF with minimal in vitro expansion. The proliferative peak of freshly isolated human ASCs and BMSCs was reached 4 and 6 days after plating, respectively. By performing retroviral vector transduction at this time point, >90% efficiency was routinely achieved before the first passage. MSCs were transduced with vectors expressing rat VEGF164 quantitatively linked to a syngenic cell surface marker (truncated rat CD8). Retroviral transduction and VEGF expression did not affect MSC phenotype nor impair their in vitro proliferation and differentiation potential. Transgene expression was also maintained during in vitro differentiation. Furthermore, three subpopulations of transduced BMSCs homogeneously producing specific low, medium, and high VEGF doses could be prospectively isolated by flow cytometry based on the intensity of their CD8 expression already at the first passage. In conclusion, this optimized platform allowed the generation of populations of genetically modified MSCs, expressing specific levels of a therapeutic transgene, already at the first passage, thereby minimizing in vitro expansion and loss of regenerative potential. PMID:22070632

Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn’s disease and contributes to accelerated epithelial wound healing in vitro

PubMed Central

Hafner, Christian; Meyer, Stefanie; Langmann, Thomas; Schmitz, Gerd; Bataille, Frauke; Hagen, Ilja; Becker, Bernd; Roesch, Alexander; Rogler, Gerhard; Landthaler, Michael; Vogt, Thomas

2005-01-01

AIM: Eph receptor tyrosine kinases and their membrane bound receptor-like ligands, the ephrins, represent a bi-directional cell-cell contact signaling system that directs epithelial movements in development. The meaning of this system in the adult human gut is unknown. We investigated the Eph/ephrin mRNA expression in the intestinal epithelium of healthy controls and patients with inflammatory bowel disease (IBD). METHODS: mRNA expression profiles of all Eph/ephrin family members in normal small intestine and colon were established by real-time RT-PCR. In addition, differential expression in IBD was investigated by cDNA array technology, and validated by both real-time RT-PCR and immunohistochemistry. Potential effects of enhanced EphB/ephrin-B signaling were analyzed in an in vitro IEC-6 cell scratch wound model. RESULTS: Human adult intestinal mucosa exhibits a complex pattern of Eph receptors and ephrins. Beside the known prominent co-expression of EphA2 and ephrinA1, we found abundantly co-expressed EphB2 and ephrin-B1/2. Interestingly, cDNA array data, validated by real-time PCR and immunohistochemistry, showed upregulation of ephrin-B2 in both perilesional and lesional intestinal epithelial cells of IBD patients, suggesting a role in epithelial homeostasis. Stimulation of ephrin-B signaling in ephrin-B1/2 expressing rat IEC-6-cells with recombinant EphB1-Fc resulted in a significant dose-dependent acceleration of wound closure. Furthermore, fluorescence microscopy showed that EphB1-Fc induced coordinated migration of wound edge cells is associated with enhanced formation of lamellipodial protrusions into the wound, increased actin stress fiber assembly and production of laminin at the wound edge. CONCLUSION: EphB/ephrin-B signaling might represent a novel protective mechanism that promotes intestinal epithelial wound healing, with potential impact on epithelial restitution in IBD. PMID:15996027

PSMB5 plays a dual role in cancer development and immunosuppression

PubMed Central

Wang, Chih-Yang; Li, Chung-Yen; Hsu, Hui-Ping; Cho, Chien-Yu; Yen, Meng-Chi; Weng, Tzu-Yang; Chen, Wei-Ching; Hung, Yu-Hsuan; Lee, Kuo-Ting; Hung, Jui-Hsiang; Chen, Yi-Ling; Lai, Ming-Derg

2017-01-01

Tumor progression and metastasis are dependent on the intrinsic properties of tumor cells and the influence of microenvironment including the immune system. It would be important to identify target drug that can inhibit cancer cell and activate immune cells. Proteasome β subunits (PSMB) family, one component of the ubiquitin-proteasome system, has been demonstrated to play an important role in tumor cells and immune cells. Therefore, we used a bioinformatics approach to examine the potential role of PSMB family. Analysis of breast TCGA and METABRIC database revealed that high expression of PSMB5 was observed in breast cancer tissue and that high expression of PSMB5 predicted worse survival. In addition, high expression of PSMB5 was observed in M2 macrophages. Based on our bioinformatics analysis, we hypothesized that PSMB5 contained immunosuppressive and oncogenic characteristics. To study the effects of PSMB5 on the cancer cell and macrophage in vitro, we silenced PSMB5 expression with shRNA in THP-1 monocytes and MDA-MB-231 cells respectively. Knockdown of PSMB5 promoted human THP-1 monocyte differentiation into M1 macrophage. On the other hand, knockdown PSMB5 gene expression inhibited MDA-MB-231 cell growth and migration by colony formation assay and boyden chamber. Collectively, our data demonstrated that delivery of PSMB5 shRNA suppressed cell growth and activated defensive M1 macrophages in vitro. Furthermore, lentiviral delivery of PSMB5 shRNA significantly decreased tumor growth in a subcutaneous mouse model. In conclusion, our bioinformatics study and functional experiments revealed that PSMB5 served as novel cancer therapeutic targets. These results also demonstrated a novel translational approach to improve cancer immunotherapy. PMID:29218236

K+ channels expression in hypertension after arterial injury, and effect of selective Kv1.3 blockade with PAP-1 on intimal hyperplasia formation.

PubMed

Cidad, P; Novensà, L; Garabito, M; Batlle, M; Dantas, A P; Heras, M; López-López, J R; Pérez-García, M T; Roqué, M

2014-12-01

K(+) channels are central to vascular pathophysiology. Previous results demonstrated that phenotypic modulation associates with a change in Kv1.3 to Kv1.5 expression, and that Kv1.3 blockade inhibits proliferation of VSMCs cultures. To explore whether the Kv1.3 to Kv1.5 switch could be a marker of the increased risk of intimal hyperplasia in essential hypertension and whether systemic treatment with Kv1.3 blockers can prevent intimal hyperplasia after endoluminal lesion . Morphometric and immunohistochemical analysis were performed in arterial segments following arterial injury and constant infusion of the Kv1.3 blocker PAP-1 during 28 days. Differential expression of K(+) channel genes was studied in VSMC from hypertensive (BPH) and normotensive (BPN) mice, both in control and after endoluminal lesion. Finally, the migration and proliferation rate of BPN and BPH VSMCs was explored in vitro. Changes in mRNA expression led to an increased Kv1.3/Kv1.5 ratio in BPH VSMC. Consistent with this, arterial injury in BPH mice induced a higher degree of luminal stenosis, (84 ± 4% vs. 70 ± 5% in BPN, p < 0.01), although no differences in migration and proliferation rate were observed in cultured VSMCs. The in vivo proliferative lesions were significantly decreased upon PAP-1 systemic infusion (18 ± 6% vs. 58 ± 20% with vehicle, p < 0.05). Hypertension leads to a higher degree of luminal stenosis in our arterial injury model, that correlates with a decreased expression of Kv1.5 channels. Kv1.3 blockers decreased in vitro VSMCs proliferation, migration, and in vivo intimal hyperplasia formation, pointing to Kv1.3 channels as promising therapeutical targets against restenosis.

Use of a dynamic in vitro attachment and invasion system (DIVAS) to determine influence of growth rate on invasion of respiratory epithelial cells by group B Streptococcus.

PubMed

Malin, G; Paoletti, L C

2001-11-06

Expression of capsular polysaccharide (CPS) and some surface proteins by group B Streptococcus (GBS) is regulated by growth rate. We hypothesized that precise control of GBS growth, and thus surface-expressed components, could modulate the ability of GBS to invade eukaryotic cells. To test this hypothesis, a dynamic in vitro attachment and invasion system (DIVAS) was developed that combines the advantages of bacterial growth in continuous culture with tissue culture. Tissue culture flasks were modified with inlet and outlet ports to permit perfusion of GBS. Encapsulated type III GBS strains M781 and COH1 and strains COH1-11 and COH1-13 (transposon mutants of COH1 that express an asialo CPS or are acapsular, respectively) were grown in continuous culture in a chemically defined medium at fast mass doubling time (t(d) = 1.8 h) and slow (t(d) = 11 h) growth rates, conditions previously shown to induce and repress, respectively, type III CPS expression. Encapsulated GBS strains invaded A549 respiratory epithelial cells 20- to 700-fold better at the fast than at the slow growth rate, suggesting a role for CPS. However, unencapsulated GBS were also invasive but only when cultured at the fast growth rate, which indicates that GBS invasion is independent of CPS expression and can be regulated by growth rate. Growth rate-dependent invasion occurred when GBS was grown in continuous culture under glucose-defined, thiamine-defined, and undefined nutrient limitations. These results suggest a growth rate-dependent regulation of GBS pathogenesis and demonstrate the usefulness of DIVAS as a tool in studies of host-microbe interactions.

Glial cell-expressed mechanosensitive channel TRPV4 mediates infrasound-induced neuronal impairment.

PubMed

Shi, Ming; Du, Fang; Liu, Yang; Li, Li; Cai, Jing; Zhang, Guo-Feng; Xu, Xiao-Fei; Lin, Tian; Cheng, Hao-Ran; Liu, Xue-Dong; Xiong, Li-Ze; Zhao, Gang

2013-11-01

Vibroacoustic disease, a progressive and systemic disease, mainly involving the central nervous system, is caused by excessive exposure to low-frequency but high-intensity noise generated by various heavy transportations and machineries. Infrasound is a type of low-frequency noise. Our previous studies demonstrated that infrasound at a certain intensity caused neuronal injury in rats but the underlying mechanism(s) is still largely unknown. Here, we showed that glial cell-expressed TRPV4, a Ca(2+)-permeable mechanosensitive channel, mediated infrasound-induced neuronal injury. Among different frequencies and intensities, infrasound at 16 Hz and 130 dB impaired rat learning and memory abilities most severely after 7-14 days exposure, a time during which a prominent loss of hippocampal CA1 neurons was evident. Infrasound also induced significant astrocytic and microglial activation in hippocampal regions following 1- to 7-day exposure, prior to neuronal apoptosis. Moreover, pharmacological inhibition of glial activation in vivo protected against neuronal apoptosis. In vitro, activated glial cell-released proinflammatory cytokines IL-1β and TNF-α were found to be key factors for this neuronal apoptosis. Importantly, infrasound induced an increase in the expression level of TRPV4 both in vivo and in vitro. Knockdown of TRPV4 expression by siRNA or pharmacological inhibition of TRPV4 in cultured glial cells decreased the levels of IL-1β and TNF-α, attenuated neuronal apoptosis, and reduced TRPV4-mediated Ca(2+) influx and NF-κB nuclear translocation. Finally, using various antagonists we revealed that calmodulin and protein kinase C signaling pathways were involved in TRPV4-triggered NF-κB activation. Thus, our results provide the first evidence that glial cell-expressed TRPV4 is a potential key factor responsible for infrasound-induced neuronal impairment.

Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

PubMed

Turunen, Tytteli Anni Kaarina; Laakkonen, Johanna Päivikki; Alasaarela, Laura; Airenne, Kari Juhani; Ylä-Herttuala, Seppo

2014-01-01

A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

Effect of estrogen on molecular and functional characteristics of the rodent vaginal muscularis

PubMed Central

Basha, Maureen E.; Chang, Shaohua; Burrows, Lara J.; Lassmann, Jenny; Wein, Alan J.; Moreland, Robert S.; Chacko, Samuel K.

2013-01-01

Introduction Vaginal atrophy is a consequence of menopause however little is known concerning the effect of a decrease in systemic estrogen on vaginal smooth muscle structure and function. As the incidence of pelvic floor disorders increases with age, it is important to determine if estrogen regulates the molecular composition and contractility of the vaginal muscularis. Aim The goal of this study was to determine the effect of estrogen on molecular and functional characteristics of the vaginal muscularis utilizing a rodent model of surgical menopause. Methods 3–4 month old Sprague Dawley rats underwent sham laparotomy (Sham, n=18) or ovariectomy (Ovx, n=39). Two weeks following surgery, animals received a subcutaneous osmotic pump containing vehicle (Sham, Ovx) or 17- β estradiol (Ovx). Animals were euthanized one week later and the proximal vagina was collected for analysis of contractile protein expression and in vitro studies of contractility. Measurements were analyzed using a one-way ANOVA followed by Tukey's post hoc analysis (α= 0.05). Main Outcome Measures Protein and mRNA transcript expression levels of contractile proteins, in vitro measurements of vaginal contractility Results Ovariectomy decreased the expression of carboxyl-terminal myosin heavy chain isoform SM1 and h-caldesmon and reduced the amplitude of contraction of the vaginal muscularis in response to KCl. Estradiol replacement reversed these changes. No differences were detected in the % vaginal muscularis, mRNA transcript expression of amino terminal MHC isoforms, l-caldesmon expression and maximal velocity of shortening. Conclusion Systemic estrogen replacement restores functional and molecular characteristics of the vaginal muscularis of ovariectomized rats. Our results indicate that menopause is associated with changes in the vaginal muscularis, which may contribute to the increased incidence of pelvic floor disorders with age. PMID:23438289

6-Gingerol Suppresses Adipocyte-Derived Mediators of Inflammation In Vitro and in High-Fat Diet-Induced Obese Zebra Fish.

PubMed

Choi, Jia; Kim, Kui-Jin; Kim, Byung-Hak; Koh, Eun-Jeong; Seo, Min-Jung; Lee, Boo-Yong

2017-02-01

The present study was performed to investigate the molecular mechanism of 6-gingerol on adipocyte-mediated systemic inflammation in vitro and in high-fat diet-induced obese zebra fish. 6-Gingerol decreased adipogenesis due to the suppression of adipocyte differentiation markers, including peroxisome proliferator-activated receptor gamma, CCAATT enhancer binding protein α , and adipocyte protein 2, and triglyceride synthesis enzymes, including sterol regulatory element-binding protein-1, fatty acid synthase, lysophosphatidic acid acyltransferase, and acyl-coA : diacylglycerol acyltransferase 1, in 3T3-L1. A coculture insert system using 3T3-L1 with RAW 264.7 (coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages) revealed that 6-gingerol increased anti-inflammatory cytokine interleukin-10. The expression of TNF α , monocyte chemotactic protein-1, interleukin-1 β , and interleukin-6 were decreased in the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages treated with 6-gingerol. Moreover, the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages treated with 6-gingerol inhibited the protein expression of TNF α and monocyte chemotactic protein-1 in RAW 264.7. 6-Gingerol decreased c-JUN N-terminal kinase and I kappa B kinase beta and its downstream target AP-1 expression in the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages. Furthermore, 6-gingerol decreased the expression of inducible nitric oxide synthase stimulated by the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages in RAW 264.7 and attenuated nitric oxide production in diet-induced obese zebra fish. Our results suggest that 6-gingerol suppresses inflammation through the regulation of the c-JUN N-terminal kinase-I kappa B kinase beta and its downstream targets. Georg Thieme Verlag KG Stuttgart · New York.

Quantitative expression patterns of GDF9 and BMP15 genes in sheep ovarian follicles grown in vivo or cultured in vitro.

PubMed

Kona, S S R; Praveen Chakravarthi, V; Siva Kumar, A V N; Srividya, D; Padmaja, K; Rao, V H

2016-01-15

Quantitative patterns of expression of the growth differentiation factor 9 (GDF9) and bone morphogenic protein 15 (BMP15) genes in different development stages of in vivo and in vitro grown ovarian follicles in sheep were studied for the first time. Both GDF9 and BMP15 were expressed in the cumulus cells and oocytes at all the development stages of in vivo and in vitro grown ovarian follicles. Growth differentiation factor 9 and bone morphogenic protein 15 exhibited stage-specific undulations in the expression in the cumulus cells and oocytes isolated from in vivo grown ovarian follicles. These undulations could be related to discrete development events during the ovarian follicle development. The expression of GDF9 and BMP15 was highest (3.38 ± 0.02 and 2.69 ± 0.06, respectively; P ≤ 0.05) in the primordial follicles compared with preantral, early antral, antral, and large antral stages. Similarly, GDF9 and BMP15 expression in the cumulus cells (0 ± 0.16 and 0 ± 0.07) and oocytes (1.47 ± 0.07 and 1.32 ± 0.03) was lowest (P ≤ 0.05) in the in vivo grown antral follicles. In the cultured follicles, the stage-specific undulations observed in the expression of GDF9 and BMP15 in the in vivo grown follicles were either different or abolished. For example, in the oocytes from in vitro grown follicles, the expression of BMP15 did not change as the development progressed all the way from preantral to large antral follicle stage although in the oocytes from in vivo grown follicles BMP15 expression exhibited stage-specific variations. It is concluded that GDF9 and BMP15 follow a stage-specific pattern of expression during the in vivo development of ovarian follicles in sheep, and in vitro culture altered the stage-specific changes in the expression of these two genes. Copyright © 2016 Elsevier Inc. All rights reserved.

Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro

PubMed Central

Garrett, I.R.; Chen, D.; Gutierrez, G.; Zhao, M.; Escobedo, A.; Rossini, G.; Harris, S.E.; Gallwitz, W.; Kim, K.B.; Hu, S.; Crews, C.M.; Mundy, G.R.

2003-01-01

We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic β subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor–1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss. PMID:12782679

Altered Gag Polyprotein Cleavage Specificity of Feline Immunodeficiency Virus/Human Immunodeficiency Virus Mutant Proteases as Demonstrated in a Cell-Based Expression System

PubMed Central

Lin, Ying-Chuan; Brik, Ashraf; de Parseval, Aymeric; Tam, Karen; Torbett, Bruce E.; Wong, Chi-Huey; Elder, John H.

2006-01-01

We have used feline immunodeficiency virus (FIV) protease (PR) as a mutational system to study the molecular basis of substrate-inhibitor specificity for lentivirus PRs, with a focus on human immunodeficiency virus type 1 (HIV-1) PR. Our previous mutagenesis studies demonstrated that discrete substitutions in the active site of FIV PR with structurally equivalent residues of HIV-1 PR dramatically altered the specificity of the mutant PRs in in vitro analyses. Here, we have expanded these studies to analyze the specificity changes in each mutant FIV PR expressed in the context of the natural Gag-Pol polyprotein ex vivo. Expression mutants were prepared in which 4 to 12 HIV-1-equivalent substitutions were made in FIV PR, and cleavage of each Gag-Pol polyprotein was then assessed in pseudovirions from transduced cells. The findings demonstrated that, as with in vitro analyses, inhibitor specificities of the mutants showed increased HIV-1 PR character when analyzed against the natural substrate. In addition, all of the mutant PRs still processed the FIV polyprotein but the apparent order of processing was altered relative to that observed with wild-type FIV PR. Given the importance of the order in which Gag-Pol is processed, these findings likely explain the failure to produce infectious FIVs bearing these mutations. PMID:16873240

Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements.

PubMed

Kelwick, Richard; Webb, Alexander J; MacDonald, James T; Freemont, Paul S

2016-11-01

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type P grac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Inhibition of rat and human steroidogenesis by triazole antifungals.

PubMed

Goetz, Amber K; Rockett, John C; Ren, Hongzu; Thillainadarajah, Inthirany; Dix, David J

2009-12-01

Environmental chemicals that alter steroid production could interfere with male reproductive development and function. Three agricultural antifungal triazoles that are known to modulate expression of cytochrome P450 (CYP) genes and enzymatic activities were tested for effects on steroidogenesis using rat in vivo (triadimefon), rat in vitro (myclobutanil and triadimefon), and human in vitro (myclobutanil, propiconazole, and triadimefon) model systems. Hormone production was measured in testis organ cultures from untreated adult and neonatal rats, following in vitro exposure to 1, 10, or 100 muM of myclobutanil or triadimefon. Myclobutanil and triadimefon reduced media levels of testosterone by 40-68% in the adult and neonatal testis culture, and altered steroid production in a manner that indicated CYP17-hydroxylase/17,20 lyase (CYP17A1) inhibition at the highest concentration tested. Rat to human comparison was explored using the H295R (human adrenal adenocarcinoma) cell line. Following 48 h exposure to myclobutanil, propiconazole, or triadimefon at 1, 3, 10, 30, or 100 muM, there was an overall decrease in estradiol, progesterone, and testosterone by all three triazoles. These data indicate that myclobutanil, propiconazole, and triadimefon are weak inhibitors of testosterone production in vitro. However, in vivo exposure of rats to triazoles resulted in increased serum and intra-testicular testosterone levels. This discordance could be due to higher concentrations of triazoles tested in vitro, and differences within an in vitro model system lacking hepatic metabolism and neuroendocrine control.

Induction of human embryonic and induced pluripotent stem cells into urothelium.

PubMed

Osborn, Stephanie L; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H; Nolta, Jan; Kurzrock, Eric A

2014-05-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.

Induction of Human Embryonic and Induced Pluripotent Stem Cells Into Urothelium

PubMed Central

Osborn, Stephanie L.; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H.; Nolta, Jan

2014-01-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium (“Uromedium”), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation. PMID:24657961

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction.

PubMed

Stoelzle, Sonja; Haythornthwaite, Alison; Kettenhofen, Ralf; Kolossov, Eugen; Bohlen, Heribert; George, Michael; Brüggemann, Andrea; Fertig, Niels

2011-09-01

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

Transcriptional organization of the DNA region controlling expression of the K99 gene cluster.

PubMed

Roosendaal, B; Damoiseaux, J; Jordi, W; de Graaf, F K

1989-01-01

The transcriptional organization of the K99 gene cluster was investigated in two ways. First, the DNA region, containing the transcriptional signals was analyzed using a transcription vector system with Escherichia coli galactokinase (GalK) as assayable marker and second, an in vitro transcription system was employed. A detailed analysis of the transcription signals revealed that a strong promoter PA and a moderate promoter PB are located upstream of fanA and fanB, respectively. No promoter activity was detected in the intercistronic region between fanB and fanC. Factor-dependent terminators of transcription were detected and are probably located in the intercistronic region between fanA and fanB (T1), and between fanB and fanC (T2). A third terminator (T3) was observed between fanC and fanD and has an efficiency of 90%. Analysis of the regulatory region in an in vitro transcription system confirmed the location of the respective transcription signals. A model for the transcriptional organization of the K99 cluster is presented. Indications were obtained that the trans-acting regulatory polypeptides FanA and FanB both function as anti-terminators. A model for the regulation of expression of the K99 gene cluster is postulated.

An Alternative Method for Long-Term Culture of Chicken Embryonic Stem Cell In Vitro.

PubMed

Zhang, Li; Wu, Yenan; Li, Xiang; Wei, Shao; Xing, Yiming; Lian, Zhengxing; Han, Hongbing

2018-01-01

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro . Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro .

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells.

PubMed

Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y; Tanaka, Minoru; Miyajima, Atsushi

2015-10-13

To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells

PubMed Central

Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y.; Tanaka, Minoru; Miyajima, Atsushi

2015-01-01

Summary To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM+ cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM+ cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM+ cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes. PMID:26365514

Manipulation of immune system via immortal bone marrow stem cells.

PubMed

Ruedl, Christiane; Khameneh, Hanif Javanmard; Karjalainen, Klaus

2008-09-01

Extensive amplification of hematopoietic stem cells (HSCs) and their multipotent primitive progenitors (MPPs) in culture would greatly benefit not only clinical transplantation but also provide a potential tool to manipulate all cellular lineages derived from these cells for gene therapy and experimental purposes. Here, we demonstrate that mouse bone marrow cultures containing cells engineered to over-express NUP98-HOXB4 fusion protein support self-renewal of physiologically normal HSC and MPP for several weeks leading practically to their unlimited expansion. This allows time consuming and cumulative in vitro experimental manipulations without sacrificing their ability to differentiate in vivo or in vitro to any hematopoietic lineage.

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

PubMed

Mayrhofer, Corina; Krieger, Sigurd; Allmaier, Günter; Kerjaschki, Dontscho

2006-01-01

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.

Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L).

PubMed

Srinivasan, C; Liu, Zongrang; Scorza, Ralph

2011-04-01

Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.

Expression of RXFP1 Is Decreased in Idiopathic Pulmonary Fibrosis. Implications for Relaxin-based Therapies

PubMed Central

Tan, Jiangning; Tedrow, John R.; Dutta, Justin A.; Juan-Guardela, Brenda; Nouraie, Mehdi; Chu, Yanxia; Trejo Bittar, Humberto; Ramani, Kritika; Biswas, Partha S.; Veraldi, Kristen L.; Kaminski, Naftali; Zhang, Yingze

2016-01-01

Rationale: Relaxin is a hormone that has been considered as a potential therapy for patients with fibrotic diseases. Objectives: To gauge the potential efficacy of relaxin-based therapies in idiopathic pulmonary fibrosis (IPF), we studied gene expression for relaxin/insulin-like family peptide receptor 1 (RXFP1) in IPF lungs and controls. Methods: We analyzed gene expression data obtained from the Lung Tissue Research Consortium and correlated RXFP1 gene expression data with cross-sectional clinical and demographic data. We also employed ex vivo donor and IPF lung fibroblasts to test RXFP1 expression in vitro. We tested CGEN25009, a relaxin-like peptide, in lung fibroblasts and in bleomycin injury. Measurements and Main Results: We found that RXFP1 is significantly decreased in IPF. In patients with IPF, the magnitude of RXFP1 gene expression correlated directly with diffusing capacity of the lung for carbon monoxide (P < 0.0001). Significantly less RXFP1 was detected in vitro in IPF fibroblasts than in donor controls. Transforming growth factor-β decreased RXFP1 in both donor and IPF lung fibroblasts. CGEN25009 was effective at decreasing bleomycin-induced, acid-soluble collagen deposition in vivo. The relaxin-like actions of CGEN25009 were abrogated by RXFP1 silencing in vitro, and, in comparison with donor lung fibroblasts, IPF lung fibroblasts exhibited decreased sensitivity to the relaxin-like effects of CGEN25009. Conclusions: IPF is characterized by the loss of RXFP1 expression. RXFP1 expression is directly associated with pulmonary function in patients with IPF. The relaxin-like effects of CGEN25009 in vitro are dependent on expression of RXFP1. Our data suggest that patients with IPF with the highest RXFP1 expression would be predicted to be most sensitive to relaxin-based therapies. PMID:27310652

Expression of RXFP1 Is Decreased in Idiopathic Pulmonary Fibrosis. Implications for Relaxin-based Therapies.

