Vinblastine and diethylstilboestrol tested in the in vitro mammalian cell micronucleus test (MNvit) at Swansea University UK in support of OECD draft Test Guideline 487.

PubMed

Johnson, George E; Jenkins, Gareth J; Thomas, Adam D; Doak, Shareen H

2010-10-29

The known aneugens vinblastine and diethylstilboestrol (DES) were tested in the in vitro micronucleus assay, with and without cytokinesis block in Chinese hamster CHO cells, at the laboratories of Swansea University, Swansea, UK. These experiments were carried out to determine the suitability of the cell death and cytostasis measures used in the assay, as recommended in the draft OECD Test Guideline 487, 2007. Both compounds were positive in the assay without cytokinesis block at concentrations giving approximately 50% or less cell death and cytostasis, using relative population doublings and relative increase in cell counts. Moreover, both compounds were positive in the assay with cytokinesis block at concentrations giving approximately 50% cell death and cytostasis, using replicative index. Vinblastine was also positive for mitotic slippage, causing micronuclei in mononucleate cells with cytokinesis block. Relative population doublings and relative increase in cell counts were appropriate measures of cell death and cytostasis for the non-cytokinesis block in vitro micronucleus assay. In the cytokinesis blocked micronucleus assay, replicative index and cytokinesis block proliferation index were suitable cell death and cytostasis measures. Copyright © 2009 Elsevier B.V. All rights reserved.

Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

PubMed Central

Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

2016-01-01

Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature. PMID:27239276

Cutaneous double-hit B-cell lymphoma: an aggressive form of B-cell lymphoma with a propensity for cutaneous dissemination.

PubMed

Magro, Cynthia M; Wang, Xuan; Subramaniyam, Shivakumar; Darras, Natasha; Mathew, Susan

2014-04-01

Diffuse large cell B-cell lymphoma of the skin is most commonly represented by diffuse large cell variants of primary cutaneous follicle center cell lymphoma and the leg-type lymphoma. In a minority of cases, the infiltrates are an expression of stage 4 disease of established extracutaneous B-cell lymphoma. We describe 3 patients with an aggressive form of B-cell lymphoma secondarily involving the skin. Two of the patients were in the ninth decade of life, whereas 1 patient was 34 years of age. In the elderly patients, there was an antecedent and/or concurrent history of follicular lymphoma, whereas in the younger patient, the tumor was a de novo presentation of this aggressive form of lymphoma. The elderly patients succumbed to their disease within less than a year from the time of diagnosis, whereas 1 patient is alive but with persistent and progressive disease despite chemotherapeutic intervention. The infiltrates in all 3 cases were diffuse and composed of large malignant hematopoietic cells that exhibited a round nucleus with a finely dispersed chromatin. Phenotypically, the tumor cells were Bcl-2 and CD10 positive, whereas Bcl-6 and Mum-1 showed variable positivity. One case showed combined Mum-1 positivity along with an acute lymphoblastic lymphoma phenotype, including the absence of CD20 expression. In each case, there was a c-MYC and BCL2/IGH rearrangement diagnostic of double-hit lymphoma. In one case, there was an additional BCL6 rearrangement, defining what is in essence triple-hit lymphoma. In conclusion, double-hit lymphoma is an aggressive form of B-cell neoplasia resistant to standard chemotherapy regimens, which in many but not all cases represents tumor progression in the setting of a lower grade B-cell malignancy.

Using an AMACR (P504S)/34betaE12/p63 cocktail for the detection of small focal prostate carcinoma in needle biopsy specimens.

PubMed

Jiang, Zhong; Li, Cuizhen; Fischer, Andrew; Dresser, Karen; Woda, Bruce A

2005-02-01

We assessed the usefulness of immunohistochemical analysis with a 3-antibody cocktail (alpha-methylacyl coenzyme A racemase [AMACR, or P504S], 34betaE12, p63) and a double-chromogen reaction for detection of limited prostate cancer in 138 needle biopsy specimens, including 82 with small foci of prostatic adenocarcinoma and 56 benign prostates. When carcinoma was present, red cytoplasmic granular staining (AMACR) in the malignant glands and cells and dark brown nuclear (p63) and cytoplasmic (34betaE12) staining in basal cells of adjacent nonmalignant glands were found. Of 82 cases of small foci of prostatic adenocarcinoma, 78 (95%) expressed AMACR; all malignant glands were negative for basal cell staining. All benign glands adjacent to malignant glands were recognized easily by basal cell marker positivity and little or no AMACR expression. No benign glands were simultaneously positive for AMACR and negative for basal cell markers (specificity, 100%). There were no differences in intensity and numbers of positive glands with double-chromogen staining compared with using 1-color staining. Our results indicate that immunohistochemistry with a 3-antibody cocktail and double chromogen is a simple and easy assay that can be used as a routine test, which overcomes the problems of studying small lesions in prostate needle biopsies with multiple immunohistochemical stains.

Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

PubMed Central

Chen, Xianling; Chen, Xiaole; Li, Ding; Fan, Yingjuan; Xu, Jianhua; Chen, Yuanzhong; Wu, Lixian

2017-01-01

Heat shock protein 90 (Hsp90) contains amino (N)–terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states. PMID:28036294

Double Aneuploidy Detected by Cell-Free DNA Testing and Confirmed by Fetal Tissue Analysis.

PubMed

Echague, Charlene G; Petersen, Scott M

2016-06-01

Double aneuploidies account for 0.21-2.8% of spontaneous abortions resulting from chromosomal abnormalities. Rarely, cell-free DNA testing detects multiple aneuploidies; however, to discern among maternal, placental, and fetal origin, further evaluation is required. A 49-year-old woman, gravida 5 para 0, underwent cell-free DNA testing at 11 4/7 weeks of gestation, which revealed a fetus that was high risk for trisomies 18 and 21. On ultrasonography at 14 weeks of gestation, she was diagnosed with a missed abortion and underwent surgical management. Fetal and placental tissues were sent for analysis and were positive for trisomies 18 and 21, confirming the results of cell-free DNA testing. Our case highlights the ability of cell-free DNA testing to recognize a double aneuploidy confirmed by fetal tissue analysis.

A1E reduces stemness and self-renewal in HPV 16-positive cervical cancer stem cells.

PubMed

Kwon, Taeho; Bak, Yesol; Ham, Sun-Young; Yu, Dae-Yeul; Yoon, Do-Young

2016-02-02

Cervical cancer is the second most common cancer in females. Recent reports have revealed the critical role of cervical cancer stem cells (CSCs) in tumorigenicity and metastasis. Previously we demonstrated that A1E exerts an anti-proliferative action, which inhibits the growth of cervical cancer cells. A1E is composed of 11 oriental medicinal herbs. Cervical cancer cell culture, wund healing and invasion assay, flow cytometry, sheroid formation assay, and wstern blot assays were performed in HPV 16-positive SiHa cell and HPV 16-negative C33A cells. A1E targets the E6 and E7 oncogenes; thus, A1E significantly inhibited proliferation of human papilloma virus (HPV) 16-positive SiHa cells, it did not inhibit the proliferation of HPV-negative C33A cells. Accordingly, we investigated whether A1E can regulate epithelial-to-mesenchymal transition (EMT), CSC self-renewal, and stemness-related gene expression in cervical cancer cells. Down rgulation of cell migration, cell invasion, and EMT was observed in A1E-treated SiHa cells. Specifically, A1E-treated SiHa cells showed significant decreases in OCT-3/4 and Sox2 expression levels and in sphere formation. Moreover, CSCs makers ALDH+ and ALDH, CD133 double positive cell were significantly decreased in A1E-treated SiHa cells. However, A1E treatment did not down regulate ALDH+ expression and the number of ALDH/CD133 double positive cells in C33A cells. Taken together, A1E can inhibit CSCs and reduce the expression of stemness markers. Treating CSCs with A1E may be a potential therapy for cervical cancer.

Identification of the soluble HVP-associated antigen of the lymphoblastoid cell line established from lymphomatous baboon (Papio hamadryas).

PubMed

Voevodin, A F; Lapin, B A; Agrba, V Z; Timanovskaya, V V

1978-01-01

A new technique (indirect double immunodiffusion) for detection of EBV-associated soluble antigen and corresponding antibodies has been developed. This technique includes three steps: 1) simple double immunodiffusion with extracts of Raji cells (or other EBV-genome positive cells) and human sera containing antibodies against EBV-associated soluble antigen; 2) extensive washing and treatment with anti-human globulin; 3) extensive washing and treatment with tannic acid. Using this test it was shown that the soluble antigen indistinguishable from EBV-associated soluble antigen was present in KMPG-1 cells producing HVP.

Intestinal double-positive CD4+CD8+ T cells of neonatal rhesus macaques are proliferating, activated memory cells and primary targets for SIVMAC251 infection

PubMed Central

Wang, Xiaolei; Das, Arpita; Lackner, Andrew A.; Veazey, Ronald S.

2008-01-01

Peripheral blood and thymic double-positive (DP) CD4+CD8+ T cells from neonates have been described earlier, but the function and immunophenotypic characteristics of other tissue-derived DP T cells are not clearly understood. Here, we demonstrate the functional and immunophenotypic characteristics of DP cells in 6 different tissues, including thymus from normal neonatal rhesus macaques (Macaca mulatta) between 0 and 21 days of age. In general, intestinal DP T cells of neonates have higher percentages of memory markers (CD28+CD95+CD45RAlowCD62Llow) and proliferation compared with single-positive (SP) CD4+ and CD8+ T cells. In addition, percentages of DP T cells increase and CD62L expression decreases as animals mature, suggesting that DP cells mature and proliferate with maturity and/or antigen exposure. Consistent with this, intestinal DP T cells in neonates express higher levels of CCR5 and are the primary targets in simian immunodeficiency virus (SIV) infection. Finally, DP T cells produce higher levels of cytokine in response to mitogen stimulation compared with SP CD4+ or CD8+ T cells. Collectively, these findings demonstrate that intestinal DP T cells of neonates are proliferating, activated memory cells and are likely involved in regulating immune responses, in contrast to immature DP T cells in the thymus. PMID:18820133

Clinicopathological and genomic analysis of double-hit follicular lymphoma: comparison with high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements.

PubMed

Miyaoka, Masashi; Kikuti, Yara Y; Carreras, Joaquim; Ikoma, Haruka; Hiraiwa, Shinichiro; Ichiki, Akifumi; Kojima, Minoru; Ando, Kiyoshi; Yokose, Tomoyuki; Sakai, Rika; Hoshikawa, Masahiro; Tomita, Naoto; Miura, Ikuo; Takata, Katsuyoshi; Yoshino, Tadashi; Takizawa, Jun; Bea, Silvia; Campo, Elias; Nakamura, Naoya

2018-02-01

Most high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements are aggressive B-cell lymphomas. Occasional double-hit follicular lymphomas have been described but the clinicopathological features of these tumors are not well known. To clarify the characteristics of double-hit follicular lymphomas, we analyzed 10 cases of double-hit follicular lymphomas and 15 cases of high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements for clinicopathological and genome-wide copy-number alterations and copy-neutral loss-of-heterozygosity profiles. For double-hit follicular lymphomas, the median age was 67.5 years (range: 48-82 years). The female/male ratio was 2.3. Eight patients presented with advanced clinical stage. The median follow-up time was 20 months (range: 1-132 months). At the end of the follow-up, 8 patients were alive, 2 patients were dead including 1 patient with diffuse large B-cell lymphoma transformation. Rearrangements of MYC/BCL2, MYC/BCL6, and MYC/BCL2/BCL6 were seen in 8, 1, and 1 cases, respectively. The partner of MYC was IGH in 6 cases. There were no cases of histological grade 1, 4 cases of grade 2, 5 cases of grade 3a, and 1 case of grade 3b. Two cases of grade 3a exhibited immunoblast-like morphology. Immunohistochemistry demonstrated 9 cases with ≥50% MYC-positive cells. There was significant difference in MYC intensity (P=0.00004) and MIB-1 positivity (P=0.001) between double-hit follicular lymphomas and high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements. The genome profile of double-hit follicular lymphomas was comparable with conventional follicular lymphomas (GSE67385, n=198) with characteristic gains of 2p25.3-p11.1, 7p22.3-q36.3, 12q11-q24.33, and loss of 18q21.32-q23 (P<0.05). In comparison with high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements, double-hit follicular lymphomas had fewer copy-number alterations and minimal common region of gain at 2p16.1 (70%), locus also significant against conventional follicular lymphomas (P=0.0001). In summary, double-hit follicular lymphomas tended to be high-grade histology, high MYC protein expression, high MYC/IGH fusion, and minimal common region of gain at 2p16.1. Double-hit follicular lymphomas seemed to be a different disease from high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements and have an indolent clinical behavior similar to follicular lymphomas without MYC rearrangement.

Important parameters affecting the cell voltage of aqueous electrical double-layer capacitors

NASA Astrophysics Data System (ADS)

Wu, Tzu-Ho; Hsu, Chun-Tsung; Hu, Chi-Chang; Hardwick, Laurence J.

2013-11-01

This study discusses and demonstrates how the open-circuit potential and charges stored in the working potential window on positive and negative electrodes affect the cell voltage of carbon-based electrical double-layer capacitors (EDLCs) in aqueous electrolytes. An EDLC consisting of two activated carbon electrodes is employed as the model system for identifying these key parameters although the potential window of water decomposition can be simply determined by voltammetric methods. First, the capacitive performances of an EDLC with the same charge on positive and negative electrodes are evaluated by cyclic voltammetric, charge-discharge, electrochemical impedance spectroscopic (EIS) analyses, and inductance-capacitance-resistance meter (LCR meter). The principles for obtaining the highest acceptable cell voltage of such symmetric ECs with excellent reversibility and capacitor-like behaviour are proposed. Aqueous charge-balanced EDLCs can be operated as high as 2.0 V with high energy efficiency (about 90%) and only 4% capacitance loss after the 600-cycle stability checking. The necessity of charge balance (but not capacitance balance) for positive and negative electrodes is substantiated from the lower acceptable cell voltage of charge-unbalanced EDLCs.

Colocalization of numerous immunoreactivities in endocrine cells of the chicken proventriculus at hatching.

PubMed

Martínez, A; Buchan, A M; López, J; Sesma, P

2000-05-01

The colocalization of regulatory peptide immunoreactivities in endocrine cells of the chicken proventriculus at hatching has been investigated using the avidin-biotin technique in serial sections and double immunofluorescence in the same section for light microscopy, and double immunogold staining for electron microscopy. In addition to the eight immunoreactivities previously described in this organ, cells immunoreactive for peptide histidine isoleucine (PHI), peptide gene product 9.5 (PGP), and the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM) were observed. All the cells immunoreactive to glucagon were also immunostained by the PHI antiserum. In addition, all the glucagon-like peptide 1, avian pancreatic polypeptide, and some of the neurotensin-like cells costored also glucagon- and PHI-immunoreactive substances. PGP- and PAM-immunoreactivities were also found in the glucagon-positive cells. A small proportion of the somatostatin-containing cells were positive for PHI but not for other regulatory peptides. These results could suggest either the existence of a very complex regulatory system or that the endocrine system of the newborn chickens is not yet fully developed.

Acceleration of astrocytic differentiation in neural stem cells surviving X-irradiation.

PubMed

Ozeki, Ayumi; Suzuki, Keiji; Suzuki, Masatoshi; Ozawa, Hiroki; Yamashita, Shunichi

2012-03-28

Neural stem cells (NSCs) are highly susceptible to DNA double-strand breaks; however, little is known about the effects of radiation in cells surviving radiation. Although the nestin-positive NSCs predominantly became glial fibrillary acidic protein (GFAP)-positive in differentiation-permissive medium, little or no cells were GFAP positive in proliferation-permissive medium. We found that more than half of the cells surviving X-rays became GFAP positive in proliferation-permissive medium. Moreover, localized irradiation stimulated differentiation of cells outside the irradiated area. These results indicate for the first time that ionizing radiation is able to stimulate astrocyte-specific differentiation of surviving NSCs, whose process is mediated both by the direct activation of nuclear factor-κB and by the indirect bystander effect induced by X-irradiation.

Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

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

Wei, Pei; Li, Li; Qi, Hui

2012-02-10

Highlights: Black-Right-Pointing-Pointer The NPPCs from mouse pancreas were isolated. Black-Right-Pointing-Pointer Tet-on system for SV40 large in NPPCs was used to get RINPPCs. Black-Right-Pointing-Pointer The RINPPCs can undergo at least 80 population doublings without senescence. Black-Right-Pointing-Pointer The RINPPCs can be induced to differentiate into insulin-producing cells. Black-Right-Pointing-Pointer The combination of GLP-1 and sodium butyrate promoted the differentiation process. -- Abstract: Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic {beta} cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas thatmore » expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic {beta} cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.« less

Rare transformation to double hit lymphoma in Waldenstrom's macroglobulinemia.

PubMed

Okolo, Onyemaechi N; Johnson, Ariel C; Yun, Seongseok; Arnold, Stacy J; Anwer, Faiz

2017-08-01

Waldenström macroglobulinemia (WM) is a lymphoproliferative lymphoma that is characterized by monoclonal immunoglobulin M (IgM) protein and bone marrow infiltration. Its incidence is rare and rarer still is its ability to transform to a B-cell lymphoma, particularly the aggressive diffuse large B-cell lymphoma, which bodes a poor prognosis. When transformation includes mutations of MYC, BCL-2 and/or BCL-6, it is known as a 'double hit' or 'triple hit' lymphoma respectively. This paper presents a rare case of WM with mutations positive for MYC and BCL2, making it a case of double hit B-cell lymphoplasmacytic lymphoma with plasmatic differentiation without morphological transformation to aggressive histology like DLBCL. The paper also broadens to include discussions on current topics in the classification, diagnosis, possible causes of transformation, and treatment of WM, including transformation to double hit lymphoma. The significance of this case lies in that the presence of double hit lymphoma-like genetic mutations in WM have not been previously described in the literature and potentially such changes are harbinger of extra-nodal presentation, aggressive growth, and possibly poor prognosis, if data from other double-hit lymphoma are extrapolated.

Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.

PubMed

Kishimoto, Masanobu; Fukui, Toshiro; Suzuki, Ryo; Takahashi, Yu; Sumimoto, Kimi; Okazaki, Takashi; Sakao, Masayuki; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2015-02-01

Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells. Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells. We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU. In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase. In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.

How I treat double-hit lymphoma.

PubMed

Friedberg, Jonathan W

2017-08-03

The 2016 revision of the World Health Organization (WHO) classification for lymphoma has included a new category of lymphoma, separate from diffuse large B-cell lymphoma, termed high-grade B-cell lymphoma with translocations involving myc and bcl-2 or bcl-6 . These lymphomas, which occur in <10% of cases of diffuse large B-cell lymphoma, have been referred to as double-hit lymphomas (or triple-hit lymphomas if all 3 rearrangements are present). It is important to differentiate these lymphomas from the larger group of double-expressor lymphomas, which have increased expression of MYC and BCL-2 and/or BCL-6 by immunohistochemistry, by using variable cutoff percentages to define positivity. Patients with double-hit lymphomas have a poor prognosis when treated with standard chemoimmunotherapy and have increased risk of central nervous system involvement and progression. Double-hit lymphomas may arise as a consequence of the transformation of the underlying indolent lymphoma. There are no published prospective trials in double-hit lymphoma, however retrospective studies strongly suggest that aggressive induction regimens may confer a superior outcome. In this article, I review my approach to the evaluation and treatment of double-hit lymphoma, with an eye toward future clinical trials incorporating rational targeted agents into the therapeutic armamentarium. © 2017 by The American Society of Hematology.

Expression of CD163 in the liver of patients with viral hepatitis.

PubMed

Hiraoka, Atsushi; Horiike, Norio; Akbar, Sk Md Fazle; Michitaka, Kojiro; Matsuyama, Takami; Onji, Morikazu

2005-01-01

CD163 is a marker of activated macrophages, and increased levels of soluble CD163 have been detected in sera obtained from patients with hepatitis. The aim of this study was to detect the expression of CD163 in the liver from patients with viral hepatitis. Frozen sections of liver specimens were obtained from 5 patients with acute viral hepatitis (AH) and from 23 patients with chronic viral hepatitis (CH). The expression of CD163 in the liver was determined immunohistochemically using monoclonal antibody to human CD163. Double immunostaining was done to assess those cell types that express CD163 in the liver. The frequencies of CD163-positive cells were significantly higher both in the portal areas and in the hepatic lobules in the liver of patients with AH compared to those with CH (p < 0.05). Double immunostaining revealed that most of the CD163-positive cells were macrophages and Kupffer cells, because they expressed CD68. The expression of CD163 was very low in endothelial cells and liver stellate cells. This study shows that macrophages are activated in hepatitis liver.

Epstein-Barr virus-positive nodal T/NK-cell lymphoma: an analysis of 15 cases with distinct clinicopathological features.

PubMed

Jeon, Yoon Kyung; Kim, Jo-Heon; Sung, Ji-Youn; Han, Jae Ho; Ko, Young-Hyeh

2015-07-01

Nodal peripheral T-cell lymphoma, not otherwise specified, is a heterogeneous entity with variable biologic behavior. We analyze the clinicopathological features of 15 patients with Epstein-Barr virus-positive (EBV+) nodal T/NK-cell lymphoma, including 9 males and 6 females, with a median age of 64 years. All patients presented with multiple lymphadenopathy with common B symptoms (80%, 12/15) at an advanced Ann Arbor stage (III, IV) (87%, 13/15). The International Prognostic Index was high or high/intermediate in 87% (13/15) of patients, and the prognostic index for peripheral T-cell lymphoma was group 3 or 4 in 73% (11/15). Spleen and liver involvement was observed in 73% (11/15) and 60% (9/15) of patients, respectively. In contrast, extranodal involvement was infrequent, with no more than 1 site in 71% (10/15) of patients. Moreover, none had nasal lesions, and only 1 had mucocutaneous involvement. The cell lineage of EBV+ tumor cells was determined to be T cell in all except 1 patient, who was NK-cell lineage. Cytotoxic molecules were expressed in all cases, and 64% (9/14) of patients expressed the αβT-cell receptor. Moreover, most patients (67%, 10/15) showed CD8 positivity, with 2 of them being CD4CD8 double positive; the others were CD4 positive (n = 2) or CD4CD8 double negative (n = 3). The clinical course was very aggressive, with a median survival time of 3.5 months, and 10 patients died within 6 months of diagnosis. Taken together, our data demonstrate that EBV+ nodal T/NK-cell lymphoma is a distinct clinicopathological entity characterized by cytotoxic molecule expression, a frequent CD8-positive αβT-cell lineage, and a very aggressive clinical behavior. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of cell types during rat liver development.

PubMed

Fiegel, Henning C; Park, Jonas J h; Lioznov, Michael V; Martin, Andreas; Jaeschke-Melli, Stefan; Kaufmann, Peter M; Fehse, Boris; Zander, Axel R; Kluth, Dietrich

2003-01-01

Hepatic stem cells have been identified in adult liver. Recently, the origin of hepatic progenitors and hepatocytes from bone marrow was demonstrated. Hematopoietic and hepatic stem cells share the markers CD 34, c-kit, and Thy1. Little is known about liver stem cells during liver development. In this study, we investigated the potential stem cell marker Thy1 and hepatocytic marker CK-18 during liver development to identify putative fetal liver stem cell candidates. Livers were harvested from embryonic and fetal day (ED) 16, ED 18, ED 20, and neonatal ED 22 stage rat fetuses from Sprague-Dawley rats. Fetal livers were digested by collagenase-DNAse solution and purified by percoll centrifugation. Magnetic cell sorting (MACS) depletion of fetal liver cells was performed using OX43 and OX44 antibodies. Cells were characterized by immunocytochemistry for Thy1, CK-18, and proliferating cell antigen Ki-67 and double labeling for Thy1 and CK-18. Thy1 expression was found at all stages of liver development before and after MACS in immunocytochemistry. Thy1 positive cells were enriched after MACS only in early developmental stages. An enrichment of CK-18 positive cells was found after MACS at all developmental stages. Cells coexpressing Thy1 and CK-18 were identified by double labeling of fetal liver cell isolates. In conclusion, hepatic progenitor cells (CK-18 positive) in fetal rat liver express Thy1. Other progenitors express only CK-18. This indicates the coexistence of different hepatic cell compartments. Isolation and further characterization of such cells is needed to demonstrate their biologic properties.

Curcumin improves the efficacy of cisplatin by targeting cancer stem-like cells through p21 and cyclin D1-mediated tumour cell inhibition in non-small cell lung cancer cell lines

PubMed Central

BAHARUDDIN, PUTERI; SATAR, NAZILAH; FAKIRUDDIN, KAMAL SHAIK; ZAKARIA, NORASHIKIN; LIM, MOON NIAN; YUSOFF, NARAZAH MOHD; ZAKARIA, ZUBAIDAH; YAHAYA, BADRUL HISHAM

2016-01-01

Natural compounds such as curcumin have the ability to enhance the therapeutic effectiveness of common chemotherapy agents through cancer stem-like cell (CSC) sensitisation. In the present study, we showed that curcumin enhanced the sensitivity of the double-positive (CD166+/EpCAM+) CSC subpopulation in non-small cell lung cancer (NSCLC) cell lines (A549 and H2170) to cisplatin-induced apoptosis and inhibition of metastasis. Our results revealed that initial exposure of NSCLC cell lines to curcumin (10–40 µM) markedly reduced the percentage of viability to an average of ~51 and ~54% compared to treatment with low dose cisplatin (3 µM) with only 94 and 86% in both the A549 and H2170 cells. Moreover, sensitisation of NSCLC cell lines to curcumin through combined treatment enhanced the single effect induced by low dose cisplatin on the apoptosis of the double-positive CSC subpopulation by 18 and 20% in the A549 and H2170 cells, respectively. Furthermore, we found that curcumin enhanced the inhibitory effects of cisplatin on the highly migratory CD166+/EpCAM+ subpopulation, marked by a reduction in cell migration to 9 and 21% in the A549 and H2170 cells, respectively, indicating that curcumin may increase the sensitivity of CSCs to cisplatin-induced migratory inhibition. We also observed that the mRNA expression of cyclin D1 was downregulated, while a substantial increased in p21 expression was noted, followed by Apaf1 and caspase-9 activation in the double-positive (CD166+/EpCAM+) CSC subpopulation of A549 cells, suggested that the combined treatments induced cell cycle arrest, therefore triggering CSC growth inhibition via the intrinsic apoptotic pathway. In conclusion, we provided novel evidence of the previously unknown therapeutic effects of curcumin, either alone or in combination with cisplatin on the inhibition of the CD166+/EpCAM+ subpopulation of NSCLC cell lines. This finding demonstrated the potential therapeutic approach of using curcumin that may enhance the effects of cisplatin by targeting the CSC subpopulation in NSCLC. PMID:26531053

Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours

PubMed Central

2013-01-01

Background Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Results Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Conclusions Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard. PMID:24252243

Double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours.

PubMed

Burke, Elinor; Grobler, Mariana; Elderfield, Kay; Bond, Frances; Crocker, Matthew; Taylor, Rohan; Bridges, Leslie R

2013-06-10

Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard.

T-cell chronic lymphocytic leukemia in a double yellow-headed Amazon parrot (Amazona ochrocephala oratrix).

PubMed

Osofsky, Anna; Hawkins, Michelle G; Foreman, Oded; Kent, Michael S; Vernau, William; Lowenstine, Linda J

2011-12-01

An adult, male double yellow-headed Amazon parrot (Amazona ochrocephala oratrix) was diagnosed with chronic lymphocytic leukemia based on results of a complete blood cell count and cytologic examination of a bone marrow aspirate. Treatment with oral chlorambucil was attempted, but no response was evident after 40 days. The bird was euthanatized, and the diagnosis of chronic lymphocytic leukemia was confirmed on gross and microscopic examination of tissues. Neoplastic lymphocytes were found in the bone marrow, liver, kidney, testes, and blood vessels. Based on CD3-positive immunocytochemical and immunohistochemical immunophenotyping, the chronic lymphocytic leukemia was determined to be of T-cell origin.

Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

PubMed

Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

2015-01-01

The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

PubMed

Takahashi, Y; Fukui, T; Kishimoto, M; Suzuki, R; Mitsuyama, T; Sumimoto, K; Okazaki, T; Sakao, M; Sakaguchi, Y; Yoshida, K; Uchida, K; Nishio, A; Matsuzaki, K; Okazaki, K

2016-01-01

The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle. © 2016 International Society for Diseases of the Esophagus.

Use of polymerase chain reaction in human African trypanosomiasis stage determination and follow-up.

PubMed Central

Truc, P.; Jamonneau, V.; Cuny, G.; Frézil, J. L.

1999-01-01

Stage determination of human African trypanosomiasis is based on the detection of parasites and measurements of biological changes in the cerebrospinal fluid (CSF) (concentration of white blood cells > 5 cells per mm3 and increased total protein levels). The patient is treated accordingly. Demonstration of the absence or presence of trypanosomes by the double centrifugation technique is still the only test available to clinicians for assessing treatment success. In this study, however, we evaluate the polymerase chain reaction (PCR) as a tool for assessing the disease stage of trypanosomiasis and for determining whether treatment has been successful. All 15 study patients considered to be in the advanced stage of the disease were PCR positive; however, trypanosomes were demonstrated by double centrifugation in only 11 patients. Of the five remaining patients, who were considered to be in the early stage, PCR and double centrifugation were negative. Following treatment, 13 of the 15 second-stage patients were found to be negative for the disease in at least two samples by PCR and double centrifugation. Two others were still positive by PCR immediately and one month after the treatment. Trypanosome DNA detection using PCR suggested that the two positive patients were not cured but that their possible relapse could not be identified by a search for parasites using the double centrifugation technique. Further evaluation of the PCR method is required, in particular to determine whether PCR assays could be used in studies on patients who fail to respond to melarsoprol, as observed in several foci. PMID:10534898

Peripheral territory and neuropeptides of the trigeminal ganglion neurons centrally projecting through the oculomotor nerve demonstrated by fluorescent retrograde double-labeling combined with immunocytochemistry.

PubMed

Bortolami, R; Calzà, L; Lucchi, M L; Giardino, L; Callegari, E; Manni, E; Pettorossi, V E; Barazzoni, A M; Lalatta Costerbosa, G

1991-04-26

The peripheral territories of sheep trigeminal neurons which send their central process to the brainstem through the oculomotor nerve were investigated by the use of fluorescent tracers in double-labeling experiments. For this purpose Diamidino yellow (DY) injection into the oculomotor nerve was combined with Fast blue (FB) injection either into the extraocular muscles (EOMs), or the cornea, or the superior eyelid. Double-labeled DY + FB cells were found in the ophthalmic region of the trigeminal ganglion in addition to single-labeled DY or FB cells. The DY and DY + FB-labeled trigeminal cells were analysed immunocytochemically for their content of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, and cholecystokinin-8 (CCK-8)-like. All single-labeled DY cells showed SP-, CGRP- or CCK-8-like immunoreactivity. Double-labeled DY + FB neurons innervating the EOMs were immunoreactive for each of the three peptides, whereas double-labeled neurons supplying the cornea were only CGRP-like positive. The findings suggest that, in the sheep, trigeminal neurons which send their process centrally through the oculomotor nerve supply the EOMs, the cornea, and the superior eyelid and contain neuropeptides which are usually associated with pain sensation.

Evidence for cis-trans isomerization of a double bond in the fatty acids of the psychrophilic bacterium Vibrio sp. strain ABE-1.

PubMed

Morita, N; Shibahara, A; Yamamoto, K; Shinkai, K; Kajimoto, G; Okuyama, H

1993-02-01

Vibrio sp. strain ABE-1 was grown in a medium that contained as its stable isotope tracer either [2,2-2H2]cis-9-hexadecenoic or [2,2-2H2]trans-9-hexadecenoic acid. Gas chromatographic-mass spectrometric analysis of the cis-9-hexadecenoic and trans-9-hexadecenoic acid fractions from the cells revealed the formation of an intracellularly isomerized 2,2-2H2-fatty acid which differed from the tracer only in the geometrical configuration of the double bond. This observation shows that cis-trans isomerization without a shift in double-bond position between these two geometric hexadecenoic acid isomers can occur in the cells.

Evidence for cis-trans isomerization of a double bond in the fatty acids of the psychrophilic bacterium Vibrio sp. strain ABE-1.

PubMed Central

Morita, N; Shibahara, A; Yamamoto, K; Shinkai, K; Kajimoto, G; Okuyama, H

1993-01-01

Vibrio sp. strain ABE-1 was grown in a medium that contained as its stable isotope tracer either [2,2-2H2]cis-9-hexadecenoic or [2,2-2H2]trans-9-hexadecenoic acid. Gas chromatographic-mass spectrometric analysis of the cis-9-hexadecenoic and trans-9-hexadecenoic acid fractions from the cells revealed the formation of an intracellularly isomerized 2,2-2H2-fatty acid which differed from the tracer only in the geometrical configuration of the double bond. This observation shows that cis-trans isomerization without a shift in double-bond position between these two geometric hexadecenoic acid isomers can occur in the cells. PMID:8423164

[Sorting role of p16(INK4a)/Ki-67 double immunostaining in the cervical cytology specimens of ASCUS and LSIL cases].

PubMed

Yu, J; Zhu, H T; Zhao, J J; Su, J Z; Xia, Y D

2017-05-08

Objective: To investigate the sorting effect of p16(INK4a)/Ki-67 double immunostaining method in patients with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) cytology results. Methods: Four-hundred and twenty cases collected during April 2014 to February 2015 of cervical cytology of ASCUS ( n =318) and LSIL ( n =102) were selected, and residual liquid-based cytology specimens were used for p16(INK4a)/Ki-67 double immunostaining. The sensitivity and specificity of the detection of cervical precancerous lesions and cervical cancer were calculated, and the results were compared with high risk HPV. Taking histological follow-up as the gold standard, the test was considered positive when at least one cell exhibited p16(INK4a)/Ki-67 co-staining, without requirement of adjunct morphologic interpretation of positive cells. Results: Further screening CIN2+ in cytology ASCUS and LSIL group , the sensitivity of p16(INK4a)/Ki-67 double immunostaining was slightly lower than high risk HPV (84.2% vs . 94.7%), while the specificity was higher (84.0% vs . 53.9%). For ASCUS patients, the sensitivity of p16(INK4a)/Ki-67 double immunostaining and high risk HPV was 82.6% and 91.3%, and the specificity was 88.8% and 63.7%, respectively. For LSIL patients, the sensitivity of p16(INK4a)/Ki-67 double immunostaining and high risk HPV was 86.7% and 100.0%, and the specificity was 67.8% and 20.7%, respectively. For patients younger and older than 30 years, specificity of p16(INK4a)/Ki-67 double immunostaining was both higher than that of high risk HPV (80.8% vs . 42.3%; 84.6% vs . 56.9%). Conclusions: p16(INK4a)/Ki-67 double immunostaining can effectively identify the high risk population in ASCUS or LSIL, with higher specificity than high risk HPV test. p16(INK4a)/Ki-67 double immunostaining may benefit patients younger than 30 years of age as a preliminary or potential cytology-combining screening tool.

Ternary complex factor SAP-1 is required for Erk-mediated thymocyte positive selection.

PubMed

Costello, Patrick S; Nicolas, Robert H; Watanabe, Yasuyuki; Rosewell, Ian; Treisman, Richard

2004-03-01

Thymocyte selection and differentiation requires extracellular signal-regulated kinase (Erk) signaling, but transcription factor substrates of Erk in thymocytes are unknown. We have characterized the function of SAP-1 (Elk4), an Erk-regulated transcription factor, in thymocyte development. Early thymocyte development was normal, but single-positive thymocyte and peripheral T cell numbers were reduced, reflecting a T cell-autonomous defect. T cell receptor-induced activation of SAP-1 target genes such as Egr1 was substantially impaired in double-positive thymocytes, although Erk activation was normal. Analysis of T cell receptor transgenes showed that positive selection was reduced by 80-90% in SAP-1-deficient mice; heterozygous mice showed a moderate defect. Negative selection was unimpaired. SAP-1 thus directly links Erk signaling to the transcriptional events required for thymocyte positive selection.

Dichroic Liquid Crystal Displays

NASA Astrophysics Data System (ADS)

Bahadur, Birendra

The following sections are included: * INTRODUCTION * DICHROIC DYES * Chemical Structure * Chemical and Photochemical Stability * THEORETICAL MODELLING * DEFECTS CAUSED BY PROLONGED LIGHT IRRADIATION * CHEMICAL STRUCTURE AND PHOTOSTABILITY * OTHER PARAMETERS AFFECTING PHOTOSTABILITY * CELL PREPARATION * DICHROIC PARAMETERS AND THEIR MEASUREMENTS * Order Parameter and Dichroic Ratio Of Dyes * Absorbance, Order Parameter and Dichroic Ratio Measurements * IMPACT OF DYE STRUCTURE AND LIQUID CRYSTAL HOST ON PHYSICAL PROPERTIES OF A DICHROIC MIXTURE * Order Parameter and Dichroic Ratio * EFFECT OF LENGTH OF DICHROIC DYES ON THE ORDER PARAMETER * EFFECT OF THE BREADTH OF DYE ON THE ORDER PARAMETER * EFFECT OF THE HOST ON THE ORDER PARAMETER * TEMPERATURE VARIATION OF THE ORDER PARAMETER OF DYES IN A LIQUID CRYSTAL HOST * IMPACT OF DYE CONCENTRATION ON THE ORDER PARAMETER * Temperature Range * Viscosity * Dielectric Constant and Anisotropy * Refractive Indices and Birefringence * solubility43,153-156 * Absorption Wavelength and Auxochromic Groups * Molecular Engineering of Dichroic Dyes * OPTICAL, ELECTRO-OPTICAL AND LIFE PARAMETERS * Colour And CIE Colour space120,160-166 * CIE 1931 COLOUR SPACE * CIE 1976 CHROMATICITY DIAGRAM * CIE UNIFORM COLOUR SPACES & COLOUR DIFFERENCE FORMULAE120,160-166 * Electro-Optical Parameters120 * LUMINANCE * CONTRAST AND CONTRAST RATIO * SWITCHING SPEED * Life Parameters and Failure Modes * DICHROIC MIXTURE FORMULATION * Monochrome Mixture * Black Mixture * ACHROMATIC BLACK MIXTURE FOR HEILMEIER DISPLAYS * Effect of Illuminant on Display Colour * Colour of the Field-On State * Effect of Dye Linewidth * Optimum Centroid Wavelengths * Effect of Dye Concentration * Mixture Formulation Using More Than Three Dyes * ACHROMATIC MIXTURE FOR WHITE-TAYLOR TYPE DISPLAYS * HEILMEIER DISPLAYS * Theoretical Modelling * Threshold Characteristic * Effects of Dye Concentration on Electro-optical Parameters * Effect of Cholesteric Doping * Effect of Alignment * Effect of Thickness * Impact of Order Parameter * Impact of the Host * Impact of Polarizer * Colour Applications * Multiplexing * QUARTER WAVE PLATE DICHROIC DISPLAYS * Operational Principle and Display Configuration11-13 * Electro-Optical Performance * DYE-DOPED TN DISPLAYS * Threshold Characteristic, Contrast Ratio and Switching Speed * PHASE CHANGE EFFECT DICHROIC LCDs * Theoretical Background * Threshold Characteristic and Molecular Orientation * MOLECULAR ORIENTATION DURING FIELD-INDUCED PHASE TRANSITION WITH HOMOGENEOUS WALL ALIGNMENT * MOLECULAR ORIENTATION DURING FIELD-INDUCED PHASE TRANSITION WITH HOMEOTROPIC WALL ALIGNMENT * Contrast Ratio, Transmission, Brightness and Switching Speed3,7,10,198-214 * Memory or Reminiscent Contrast * Electro-optical Performance vs. Temperature * Multiplexing Phase Change Dichroic LCDs * DOUBLE CELL DICHROIC LCDs3,9,14-17,232-234 * Double Cell Nematic Dichroic LCD3,8,9,14,15,233 * Double Cell One Pitch Cholesteric LCD16,17 * Double Cell Phase Change Dichroic LCD214,232 * POSITIVE MODE DICHROIC LCDS3,8,9 * Positive Mode Heilmeier Cells3,8,9,43,77,78,235-238 * USING PLEOCHROIC DYES3,8,9,43,235-238 * USING NEGATIVE DICHROIC DYES3,8,9,63,77,78156 * DUAL FREQUENCY ADDRESSED DICHROIC DISPLAYS75,238 * Positive Mode Dichroic LCDs Using λ/4 Plate * Positive Mode Double Cell Dichroic LCD * Positive Mode Dichroic LCDs Using Special Electrode patterns7,8,239-241 * Positive Mode Phase Change Dichroic LCDs3,8,9,230,243-248 * Dichroic LCDs Using an Admixture of Pleochroic and Negative Dichroic Dyes78,118 * SUPERTWIST DICHROIC EFFECT (SDE) DISPLAYS21-23 * FERROELECTRIC DICHROIC LCDs24-27 * Devices Using A Single Polarizer * Devices Using No Polarizer24-27 * POLYMER DISPERSED DICHROIC LCDs28-30,252-259 * DICHROIC POLYMER LIQUID CRYSTAL DISPLAYS * Heilmeier Type Displays * Guest-Host Cell Using an Admixture Of L.C. Polymer and Low Molecular Weight Liquid Crysta As Host * Polymeric Ferroelectric Dichroic LCDs * SMECTIC A DICHROIC LCDs * Laser Addressed Dichroic SA Displays * Thermally and Electrically Addressed Dichroic SA Displays * FLUORESCENT DICHROIC LCDs * ACKNOWLEDGEMENTS * REFERENCES

Dihydroceramide desaturase inhibition by a cyclopropanated dihydroceramide analog in cultured keratinocytes.