PubMed

Tan, Jiangning; Tedrow, John R; Dutta, Justin A; Juan-Guardela, Brenda; Nouraie, Mehdi; Chu, Yanxia; Trejo Bittar, Humberto; Ramani, Kritika; Biswas, Partha S; Veraldi, Kristen L; Kaminski, Naftali; Zhang, Yingze; Kass, Daniel J

2016-12-01

Relaxin is a hormone that has been considered as a potential therapy for patients with fibrotic diseases. To gauge the potential efficacy of relaxin-based therapies in idiopathic pulmonary fibrosis (IPF), we studied gene expression for relaxin/insulin-like family peptide receptor 1 (RXFP1) in IPF lungs and controls. We analyzed gene expression data obtained from the Lung Tissue Research Consortium and correlated RXFP1 gene expression data with cross-sectional clinical and demographic data. We also employed ex vivo donor and IPF lung fibroblasts to test RXFP1 expression in vitro. We tested CGEN25009, a relaxin-like peptide, in lung fibroblasts and in bleomycin injury. We found that RXFP1 is significantly decreased in IPF. In patients with IPF, the magnitude of RXFP1 gene expression correlated directly with diffusing capacity of the lung for carbon monoxide (P < 0.0001). Significantly less RXFP1 was detected in vitro in IPF fibroblasts than in donor controls. Transforming growth factor-β decreased RXFP1 in both donor and IPF lung fibroblasts. CGEN25009 was effective at decreasing bleomycin-induced, acid-soluble collagen deposition in vivo. The relaxin-like actions of CGEN25009 were abrogated by RXFP1 silencing in vitro, and, in comparison with donor lung fibroblasts, IPF lung fibroblasts exhibited decreased sensitivity to the relaxin-like effects of CGEN25009. IPF is characterized by the loss of RXFP1 expression. RXFP1 expression is directly associated with pulmonary function in patients with IPF. The relaxin-like effects of CGEN25009 in vitro are dependent on expression of RXFP1. Our data suggest that patients with IPF with the highest RXFP1 expression would be predicted to be most sensitive to relaxin-based therapies.

Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a.

PubMed

Xin, Xiaoru; Wu, Mengying; Meng, Qiuyu; Wang, Chen; Lu, Yanan; Yang, Yuxin; Li, Xiaonan; Zheng, Qidi; Pu, Hu; Gui, Xin; Li, Tianming; Li, Jiao; Jia, Song; Lu, Dongdong

2018-06-12

Long noncoding RNA HULC is highly up-regulation in human hepatocellular carcinoma (HCC). However, the functions of HULC in hepatocarcinogenesis remains unclear. RT-PCR, Western blotting, Chromatin immunoprecipitation (CHIP) assay, RNA Immunoprecipitation (RIP) and tumorignesis test in vitro and in vivo were performed. HULC is negatively associated with expression of PTEN or miR15a in patients of human liver cancer. Moreover, HULC accelerates malignant progression of liver cancer cells in vitro and in vivo. Mechanistically, HULC increasesthe expression of P62 via decreasing mature miR15a. On the other hand, excessive HULC increases the expression of LC3 on the level of transcription and then activates LC3 through Sirt1 (a deacetylase). Notably, HULC enhanced the interplay between LC3 and ATG3. Furthermore, HULC also increases the expression of becline-1(autophagy related gene). Therefore, HULC increases the cellular autophagy by increasing LC3II dependent on Sirt1.Noteworthy, excessive HULC reduces the expression of PTEN, β-catenin and enhances the expression of SAPK/JUNK, PKM2, CDK2, NOTCH1, C-Jun in liver cancer cells. Of significance, our observations also revealed that HULC inhibited PTEN through ubiquitin-proteasome system mediated by autophagy-P62.Ultimately,HULC activates AKT-PI3K-mTOR pathway through inhibiting PTEN in human liver cancer cells. This study elucidates a novel mechanism that lncRNA HULC produces a vital function during hepatocarcinogenesis.

A plasmid-based reverse genetics system for influenza A virus.

PubMed Central

Pleschka, S; Jaskunas, R; Engelhardt, O G; Zürcher, T; Palese, P; García-Sastre, A

1996-01-01

A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system. PMID:8648766

Novel approach to study the cardiovascular effects and mechanism of action of urban particulate matter using lung epithelial-endothelial tetra-culture system.

PubMed

Kim, Ha Ryong; Cho, Han Soo; Shin, Da Young; Chung, Kyu Hyuck

2017-02-01

In vitro models have become increasingly sophisticated, and their usefulness in supporting toxicity testing is well established. The present study was designed to establish a novel in vitro model that mimics the cellular network surrounding airways and pulmonary blood vessels, to study the cardiovascular toxic effects of particulate matter (PM). Transwell culture method was used to develop a novel tetra-culture system consisting of tri-cultures (one lung epithelial and two immune cell lines) in the apical chamber and endothelial cells in the basolateral chamber. Tri-cultures were exposed to standard reference material (SRM) 1648a, an urban PM. SRM 1648a did not show cytotoxic effects; however, it increased IL-6 level in apical and basolateral chambers. The cells in the basolateral chamber showed increased monocyte adhesion. Furthermore, exposure of tri-cultured cells to SRM 1648a in the apical chamber induced ICAM-1 expression in endothelial cells in the basolateral chamber by activating the IL-6/STAT3 pathway. In conclusion, a tetra-culture system was established to facilitate the identification of cellular adhesion molecule expression induced by the interaction between pulmonary epithelial and endothelial cells. The tetra-culture system will contribute to elucidation of the relationships between inhalable PM and cardiovascular diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression Analysis of a Highly Adherent and Cytotoxic Small Colony Variant of Pseudomonas aeruginosa Isolated from a Lung of a Patient with Cystic Fibrosis†

PubMed Central

von Götz, Franz; Häussler, Susanne; Jordan, Doris; Saravanamuthu, Senthil Selvan; Wehmhöner, Dirk; Strüßmann, André; Lauber, Joerg; Attree, Ina; Buer, Jan; Tümmler, Burkhard; Steinmetz, Ivo

2004-01-01

The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms. In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P. aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population. Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins. This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type. The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS. Thus, the prevailing assumption that P. aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants. PMID:15175297

Mice with GFAP-targeted loss of neurofibromin demonstrate increased axonal MET expression with aging.

PubMed

Su, Weiping; Xing, Rubing; Guha, Abhijit; Gutmann, David H; Sherman, Larry S

2007-05-01

Neurofibromatosis 1 (NF1) is a common genetic disease that predisposes patients to peripheral nerve tumors and central nervous system (CNS) abnormalities including low-grade astrocytomas and cognitive disabilities. Using mice with glial fibrillary acidic protein (GFAP)-targeted Nf1 loss (Nf1(GFAP)CKO mice), we found that Nf1(-/-) astrocytes proliferate faster and are more invasive than wild-type astrocytes. In light of our previous finding that aberrant expression of the MET receptor tyrosine kinase contributes to the invasiveness of human NF1-associated malignant peripheral nerve sheath tumors, we sought to determine whether MET expression is aberrant in the brains of Nf1 mutant mice. We found that Nf1(-/-) astrocytes express slightly more MET than wild-type cells in vitro, but do not express elevated MET in situ. However, fiber tracts containing myelinated axons in the hippocampus, midbrain, cerebral cortex, and cerebellum express higher than normal levels of MET in older (> or =6 months) Nf1(GFAP)CKO mice. Both Nf1(GFAP)CKO and wild-type astrocytes induced MET expression in neurites of wild-type hippocampal neurons in vitro, suggesting that astrocyte-derived signals may induce MET in Nf1 mutant mice. Because the Nf1 gene product functions as a RAS GTPase, we examined MET expression in the brains of mice with GFAP-targeted constitutively active forms of RAS. MET was elevated in axonal fiber tracts in mice with active K-RAS but not H-RAS. Collectively, these data suggest that loss of Nf1 in either astrocytes or GFAP(+) neural progenitor cells results in increased axonal MET expression, which may contribute to the CNS abnormalities in children and adults with NF1. (c) 2007 Wiley-Liss, Inc.

Effect of triiodothyronine on developmental competence of bovine oocytes.

PubMed

Costa, N N; Cordeiro, M S; Silva, T V G; Sastre, D; Santana, P P B; Sá, A L A; Sampaio, R V; Santos, S S D; Adona, P R; Miranda, M S; Ohashi, O M

2013-09-01

Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

A simple and robust vector-based shRNA expression system used for RNA interference.

PubMed

Wang, Xue-jun; Li, Ying; Huang, Hai; Zhang, Xiu-juan; Xie, Pei-wen; Hu, Wei; Li, Dan-dan; Wang, Sheng-qi

2013-01-01

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks

PubMed Central

Nakahata, Yasukazu; Akashi, Makoto; Trcka, Daniel; Yasuda, Akio; Takumi, Toru

2006-01-01

Background Circadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. The circadian organization of these processes in mammals is governed by the master oscillator within the suprachiasmatic nuclei (SCN) of the hypothalamus. Recent findings revealed that circadian oscillators exist in most organs, tissues, and even in immortalized cells, and that the oscillators in peripheral tissues are likely to be coordinated by SCN, the master oscillator. Some candidates for endogenous entrainment factors have sporadically been reported, however, their details remain mainly obscure. Results We developed the in vitro real-time oscillation monitoring system (IV-ROMS) by measuring the activity of luciferase coupled to the oscillatory gene promoter using photomultiplier tubes and applied this system to screen and identify factors able to influence circadian rhythmicity. Using this IV-ROMS as the primary screening of entrainment factors for circadian clocks, we identified 12 candidates as the potential entrainment factor in a total of 299 peptides and bioactive lipids. Among them, four candidates (endothelin-1, all-trans retinoic acid, 9-cis retinoic acid, and 13-cis retinoic acid) have already been reported as the entrainment factors in vivo and in vitro. We demonstrated that one of the novel candidates, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a natural ligand of the peroxisome proliferator-activated receptor-γ (PPAR-γ), triggers the rhythmic expression of endogenous clock genes in NIH3T3 cells. Furthermore, we showed that 15d-PGJ2 transiently induces Cry1, Cry2, and Rorα mRNA expressions and that 15d-PGJ2-induced entrainment signaling pathway is PPAR-γ – and MAPKs (ERK, JNK, p38MAPK)-independent. Conclusion Here, we identified 15d-PGJ2 as an entrainment factor in vitro. Using our developed IV-ROMS to screen 299 compounds, we found eight novel and four known molecules to be potential entrainment factors for circadian clocks, indicating that this assay system is a powerful and useful tool in initial screenings. PMID:16483373

Three-dimensional hydrogel scaffolds facilitate in vitro self-renewal of human skin-derived precursors.

PubMed

Wang, Xinyue; Liu, Shu; Zhao, Qian; Li, Na; Zhang, Huishan; Zhang, Xudong; Lei, Xiaohua; Zhao, Huashan; Deng, Zhili; Qiao, Jingqiao; Cao, Yujing; Ning, Lina; Liu, Shuang; Duan, Enkui

2014-07-01

Skin-derived precursors (SKPs) are multipotent cells with dermal stem cell properties. These easily available cells possess the capacity to reconstitute the skin in vivo, as well as a broader differentiation potential in vitro, which endows them with great prospects in regenerative medicine. However, the present authors' group and others previously found that adult human SKPs (hSKPs) expanded deficiently in vitro, which largely counteracted their research and practical values. Taking the physiological micro-environment of hSKPs into consideration, the authors sought to establish a hydrogel scaffold-based three-dimensional (3-D) culture system for hSKPs in the present study. After comparing their morphology, growth characteristics, signature gene expression and differentiation potential in different hydrogels, the present authors found that a chemically defined hyaluronic acid and denatured collagen-based hydrogel system that mimicked the natural niche of hSKPs in the dermis could alleviate hSKP senescence, support hSKP proliferation as spheres, while largely retaining their properties and potential. This study suggested that recapitulating the in vivo stem cell niche by providing them with 3-D extracellular matrix environments could help them achieve better self-renewal in vitro. In addition, the animal-origin-free and biocompatible 3-D hydrogel system will certainly benefit fundamental research and clinical applications of hSKPs in the near future. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Anti-Müllerian hormone is produced heterogeneously in primate preantral follicles and is a potential biomarker for follicle growth and oocyte maturation in vitro.

PubMed

Xu, Jing; Xu, Fuhua; Letaw, John H; Park, Byung S; Searles, Robert P; Ferguson, Betsy M

2016-12-01

The main goals of this study were to investigate the expression of anti-Müllerian hormone (AMH) and its receptor (AMHR2) during follicular development in primates, and to evaluate the potential of AMH as a biomarker for follicle growth and oocyte maturation in vitro. The mRNA and protein expression of AMH and AMHR2 were determined using isolated follicles and ovarian sections from rhesus macaques (n = 4) by real-time PCR and immunohistochemistry, respectively. Isolated secondary follicles were cultured individually. Follicle growth and media AMH concentrations were assessed by ELISA. The mRNA expression profiles, obtained from RNA sequencing, of in vitro- and in vivo-developed antral follicles were compared. Secondary follicles from additional animals (n = 35) were cultured. Follicle growth, oocyte maturation, and media AMH concentrations were evaluated for forecasting follicular development in vitro by AMH levels. AMH immunostaining was heterogeneous in the population of preantral follicles that were also stained for AMHR2. The mRNA expression profiles were comparable between in vivo- and in vitro-developed follicles. AMH levels produced by growing follicles were higher than those of nongrowing follicles in culture. With a cutoff value of 1.40 ng/ml, 85 % of nongrowing follicles could be identified while eliminating only 5 % of growing follicles. Growing follicles that generated metaphase II-stage oocytes secreted greater amounts of AMH than did those yielding immature germinal vesicle-stage oocytes. AMH, co-expressed with AMHR2, was produced heterogeneously by preantral follicles in macaques with levels correlated positively with follicle growth and oocyte maturation. AMH may serve as a biomarker for primate follicular development in vitro.

Minimum Transendothelial Electrical Resistance Thresholds for the Study of Small and Large Molecule Drug Transport in a Human in Vitro Blood-Brain Barrier Model.

PubMed

Mantle, Jennifer L; Min, Lie; Lee, Kelvin H

2016-12-05

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here, human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ω·cm 2 . By assessing the permeabilities of several known drugs, a benchmarking system to evaluate brain permeability of drugs was established. Furthermore, relationships between TEER and permeability to both small and large molecules were established, demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype, and the benchmarks established here are useful for assessing functionality of other in vitro BBB models.

In Vitro Formation of Plant RNA-Induced Silencing Complexes Using an Extract of Evacuolated Tobacco Protoplasts.

PubMed

Iki, Taichiro; Ishikawa, Masayuki; Yoshikawa, Manabu

2017-01-01

Small RNA-mediated gene silencing is involved in a variety of biological processes among many eukaryotic organisms. The silencing effector, generally referred to as RNA-induced silencing complex (RISC), comprises an ARGONAUTE (AGO) protein and a small single-stranded guide RNA in its core. RISCs recognize target genes containing sequences complementary to the guide RNA and repress their expression transcriptionally or posttranscriptionally. In vitro systems that recapitulate RISC assembly are useful not only to decipher the molecular mechanisms underlying the assembly process itself but also to dissect the downstream silencing pathways mediated by RISCs. Here, we describe a method for in vitro plant RISC assembly, which relies on an extract of evacuolated protoplasts derived from Nicotiana tabacum BY-2 suspension-cultured cells. In this extract, synthetic duplexes of small RNAs are incorporated into AGO proteins that are synthesized by in vitro translation, and then duplex unwinding and selective strand elimination result in formation of mature RISCs.

IL-1β and IL-6 activate inflammatory responses of astrocytes against Naegleria fowleri infection via the modulation of MAPKs and AP-1.

PubMed

Kim, J-H; Song, A-R; Sohn, H-J; Lee, J; Yoo, J-K; Kwon, D; Shin, H-J

2013-01-01

Naegleria fowleri, a free-living amoeba, has been found in diverse habitats throughout the world. It causes primary amoebic meningoencephalitis in children and young adults. The amoeba attaches to nasal mucosa, migrates along olfactory nerves and enters the brain. Astrocytes are involved in the defence against infection and produce inflammatory responses. In this study, we focus on the mechanism of immune responses in astrocytes. We showed, using RNase protection assay, RT-PCR and ELISA in an in vitro culture system, that N. fowleri lysates induce interleukin-1beta (IL-1β) and IL-6 expression of astrocytes. In addition, cytokine levels of astrocytes gradually decreased due to extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 inhibitors. To determine the transcription factor, we used transcription inhibitor (AP-1 inhibitor), which downregulated IL-1β and IL-6 expression. These results show that AP-1 is related to IL-1β and IL-6 production. N. fowleri-mediated IL-1β and IL-6 expression requires ERK, JNK and p38 mitogen-activated protein kinases (MAPKs) activation in astrocytes. These findings show that N. fowleri-stimulated astrocytes in an in vitro culture system lead to AP-1 activation and the subsequent expressions of IL-1β and IL-6, which are dependent on ERK, JNK and p38 MAPKs activation. These results may imply that proinflammatory cytokines have important roles in inflammatory responses to N. fowleri infection. © 2012 Blackwell Publishing Ltd.

Induction of IL-10 gene expression in human keratinocytes by UVB exposure in vivo and in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Enk, C.D.; Blauvelt, A.; Katz, S.I.

Numerous studies have demonstrated that ultraviolet B (UVB) irradiation has profound effects on the skin and systemic immune systems. Because many of the effects of UVB result in suppression of contact sensitivity responses and because IL-10 induces a Th2 rather than a Th1 response, we sought to determine whether UVB irradiation induces IL-10 transcription and subsequent protein secretion by human epidermal cells. Skin of nine volunteers was exposed to UVB or sham irradiation, and epidermal cell suspensions were prepared from suction blister roofs 24 h thereafter. mRNA was extracted using oligo dT-coated magnetic beads, and IL-10 cDNA was amplified withmore » a sensitive RT-PCR technique. We found that IL-10 was constitutively expressed by epidermal cells in 5 of 9 volunteers and that IL-10 message was up-regulated by UVB exposure in all experiments. Since epidermis consists of a heterogeneous cell population with distinct cytokine profiles, we determined whether UVB caused enhanced IL-10 transcription and protein secretion in human keratinocyte cultures. In these experiments, IL-10 was constitutively expressed by keratinocytes and UVB up-regulated IL-10 gene expression in a dose-dependent manner 24 h after in vitro irradiation, coinciding with IL-10 protein secretion into the culture supernatants. Taken together, the findings indicate that UVB irradiation induces IL-10 in human keratinocytes and suggest that keratinocyte-derived IL-10 may be an important component of the immunosuppression that results from UVB irradiation. 55 refs., 5 figs.« less

A triplex ribozyme expression system based on a single hairpin ribozyme.

PubMed

Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A; Alvarez-Salas, Luis M

2008-09-01

Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.

Developmental gene regulation during tomato fruit ripening and in-vitro sepal morphogenesis

PubMed Central

Bartley, Glenn E; Ishida, Betty K

2003-01-01

Background Red ripe tomatoes are the result of numerous physiological changes controlled by hormonal and developmental signals, causing maturation or differentiation of various fruit tissues simultaneously. These physiological changes affect visual, textural, flavor, and aroma characteristics, making the fruit more appealing to potential consumers for seed dispersal. Developmental regulation of tomato fruit ripening has, until recently, been lacking in rigorous investigation. We previously indicated the presence of up-regulated transcription factors in ripening tomato fruit by data mining in TIGR Tomato Gene Index. In our in-vitro system, green tomato sepals cultured at 16 to 22°C turn red and swell like ripening tomato fruit while those at 28°C remain green. Results Here, we have further examined regulation of putative developmental genes possibly involved in tomato fruit ripening and development. Using molecular biological methods, we have determined the relative abundance of various transcripts of genes during in vitro sepal ripening and in tomato fruit pericarp at three stages of development. A number of transcripts show similar expression in fruits to RIN and PSY1, ripening-associated genes, and others show quite different expression. Conclusions Our investigation has resulted in confirmation of some of our previous database mining results and has revealed differences in gene expression that may be important for tomato cultivar variation. We present new and intriguing information on genes that should now be studied in a more focused fashion. PMID:12906715

Immunostimulatory activity of water-extractable polysaccharides from Cistanche deserticola as a plant adjuvant in vitro and in vivo

PubMed Central

Yang, Xiumei; Yang, Yu; Zhao, Gan; Wang, Bin; Wu, Daocheng

2018-01-01

A safe and effective vaccine adjuvant is important in modern vaccines. Various Chinese herbal polysaccharides can activate the immune system. Cistanche deserticola (CD) is a traditional Chinese herb and an adjuvant candidate. Here, we confirmed that water-extractable polysaccharides of CD (WPCD) could modulate immune responses in vitro and in vivo. In a dose-dependent manner, WPCD significantly promoted the maturation and function of murine marrow-derived dendritic cells (BM-DCs) through up-regulating the expression levels of MHC-II, CD86, CD80, and CD40, allogenic T cell proliferation, and the yields of IL-12 and TNF-α via toll-like receptor4 (TLR4), as indicated by in vitro experiments. In addition, its immunomodulatory activity was also observed in mice. WPCD effectively improved the titers of IgG, IgG1 and IgG2a and markedly enhanced the proliferation of T and B cells, the production of IFN-γ and IL-4 in CD4+ T cells and the expression level of IFN-γ in CD8+ T cells better than Alum. Furthermore, WPCD could markedly up-regulate the expression levels of CD40 and CD80 on DCs in spleen and down-regulate the Treg frequency. The study suggests that polysaccharides of Cistanche deserticola are a safe and effective vaccine adjuvant for eliciting both humoral immunity and cellular immunity by activating DCs via TLR4 signaling pathway. PMID:29360858

Selection of the in vitro culture media influences mRNA expression of Hedgehog genes, Il-6, and important genes regarding reactive oxygen species in single murine preimplantation embryos.

PubMed

Pfeifer, N; Baston-Büst, D M; Hirchenhain, J; Friebe-Hoffmann, U; Rein, D T; Krüssel, J S; Hess, A P

2012-01-01

The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

Selection of the In Vitro Culture Media Influences mRNA Expression of Hedgehog Genes, Il-6, and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

PubMed Central

Pfeifer, N.; Baston-Büst, D. M.; Hirchenhain, J.; Friebe-Hoffmann, U.; Rein, D. T.; Krüssel, J. S.; Hess, A. P.

2012-01-01

Background. The aim of this paper was to determine the influence of different in vitro culture media on mRNA expression of Hedgehog genes, il-6, and important genes regarding reactive oxygen species in single mouse embryos. Methods. Reverse transcription of single embryos either cultured in vitro from day 0.5 until 3.5 (COOK's Cleavage medium or Vitrolife's G-1 PLUS medium) or in vivo until day 3.5 post coitum. PCR was carried out for β-actin followed by nested-PCR for shh, ihh, il-6, nox, gpx4, gpx1, and prdx2. Results. The number of murine blastocysts cultured in COOK medium which expressed il-6, gpx4, gpx1, and prdx2 mRNA differed significantly compared to the in vivo group. Except for nox, the mRNA profile of the Vitrolife media group embryos varied significantly from the in vivo ones regarding the number of blastocysts expressing the mRNA of shh, ihh, il-6, gpx4, gpx1 and prdx2. Conclusions. The present study shows that different in vitro culture media lead to different mRNA expression profiles during early development. Even the newly developed in vitro culture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies. PMID:22919324

Bacteriophage T4 capsid packaging and unpackaging of DNA and proteins.

PubMed

Mullaney, Julienne M; Black, Lindsay W

2014-01-01

Bacteriophage T4 has proven itself readily amenable to phage-based DNA and protein packaging, expression, and display systems due to its physical resiliency and genomic flexibility. As a large dsDNA phage with dispensable internal proteins and dispensable outer capsid proteins it can be adapted to package both DNA and proteins of interest within the capsid and to display peptides and proteins externally on the capsid. A single 170 kb linear DNA, or single or multiple copies of shorter linear DNAs, of any sequence can be packaged by the large terminase subunit in vitro into protein-containing proheads and give full or partially full capsids. The prohead receptacles for DNA packaging can also display peptides or full-length proteins from capsid display proteins HOC and SOC. Our laboratory has also developed a protein expression, packaging, and processing (PEPP) system which we have found to have advantages over mammalian and bacterial cell systems, including high yield, increased stability, and simplified downstream processing. Proteins that we have produced by the phage PEPP platform include human HIV-1 protease, micrococcal endonuclease from Staphylococcus aureus, restriction endonuclease EcoRI, luciferase, human granulocyte colony stimulating factor (GCSF), green fluorescent protein (GFP), and the 99 amino acid C-terminus of amyloid precursor protein (APP). Difficult to produce proteins that are toxic in mammalian protein expression systems are easily produced, packaged, and processed with the PEPP platform. APP is one example of such a highly refractory protein that has been produced successfully. The methods below describe the procedures for in vitro packaging of proheads with DNA and for producing recombinant T4 phage that carry a gene of interest in the phage genome and produce and internally package the corresponding protein of interest.

Cardiovascular tissues contain independent circadian clocks

NASA Technical Reports Server (NTRS)

Davidson, A. J.; London, B.; Block, G. D.; Menaker, M.