PubMed

Brodesser, Susanne; Kolter, Thomas

2011-01-01

Most mammalian sphingolipids contain a 4,5-(E)-double bond. We report on the chemical synthesis of a dihydroceramide derivative that prevents the introduction of the double bond into sphingolipids. Minimal alteration of the parent structure by formally replacing the hydrogen atoms in the 5- and in the 6-position of the sphinganine backbone by a methylene group leads to an inhibitor of dihydroceramide desaturase in cultured cells. In the presence of 10-50 μM of compound (1), levels of biosynthetically formed dihydroceramide and-surprisingly-also of phytoceramide are elevated at the expense of ceramide. The cells respond to the lack of unsaturated sphingolipids by an elevation of mRNAs of enzymes required for sphingosine formation. At the same time, the analysis of proliferation and differentiation markers indicates that the sphingolipid double bond is required to keep the cells in a differentiated state.

Dihydroceramide Desaturase Inhibition by a Cyclopropanated Dihydroceramide Analog in Cultured Keratinocytes

PubMed Central

Brodesser, Susanne; Kolter, Thomas

2011-01-01

Most mammalian sphingolipids contain a 4,5-(E)-double bond. We report on the chemical synthesis of a dihydroceramide derivative that prevents the introduction of the double bond into sphingolipids. Minimal alteration of the parent structure by formally replacing the hydrogen atoms in the 5- and in the 6-position of the sphinganine backbone by a methylene group leads to an inhibitor of dihydroceramide desaturase in cultured cells. In the presence of 10–50 μM of compound (1), levels of biosynthetically formed dihydroceramide and—surprisingly—also of phytoceramide are elevated at the expense of ceramide. The cells respond to the lack of unsaturated sphingolipids by an elevation of mRNAs of enzymes required for sphingosine formation. At the same time, the analysis of proliferation and differentiation markers indicates that the sphingolipid double bond is required to keep the cells in a differentiated state. PMID:21490810

Manipulating motions of targeted single cells in solution by an integrated double-ring magnetic tweezers imaging microscope.

PubMed

Wu, Meiling; Yadav, Rajeev; Pal, Nibedita; Lu, H Peter

2017-07-01

Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.

Manipulating motions of targeted single cells in solution by an integrated double-ring magnetic tweezers imaging microscope

NASA Astrophysics Data System (ADS)

Wu, Meiling; Yadav, Rajeev; Pal, Nibedita; Lu, H. Peter

2017-07-01

Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.

Interleukin-7 negatively regulates the development of mature T cells in fetal thymus organ cultures.

PubMed

DeLuca, Dominick; Clark, Dawn R

2002-05-01

We added antibody specific for interleukin-7 (IL-7) to chimeric fetal thymus organ cultures (FTOC) to investigate the involvement of this cytokine at distinct stages of T cell development. We report that the neutralization of IL-7 early in fetal T cell development results in a decrease in the production of mature CD4 or CD8 ('single positive', SP) or CD4/8 negative ('double negative', DN) T cell phenotypes, as defined by their expression of CD3. This loss of T cell development was not complete, but it did include the development of gammadelta T cells. However, if IL-7 was neutralized at later stages of FTOC, the production of CD4/8 positive ('double positive', DP) T cells was increased, and if the addition of the antibody was delayed further, the production of mature SP T cells was increased. This last result could be extended to both alphabeta and gammadelta T cells. These data suggested that IL-7 played a negative regulatory role in the development of progressively mature T cells. Tissue sections of FTOC showed that IL-7 was expressed in the subcapsular region of the tissue where immature T cells reside. However, IL-7 was not detected in the medullary region where mature T cells are located. These data suggest that IL-7 not only supports the development of immature fetal T cells, but it may inhibit the development of mature T cells. The production of mature fetal T cells may, therefore, be delayed until their precursors enter the medullary microenvironment, where IL-7 production is low. In this way, T cells may be prevented from maturing until negative selection or anergy events eliminate or inactivate autoreactive clones.

Localization of P2X receptor subtypes 2, 3 and 7 in human urinary bladder.

PubMed

Svennersten, Karl; Hallén-Grufman, Katarina; de Verdier, Petra J; Wiklund, N Peter; Poljakovic, Mirjana

2015-08-08

Voiding dysfunctions are a common problem that has a severe negative impact on the quality of life. Today there is a need for new drug targets for these conditions. The role of ATP receptors in bladder physiology has been studied for some time, primarily in animal models. The aim of this work is to investigate the localization of the ATP receptors P2X2, P2X3 and P2X7 and their colocalization with vimentin and actin in the human urinary bladder. Immunohistochemical analysis was conducted on full-thickness bladder tissues from fundus and trigonum collected from 15 patients undergoing open radical cystectomy due to chronic cystitis, bladder cancer or locally advanced prostate cancer. Colocalization analyses were performed between the three different P2X subtypes and the structural proteins vimentin and actin. Specimens were examined using epifluorescence microscopy and correlation coefficients were calculated for each costaining as well as the mean distance from the laminin positive basal side of the urothelium to the vimentin positive cells located in the suburothelium. P2X2 was expressed in vimentin positive cells located in the suburothelium. Less distinct labelling of P2X2 was also observed in actin positive smooth muscle cells and in the urothelium. P2X3 was expressed in vimentin positive cells surrounding the smooth muscle, and in vimentin positive cells located in the suburothelium. Weaker P2X3 labelling was seen in the urothelium. P2X7 was expressed in the smooth muscle cells and the urothelium. In the suburothelium, cells double positive for P2X2 and vimentin where located closer to the urothelium while cells double positive for P2X3 and vimentin where located further from the urothelium. The results from this study demonstrate that there is a significant difference in the expression of the purinergic P2X2, P2X3 and P2X7 receptors in the different histological layers of the human urinary bladder.

The role of bone marrow-derived cells during the bone healing process in the GFP mouse bone marrow transplantation model.

PubMed

Tsujigiwa, Hidetsugu; Hirata, Yasuhisa; Katase, Naoki; Buery, Rosario Rivera; Tamamura, Ryo; Ito, Satoshi; Takagi, Shin; Iida, Seiji; Nagatsuka, Hitoshi

2013-03-01

Bone healing is a complex and multistep process in which the origin of the cells participating in bone repair is still unknown. The involvement of bone marrow-derived cells in tissue repair has been the subject of recent studies. In the present study, bone marrow-derived cells in bone healing were traced using the GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) were transplanted into C57BL/6 J wild mice. After transplantation, bone injury was created using a 1.0-mm drill. Bone healing was histologically assessed at 3, 7, 14, and 28 postoperative days. Immunohistochemistry for GFP; double-fluorescent immunohistochemistry for GFP-F4/80, GFP-CD34, and GFP-osteocalcin; and double-staining for GFP and tartrate-resistant acid phosphatase were performed. Bone marrow transplantation successfully replaced the hematopoietic cells into GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts or osteocytes in the repair stage were GFP-negative, whereas osteoclasts in the repair and remodeling stages and hematopoietic cells were GFP-positive. The results indicated that bone marrow-derived cells might not differentiate into osteoblasts. The role of bone marrow-derived cells might be limited to adjustment of the microenvironment by differentiating into inflammatory cells, osteoclasts, or endothelial cells in immature blood vessels.

Expression of interleukin-33 and its receptor ST2 in periapical granulomas and radicular cysts.

PubMed

Velickovic, Milena; Pejnovic, Nada; Petrovic, Renata; Mitrovic, Slobodanka; Jeftic, Ilija; Kanjevac, Tatjana; Lukic, Aleksandra

2016-01-01

Interleukin-33 (IL-33) is a recently identified cytokine belonging to the IL-1 family and ligand for the IL-1 receptor-related protein ST2. IL-33/ST2 signaling plays a critical role in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. We aimed to investigate the expression patterns of IL-33 and ST2 in human periapical lesions. Periapical lesions (n = 36) and healthy periapical tissues (n = 10) were evaluated by immunohistochemistry using antibodies specific for human IL-33 and ST2. Lesion samples were further analyzed by double immunofluorescence to assess IL-33/ST2 co-expression. The numbers of IL-33- and ST2-positive fibroblasts were significantly higher in periapical lesions compared to healthy periapical tissues (both P < 0.05), while the numbers of IL-33- and ST2-positive endothelial cells were similar (both P > 0.05). There were no significant differences in the numbers of IL-33- and ST2-positive fibroblasts and endothelial cells between periapical granulomas and radicular cysts (all P > 0.05). Similarly, numbers of ST2-positive mononuclear cells did not differ between periapical granulomas and radicular cysts (P > 0.05). The majority of epithelial cells in radicular cysts were IL-33 positive, while the small proportion of epithelial cells was ST2 positive. Double immunofluorescence analysis revealed IL-33/ST2 co-expression in fibroblasts and endothelial cells. IL-33 and ST2 are expressed in periapical granulomas and radicular cysts. Increased numbers of IL-33- and ST2-positive fibroblasts in periapical lesions when compared to healthy periapical tissues suggest that IL-33/ST2 signaling may be involved in periapical inflammation and tissue fibrosis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Self-organization and positioning of bacterial protein clusters

NASA Astrophysics Data System (ADS)

Murray, Seán M.; Sourjik, Victor

2017-10-01

Many cellular processes require proteins to be precisely positioned within the cell. In some cases this can be attributed to passive mechanisms such as recruitment by other proteins in the cell or by exploiting the curvature of the membrane. However, in bacteria, active self-positioning is likely to play a role in multiple processes, including the positioning of the future site of cell division and cytoplasmic protein clusters. How can such dynamic clusters be formed and positioned? Here, we present a model for the self-organization and positioning of dynamic protein clusters into regularly repeating patterns based on a phase-locked Turing pattern. A single peak in the concentration is always positioned at the midpoint of the model cell, and two peaks are positioned at the midpoint of each half. Furthermore, domain growth results in peak splitting and pattern doubling. We argue that the model may explain the regular positioning of the highly conserved structural maintenance of chromosomes complexes on the bacterial nucleoid and that it provides an attractive mechanism for the self-positioning of dynamic protein clusters in other systems.

Chaotic and stable perturbed maps: 2-cycles and spatial models

NASA Astrophysics Data System (ADS)

Braverman, E.; Haroutunian, J.

2010-06-01

As the growth rate parameter increases in the Ricker, logistic and some other maps, the models exhibit an irreversible period doubling route to chaos. If a constant positive perturbation is introduced, then the Ricker model (but not the classical logistic map) experiences period doubling reversals; the break of chaos finally gives birth to a stable two-cycle. We outline the maps which demonstrate a similar behavior and also study relevant discrete spatial models where the value in each cell at the next step is defined only by the values at the cell and its nearest neighbors. The stable 2-cycle in a scalar map does not necessarily imply 2-cyclic-type behavior in each cell for the spatial generalization of the map.

Conditional deletion reveals a cell-autonomous requirement of SLP-76 for thymocyte selection.

PubMed

Maltzman, Jonathan S; Kovoor, Lisa; Clements, James L; Koretzky, Gary A

2005-10-03

The SH2 domain containing leukocyte phosphoprotein of 76 kD (SLP-76) is critical for pre-TCR-mediated maturation to the CD4+CD8+ double positive (DP) stage in the thymus. The absolute block in SLP-76null mice at the CD4-CD8-CD44-CD25+ (double-negative 3, DN3) stage has hindered our understanding of the role of this adaptor in alphabeta TCR-mediated signal transduction in primary thymocytes and peripheral T lymphocytes. To evaluate the requirements for SLP-76 in these events, we used a cre-loxP approach to generate mice that conditionally delete SLP-76 after the DN3 checkpoint. These mice develop DP thymocytes that express the alphabeta TCR on the surface, but lack SLP-76 at the genomic DNA and protein levels. The DP compartment has reduced cellularity in young mice and fails to undergo positive selection to CD4+ or CD8+ single positive (SP) cells in vivo or activation-induced cell death in vitro. A small number of CD4+SP thymocytes are generated, but these cells fail to flux calcium in response to an alphabeta TCR-generated signal. Peripheral T cells are reduced in number, lack SLP-76 protein, and have an abnormal surface phenotype. These studies show for the first time that SLP-76 is required for signal transduction through the mature alphabeta TCR in primary cells of the T lineage.

TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells.

PubMed

Tsagaratou, Ageliki; González-Avalos, Edahí; Rautio, Sini; Scott-Browne, James P; Togher, Susan; Pastor, William A; Rothenberg, Ellen V; Chavez, Lukas; Lähdesmäki, Harri; Rao, Anjana

2017-01-01

TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4 + CD8 + double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

Digoxigenylated wheat germ agglutinin visualized with alkaline phosphatase-labeled anti-digoxigenin antibodies--a new, sensitive technique with the potential for single and double tracing of neuronal connections.

PubMed

Veh, R W

1991-01-02

For double tracing experiments, wheat germ agglutinin (WGA) molecules labeled with two different haptens are desirable. In the present report the suitability of digoxigenylated WGA (DIG-WGA) for retrograde tracing was investigated. For this purpose the new tracer was pressure injected into rat brains and the transported DIG-WGA visualized via its digoxigenyl group with an alkaline phosphatase linked anti DIG antibody in permanently stained sections of high quality. With fixatives containing 2.5% glutaraldehyde only few positive cells were found. However, at milder fixation conditions (4% paraformaldehyde, 0.05% glutaraldehyde 0.2% picric acid, 30 min) retrogradely labeled cells were detected with a sensitivity comparable to tetramethylbenzidine protocols for conventional WGA-HRP (horseradish peroxidase) tracing. Preliminary experiments suggest excellent suitability for double labeling.

SOX10-positive cells emerge in the rat pituitary gland during late embryogenesis and start to express S100β.

PubMed

Ueharu, Hiroki; Yoshida, Saishu; Kanno, Naoko; Horiguchi, Kotaro; Nishimura, Naoto; Kato, Takako; Kato, Yukio

2018-04-01

In the pituitary gland, S100β-positive cells localize in the neurohypophysis and adenohypophysis but the lineage of the two groups remains obscure. S100β is often observed in many neural crest-derived cell types. Therefore, in this study, we investigate the origin of pituitary S100β-positive cells by immunohistochemistry for SOX10, a potent neural crest cell marker, using S100β-green fluorescence protein-transgenic rats. On embryonic day 21.5, a SOX10-positive cell population, which was also positive for the stem/progenitor cell marker SOX2, emerged in the pituitary stalk and posterior lobe and subsequently expanded to create a rostral-caudal gradient on postnatal day 3 (P3). Thereafter, SOX10-positive cells appeared in the intermediate lobe by P15, localizing to the boundary facing the posterior lobe, the gap between the lobule structures and the marginal cell layer, a pituitary stem/progenitor cell niche. Subsequently, there was an increase in SOX10/S100β double-positive cells; some of these cells in the gap between the lobule structures showed extended cytoplasm containing F-actin, indicating a feature of migration activity. The proportion of SOX10-positive cells in the postnatal anterior lobe was lower than 0.025% but about half of them co-localized with the pituitary-specific progenitor cell marker PROP1. Collectively, the present study identified that one of the lineages of S100β-positive cells is a SOX10-positive one and that SOX10-positive cells express pituitary stem/progenitor cell marker genes.

Probing phospholipase a(2) with fluorescent phospholipid substrates.

PubMed

Wichmann, Oliver; Gelb, Michael H; Schultz, Carsten

2007-09-03

The Foerster resonance energy transfer-based sensor, PENN, measures intracellular phospholipase A(2) (PLA(2)) activity in living cells and small organisms. In an attempt to modify the probe for the detection of particular isoforms, we altered the sn-2 fatty acid in such a way that either one or three of the Z double bonds in arachidonic acid were present in the sensor molecule. Arachidonic-acid-mimicking fatty acids were prepared by copper-mediated coupling reactions. Probes with a single double bond in the 5-position exhibited favorable substrate properties for secretory PLA(2)s. In vitro experiments with the novel unsaturated doubly labeled phosphatidylethanolamine derivatives showed preferred cleavage of the sensor PENN2 (one double bond) by the physiologically important group V sPLA(2), while the O-methyl-derivative PMNN2 was accepted best by the isoform from hog pancreas. For experiments in living cells, we demonstrated that bioactivation via S-acetylthioethyl (SATE) groups is essential for probe performance. Surprisingly, membrane-permeant versions of the new sensors that contained double bonds, PENN2 and PENN3, were only cleaved to a minor extent in HeLa cells while the saturated form, PENN, was well accepted.

Rosuvastatin reduces atherosclerotic lesions and promotes progenitor cell mobilisation and recruitment in apolipoprotein E knockout mice.

PubMed

Schroeter, Marco R; Humboldt, Tim; Schäfer, Katrin; Konstantinides, Stavros

2009-07-01

Statins enhance incorporation of bone marrow-derived cells into experimental neointimal lesions. However, the contribution of progenitor cells to progression of spontaneous atherosclerotic plaques, and the possible modulatory role of statins in this process, remain poorly understood. We compared the effects of rosuvastatin (1 and 10mg/kg BW) and pravastatin (10mg/kg) on progenitor cell mobilisation, recruitment into atherosclerotic plaques, and lesion growth. Statins were administered over 8 weeks to apolipoprotein E knockout mice on atherogenic diet. In addition, mice were lethally irradiated, followed by transplantation of bone marrow from LacZ transgenic mice. Rosuvastatin reduced lesion area and intima-to-media ratio at the brachiocephalic artery compared to vehicle, while both parameters were not significantly altered by pravastatin. Rosuvastatin also augmented endothelialisation (P<0.05) and reduced the smooth muscle cells (SMC) content (P=0.042) of lesions. Numbers of c-kit, sca-1 and flk-1, sca-1 double-positive progenitor cells were significantly increased in rosuvastatin compared to control-treated mice, both in the bone marrow and the peripheral blood. Similarly, the number of spleen-derived acLDL, lectin double-positive progenitor cells (P=0.001) and colony-forming units (P=0.0104) was significantly increased in mice treated with rosuvastatin compared to vehicle alone. In the bone marrow, increased Akt and p42/44 MAP kinase phosphorylation and upregulated SDF1alpha mRNA expression were observed. Importantly, rosuvastatin treatment also increased the plasma levels of c-kit ligand (P=0.003), and the number of c-kit-positive cells within atherosclerotic lesions (P=0.041). Our findings suggest that rosuvastatin reduces the size of atherosclerotic plaques, and this effect appears to involve progenitor cell mobilisation and recruitment into vascular lesions.

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma.

PubMed

Sun, Xi-Tai; Yuan, Xian-Wen; Zhu, Hai-Tao; Deng, Zheng-Ming; Yu, De-Cai; Zhou, Xiang; Ding, Yi-Tao

2012-09-21

To investigate the role of bone marrow-derived endothelial progenitor cells (EPCs) in the angiogenesis of hepatocellular carcinoma (HCC). The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein (GFP) + bone marrow cells. The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting. Serum and tissue levels of vascular endothelial growth factor (VEGF) and colony-stimulating factor (CSF) were quantified by enzyme-linked immunosorbent assay. The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction. The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry. The proportion of EPCs in vessels was then calculated. The HCC model was successful established. The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively. These values are much higher than in the sham-operation group (0.11% ± 0.13%, 0.05% ± 0.11%, n = 9) at 14 d after modeling. At 21 d, the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%, 0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%, 0.12% ± 0.11% in control. Compared to the transient increase observed in controls, the higher level of circulating EPCs were induced by HCC. In addition, the level of serum VEGF and CSF increased gradually in HCC, reaching its peak 14 d after modeling, then slowly decreased. Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels. Under fluorescence microscopy, the bone-marrow (BM)-derived cells labeled with GFP were concentrated in the same area. The relative levels of CD133 and CD34 gene expression were elevated in tumors, around 5.0 and 3.8 times that of the tumor free area. In frozen liver sections from HCC mice, cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies. In tumor tissue, the double-positive cells were incorporated into vessel walls. In immunofluorescent staining. These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells (VECs) come partly from BM-derived EPCs. The proportion of GFP CD31 double positive VECs (out of all VECs) on day 21 was around 35.3% ± 21.2%. This is much higher than the value recorded on day 7 group (17.1% ± 8.9%). The expression of intercellular adhesion molecule 1, vascular adhesion molecule 1, and VEGF was higher in tumor areas than in tumor-free tissues. Mobilized EPCs were found to participate in tumor vasculogenesis of HCC. Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.

Chronology of Islet Differentiation Revealed By Temporal Cell Labeling

PubMed Central

Miyatsuka, Takeshi; Li, Zhongmei; German, Michael S.

2009-01-01

OBJECTIVE Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene. RESEARCH DESIGN AND METHODS In order to separate the transient neurogenin 3 –expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus. RESULTS In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas. CONCLUSIONS The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells. PMID:19478145

Double immunohistochemical staining with MUC4/p53 is useful in the distinction of pancreatic adenocarcinoma from chronic pancreatitis: a tissue microarray-based study.