2005-01-01

Acute cardiovascular events exhibit a circadian rhythm in the frequency of occurrence. The mechanisms underlying these phenomena are not yet fully understood, but they may be due to rhythmicity inherent in the cardiovascular system. We have begun to characterize rhythmicity of the clock gene mPer1 in the rat cardiovascular system. Luciferase activity driven by the mPer1 gene promoter is rhythmic in vitro in heart tissue explants and a wide variety of veins and arteries cultured from the transgenic Per1-luc rat. The tissues showed between 3 and 12 circadian cycles of gene expression in vitro before damping. Whereas peak per1-driven bioluminescence consistently occurred during the late night in the heart and all arteries sampled, the phases of the rhythms in veins varied significantly by anatomical location. Varying the time of the culture procedure relative to the donor animal's light:dark cycle revealed that, unlike some other rat tissues such as liver, the phases of in vitro rhythms of arteries, veins, and heart explants were affected by culture time. However, phase relationships among tissues were consistent across culture times; this suggests diversity in circadian regulation among components of the cardiovascular system.

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inadera, Hidekuni; Shimomura, Akiko; Tachibana, Shinjiro

2009-02-20

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein {delta} expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activatormore » of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor {gamma} expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-{alpha} did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.« less

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway*

PubMed Central

Henrich, Erik; Ma, Yi; Engels, Ina; Münch, Daniela; Otten, Christian; Schneider, Tanja; Henrichfreise, Beate; Sahl, Hans-Georg; Dötsch, Volker; Bernhard, Frank

2016-01-01

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin. PMID:26620564

Recombinant raccoon pox vaccine protects mice against lethal plague

USGS Publications Warehouse

Osorio, J.E.; Powell, T.D.; Frank, R.S.; Moss, K.; Haanes, E.J.; Smith, S.R.; Rocke, T.E.; Stinchcomb, D.T.

2003-01-01

Using a raccoon poxvirus (RCN) expression system, we have developed new recombinant vaccines that can protect mice against lethal plague infection. We tested the effects of a translation enhancer (EMCV-IRES) in combination with a secretory (tPA) signal or secretory (tPA) and membrane anchoring (CHV-gG) signals on in vitro antigen expression of F1 antigen in tissue culture and the induction of antibody responses and protection against Yersinia pestis challenge in mice. The RCN vector successfully expressed the F1 protein of Y. pestis in vitro. In addition, the level of expression was increased by the insertion of the EMCV-IRES and combinations of this and the secretory signal or secretory and anchoring signals. These recombinant viruses generated protective immune responses that resulted in survival of 80% of vaccinated mice upon challenge with Y. pestis. Of the RCN-based vaccines we tested, the RCN-IRES-tPA-YpF1 recombinant construct was the most efficacious. Mice vaccinated with this construct withstood challenge with as many as 1.5 million colony forming units of Y. pestis (7.7×104 LD50). Interestingly, vaccination with F1 fused to the anchoring signal (RCN-IRES-tPA-YpF1-gG) elicited significant anti-F1 antibody titers, but failed to protect mice from plague challenge. Our studies demonstrate, in vitro and in vivo, the potential importance of the EMCV-IRES and secretory signals in vaccine design. These molecular tools provide a new approach for improving the efficacy of vaccines. In addition, these novel recombinant vaccines could have human, veterinary, and wildlife applications in the prevention of plague.

mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

PubMed

Danesh Mesgaran, Sadjad; Sharbati, Jutta; Einspanier, Ralf; Gabler, Christoph

2016-08-15

The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation to the new in vitro environment and during consecutive passages. The consequence of cell culture passaging on BOEC ability to release bioactive compounds should be considered.

Natural products that reduce rotavirus infectivity identified by a cell-based moderate-throughput screening assay.

PubMed

Shaneyfelt, Mark E; Burke, Anna D; Graff, Joel W; Jutila, Mark A; Hardy, Michele E

2006-09-01

There is widespread interest in the use of innate immune modulators as a defense strategy against infectious pathogens. Using rotavirus as a model system, we developed a cell-based, moderate-throughput screening (MTS) assay to identify compounds that reduce rotavirus infectivity in vitro, toward a long-term goal of discovering immunomodulatory agents that enhance innate responses to viral infection. A natural product library consisting of 280 compounds was screened in the assay and 15 compounds that significantly reduced infectivity without cytotoxicity were identified. Time course analysis of four compounds with previously characterized effects on inflammatory gene expression inhibited replication with pre-treatment times as minimal as 2 hours. Two of these four compounds, alpha-mangostin and 18-beta-glycyrrhetinic acid, activated NFkappaB and induced IL-8 secretion. The assay is adaptable to other virus systems, and amenable to full automation and adaptation to a high-throughput format. Identification of several compounds with known effects on inflammatory and antiviral gene expression that confer resistance to rotavirus infection in vitro suggests the assay is an appropriate platform for discovery of compounds with potential to amplify innate antiviral responses.

In vivo system for analyzing the function of the PsbP protein using Chlamydomonas reinhardtii.

PubMed

Nishimura, Taishi; Sato, Fumihiko; Ifuku, Kentaro

2017-09-01

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically developed in green-plant species including land plants and green algae. The protein-protein interactions involving PsbP and its effect on oxygen evolution have been investigated in vitro using isolated PSII membranes. However, the importance of those interactions needs to be examined at the cellular level. To this end, we developed a system expressing exogenous PsbP in the background of the Chlamydomonas BF25 mutant lacking native PsbP. Expression of His-tagged PsbP successfully restored the oxygen-evolving activity and photoautotrophic growth of the mutant, while PsbP-∆15 lacking the N-terminal 15 residues, which are crucial for the oxygen-evolving activity of spinach PSII in vitro, only partially did. This demonstrated the importance of N-terminal sequence of PsbP for the photosynthetic activity in vivo. Furthermore, the PSII-LHCII supercomplex can be specifically purified from the Chlamydomonas cells having His-tagged PsbP using a metal affinity chromatography. This study provides a platform not only for the functional analysis of PsbP in vivo but also for structural analysis of the PSII-LHCII supercomplex from green algae.

Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures.

PubMed

Roy, A; Krzykwa, E; Lemieux, R; Néron, S

2001-12-01

Several normal human cells, such as hematopoietic stem cells, dendritic cells, and B cells, can be cultured in vitro in defined optimal conditions. Several ex vivo culture systems require the use of feeder cells to support the growth of target cells. In such systems, proliferation of feeder cells has to be stopped, so that they can be used as nonreplicating viable support cells. Because feeder cells need to provide one or few active signals, it is important to maintain them in an metabolically active state, allowing continued expression of specific ligands or cytokines. Mitomycin C and gamma-irradiation treatments are commonly used to prepare nonproliferating feeder cells and are usually considered to be equivalent. Normal human B lymphocytes can be expanded in vitro in the presence of feeder cells expressing the CD40 ligand CD154. Here we compared the ability of gamma-irradiation- and mitomycin C-treated feeder cells to support the expansion of normal human B lymphocytes. The results indicate that expansion of B cells during a long-term culture was 100 times more potent using gamma-irradiated feeder cells compared to mitomycin C-treated cells. This difference could be related to a significant reduction in both cellular metabolism and level of CD154 expression observed in mitomycin C-treated feeder cells, but not in gamma-irradiated cells nor in control untreated cells. These results indicate that mitomycin C-treated feeder cells are metabolically altered, and consequently less efficient at maintaining cell expansion in the long-term cell culture system used.

Human peripheral blood mononuclear cell in vitro system to test the efficacy of food bioactive compounds: Effects of polyunsaturated fatty acids and their relation with BMI.

PubMed

Cifre, Margalida; Díaz-Rúa, Rubén; Varela-Calviño, Rubén; Reynés, Bàrbara; Pericás-Beltrán, Jordi; Palou, Andreu; Oliver, Paula

2017-04-01

To analyse the usefulness of isolated human peripheral blood mononuclear cells (PBMC) to rapidly/easily reflect n-3 long-chain polyunsaturated fatty acid (LCPUFA) effects on lipid metabolism/inflammation gene profile, and evaluate if these effects are body mass index (BMI) dependent. PBMC from normoweight (NW) and overweight/obese (OW/OB) subjects were incubated with physiological doses of docosahexaenoic (DHA), eicosapentaenoic acid (EPA), or their combination. PBMC reflected increased beta-oxidation-like capacity (CPT1A expression) in OW/OB but only after DHA treatment. However, insensitivity to n-3 LCPUFA was evident in OW/OB for lipogenic genes: both PUFA diminished FASN and SREBP1C expression in NW, but no effect was observed for DHA in PBMC from high-BMI subjects. This insensitivity was also evident for inflammation gene profile: all treatments inhibited key inflammatory genes in NW; nevertheless, no effect was observed in OW/OB after DHA treatment, and EPA effect was impaired. SLC27A2, IL6 and TNFα PBMC expression analysis resulted especially interesting to determine obesity-related n-3 LCPUFA insensitivity. A PBMC-based human in vitro system reflects n-3 LCPUFA effects on lipid metabolism/inflammation which is impaired in OW/OB. These results confirm the utility of PBMC ex vivo systems for bioactive-compound screening to promote functional food development and to establish appropriate dietary strategies for obese population. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo.

PubMed

Aldrian, Gudrun; Vaissière, Anaïs; Konate, Karidia; Seisel, Quentin; Vivès, Eric; Fernandez, Frédéric; Viguier, Véronique; Genevois, Coralie; Couillaud, Franck; Démèné, Héléne; Aggad, Dina; Covinhes, Aurélie; Barrère-Lemaire, Stéphanie; Deshayes, Sébastien; Boisguerin, Prisca

2017-06-28

Small interfering RNAs (siRNAs) present a strong therapeutic potential because of their ability to inhibit the expression of any desired protein. Recently, we developed the retro-inverso amphipathic RICK peptide as novel non-covalent siRNA carrier. This peptide is able to form nanoparticles (NPs) by self-assembling with the siRNA resulting in the fully siRNA protection based on its protease resistant peptide sequence. With regard to an in vivo application, we investigated here the influence of the polyethylene glycol (PEG) grafting to RICK NPs on their in vitro and in vivo siRNA delivery properties. A detailed structural study shows that PEGylation did not alter the NP formation (only decrease in zeta potential) regardless of the used PEGylation rates. Compared to the native RICK:siRNA NPs, low PEGylation rates (≤20%) of the NPs did not influence their cellular internalization capacity as well as their knock-down specificity (over-expressed or endogenous system) in vitro. Because the behavior of PEGylated NPs could differ in their in vivo application, we analyzed the repartition of fluorescent labeled NPs injected at the one-cell stage in zebrafish embryos as well as their pharmacokinetic (PK) profile after administration to mice. After an intra-cardiac injection of the PEGylated NPs, we could clearly determine that 20% PEG-RICK NPs reduce significantly liver and kidney accumulation. NPs with 20% PEGylation constitutes a modular, easy-to-handle drug delivery system which could be adapted to other types of functional moieties to develop safe and biocompatible delivery systems for the clinical application of RNAi-based cancer therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

[Differentiation of bone marrow derived from mesenchymal stem cells into cardiomyocyte-like cells induced by co-culture with rat myocardial cells].

PubMed

Zhang, Rong-Li; Jiang, Er-Lie; Wang, Mei; Zhou, Zheng; Zhai, Wen-Jing; Zhai, Wei-Hua; Wang, Hua; Wang, Zhi-Yong; Bao, Yu-Shi; DU, Hong; Han, Ming-Zhe

2008-10-01

The study was purposed to investigate the differentiation ability of mesenchymal stem cells (MSCs) into myocardial cells in vitro. Rat bone marrow-derived MSCs were labeled and co-cultured with neonatal rat cardiomyocytes (CM) for 5 - 7 days. The expression of cell surface antigens was detected by flow cytometry, and the expression of muscle-specific marker myosin and troponin T in labeled cells was detected by immunofluorescence. The results showed that in vitro cultured MSCs expressed CD90, CD44, CD105, CD54, not expressed CD34, CD45, CD31. After co-cultured with neonatal rat CM, labeled MSCs differentiated into cardiomyocyte-like cells expressing myosin and troponin T. It is concluded that MSCs can differentiate into cardiomyocyte-like cells when co-cultured with neonatal myocardial cells in vitro. In co-culture of two kind of cells in ratio of four to one showed obvious efficacy differentiating MSCs into CMs.

Chronology and regulation of gene expression of RANKL in the rat dental follicle.

PubMed

Liu, D; Yao, S; Pan, F; Wise, G E

2005-10-01

Tooth eruption in the rat requires bone resorption resulting from a major burst of osteoclastogenesis on postnatal day 3 and a minor burst of osteoclastogenesis on postnatal day 10 in the alveolar bone of the first mandibular molar. The dental follicle regulates the major burst on postnatal day 3 by down-regulating its osteoprotegerin (OPG) gene expression to enable osteoclastogenesis to occur. To determine the role of receptor activator of nuclear factor-kappa B ligand (RANKL) in tooth eruption, its gene expression was measured on postnatal days 1-11 in the dental follicle. The results show that RANKL expression was significantly elevated on postnatal days 9-11 in comparison to low expression levels at earlier time-points. As OPG expression is high at this latter time-point, this increase in RANKL expression would be needed for stimulating the minor burst of osteoclastogenesis. Tumor necrosis factor-alpha enhances RANKL gene expression in vitro and it may be responsible for up-regulating RANKL in vivo. Transforming growth factor-beta1 and interleukin-1alpha also enhance RANKL gene expression in vitro but probably have no effect in vivo because they are maximally expressed early. Bone morphogenetic protein-2 acts to down-regulate RANKL expression in vitro and, in vivo, may promote alveolar bone growth in the basal region of the tooth.

Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.

PubMed

Morsczeck, C

2006-02-01

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.

C-type natriuretic peptide suppresses mesangial proliferation and matrix expression via a MMPs/TIMPs-independent pathway in vitro.

PubMed

Huang, Bao Yu; Hu, Peng; Zhang, Dong Dong; Jiang, Guang Mei; Liu, Si Yan; Xu, Yao; Wu, Yang Fang; Xia, Xun; Wang, Ya

2017-08-01

C-type natriuretic peptide (CNP) acts mainly in a local, paracrine fashion to regulate vascular tone and cell proliferation. Although several in vivo studies have demonstrated that CNP exerts an inhibitory effect on mesangial matrix generation, a limited number of reports exist about the anti-extracellular matrix (ECM) accumulation effect of CNP and its underlying mechanisms in mesangial cells (MCs) in vitro. In this study, human MCs were incubated in serum-containing medium in the absence or presence of CNP (0, 10 and 100 pM) for 24, 48 and 72 h, respectively. CNP administration significantly suppresses MCs proliferation and collagen (Col)-IV expression in a time- and dose-dependent manner. In addition, the study presented herein was designed as a first demonstration of the regulative effects of CNP on the metabolisms of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in MCs in vitro, and found that: (1) CNP administration significantly decreased the secretion and expression of MMP-2 and MMP-9 in the cultured MCs; (2) the secretion and expression of TIMP-1 progressively elevated after treatment with CNP for 24 and 48 h, whereas declined at later time point; (3) CNP expression was negatively correlated with MMP-2 and MMP-9 expression; (4) the balance of MMPs/TIMPs was shifted toward the reduction in MMP-2 and MMP-9 activity and/or the increment in TIMP-1 expression, which could not account for the down-regulation of Col-IV expression in CNP-treated MCs. In conclusion, CNP suppresses mesangial proliferation and ECM expression via a MMPs/TIMPs-independent pathway in vitro.

Viral delivery of genome-modifying proteins for cellular reprogramming.

PubMed

Mikkelsen, Jacob Giehm

2018-06-18

Following the successful development of virus-based gene vehicles for genetic therapies, exploitation of viruses as carriers of genetic tools for cellular reprogramming and genome editing should be right up the street. However, whereas persistent, potentially life-long gene expression is the main goal of conventional genetic therapies, tools and bits for genome engineering should ideally be short-lived and active only for a limited time. Although viral vector systems have already been adapted for potent genome editing both in vitro and in vivo, regulatable gene expression systems or self-limiting expression circuits need to be implemented limiting exposure of chromatin to genome-modifying enzymes. As an alternative approach, emerging virus-based protein delivery technologies support direct protein delivery, providing a short, robust boost of enzymatic activity in transduced cells. Is this potentially the perfect way of shipping loads of cargo to many recipients and still maintain short-term activity? Copyright © 2018 Elsevier Ltd. All rights reserved.

SOX2 expression levels distinguish between neural progenitor populations of the developing dorsal telencephalon.

PubMed

Hutton, Scott R; Pevny, Larysa H

2011-04-01

The HMG-Box transcription factor SOX2 is expressed in neural progenitor populations throughout the developing and adult central nervous system and is necessary to maintain their progenitor identity. However, it is unclear whether SOX2 levels are uniformly expressed across all neural progenitor populations. In the developing dorsal telencephalon, two distinct populations of neural progenitors, radial glia and intermediate progenitor cells, are responsible for generating a majority of excitatory neurons found in the adult neocortex. Here we demonstrate, using both cellular and molecular analyses, that SOX2 is differentially expressed between radial glial and intermediate progenitor populations. Moreover, utilizing a SOX2(EGFP) mouse line, we show that this differential expression can be used to prospectively isolate distinct, viable populations of radial glia and intermediate cells for in vitro analysis. Given the limited repertoire of cell-surface markers currently available for neural progenitor cells, this provides an invaluable tool for prospectively identifying and isolating distinct classes of neural progenitor cells from the central nervous system. Copyright © 2011 Elsevier Inc. All rights reserved.

Solar-simulating irradiation of the skin of human subjects in vivo produces Langerhans cell responses distinct from irradiation ex vivo and in vitro.

PubMed

Laihia, J K; Jansen, C T

2000-08-01

It has been postulated that Langerhans cells (LC) provide tolerogenic signals in the local impairment of cutaneous immune functions and antigen-specific tolerance induced by UV radiation. Studies in vitro and ex vivo have indicated that UV radiation may down-regulate the expression of costimulatory molecules on LC, leading to reduced antigen-presenting function. In contrast, we recently observed an up-regulatory stage in the number of human epidermal LC with induced expression of B7 costimulatory molecules 12-24 h after solar-simulating UV radiation (SSR) in vivo. To examine the apparent discrepancy between the observed human LC responses in vitro, ex vivo and in vivo, we compared the three protocols in a parallel fashion. The intact skin as well as skin explants and epidermal cell suspensions from the same individuals were irradiated with a single erythematogenic dose of SSR. The expression of cell surface markers in the epidermal cells was analysed with flow cytometry 24 h later. The number of CD1a+/HLA-DR+ LC increased post-SSR in vivo by a factor of 2.8+/-0.4, whereas in irradiated skin explants ex vivo or in cell suspensions in vitro, reduced numbers were seen. HLA-DR expression intensities were found to have increased on DR+ and CD1a+/DR+ cells in vivo. Similarly, SSR induced B7-2 (CD86) expression in CD1a+ cells significantly in vivo (P=0.031) but reduced the expression ex vivo or in vitro. We conclude that the early up-regulatory stage of human LC number and membrane markers, recorded at 24 h after a single exposure to SSR, is exclusively an in vivo phenomenon.

Temporal and Embryonic Lineage-Dependent Regulation of Human Vascular SMC Development by NOTCH3

PubMed Central

Granata, Alessandra; Bernard, William G.; Zhao, Ning; Mccafferty, John; Lilly, Brenda

2015-01-01

Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8–9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner. PMID:25539150

Simvastatin prevents triple-negative breast cancer metastasis in pre-clinical models through regulation of FOXO3a

PubMed Central

Wolfe, Adam R.; Debeb, Bisrat G.; Lacerda, Lara; Larson, Richard; Bambhroliya, Arvind; Huang, Xuelin; Bertucci, Francois; Finetti, Pascal; Birnbaum, Daniel; Van Laere, Steven; Diagaradjan, Parmeswaran; Ruffell, Brian; Trenton, Nicholaus J.; Chu, Khoi; Hittelman, Walter; Diehl, Michael; Levental, Ilya; Ueno, Naoto T.; Woodward, Wendy A.

2016-01-01

Purpose We previously reported using statins was correlated with improved metastasis free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple negative breast cancer (TNBC) cells. Experimental Design TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in 8 public breast cancer gene expression data sets including 1,479 patients. Results Simvastatin increased G1/S phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pretreatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Conclusions Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted. PMID:26590814

Simvastatin prevents triple-negative breast cancer metastasis in pre-clinical models through regulation of FOXO3a.

PubMed

Wolfe, Adam R; Debeb, Bisrat G; Lacerda, Lara; Larson, Richard; Bambhroliya, Arvind; Huang, Xuelin; Bertucci, Francois; Finetti, Pascal; Birnbaum, Daniel; Van Laere, Steven; Diagaradjan, Parmeswaran; Ruffell, Brian; Trenton, Nicholaus J; Chu, Khoi; Hittelman, Walter; Diehl, Michael; Levental, Ilya; Ueno, Naoto T; Woodward, Wendy A

2015-12-01

We previously reported using statins was correlated with improved metastasis-free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple-negative breast cancer (TNBC) cells. TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in eight public breast cancer gene expression datasets including 1479 patients. Simvastatin increased G1/S-phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pre-treatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted.

Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

PubMed

Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

2014-09-01

This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

7 CFR 331.3 - PPQ select agents and toxins.

Code of Federal Regulations, 2011 CFR

2011-01-01

... listed in paragraph (b) of this section if the nucleic acids: (i) Can be expressed in vivo or in vitro; or (ii) Are in a vector or recombinant host genome and can be expressed in vivo or in vitro. (3... select agents and toxins. (a) Except as provided in paragraphs (d) and (e) of this section, the...

A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system.

PubMed

Zhang, Li; Li, Qianqian; Liu, Qiang; Huang, Weijin; Nie, Jianhui; Wang, Youchun

2017-08-03

Marburg virus (MARV) can cause lethal hemorrhagic fever in humans. Handling of MARV is restricted to high-containment biosafety level 4 (BSL-4) facilities, which greatly impedes research into this virus. In this study, a high titer of MARV pseudovirus was generated through optimization of the HIV backbone vectors, the ratio of backbone vector to MARV glycoprotein expression vector, and the transfection reagents. An in vitro neutralization assay and an in vivo bioluminescent imaging mouse model for MARV were developed based on the pseudovirus. Protective serum against MARV was successfully induced in guinea pigs, which showed high neutralization activity in vitro and could also protect Balb/c mice from MARV pseudovirus infection in vivo. This system could be a convenient tool to enable the evaluation of vaccines and therapeutic drugs against MARV in non-BSL-4 laboratories.

Peroxidases, lignin and anatomy during in vitro and ex vitro rooting of gardenia (Gardenia jasminoides Ellis) microshoots.

PubMed

Hatzilazarou, Stefanos P; Syros, Thomas D; Yupsanis, Traianos A; Bosabalidis, Artemios M; Economou, Athanasios S

2006-07-01

In vitro and ex vitro rooting of gardenia (Gardenia jasminoides Ellis) microshoots with or without indolic-3-butyric acid (IBA) was studied in order to improve acclimatization of microplants after root formation and transplantation. Peroxidase (POD) activity and isoforms, lignin content and anatomical observations were evaluated in the course of the three interdependent phases (induction, initiation and expression) of microshoot rooting. Microshoots treated or not treated with IBA achieved high rooting percentages both in vitro and ex vitro. At the end of the 2-week acclimatization period, the percentage of surviving microplants ranged from 80% to 100%, for in vitro and ex vitro rooted microshoots, respectively. Microshoots rooted in vitro and ex vitro showed a relationship between rooting and POD activity but in a different time course. It appeared that root formation occurred after the microshoots had reached and passed a peak of maximum enzyme activity. In all treatments, electrophoretic analysis (native PAGE) of PODs revealed the appearance of one anionic and three cationic POD isoforms (C(1), C(3) and C(4)). An additional cationic POD isoform (C(2)) appeared only in the ex vitro rooting. The lignin content was similar in microshoots rooted both in vitro and ex vitro. The sequential anatomical changes during the rooting process were similar in both in vitro and ex vitro rooting treatments. In the case of in vitro rooting, pith cells had vacuoles entirely filled with a dark substance, while in the case of ex vitro rooting, pith cells contained many amyloplasts. The origin of the adventitious roots, in both rooting conditions, was located in the cambial ring. Roots with organized tissue systems emerged from the microshoot stem 10-14 days after the root induction treatments; on day 10 for rooting in vitro, while a 4-day delay was noted in microshoots rooted ex vitro.

Net Expression Inhibits the Growth of Pancreatic Ductal Adenocarcinoma Cell PL45 In Vitro and In Vivo

PubMed Central

Li, Baiwen; Wan, Xinjian; Zhu, Qi; Li, Lei; Zeng, Yue; Hu, Duanmin; Qian, Yueqin; Lu, Lungen; Wang, Xingpeng; Meng, Xiangjun

2013-01-01

Pancreatic ductal adenocarcinoma has a poor prognosis due to late diagnosis and a lack of effective therapeutic options. Thus, it is important to better understand its molecular mechanisms and to develop more effective treatments for the disease. The ternary complex factor Net, which exerts its strong inhibitory function on transcription of proto-oncogene gene c-fos by forming ternary complexes with a second transcription factor, has been suspected of being involved in pancreatic cancer and other tumors biology. In this study, we found that the majority of pancreatic ductal adenocarcinoma tissues and cell lines had weak or no expression of Net, whereas significantly high level of Net expression occurred in paired adjacent normal tissues we studied. Furthermore, using in vitro and in vivo model systems, we found that overexpression of Net inhibited cell growth and survival and induced cell apoptosis in human pancreatic ductal adenocarcinoma cell PL45; the mechanisms by which Net inhibited the cell cycle progression were mainly through P21-Cyclin D1/CDK4 Pathway. Our data thus suggested that Net might play an important role in pancreatic carcinogenesis, possibly by acting as a tumor suppressor gene. PMID:23469073

Circadian clock gene plays a key role on ovarian cycle and spontaneous abortion.