PubMed

Bhardwaj, Atul; Marsh, William L; Nash, Jason W; Barbacioru, Catalin C; Jones, Susie; Frankel, Wendy L

2007-04-01

Immunohistochemical stains have been used for the distinction of pancreatic adenocarcinoma from chronic pancreatitis. To determine if a double stain for MUC/p53 improved specificity and sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis by comparing maspin, mucin 4 (MUC4), p53, Smad4, and the double stain MUC4/p53. Seventy-four pancreatic adenocarcinomas and 19 chronic pancreatitis cases were retrieved from archival files. Tissue cores were arrayed to create a tissue microarray of 2-mm cores. Sections were stained with antibodies against maspin, MUC4, p53, and Smad4. Additionally, a 2-color, double stain for MUC4 and p53 was developed and evaluated. Five percent or greater staining in either of the cores was considered positive. Intensity (0, 1, 2) and extent (%) of tumor cells staining was also determined. The sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis with maspin, MUC4, p53, and Smad4 was 90%, 77%, 60%, and 63%, respectively; the specificity was 67%, 78%, 88%, and 88%, respectively. When MUC4 and p53 were combined in a double stain, and positive staining for either considered a positive result, the sensitivity increased to 96% but specificity was 73%. When immunoreactivity for both antibodies was necessary for a positive result, sensitivity fell to 39% but specificity was 100%. No correlation was found between intensity or extent of staining with any of the individual stains and tumor differentiation. The double immunohistochemical stain for MUC4/p53 can be a useful diagnostic tool in conjunction with the hematoxylin-eosin-stained section for pancreatic adenocarcinoma, particularly when limited tumor is available for multiple stains.

Immunohistochemical studies for the neuronal elements in the vomeronasal organ of the one-humped camel

PubMed Central

IBRAHIM, Dalia; ABDEL-MAKSOUD, Fatma; TANIGUCHI, Kazumi; YAMAMOTO, Yoshio; TANIGUCHI, Kazuyuki; NAKAMUTA, Nobuaki

2014-01-01

The neuronal elements of the vomeronasal organ (VNO) of camel were investigated immunohistochemically. PGP 9.5 labeled the receptor cells in the vomeronasal sensory epithelium, but not the supporting or basal cells. OMP stained some receptor cells, but no immunoreactive signals for OMP were detected in the non-sensory epithelium. PLCβ2 labeled scattered cells in the sensory epithelium and a larger number of cells in the non-sensory epithelium. Double labeling immunohistochemistry revealed that the PLCβ2-positive cells were surrounded by substance P-positive nerve fibers. Collectively, these data suggest that the camel VNO bears, in addition to the mature vomeronasal receptor cells, trigeminally-innervated solitary chemosensory cells which are expected to play a substantial role in the control of stimulus access to the VNO. PMID:25319516

DNA double strand break repair defect and sensitivity to poly ADP-ribose polymerase (PARP) inhibition in human papillomavirus 16-positive head and neck squamous cell carcinoma

PubMed Central

Weaver, Alice N.; Cooper, Tiffiny S.; Rodriguez, Marcela; Trummell, Hoa Q.; Bonner, James A.; Rosenthal, Eben L.; Yang, Eddy S.

2015-01-01

Patients with human papillomavirus-positive (HPV+) head and neck squamous cell carcinomas (HNSCCs) have increased response to radio- and chemotherapy and improved overall survival, possibly due to an impaired DNA damage response. Here, we investigated the correlation between HPV status and repair of DNA damage in HNSCC cell lines. We also assessed in vitro and in vivo sensitivity to the PARP inhibitor veliparib (ABT-888) in HNSCC cell lines and an HPV+ patient xenograft. Repair of DNA double strand breaks (DSBs) was significantly delayed in HPV+ compared to HPV− HNSCCs, resulting in persistence of γH2AX foci. Although DNA repair activators 53BP1 and BRCA1 were functional in all HNSCCs, HPV+ cells showed downstream defects in both non-homologous end joining and homologous recombination repair. Specifically, HPV+ cells were deficient in protein recruitment and protein expression of DNA-Pk and BRCA2, key factors for non-homologous end joining and homologous recombination respectively. Importantly, the apparent DNA repair defect in HPV+ HNSCCs was associated with increased sensitivity to the PARP inhibitor veliparib, resulting in decreased cell survival in vitro and a 10–14 day tumor growth delay in vivo. These results support the testing of PARP inhibition in combination with DNA damaging agents as a novel therapeutic strategy for HPV+ HNSCC. PMID:26336991

Mitosis, double strand break repair, and telomeres: a view from the end: how telomeres and the DNA damage response cooperate during mitosis to maintain genome stability.

PubMed

Cesare, Anthony J

2014-11-01

Double strand break (DSB) repair is suppressed during mitosis because RNF8 and downstream DNA damage response (DDR) factors, including 53BP1, do not localize to mitotic chromatin. Discovery of the mitotic kinase-dependent mechanism that inhibits DSB repair during cell division was recently reported. It was shown that restoring mitotic DSB repair was detrimental, resulting in repair dependent genome instability and covalent telomere fusions. The telomere DDR that occurs naturally during cellular aging and in cancer is known to be refractory to G2/M checkpoint activation. Such DDR-positive telomeres, and those that occur as part of the telomere-dependent prolonged mitotic arrest checkpoint, normally pass through mitosis without covalent ligation, but result in cell growth arrest in G1 phase. The discovery that suppressing DSB repair during mitosis may function primarily to protect DDR-positive telomeres from fusing during cell division reinforces the unique cooperation between telomeres and the DDR to mediate tumor suppression. © 2014 The Author. Bioessays published by WILEY Periodicals, Inc.

Determining Double Bond Position in Lipids Using Online Ozonolysis Coupled to Liquid Chromatography and Ion Mobility-Mass Spectrometry.

PubMed

Harris, Rachel A; May, Jody C; Stinson, Craig A; Xia, Yu; McLean, John A

2018-02-06

The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20-95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species.

Deletion of Pten Expands Lung Epithelial Progenitor Pools and Confers Resistance to Airway Injury

PubMed Central

Tiozzo, Caterina; De Langhe, Stijn; Yu, Mingke; Londhe, Vedang A.; Carraro, Gianni; Li, Min; Li, Changgong; Xing, Yiming; Anderson, Stewart; Borok, Zea; Bellusci, Saverio; Minoo, Parviz

2009-01-01

Rationale: Pten is a tumor-suppressor gene involved in stem cell homeostasis and tumorigenesis. In mouse, Pten expression is ubiquitous and begins as early as 7 days of gestation. Pten−/− mouse embryos die early during gestation indicating a critical role for Pten in embryonic development. Objectives: To test the role of Pten in lung development and injury. Methods: We conditionally deleted Pten throughout the lung epithelium by crossing Ptenflox/flox with Nkx2.1-cre driver mice. The resulting PtenNkx2.1-cre mutants were analyzed for lung defects and response to injury. Measurements and Main Results: PtenNkx2.1-cre embryonic lungs showed airway epithelial hyperplasia with no branching abnormalities. In adult mice, PtenNkx2.1-cre lungs exhibit increased progenitor cell pools composed of basal cells in the trachea, CGRP/CC10 double-positive neuroendocrine cells in the bronchi, and CC10/SPC double-positive cells at the bronchioalveolar duct junctions. Pten deletion affected differentiation of various lung epithelial cell lineages, with a decreased number of terminally differentiated cells. Over time, PtenNxk2.1-cre epithelial cells residing in the bronchioalveolar duct junctions underwent proliferation and formed uniform masses, supporting the concept that the cells residing in this distal niche may also be the source of procarcinogenic stem cells. Finally, increased progenitor cells in all the lung compartments conferred an overall selective advantage to naphthalene injury compared with wild-type control mice. Conclusions: Pten has a pivotal role in lung stem cell homeostasis, cell differentiation, and consequently resistance to lung injury. PMID:19574443

EBV-associated but HHV8-unrelated double-hit effusion-based lymphoma.

PubMed

Chen, Bo-Jung; Chen, David Yen-Ting; Kuo, Chun-Chi; Chuang, Shih-Sung

2017-03-01

Effusion-based lymphoma is a rare and unique type of large B-cell lymphoma presenting in effusion without a mass lesion. It shares many clinicopathological features with primary effusion lymphoma (PEL), but is distinct from PEL by the absence of HHV8 association. Double hit lymphoma (DHL) is an aggressive B-cell lymphoma, defined by concurrent rearrangement of MYC and BCL2 or BCL6. DHL often presents as lymphadenopathy or an extranodal mass, but rarely occurs in effusion. Here we report a 61-year-old male with alcoholic cirrhosis presenting as massive ascites and left pleural effusion. He has no HIV, HBV or HCV infection and no mass lesion by CT scans. Cytology of both pleural effusion and ascites show large lymphoma cells with plasmablastic morphology characterized by pleomorphic and eccentric nuclei, prominent nucleoli and frequent mitoses. Immunohistochemical study with cell block shows that the lymphoma cells express plasma cell-related markers (CD138, MUM-1 and EMA), but not CD3, CD30, CD45, B-cell markers (CD19, CD20, CD79a, and PAX5), HHV8, ALK or cytokeratin. EBER is positive in most lymphoma cells. Fluorescence in situ hybridization reveals rearrangement at the IGH, BCL2, and MYC loci, but not at BCL6. It is diagnosed as an EBV-associated but HHV8-unrelated double hit effusion-based lymphoma with plasmablastic features. The patient passed away soon after diagnosis without chemotherapy. This is the first reported case of double-hit effusion-based lymphoma with MYC and BCL2 rearrangement. This case illustrates the importance of integrating clinical, cytological, immunophenotypical, and molecular findings to reach a correct diagnosis. Diagn. Cytopathol. 2017;45:257-261. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A retrospective evaluation method for in vitro mammalian genotoxicity tests using cytotoxicity index transformation formulae.

PubMed

Fujita, Yurika; Kasamatsu, Toshio; Ikeda, Naohiro; Nishiyama, Naohiro; Honda, Hiroshi

2016-01-15

Although in vitro chromosomal aberration tests and micronucleus tests have been widely used for genotoxicity evaluation, false-positive results have been reported under strong cytotoxic conditions. To reduce false-positive results, the new Organization for Economic Co-operation and Development (OECD) test guideline (TG) recommends the use of a new cytotoxicity index, relative increase in cell count or relative population doubling (RICC/RPD), instead of the traditionally used index, relative cell count (RCC). Although the use of the RICC/RPD may result in different outcomes and require re-evaluation of tested substances, it is impractical to re-evaluate all existing data. Therefore, we established a method to estimate test results from existing RCC data. First, we developed formulae to estimate RICC/RPD from RCC without cell counts by considering cell doubling time and experiment time. Next, the accuracy of the cytotoxicity index transformation formulae was verified by comparing estimated RICC/RPD and measured RICC/RPD for 3 major chemicals associated with false-positive genotoxicity test results: ethyl acrylate, eugenol and p-nitrophenol. Moreover, 25 compounds with false-positive in vitro chromosomal aberration (CA) test results were re-evaluated to establish a retrospective evaluation method based on derived estimated RICC/RPD values. The estimated RICC/RPD values were in good agreement with the measured RICC/RPD values for every concentration and chemical, and the estimated RICC suggested the possibility that 12 chemicals (48%) with previously judged false-positive results in fact had negative results. Our method enables transformation of RCC data into RICC/RPD values with a high degree of accuracy and will facilitate comprehensive retrospective evaluation of test results. Copyright © 2015 Elsevier B.V. All rights reserved.

Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.

PubMed

He, Jing; Xiu, Bingshui; Wang, Guohua; Chen, Kun; Feng, Xiaoyan; Song, Xiaoguo; Zhu, Cuixia; Yang, Xiqin; Bai, Guanzhong; Ling, Shigan; Zhang, Heqiu

2011-08-01

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

Molecular Mechanisms of Particle Ration Induced Apoptosis in Lymphocyte

NASA Astrophysics Data System (ADS)

Shi, Yufang

Space radiation, composed of high-energy charged nuclei (HZE particles) and protons, has been previously shown to severely impact immune homeostasis in mice. To determine the molecular mechanisms that mediate acute lymphocyte depletion following exposure to HZE particle radiation mice were exposed to particle radiation beams at Brookhaven National Laboratory. We found that mice given whole body 5 6Fe particle irradiation (1GeV /n) had dose-dependent losses in total lymphocyte numbers in the spleen and thymus (using 200, 100 and 50 cGy), with thymocytes being more sensitive than splenocytes. All phenotypic subsets were reduced in number. In general, T cells and B cells were equally sensitive, while CD8+ T cells were more senstive than CD4+ T cells. In the thymus, immature CD4+CD8+ double-positive thymocytes were exquisitely sensitive to radiation-induced losses, single-positive CD4 or CD8 cells were less sensitive, and the least mature double negative cells were resistant. Irradiation of mice deficient in genes encoding essential apoptosis-inducing proteins revealed that the mechanism of lymphocyte depletion is independent of Fas ligand and TRAIL (TNF-ralated apoptosis-inducing ligand), in contrast to γ-radiation-induced lymphocyte losses which require the Fas-FasL pathway. Using inhibitors in vitro, lymphocyte apoptosis induced by HZE particle radiation was found to be caspase dependent, and not involve nitric oxide or oxygen free radicals.

Presence of S100A8/Gr1-Positive Myeloid-Derived Suppressor Cells in Primary Tumors and Visceral Organs Invaded by Breast Carcinoma Cells.

PubMed

Tanriover, Gamze; Eyinc, Mehmet Berk; Aliyev, Elnur; Dilmac, Sayra; Erin, Nuray

2018-04-26

Increased S100A8/A9 expression in Gr1-positive cells has been shown in myeloid-derived suppressor cells and may play a role in the formation of a metastatic milieu. We aimed to determine S100A8/A9 expression alone and with coexpression of Gr1 (a myeloid marker) in primary tumor and visceral tissues invaded by metastatic breast carcinoma. Female BALB/c mice were injected with 4TLM, 4THM, and 67NR orthotopically. Confluent cells (75%-80%) were used. Primary tumor, lung, liver, and spleen tissue samples were removed 26 days after injection. Peripheral blood smears and metastasis assay were performed, as was immunohistochemistry and staining. S100A8/A9 immunoreactivity alone or coexpressed with Gr1 was found in primary tumors formed by 4TLM and 4THM cells, which was markedly higher than in primary tumors formed by nonmetastatic 67NR cells. Similarly, liver and lung tissues obtained from mice injected with 4TLM or 4THM cells were invaded by S100A8/A9-positive and Gr1-positive cells. Double-positive cells were markedly fewer in liver and lung tissues of animals injected with 67NR cells. S100A8/A9-positive cells were mostly localized in red pulp of spleens. We observed an increased number of neutrophils in the peripheral blood of mice injected with metastatic breast carcinoma cells. Tumor-derived factors may increase S100A8/A9-positive cells locally and systemically, and S100A8/A9-positive cells may provide an appropriate milieu for the formation of metastasis. Copyright © 2018 Elsevier Inc. All rights reserved.

Towards high-throughput automated targeted femtosecond laser-based transfection of adherent cells

NASA Astrophysics Data System (ADS)

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Gunn-Moore, Frank; Dholakia, Kishan

2011-03-01

Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Zhang, Meixiang; Ngo, Justine; Pirozzi, Filomena; Sun, Ying-Pu; Wynshaw-Boris, Anthony

2018-03-15

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically significant differences in the variation of PAX6 and SOX1-positive NPCs between the two human pluripotent cell-derived methods; therefore, both methods are suitable for producing stable dorsal NPCs. When further differentiated into mature neurons, NPCs gave rise to a population of almost exclusively forebrain cortical neurons, confirming the dorsal fate commitment of the progenitors. The methods described in this study show improvements over previously published studies and are highly efficient at differentiating human and mouse pluripotent cell types into dorsal PAX6-positive NPCs and eventually into forebrain cortical neurons.

In silico characterization of cell-cell interactions using a cellular automata model of cell culture.

PubMed

Kihara, Takanori; Kashitani, Kosuke; Miyake, Jun

2017-07-14

Cell proliferation is a key characteristic of eukaryotic cells. During cell proliferation, cells interact with each other. In this study, we developed a cellular automata model to estimate cell-cell interactions using experimentally obtained images of cultured cells. We used four types of cells; HeLa cells, human osteosarcoma (HOS) cells, rat mesenchymal stem cells (MSCs), and rat smooth muscle A7r5 cells. These cells were cultured and stained daily. The obtained cell images were binarized and clipped into squares containing about 10 4 cells. These cells showed characteristic cell proliferation patterns. The growth curves of these cells were generated from the cell proliferation images and we determined the doubling time of these cells from the growth curves. We developed a simple cellular automata system with an easily accessible graphical user interface. This system has five variable parameters, namely, initial cell number, doubling time, motility, cell-cell adhesion, and cell-cell contact inhibition (of proliferation). Within these parameters, we obtained initial cell numbers and doubling times experimentally. We set the motility at a constant value because the effect of the parameter for our simulation was restricted. Therefore, we simulated cell proliferation behavior with cell-cell adhesion and cell-cell contact inhibition as variables. By comparing growth curves and proliferation cell images, we succeeded in determining the cell-cell interaction properties of each cell. Simulated HeLa and HOS cells exhibited low cell-cell adhesion and weak cell-cell contact inhibition. Simulated MSCs exhibited high cell-cell adhesion and positive cell-cell contact inhibition. Simulated A7r5 cells exhibited low cell-cell adhesion and strong cell-cell contact inhibition. These simulated results correlated with the experimental growth curves and proliferation images. Our simulation approach is an easy method for evaluating the cell-cell interaction properties of cells.

Establishment of primary cultures of craniopharyngioma cells★

PubMed Central

Liu, Hao; Liu, Liang; Liu, Zhiyong; Li, Qiang; You, Chao; Xu, Jianguo

2012-01-01

Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research. PMID:25745451

Presence of Epstein-Barr virus-infected B lymphocytes with thyrotropin receptor antibodies on their surface in Graves' disease patients and in healthy individuals.

PubMed

Nagata, Keiko; Higaki, Katsumi; Nakayama, Yuji; Miyauchi, Hiromi; Kiritani, Yui; Kanai, Kyosuke; Matsushita, Michiko; Iwasaki, Takeshi; Sugihara, Hirotsugu; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Nanba, Eiji; Kimura, Hiroshi; Hayashi, Kazuhiko

2014-05-01

Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein-Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves' disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves' disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves' disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves' disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves' disease.

Presence of Epstein–Barr virus-infected B lymphocytes with thyrotropin receptor antibodies on their surface in Graves’ disease patients and in healthy individuals

PubMed Central

Nagata, Keiko; Higaki, Katsumi; Nakayama, Yuji; Miyauchi, Hiromi; Kiritani, Yui; Kanai, Kyosuke; Matsushita, Michiko; Iwasaki, Takeshi; Sugihara, Hirotsugu; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Nanba, Eiji; Kimura, Hiroshi; Hayashi, Kazuhiko

2014-01-01

Abstract Graves’ disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein–Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves’ disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves’ disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves’ disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves’ disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves’ disease. PMID:24467196

Distinct CD4+CD8+ Double-Positive T Cells in the Blood and Liver of Patients during Chronic Hepatitis B and C

PubMed Central

Nascimbeni, Michelina; Pol, Stanislas; Saunier, Bertrand

2011-01-01

CD4+ and CD8+ T cells, the main effectors of adaptive cellular immune responses, differentiate from immature, non-functional CD4+CD8+ double-positive T (DPT) cells in the thymus. Increased proportions of circulating DPT lymphocytes have been observed during acute viral infections; in chronic viral diseases, the role and repartition of extra-thymic DPT cells remain largely uncharacterized. We performed a phenotypic analysis of DPT cells in blood and liver from patients chronically infected by hepatitis C (HCV) or B (HBV) viruses. The highest percentages of DPT cells, predominantly CD4highCD8low, were observed in patients infected by HCV, while HBV-infected patients mostly displayed CD4lowCD8high and CD4highCD8high DPT cells. All proportions of DPT cells were higher in liver than in blood with, for each subpopulation referred to above, a correlation between their frequencies in these two compartments. In HCV patients, intra-hepatic DPT cells displayed more heterogeneous activation, differentiation and memory phenotypes than in the blood; most of them expressed CD1a, a marker of T cell development in the thymus. Ex vivo, the inoculation of liver slices with HCV produced in cell culture was accompanied by a disappearance of CD8high cells, suggesting a direct effect of the virus on the phenotype of DPT cells in the liver. Our results suggest that, in half of the patients, chronic HCV infection promotes the production of DPT cells, perhaps by their re-induction in the thymus and selection in the liver. PMID:21647449

Selective Cell Adhesion and Biosensing Applications of Bio-Active Block Copolymers Prepared by CuAAC/Thiol-ene Double Click Reactions.