PubMed

Li, Ruiwen; Cheng, Shuting; Wang, Zhengrong

2015-01-01

Circadian locomotor output cycles protein kaput (CLOCK) plays a key role in maintaining circadian rhythms and activation of downstream elements. However, its function on human female reproductive system remains unknown. To investigate the potential role of CLOCK, CLOCK-shRNAs were transfected into mouse 129 ES cells or injected into the ovaries of adult female mice. Western blotting was utilized to analyze the protein interactions and flow cytometry was used to assess apoptosis. The expression of CLOCK peaked at the 6th week in the healthy fetuses. However, an abnormal expression of CLOCK was detected in fetuses from spontaneous miscarriage. To determine the effect of CLOCK on female fertility, a small hairpin RNA (shRNA) strategy was used to specifically knockdown the CLOCK gene expression in vitro and in vivo. Knockdown of CLOCK induced apoptosis in mouse embryonic stem (mES) cells and inhibited the proliferation in mES cells in vitro. CLOCK knockdown also led to decreased release of oocytes and smaller litter size compared with control in vivo. Collectively, theses findings indicate that CLOCK plays an important role in fertility and that the CLOCK knockdown leads to reduction in reproduction and increased miscarriage risk. © 2015 S. Karger AG, Basel.

Monoamine oxidase A gene promoter methylation and transcriptional downregulation in an offender population with antisocial personality disorder.

PubMed

Checknita, D; Maussion, G; Labonté, B; Comai, S; Tremblay, R E; Vitaro, F; Turecki, N; Bertazzo, A; Gobbi, G; Côté, G; Turecki, G

2015-03-01

Antisocial personality disorder (ASPD) is characterised by elevated impulsive aggression and increased risk for criminal behaviour and incarceration. Deficient activity of the monoamine oxidase A (MAOA) gene is suggested to contribute to serotonergic system dysregulation strongly associated with impulsive aggression and antisocial criminality. To elucidate the role of epigenetic processes in altered MAOA expression and serotonin regulation in a population of incarcerated offenders with ASPD compared with a healthy non-incarcerated control population. Participants were 86 incarcerated participants with ASPD and 73 healthy controls. MAOA promoter methylation was compared between case and control groups. We explored the functional impact of MAOA promoter methylation on gene expression in vitro and blood 5-HT levels in a subset of the case group. Results suggest that MAOA promoter hypermethylation is associated with ASPD and may contribute to downregulation of MAOA gene expression, as indicated by functional assays in vitro, and regression analysis with whole-blood serotonin levels in offenders with ASPD. These results are consistent with prior literature suggesting MAOA and serotonergic dysregulation in antisocial populations. Our results offer the first evidence suggesting epigenetic mechanisms may contribute to MAOA dysregulation in antisocial offenders. Royal College of Psychiatrists.

Modulation of human cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) in Caco-2 cell monolayers by selected commercial-source milk thistle and goldenseal products.

PubMed

Budzinski, Jason W; Trudeau, Vance L; Drouin, Cathy E; Panahi, Mitra; Arnason, J Thor; Foster, Brian C

2007-09-01

In this study, we used an in vitro Caco-2 cell monolayer model to evaluate aqueous extracts of commercial-source goldenseal (Hydrastis canadensis) and milk thistle (Silybum marianum) capsule formulations, their marker phytochemicals (berberine and silibinin, respectively), as well as dillapiol, vinblastine, and the HIV protease inhibitor saquinavir for their ability to modulate CYP3A4 and ABCB1 expression after short-term exposure (48 h). Both upregulation and downregulation of CYP3A4 expression was observed with extracts of varying concentrations of the two natural health products (NHPs). CYP3A4 was highly responsive in our system, showing a strong dose-dependent modulation by the CYP3A4 inhibitor dillapiol (upregulation) and the milk thistle flavonolignan silibinin (downregulation). ABCB1 was largely unresponsive in this cellular model and appears to be of little value as a biomarker under our experimental conditions. Therefore, the modulation of CYP3A4 gene expression can serve as an important marker for the in vitro assessment of NHP-drug interactions.

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle.

PubMed

Chiba, Eriko; Villena, Julio; Hosoya, Shoichi; Takanashi, Naoya; Shimazu, Tomoyuki; Aso, Hisashi; Tohno, Masanori; Suda, Yoshihito; Kawai, Yasushi; Saito, Tadao; Miyazawa, Kenji; He, Fang; Kitazawa, Haruki

2012-10-01

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts. Copyright © 2011 Elsevier Ltd. All rights reserved.

Reproducibility and Consistency of In Vitro Nucleosome Reconstitutions Demonstrated by Invitrosome Isolation and Sequencing

PubMed Central

Kempton, Colton E.; Heninger, Justin R.; Johnson, Steven M.

2014-01-01

Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reasons for performing in vitro nucleosome reconstitution experiments is to identify how the underlying DNA sequence will influence a nucleosome's position in the absence of other compounding cellular factors. However, concerns have been raised about the reproducibility of data generated from these kinds of experiments. Here we present data for in vitro nucleosome reconstitution experiments performed on linear plasmid DNA that demonstrate that, when coverage is deep enough, these reconstitution experiments are exquisitely reproducible and highly consistent. Our data also suggests that a coverage depth of 35X be maintained for maximal confidence when assaying nucleosome positions, but lower coverage levels may be generally sufficient. These coverage depth recommendations are sufficient in the experimental system and conditions used in this study, but may vary depending on the exact parameters used in other systems. PMID:25093869

Gene expression platform for synthetic biology in the human pathogen Streptococcus pneumoniae.

PubMed

Sorg, Robin A; Kuipers, Oscar P; Veening, Jan-Willem

2015-03-20

The human pathogen Streptococcus pneumoniae (pneumococcus) is a bacterium that owes its success to complex gene expression regulation patterns on both the cellular and the population level. Expression of virulence factors enables a mostly hazard-free presence of the commensal, in balance with the host and niche competitors. Under specific circumstances, changes in this expression can result in a more aggressive behavior and the reversion to the invasive form as pathogen. These triggering conditions are very difficult to study due to the fact that environmental cues are often unknown or barely possible to simulate outside the host (in vitro). An alternative way of investigating expression patterns is found in synthetic biology approaches of reconstructing regulatory networks that mimic an observed behavior with orthogonal components. Here, we created a genetic platform suitable for synthetic biology approaches in S. pneumoniae and characterized a set of standardized promoters and reporters. We show that our system allows for fast and easy cloning with the BglBrick system and that reliable and robust gene expression after integration into the S. pneumoniae genome is achieved. In addition, the cloning system was extended to allow for direct linker-based assembly of ribosome binding sites, peptide tags, and fusion proteins, and we called this new generally applicable standard "BglFusion". The gene expression platform and the methods described in this study pave the way for employing synthetic biology approaches in S. pneumoniae.

ZENK expression of retinal glucagon amacrine cells in chicks: the effect of defocus presented in vivo, in vitro and under anesthesia.

PubMed

Bitzer, Michaela; Schaeffel, Frank

2006-03-01

The transcription factor ZENK, expressed in glucagon amacrine cells in the retina in the chicken, has been shown to respond selectively to the sign of imposed defocus. To find out whether the retina by itself is able to perform the underlying image processing, we used ZENK to probe responses of amacrine cells to defined amounts of imposed defocus, both in vitro and in vivo. To stimulate in vitro, retinal punches (8mm diameter), whole eyecups, or complete excised globes were stimulated with digitally generated video movies that were presented with defined amounts of defocus for 15, 30 or 45 min. Infrared photoretinoscopy was used to control the defocus of the projected movies. Furthermore, both anesthetized and alert chickens were stimulated with the differently focused video movies, or they were exposed to the visual environment of the laboratory for 4 days, wearing spectacle lenses for 45 or 90 min per day. Anesthetic effects per se were determined by using two different anesthetic protocols. After the stimulation, ZENK-immunoreactivity in glucagon amacrine cells was quantified. Eye growth and refractive development were recorded by A-scan ultrasound and infrared photoretinoscopy, respectively. In the isolated retina (stimulated in vitro), no consistent changes in ZENK expression could be induced by defocus, with a baseline expression similar in alert chicks. Placing a diffuser into the light path during optical stimulation did not lower ZENK expression, although diffusers are known to reduce ZENK expression in alert chicks. Imposed defocus in vitro caused merely a higher variability in ZENK expression in some cases. Defocus produced significant changes in ZENK expression only in the case of 4D of imposed hyperopia, but these changes were in the opposite direction to what was previously observed in vivo. Also in isolated eye cup or eye balls, no changes in ZENK expression could be induced by defocus. Most strikingly, neither ZENK expression, nor eye growth were affected by imposing defocus under anesthesia. Only alert chickens displayed the expected changes in ZENK expression and eye growth during imposed defocus, both with video stimulation and the "real world". In vitro, the chicken retina can still process some spatial features (since ZENK expression in the isolated retina was changed by imposed defocus with video stimulation), but the directions of the changes were no longer related to the sign of defocus. Also in intact animals, though under anesthesia, ZENK and eye growth did not respond to the sign of imposed defocus. Some stimulus for emmetropization appears to be important which is available only in alert animals.

Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines.

PubMed

Kowarz, Eric; Löscher, Denise; Marschalek, Rolf

2015-04-01

Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of retro-/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium-throughput setting (three to five days). Cell lines robustly express any gene-of-interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generation of data in a rapid and robust manner. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nupr1 Modulates Methamphetamine-Induced Dopaminergic Neuronal Apoptosis and Autophagy through CHOP-Trib3-Mediated Endoplasmic Reticulum Stress Signaling Pathway

PubMed Central

Xu, Xiang; Huang, Enping; Tai, Yunchun; Zhao, Xu; Chen, Xuebing; Chen, Chuanxiang; Chen, Rui; Liu, Chao; Lin, Zhoumeng; Wang, Huijun; Xie, Wei-Bing

2017-01-01

Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH exposure causes detrimental effects on multiple organ systems, primarily the nervous system, especially dopaminergic pathways, in both laboratory animals and humans. In this study, we hypothesized that Nuclear protein 1 (Nupr1/com1/p8) is involved in METH-induced neuronal apoptosis and autophagy through endoplasmic reticulum (ER) stress signaling pathway. To test this hypothesis, we measured the expression levels of Nupr1, ER stress protein markers CHOP and Trib3, apoptosis-related protein markers cleaved-caspase3 and PARP, as well as autophagy-related protein markers LC3 and Beclin-1 in brain tissues of adult male Sprague-Dawley (SD) rats, rat primary cultured neurons and the rat adrenal pheochromocytoma cells (PC12 cells) after METH exposure. We also determined the effects of METH exposure on the expression of these proteins after silencing Nupr1, CHOP, or Trib3 expression with synthetic small hairpin RNA (shRNA) or siRNA in vitro, and after silencing Nupr1 in the striatum of rats by injecting lentivirus containing shRNA sequence targeting Nupr1 gene to rat striatum. The results showed that METH exposure increased Nupr1 expression that was accompanied with increased expression of ER stress protein markers CHOP and Trib3, and also led to apoptosis and autophagy in rat primary neurons and in PC12 cells after 24 h exposure (3.0 mM), and in the prefrontal cortex and striatum of rats after repeated intraperitoneal injections (15 mg/kg × 8 injections at 12 h intervals). Silencing of Nupr1 expression partly reduced METH-induced apoptosis and autophagy in vitro and in vivo. These results suggest that Nupr1 plays an essential role in METH-caused neuronal apoptosis and autophagy at relatively higher doses and may be a potential therapeutic target in high-dose METH-induced neurotoxicity. PMID:28694771

Construction of an agglutination tool: recombinant Fab fragments biotinylated in vitro.

PubMed

Czerwinski, Marcin; Krop-Watorek, Anna; Wasniowska, Kazimiera; Smolarek, Dorota; Spitalnik, Steven L

2009-11-30

The pComb3H vector system is used for constructing and panning recombinant antibody libraries. It allows for expression of monovalent Fab fragments, either on the surface of M13 phage, or in the form of soluble proteins secreted into the periplasmic space of bacteria. We constructed a modified pComb3H vector containing cDNA encoding for a 23-amino acid fragment of the Escherichia coli biotin carboxy carrier protein (BCCP), which is an acceptor sequence for biotinylation. The vector was used to express the Fab fragment recognizing human glycophorin A. The purified Fab fragment containing this biotin acceptor sequence was effectively biotinylated in vitro using biotin ligase (BirA). The specificity and avidity of the biotinylated Fab fragments were similar to the previously produced, unmodified Fab fragments. An avidin-alkaline phosphatase conjugate was used to detect the recombinant Fab fragments, instead of secondary antibody. In addition, when biotinylated Fab fragments were mixed with avidin, red blood cells were directly agglutinated.

Antitumor activity of EGFR-specific CAR T cells against non-small-cell lung cancer cells in vitro and in mice.

PubMed

Li, He; Huang, Yao; Jiang, Du-Qing; Cui, Lian-Zhen; He, Zhou; Wang, Chao; Zhang, Zhi-Wei; Zhu, Hai-Li; Ding, Yong-Mei; Li, Lin-Fang; Li, Qiang; Jin, Hua-Jun; Qian, Qi-Jun

2018-02-07

Effective control of non-small-cell lung cancer (NSCLC) remains clinically challenging, especially during advanced stages of the disease. This study developed an adoptive T-cell treatment through expression of a chimeric antigen receptor (CAR) to target human epidermal growth factor receptor (EGFR) in NSCLC. We optimized the non-viral piggyBac transposon system to engineer human T cells for the expression of EGFR-CAR, consisting of EGFR scFv, transmembrane domain, and intracellular 4-1BB-CD3ζ signaling domains. The modified CAR T cells exhibited expansion capability and anticancer efficacy in a time- and antigen-dependent manner in vitro as well as regression of EGFR-positive human lung cancer xenografts in vivo. EGFR-CAR T therapy is a promising strategy to improve the efficacy and potency of the adoptive immunotherapy in NSCLC. Moreover, EGFR-CAR T therapy could become a clinical application for NSCLC patients in the future.

TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

2012-06-01

Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

The Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone enhances keratinocyte migration and induces Mmp13 gene expression in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paes, Camila, E-mail: camilaquinetti@gmail.com; Nakagami, Gojiro, E-mail: gojiron-tky@umin.ac.jp; Minematsu, Takeo, E-mail: tminematsu-tky@umin.ac.jp

2012-10-19

Highlights: Black-Right-Pointing-Pointer An evidence of the positive effect of AHL on epithelialization process is provided. Black-Right-Pointing-Pointer AHL enhances keratinocyte's ability to migrate in an in vitro scratch wound model. Black-Right-Pointing-Pointer AHL induces the expression of Mmp13. Black-Right-Pointing-Pointer Topical application of AHL represents a possible strategy to treat chronic wounds. -- Abstract: Re-epithelialization is an essential step of wound healing involving three overlapping keratinocyte functions: migration, proliferation and differentiation. While quorum sensing (QS) is a cell density-dependent signaling system that enables bacteria to regulate the expression of certain genes, the QS molecule N-(3-oxododecanoyl) homoserine lactone (AHL) exerts effects also on mammalianmore » cells in a process called inter-kingdom signaling. Recent studies have shown that AHL improves epithelialization in in vivo wound healing models but detailed understanding of the molecular and cellular mechanisms are needed. The present study focused on the AHL as a candidate reagent to improve wound healing through direct modulation of keratinocyte's activity in the re-epithelialization process. Results indicated that AHL enhances the keratinocyte's ability to migrate in an in vitro scratch wound healing model probably due to the high Mmp13 gene expression analysis after AHL treatment that was revealed by real-time RT-PCR. Inhibition of activator protein 1 (AP-1) signaling pathway completely prevented the migration of keratinocytes, and also resulted in a diminished Mmp13 gene expression, suggesting that AP-1 might be essential in the AHL-induced migration. Taken together, these results imply that AHL is a promising candidate molecule to improve re-epithelialization through the induction of migration of keratinocytes. Further investigation is needed to clarify the mechanism of action and molecular pathway of AHL on the keratinocyte migration process.« less

Effect of intermittent shear stress on corneal epithelial cells using an in vitro flow culture model.

PubMed

Hampel, Ulrike; Garreis, Fabian; Burgemeister, Fabian; Eßel, Nicole; Paulsen, Friedrich

2018-04-27

The aim of this study was to establish and to evaluate an in vitro model for culturing human telomerase-immortalized corneal epithelial (hTCEpi) cells under adjustable medium flow mimicking the movements of the tear film on the ocular surface. Using an IBIDI pump system, cells were cultured under unidirectional, continuous or oscillating, discontinuous medium flow. Cell surface and cytoskeletal architecture were investigated by scanning electron microscopy and immunofluorescence. Gene expression of e-cadherin, occludin, tight junction protein (TJP), desmoplakin, desmocollin and mucins was investigated by real-time PCR. Protein expression of desmoplakin, TJP, occludin and e-cadherin was analyzed by western blot and localization was detected by immunofluorescence. Rose bengal staining was used to assess mucin (MUC) barrier integrity. MUC1, -4 and -16 proteins were localized by immunofluorescence. Medium flow-induced shear stress dramatically changed cellular morphology of hTCEpi. Cells subjected to discontinuous shear stress displayed the typical flattened, polygonal cell shape of the superficial layer of stratified squamous epithelia. Cell surfaces showed less bulging under shear stress and less extracellular gaps. The mRNA expression of E-cadherin, occludin and TJP were increased under oscillatory medium flow. Desmoplakin and occludin protein were upregulated under oscillatory shear stress. Stress fiber formation was not aligned to flow direction. MUC1, -4, and -16 protein were localized under all culture conditions, a regulation on mRNA expression was not detectable. Rose Bengal uptake was diminished under unidirectional conditions. Our findings suggest that shear stress as it occurs at the ocular surface during blinking exerts marked effects on corneal epithelial cells, such as changes in cellular morphology and expression of cell junctions. The described model may be useful for in vitro investigations of ocular surface epithelia as it represents a much more physiologic reproduction of the in vivo situation than the commonly applied static culture conditions. Copyright © 2018. Published by Elsevier Inc.

Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters

PubMed Central

Wang, Yingying; Ji, Teng; Sun, Shujuan; Mo, Qingqing; Chen, Pingbo; Fang, Yong; Liu, Jia; Wang, Beibei; Zhou, Jianfeng; Ma, Ding; Wu, Peng

2013-01-01

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi) -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA) by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells. PMID:24367661

Rapid discovery of protein interactions by cell-free protein technologies.

PubMed

He, M; Taussig, M J

2007-11-01

Cell-free transcription and translation provides an open, controllable environment for production of correctly folded, soluble proteins and allows the rapid generation of proteins from DNA without the need for cloning. Thus it is becoming an increasingly attractive alternative to conventional in vivo expression systems, especially when parallel expression of multiple proteins is required. Through novel design and exploitation, powerful cell-free technologies of ribosome display and protein in situ arrays have been developed for in vitro production and isolation of protein-binding molecules from large libraries. These technologies can be combined for rapid detection of protein interactions.

Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4.

PubMed

Cao, Qing-Hua; Shao, Huan-Huan; Qiu, Hui; Li, Tao; Zhang, Yi-Zheng; Tan, Xue-Mei

2017-03-01

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.

Development of wound healing models to study TGFβ3's effect on SMA.

PubMed

Sriram, Sriniwas; Tran, Jennifer A; Guo, Xiaoqing; Hutcheon, Audrey E K; Kazlauskas, Andrius; Zieske, James D

2017-08-01

The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFβ3) in reducing α-smooth muscle actin (SMA) expression in two models-an ex vivo organ culture and an in vitro 3D cell construct-both of which closely mimic an in vivo environment. For the ex vivo organ culture system, a central 6.0 mm corneal keratectomy was performed on freshly excised rabbit globes The corneas were then excised, segregated into groups treated with 1.0 ng/ml TGFβ1 or β3 (T1 or T3, respectively), and cultured for 2 weeks. The corneas were assessed for levels of haze and analyzed for SMA mRNA levels. For the 3D in vitro model, rabbit corneal fibroblasts (RbCFs) were cultured for 4 weeks on poly-transwell membranes in Eagle's minimum essential media (EMEM) + 10% FBS + 0.5 mM vitamin C ± 0.1 ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and RT-qPCR. The RT-qPCR data showed that SMA mRNA expression in T3 samples for both models was significantly lower (p < 0.05) than T1 treatment (around 3-fold in ex vivo and 2-fold in constructs). T3 also reduced the amount of scarring in ex vivo corneas as compared with the T1 samples. IF data from RbCF constructs confirmed that T3-treated samples had up to 4-fold (p < 0.05) lower levels of SMA protein expression than samples treated with T1. These results show that T3 when compared to T1 decreases the expression of SMA in both ex vivo organ culture and in vitro 3D cell construct models. Understanding the mechanism of T3's action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti-scarring therapy. Copyright © 2017. Published by Elsevier Ltd.

CD4+ Memory Stem Cells Are Infected by HIV-1 in a Manner Regulated in Part by SAMHD1 Expression

PubMed Central

Tabler, Caroline O.; Lucera, Mark B.; Haqqani, Aiman A.; McDonald, David J.; Migueles, Stephen A.; Connors, Mark

2014-01-01

ABSTRACT CD4+ and CD8+ memory T cells with stem cell-like properties (TSCM cells) have been identified in mice, humans, and nonhuman primates and are being investigated for antitumor and antiviral vaccines and immunotherapies. Whether CD4+ TSCM cells are infected by human immunodeficiency virus (HIV) was investigated by using a combination HIV reporter virus system in vitro and by direct staining for HIV p24 antigen ex vivo. A proportion of TSCM cells were found to express the HIV coreceptors CCR5 and CXCR4 and were infected by HIV both in vitro and in vivo. Analysis of viral outcome following fusion using the combination reporter virus system revealed that TSCM cells can become productively or latently infected, although the vast majority of TSCM cells are abortively infected. Knockdown of the HIV restriction factor SAMHD1 using Vpx-containing simian immunodeficiency virus (SIV) virion-like particles enhanced the productive infection of TSCM cells, indicating that SAMHD1 contributes to abortive infection in these cells. These results demonstrate that CD4+ TSCM cells are targets for HIV infection, that they become productively or latently infected at low levels, and that SAMHD1 expression promotes abortive infection of this important memory cell subset. IMPORTANCE Here we demonstrate the susceptibility of CD4+ memory stem cells (TSCM cells) to infection by HIV in vitro and in vivo, provide an in-depth analysis of coreceptor expression, demonstrate the infection of naïve and memory CD4+ T cell subsets with both CCR5- and CXCR4-tropic HIV, and also perform outcome analysis to calculate the percentage of cells that are productively, latently, or abortively infected. Through these outcome studies, we determined that the vast majority of TSCM cells are abortively infected by HIV, and we demonstrate that knockdown of SAMHD1 significantly increases the frequency of infection of this CD4+ T cell subset, indicating that SAMHD1 is an active restriction factor in TSCM cells. PMID:24554663

Transcriptional Profiling of the Iron Starvation Response in Bordetella pertussis Provides New Insights into Siderophore Utilization and Virulence Gene Expression ▿ §

PubMed Central

Brickman, Timothy J.; Cummings, Craig A.; Liew, Sin-Yee; Relman, David A.; Armstrong, Sandra K.

2011-01-01

Serological studies of patients with pertussis and the identification of antigenic Bordetella pertussis proteins support the hypothesis that B. pertussis perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these B. pertussis iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in B. pertussis gene transcript abundance in response to iron starvation in vitro. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system, fbpABC, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both B. pertussis strain Tohama I and Bordetella bronchiseptica strain RB50. In a survey of type III secretion system protein production by an assortment of B. pertussis laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only Bordetella iron transport gene expression but also the expression of other important virulence-associated genes. PMID:21742863

Grazing dairy cows had decreased interferon-γ, tumor necrosis factor, and interleukin-17, and increased expression of interleukin-10 during the first week after calving.

PubMed

Heiser, Axel; McCarthy, Allison; Wedlock, Neil; Meier, Susanne; Kay, Jane; Walker, Caroline; Crookenden, Mallory A; Mitchell, Murray D; Morgan, Stuart; Watkins, Kate; Loor, Juan J; Roche, John R

2015-02-01

Peripartum, and especially during the transition period, dairy cows undergo dramatic physiological changes. These coincide with an increased risk of disease during the first 2 wk after calving and have been linked to dairy cows failing to achieve production as well as reproductive targets. Previous evidence suggests that these physiological changes affect the immune system and that transition dairy cows experience some form of reduced immunocompetence. However, almost all of these studies were undertaken in high-production, housed dairy cows. Grazing cows have much lower levels of production and this study aimed to provide clarity whether or not the dysfunctional attributes of the peripartum immune system reported in high production housed cows are evident in these animals. Therefore, cell culture techniques, flow cytometry, and quantitative PCR were applied to analyze the cellular composition of peripheral blood mononuclear cells from transition dairy cows as well as the performance of these cells in an in vitro assay. First, a combination of in vitro stimulation and quantitative PCR for cytokines was validated as a quantifiable immunocompetence assay in 29 cattle and a correlation of quantitative PCR and ELISA demonstrated. Second, the relative number of T helper cells, cytotoxic T cells, B cells, γδ T cells, natural killer cells, and monocytes in peripheral blood was measured, of which B cells and natural killer cells increased in number postcalving (n=29) compared with precalving. Third, following in vitro stimulation cytokine profiles indicated decreased expression of IFNγ, tumor necrosis factor, and IL-17 and increased expression of IL-10 wk 1 after calving, which later all returned to precalving values (n=39). Additionally, treatment of transition cows with a nonsteroidal anti-inflammatory drug (i.e., carprofen) administered on d 1, 3, and 5 postcalving (n=19; untreated control n=20) did not affect the cytokine expression at any time point. In conclusion, an immunocompetence assay has been developed that highlights a characteristic expression pattern for IFNγ, tumor necrosis factor, IL-17, and IL-10 that reflects a state of reduced immunocompetence in moderate-yielding pasture-based transition cows after calving, which is similar to that described for higher-yielding housed cows. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[Effect of Leonurus Heterophyllus Sweet on tissue factor transcription and expression in human umbilical vein endothelial cells in vitro].

PubMed

Zheng, Lian; Fang, Chi-hua

2007-06-01

To investigate the effect of Leonurus Heterophyllus Sweet, (LHS) on tissue factor (TF) transcription and expression induced by thrombin in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with different concentrations of LHS and the TF mRNA expression was detected by reverse transcript-polymerase chain reaction (RT-PCR). LHS treatment of HUVECs at different concentrations and for different times resulted in significant differences in TF expression (Plt;0.01). The transcription of TF in LHS-treated cells was significantly different from that of the blank control group (Plt;0.01). LHS can decrease the expression of TF and intervene with TF transcription in HUVECs in vitro.

Expression of cholera toxin under non-AKI conditions in Vibrio cholerae El Tor induced by increasing the exposed surface of cultures.

PubMed

Sánchez, Joaquín; Medina, Gerardo; Buhse, Thomas; Holmgren, Jan; Soberón-Chavez, Gloria

2004-03-01

The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

PubMed

Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

2016-09-01

Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. None. This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture

PubMed Central

Reda, A.; Hou, M.; Winton, T.R.; Chapin, R.E.; Söder, O.; Stukenborg, J.-B.

2016-01-01

STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA None. STUDY FUNDING AND COMPETING INTEREST(S) This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests. PMID:27430551

Ex vitro composite plants: an inexpensive, rapid method for root biology.

PubMed

Collier, Ray; Fuchs, Beth; Walter, Nathalie; Kevin Lutke, William; Taylor, Christopher G

2005-08-01

Plant transformation technology is frequently the rate-limiting step in gene function analysis in non-model plants. An important tool for root biologists is the Agrobacterium rhizogenes-derived composite plant, which has made possible genetic analyses in a wide variety of transformation recalcitrant dicotyledonous plants. The novel, rapid and inexpensive ex vitro method for producing composite plants described in this report represents a significant advance over existing composite plant induction protocols, which rely on expensive and time-consuming in vitro conditions. The utility of the new system is validated by expression and RNAi silencing of GFP in transgenic roots of composite plants, and is bolstered further by experimental disruption, via RNAi silencing, of endogenous plant resistance to the plant parasitic nematode Meloidogyne incognita in transgenic roots of Lycopersicon esculentum cv. Motelle composite plants. Critical parameters of the method are described and discussed herein.

Models and methods for in vitro testing of hepatic gap junctional communication.

PubMed

Maes, Michaël; Yanguas, Sara Crespo; Willebrords, Joost; Vinken, Mathieu

2015-12-25

Inherent to their pivotal roles in controlling all aspects of the liver cell life cycle, hepatocellular gap junctions are frequently disrupted upon impairment of the homeostatic balance, as occurs during liver toxicity. Hepatic gap junctions, which are mainly built up by connexin32, are specifically targeted by tumor promoters and epigenetic carcinogens. This renders inhibition of gap junction functionality a suitable indicator for the in vitro detection of nongenotoxic hepatocarcinogenicity. The establishment of a reliable liver gap junction inhibition assay for routine in vitro testing purposes requires a cellular system in which gap junctions are expressed at an in vivo-like level as well as an appropriate technique to probe gap junction activity. Both these models and methods are discussed in the current paper, thereby focusing on connexin32-based gap junctions. Copyright © 2015 Elsevier B.V. All rights reserved.

18β-Glycyrrhetinic Acid, a Novel Naturally Derived Agent, Suppresses Prolactin Hyperactivity and Reduces Antipsychotic-Induced Hyperprolactinemia in In Vitro and In Vivo Models.

PubMed

Wang, Di; Zhang, Yongfeng; Wang, Chunyue; Jia, Dongxu; Cai, Guangsheng; Lu, Jiahui; Wang, Di; Zhang, Zhang-Jin

2016-09-01

The purpose of this study was to examine the effects of 18β-glycyrrhetinic acid (GA), a novel naturally derived agent, in suppressing prolactin (PRL) hyperactivity and reducing antipsychotic-induced hyperprolactinemia (hyperPRL) and the underlying mechanisms in in vitro and in vivo models. GA treatment for 24 h inhibited PRL synthesis and secretion in MMQ cells and cultured pituitary cells in a dose-dependent fashion; but this effect was not reproduced in GH3 cells that lack the expression of functional dopamine D2 receptors. GA suppressed elevated PRL level and growth hormone, and normalized several sex hormones in a rat model of hyperPRL, produced by repeated injection of the dopamine blocker metoclopramide. GA also modulated the expression 5-HT1A and 5-HT2A receptors in both in vivo and in vitro models. These results indicate that GA is effective in suppressing PRL hyperactivity caused by the blockade of dopamine D2 receptors. This suppressive effect of GA may be related to its modulation of the serotonergic system. This study provides additional evidence in support of GA as an adjunct for the treatment of hyperPRL.

Emodin ameliorates high glucose induced-podocyte epithelial-mesenchymal transition in-vitro and in-vivo.

PubMed

Chen, Tingfang; Zheng, Li Yang; Xiao, Wenzhen; Gui, Dingkun; Wang, Xiaoxia; Wang, Niansong

2015-01-01

Epithelial-to-mesenchymal transition (EMT) is a potential pathway leading to podocyte depletion and proteinuria in diabetic kidney disease (DKD). Here, we investigated the protective effects of Emodin (EMO) on high glucose (HG) induced-podocyte EMT in-vitro and in-vivo. Conditionally immortalized mouse podocytes were exposed to HG with 30 μg /ml of EMO and 1 μmol/ml of integrin-linked kinase (ILK) inhibitor QLT0267 for 24 h. Streptozotocin (STZ)-induced diabetic rats were treated with EMO at 20 mg· kg(-1)· d(-1) and QLT0267 at 10 mg· kg(-1)· w(-1) p.o., for 12 weeks. Albuminuria and blood glucose level were measured. Immunohistochemistry, immunofluorescence, western blotting and real-time PCR were used to detect expression of ILK, the epithelial marker of nephrin and the mesenchymal marker of desmin in-vitro and in-vivo. HG increased podocyte ILK and desmin expression while decreased nephrin expression. However, EMO significantly inhibited ILK and desmin expression and partially restored nephrin expression in HG-stimulated podocytes. These in-vitro observations were further confirmed in-vivo. Treatment with EMO for 12 weeks attenuated albuminuria, renal histopathology and podocyte foot process effacement in diabetic rats. EMO also repressed renal ILK and desmin expression, preserved nephrin expression, as well as ameliorated albuminuria in STZ-induced diabetic rats. EMO ameliorated glucose-induced EMT and subsequent podocyte dysfunction partly through ILK and desmin inhibition as well as nephrin upregulatiotion, which might provide a potential novel therapeutic option for DKD. © 2015 S. Karger AG, Basel.

Cortisol increases CXCR4 expression but does not affect CD62L and CCR7 levels on specific T cell subsets in humans.

PubMed

Besedovsky, Luciana; Linz, Barbara; Dimitrov, Stoyan; Groch, Sabine; Born, Jan; Lange, Tanja

2014-06-01

Glucocorticoids are well known to affect T cell migration, leading to a redistribution of the cells from blood to the bone marrow, accompanied by a concurrent suppression of lymph node homing. Despite numerous studies in this context, with most of them employing synthetic glucocorticoids in nonphysiological doses, the mechanisms of this redistribution are not well understood. Here, we investigated in healthy men the impact of cortisol at physiological concentrations on the expression of different migration molecules on eight T cell subpopulations in vivo and in vitro. Hydrocortisone (cortisol, 22 mg) infused during nocturnal rest when endogenous cortisol levels are low, compared with placebo, differentially reduced numbers of T cell subsets, with naive CD4(+) and CD8(+) subsets exhibiting the strongest reduction. Hydrocortisone in vivo and in vitro increased CXCR4 expression, which presumably mediates the recruitment of T cells to the bone marrow. Expression of the lymph node homing receptor CD62L on total CD3(+) and CD8(+) T cells appeared reduced following hydrocortisone infusion. However, this was due to a selective extravasation of CD62L(+) T cell subsets, as hydrocortisone affected neither CD62L expression on a subpopulation level nor CD62L expression in vitro. Corresponding results in the opposite direction were observed after blocking of endogenous cortisol synthesis by metyrapone. CCR7, another lymph node homing receptor, was also unaffected by hydrocortisone in vitro. Thus, cortisol seems to redirect T cells to the bone marrow by upregulating their CXCR4 expression, whereas its inhibiting effect on T cell homing to lymph nodes is apparently regulated independently of the expression of classical homing receptors. Copyright © 2014 the American Physiological Society.

Oocyte formation by mitotically-active germ cells purified from ovaries of reproductive age women

PubMed Central

White, Yvonne A. R.; Woods, Dori C.; Takai, Yasushi; Ishihara, Osamu; Seki, Hiroyuki; Tilly, Jonathan L.

2012-01-01

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a FACS-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically-active cells that exhibit a gene expression profile consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and spontaneously generate 35–50 µm oocytes, as determined by morphology, gene expression and attainment of haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, like adult mice, possess rare mitotically-active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo. PMID:22366948

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women.

PubMed

White, Yvonne A R; Woods, Dori C; Takai, Yasushi; Ishihara, Osamu; Seki, Hiroyuki; Tilly, Jonathan L

2012-02-26

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

Cross-study projections of genomic biomarkers: an evaluation in cancer genomics.

PubMed

Lucas, Joseph E; Carvalho, Carlos M; Chen, Julia Ling-Yu; Chi, Jen-Tsan; West, Mike

2009-01-01

Human disease studies using DNA microarrays in both clinical/observational and experimental/controlled studies are having increasing impact on our understanding of the complexity of human diseases. A fundamental concept is the use of gene expression as a "common currency" that links the results of in vitro controlled experiments to in vivo observational human studies. Many studies--in cancer and other diseases--have shown promise in using in vitro cell manipulations to improve understanding of in vivo biology, but experiments often simply fail to reflect the enormous phenotypic variation seen in human diseases. We address this with a framework and methods to dissect, enhance and extend the in vivo utility of in vitro derived gene expression signatures. From an experimentally defined gene expression signature we use statistical factor analysis to generate multiple quantitative factors in human cancer gene expression data. These factors retain their relationship to the original, one-dimensional in vitro signature but better describe the diversity of in vivo biology. In a breast cancer analysis, we show that factors can reflect fundamentally different biological processes linked to molecular and clinical features of human cancers, and that in combination they can improve prediction of clinical outcomes.

Annexin A2 in Proliferative Vitreoretinopathy

DTIC Science & Technology

2016-10-01

migrate in the presence of macrophages in an in vitro system. In addition, analysis of human retinal tissue from subjects undergoing ocular surgery... tissue from subjects undergoing ocular surgery for PVR reveals the presence of A2- immunoreactive cells that express both macrophage and RPE cell...greatly attenuated in the absence of annexin A2. Task 2: Macrophage depletion and tissue specific knockout. We have completed the characterization

Presence of encircling granulosa cells protects against oxidative stress-induced apoptosis in rat eggs cultured in vitro.

PubMed

Tiwari, Meenakshi; Tripathi, Anima; Chaube, Shail K

2017-01-01

Increased oxidative stress (OS) due to in vitro culture conditions can affect the quality of denuded eggs during various assisted reproductive technologies (ARTs). Presence of intact granulosa cells may protect eggs from OS damage under in vitro culture conditions. The present study was aimed to investigate whether encircling granulosa cells could protect against hydrogen peroxide (H 2 O 2 )-induced egg apoptosis in ovulated cumulus oocyte complexes (COCs) cultured in vitro. The OS was induced by exposing COCs as well as denuded eggs with various concentrations of H 2 O 2 for 3 h in vitro. The morphological changes, total reactive oxygen species (ROS) as well as catalase expression, Bax/Bcl-2, cytochrome c levels and DNA fragmentation were analysed in COCs as well as denuded eggs. Our results suggest that H 2 O 2 treatment induced morphological apoptotic features in a concentration-dependent manner in denuded eggs cultured in vitro. The 20 µM of H 2 O 2 treatment induced OS by elevating total ROS level, reduced catalase and Bcl-2 expression levels with overexpression of Bax and cytochrome c and induced DNA fragmentation in denuded eggs cultured in vitro. The presence of encircling granulosa cells protected H 2 O 2 -induced morphological apoptotic features by preventing the increase of Bax, cytochrome c expression levels and DNA fragmentation in associated egg. However, 20 µM of H 2 O 2 was sufficient to induce peripheral granulosa cell apoptosis in COCs and degeneration in few denuded eggs cultured in vitro. Taken together our data suggest that the presence of encircling granulosa cells could be beneficial to protect ovulated eggs from OS damage under in vitro culture conditions during various ART programs.

Glucose-induced gradual phenotypic modulation of cultured human glomerular epithelial cells may be independent of Wilms’ tumor 1 (WT1)

PubMed Central

2013-01-01

Background Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. We used an in vitro model of human glomerular epithelial cells (HGEC) to investigate the role of high glucose in dysregulating the podocytic epithelial phenotype and determined the time needed for this change to occur. Results In our in vitro podocyte system changes indicating podocyte dedifferentiation in the prolonged presence of high glucose included loss of podocalyxin, nephrin and CD10/CALLA concomitant with upregulation of mesenchymal vimentin. Our study demonstrates for the first time that podocyte-specific markers undergo changes of expression at different time intervals, since glucose-mediated podocalyxin downregulation is a progressive process that precedes downregulation of nephrin expression. Finally we demonstrate that high glucose permanently impaired WT1 binding to the podocalyxin gene promoter region but did not affect WT1 binding on the nephrin gene promoter region. Conclusion The presence of high glucose induced a phenotypic conversion of podocytes resembling partial dedifferentiation. Our study demonstrates that dysregulation of the normal podocytic phenotype is an event differentially affecting the expression of function-specific podocytic markers, exhibiting downregulation of the epithelial marker CD10/CALLA and PC first, followed by stably downregulated nephrin. Furthermore, it is herein suggested that WT1 may not be directly involved with upregulation of previously reduced PC and nephrin expression. PMID:23768159

Establishment and culture optimization of a new type of pituitary immortalized cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kokubu, Yuko; Asashima, Makoto; Life Science Center of TARA, The University of Tsukuba, Ibaraki-ken 305-8577

The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells undermore » sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. - Highlights: • Mouse pituitary cell lines were immortalized by introducing TERT, E6, and E7. • The immortalized cell lines mainly expressed thyroid stimulating hormone beta. • The cell lines responded to PKA or PKC pathway activators, and induced Tshb.« less

Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints.

PubMed

Terrasso, Ana Paula; Pinto, Catarina; Serra, Margarida; Filipe, Augusto; Almeida, Susana; Ferreira, Ana Lúcia; Pedroso, Pedro; Brito, Catarina; Alves, Paula Marques

2015-07-10

There is an urgent need for new in vitro strategies to identify neurotoxic agents with speed, reliability and respect for animal welfare. Cell models should include distinct brain cell types and represent brain microenvironment to attain higher relevance. The main goal of this study was to develop and validate a human 3D neural model containing both neurons and glial cells, applicable for toxicity testing in high-throughput platforms. To achieve this, a scalable bioprocess for neural differentiation of human NTera2/cl.D1 cells in stirred culture systems was developed. Endpoints based on neuronal- and astrocytic-specific gene expression and functionality in 3D were implemented in multi-well format and used for toxicity assessment. The prototypical neurotoxicant acrylamide affected primarily neurons, impairing synaptic function; our results suggest that gene expression of the presynaptic marker synaptophysin can be used as sensitive endpoint. Chloramphenicol, described as neurotoxicant affected both cell types, with cytoskeleton markers' expression significantly reduced, particularly in astrocytes. In conclusion, a scalable and reproducible process for production of differentiated neurospheres enriched in mature neurons and functional astrocytes was obtained. This 3D approach allowed efficient production of large numbers of human differentiated neurospheres, which in combination with gene expression and functional endpoints are a powerful cell model to evaluate human neuronal and astrocytic toxicity. Copyright © 2014 Elsevier B.V. All rights reserved.

Increase of CD69, CD161 and CD94 on NK cells in women with recurrent spontaneous abortion and in vitro fertilization failure.

PubMed

Ghafourian, Mehri; Karami, Najmeh; Khodadadi, Ali; Nikbakht, Roshan

2014-06-01

Recurrent spontaneous abortion (RSA) and in vitro fertilization (IVF) failure with unknown causes are the controversial issues that are probably related to the immune system. To compare circulating NK cells expressing activation and inhibition surface markers between patients with RSA and IVF failure with those of healthy multiparous and successful IVF control women, respectively. In this case-control study peripheral blood samples were collected from 43 patients who included 23 women with RSA and 20 with IVF failure, plus 43 healthy control women comprising of 36 normal multiparous women and seven women with successful IVF. The expression of CD69, CD94 and CD161 surface markers on CD56+NK cells were assessed using specific monoclonal antibodies by flowcytometry. The percentage of NK cells increased significantly in patients with RSA and in women with IVF failure in comparison to healthy multiparous and successful IVF control groups (p<0.001). The overall expression of CD69, CD94, CD161 were also increased significantly on NK cells in both patient groups compared to control groups (p<0.001). Elevated expression of CD69 and CD161 on NK cells can be considered as immunological risk markers in RSA and IVF failure. However, it is not clear if high expression of CD94 on peripheral blood NK cells is related to abnormal activity of endometrial NK cells.

Resveratrol-cyclodextrin complex affects the expression of genes associated with lipid metabolism in bovine in vitro produced embryos.

PubMed

Torres, V; Hamdi, M; Millán de la Blanca, M G; Urrego, R; Echeverri, J; López-Herrera, A; Rizos, D; Gutiérrez-Adán, A; Sánchez-Calabuig, M J

2018-03-26

Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol-methyl β-cyclodextrin (RV-CD) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV-CD during in vitro oocyte maturation (IVM) or in vitro embryo culture (IVC) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV-CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT-qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV-CD and embryo development and blastocysts gene expression by RT-qPCR were studied. A group without RV-CD (control - ) and a group with cyclodextrin (control + ) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p < .05). Blastocysts produced by IVC with resveratrol showed that RV-CD could modify the expression of genes related to lipid metabolism (CYP51A1, PNPLA2 and MTORC1) compared with control groups (p < .05). RV-CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts. © 2018 Blackwell Verlag GmbH.

Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang

2011-07-15

Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blotmore » and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.« less

Knockdown of RhoA expression alters ovarian cancer biological behavior in vitro and in nude mice.

PubMed

Wang, Xiaoxia; Jiang, Wenyan; Kang, Jiali; Liu, Qicai; Nie, Miaoling

2015-08-01

RhoA regulates cell proliferation, migration, angiogenesis and gene expression. Altered RhoA activity contributes to cancer progression. The present study investigated the effects of RhoA knockdown on the regulation of ovarian cancer biological behavior in vitro and in nude mice. The expression of RhoA was knocked down using a lentivirus carrying RhoA short hairpin RNA (shRNA) in ovarian cancer cells and was confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The altered ovarian cancer biological behaviors were assayed by cell viability, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL), migration, invasion, and nude mice tumorigenicity assays, while the altered gene expression was detected by RT-qPCR and western blot analysis. The results showed that lentivirus-carrying RhoA shRNA significantly suppressed RhoA expression in ovarian cancer cells, which suppressed tumor cell viability, migration, invasion and adhesion in vitro. RhoA silencing also inhibited the tumorigenicity of ovarian cancer cells in nude mice, which was characterized by the suppression of tumor xenograft formation and growth and induction of tumor cell apoptosis. The results of the present study demonstrated that knockdown of RhoA expression had a significant antitumor effect on ovarian cancer cells in vitro and in nude mice, suggesting that RhoA may be a target for the development of a novel therapeutic strategy in the control of ovarian cancer.

Targeting FR-expressing cells in ovarian cancer with Fab-functionalized nanoparticles: a full study to provide the proof of principle from in vitro to in vivo

NASA Astrophysics Data System (ADS)

Quarta, Alessandra; Bernareggi, Davide; Benigni, Fabio; Luison, Elena; Nano, Giuseppe; Nitti, Simone; Cesta, Maria Candida; di Ciccio, Luciano; Canevari, Silvana; Pellegrino, Teresa; Figini, Mariangela

2015-01-01

Efficient targeting in tumor therapies is still an open issue: systemic biodistribution and poor specific accumulation of drugs weaken efficacy of treatments. Engineered nanoparticles are expected to bring benefits by allowing specific delivery of drug to the tumor or acting themselves as localized therapeutic agents. In this study we have targeted epithelial ovarian cancer with inorganic nanoparticles conjugated to a human antibody fragment against the folate receptor over-expressed on cancer cells. The conjugation approach is generally applicable. Indeed several types of nanoparticles (either magnetic or fluorescent) were engineered with the fragment, and their biological activity was preserved as demonstrated by biochemical methods in vitro. In vivo studies with mice bearing orthotopic and subcutaneous tumors were performed. Elemental and histological analyses showed that the conjugated magnetic nanoparticles accumulated specifically and were retained at tumor sites longer than the non-conjugated nanoparticles.Efficient targeting in tumor therapies is still an open issue: systemic biodistribution and poor specific accumulation of drugs weaken efficacy of treatments. Engineered nanoparticles are expected to bring benefits by allowing specific delivery of drug to the tumor or acting themselves as localized therapeutic agents. In this study we have targeted epithelial ovarian cancer with inorganic nanoparticles conjugated to a human antibody fragment against the folate receptor over-expressed on cancer cells. The conjugation approach is generally applicable. Indeed several types of nanoparticles (either magnetic or fluorescent) were engineered with the fragment, and their biological activity was preserved as demonstrated by biochemical methods in vitro. In vivo studies with mice bearing orthotopic and subcutaneous tumors were performed. Elemental and histological analyses showed that the conjugated magnetic nanoparticles accumulated specifically and were retained at tumor sites longer than the non-conjugated nanoparticles. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr04426f

Impact of combined sodium chloride and saturated long-chain fatty acid challenge on the differentiation of T helper cells in neuroinflammation.

PubMed

Hammer, Anna; Schliep, Anne; Jörg, Stefanie; Haghikia, Aiden; Gold, Ralf; Kleinewietfeld, Markus; Müller, Dominik N; Linker, Ralf A

2017-09-12

There has been a marked increase in the incidence of autoimmune diseases like multiple sclerosis (MS) in the last decades which is most likely driven by a change in environmental factors. Here, growing evidence suggests that ingredients of a Western diet like high intake of sodium chloride (NaCl) or saturated fatty acids may impact systemic immune responses, thus increasing disease susceptibility. Recently, we have shown that high dietary salt or long-chain fatty acid (LCFA) intake indeed aggravates T helper (Th) cell responses and neuroinflammation. Naïve CD4 + T cells were treated with an excess of 40 mM NaCl and/or 250 μM lauric acid (LA) in vitro to analyze effects on Th cell differentiation, cytokine secretion, and gene expression. We employed ex vivo analyses of the model disease murine experimental autoimmune encephalomyelitis (EAE) to investigate whether salt and LCFA may affect disease severity and T cell activation in vivo. LCFA, like LA, together with NaCl enhance the differentiation of Th1 and Th17 cells as well as pro-inflammatory cytokine and gene expression in vitro. In cell culture, we observed an additive effect of LA and hypertonic extracellular NaCl (NaCl + LA) in Th17 differentiation assays as well as on IL-17, GM-CSF, and IL-2 gene expression. In contrast, NaCl + LA reduced Th2 frequencies. We employed EAE as a model of Th1/Th17 cell-mediated autoimmunity and show that the combination of a NaCl- and LA-rich diet aggravated the disease course and increased T cell infiltration into the central nervous system (CNS) to the same extent as dietary NaCl. Our findings demonstrate a partially additive effect of NaCl and LA on Th cell polarization in vitro and on Th cell responses in autoimmune neuroinflammation. These data may help to better understand the pathophysiology of autoimmune diseases such as MS.

Mechanical tensile stress effects on the expression of bone sialoprotein in bovine cementoblasts.

PubMed

Yu, Hongyou; Ren, Yijin; Sandham, Andrew; Ren, Aishu; Huang, Lan; Bai, Ding

2009-03-01

To develop a new cementoblast culture method and to detect bone sialoprotein (BSP) expression in response to high and low mechanical tensile stress in cementoblast in vitro. Cementoblasts were collected from the roots of newborn bovine teeth and were identified with cementum-derived attachment protein (CAP) antibody 3G9. Cell proliferation was evaluated by MTT [3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, and mineralization was confirmed by von Kossa staining. Mechanical tensile stress was applied in vitro to the cementoblast with the use of a uniaxial four-point bending system with 2000 or 4000 microstrains, at a frequency of 0.5 Hz for 3, 6, 12, 24, or 36 hours. BSP mRNA level was quantified by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). A large amount of cementoblast was observed to be expressing CAP. Cementoblasts had a proliferation tendency similar to that of osteoblasts but different from that of periodontal ligament (PDL) cells. Cementoblasts had the ability to become mineralized between osteoblasts and PDL cells. The mechanical tensile stress significantly up-regulated BSP mRNA expression, which reached a peak at 24 hours in both 2000 and 4000 microstrain groups (P < .01) and was tenfold and sixfold higher than that of controls, respectively. BSP expression dropped toward baseline levels at 36 hours in both groups. Mechanical tensile stress up-regulated the expression of BSP. Low mechanical tensile stress induced earlier and more intensive up-regulation of BSP mRNA; this might represent the optimal stimuli for cementoblast activity.

Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique

PubMed Central

Auth, Marcus K.H.; Boost, Kim A.; Leckel, Kerstin; Beecken, Wolf-Dietrich; Engl, Tobias; Jonas, Dietger; Oppermann, Elsie; Hilgard, Philip; Markus, Bernd H.; Bechstein, Wolf-Otto; Blaheta, Roman A.

2005-01-01

AIM: Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors. PMID:15810072

Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique.

PubMed

Auth, Marcus-K H; Boost, Kim A; Leckel, Kerstin; Beecken, Wolf-Dietrich; Engl, Tobias; Jonas, Dietger; Oppermann, Elsie; Hilgard, Philip; Markus, Bernd H; Bechstein, Wolf-Otto; Blaheta, Roman A

2005-04-14

Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors.