PubMed

Oyman Eyrilmez, Gizem; Doran, Sean; Murtezi, Eljesa; Demir, Bilal; Odaci Demirkol, Dilek; Coskunol, Hakan; Timur, Suna; Yagci, Yusuf

2015-09-01

N-Acetyl-l-cysteine (NAC)-capped poly(methyl methacrylate)-b-polycaprolactone block copolymer (PMMA-b-PCL-NAC) was prepared using the previously described one-pot photoinduced sequential CuAAC/thiol-ene double click procedure. PMMA-b-PCL-NAC had previously shown good applicability as a matrix for cell adhesion of cells from the Vero cell line (African green monkey kidney epithelial). Here, in this work, PMMA-b-PCL-NAC served as an excellent immobilization matrix for biomolecule conjugation. Covalent binding of RGD (R: arginine, G: glycine, and D: aspartic acid) peptide sequence onto the PMMA-b-PCL-NAC-coated surface was performed via EDC chemistry. RGD-modified PMMA-b-PCL-NAC (PMMA-b-PCL-NAC-RGD) as a non-toxic cell proliferation platform was used for selective "integrin αvβ3-mediated cell adhesion and biosensing studies. Both optical and electrochemical techniques were used to monitor the adhesion differences between "integrin αvβ3" receptor positive and negative cell lines on to the designed biofunctional surfaces. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Abundant immunoglobulin E-positive cells in skin lesions support an allergic etiology of atopic dermatitis in the elderly

PubMed Central

Tanei, R; Hasegawa, Y; Sawabe, M

2013-01-01

Background/Objectives Atopic dermatitis (AD) in the elderly is gradually increasing in industrialized countries in association with the aging of society. We report herein four cases of elderly AD {three extrinsic [immunoglobulin (Ig)E-mediated allergy]; one intrinsic (non-IgE-allergy)} in which we investigated the presence of IgE+ cells in lesional skin. Methods/Results Single immunohistochemical and double immunofluorescence stainings were performed for skin biopsy specimens from AD patients and non-atopic control subjects with chronic eczema. In the lesional lichenified skin of patients with extrinsic elderly AD, numerous IgE+ cells were found among inflammatory cells infiltrates in the upper dermis. Comparative analysis of single immunohistochemistry results using serial paraffin and/or frozen sections found that many IgE+ cells showed identical distributions to tryptase+ mast cells. IgE+ cells coincident with CD1a+ Langerhans cells in the epidermis were found in small numbers only in frozen sections. Double immunofluorescence staining for IgE and CD11c revealed cells coexpressing IgE and CD11c with a dendritic morphology in the papillary and upper dermis. These IgE+ mast cells and IgE+ CD11c+ cells were also found in cured normal-looking skin from a patient with extrinsic elderly AD after successful treatment. Although only a few weakly positive IgE+ cells were detected, no IgE+CD11c+ cells were found in specimens from patients with intrinsic elderly AD or non-atopic chronic eczema. Conclusion IgE-mediated allergic inflammation may play an important role in the pathobiology of elderly AD, similar to other age groups of AD. PMID:22702954

A Co-Receptor Independent Transgenic Human TCR Mediates Anti-Tumor and Anti-Self Immunity in Mice

PubMed Central

Mehrotra, Shikhar; Al-Khami, Amir A.; Klarquist, Jared; Husain, Shahid; Naga, Osama; Eby, Jonathan M.; Murali, Anuradha K.; Lyons, Gretchen E.; Li, Mingli; Spivey, Natali D.; Norell, Håkan; Martins da Palma, Telma; Onicescu, Georgiana; Diaz-Montero, C. Marcela; Garrett-Mayer, Elizabeth; Cole, David J.; Le Poole, I. Caroline; Nishimura, Michael I.

2013-01-01

Recent advancements in T cell immunotherapy suggest that T cells engineered with high affinity T cell receptors (TCR) can offer better tumor regression. However, whether a high affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope reactive, CD8 independent, high affinity TCR isolated from MHC class-I restricted CD4+ T cells obtained from tumor infiltrating lymphocytes of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2 restricted TCR was positively selected on both CD4+ and CD8+ single-positive (SP) cells. However, when the TCR transgenic mouse was developed with an HLA-A2 background, the transgenic TCR was primarily expressed by CD3+CD4-CD8- double-negative (DN) T cells. TIL 1383I TCR transgenic CD4+, CD8+ and CD4-CD8- T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2+/human tyrosinase TCR double transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high affinity TIL 1383I TCR alone in CD3+ T cells is sufficient to control the growth of murine and human melanoma and the presence or absence of CD4 and CD8 co-receptors had little effect on its functional capacity. PMID:22798675

Genotoxicity induced by monomethylarsonous acid (MMA+3) in mouse thymic developing T cells.

PubMed

Xu, Huan; Medina, Sebastian; Lauer, Fredine T; Douillet, Christelle; Liu, Ke Jian; Stýblo, Miroslav; Burchiel, Scott W

2017-09-05

Drinking water exposure to arsenic is known to cause immunotoxicity. Our previous studies demonstrated that monomethylarsonous acid (MMA +3 ) was the major arsenical species presented in mouse thymus cells after a 30 d drinking water exposure to arsenite (As +3 ). MMA +3 was also showed to be ten times more toxic than As +3 on the suppression of IL-7/STAT5 signaling in the double negative (DN) thymic T cells. In order to examine the genotoxicity induced by low to moderate doses of MMA +3 , isolated mouse thymus cells were treated with 5, 50 and 500nMMMA +3 for 18h in vitro. MMA +3 suppressed the proliferation of thymus cells in a dose dependent manner. MMA +3 at 5nM induced DNA damage in DN not double positive (DP) cells. Differential sensitivity to double strand breaks and reactive oxygen species generation was noticed between DN and DP cells at 50nM, but the effects were not seen at the high dose (500nM). A stronger apoptotic effect induced by MMA +3 was noticed in DN cells than DP cells at low doses (5 and 50nM), which was negated by the strong apoptosis induction at the high dose (500nM). Analysis of intracellular MMA +3 concentrations in DN and DP cells, revealed that more MMA +3 accumulated in the DN cells after the in vitro treatment. Collectively, these results suggested that MMA +3 could directly induce strong genotoxicity in the early developing T cells in the thymus. The DN cells were much more sensitive to MMA +3 induced genotoxicity and apoptosis than DP cells, probably due to the higher intracellular levels of MMA +3 . Copyright © 2017 Elsevier B.V. All rights reserved.

Moderate temperature sodium cells. V - Discharge reactions and rechargeability of NiS and NiS2 positive electrodes in molten NaAlCl4

NASA Technical Reports Server (NTRS)

Abraham, K. M.; Elliot, J. E.

1984-01-01

NiS2 and NiS have been characterized as high energy density rechargeable positive electrodes for moderate-temperature Na batteries of the configuration, Na(1)/beta double prime-Al2O3/NaAlCl4(1), NiSx. The batteries operate in the temperature range 170 - 190 C. Positive electrode reactions during discharge/charge cycles have been characterized. Excellent rechargeability of the batteries has been demonstrated by extended cell cycling. A Na/NiS2 cell, operating at 190 C, exceeded 600 deep discharge/charge cycles with practically no capacity deterioration. The feasibility of secondary Na/NiSx batteries with specific energies equal to or greater than 50 Wh/lb and cycle lifes exceeding 1000 deep discharge/charge cycles has been demonstrated.

Deletion of Cytoplasmic Double-Stranded RNA Sensors Does Not Uncover Viral Small Interfering RNA Production in Human Cells.

PubMed

Schuster, Susan; Tholen, Lotte E; Overheul, Gijs J; van Kuppeveld, Frank J M; van Rij, Ronald P

2017-01-01

Antiviral immunity in insects and plants is mediated by the RNA interference (RNAi) pathway in which viral long double-stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer enzymes. Although this pathway is evolutionarily conserved, its involvement in antiviral defense in mammals is the subject of debate. In vertebrates, recognition of viral RNA induces a sophisticated type I interferon (IFN)-based immune response, and it has been proposed that this response masks or inhibits antiviral RNAi. To test this hypothesis, we analyzed viral small RNA production in differentiated cells deficient in the cytoplasmic RNA sensors RIG-I and MDA5. We did not detect 22-nucleotide (nt) viral siRNAs upon infection with three different positive-sense RNA viruses. Our data suggest that the depletion of cytoplasmic RIG-I-like sensors is not sufficient to uncover viral siRNAs in differentiated cells. IMPORTANCE The contribution of the RNA interference (RNAi) pathway in antiviral immunity in vertebrates has been widely debated. It has been proposed that RNAi possesses antiviral activity in mammalian systems but that its antiviral effect is masked by the potent antiviral interferon response in differentiated mammalian cells. In this study, we show that inactivation of the interferon response is not sufficient to uncover antiviral activity of RNAi in human epithelial cells infected with three wild-type positive-sense RNA viruses.

The effect of low static magnetic field on osteogenic and adipogenic differentiation potential of human adipose stromal/stem cells

NASA Astrophysics Data System (ADS)

Marędziak, Monika; Śmieszek, Agnieszka; Tomaszewski, Krzysztof A.; Lewandowski, Daniel; Marycz, Krzysztof

2016-01-01

The aim of this work was to investigate the effects of static magnetic field (SMF) on the osteogenic properties of human adipose derived mesenchymal stem cells (hASCs). In this study in seven days viability assay we examined the impact of SMF on cells proliferation rate, population doubling time, and ability to form single-cell derived colonies. We have also examined cells' morphology, ultrastructure and osteogenic properties on the protein as well as mRNA level. We established a complex approach, which enabled us to obtain information about SMF and hASCs potential in the context of differentiation into osteogenic and adipogenic lineages. We demonstrated that SMF enhances both viability and osteogenic properties of hASCs through higher proliferation factor and shorter population doubling time. We have also observed asymmetrically positioned nuclei and organelles after SMF exposition. With regards to osteogenic properties we observed increased levels of osteogenic markers i.e. osteopontin, osteocalcin and increased ability to form osteonodules with positive reaction to Alizarin Red dye. We have also shown that SMF besides enhancing osteogenic properties of hASCs, simultaneously decreases their ability to differentiate into adipogenic lineage. Our results clearly show a direct influence of SMF on the osteogenic potential of hASCs. These results provide key insights into the role of SMF on their cellular fate and properties.

Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation

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

Cheng, Xiaofei; College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036; Deng, Ping

Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings themore » split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells. - Highlights: • A live-cell imaging system was developed for visualizing dsRNA in vivo. • It uses dsRNA binding proteins fused with two halves of a fluorescent protein. • Binding to a common dsRNA enables the reporter to become fluorescent. • The system can efficiently monitor viral RNA replication in living cells.« less

Three-dimensional Invasion of Human Glioblastoma Cells Remains Unchanged by X-ray and Carbon Ion Irradiation In Vitro

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

Eke, Iris; Storch, Katja; Kaestner, Ina

Purpose: Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. Methods and Materials: Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks ({gamma}H2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg,more » {beta}1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. Results: Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the {beta}1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. Conclusions: These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy.« less

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

PubMed Central

Kutleša, Snježana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B.; Jurecic, Roland

2011-01-01

Objective Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. Materials and Methods EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. Results The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Tα, RAG-1, and T-cell receptor – Vβ genes; and 3) produced interferon-γ in response to T-cell receptor stimulation. Conclusions These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation. PMID:19447159

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

PubMed

Kutlesa, Snjezana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland

2009-08-01

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

Regional expression patterns of taste receptors and gustducin in the mouse tongue.

PubMed

Kim, Mi-Ryung; Kusakabe, Yuko; Miura, Hirohito; Shindo, Yoichiro; Ninomiya, Yuzo; Hino, Akihiro

2003-12-12

In order to understand differences in taste sensitivities of taste bud cells between the anterior and posterior part of tongue, it is important to analyze the regional expression patterns of genes related to taste signal transduction on the tongue. Here we examined the expression pattern of a taste receptor family, the T1r family, and gustducin in circumvallate and fungiform papillae of the mouse tongue using double-labeled in situ hybridization. Each member of the T1r family was expressed in both circumvallate and fungiform papillae with some differences in their expression patterns. The most striking difference between fungiform and circumvallate papillae was observed in their co-expression patterns of T1r2, T1r3, and gustducin. T1r2-positive cells in fungiform papillae co-expressed T1r3 and gustducin, whereas T1r2 and T1r3 double-positive cells in circumvallate papillae merely expressed gustducin. These results suggested that in fungiform papillae, gustducin might play a role in the sweet taste signal transduction cascade mediated by a sweet receptor based on the T1r2 and T1r3 combination, in fungiform papillae.

Cordyceps sinensis promotes immune regulation and enhances bacteriostatic activity of PA-824 via IL-10 in Mycobacterium tuberculosis disease

PubMed Central

Li, D.G.; Ren, Z.X.

2017-01-01

PA-824 is a novel bicyclic nitroimidazole anti-tuberculosis (TB) drug. Cordyceps sinensis (Berk.) Sacc. (CS) was proven to be a good immunomodulatory compound. This research aimed to investigate the effect of CS on PA-824 in Mycobacterium tuberculosis (M.tb) infected mice (female CBA/J mice, 6 to 8 weeks of age and 20±2 g of weight). Mice were randomly assigned to 4 groups: PA-824, CS, PA-824+CS, and control. To verify the effect of PA-824 and CS on M.tb, after drug administration, mice lungs were harvested and bacterial colony formations were measured. Cells were isolated from infected lungs and spleens to analyze the percentage of CD4+ T cells (CD11a positive). Lung cells were cultured to detect the secretion of interferon-γ (IFN-γ) and interleukin-10 (IL-10) by ELISA. IFN-γ and IL-10 double-positive CD4+ cells in peripheral blood were measured by flow cytometry. The expression levels of IL-2 and IL-10 in mice lungs were analyzed by real-time PCR and western blot. Results showed that PA-824 combined with CS led to the lowest lung colony-forming units (CFU) counts among treated groups. Furthermore, this beneficial outcome might be associated with the decreased CD11a on CD4+ cells in mice lungs and spleens. Moreover, the suppressed secretion of IFN-γ and IL-10, and IL-10 expressions, as well as the decreased IFN-γ and IL-10 double-positive CD4+ cells in blood, could also be associated with the positive effect. However, no significant effect on IL-2 production was found. The combination of PA-824 and CS had more effective bacteriostatic and immunomodulatory effects on M.tb infected mice than PA-824 alone. In conclusion, CS has the potential to be an effective adjuvant in TB treatment. PMID:28793052

Cordyceps sinensis promotes immune regulation and enhances bacteriostatic activity of PA-824 via IL-10 in Mycobacterium tuberculosis disease.

PubMed

Li, D G; Ren, Z X

2017-08-07

PA-824 is a novel bicyclic nitroimidazole anti-tuberculosis (TB) drug. Cordyceps sinensis (Berk.) Sacc. (CS) was proven to be a good immunomodulatory compound. This research aimed to investigate the effect of CS on PA-824 in Mycobacterium tuberculosis (M.tb) infected mice (female CBA/J mice, 6 to 8 weeks of age and 20±2 g of weight). Mice were randomly assigned to 4 groups: PA-824, CS, PA-824+CS, and control. To verify the effect of PA-824 and CS on M.tb, after drug administration, mice lungs were harvested and bacterial colony formations were measured. Cells were isolated from infected lungs and spleens to analyze the percentage of CD4+ T cells (CD11a positive). Lung cells were cultured to detect the secretion of interferon-γ (IFN-γ) and interleukin-10 (IL-10) by ELISA. IFN-γ and IL-10 double-positive CD4+ cells in peripheral blood were measured by flow cytometry. The expression levels of IL-2 and IL-10 in mice lungs were analyzed by real-time PCR and western blot. Results showed that PA-824 combined with CS led to the lowest lung colony-forming units (CFU) counts among treated groups. Furthermore, this beneficial outcome might be associated with the decreased CD11a on CD4+ cells in mice lungs and spleens. Moreover, the suppressed secretion of IFN-γ and IL-10, and IL-10 expressions, as well as the decreased IFN-γ and IL-10 double-positive CD4+ cells in blood, could also be associated with the positive effect. However, no significant effect on IL-2 production was found. The combination of PA-824 and CS had more effective bacteriostatic and immunomodulatory effects on M.tb infected mice than PA-824 alone. In conclusion, CS has the potential to be an effective adjuvant in TB treatment.

Ectopic transgene expression in the retina of four transgenic mouse lines

PubMed Central

Gábriel, Robert; Erdélyi, Ferenc; Szabó, Gábor; Lawrence, J. Josh

2017-01-01

Retinal expression of transgenes was examined in four mouse lines. Two constructs were driven by the choline acetyltransferase (ChAT) promoter: green fluorescent protein conjugated to tau protein (tau-GFP) or cytosolic yellow fluorescent protein (YFP) generated through CRE recombinase-induced expression of Rosa26 (ChAT-CRE/ Rosa26YFP). Two other constructs targeted inhibitory interneurons: GABAergic horizontal and amacrine cells identified by glutamic acid decarboxylase (GAD65-GFP) or parvalbumin (PV) cells (PV-CRE/Rosa26YFP). Animals were transcardially perfused and retinal sections prepared. Antibodies against PV, calretinin (CALR), calbindin (CALB), and tyrosine hydroxylase (TH) were used to counterstain transgene-expressing cells. In PVxRosa and ChAT-tauGFP constructs, staining appeared in vertically oriented row of processes resembling Müller cells. In the ChATxRosa construct, populations of amacrine cells and neurons in the ganglion cell layer were labeled. Some cones also exhibited GFP fluorescence. CALR, PV and TH were found in none of these cells. Occasionally, we found GFP/ CALR and GFP/PV double-stained cells in the ganglion cell layer (GCL). In the GAD65-GFP construct, all layers of the neuroretina were labeled, except photoreceptors. Not all horizontal cells expressed GFP. We did not find GFP/TH double-labeled cells and GFP was rarely present in CALR-and CALB-containing cells. Many PV-positive neurons were also labeled for GFP, including small diameter amacrines. In the GCL, single labeling for GFP and PV was ascertained, as well as several CALR/PV double-stained neurons. In the GCL, cells triple labeled with GFP/CALR/ CALB were sparse. In conclusion, only one of the four transgenic constructs exhibited an expression pattern consistent with endogenous retinal protein expression, while the others strongly suggested ectopic gene expression. PMID:26563404

A prospective, randomized, double-blind study, comparing unirradiated to irradiated white blood cell transfusions in acute leukemia patients

PubMed Central

Freireich, E J; Lichtiger, B; Mattiuzzi, G; Martinez, F; Reddy, V; Kyle Wathen, J

2013-01-01

A prospective, randomized double-blind study comparing the effects of irradiated and unirradiated white blood cells was conducted in 108 acute leukemia patients with life-threatening infections, refractory to antibiotics. The study demonstrated no significant improvement in 30-day survival or overall survival. Transfusion of unirradiated white cells did not compromise the patient's opportunity to undergo allogeneic stem cell transplant, nor the success rate or overall survival after allogeneic transplant. The important positive finding in this study was that the unirradiated white cells produced a significantly higher increment in circulating granulocytes and in a higher proportion of patients granulocyte count exceeded 1000 per microliter, approaching normal concentrations. The increase in the number and the improved survival of the unirradiated granulocytes suggest that this procedure might potentially be a method to improve the utility of granulocyte transfusions and merits further investigation. The study demonstrated non-inferiority for unirradiated white cells. There were no harmful effects such as graft-versus-host disease, indicating that such studies would be safe to conduct in the future. PMID:23072780

Sex differences in the expression of estrogen receptor alpha within noradrenergic neurons in the sheep brain stem.

PubMed

Rose, J L; Hamlin, A S; Scott, C J

2014-10-01

In female sheep, high levels of estrogen exert a positive feedback action on gonadotropin releasing hormone (GnRH) secretion to stimulate a surge in luteinizing hormone (LH) secretion. Part of this action appears to be via brain stem noradrenergic neurons. By contrast, estrogen action in male sheep has a negative feedback action to inhibit GnRH and LH secretion. To investigate whether part of this sex difference is due to differences in estrogen action in the brain stem, we tested the hypothesis that the distribution of estrogen receptor α (ERα) within noradrenergic neurons in the brain stem differs between rams and ewes. To determine the distribution of ERα, we used double-label fluorescence immunohistochemistry for dopamine β-Hydroxylase, as a marker for noradrenergic and adrenergic cells, and ERα. In the ventrolateral medulla (A1 region), most ERα-immunoreactive (-ir) cells were located in the caudal part of the nucleus. Overall, there were more ERα-ir cells in rams than ewes, but the proportion of double-labeled cells was did not differ between sexes. Much greater numbers of ERα-ir cells were found in the nucleus of the solitary tract (A2 region), but <10% were double labeled and there were no sex differences. The majority of ERα-labeled cells in this nucleus was located in the more rostral areas. ERα-labeled cells were found in several rostral brain stem regions but none of these were double labeled and so were not quantified. Because there was no sex difference in the number of ERα-ir cells in the brain stem that were noradrenergic, the sex difference in the action of estrogen on gonadotropin secretion in sheep is unlikely to involve actions on brain stem noradrenergic cells. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

MYC immunohistochemical and cytogenetic analysis are required for identification of clinically relevant aggressive B cell lymphoma subtypes.

PubMed

Raess, Philipp W; Moore, Stephen R; Cascio, Michael J; Dunlap, Jennifer; Fan, Guang; Gatter, Ken; Olson, Susan B; Braziel, Rita M

2018-06-01

Accurate subclassification of aggressive B cell lymphomas (ABCLs) requires integration of morphologic, immunohistochemical (IHC), and cytogenetic information. Optimal strategies have not been well defined for diagnosis of high grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBLwR) and double expressor lymphomas with MYC and BCL2 protein overexpression. One hundred and eighty seven ABCLs were investigated with complete IHC and FISH analysis. Morphologic and IHC analysis was insufficient to identify clinically relevant HGBLwR. Approximately, 75% of cases classified as HGBLwR showed conventional DLBCL morphologic features. Fourteen percent of MYC-rearranged cases were negative by IHC. Conversely, 60% of cases positive for MYC by IHC did not demonstrate a MYC rearrangement. Analysis by FISH without MYC and BCL2 IHC would miss 41 cases of double expressor lymphoma. Complete IHC and FISH analysis is recommended in the evaluation of all ABCLs.

The Sda/GM2-glycan is a carbohydrate marker of porcine primordial germ cells and of a subpopulation of spermatogonia in cattle, pigs, horses and llama.

PubMed

Klisch, K; Contreras, D A; Sun, X; Brehm, R; Bergmann, M; Alberio, R

2011-11-01

Spermatogonia are a potential source of adult pluripotent stem cells and can be used for testis germ cell transplantation. Markers for the isolation of these cells are of great importance for biomedical applications. Primordial germ cells and prepubertal spermatogonia in many species can be identified by their binding of Dolichos biflorus agglutinin (DBA). This lectin binds to two different types of glycans, which are α-linked N-acetylgalactosamine (GalNac) and β-linked GalNac, if this is part of the Sda or GM2 glycotopes. We used the MAB CT1, which is specific for the trisaccharides motif NeuAcα2-3(GalNAcβ1-4)Galβ1-, which is common to both Sda and GM2 glycotopes, to further define the glycosylation of DBA binding germ cells. In porcine embryos, CT1 bound to migratory germ cells and gonocytes. CT1/DBA double staining showed that the mesonephros was CT1 negative but contained DBA-positive cells. Gonocytes in the female gonad became CT1 negative, while male gonocytes remained CT1 positive. In immunohistological double staining of cattle, pig, horse and llama testis, DBA and CT1 staining was generally colocalised in a subpopulation of spermatogonia. These spermatogonia were mainly single, sometimes paired or formed chains of up to four cells. Our data show that the Sda/GM2 glycotope is present in developing germ cells and spermatogonia in several species. Owing to the narrower specificity of the CT1 antibody, compared with DBA, the former is likely to be a useful tool for labelling and isolation of these cells.

Synthesising methods of layered double hydroxides and its use in the fabrication of dye Sensitised solar cell (DSSC): A short review

NASA Astrophysics Data System (ADS)

George, Giphin; Saravanakumar, M. P.

2017-11-01

The layered double hydroxides (LDH) which are anionic clay substances comprising of stacked cationic layers and interlayer anions. The cationic sheets contain octahedral structure consisting the divalent and trivalent ions in the center and hydroxyl bunches in the corners, gathered by three bonding with the neighbouring octahedra on every side of the layer. The ratio between the quantity of cations and OH- ions is 2:1, so a positive charge shows up on the layer because of the presence of trivalent cations. The interlayer space gives the compensation anions and water molecules, assuring a balanced out layered structure. The LDH materials were successfully synthesised from magnesium, aluminium, zinc and chromium chloride salts utilizing the co-precipitation technique. A Zn-Al LDH was researched as a potential sorbent material. This article reviews the recent advances in the preparation and intercalation of layered double hydroxides and its application in the fabrication of Dye Sensitized Solar Cell (DSSC).

Monoclonal gammopathy with double M-bands: An atypical presentation on serum protein electrophoresis simulating biclonal gammopathy.

PubMed

Bora, Kaustubh; Das, Umesh; Barman, Bhupen; Ruram, Alice Abraham

2017-01-01

Monoclonal gammopathies, such as multiple myeloma, typically exhibit high levels of a monoclonal immunoglobulin (M-protein), produced by a clone of abnormally proliferating B-lymphocytes and/or plasma cells. The M-protein can be evaluated by serum protein electrophoresis (SPEP), which yields a single discrete band (M-band), usually in the γ-globulin region. Rarely, two M-bands appear simultaneously at different positions during SPEP - a condition known as biclonal gammopathy, which is a result of clonal expansion of two different neoplastic cell lines. Here, we describe an atypical case of IgA-λ multiple myeloma, where double M-bands (one in β- and the other in γ-globulin region) were found during SPEP simulating biclonal gammopathy, although it was monoclonal in nature. This peculiar presentation of double M-bands in monoclonal gammopathy was attributed to polymeric forms of IgA by systematic workup. Further, we discuss how true and apparent biclonality can be distinguished by inexpensive analytical techniques in resource-constrained settings.

Lack of spontaneous and radiation-induced chromosome breakage at interstitial telomeric sites in murine scid cells.

PubMed

Wong, H-P; Mozdarani, H; Finnegan, C; McIlrath, J; Bryant, P E; Slijepcevic, P

2004-01-01

Interstitial telomeric sites (ITSs) in chromosomes from DNA repair-proficient mammalian cells are sensitive to both spontaneous and radiation-induced chromosome breakage. Exact mechanisms of this chromosome breakage sensitivity are not known. To investigate factors that predispose ITSs to chromosome breakage we used murine scid cells. These cells lack functional DNA-PKcs, an enzyme involved in the repair of DNA double-strand breaks. Interestingly, our results revealed lack of both spontaneous and radiation-induced chromosome breakage at ITSs found in scid chromosomes. Therefore, it is possible that increased sensitivity of ITSs to chromosome breakage is associated with the functional DNA double-strand break repair machinery. To investigate if this is the case we used scid cells in which DNA-PKcs deficiency was corrected. Our results revealed complete disappearance of ITSs in scid cells with functional DNA-PKcs, presumably through chromosome breakage at ITSs, but their unchanged frequency in positive and negative control cells. Therefore, our results indicate that the functional DNA double-strand break machinery is required for elevated sensitivity of ITSs to chromosome breakage. Interestingly, we observed significant differences in mitotic chromosome condensation between scid cells and their counterparts with restored DNA-PKcs activity suggesting that lack of functional DNA-PKcs may cause a defect in chromatin organization. Increased condensation of mitotic chromosomes in the scid background was also confirmed in vivo. Therefore, our results indicate a previously unanticipated role of DNA-PKcs in chromatin organisation, which could contribute to the lack of ITS sensitivity to chromosome breakage in murine scid cells. Copyright 2003 S. Karger AG, Basel

Isolation and characterization of ventricular-like cells derived from NKX2-5eGFP/w and MLC2vmCherry/w double knock-in human pluripotent stem cells.

PubMed

Yamauchi, Kaori; Li, Junjun; Morikawa, Kumi; Liu, Li; Shirayoshi, Yasuaki; Nakatsuji, Norio; Elliott, David A; Hisatome, Ichiro; Suemori, Hirofumi

2018-01-01

Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are a promising source for cell transplantation into the damaged heart, which has limited regenerative ability. Many methods have been developed to obtain large amounts of functional CMs from hPSCs for therapeutic applications. However, during the differentiation process, a mixed population of various cardiac cells, including ventricular, atrial, and pacemaker cells, is generated, which hampers the proper functional analysis and evaluation of cell properties. Here, we established NKX2-5 eGFP/w and MLC2v mCherry/w hPSC double knock-ins that allow for labeling, tracing, purification, and analysis of the development of ventricular cells from early to late stages. As with the endogenous transcriptional activities of these genes, MLC2v-mCherry expression following NKX2-5-eGFP expression was observed under previously established culture conditions, which mimic the in vivo cardiac developmental process. Patch-clamp and microelectrode array electrophysiological analyses showed that the NKX2-5 and MLC2v double-positive cells possess ventricular-like properties. The results demonstrate that the NKX2-5 eGFP/w and MLC2v mCherry/w hPSCs provide a powerful model system to capture region-specific cardiac differentiation from early to late stages. Our study would facilitate subtype-specific cardiac development and functional analysis using the hPSC-derived sources. Copyright © 2017 Elsevier Inc. All rights reserved.

The role of exogenous neural stem cells transplantation in cerebral ischemic stroke.

PubMed

Chen, Lukui; Qiu, Rong; Li, Lushen; He, Dan; Lv, Haiqin; Wu, Xiaojing; Gu, Ning

2014-11-01

To observe the effects of neural stem cells (NSCs) transplantation in rats' striatum and subventricular zone (SVZ) in rat models of focal cerebral ischemia and reperfusion. Hippocampus was extracted from fetal rats with 14 days of gestation. Suspension culture was used to isolate and culture the rat's NSCs. A cerebral ischemia and reperfusion rat's model was made on the left side of the brain through occlusion of the left middle cerebral artery. Neurological signs were assessed by Zea Longa's five-grade scale, with scores 1, 2, and 3 used to determine the successful establishment of the rat's model. The NSCs were stereotaxically injected into the left striatum 24 hours after the successful rat's model was built. Rats were then randomly divided into 5 groups, namely, normal group, sham operation group, ischemia group, PBS transplantation group, and NSCs transplantation group, each of which was observed on day 3, day 7, and day 14. The ischemia-related neurological deficits were assessed by using a 7-point evaluation criterion. Forelimb injuries were evaluated in all rats using the foot-fault approach. Infarct size changes were observed through TTC staining and cell morphology and structure in the infarct region were investigated by Nissl staining. Apoptosis and apoptosis-positive cell counts were studied by Tunel assay. Expressions of double-labeling positive cells in the striatum and subventricular zone (SVZ) were observed by BrdU/NeuN and BrdU/GFAP fluorescent double-labeling method and the number of positive cells in the striatum and SVZ was counted. Results from the differently treated groups showed that right hemiplegia occurred in the ischemia group, PBS transplantation group, and NSCs transplantation group in varying degrees. Compared with the former two groups, there was least hemiplegia in the NSCs transplantation group. The TTC staining assay showed that rats in the NSCs transplantation group had smaller infarct volume than those from the PBS transplantation group. The Nissl dyeing showed that there was a large area of neuronal necrosis and apoptosis in the ischemia and PBS transplantation groups, and damage was mainly focused in the striatum. Degeneration and damage of nerve cells were significantly reduced in the NSCs transplantation group. The Tunel assay showed that the number of apoptosis-positive cells in the NSCs transplantation group was less than that in the PBS transplantation group at each time point. Double immunofluorescent labeling showed that the proliferation of endogenous neural stem cells began at the third day, reaching the peak at the 7th day, and was significantly reduced at the 14th day in the SVZ. The number of BrdU/NeuN increased significantly in the NSCs transplantation group compared to that in the PBS transplantation group (P < 0.05). The number of BrdU/GFAP decreased significantly in the NSCs transplantation group compared to that of PBS transplantation group (P < 0.05). The number of BrdU/GFAP-positive cells in the striatum was observed to be much more in the PBS transplantation group than in the NSCs transplantation group. Both neurological deficits and coordination capacity of rats with cerebral ischemia were significantly improved via transplantation of the neural stem cells. In conclusion, transplantation of neural stem cells can therefore possibly promote the differentiation of endogenous NSCs into neurons and reduce their differentiation towards glial cells. Transplantation of the neural stem cells may also change the ischemic microenvironment of striatum, possibly inhibiting the proliferation of glial cells.

Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

PubMed

Ito, Sayaka; Kobayashi, Yoshihiko; Yamamoto, Yuki; Kimura, Koji; Okuda, Kiyoshi

2016-11-01

Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

[Application and usefulness of flowcytometry in the haematology laboratory].

PubMed

Kubota, K; Makino, M

1991-02-01

Recent technological advances, in both hardware and software, and availability of various monoclonal antibodies (MoAb) for membrane antigens of blood cells have expanded the application of flow cytometry (FCM) in medicine. In the haematology laboratory, FCM has been used mainly for assessment of leukemia and lymphoma and for determination of lymphocyte subsets. In acute leukemia, FCM is useful to classify ALL accurately, particularly for bi phenotypic or mixed lineage leukemia. In lymphocyte subset determination, we found that the use of magnetic beads to remove contaminating monocytes and some granulocytes to purify the lymphocyte-population is helpful in clarify the subsets. We present data describing the age dependent variation in lymphocyte subsets in the pediatric population. In early life (up to 2 years old), CD4 (+) 2H4 (+) lymphocyte overwhelmed CD4 (+) 2H4 (-) cells, implying predominance of suppressor-inducer activity. We also presented some cases of markedly increased double negative T cells (gamma/delta TCR) and a rare case of double positive (CD4+, CD8+) T cells.

Cell type specific gene expression analysis of prostate needle biopsies resolves tumor tissue heterogeneity

PubMed Central

Krönig, Malte; Walter, Max; Drendel, Vanessa; Werner, Martin; Jilg, Cordula A.; Richter, Andreas S.; Backofen, Rolf; McGarry, David; Follo, Marie; Schultze-Seemann, Wolfgang; Schüle, Roland

2015-01-01

A lack of cell surface markers for the specific identification, isolation and subsequent analysis of living prostate tumor cells hampers progress in the field. Specific characterization of tumor cells and their microenvironment in a multi-parameter molecular assay could significantly improve prognostic accuracy for the heterogeneous prostate tumor tissue. Novel functionalized gold-nano particles allow fluorescence-based detection of absolute mRNA expression levels in living cells by fluorescent activated flow cytometry (FACS). We use of this technique to separate prostate tumor and benign cells in human prostate needle biopsies based on the expression levels of the tumor marker alpha-methylacyl-CoA racemase (AMACR). We combined RNA and protein detection of living cells by FACS to gate for epithelial cell adhesion molecule (EPCAM) positive tumor and benign cells, EPCAM/CD45 double negative mesenchymal cells and CD45 positive infiltrating lymphocytes. EPCAM positive epithelial cells were further sub-gated into AMACR high and low expressing cells. Two hundred cells from each population and several biopsies from the same patient were analyzed using a multiplexed gene expression profile to generate a cell type resolved profile of the specimen. This technique provides the basis for the clinical evaluation of cell type resolved gene expression profiles as pre-therapeutic prognostic markers for prostate cancer. PMID:25514598

Changes in cell death of peripheral blood lymphocytes isolated from children with acute lymphoblastic leukemia upon stimulation with 7 Hz, 30 mT pulsed electromagnetic field.

PubMed

Kaszuba-Zwoińska, Jolanta; Ćwiklińska, Magdalena; Balwierz, Walentyna; Chorobik, Paulina; Nowak, Bernadeta; Wójcik-Piotrowicz, Karolina; Ziomber, Agata; Malina-Novak, Kinga; Zaraska, Wiesław; Thor, Piotr J

2015-03-01

Pulsed electromagnetic field (PEMF) influenced the viability of proliferating in vitro peripheral blood mononuclear cells (PBMCs) isolated from Crohn's disease patients as well as acute myeloblastic leukemia (AML) patients by induction of cell death, but did not cause any vital changes in cells from healthy donors. Experiments with lymphoid U937 and monocytic MonoMac6 cell lines have shown a protective effect of PEMF on the death process in cells treated with death inducers. The aim of the current study was to investigate the influence of PEMF on native proliferating leukocytes originating from newly diagnosed acute lymphoblastic leukemia (ALL) patients. The effects of exposure to PEMF were studied in PBMCs from 20 children with ALL. PBMCs were stimulated with three doses of PEMF (7 Hz, 30 mT) for 4 h each with 24 h intervals. After the last stimulation, the cells were double stained with annexin V and propidium iodide dye to estimate viability by flow cytometric analysis. The results indicated an increase of annexin V positive as well as double stained annexin V and propidium iodide positive cells after exposure to threefold PEMF stimulation. A low-frequency pulsed electromagnetic field induces cell death in native proliferating cells isolated from ALL patients. The increased vulnerability of proliferating PBMCs to PEMF-induced interactions may be potentially applied in the therapy of ALL. The analysis of expression of apoptosis-related genes revealed changes in mRNA of some genes engaged in the intrinsic apoptotic pathway belonging to the Bcl-2 family and the pathway with apoptosis-inducing factor (AIF) abundance upon PEMF stimulation of PBMCs.

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

PubMed

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

2012-03-01

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

Synthesis, characterization, and efficacy of antituberculosis isoniazid zinc aluminum-layered double hydroxide based nanocomposites

PubMed Central

Saifullah, Bullo; El Zowalaty, Mohamed Ezzat; Arulselvan, Palanisamy; Fakurazi, Sharida; Webster, Thomas J; Geilich, Benjamin Mahler; Hussein, Mohd Zobir

2016-01-01

The chemotherapy for tuberculosis (TB) is complicated by its long-term treatment, its frequent drug dosing, and the adverse effects of anti-TB drugs. In this study, we have developed two nanocomposites (A and B) by intercalating the anti-TB drug isoniazid (INH) into Zn/Al-layered double hydroxides. The average size of the nanocomposites was found to bê164 nm. The efficacy of the Zn/Al-layered double hydroxides intercalated INH against Mycobacterium tuberculosis was increased by approximately three times more than free INH. The nanocomposites were also found to be active against Gram-positive and -negative bacteria. Compared to the free INH, the nanodelivery formulation was determined to be three times more biocompatible with human normal lung fibroblast MRC-5 cells and 3T3 fibroblast cells at a very high concentration of 50 µg/mL for up to 72 hours. The in vitro release of INH from the Zn/Al-layered double hydroxides was found to be sustained in human body-simulated buffer solutions of pH 4.8 and 7.4. This research is a step forward in making the TB chemotherapy patient friendly. PMID:27486322

TopBP1 deficiency impairs V(D)J recombination during lymphocyte development

PubMed Central

Kim, Jieun; Kyu Lee, Sung; Jeon, Yoon; Kim, Yehyun; Lee, Changjin; Ho Jeon, Sung; Shim, Jaegal; Kim, In-Hoo; Hong, Seokmann; Kim, Nayoung; Lee, Ho; Seong, Rho Hyun

2014-01-01

TopBP1 was initially identified as a topoisomerase II-β-binding protein and it plays roles in DNA replication and repair. We found that TopBP1 is expressed at high levels in lymphoid tissues and is essential for early lymphocyte development. Specific abrogation of TopBP1 expression resulted in transitional blocks during early lymphocyte development. These defects were, in major part, due to aberrant V(D)J rearrangements in pro-B cells, double-negative and double-positive thymocytes. We also show that TopBP1 was located at sites of V(D)J rearrangement. In TopBP1-deficient cells, γ-H2AX foci were found to be increased. In addition, greater amount of γ-H2AX product was precipitated from the regions where TopBP1 was localized than from controls, indicating that TopBP1 deficiency results in inefficient DNA double-strand break repair. The developmental defects were rescued by introducing functional TCR αβ transgenes. Our data demonstrate a novel role for TopBP1 as a crucial factor in V(D)J rearrangement during the development of B, T and iNKT cells. PMID:24442639

Double-hit lymphoma demonstrating t(6;14;18)(p25;q32;q21), suggesting two independent dual-hit translocations, MYC/BCL-2 and IRF4/BCL-2.

PubMed

Tabata, Rie; Yasumizu, Ryoji; Tabata, Chiharu; Kojima, Masaru

2013-01-01

Here, we report a rare case of double-hit lymphoma, demonstrating t(6;14;18)(p25;q32;q21), suggesting two independent dual-translocations, c-MYC/BCL-2 and IRF4/BCL-2. The present case had a rare abnormal chromosome, t(6;14;18)(p25;q32;q21), independently, in addition to known dual-hit chromosomal abnormalities, t(14;18)(q32;q21) and t(8;22)(q24;q11.2). Lymph node was characterized by a follicular and diffuse growth pattern with variously sized neoplastic follicles. The intrafollicular area was composed of centrocytes with a few centroblasts and the interfollicular area was occupied by uniformly spread medium- to large-sized lymphocytes. CD23 immunostaining demonstrated a disrupted follicular dendritic cell meshwork. The intrafollicular tumor cells had a germinal center phenotype with the expression of surface IgM, CD10, Bcl-2, Bcl-6, and MUM1/IRF4. However, the interfollicular larger cells showed plasmacytic differentiation with diminished CD20, Bcl-2, Bcl-6, and positive intracytoplasmic IgM, and co-expression of MUM1/IRF4 and CD138 with increased Ki-67-positive cells (> 90%). MUM1/IRF4 has been found to induce c-MYC expression, and in turn, MYC transactivates MUM1/IRF4, creating a positive autoregulatory feedback loop. On the other hand, MUM1/IRF4 functions as a tumor suppressor in c-MYC-induced B-cell leukemia. The present rare case arouses interest in view of the possible "dual" activation of both c-MYC and MUM1/IRF4 through two independent dual-translocations, c-MYC/BCL-2 and IRF4/BCL-2.

Micro-abrasion package capture cell experiment on the trailing edge of LDEF: Impactor chemistry and whipple bumper shield efficiencies

NASA Technical Reports Server (NTRS)

Fitzgerald, Howard J.; Yano, Hajime

1995-01-01

Four of the eight available double layer microparticle capture cells, flown as the experiment A0023 on the trailing (West) face of LDEF, have been extensively studied. An investigation of the chemistry of impactors has been made using SEM/EDX techniques and the effectiveness of the capture cells as bumper shields has also been examined. Studies of these capture cells gave positive EDX results, with 53 percent of impact sites indicating the presence of some chemical residues, the predominant residue identified as being silicon in varying quantities.

Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast

PubMed Central

Bendezú, Felipe O.; Martin, Sophie G.