Immunostimulant effects and potential application of β-glucans derived from marine yeast Debaryomyces hansenii in goat peripheral blood leucocytes.

PubMed

Medina-Córdova, Noé; Reyes-Becerril, Martha; Ascencio, Felipe; Castellanos, Thelma; Campa-Córdova, Angel I; Angulo, Carlos

2018-05-12

Debaryomyces hansenii has been described to be effective probiotic and immunostimulatory marine yeast in fish. Nonetheless, to the best of our knowledge, it has been not assayed in ruminants. This study attempts to describe the immunostimulatory effects of its β-glucan content through in vitro assays using goat peripheral blood leukocytes at 24 h of stimulation. The structural characterization of yeast glucans by proton nuclear magnetic resonance indicated structures containing (1-6)-branched (1-3)-β-D-glucan. In vitro assays using peripheral blood leukocytes stimulated with β-glucans derived from three D. hansenii strains and zymosan revealed that β-glucans significantly increased cell immune parameters, such as phagocytic ability, reactive oxygen species production (respiratory burst), peroxidase activity and nitric oxide production. Antioxidant enzymes revealed an increase in superoxide dismutase and catalase activities in leukocytes stimulated with yeast β-glucans. This study revealed that yeast β-glucans were able to activate dectin-1 mRNA gene expression in leukocytes. The TLR4 gene expression was up-regulated in leukocytes after stimulation with yeast β-glucans. In conclusion, β-glucans were able to modulate the immune system by promoting cell viability, phagocytic activity, antioxidant immune response and immune-related gene expression in leukocytes. Therefore, β-glucans derived from Debaryomyces hansenii should be considered a potential immunostimulant for goat production systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Endosulfan inhibiting the meiosis process via depressing expressions of regulatory factors and causing cell cycle arrest in spermatogenic cells.

PubMed

Guo, Fang-Zi; Zhang, Lian-Shuang; Wei, Jia-Liu; Ren, Li-Hua; Zhang, Jin; Jing, Li; Yang, Man; Wang, Ji; Sun, Zhi-Wei; Zhou, Xian-Qing

2016-10-01

Endosulfan is a persistent organic pollutant and widely used in agriculture as a pesticide. It is present in air, water, and soil worldwide; therefore, it is a health risk affecting especially the reproductive system. The aim of this study was to evaluate the toxicity of endosulfan in the reproductive system. To investigate the effect of endosulfan on meiosis process, 32 rats were divided into four groups, treated with 0, 1, 5, and 10 mg/kg/day endosulfan, respectively, and sacrificed after the 21 days of treatments. Results show that endosulfan caused the reductions in sperm concentration and motility rate, which resulted into an increased in sperm abnormality rate; further, endosulfan induced downregulation of spermatogenesis- and oogenesis-specific basic helix-loop-helix transcription factor (Sohlh1) which controls the switch on meiosis in mammals, as well cyclin A1, cyclin-dependent kinases 1 (CDK1), and cyclin-dependent kinases 2 (CDK2). In vitro, endosulfan induced G2/M phase arrest in the spermatogenic cell cycle and caused proliferation inhibition. Moreover, endosulfan induced oxidative stress and DNA damage in vivo and vitro. The results suggested that endosulfan could inhibit the start of meiosis by downregulating the expression of Sohlh1 and induce G2/M phase arrest of cell cycle by decreasing the expression of cyclin A1, CDK1, and CDK2 via oxidative damage, which inhibits the meiosis process, and therefore decrease the amount of sperm.

3,3',5-Triiodo-L-thyronine-like activity in effluents from domestic sewage treatment plants detected by in vitro and in vivo bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murata, Tomonori; Yamauchi, Kiyoshi

2008-02-01

Thyroid system-disrupting activity in effluents from municipal domestic sewage treatment plants was detected using three in vitro assays and one in vivo assay. Contaminants in the effluents were extracted by solid-phase extraction (SPE) and eluted stepwise with different organic solvents. The majority of the thyroid system-disrupting activity was detected in the dichloromethane/methanol (1/1) fraction after SPE in all three in vitro assays: competitive assays of 3,3',5-[{sup 125}I]triiodo-L-thyronine ([{sup 125}I]T{sub 3}) binding to the plasma protein transthyretin (TTR assay) and thyroid hormone receptor (TR assay) and T{sub 3}-dependent luciferase assay (Luc assay). Subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) of the dichloromethane/methanolmore » (1/1) fraction separated contaminants potent in the TR and Luc assays from those potent in the TTR assay. The contaminants potent in the TR and Luc assays were also potent in an in vivo short-term gene expression assay in Xenopus laevis (Tadpole assay). The present study demonstrated that the effluents from domestic sewage treatment plants contain contaminants with T{sub 3}-like activity of {approx} 10{sup -10} M T{sub 3}-equivalent concentration (T{sub 3}EQ) and that the TR and Luc assays are powerful in vitro bioassays for detecting thyroid system-disrupting activity in effluents. The availability and applicability of these bioassays for screening contaminants with thyroid system-disrupting activity in the water environment are discussed.« less

In Vitro Interleukin-1 and 2 Production and Interleukin 2 Receptor Expression in the Rhesus Monkey

NASA Technical Reports Server (NTRS)

Schmitt, Didier A.; Sonnenfeld, Gerald; Husson, David; Tkaczuk, Jean; Andre, Eric; Schaffar, Laurance

1996-01-01

Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.

In vitro decidualisation of canine uterine stromal cells.

PubMed

Kautz, Ewa; de Carvalho Papa, Paula; Reichler, Iris M; Gram, Aykut; Boos, Alois; Kowalewski, Mariusz P

2015-08-05

The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology. Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n = 6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated. The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ERα were observed in decidualised cells; PRL and ERβ remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle α actin (αSMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable. An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.

RNA-Seq transcriptome profiling of mouse oocytes after in vitro maturation and/or vitrification.

PubMed

Gao, Lei; Jia, Gongxue; Li, Ai; Ma, Haojia; Huang, Zhengyuan; Zhu, Shien; Hou, Yunpeng; Fu, Xiangwei

2017-10-16

In vitro maturation (IVM) and vitrification have been widely used to prepare oocytes before fertilization; however, potential effects of these procedures, such as expression profile changes, are poorly understood. In this study, mouse oocytes were divided into four groups and subjected to combinations of in vitro maturation and/or vitrification treatments. RNA-seq and in silico pathway analysis were used to identify differentially expressed genes (DEGs) that may be involved in oocyte viability after in vitro maturation and/or vitrification. Our results showed that 1) 69 genes were differentially expressed after IVM, 66 of which were up-regulated. Atp5e and Atp5o were enriched in the most significant gene ontology term "mitochondrial membrane part"; thus, these genes may be promising candidate biomarkers for oocyte viability after IVM. 2) The influence of vitrification on the transcriptome of oocytes was negligible, as no DEGs were found between vitrified and fresh oocytes. 3) The MII stage is more suitable for oocyte vitrification with respect to the transcriptome. This study provides a valuable new theoretical basis to further improve the efficiency of in vitro maturation and/or oocyte vitrification.

Prediction of in vivo hepatotoxicity effects using in vitro transcriptomics data (SOT)

EPA Science Inventory

High-throughput in vitro transcriptomics data support molecular understanding of chemical-induced toxicity. Here, we evaluated the utility of such data to predict liver toxicity. First, in vitro gene expression data for 93 genes was generated following exposure of metabolically c...

Activated entomopathogenic nematode infective juveniles release lethal venom proteins

PubMed Central

Macchietto, Marissa; Baldwin, James; Mortazavi, Ali

2017-01-01

Entomopathogenic nematodes (EPNs) are unique parasites due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. It is widely believed that EPNs rely on their bacterial partners for killing hosts. Here we disproved this theory by demonstrating that the in vitro activated infective juveniles (IJs) of Steinernema carpocapsae (a well-studied EPN species) release venom proteins that are lethal to several insects including Drosophila melanogaster. We confirmed that the in vitro activation is a good approximation of the in vivo process by comparing the transcriptomes of individual in vitro and in vivo activated IJs. We further analyzed the transcriptomes of non-activated and activated IJs and revealed a dramatic shift in gene expression during IJ activation. We also analyzed the venom proteome using mass spectrometry. Among the 472 venom proteins, proteases and protease inhibitors are especially abundant, and toxin-related proteins such as Shk domain-containing proteins and fatty acid- and retinol-binding proteins are also detected, which are potential candidates for suppressing the host immune system. Many of the venom proteins have conserved orthologs in vertebrate-parasitic nematodes and are differentially expressed during IJ activation, suggesting conserved functions in nematode parasitism. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of excretory/secretory products. PMID:28426766

CSAHi study-2: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information.

PubMed

Nozaki, Yumiko; Honda, Yayoi; Watanabe, Hitoshi; Saiki, Shota; Koyabu, Kiyotaka; Itoh, Tetsuji; Nagasawa, Chiho; Nakamori, Chiaki; Nakayama, Chiaki; Iwasaki, Hiroshi; Suzuki, Shinobu; Tanaka, Kohji; Takahashi, Etsushi; Miyamoto, Kaori; Morimura, Kaoru; Yamanishi, Atsuhiro; Endo, Hiroko; Shinozaki, Junko; Nogawa, Hisashi; Shinozawa, Tadahiro; Saito, Fumiyo; Kunimatsu, Takeshi

2017-08-01

With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lot-to-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period. The hiPS-CMs/MEA assay accurately predicted reference compounds potential for arrhythmogenesis, and yielded results that showed better correlation with target concentrations of QTc prolongation or TdP in clinical setting than other current in vitro and in vivo assays. Gene expression analyses revealed consistent profiles in all samples within and among the testing facilities. This report would provide CiPA with informative guidance on the use of the hiPS-CMs/MEA assay, and promote the establishment of a new paradigm, beyond conventional in vitro and in vivo assays for cardiac safety assessment of new drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

Sulforaphane alleviates scopolamine-induced memory impairment in mice.

PubMed

Lee, Siyoung; Kim, Jisung; Seo, Sang Gwon; Choi, Bo-Ryoung; Han, Jung-Soo; Lee, Ki Won; Kim, Jiyoung

2014-07-01

Sulforaphane, an organosulfur compound present in cruciferous vegetables, has been shown to exert neuroprotective effects in experimental in vitro and in vivo models of neurodegeneration. To determine whether sulforaphane can preserve cognitive function, we examined its effects on scopolamine-induced memory impairment in mice using the Morris water maze test. Sulforaphane (10 or 50mg/kg) was administered to C57BL/6 mice by oral gavage for 14 days (days 1-14), and memory impairment was induced by intraperitoneal injection of scopolamine (1mg/kg) for 7 days (days 8-14). Mice that received scopolamine alone showed impaired learning and memory retention and considerably decreased cholinergic system reactivity in the hippocampus and frontal cortex, as indicated by a decreased acetylcholine (ACh) level and an increased acetylcholinesterase (AChE) activity. Sulforaphane significantly attenuated the scopolamine-induced memory impairment and improved cholinergic system reactivity, as indicated by an increased ACh level, decreased AChE activity, and increased choline acetyltransferase (ChAT) expression in the hippocampus and frontal cortex. These effects of sulforaphane on cholinergic system reactivity were confirmed in vitro. Sulforaphane (10 or 20μM) increased the ACh level, decreased the AChE activity, and increased ChAT expression in scopolamine-treated primary cortical neurons. These observations suggest that sulforaphane might exert a significant neuroprotective effect on cholinergic deficit and cognitive impairment. Copyright © 2014. Published by Elsevier Ltd.

Three-dimensional culture of a mixed mullerian tumor of the ovary: expression of in vivo characteristics

NASA Technical Reports Server (NTRS)

Goodwin, T. J.; Prewett, T. L.; Spaulding, G. F.; Becker, J. L.

1997-01-01

The Rotating-Wall Vessel (RWV) is a novel in vitro cell culture system used to successfully culture a cell line derived from a heterologous mixed mullerian tumor cell of the ovary. Although the original tumor was comprised of both epithelial and mesodermal components, long-term culture in conventional flasks established a cell line from this tumor with homogeneous epitheliallike growth characteristics (1). Cells from Passage 36 were seeded into a Rotating-Wall Vessel containing Cytodex-3 microcarrier beads. Scanning electron micrographs of tumor cells cultured for 32 d in the RWV showed the presence of heterogeneous cell populations organized into three-dimensional tissuelike architecture. Immunocytochemical analysis confirmed the cellular heterogeneity, as demonstrated by expression of both epithelial and mesenchymal antigens. Reverse transcription polymerase chain reaction amplification demonstrated the presence of mRNA for cellular oncogenes HER-2/neu, H-ras, K-ras, and tumor suppressor p53. Thus, there are two advantages to propagation of tissue in the RWV culture system:(a) tissue diversification representing populations present in the original tumor, and (b) the three-dimensional freedom to organize tissues morphologically akin to those observed in vivo. These data indicate that the RWV culture system is suitable for generating large quantities of ovarian tumor cells in vitro that are amenable to immunocytochemical, oncogenic, morphologic characteristics demonstrated in vivo.

New insights into the reduction systems of plastidial thioredoxins point out the unique properties of thioredoxin z from Arabidopsis.

PubMed

Bohrer, Anne-Sophie; Massot, Vincent; Innocenti, Gilles; Reichheld, Jean-Philippe; Issakidis-Bourguet, Emmanuelle; Vanacker, Hélène

2012-11-01

In plants, thioredoxins (TRX) constitute a large protein disulphide oxidoreductase family comprising 10 plastidial members in Arabidopsis thaliana and subdivided in five types. The f- and m-types regulate enzymes involved mainly in carbon metabolism whereas the x, y, and z types have an antioxidant function. The reduction of TRXm and f in chloroplasts is performed in the light by ferredoxin:thioredoxin reductase (FTR) that uses photosynthetically reduced ferredoxin (Fd) as a reductant. The reduction system of Arabidopsis TRXx, y, and z has never been demonstrated. Recently, a gene encoding an atypical plastidial NADPH-dependent TRX reductase (NTRC) was found. In the present study, gene expression analysis revealed that both reductases are expressed in all organs of Arabidopsis and could potentially serve as electron donors to plastidial TRX. This ability was tested in vitro either with purified NTRC in presence of NADPH or with a light-driven reconstituted system comprising thylakoids and purified Fd and FTR. The results demonstrate that FTR reduces the x and y TRX isoforms but not the recently identified TRXz. Moreover, the results show that NTRC cannot be an efficient alternative reducing system, neither for TRXz nor for the other plastidial TRX. The data reveal that TRXf, m, x, and y, known as redox regulators in the chloroplast, have also the ability to reduce TRXz in vitro. Overall, the present study points out the unique properties of TRXz among plastidial TRX.

Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: preparation, characterization, intracellular trafficking, and cellular distribution

PubMed Central

Fan, Wei; Wu, Xin; Ding, Baoyue; Gao, Jing; Cai, Zhen; Zhang, Wei; Yin, Dongfeng; Wang, Xiang; Zhu, Quangang; Liu, Jiyong; Ding, Xueying; Gao, Shen

2012-01-01

Background Cationic copolymers consisting of polycations linked to nonionic amphiphilic block polymers have been evaluated as nonviral gene delivery systems, and a large number of different polymers and copolymers of linear, branched, and dendrimeric architectures have been tested in terms of their suitability and efficacy for in vitro and in vivo transfection. However, the discovery of new potent materials still largely relies on empiric approaches rather than a rational design. The authors investigated the relationship between the polymers’ structures and their biological performance, including DNA compaction, toxicity, transfection efficiency, and the effect of cellular uptake. Methods This article reports the synthesis and characterization of a series of cationic copolymers obtained by grafting polyethyleneimine with nonionic amphiphilic surfactant polyether-Pluronic® consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks. Transgene expression, cytotoxicity, localization of plasmids, and cellular uptake of these copolymers were evaluated following in vitro transfection of HeLa cell lines with various individual components of the copolymers. Results Pluronics can exhibit biological activity including effects on enhancing DNA cellular uptake, nuclear translocation, and gene expression. The Pluronics with a higher hydrophilic-lipophilic balance value lead to homogeneous distribution in the cytoplasm; those with a lower hydrophilic-lipophilic balance value prefer to localize in the nucleus. Conclusion This Pluronic-polyethyleneimine system may be worth exploring as components in the cationic copolymers as the DNA or small interfering RNA/microRNA delivery system in the near future. PMID:22403492

Expression of membrane-associated proteins within single emulsion cell facsimiles.

PubMed

Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

2012-07-07

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

LIN28A enhances the therapeutic potential of cultured neural stem cells in a Parkinson's disease model.

PubMed

Rhee, Yong-Hee; Kim, Tae-Ho; Jo, A-Young; Chang, Mi-Yoon; Park, Chang-Hwan; Kim, Sang-Mi; Song, Jae-Jin; Oh, Sang-Min; Yi, Sang-Hoon; Kim, Hyeon Ho; You, Bo-Hyun; Nam, Jin-Wu; Lee, Sang-Hun

2016-10-01

The original properties of tissue-specific stem cells, regardless of their tissue origins, are inevitably altered during in vitro culturing, lessening the clinical and research utility of stem cell cultures. Specifically, neural stem cells derived from the ventral midbrain lose their dopamine neurogenic potential, ventral midbrain-specific phenotypes, and repair capacity during in vitro cell expansion, all of which are critical concerns in using the cultured neural stem cells in therapeutic approaches for Parkinson's disease. In this study, we observed that the culture-dependent changes of neural stem cells derived from the ventral midbrain coincided with loss of RNA-binding protein LIN28A expression. When LIN28A expression was forced and sustained during neural stem cell expansion using an inducible expression-vector system, loss of dopamine neurogenic potential and midbrain phenotypes after long-term culturing was blocked. Furthermore, dopamine neurons that differentiated from neural stem cells exhibited remarkable survival and resistance against toxic insults. The observed effects were not due to a direct action of LIN28A on the differentiated dopamine neurons, but rather its action on precursor neural stem cells as exogene expression was switched off in the differentiating/differentiated cultures. Remarkable and reproducible behavioural recovery was shown in all Parkinson's disease rats grafted with neural stem cells expanded with LIN28A expression, along with extensive engraftment of dopamine neurons expressing mature neuronal and midbrain-specific markers. These findings suggest that LIN28A expression during stem cell expansion could be used to prepare therapeutically competent donor cells. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Precision-cut liver slices of Atlantic cod (Gadus morhua): an in vitro system for studying the effects of environmental contaminants.

PubMed

Eide, M; Karlsen, O A; Kryvi, H; Olsvik, P A; Goksøyr, A

2014-08-01

The Atlantic cod (Gadus morhua) is an economically important species commonly consumed by humans. The widespread distribution of cod in the North Atlantic Ocean makes it vulnerable to effluents from human activities, such as coastal industries and offshore petroleum exploration. It has been demonstrated that many effluents have adverse effects on cod reproduction and health, e.g. by disrupting endocrine signaling pathways. The liver, expressing important components of the biotransformation and the endocrine system, is one of the main target organs. Thus, reliable and reproducible in vitro systems of the liver are important for studying effects of environmental contaminants. The aim of this study was to investigate precision-cut liver slices (PCLS) as an alternative in vitro system for toxicological studies of the Atlantic cod liver. Slices of 8 mm in diameter and 250 μm thickness were prepared and cultivated from immature cod. Several analyses to measure the liver slice viability were performed: enzyme assays, histology, and morphometric analysis, all confirming cell viability for up to 72 h in culture. The liver slices were also exposed to two well-known model environmental contaminants, β-naphthoflavone (BNF) and 17α-ethynylestradiol (EE2), representing established agonists for the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER), respectively. The results showed increased transcription of the target genes cytochrome P450 1A (CYP1A) and vitellogenin (VTG), both well-established biomarkers for exposure of fish to the selected compounds. In conclusion, PCLS is a promising in vitro system for toxicological studies of cod liver cells. The liver slices are viable in culture for several days and respond to environmental contaminants in a dose- and time-specific manner. Copyright © 2013 Elsevier B.V. All rights reserved.

MiR-26b Mimic Inhibits Glioma Proliferation In Vitro and In Vivo Suppressing COX-2 Expression.

PubMed

Chen, Zheng-Gang; Zheng, Chuan-Yi; Cai, Wang-Qing; Li, Da-Wei; Ye, Fu-Yue; Zhou, Jian; Wu, Ran; Yang, Kun

2017-08-11

Glioma is the most common malignant tumor of the nervous system. Studies have shown the microRNA (miR)-26b/cyclooxygenase (COX)-2 axis in the development and progression in many tumor cells. Our study aims to investigate the effect and mechanism of miR-26b/COX-2 axis in glioma. Decreased expression of miR-26b with increased level of COX-2 was found in glioma tissues compared with matched normal tissues. A strong negative correlation was observed between the level of miR-26b and COX-2 in 30 glioma tissues. The miR-26b was then overexpressed by transfecting miR-26b mimic into U-373 cells. The invasive cell number and wounld closing rate were reduced in U-373 cells transfected with miR-26b mimic. Besides, COX2 siRNA enhanced the effect of miR-26b mimic in suppressing the expression of p-ERK1 and p-JNK. Finally, the in vivo experiment revealed that miR-26b mimic transfection strongly reduced the tumor growth, tumor volume and the expression of matrix metalloproteinase (MMP)-2, MMP-9). Taken together, our research indicated a miR-26b/COX-2/ERK/JNK axis in regulating the motility of glioma in vitro and in vivo, providing a new sight for treatment of glioma.

Complete TCRα gene locus control region activity in T cells derived in vitro from embryonic stem cells

PubMed Central

Lahiji, Armin; Kučerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D.

2013-01-01

Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin’s gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein we report manifestation of all key features of mouse T cell receptor (TCR)-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC). High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and upregulated during the expected stage transition of T cell development. We further report that de novo introduction of TCRα LCR linked transgenes into existing T cell lines yields incomplete LCR activity. Together, these data indicate that establishing full TCRα LCR activity requires critical molecular events occurring prior to final T-lineage determination. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering. PMID:23720809

A new and efficient culture method for porcine bone marrow-derived M1- and M2-polarized macrophages.

PubMed

Gao, Jiye; Scheenstra, Maaike R; van Dijk, Albert; Veldhuizen, Edwin J A; Haagsman, Henk P

2018-06-01

Macrophages play an important role in the innate immune system as part of the mononuclear phagocyte system (MPS). They have a pro-inflammatory signature (M1-polarized macrophages) or anti-inflammatory signature (M2-polarized macrophages) based on expression of surface receptors and secretion of cytokines. However, very little is known about the culture of macrophages from pigs and more specific about the M1 and M2 polarization in vitro. Porcine monocytes or mononuclear bone marrow cells were used to culture M1- and M2-polarized macrophages in the presence of GM-CSF and M-CSF, respectively. Surface receptor expression was measured with flow cytometry and ELISA was used to quantify cytokine secretion in response to LPS and PAM 3 CSK 4 stimulation. Human monocyte-derived macrophages were used as control. Porcine M1- and M2-polarized macrophages were cultured best using porcine GM-CSF and murine M-CSF, respectively. Cultures from bone marrow cells resulted in a higher yield M1- and M2-polarized macrophages which were better comparable to human monocyte-derived macrophages than cultures from porcine monocytes. Porcine M1-polarized macrophages displayed the characteristic fried egg shape morphology, lower CD163 expression and low IL-10 production. Porcine M2-polarized macrophages contained the spindle-like morphology, higher CD163 expression and high IL-10 production. Porcine M1- and M2-polarized macrophages can be most efficiently cultured from mononuclear bone marrow cells using porcine GM-CSF and murine M-CSF. The new culture method facilitates more refined studies of porcine macrophages in vitro, important for both porcine and human health since pigs are increasingly used as model for translational research. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Balance between activating NKG2D, DNAM-1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts.

PubMed

Nielsen, Natasja; Pascal, Veronique; Fasth, Andreas E R; Sundström, Yvonne; Galsgaard, Elisabeth D; Ahern, David; Andersen, Martin; Baslund, Bo; Bartels, Else M; Bliddal, Henning; Feldmann, Marc; Malmström, Vivianne; Berg, Louise; Spee, Pieter; Söderström, Kalle

2014-08-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast-like synoviocytes (FLS) are central components of the aggressive, tumour-like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA-FLS and NK cells. We used cultured RA-FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA-FLS/NK cell cross-talk. We show that RA-FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM-1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA-FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA-E expressed on RA-FLS further enhanced Nishi cell degranulation in co-culture with RA-FLS. Using cultured RA-FLS and the human NK cell line Nishi as an in vitro model system of RA-FLS/NK cell cross-talk, our results suggest that cell-mediated cytotoxicity of RA-FLS may be one mechanism by which NK cells influence local joint inflammation in RA. © 2014 John Wiley & Sons Ltd.

Balance between activating NKG2D, DNAM-1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

PubMed Central

Nielsen, Natasja; Pascal, Veronique; Fasth, Andreas E R; Sundström, Yvonne; Galsgaard, Elisabeth D; Ahern, David; Andersen, Martin; Baslund, Bo; Bartels, Else M; Bliddal, Henning; Feldmann, Marc; Malmström, Vivianne; Berg, Louise; Spee, Pieter; Söderström, Kalle

2014-01-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast-like synoviocytes (FLS) are central components of the aggressive, tumour-like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA-FLS and NK cells. We used cultured RA-FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA-FLS/NK cell cross-talk. We show that RA-FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM-1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA-FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA-E expressed on RA-FLS further enhanced Nishi cell degranulation in co-culture with RA-FLS. Using cultured RA-FLS and the human NK cell line Nishi as an in vitro model system of RA-FLS/NK cell cross-talk, our results suggest that cell-mediated cytotoxicity of RA-FLS may be one mechanism by which NK cells influence local joint inflammation in RA. PMID:24673109

Evaluating antithrombotic activity of HY023016 on rat hypercoagulable model.