2011-01-01

Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)–dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP2-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types. PMID:21148300

Layered Double Hydroxide Nanotransporter for Molecule Delivery to Intact Plant Cells

PubMed Central

Bao, Wenlong; Wang, Junya; Wang, Qiang; O’Hare, Dermot; Wan, Yinglang

2016-01-01

Here we report a powerful method that facilitates the transport of biologically active materials across the cell wall barrier in plant cells. Positively charged delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) with a 0.5‒2 nm thickness and 30‒60 nm diameter exhibit a high adsorptive capacity for negatively charged biomolecules, including fluorescent dyes such as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate isomer I(FITC) and DNA molecules, forming neutral LDH-nanosheet conjugates. These neutral conjugates can shuttle the bound fluorescent dye into the cytosol of intact plant cell very efficiently. Furthermore, typical inhibitors of endocytosis and low temperature incubation did not prevent LDH-lactate-NS internalization, suggesting that LDH-lactate-NS penetrated the plasma membrane via non-endocytic pathways, which will widen the applicability to a variety of plant cells. Moreover, the absence of unwanted side effects in our cytological studies, and the nuclear localization of ssDNA-FITC suggest that nano-LDHs have potential application as a novel gene carrier to plants. PMID:27221055

Immune system cells in healthy ferrets: an immunohistochemical study.

PubMed

Vidaña, B; Majó, N; Pérez, M; Montoya, M; Martorell, J; Martínez, J

2014-07-01

The ferret has emerged as an excellent animal model to characterize several physiologic and pathologic conditions. The distribution and characterization of different types of immune system cells were studied in healthy ferret tissues. Eight primary antibodies were tested for immunohistochemistry in formalin-fixed tissues: anti-CD3, anti-CD79α, anti-CD20, anti-HLA-DR, anti-lysozyme, anti-CD163, anti-SWC3, and anti-Mac387. The anti-CD3 antibody labeled T cells mainly in interfollicular and paracortical areas of lymph nodes, cortex and thymic medulla, and periarteriolar lymphoid sheaths in the spleen. The anti-CD79α and anti-CD20 antibodies immunolabeled B cells located in lymphoid follicles at lymph nodes, spleen, and Peyer patches. The CD79α and CD20 antibodies also labeled cells with nonlymphoid morphology in atypical B-cell locations. The anti-HLA-DR antibody labeled macrophages, some populations of B and T lymphocytes, and different populations of dendritic cells in lymph nodes, Peyer patches, spleen, and thymus. The anti-lysozyme antibody immunolabeled macrophages in the liver, lymph nodes, spleen, and thymus. The Mac-387, CD163, and SWC3 antibodies did not show any positive reaction in formalin-fixed or frozen tissues. To elucidate the origin of the uncommon CD79α/CD20 positive cells, a double immunohistochemistry was carried out using the anti-HLA-DR + the anti-CD79α, the anti-HLA-DR + the anti-CD20, and the anti-lysozyme + the anti-CD79α antibodies. Double labeling was mainly observed when the anti-HLA-DR + the anti-CD79α antibodies were combined. The immunohistologic characterization and distribution of these immune system cells in healthy ferret tissues should be of value in future comparative studies of diseases in ferrets. © The Author(s) 2013.

Thymocyte Maturation Is Regulated by the Activity of the Helix-Loop-Helix Protein, E47

PubMed Central

Bain, Gretchen; Quong, Melanie W.; Soloff, Rachel S.; Hedrick, Stephen M.; Murre, Cornelis

1999-01-01

The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I– and class II–restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I–restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection. PMID:10587351

Histochemical and cellular changes accompanying the appearance of lung fibrosis in an experimental mouse model for Hermansky Pudlak syndrome

PubMed Central

Lyerla, Timothy

2010-01-01

Hermansky Pudlak syndrome (HPS) is a heterogeneous recessive genetic disease with a tendency to develop lung fibrosis with aging. A mouse strain with two mutant HPS genes affecting separate vesicle trafficking pathways, C57BL/6-Hps1ep-Ap3b1pe, exhibits severe lung abnormalities at young ages, including enlarged alveolar type II (ATII) cells with giant lamellar bodies and foamy alveolar macrophages (AMs), which are readily identified histologically. In this study, the appearance of lung fibrosis in older animals was studied using classical histological and biochemical methods. The HPS double mutant mice, but not Chediak Higashi syndrome (C57BL/6-Lystbg-J-J, CHS) or C57BL/6J black control (WT) mice, were found to develop lung fibrosis at about 17 months of age using Masson trichrome staining, which was confirmed by hydroxyproline analysis. TGF β1 levels were elevated in bronchial alveolar lavage samples at all ages tested in the double mutant, but not WT or CHS mice, indicative of a prefibrotic condition in this experimental strain; and AMs were highly positive for this cytokine using immunohistochemistry staining. Prosurfactant protein C staining for ATII cells showed redistribution and dysmorphism of these cells with aging, but there was no evidence for epithelial-mesenchymal transition of ATII cells by dual staining for prosurfactant C protein and α-smooth muscle actin. This investigation showed that the HPS double mutant mouse strain develops interstitial pneumonia (HPSIP) past 1 year of age, which may be initiated by abnormal ATII cells and exacerbated by AM activation. With prominent prefibrotic abnormalities, this double mutant may serve as a model for interventive therapy in HPS. PMID:20603711

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.

PubMed

Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

2010-04-07

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development

PubMed Central

Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

2010-01-01

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ɛ proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development. PMID:20150895

False Position, Double False Position and Cramer's Rule

ERIC Educational Resources Information Center

Boman, Eugene

2009-01-01

We state and prove the methods of False Position (Regula Falsa) and Double False Position (Regula Duorum Falsorum). The history of both is traced from ancient Egypt and China through the work of Fibonacci, ending with a connection between Double False Position and Cramer's Rule.

A television scanner for the ultracentrifuge. II. Multiple cell operation.

PubMed

Rockholt, D L; Royce, C R; Richards, E G

1976-07-01

The "Optical Multichannel Analyzer" (OMA) is a commercially available instrument that with the absorption optical system of the ultracentrifuge, provides an entire 500 channel intensity profile of a cell in real time. With its own analog-todigital converter, the OMA integrates a selectable number of 32.8 msec scans to provide a time-averaged image in digital form. This paper describes an interface-controller for operation of the OMA with single- and double-sector cells in multi-cell rotors, simulating double-beam measurement required for absorbance determinations. The desired sector is selected by "gating" the intensifier stage of a "Silicon Intensified Target" vidicon (SIT) used as the light detector. The cell location in the rotor and the position of the gate relative to the cell centerline is obtained from a phase-locked loop circuit which divides each rotation of the rotor into 3600 parts independent of rotor speed. (This circuit employed with photo-multiplier scanners would select the gate position for integration of photomultiplier pulses.) From examination of appropriate signals with an oscilloscope, it was verified that gate positions and widths are located with an accuracy of 0.1degree or better and with a precision of +/- 0.1 mus. The light intensity profile for any desired cell can be examined in "real time", even during acceleration of the rotor. Additional circuits employing a 10 MHz crystal clock 1) control the automatic collection of data for all sectors in multicell rotors at digitally selected time intervals, 2) display the rotor speed, and 3) indicate the elapsed time of the experiment. Constructed but not tested are additional circuits for pulsing a laser into the absorption or Rayleigh optical system. The accuracy of the pulsed SIT has been demonstrated by measurement of absorbances of solutions and also by sedimentation equilibrium experiments with myoglobin. The estimated error is 0.003 for absorbances ranging from 0 to 1. The interface-controller operates extremely well, but problems related to the pulsed SIT (optimum gate position relative to the sector opening shape of high-voltage pulse, slight pincushion distortion) require more work.

c-Myb promotes the survival of CD4+CD8+ double positive thymocytes through up-regulation of Bcl-xL1

PubMed Central

Yuan, Joan; Crittenden, Rowena B.; Bender, Timothy P.

2010-01-01

Mechanisms that regulate the lifespan of CD4+CD8+ double positive (DP) thymocytes help shape the peripheral T cell repertoire. However, the molecular mechanisms that control DP thymocyte survival remain poorly understood. The Myb proto-oncogene encodes a transcription factor required during multiple stages of T cell development. We demonstrate that Myb mRNA expression is up-regulated in the small, pre-selection DP stage during T cell development. Using a conditional deletion mouse model, we demonstrate that Myb deficient DP thymocytes undergo premature apoptosis, resulting in a limited Tcrα repertoire biased towards 5’ Jα segment usage. Premature apoptosis occurs in the small pre-selection DP compartment in an αβTCR independent manner and is a consequence of decreased Bcl-xL expression. Forced Bcl-xL expression is able to rescue survival and re-introduction of c-Myb restores both Bcl-xL expression and the small pre-selection DP compartment. We further demonstrate that thymocytes become dependent on Bcl-xL for survival upon entering the quiescent, small pre-selection DP stage and c-Myb promotes transcription at the Bclx locus via a genetic pathway that is independent of the expression of TCF-1 or RORγt, two transcription factors that induce Bcl-xL expression in T cell development. Thus, Bcl-xL is a novel mediator of c-Myb activity during normal T cell development. PMID:20142358

The use of antibody D8/17 to identify B cells in adults with obsessive-compulsive disorder.

PubMed

Eisen, J L; Leonard, H L; Swedo, S E; Price, L H; Zabriskie, J B; Chiang, S Y; Karitani, M; Rasmussen, S A

2001-11-30

Compared with healthy control subjects, individuals with childhood-onset obsessive-compulsive disorder (OCD) have been reported to have a higher percentage of B cells that react with the monoclonal antibody D8/17, a marker for rheumatic fever. This study sought to replicate these findings in adults with OCD. Double-blind analyses of blood samples from 29 consecutive adults with primary OCD and 26 healthy control subjects were conducted to determine the percentage of B cells identified by D8/17. Using a standard criterion of > or =12% labeled B cells to denote positivity, rates of D8/17 positive individuals did not significantly differ between the OCD (58.6%) and control (42.3%) groups. Early age of onset was not a predictor of D8/17 positivity in the OCD group. The percentage of B cells identified by the monoclonal antibody marker D8/17 did not distinguish adults with OCD from control subjects, nor did it distinguish a sub-group of adults with OCD who described pre-pubertal onset of their OCD symptoms.

Both cell fusion and transdifferentiation account for the transformation of human peripheral blood CD34-positive cells into cardiomyocytes in vivo.

PubMed

Zhang, Sui; Wang, Dachun; Estrov, Zeev; Raj, Sean; Willerson, James T; Yeh, Edward T H

2004-12-21

Adult human peripheral blood CD34-positive (CD34+) cells appear to transform into cardiomyocytes in the injured hearts of severe combined immunodeficient mice. It remains unclear, however, whether the apparent transformation is the result of transdifferentiation of the donor stem cells or of fusion of the donor cell with the cardiomyocyte of the recipients. We performed flow cytometry analyses of cells isolated from the hearts of mice that received human CD34+ cells. Human HLA-ABC antigen and cardiac troponin T or Nkx2.5 were used as markers for cardiomyocytes derived from human CD34+ cells, and HLA-ABC and VE-cadherin were used to identify the transformed endothelial cells. The double-positive cells were collected and interphase fluorescence in situ hybridization was used to detect the expression of human and mouse X chromosomes in these cells. We found that 73.3% of nuclei derived from HLA+ and troponin T+ or Nkx2.5+ cardiomyocytes contain both human and mouse X chromosomes and 23.7% contain only human X chromosome. In contrast, the nuclei of HLA-, troponin T+ cells contain only mouse X chromosomes. Furthermore, 97.3% of endothelial cells derived from CD34+ cells contained human X chromosome only. Thus, both cell fusion and transdifferentiation may account for the transformation of peripheral blood CD34+ cells into cardiomyocytes in vivo.

Novel Derivative of Benzofuran Induces Cell Death Mostly by G2/M Cell Cycle Arrest through p53-dependent Pathway but Partially by Inhibition of NF-κB*

PubMed Central

Manna, Sunil K.; Bose, Julie S.; Gangan, Vijay; Raviprakash, Nune; Navaneetha, Thota; Raghavendra, Pongali B.; Babajan, Banaganapalli; Kumar, Chitta S.; Jain, Swatantra K.

2010-01-01

The Dracaena resin is widely used in traditional medicine as an anticancer agent, and benzofuran lignan is the active component. In this report, we provide evidence that the synthetic derivative of benzofuran lignan (Benfur) showed antitumor activities. It induced apoptosis in p53-positive cells. Though it inhibited endotoxin-induced nuclear factor κB (NF-κB) activation in both p53-positive and -negative cells, the activation of caspase 3 was observed in p53-positive cells. It showed partial cell death effect in both p53-positive and -negative cells through inhibition of NF-κB. Cell cycle analysis using flow cytometry showed that treatment with this novel benozofuran lignan derivative to Jurkat T-cells, but not U-937 cells, resulted in a G2/M arrest in a dose- and time-dependent manner. It increased amounts of p21, p27, and cyclin B, but not phospho-Rb through p53 nuclear translocation in Jurkat T-cells, but not in U-937 cells. It inhibited amounts of MDM2 (murine double minute 2) by repressing the transcription factor Sp1, which was also proved in silico. It induced cell death in tumor cells, but not in primary T-cells. Overall, our data suggest that Benfur-mediated cell death is partially dependent upon NF-κB, but predominantly dependent on p53. Thus, this novel benzofuran lignan derivative can be effective chemopreventive or chemotherapeutic agent against malignant T-cells. PMID:20472557

In vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function

PubMed Central

Lew, Matthew D.; Thompson, Michael A.; Badieirostami, Majid; Moerner, W. E.

2010-01-01

The point spread function (PSF) of a widefield fluorescence microscope is not suitable for three-dimensional super-resolution imaging. We characterize the localization precision of a unique method for 3D superresolution imaging featuring a double-helix point spread function (DH-PSF). The DH-PSF is designed to have two lobes that rotate about their midpoint in any transverse plane as a function of the axial position of the emitter. In effect, the PSF appears as a double helix in three dimensions. By comparing the Cramer-Rao bound of the DH-PSF with the standard PSF as a function of the axial position, we show that the DH-PSF has a higher and more uniform localization precision than the standard PSF throughout a 2 μm depth of field. Comparisons between the DH-PSF and other methods for 3D super-resolution are briefly discussed. We also illustrate the applicability of the DH-PSF for imaging weak emitters in biological systems by tracking the movement of quantum dots in glycerol and in live cells. PMID:20563317

Immunohistochemical characterisation of the hepatic stem cell niche in feline hepatic lipidosis: a preliminary morphological study.

PubMed

Valtolina, Chiara; Robben, Joris H; Favier, Robert P; Rothuizen, Jan; Grinwis, Guy Cm; Schotanus, Baukje A; Penning, Louis C

2018-05-01

Objectives The aim of this study was to describe the cellular and stromal components of the hepatic progenitor cell niche in feline hepatic lipidosis (FHL). Methods Immunohistochemical staining for the progenitor/bile duct marker (K19), activated Kupffer cells (MAC387), myofibroblasts (alpha-smooth muscle actin [α-SMA]) and the extracellular matrix component laminin were used on seven liver biopsies of cats with FHL and three healthy cats. Double immunofluorescence stainings were performed to investigate co-localisation of different cell types in the hepatic progenitor cell (HPC) niche. Results HPCs, Kupffer cells, myofibroblasts and laminin deposition were observed in the liver samples of FHL, although with variability in the expression and positivity of the different immunostainings between different samples. When compared with the unaffected cats where K19 positivity and minimal α-SMA and laminin positivity were seen mainly in the portal area, in the majority of FHL samples K19 and α-SMA-positive cells and laminin positivity were seen also in the periportal and parenchymatous area. MAC387-positive cells were present throughout the parenchyma. Conclusions and relevance This is a preliminary morphological study to describe the activation and co-localisation of components of the HPC niche in FHL. Although the HPC niche in FHL resembles that described in hepatopathies in dogs and in feline lymphocytic cholangitis, the expression of K19, α-SMA, MAC387 and lamin is more variable in FHL, and a common pattern of activation could not be established. Nevertheless, when HPCs were activated, a spatial association between HPCs and their niche could be demonstrated.

Role of positive selection of thymoma-associated T cells in the pathogenesis of myasthenia gravis.

PubMed

Inada, Keiji; Okumura, Meinoshin; Shiono, Hiroyuki; Inoue, Masayoshi; Kadota, Yoshihisa; Ohta, Mitsunori; Matsuda, Hikaru

2005-06-01

A human thymoma is a thymic epithelial neoplasm and is characterized by its frequent association with myasthenia gravis. The histological characteristic of thymoma is coexistence of a large number of lymphocytes, including CD4(+)CD8(+) double positive T cells, phenotypes of the cortical thymocytes. To elucidate the role of these T lymphocytes in the pathogenesis of thymoma-associated myasthenia gravis, we examined the usage of alphabeta or gammadelta T cell receptor of the T lymphocytes in thymoma in conjunction with the positive selection event. Thymomas were obtained from 28 patients. Nine patients were associated with myasthenia gravis. Lymphocytes were freshly isolated from the tumor tissue and were subjected to four-color flow cytometric analysis. The average proportion of TCRalphabeta(+) cells in thymomas associated with myasthenia gravis was 47.0% and was significantly higher (P = 0.0008) than that without myasthenia gravis (23.4%). Positive selection event was then examined in terms of CD69, a positive selection marker. The mean proportion of TCRalphabeta(+)CD69(+)CD4(+)CD8(-) cells in the myasthenic thymomas (8.22%) was significantly greater (P = 0.015) than the nonmyasthenic thymomas (2.99%). On the other hand, there was not a significant difference in the mean proportion of TCRalphabeta(+)CD69(+)CD4(-)CD8(+) cells between the myasthenic and the nonmyasthenic thymomas. The possible role of development of TCRalphabeta(+) T cells, especially the role of positive selection of TCRalphabeta(+)CD4(+)CD8(-) T cells in thymoma, was suggested in the pathogenesis of thymoma-associated myasthenia gravis.

Intracerebral estrogen provision increases cytogenesis and neurogenesis in the injured zebra finch brain

PubMed Central

Walters, Bradley J.; Alexiades, Nikita G.; Saldanha, Colin J.

2010-01-01

To determine whether or not local, injury-induced aromatization and/orestrogen provision can affect cyto-or neuro-genesis following mechanical brain damage, two groups of adult male zebra finches sustained bilateral penetrating brain injuries. The first received contralateral injections of vehicle or the aromatase inhibitor fadrozole. The second group received contalateral injections of fadrozole, or fadrozole with 17β-estradiol. Subsequent to injury, birds were injected with the thymidine analog 5-Bromo-2′-deoxyuridine (BrdU). Two weeks following injury, the birds were perfused, and coronal sections were labeled using antibodies against BrdU and the neuronal proteins HuC/HuD. In a double blind fashion, BrdU positive cells and BrdU/Hu double-labeled cells in the subventricular zone (SVZ) and at the injury site (INJ) were imaged and sampled. The average numbers of cells per image were compared across brain regions and treatments using repeated measures ANOVAs and, where applicable, post-hoc, pairwise comparisons. Fadrozole administration had no detectable effect on cytogenesis or neurogenesis, however, fadrozole coupled with estradiol significantly increased both measures. The dorsal SVZ had the greatest proportion of new cells that differentiated into neurons, though the highest numbers of BrdU labeled and BrdU, Hu double-labeled cells were detected at the injury site. In the adult zebra finch brain, local estradiol provision can increase cytogenesis and neurogenesis, however, whether or not endogenous glial aromatization is sufficient to similarly affect these processes remains to be seen. PMID:20878945

A large, single-center, real-world study of clinicopathological characteristics and treatment in advanced ALK-positive non-small-cell lung cancer.

PubMed

Chen, Gang; Chen, Xi; Zhang, Yaxiong; Yan, Fang; Fang, Wenfeng; Yang, Yunpeng; Hong, Shaodong; Miao, Siyu; Wu, Manli; Huang, Xiaodan; Luo, Youli; Zhou, Cong; Gong, Run; Huang, Yan; Zhou, Ningning; Zhao, Hongyun; Zhang, Li

2017-05-01

Crizotinib has achieved astonishing success in advanced non-small-cell lung cancer (NSCLC) patients harboring anaplastic lymphoma kinase (ALK) rearrangement. However, no real-world studies described the clinicopathological characteristics and treatment of such patients in China. Patients were consecutively collected from Sun Yat-sen University Cancer Center. Chi-square test was applied to explore the relationship between ALK fusion status and metastasis sites. Kaplan-Meier methods and multivariable analyses were used to estimate progression-free survival (PFS). A total of 291 advanced NSCLC patients (ALK (+), N = 97; both ALK & epidermal growth factor receptor (EGFR) (-), N = 194) were enrolled. The occurrence of brain metastasis in ALK-positive patients was significantly higher than double-negative ones both at baseline (26.5% vs. 16.5%, P = 0.038) and during treatment (25.8% vs. 11.9%, P = 0.003), but opposite for pleural effusion (6.2% vs. 26.9%, P < 0.001 at baseline; 3.1% vs. 10.3%, P = 0.031 during treatment). ALK-positive patients of 53.6% used crizotinib, whereas others only received chemotherapy (37.1%) or supportive care (9.3%). Usage of crizotinib prolonged PFS compared with chemotherapy in ALK-positive patients (median PFS 17.6 m vs. 4.8 m, P < 0.001). ALK-positive NSCLC had more brain metastasis and less pleural effusion than double-negative ones. Crizotinib showed better PFS than chemotherapy in advanced ALK-positive NSCLC at any line. However, half advanced ALK-positive patients never received crizotinib, which was grim and need improving. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Double-hit follicular lymphoma with MYC and BCL2 translocations: a study of 7 cases with a review of literature.