PubMed

Chen, Qiu-Fang; Li, Yun-Zhan; Wang, Xin-Hui; Su, You-Rui; Cui, Shuang; Miao, Ming-Xing; Jiang, Zhen-Zhou; Jiang, Mei-Ling; Jiang, Ai-Dou; Chen, Xiang; Xu, Yun-Gen; Gong, Guo-Qing

2016-06-15

The generation of thrombus is not considered as an isolated progression without other pathologic processes, which may also enhance procoagulant state. The purpose of this study was to assess whether HY023016, a novel dabigatran prodrug and an oral direct thrombin inhibitor, or dabigatran etexilate, another thrombin inhibitor can improve the state of whole blood hypercoagulability in vitro/vivo. By using whole blood flow cytometry we explored the effects of HY023016 and dabigatran etexilate on thrombin and ADP-induced human platelet-leukocyte aggregation generated in vitro. With the method of continuous infusion of thrombin intravenous, we successfully established a rat hypercoagulable model and evaluated the effect of HY023016 or dabigatran etexilate in vivo. HY023016 was able to inhibit thrombin- or ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates in dose-dependent manner. Dabigatran etexilate was unable to affect ADP-induced platelet P-selectin or CD40L expression, leukocyte CD11b expression and formation of platelet-leukocyte aggregates. Based on rat hypercoagulable model, dabigatran etexilate could reverse thrombin-induced circulatory system hypercoagulable state in a concentration-dependent manner. Dabigatran etexilate also inhibited electrical stimulation induced formation of arterial thrombus in rat under hypercoagulable state, and extracorporal circulation-induced formation of thrombus in dose-dependent manner. Compared with dabigatran etexilate, HY023016 showed nearly equal or even better antithrombotic activity, regardless of reversing the cycle of rat hypercoagulable state or inhibiting platelet-leukocyte aggregation. In surrmary, HY023016 could effectively improve hypercoagulable state of circulatory system. Copyright © 2016. Published by Elsevier B.V.

Bovine oocyte meiotic inhibition before in vitro maturation and its value to in vitro embryo production: does it improve developmental competence?

PubMed

Bilodeau-Goeseels, S

2012-08-01

The efficiency of bovine in vitro embryo production has remained low despite extensive effort to understand the effects of culture conditions, media composition and supplementation. As bovine oocytes resume meiosis spontaneously when cultured, it was hypothesized that preventing meiosis in vitro before in vitro maturation (IVM) and in vitro fertilization (IVF) would allow more oocytes to acquire developmental competence. This article reviews some of the factors involved in meiotic arrest as well as the effects of meiotic inhibition before IVM on bovine oocytes developmental competence following IVF. Follicular components and cAMP-elevating agents can delay or inhibit meiosis in various proportions of oocytes; however, few studies have examined their effects on development following IVM and IVF because they are not practical (follicular components) or have a transient effect on meiosis (cAMP-elevating agents). Protein synthesis or phosphorylation inhibition prevented meiosis in high percentages of oocytes; however, these non-specific inhibitions led to lower developmental competence compared with non-arrested oocytes. Maturation promoting factor (MPF) inhibition with specific inhibitors has been examined in several studies. Despite faster maturation following removal from inhibition and some structural damage to the oocytes, MPF inhibition generally led to blastocyst rates similar to control, non-arrested oocytes. Future work will involve evaluating the effects on arrested oocytes of molecules that can improve developmental competence in non-arrested oocytes. It is also anticipated that new IVM systems that take into consideration new knowledge of the mechanisms involved in the control of meiosis will be developed. Moreover, global gene expression analysis studies will also provide clues to the culture conditions required for optimal expression of developmental competence. © Her Majesty the Queen in Right of Canada 2011. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

Complete In Vitro Life Cycle of Trypanosoma congolense: Development of Genetic Tools

PubMed Central

Plazolles, Nicolas; Baltz, Théo

2010-01-01

Background Animal African trypanosomosis, a disease mainly caused by the protozoan parasite Trypanosoma congolense, is a major constraint to livestock productivity and has a significant impact in the developing countries of Africa. RNA interference (RNAi) has been used to study gene function and identify drug and vaccine targets in a variety of organisms including trypanosomes. However, trypanosome RNAi studies have mainly been conducted in T. brucei, as a model for human infection, largely ignoring livestock parasites of economical importance such as T. congolense, which displays different pathogenesis profiles. The whole T. congolense life cycle can be completed in vitro, but this attractive model displayed important limitations: (i) genetic tools were currently limited to insect forms and production of modified infectious BSF through differentiation was never achieved, (ii) in vitro differentiation techniques lasted several months, (iii) absence of long-term bloodstream forms (BSF) in vitro culture prevented genomic analyses. Methodology/Principal Findings We optimized culture conditions for each developmental stage and secured the differentiation steps. Specifically, we devised a medium adapted for the strenuous development of stable long-term BSF culture. Using Amaxa nucleofection technology, we greatly improved the transfection rate of the insect form and designed an inducible transgene expression system using the IL3000 reference strain. We tested it by expression of reporter genes and through RNAi. Subsequently, we achieved the complete in vitro life cycle with dramatically shortened time requirements for various wild type and transgenic strains. Finally, we established the use of modified strains for experimental infections and underlined a host adaptation phase requirement. Conclusions/Significance We devised an improved T. congolense model, which offers the opportunity to perform functional genomics analyses throughout the whole life cycle. It represents a very useful tool to understand pathogenesis mechanisms and to study potential therapeutic targets either in vitro or in vivo using a mouse model. PMID:20209144

The role of the obestatin/GPR39 system in human gastric adenocarcinomas.

PubMed

Alén, Begoña O; Leal-López, Saúl; Alén, María Otero; Viaño, Patricia; García-Castro, Victoria; Mosteiro, Carlos S; Beiras, Andrés; Casanueva, Felipe F; Gallego, Rosalía; García-Caballero, Tomás; Camiña, Jesús P; Pazos, Yolanda

2016-02-02

Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer.

The role of the obestatin/GPR39 system in human gastric adenocarcinomas

PubMed Central

Alén, Begoña O.; Leal-López, Saúl; Alén, María Otero; Viaño, Patricia; García-Castro, Victoria; Mosteiro, Carlos S.; Beiras, Andrés; Casanueva, Felipe F.; Gallego, Rosalía; García-Caballero, Tomás; Camiña, Jesús P.; Pazos, Yolanda

2016-01-01

Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer. PMID:26716511

In Vitro Expression of the Extracellular Matrix Components Aggrecan, Collagen Types I and II by Articular Cartilage-Derived Chondrocytes.

PubMed

Schneevoigt, J; Fabian, C; Leovsky, C; Seeger, J; Bahramsoltani, M

2017-02-01

The extracellular matrix (ECM) of hyaline cartilage is perfectly suited to transmit articular pressure load to the subchondral bone. Pressure is transferred by a high amount of aggrecan-based proteoglycans and collagen type II fibres in particular. After any injury, the hyaline cartilage is replaced by fibrocartilage, which is low in proteoglycans and contains collagen type I predominantly. Until now, long-term results of therapeutic procedures including cell-based therapies like autologous chondrocyte transplantation (ACT) lead to a replacement tissue meeting the composition of fibrocartilage. Therefore, it is of particular interest to discover how and to what extent isolation and in vitro cultivation of chondrocytes affect the cells and their expression of ECM components. Hyaline cartilage-derived chondrocytes were cultivated in vitro and observed microscopically over a time period of 35 days. The expression of collagen type I, collagen type II and aggrecan was analysed using RT-qPCR and Western blot at several days of cultivation. Chondrocytes presented a longitudinal shape for the entire cultivation period. While expression of collagen type I prevailed within the first days, only prolonged cultivation led to an increase in collagen type II and aggrecan expression. The results indicate that chondrocyte isolation and in vitro cultivation lead to a dedifferentiation at least to the stage of chondroprogenitor cells. © 2016 Blackwell Verlag GmbH.

Pachymic Acid Sensitizes Gastric Cancer Cells to Radiation Therapy by Upregulating Bax through Hypoxia.

PubMed

Lu, Chunwei; Cai, Dingfang; Ma, Jun

2018-05-08

We have previously shown that pachymic acid (PA) inhibited tumorigenesis of gastric cancer (GC) cells. However, the exact mechanism underlying the radiation response of GC was still elusive. To evaluate the effects of PA treatment on radiation response of GC cell lines both in vitro and in vivo, a colony formation assay and xenograft mouse model were employed. Changes in Bax and HIF1[Formula: see text] expressions were assessed in GC cells following PA treatment. Luciferase reporter and chromatin immune-precipitation assays were carried out to investigate the regulation of Bax through HIF1[Formula: see text]. Stable HIF1[Formula: see text] knockdown was introduced into GC cells to further study the mechanism underlying PA-enhanced response to radiation both in vitro and in vivo. PA greatly enhanced the sensitivity of GC cells to radiation in vitro and in vivo, upregulated Bax expression and inhibited hypoxia. Bax expression was under hypoxia inhibition, and PA increased Bax expression through repressing HIF1[Formula: see text]. Stable HIF1[Formula: see text] overexpression in GC cells abolished the sensitizing effect of PA on GC cells to radiation both in vitro and in vivo. PA functions as a radiation sensitizing compound in GC. PA treatment induces the expression of pro-apoptotic factor Bax by inhibiting hypoxia/HIF1[Formula: see text], supporting the therapeutic potential of PA in radiation therapy against GC.

Modeling the functional genomics of autism using human neurons.

PubMed

Konopka, G; Wexler, E; Rosen, E; Mukamel, Z; Osborn, G E; Chen, L; Lu, D; Gao, F; Gao, K; Lowe, J K; Geschwind, D H

2012-02-01

Human neural progenitors from a variety of sources present new opportunities to model aspects of human neuropsychiatric disease in vitro. Such in vitro models provide the advantages of a human genetic background combined with rapid and easy manipulation, making them highly useful adjuncts to animal models. Here, we examined whether a human neuronal culture system could be utilized to assess the transcriptional program involved in human neural differentiation and to model some of the molecular features of a neurodevelopmental disorder, such as autism. Primary normal human neuronal progenitors (NHNPs) were differentiated into a post-mitotic neuronal state through addition of specific growth factors and whole-genome gene expression was examined throughout a time course of neuronal differentiation. After 4 weeks of differentiation, a significant number of genes associated with autism spectrum disorders (ASDs) are either induced or repressed. This includes the ASD susceptibility gene neurexin 1, which showed a distinct pattern from neurexin 3 in vitro, and which we validated in vivo in fetal human brain. Using weighted gene co-expression network analysis, we visualized the network structure of transcriptional regulation, demonstrating via this unbiased analysis that a significant number of ASD candidate genes are coordinately regulated during the differentiation process. As NHNPs are genetically tractable and manipulable, they can be used to study both the effects of mutations in multiple ASD candidate genes on neuronal differentiation and gene expression in combination with the effects of potential therapeutic molecules. These data also provide a step towards better understanding of the signaling pathways disrupted in ASD.

Adapting in vitro embryonic stem cell differentiation to the study of locus control regions.

PubMed

Lahiji, Armin; Kučerová-Levisohn, Martina; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D

2014-05-01

Numerous locus control region (LCR) activities have been discovered in gene loci important to immune cell development and function. LCRs are a distinct class of cis-acting gene regulatory elements that appear to contain all the DNA sequence information required to establish an independently and predictably regulated gene expression program at any genomic site in native chromatin of a whole animal. As such, LCR-regulated transgenic reporter systems provide invaluable opportunities to investigate the mechanisms of gene regulatory DNA action during development. Furthermore the qualities of LCR-driven gene expression, including spatiotemporal specificity and "integration site-independence" would be highly desirable to incorporate into vectors used in therapeutic genetic engineering. Thus, advancement in the methods used to investigate LCRs is of considerable basic and translational significance. We study the LCR present in the mouse T cell receptor (TCR)-α gene locus. Until recently, transgenic mice provided the only experimental model capable of supporting the entire spectrum of LCR activities. We have recently reported complete manifestation of TCRα LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC), thus validating a complete cell culture model for the full range of LCR activities seen in transgenic mice. Here we discuss the critical parameters involved in studying LCR-regulated gene expression during in vitro hematopoietic differentiation from ESCs. This advance provides an approach to speed progress in the LCR field, and facilitate the clinical application of its findings, particularly to the genetic engineering of T cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression of a model gene in prostate cancer cells lentivirally transduced in vitro and in vivo.

PubMed

Bastide, C; Maroc, N; Bladou, F; Hassoun, J; Maitland, N; Mannoni, P; Bagnis, C

2003-01-01

In a preclinical model for prostate cancer gene therapy, we have tested lentiviral vectors as a practical possibility for the transfer and long-term expression of the EGFP gene both in vitro and in vivo. The human prostate cancer cell lines DU145 and PC3 were transduced using experimental conditions which permitted analysis of the expression from a single proviral vector per cell. The transduced cells stably expressed the EGFP transgene for 4 months. After injection of the transduced cell populations into Nod-SCID mice a decrease in EGFP was only observed in a minority of cases, while the majority of tumors maintained transgene expression at in vitro levels. In vivo injection of viral vector preparations directly into pre-established subcutaneous or orthotopic tumor masses, obtained by implantation of untransduced PC3 and DU145 cells led to a high transduction efficiency. While the efficiency of direct intratumoral transduction was proportional to the dose of virus injected, the results indicated some technical limitations inherent in these approaches to prostate cancer gene therapy.

Protein and genome evolution in Mammalian cells for biotechnology applications.

PubMed

Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J

2009-06-01

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.

Effective mRNA Inhibition in PANC-1 Cells in Vitro Mediated via an mPEG-SeSe-PEI Delivery System.

PubMed

Zhang, Yuefeng; Yang, Bin; Liu, Yajie; Qin, Wenjie; Li, Chao; Wang, Lantian; Zheng, Wen; Wu, Yulian

2016-05-01

RNA interference (RNAi)-mediated gene therapy is a promising approach to cure various diseases. However, developing an effective, safe, specific RNAi delivery system remains a major challenge. In this study, a novel redox-responsive polyetherimide (PEI)-based nanovector, mPEG-SeSe-PEI, was developed and its efficacy evaluated. We prepared three mPEG-SeSe-PEI vector candidates for small interfering glyceraldehyde-3-phosphate dehydrogenase (siGADPH) and determined their physiochemical properties and transfection efficiency using flow cytometry and PEG11.6-SeSe-PEI polymer. We investigated the silencing efficacy of GADPH mRNA expression in PANC-1 cells and observed that PEG11.6-SeSe-PEI/siGADPH (N/P ratio=10) polyplexes possessed the appropriate size and zeta-potential and exhibited excellent in vitro gene silencing effects with the least cytotoxicity in PANC-1 cells. In conclusion, we present PEG11.6-SeSe-PEI as a potential therapeutic gene delivery system for small interfering RNA (siRNA).

Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

PubMed

Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

2007-07-30

Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

The bovine endometrial epithelial cells promote the differentiation of trophoblast stem-like cells to binucleate trophoblast cells.

PubMed

Li, Xiawei; Li, Zhiying; Hou, Dongxia; Zhao, Yuhang; Wang, Chen; Li, Xueling

2016-12-01

Endometrial epithelial cells (EECs) cultured in vitro are valuable tools for investigating embryo implantation and trophoblast differentiation. In this study, we have established the bovine EECs and trophoblast stem-like (TS) coculture system, and used it to investigate the binucleate cell formation of ungulates. The EECs was derived from the uterine horn ipsilateral to the corpus luteum by using collagenase I and deoxyribonuclease I, which exhibited typical epithelial morphology and were expressing bovine uterine epithelial marker such as IFNAR1, IFNAR2, Erα, PGR, ESR1 and KRT18. The cells immunostained positively by epithelial and trophectoderm marker cytokeratin 18 (KRT18) and stromal marker vimentin antibodies, and the KRT18 positive cells reached 99 %. The EECs can be cultured for up to 20 passages in vitro with no significant morphology changes and uterine epithelial marker gene expression alteration. The bTS cells were established in a dual inhibitor system and exhibited typical trophoblast stem cell characteristics. When bTS cells were cultured with EECs, the bTS cells adhered to the EECs as adhering to feeder cells. Binucleate cells began appearing on day 4 of coculture and reached approximately 18.47 % of the differentiated cells. Quantitative real-time PCR or immunofluorescence analyses were performed on bTS cells cocultured at day 6 and day 12. The results showed that the expression level of KRT18 was down-regulated while the expression level of trophoblast differentiation marker MASH2, HAND1, GCM1 and CDX2 was up-regulated in bTS cells. In conclusion, bovine EECs can be obtained from the uterine horn ipsilateral to the corpus luteum via treatment with collagenase I and deoxyribonuclease I, and the EECs-bTS cells coculture system presents an ideal tool for studying the differentiation of bTS cells to trophoblast binucleate cells.

An in vitro ES cell imprinting model shows that imprinted expression of the Igf2r gene arises from an allele-specific expression bias

PubMed Central

Latos, Paulina A.; Stricker, Stefan H.; Steenpass, Laura; Pauler, Florian M.; Huang, Ru; Senergin, Basak H.; Regha, Kakkad; Koerner, Martha V.; Warczok, Katarzyna E.; Unger, Christine; Barlow, Denise P.

2010-01-01

Genomic imprinting is an epigenetic process that results in parental-specific gene expression. Advances in understanding the mechanism that regulates imprinted gene expression in mammals have largely depended on generating targeted manipulations in embryonic stem (ES) cells that are analysed in vivo in mice. However, genomic imprinting consists of distinct developmental steps, some of which occur in post-implantation embryos, indicating that they could be studied in vitro in ES cells. The mouse Igf2r gene shows imprinted expression only in post-implantation stages, when repression of the paternal allele has been shown to require cis-expression of the Airn non-coding (nc) RNA and to correlate with gain of DNA methylation and repressive histone modifications. Here we follow the gain of imprinted expression of Igf2r during in vitro ES cell differentiation and show that it coincides with the onset of paternal-specific expression of the Airn ncRNA. Notably, although Airn ncRNA expression leads, as predicted, to gain of repressive epigenetic marks on the paternal Igf2r promoter, we unexpectedly find that the paternal Igf2r promoter is expressed at similar low levels throughout ES cell differentiation. Our results further show that the maternal and paternal Igf2r promoters are expressed equally in undifferentiated ES cells, but during differentiation expression of the maternal Igf2r promoter increases up to 10-fold, while expression from the paternal Igf2r promoter remains constant. This indicates, contrary to expectation, that the Airn ncRNA induces imprinted Igf2r expression not by silencing the paternal Igf2r promoter, but by generating an expression bias between the two parental alleles. PMID:19141673

Expression and in vitro regulation of integrins by normal human urothelial cells.

PubMed

Southgate, J; Kennedy, W; Hutton, K A; Trejdosiewicz, L K

1995-08-01

Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.

Expression of microRNAs in bovine and human pre-implantation embryo culture media

PubMed Central

Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

2014-01-01

MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

Hypoxia-inducible vascular endothelial growth factor gene therapy using the oxygen-dependent degradation domain in myocardial ischemia.

PubMed

Kim, Hyun Ah; Lim, Soyeon; Moon, Hyung-Ho; Kim, Sung Wan; Hwang, Ki-Chul; Lee, Minhyung; Kim, Sun Hwa; Choi, Donghoon

2010-10-01

A hypoxia-inducible VEGF expression system with the oxygen-dependent degradation (ODD) domain was constructed and tested to be used in gene therapy for ischemic myocardial disease. Luciferase and VEGF expression vector systems were constructed with or without the ODD domain: pEpo-SV-Luc (or pEpo-SV-VEGF) and pEpo-SV-Luc-ODD (or pEpo-SV-VEGF-ODD). In vitro gene expression efficiency of each vector type was evaluated in HEK 293 cells under both hypoxic and normoxic conditions. The amount of VEGF protein was estimated by ELISA. The VEGF expression vectors with or without the ODD domain were injected into ischemic rat myocardium. Fibrosis, neovascularization, and cardiomyocyte apoptosis were assessed using Masson's trichrome staining, α-smooth muscle actin (α-SMA) immunostaining, and the TUNEL assay, respectively. The plasmid vectors containing ODD significantly improved the expression level of VEGF protein in hypoxic conditions. The enhancement of VEGF protein production was attributed to increased protein stability due to oxygen deficiency. In a rat model of myocardial ischemia, the pEpo-SV-VEGF-ODD group exhibited less myocardial fibrosis, higher microvessel density, and less cardiomyocyte apoptosis compared to the control groups (saline and pEpo-SV-VEGF treatments). An ODD-mediated VEGF expression system that facilitates VEGF-production under hypoxia may be useful in the treatment of ischemic heart disease.

Genes and Proteins Differentially Expressed during In Vitro Malignant Transformation of Bovine Pancreatic Duct Cells1

PubMed Central

Jesnowski, R; Zubakov, Dmitri; Faissner, Ralf; Ringel, Jörg; Hoheisel, Jörg D; Lösel, Ralf; Schnölzer, Martina; Löhr, Matthias

2007-01-01

Abstract Pancreatic carcinoma has an extremely bad prognosis due to lack of early diagnostic markers and lack of effective therapeutic strategies. Recently, we have established an in vitro model recapitulating the first steps in the carcinogenesis of the pancreas. SV40 large T antigen-immortalized bovine pancreatic duct cells formed intrapancreatic adenocarcinoma tumors on k-rasmut transfection after orthotopic injection in the nude mouse pancreas. Here we identified genes and proteins differentially expressed in the course of malignant transformation using reciprocal suppression subtractive hybridization and 2D gel electrophoresis and mass spectrometry, respectively. We identified 34 differentially expressed genes, expressed sequence tags, and 15 unique proteins. Differential expression was verified for some of the genes or proteins in samples from pancreatic carcinoma. Among these genes and proteins, the majority had already been described either to be influenced by a mutated ras or to be differentially expressed in pancreatic adenocarcinoma, thus proving the feasibility of our model. Other genes and proteins (e.g., BBC1, GLTSCR2, and rhoGDIα), up to now, have not been implicated in pancreatic tumor development. Thus, we were able to establish an in vitro model of pancreatic carcinogenesis, which enabled us to identify genes and proteins differentially expressed during the early steps of malignant transformation. PMID:17356710

AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED CU,ZN-SOD AND MN-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO

EPA Science Inventory

AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED Cu,Zn-SOD AND Mn-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO. JB Smith1, PC Hartig3, MR Blanton3, KK Sulik1,2, and ES Hunter3. 1Department of Cell and Developmental Biology and 2Bowles Cente...

Cocaine and sigma-1 receptors modulate HIV infection, chemokine receptors, and the HPA axis in the huPBL-SCID model.

PubMed

Roth, Michael D; Whittaker, Katherine M; Choi, Ruth; Tashkin, Donald P; Baldwin, Gayle Cocita

2005-12-01

Cocaine is associated with an increased risk for, and progression of, clinical disease associated with human immunodeficiency virus (HIV) infection. A human xenograft model, in which human peripheral blood mononuclear cells were implanted into severe combined immunodeficiency mice (huPBL-SCID) and infected with a HIV reporter virus, was used to investigate the biological interactions between cocaine and HIV infection. Systemic administration of cocaine (5 mg/kg/d) significantly increased the percentage of HIV-infected PBL (two- to threefold) and viral load (100- to 300-fold) in huPBL-SCID mice. Despite the capacity for cocaine to increase corticosterone and adrenocorticotropic hormone levels in control mice, the hypothalamic-pituitary-adrenal axis was suppressed in HIV-infected animals, and corticosterone levels were further decreased when animals were exposed to HIV and cocaine. Activating huPBL in vitro in the presence of 10(-8) M cocaine increased expression of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) coreceptors. Expression of CCR5 was also increased at early time-points in the huPBL-SCID model following systemic exposure to cocaine (54.1+/-9.4% increase over control, P<0.01). This effect preceded the boost in viral infection and waned as HIV infection progressed. Cocaine has been shown to mediate immunosuppressive effects by activating sigma-1 receptors in immune cells in vitro and in vivo. Consistent with these reports, a selective sigma-1 antagonist, BD1047, blocked the effects of cocaine on HIV replication in the huPBL-SCID mouse. Our results suggest that systemic exposure to cocaine can enhance HIV infection in vivo by activating sigma-1 receptors and by modulating the expression of HIV coreceptors.

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform.

PubMed

Candiello, Joseph; Grandhi, Taraka Sai Pavan; Goh, Saik Kia; Vaidya, Vimal; Lemmon-Kishi, Maya; Eliato, Kiarash Rahmani; Ros, Robert; Kumta, Prashant N; Rege, Kaushal; Banerjee, Ipsita

2018-05-25

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering. Copyright © 2018. Published by Elsevier Ltd.

Use of a collagen-elastin matrix as transport carrier system to transfer proliferating epidermal cells to human dermis in vitro.

PubMed

Waaijman, Taco; Breetveld, Melanie; Ulrich, Magda; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

2010-01-01

This in vitro study describes a novel cell culture, transport, and transfer protocol that may be highly suitable for delivering cultured proliferating keratinocytes and melanocytes to large open skin wounds (e.g., burns). We have taken into account previous limitations identified using other keratinocyte transfer techniques, such as regulatory issues, stability of keratinocytes during transport (single cell suspensions undergo terminal differentiation), ease of handling during application, and the degree of epidermal blistering resulting after transplantation (both related to transplanting keratinocyte sheets). Large numbers of proliferating epidermal cells (EC) (keratinocytes and melanocytes) were generated within 10-14 days and seeded onto a three-dimensional matrix composed of elastin and collagen types I, III, and V (Matriderm®), which enabled easy and stable transport of the EC for up to 24 h under ambient conditions. All culture conditions were in accordance with the regulations set by the Dutch Central Committee on Research Involving Human Subjects (CCMO). As an in vitro model system for clinical in vivo transfer, the EC were then transferred from Matriderm onto human acellular dermis during a period of 3 days. After transfer the EC maintained the ability to regenerate into a fully differentiated epidermis containing melanocytes on the human dermis. Proliferating keratinocytes were located in the basal layer and keratin-10 expression was located in differentiating suprabasal layers similar to that found in human epidermis. No blistering was observed (separation of the epidermis from the basement membrane). Keratin-6 expression was strongly upregulated in the regenerating epidermis similar to normal wound healing. In summary, we show that EC-Matriderm contains viable, metabolically active keratinocytes and melanocytes cultured in a manner that permits easy transportation and contains epidermal cells with the potential to form a pigmented reconstructed epidermis. This in vitro study has produced a robust protocol that is ready for clinical studies in the future.

Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells

PubMed Central

Ruedel, Anke; Hofmeister, Simone; Bosserhoff, Anja-Katrin

2013-01-01

High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on “hanging drop” cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies. PMID:24294400

Microencapsulated equine mesenchymal stromal cells promote cutaneous wound healing in vitro.

PubMed

Bussche, Leen; Harman, Rebecca M; Syracuse, Bethany A; Plante, Eric L; Lu, Yen-Chun; Curtis, Theresa M; Ma, Minglin; Van de Walle, Gerlinde R

2015-04-11

The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailable. The present study was designed to provide answers to these questions by using the horse as a translational model. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC-derived conditioned medium (CM), containing all factors secreted by MSCs, on equine dermal fibroblasts, a cell type critical for successful wound healing, and (ii) explore the potential of microencapsulated equine MSCs to deliver CM to wounded cells in vitro. MSCs were isolated from the peripheral blood of healthy horses. Equine dermal fibroblasts from the NBL-6 (horse dermal fibroblast cell) line were wounded in vitro, and cell migration and expression levels of genes involved in wound healing were evaluated after treatment with MSC-CM or NBL-6-CM. These assays were repeated by using the CM collected from MSCs encapsulated in core-shell hydrogel microcapsules. Our salient findings were that equine MSC-derived CM stimulated the migration of equine dermal fibroblasts and increased their expression level of genes that positively contribute to wound healing. In addition, we found that equine MSCs packaged in core-shell hydrogel microcapsules had similar effects on equine dermal fibroblast migration and gene expression, indicating that microencapsulation of MSCs does not interfere with the release of bioactive factors. Our results demonstrate that the use of CM from MSCs might be a promising new therapy for impaired cutaneous wounds and that encapsulation may be a suitable way to effectively deliver CM to wounded cells in vivo.

Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

PubMed Central

Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

2015-01-01

From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field. PMID:26478227

A controlled double-duration inducible gene expression system for cartilage tissue engineering.

PubMed

Ma, Ying; Li, Junxiang; Yao, Yi; Wei, Daixu; Wang, Rui; Wu, Qiong

2016-05-25

Cartilage engineering that combines competent seeding cells and a compatible scaffold is increasingly gaining popularity and is potentially useful for the treatment of various bone and cartilage diseases. Intensive efforts have been made by researchers to improve the viability and functionality of seeding cells of engineered constructs that are implanted into damaged cartilage. Here, we designed an integrative system combining gene engineering and the controlled-release concept to solve the problems of both seeding cell viability and functionality through precisely regulating the anti-apoptotic gene bcl-2 in the short-term and the chondrogenic master regulator Sox9 in the long-term. Both in vitro and in vivo experiments demonstrated that our system enhances the cell viability and chondrogenic effects of the engineered scaffold after introduction of the system while restricting anti-apoptotic gene expression to only the early stage, thereby preventing potential oncogenic and overdose effects. Our system was designed to be modular and can also be readily adapted to other tissue engineering applications with minor modification.

An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5, a viral neurovirulence factor.

PubMed

Tang, Shuang; Bertke, Andrea S; Patel, Amita; Wang, Kening; Cohen, Jeffrey I; Krause, Philip R

2008-08-05

Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo. miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.

Dexpanthenol modulates gene expression in skin wound healing in vivo.

PubMed

Heise, R; Skazik, C; Marquardt, Y; Czaja, K; Sebastian, K; Kurschat, P; Gan, L; Denecke, B; Ekanayake-Bohlig, S; Wilhelm, K-P; Merk, H F; Baron, J M

2012-01-01

Topical application of dexpanthenol is widely used in clinical practice for the improvement of wound healing. Previous in vitro experiments identified a stimulatory effect of pantothenate on migration, proliferation and gene regulation in cultured human dermal fibroblasts. To correlate these in vitro findings with the more complex in vivo situation of wound healing, a clinical trial was performed in which the dexpanthenol-induced gene expression profile in punch biopsies of previously injured and dexpanthenol-treated skin in comparison to placebo-treated skin was analyzed at the molecular level by Affymetrix® GeneChip analysis. Upregulation of IL-6, IL-1β, CYP1B1, CXCL1, CCL18 and KAP 4-2 gene expression and downregulation of psorasin mRNA and protein expression were identified in samples treated topically with dexpanthenol. This in vivo study might provide new insight into the molecular mechanisms responsible for the effect of dexpanthenol in wound healing and shows strong correlations to previous in vitro data using cultured dermal fibroblasts. Copyright © 2012 S. Karger AG, Basel.

Novel murine clonal cell lines either express slow or mixed (fast and slow) muscle markers following differentiation in vitro.

PubMed

Peltzer, J; Colman, L; Cebrian, J; Musa, H; Peckham, M; Keller, A

2008-05-01

We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6- to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic alpha- toward muscle-specific beta-enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster.

PubMed

Cantera, Rafael; Lüer, Karin; Rusten, Tor Erik; Barrio, Rosa; Kafatos, Fotis C; Technau, Gerhard M

2002-12-01

The gene spalt is expressed in the embryonic central nervous system of Drosophila melanogaster but its function in this tissue is still unknown. To investigate this question, we used a combination of techniques to analyse spalt mutant embryos. Electron microscopy showed that in the absence of spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the beta-catenin Armadillo, its ligand N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organisation of the central nervous system.

Exploration of protein-protein interaction effects on α-2-macroglobulin in an inhibition of serine protease through gene expression and molecular simulations studies.

PubMed

Sivakamavalli, Jeyachandran; Selvaraj, Chandrabose; Singh, Sanjeev Kumar; Vaseeharan, Baskaralingam

2014-01-01

In Prophenoloxidase (ProPO) cascade, two targets namely serine protease and α-2-macroglobulin are key regulators involved in the defense system of crustaceans. In biological systems, routine role of cell systems requires the understanding in protein-protein interactions through experimental and theoretical concepts, which might yield useful insights into the cellular responses. Response of cells to regulating the immune system is governed by the interactions-involved biomolecular simulations. Unfortunately, studies on the inhibitors (SP and α-2M) that negatively regulate the proPO system or melanization in penaeid shrimp are not yet available. In order to understand how these interactions change the proPO mechanism in Indian white shrimp Fenneropenaeus indicus was determined. In F. indicus, innate immune system is in a sensitive balance of intricate interactions; elucidating these interactions by the integration of in silico and in vitro has great potential. We have determined the expression of both the SP and α-2M enzymes in regulatory mechanism, which are analyzed through qRT-PCR, protein-protein docking, and simulation studies. From this work, we propose a novel approach for studying an organism at the systems level by integrating genome-wide computational analysis and the gene expression data.

CD137+CD154− Expression As a Regulatory T Cell (Treg)-Specific Activation Signature for Identification and Sorting of Stable Human Tregs from In Vitro Expansion Cultures

PubMed Central

Nowak, Anna; Lock, Dominik; Bacher, Petra; Hohnstein, Thordis; Vogt, Katrin; Gottfreund, Judith; Giehr, Pascal; Polansky, Julia K.; Sawitzki, Birgit; Kaiser, Andrew; Walter, Jörn; Scheffold, Alexander

2018-01-01

Regulatory T cells (Tregs) are an attractive therapeutic tool for several different immune pathologies. Therapeutic Treg application often requires prolonged in vitro culture to generate sufficient Treg numbers or to optimize their functionality, e.g., via genetic engineering of their antigen receptors. However, purity of clinical Treg expansion cultures is highly variable, and currently, it is impossible to identify and separate stable Tregs from contaminating effector T cells, either ex vivo or after prior expansion. This represents a major obstacle for quality assurance of expanded Tregs and raises significant safety concerns. Here, we describe a Treg activation signature that allows identification and sorting of epigenetically imprinted Tregs even after prolonged in vitro culture. We show that short-term reactivation resulted in expression of CD137 but not CD154 on stable FoxP3+ Tregs that displayed a demethylated Treg-specific demethylated region, high suppressive potential, and lack of inflammatory cytokine expression. We also applied this Treg activation signature for rapid testing of chimeric antigen receptor functionality in human Tregs and identified major differences in the signaling requirements regarding CD137 versus CD28 costimulation. Taken together, CD137+CD154− expression emerges as a universal Treg activation signature ex vivo and upon in vitro expansion allowing the identification and isolation of epigenetically stable antigen-activated Tregs and providing a means for their rapid functional testing in vitro. PMID:29467769

CD137+CD154- Expression As a Regulatory T Cell (Treg)-Specific Activation Signature for Identification and Sorting of Stable Human Tregs from In Vitro Expansion Cultures.

PubMed

Nowak, Anna; Lock, Dominik; Bacher, Petra; Hohnstein, Thordis; Vogt, Katrin; Gottfreund, Judith; Giehr, Pascal; Polansky, Julia K; Sawitzki, Birgit; Kaiser, Andrew; Walter, Jörn; Scheffold, Alexander

2018-01-01

Regulatory T cells (Tregs) are an attractive therapeutic tool for several different immune pathologies. Therapeutic Treg application often requires prolonged in vitro culture to generate sufficient Treg numbers or to optimize their functionality, e.g., via genetic engineering of their antigen receptors. However, purity of clinical Treg expansion cultures is highly variable, and currently, it is impossible to identify and separate stable Tregs from contaminating effector T cells, either ex vivo or after prior expansion. This represents a major obstacle for quality assurance of expanded Tregs and raises significant safety concerns. Here, we describe a Treg activation signature that allows identification and sorting of epigenetically imprinted Tregs even after prolonged in vitro culture. We show that short-term reactivation resulted in expression of CD137 but not CD154 on stable FoxP3+ Tregs that displayed a demethylated Treg-specific demethylated region, high suppressive potential, and lack of inflammatory cytokine expression. We also applied this Treg activation signature for rapid testing of chimeric antigen receptor functionality in human Tregs and identified major differences in the signaling requirements regarding CD137 versus CD28 costimulation. Taken together, CD137+CD154- expression emerges as a universal Treg activation signature ex vivo and upon in vitro expansion allowing the identification and isolation of epigenetically stable antigen-activated Tregs and providing a means for their rapid functional testing in vitro .

In vitro characterization of human hair follicle dermal sheath mesenchymal stromal cells and their potential in enhancing diabetic wound healing.

PubMed

Ma, Dongrui; Kua, Jonah Ee Hsiang; Lim, Wee Keng; Lee, Seng Teik; Chua, Alvin Wen Choong

2015-08-01

Little is published on the characterization and therapeutic potential of human mesenchymal cells derived from hair follicle (HF) dermal sheath (DS). In this study, we isolated and characterized HF DS-mesenchymal stromal cells (DS-MSCs) with respect to the bone marrow mesenchymal stromal cells (BM-MSCs). We further tested if DS-MSC-conditioned medium (CM), like what was previously reported for BM-MSC CM, has superior wound-healing properties, in both in vitro and in vivo wound models compared with skin fibroblast CM. DS-MSCs were isolated from HF and cultured in vitro to assess long-term growth potential, colony-forming efficiency (CFE), expression of CD surface markers and differentiation potential. The cytokine expression of DS-MSC CM was determined through an antibody-based protein array analysis. The wound-healing effects of the CM were tested in vitro with the use of human cell cultures and in vivo with the use of a diabetic mouse wound model. In vitro results revealed that DS-MSCs have high growth capacity and CFE while displaying some phenotypes similar to BM-MSCs. DS-MSCs strongly expressed many surface markers expressed in BM-MSCs and could also differentiate into osteoblasts, chondrocytes and adipocytes. DS-MSCs secreted significantly higher proportions of paracrine factors such as interleukin-6 (IL-6), IL-8 and growth-related oncogene. DS-MSC-CM demonstrated enhanced wound-healing effects on human skin keratinocytes, fibroblasts and endothelial cells in vitro, and the wound-healing time in diabetic mice was found to be shorter, compared with vehicle controls. Human HF DS stromal cells demonstrated MSC-like properties and might be an alternative source for therapeutic use in wound healing. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Tilmicosin reduces lipopolysaccharide-stimulated bovine alveolar macrophage prostaglandin E(2) production via a mechanism involving phospholipases.

PubMed

Lakritz, Jeffrey; Tyler, Jeff W; Marsh, Antoinette E; Romesburg-Cockrell, Mary; Smith, Kathy; Holle, Julie M

2002-01-01

Tilmicosin is a potent antimicrobial with broad-spectrum activity against the bacterial agents involved in the bovine respiratory disease complex. Recent studies indicate that in addition to being bactericidal, tilmicosin is capable of modulating inflammation in the lung. A series of experiments were designed to determine whether tilmicosin alters alveolar macrophage-prostaglandin E(2) (PGE(2)) production induced by Escherichia coli (O55:B5) lipopolysaccharide (LPS). Twenty-two healthy Holstein bull calves were used to study the effects of LPS-induced PGE(2) production of alveolar macrophages after in vivo or in vitro treatment with tilmicosin. In Experiment 1, tilmicosin was given by subcutaneous injection (15 mg/kg) twice, 48 hours apart, to four calves; four control calves received no treatment. Twenty-four hours after the second treatment, alveolar macrophages were stimulated with LPS in vitro. In Experiment 2, alveolar macrophages from five untreated calves were harvested and treated in vitro with tilmicosin, followed by LPS stimulation. In Experiment 3, the ability of in vitro tilmicosin treatment to alter the expression of LPS-induced cyclooxygenase-2 (COX-2) mRNA was evaluated. In Experiments 4 and 5, secretory phospholipase A(2) activity was examined in untreated calves. Treatment of calves with tilmicosin resulted in reduced LPS-induced alveolar macrophage PGE(2) production. Similar reductions in PGE(2) by LPS-stimulated alveolar macrophages after in vitro tilmicosin treatment were noted. This in vitro tilmicosin treatment was not associated with reduction of the expression of LPS-induced COX-2. Alveolar macrophage phospholipase A(2) activity induced by LPS was significantly reduced by prior tilmicosin treatment in vitro. Tilmicosin (in vivo and in vitro) appears to reduce the PGE(2) eicosanoid response of LPS-stimulated alveolar macrophages by reducing the in vitro substrate availability without altering in vitro COX-2 mRNA expression.

The effects of small interfering RNA–targeting tissue factor on an in vitro model of neovascularization

PubMed Central

Peng, Wenyan; Yu, Ying; Li, Tiejun; Zhu, Yuanyuan

2013-01-01

Purpose Tissue factor (TF) plays an important role in neovascularization (NV). This study aimed to determine whether small interfering RNA–targeting TF (TF-siRNA) could knock down TF expression and inhibit cell proliferation, cell migration, and tube formation in an in vitro model of NV. Methods Lipopolysaccharide (LPS) was used to stimulate human umbilical vein endothelial cell (HUVEC) lines to express TF and mimic certain phenotypes of NV in vitro. HUVECs were transfected with TF-siRNAs and control siRNAs using LipofectamineTM 2000. The inhibitory effect of the siRNAs on the expression of TF mRNA and protein was evaluated by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and western blot analysis. The effects on the cell viability, migration, and tube formation of siRNA-treated cells were examined by MTT assay, wound-healing assay, and Matrigel-induced capillary tube formation. Results Lipopolysaccharide treatment increased the expression of TF. TF-siRNAs effectively knocked down TF expression, with the most efficient TF-siRNA reducing 78.9% of TF expression. TF protein was also notably curtailed by TF-siRNA. The MTT and wound-healing assays showed that the TF-siRNA substantially inhibited the proliferation and migration of HUVECs. Tube formation was decreased by 47.4% and 59.4% in cells treated with the TF-siRNA and vascular endothelial growth factor–siRNA, respectively, compared with the blank control. Conclusions TF-siRNA can knockdown TF expression and inhibit cell proliferation, migration, and tube formation in vitro. TF-siRNA may provide a novel therapeutic candidate for NV-related diseases. PMID:23805036

Alphavirus replicon approach to promoterless analysis of IRES elements.

PubMed

Kamrud, K I; Custer, M; Dudek, J M; Owens, G; Alterson, K D; Lee, J S; Groebner, J L; Smith, J F

2007-04-10

Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced >95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.

Alphavirus Replicon Approach to Promoterless Analysis of IRES Elements

PubMed Central

Kamrud, K.I.; Custer, M.; Dudek, J.M.; Owens, G.; Alterson, K.D.; Lee, J.S.; Groebner, J.L.; Smith, J.F.

2007-01-01

Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (Family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced > 95 % compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in-vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development. PMID:17156813

The TGF-beta-Pseudoreceptor BAMBI is strongly expressed in COPD lungs and regulated by nontypeable Haemophilus influenzae

PubMed Central

2010-01-01

Background Nontypeable Haemophilus influenzae (NTHI) may play a role as an infectious trigger in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available regarding the influence of acute and persistent infection on tissue remodelling and repair factors such as transforming growth factor (TGF)-β. Methods NTHI infection in lung tissues obtained from COPD patients and controls was studied in vivo and using an in vitro model. Infection experiments were performed with two different clinical isolates. Detection of NTHI was done using in situ hybridization (ISH) in unstimulated and in in vitro infected lung tissue. For characterization of TGF-β signaling molecules a transcriptome array was performed. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-α and TGF-β expression were evaluated in lung tissue and cell culture using ELISA. Results In 38% of COPD patients infection with NTHI was detected in vivo in contrast to 0% of controls (p < 0.05). Transcriptome arrays showed no significant changes of TGF-β receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after in vitro infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-β (p < 0.05) in combination with a strong proinflammatory response (p < 0.01). Conclusions We show for the first time the expression of the TGF pseudoreceptor BAMBI in the human lung, which is upregulated in response to NTHI infection in COPD lung tissue in vivo and in vitro. The combination of NTHI-mediated induction of proinflammatory cytokines and inhibition of TGF-β expression may influence inflammation induced tissue remodeling. PMID:20513241

The TGF-beta-pseudoreceptor BAMBI is strongly expressed in COPD lungs and regulated by nontypeable Haemophilus influenzae.

PubMed

Drömann, Daniel; Rupp, Jan; Rohmann, Kristina; Osbahr, Sinia; Ulmer, Artur J; Marwitz, Sebastian; Röschmann, Kristina; Abdullah, Mahdi; Schultz, Holger; Vollmer, Ekkehard; Zabel, Peter; Dalhoff, Klaus; Goldmann, Torsten

2010-05-31

Nontypeable Haemophilus influenzae (NTHI) may play a role as an infectious trigger in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available regarding the influence of acute and persistent infection on tissue remodelling and repair factors such as transforming growth factor (TGF)-beta. NTHI infection in lung tissues obtained from COPD patients and controls was studied in vivo and using an in vitro model. Infection experiments were performed with two different clinical isolates. Detection of NTHI was done using in situ hybridization (ISH) in unstimulated and in in vitro infected lung tissue. For characterization of TGF-beta signaling molecules a transcriptome array was performed. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-alpha and TGF-beta expression were evaluated in lung tissue and cell culture using ELISA. In 38% of COPD patients infection with NTHI was detected in vivo in contrast to 0% of controls (p < 0.05). Transcriptome arrays showed no significant changes of TGF-beta receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after in vitro infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-beta (p < 0.05) in combination with a strong proinflammatory response (p < 0.01). We show for the first time the expression of the TGF pseudoreceptor BAMBI in the human lung, which is upregulated in response to NTHI infection in COPD lung tissue in vivo and in vitro. The combination of NTHI-mediated induction of proinflammatory cytokines and inhibition of TGF-beta expression may influence inflammation induced tissue remodeling.

Influence of 1α, 25-dihydroxyvitamin D3 [1, 25(OH)2D3] on the expression of Sox 9 and the transient receptor potential vanilloid 5/6 ion channels in equine articular chondrocytes.

PubMed

Hdud, Ismail M; Loughna, Paul T

2014-01-01

Sox 9 is a major marker of chondrocyte differentiation. When chondrocytes are cultured in vitro they progressively de-differentiate and this is associated with a decline in Sox 9 expression. The active form of vitamin D, 1, 25 (OH)2D3 has been shown to be protective of cartilage in both humans and animals. In this study equine articular chondrocytes were grown in culture and the effects of 1, 25 (OH)2D3 upon Sox 9 expression examined. The expression of the transient receptor potential vanilloid (TRPV) ion channels 5 and 6 in equine chondrocytes in vitro, we have previously shown, is inversely correlated with de-differentiation. The expression of these channels in response to 1, 25 (OH)2D3 administration was therefore also examined. The active form of vitamin D (1, 25 (OH)2D3) when administered to cultured equine chondrocytes at two different concentrations significantly increased the expression of Sox 9 at both. In contrast 1, 25 (OH)2D3 had no significant effect upon the expression of either TRPV 5 or 6 at either the protein or the mRNA level. The increased expression of Sox 9, in equine articular chondrocytes in vitro, in response to the active form of vitamin D suggests that this compound could be utilized to inhibit the progressive de-differentiation that is normally observed in these cells. It is also supportive of previous studies indicating that 1α, 25-dihydroxyvitamin D3 can have a protective effect upon cartilage in animals in vivo. The previously observed correlation between the degree of differentiation and the expression levels of TRPV 5/6 had suggested that these ion channels may have a direct involvement in, or be modulated by, the differentiation process in vitro. The data in the present study do not support this.

Development of an efficient E. coli expression and purification system for a catalytically active, human Cullin3-RINGBox1 protein complex and elucidation of its quaternary structure with Keap1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Small, Evan; Eggler, Aimee; Mesecar, Andrew D., E-mail: amesecar@purdue.edu

2010-10-01

Research highlights: {yields} A novel expression strategy was used to purify Cul3-Rbx1 from E. coli. {yields} The Cul3-Rbx1 complex is fully active and catalyzes ubiquitination of Nrf2 in vitro. {yields} Cul3, Rbx1, and Keap1 form a complex with unique stoichiometry of 1:1:2. -- Abstract: The Cullin3-based E3 ubiquitin ligase complex is thought to play an important role in the cellular response to oxidative stress and xenobiotic assault. While limited biochemical studies of the ligase's role in these complex signaling pathways are beginning to emerge, structural studies are lagging far behind due to the inability to acquire sufficient quantities of full-length,more » highly pure and active Cullin3. Here we describe the design and construction of an optimized expression and purification system for the full-length, human Cullin3-RINGBox 1 (Rbx1) protein complex from Escherichia coli. The dual-expression system is comprised of codon-optimized Cullin3 and Rbx1 genes co-expressed from a single pET-Duet-1 plasmid. Rapid purification of the Cullin3-Rbx1 complex is achieved in two steps via an affinity column followed by size-exclusion chromatography. Approximately 15 mg of highly pure and active Cullin3-Rbx1 protein from 1 L of E. coli culture can be achieved. Analysis of the quaternary structure of the Cullin3-Rbx1 and Cullin3-Rbx1-Keap1 complexes by size-exclusion chromatography and analytical ultracentrifugation indicates a 1:1 stoichiometry for the Cullin3-Rbx1 complex (MW = 111 kDa), and a 1:1:2 stoichiometry for the Cullin3-Rbx1-Keap1 complex (MW = 280 kDa). This latter complex has a novel quaternary structural organization for cullin E3 ligases, and it is fully active based on an in vitro Cullin3-Rbx1-Keap1-Nrf2 ubiquitination activity assay that was developed and optimized in this study.« less

Functional Characterization of Paralogous Gonadotropin-Releasing Hormone-Type and Corazonin-Type Neuropeptides in an Echinoderm

PubMed Central

Tian, Shi; Egertová, Michaela; Elphick, Maurice R.

2017-01-01

Homologs of the vertebrate neuropeptide gonadotropin-releasing hormone (GnRH) have been identified in invertebrates, including the insect neuropeptide corazonin (CRZ). Recently, we reported the discovery of GnRH-type and CRZ-type signaling systems in an echinoderm, the starfish Asterias rubens, demonstrating that the evolutionary origin of paralogous GnRH-type and CRZ-type neuropeptides can be traced back to the common ancestor of protostomes and deuterostomes. Here, we have investigated the physiological roles of the GnRH-type (ArGnRH) and the CRZ-type (ArCRZ) neuropeptides in A. rubens, using mRNA in situ hybridization, immunohistochemistry and in vitro pharmacology. ArGnRH precursor (ArGnRHP)-expressing cells and ArGnRH-immunoreactive cells and/or processes are present in the radial nerve cords, circumoral nerve ring, digestive system (e.g., cardiac stomach and pyloric stomach), body wall-associated muscle (apical muscle), and appendages (tube feet, terminal tentacle). The general distribution of ArCRZ precursor (ArCRZP)-expressing cells is similar to that of ArGnRHP, but with specific local differences. For example, cells expressing ArGnRHP are present in both the ectoneural and hyponeural regions of the radial nerve cords and circumoral nerve ring, whereas cells expressing ArCRZP were only observed in the ectoneural region. In vitro pharmacological experiments revealed that both ArGnRH and ArCRZ cause contraction of cardiac stomach, apical muscle, and tube foot preparations. However, ArGnRH was more potent/effective than ArCRZ as a contractant of the cardiac stomach, whereas ArCRZ was more potent/effective than ArGnRH as a contractant of the apical muscle. These findings demonstrate that both ArGnRH and ArCRZ are myoexcitatory neuropeptides in starfish, but differences in their expression patterns and pharmacological activities are indicative of distinct physiological roles. This is the first study to investigate the physiological roles of both GnRH-type and CRZ-type neuropeptides in a deuterostome, providing new insights into the evolution and comparative physiology of these paralogous neuropeptide signaling systems in the Bilateria. PMID:29033898