PubMed

Miao, Yuan; Hu, Shimin; Lu, Xinyan; Li, Shaoying; Wang, Wei; Medeiros, L Jeffrey; Lin, Pei

2016-12-01

Follicular lymphoma with MYC and BCL2 translocations, so-called double-hit follicular lymphoma (DH-FL), is rare. Here, we report the clinicopathological features of 7 cases of DH-FL. All neoplasms had a follicular pattern (1 partially diffuse). Five cases were predominantly low grade, 4 of which had focal (≤20%) grade 3A areas, and 2 cases were of grade 3. All cases were positive for pan-B-cell antigens, CD10, and BCL6; 6 cases were positive for BCL2. Ki-67 was less than or equal to 50% in 6 cases and 90% in 1 grade 3 case. Three patients presented with stage IV disease and 3 had a Follicular Lymphoma International Prognostic Index score of greater than 2. Six patients received immunochemotherapy, and 1 is still under induction therapy with rituximab, ibrutinib, and lenalidomide. Four achieved complete remission and two had a partial response with persistent or refractory disease. The median follow-up time was 25 months (range, 8.5-53.7 months). Two patients treated with standard regimen for follicular lymphoma had relapsed or refractory disease, and 1 died from complications of allogeneic stem cell transplant administered for relapse. In contrast, all 4 patients treated with more intensive regimen for double-hit lymphoma achieved complete remission. In summary, despite predominantly low-grade histology, cases of DH-FL in this study were aggressive and responded better to more intensive than standard treatment regimens, suggesting DH-FL is part of the spectrum of double-hit high-grade lymphoma. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanisms of urodele limb regeneration

PubMed Central

2017-01-01

Abstract This review explores the historical and current state of our knowledge about urodele limb regeneration. Topics discussed are (1) blastema formation by the proteolytic histolysis of limb tissues to release resident stem cells and mononucleate cells that undergo dedifferentiation, cell cycle entry and accumulation under the apical epidermal cap. (2) The origin, phenotypic memory, and positional memory of blastema cells. (3) The role played by macrophages in the early events of regeneration. (4) The role of neural and AEC factors and interaction between blastema cells in mitosis and distalization. (5) Models of pattern formation based on the results of axial reversal experiments, experiments on the regeneration of half and double half limbs, and experiments using retinoic acid to alter positional identity of blastema cells. (6) Possible mechanisms of distalization during normal and intercalary regeneration. (7) Is pattern formation is a self‐organizing property of the blastema or dictated by chemical signals from adjacent tissues? (8) What is the future for regenerating a human limb? PMID:29299322

Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells

PubMed Central

Hashimoto, Kiyohiro; Sharma, Vyom; Sasanuma, Hiroyuki; Tian, Xu; Takata, Minoru; Takeda, Shunichi; Swenberg, James A.; Nakamura, Jun

2016-01-01

Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS. PMID:27486975

Targeting CD147 for T to NK Lineage Reprogramming and Tumor Therapy.

PubMed

Geng, Jie-Jie; Tang, Juan; Yang, Xiang-Min; Chen, Ruo; Zhang, Yang; Zhang, Kui; Miao, Jin-Lin; Chen, Zhi-Nan; Zhu, Ping

2017-06-01

CD147 is highly expressed on the surface of numerous tumor cells to promote invasion and metastasis. Targeting these cells with CD147-specific antibodies has been validated as an effective approach for lung and liver cancer therapy. In the immune system, CD147 is recognized as a co-stimulatory receptor and impacts the outcome of thymic selection. Using T cell-specific deletion, we showed here that in thymus CD147 is indispensable for the stable αβ T cell lineage commitment: loss of CD147 biases both multipotent DN (double negative) and fully committed DP (double positive) cells into innate NK-like lineages. Mechanistically, CD147 deficiency results in impaired Wnt signaling and expression of BCL11b, a master transcription factor in determining T cell identity. In addition, functional blocking of CD147 by antibody phenocopies genetic deletion to enrich NK-like cells in the periphery. Furthermore, using a melanoma model and orthotopic liver cancer transplants, we showed that the augmentation of NK-like cells strongly associates with resistance against tumor growth upon CD147 suppression. Therefore, besides its original function in tumorigenesis, CD147 is also an effective surface target for immune modulation in tumor therapy. Copyright © 2017. Published by Elsevier B.V.

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

Becker, Ines; Schillig, Cora

A double-sided adhesive metal-based tape for use as contacting aid for SOFC fuel cells is provided. The double-sided metal-based adhesive tape is suitable for simplifying the construction of cell bundles. The double-sided metal-based adhesive tape is used for electrical contacting of the cell connector with the anode and for electrical contacting of the interconnector of the fuel cells with the cell connector. A method for producing the double-sided adhesive metal-base tape is also provided.

Characterizing invading glioma cells based on IDH1-R132H and Ki-67 immunofluorescence.

PubMed

Sabit, Hemragul; Nakada, Mitsutoshi; Furuta, Takuya; Watanabe, Takuya; Hayashi, Yutaka; Sato, Hiroshi; Kato, Yukinari; Hamada, Jun-ichiro

2014-10-01

Glioma, the most common primary brain tumor, is characterized by proliferative-invasive growth. However, the detailed biological characteristics of invading glioma cells remain to be elucidated. A monoclonal antibody (clone HMab-1) that specifically and sensitively recognizes the isocitrate dehydrogenase-1 (IDH1) protein carrying the R132H mutation can identify invading glioma cells by immunostaining. To investigate the degree of invasion in gliomas of distinct grades and the proliferative capacity of the invading cells, immunofluorescent staining was conducted using antibodies against IDH1-R132H and Ki-67 on 11 surgical and autopsy specimens of the tumor core and the invading area. Higher numbers of IDH1-R132H-positive cells in the invading area correlated with a higher tumor grade. Double staining for IDH1-R132H and Ki-67 demonstrated that most invading cells that expressed IDH1-R132H were not stained by the Ki-67 antibody, and the ratio of Ki-67-positive cells among IDH1-R132H-positive cells was significantly lower in the invasion area than in the tumor core in all grades of glioma. These data suggest that higher grade gliomas have a greater invasive potential and that invading cells possess low proliferative capacity.

Effect of Arnica D30 in marathon runners. Pooled results from two double-blind placebo controlled studies.

PubMed

Tveiten, D; Bruset, S

2003-10-01

To examine whether the homeopathic medicine Arnica D30 has an effect on muscle soreness and cell damage after marathon running. The subjects were 82 marathon runners from two separate randomised double-blind placebo controlled trials participating in the Oslo Marathon in 1990 and 1995. Five pills of Arnica D30 or placebo were given morning and evening. Treatment started on the evening before the marathon and continued on day of the race and the three following days. The runners assessed muscular soreness on a visual analogue scale. Muscle enzymes, electrolytes and creatinine were measured before and after the marathon. Muscle soreness immediately after the marathon run was lower in the Arnica group than in the placebo group (P = 0.04). Cell damage measured by enzymes was similar in the Arnica and the placebo group. These pooled results suggest that Arnica D30 has a positive effect on muscle soreness after marathon running, but not on cell damage measured by enzymes.

Anticancer Activity of Ferulic Acid-Inorganic Nanohybrids Synthesized via Two Different Hybridization Routes, Reconstruction and Exfoliation-Reassembly

PubMed Central

Choi, Ae-Jin; Oh, Jae-Min

2013-01-01

We have successfully prepared nanohybrids of biofunctional ferulic acid and layered double hydroxide nanomaterials through reconstruction and exfoliation-reassembly routes. From X-ray diffraction and infrared spectroscopy, both nanohybrids were determined to incorporate ferulic acid molecules in anionic form. Micrsocopic results showed that the nanohybrids had average particle size of 150 nm with plate-like morphology. As the two nanohybridization routes involved crystal disorder and random stacking of layers, the nanohybrids showed slight alteration in z-axis crystallinity and particle size. The zeta potential values of pristine and nanohybrids in deionized water were determined to be positive, while those in cell culture media shifted to negative values. According to the in vitro anticancer activity test on human cervical cancer HeLa cells, it was revealed that nanohybrids showed twice anticancer activity compared with ferulic acid itself. Therefore we could conclude that the nanohybrids of ferulic acid and layered double hydroxide had cellular delivery property of intercalated molecules on cancer cell lines. PMID:24453848

Anticancer activity of ferulic acid-inorganic nanohybrids synthesized via two different hybridization routes, reconstruction and exfoliation-reassembly.

PubMed

Kim, Hyoung-Jun; Ryu, Kitae; Kang, Joo-Hee; Choi, Ae-Jin; Kim, Tae-il; Oh, Jae-Min

2013-01-01

We have successfully prepared nanohybrids of biofunctional ferulic acid and layered double hydroxide nanomaterials through reconstruction and exfoliation-reassembly routes. From X-ray diffraction and infrared spectroscopy, both nanohybrids were determined to incorporate ferulic acid molecules in anionic form. Microscopic results showed that the nanohybrids had average particle size of 150 nm with plate-like morphology. As the two nanohybridization routes involved crystal disorder and random stacking of layers, the nanohybrids showed slight alteration in z-axis crystallinity and particle size. The zeta potential values of pristine and nanohybrids in deionized water were determined to be positive, while those in cell culture media shifted to negative values. According to the in vitro anticancer activity test on human cervical cancer HeLa cells, it was revealed that nanohybrids showed twice anticancer activity compared with ferulic acid itself. Therefore we could conclude that the nanohybrids of ferulic acid and layered double hydroxide had cellular delivery property of intercalated molecules on cancer cell lines.

An Hsp70 peptide initiates NK cell killing of leukemic blasts after stem cell transplantation.

PubMed

Gross, Catharina; Holler, Ernst; Stangl, Stefan; Dickinson, Anne; Pockley, A Graham; Asea, Alexzander A; Mallappa, Nagaraja; Multhoff, Gabriele

2008-04-01

In contrast to solid tumors, leukemic blasts frequently present both Hsp70 and HLA-E on their cell surface and thereby present activating and inhibitory signals to CD94(+) NK cells. In the first 12 months after stem cell transplantation (SCT) CD94(+) NK cells clearly dominate over CD3(+)/CD16(-)/56(-) T and CD3(+)/CD16(+)/56(+) NK-like T cells. An incubation of post-SCT-derived peripheral blood lymphocytes with the Hsp70 peptide TKD and IL-15 enhances the cell surface density of CD56/CD94 and initiates the cytolytic activity of NK cells against Hsp70/HLA-E double-positive autologous and allogeneic leukemic blasts. Hsp70 was identified as the target structure for TKD-activated NK cells.

Interstitial inflammation in Alport syndrome.

PubMed

Jedlicka, Jan; Soleiman, Afschin; Draganovici, Dan; Mandelbaum, Jana; Ziegler, Urs; Regele, Heinz; Wüthrich, Rudolf P; Gross, Oliver; Anders, Hans-Joachim; Segerer, Stephan

2010-04-01

The Alport syndrome is a hereditary glomerular disease linked to structural abnormalities of collagen IV. In a mouse model of Alport syndrome, the interstitial lymphocyte influx was important for disease progression. CXCR3 is a chemokine receptor involved in lymphocyte recruitment to the kidney. We hypothesized that CXCR3-positive T cells might be involved in human Alport syndrome. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded biopsies from 17 patients with Alport syndrome, 10 with immunoglobulin A (IgA) nephropathy, and 11 healthy donor kidneys. We investigated the expression of the alpha5 chain of collagen IV to confirm the morphologic diagnosis, the chemokine receptor CXCR3 and CD3-positive T cells. Alport syndrome biopsies demonstrated a complete loss of the alpha5 chain of collagen IV from the glomerular basement membrane and the morphologic features consistent with Alport syndrome on electron microscopy. A prominent number of CXCR3-positive cells were found in the tubulointerstitium. Most of the CXCR3-positive cells were CD3-positive T cells, demonstrated by double-labeling in selected biopsies. The number of CXCR3-positive cells in kidneys with Alport syndrome correlated with serum creatinine (P < .05) and with morphologic features of a progressive disease (eg, interstitial fibrosis, glomerulosclerosis, and tubular atrophy). The severity of interstitial CXCR3-positive cell influx was similar in Alport syndrome as compared to immunoglobulin A nephropathy. The noninflammatory glomerular lesion of Alport syndrome is associated with prominent interstitial accumulation of CD3- and CXCR3-positive lymphocytes. The degree of infiltration correlated with renal function. We speculate that targeting T lymphocytes, for example, by CXCR3 blocking agents, might be a novel approach to inhibit disease progression in patients with Alport syndrome. Copyright 2010 Elsevier Inc.

The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in stomach; particularly increasing in mucosae of Helicobacter-associated gastritis.

PubMed

Fukui, Toshiro; Kishimoto, Masanobu; Nakajima, Atsushi; Yamashina, Masao; Nakayama, Shinji; Kusuda, Takeo; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2011-04-01

The gastric corpus and antrum are believed to contain epithelial stem cells in the isthmus. However, the lack of useful markers has hindered studies of their origin. We explored whether Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), could serve as a marker for stem cells. Stomachs, small intestines, and colons from Helicobacter felis-infected and noninfected C57BL/6 mice were examined. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, or doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) was performed, and pSmad2/3L-Thr immunostaining-positive cells were counted. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. In infected mice, pSmad2/3L-Thr immunostaining-positive cells were significantly increased in the corpus and antrum compared with those of noninfected mice (p < 0.0001). The number of Ki67 immunostaining-positive cells in the corpus and antrum of infected mice was also much greater than in the noninfected mice. Although pSmad2/3L-Thr immunostaining-positive cells were detected among the Ki67 cells, immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr immunostaining-positive cells showed immunohistochemical co-localization with cytokeratin 8, but some of them showed co-localization or adjacent localization with DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features and were confirmed in the isthmus. In small intestines and colons, pSmad2/3L-Thr immunostaining-positive cells were detected in specific epithelial cells around crypt bases, where the respective putative stem cells are thought to exist. We have identified the significant expression of pSmad2/3L-Thr in specific epithelial cells of the murine stomach and have suggested these cells to be epithelial stem cells.

UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, γ-H2AX formation, and Mre11 relocalization

PubMed Central

Limoli, Charles L.; Giedzinski, Erich; Bonner, William M.; Cleaver, James E.

2002-01-01

UV-induced replication arrest in the xeroderma pigmentosum variant (XPV) but not in normal cells leads to an accumulation of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX (γ-H2AX) in large nuclear foci at sites of stalled replication forks. These complexes have been shown to signal the presence of DNA damage, in particular, double-strand breaks (DSBs). This finding suggests that UV damage leads to the formation of DSBs during the course of replication arrest. After UV irradiation, XPV cells showed a fluence-dependent increase in the yield of γ-H2AX foci that paralleled the production of Mre11 foci. The percentage of foci-positive cells increased rapidly (10–15%) up to fluences of 10 J⋅m−2 before saturating at higher fluences. Frequencies of γ-H2AX and Mre11 foci both reached maxima at 4 h after UV irradiation. This pattern contrasts sharply to the situation observed after x-irradiation, where peak levels of γ-H2AX foci were found to precede the formation of Mre11 foci by several hours. The nuclear distributions of γ-H2AX and Mre11 were found to colocalize spatially after UV- but not x-irradiation. UV-irradiated XPV cells showed a one-to-one correspondence between Mre11 and γ-H2AX foci-positive cells. These results show that XPV cells develop DNA DSBs during the course of UV-induced replication arrest. These UV-induced foci occur in cells that are unable to carry out efficient bypass replication of UV damage and may contribute to further genetic variation. PMID:11756691

Defective thymic progenitor development and mature T-cell responses in a mouse model for Down syndrome

PubMed Central

Lorenzo, Laureanne P E; Shatynski, Kristen E; Clark, Sarah; Yarowsky, Paul J; Williams, Mark S

2013-01-01

In addition to archetypal cognitive defects, Down syndrome (DS) is characterized by altered lymphocyte development and function, including premature thymic involution and increased incidence of infections. However, the potential mechanisms for these changes have not been fully elucidated. The current study used the Ts65Dn mouse model of DS to assess deficiencies in T-cell development and possible molecular alterations. Ts65Dn mice exhibited premature thymic involution and a threefold to fourfold decrease in the number and proportion of immature, double-negative thymocyte progenitors. In addition, there were twofold fewer double-positive and CD4 single-positive thymocytes in Ts65Dn thymuses. Reflecting this deficient thymic function, there were fewer naive T cells in the spleen and polyclonal stimulation of peripheral T cells exhibited a marked reduction in proliferation, suggesting a senescent phenotype. In contrast, B-cell progenitors were unchanged in the bone marrow of Ts65Dn mice, but in the spleen, there were decreased transitional and follicular B cells and these cells proliferated less upon antigen receptor stimulus but not in response to lipopolysaccharide. As a potential mechanism for diminished thymic function, immature thymocyte populations expressed diminished levels of the cytokine receptor interleukin-7Rα, which was associated with decreased proliferation and increased apoptosis. Increased oxidative stress and inhibition of the Notch pathway were identified as possible mediators of decreased interleukin-7Rα expression in Ts65Dn mice. The data suggest that immature thymocyte defects underlie immune dysfunction in DS and that increased oxidative stress and reduced cytokine signalling may alter lymphocyte development in Ts65Dn mice. PMID:23432468

PAI-1, a target gene of miR-143, regulates invasion and metastasis by upregulating MMP-13 expression of human osteosarcoma.

PubMed

Hirahata, Mio; Osaki, Mitsuhiko; Kanda, Yusuke; Sugimoto, Yui; Yoshioka, Yusuke; Kosaka, Nobuyoshi; Takeshita, Fumitaka; Fujiwara, Tomohiro; Kawai, Akira; Ito, Hisao; Ochiya, Takahiro; Okada, Futoshi

2016-05-01

Despite recent improvements in the therapy for osteosarcoma, 30-40% of osteosarcoma patients die of this disease, mainly due to its lung metastasis. We have previously reported that intravenous injection of miR-143 significantly suppresses lung metastasis of human osteosarcoma cells (143B) in a mouse model. In this study, we examined the biological role and mechanism of miR-143 in the metastasis of human osteosarcoma cells. We identified plasminogen activator inhibitor-1 (PAI-1) as a direct target gene of miR-143. To determine the role of PAI-1 in human osteosarcoma cells, siRNA was transfected into 143B cells for knockdown of PAI-1 expression. An in vitro study showed that downregulation of PAI-1 suppressed cell invasion activity, but not proliferation. Moreover, injection of PAI-1 siRNA into a primary lesion in the osteosarcoma mouse model inhibited lung metastasis compared to control siRNA-injected mice, without influencing the proliferative activity of the tumor cells. Subsequent examination using 143B cells revealed that knockdown of PAI-1 expression resulted in downregulation of the expression and secretion of matrix metalloproteinase-13 (MMP-13), which is also a target gene of miR-143 and a proteolytic enzyme that regulates tumor-induced osteolysis. Immunohistochemical analysis using clinical samples showed that higher miR-143 expressing cases showed poor expression of PAI-1 in the primary tumor cells. All such cases belonged to the lung metastasis-negative group. Moreover, the frequency of lung metastasis-positive cases was significantly higher in PAI-1 and MMP-13 double-positive cases than in PAI-1 or MMP-13 single-positive or double-negative cases (P < 0.05). These results indicated that PAI-1, a target gene of miR-143, regulates invasion and lung metastasis via enhancement of MMP-13 expression and secretion in human osteosarcoma cells, suggesting that these molecules could be potential therapeutic target genes for preventing lung metastasis in osteosarcoma patients. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Inversin modulates the cortical actin network during mitosis

PubMed Central

Werner, Michael E.; Ward, Heather H.; Phillips, Carrie L.; Miller, Caroline; Gattone, Vincent H.

2013-01-01

Mutations in inversin cause nephronophthisis type II, an autosomal recessive form of polycystic kidney disease associated with situs inversus, dilatation, and kidney cyst formation. Since cyst formation may represent a planar polarity defect, we investigated whether inversin plays a role in cell division. In developing nephrons from inv−/− mouse embryos we observed heterogeneity of nuclear size, increased cell membrane perimeters, cells with double cilia, and increased frequency of binuclear cells. Depletion of inversin by siRNA in cultured mammalian cells leads to an increase in bi- or multinucleated cells. While spindle assembly, contractile ring formation, or furrow ingression appears normal in the absence of inversin, mitotic cell rounding and the underlying rearrangement of the cortical actin cytoskeleton are perturbed. We find that inversin loss causes extensive filopodia formation in both interphase and mitotic cells. These cells also fail to round up in metaphase. The resultant spindle positioning defects lead to asymmetric division plane formation and cell division. In a cell motility assay, fibroblasts isolated from inv−/− mouse embryos migrate at half the speed of wild-type fibroblasts. Together these data suggest that inversin is a regulator of cortical actin required for cell rounding and spindle positioning during mitosis. Furthermore, cell division defects resulting from improper spindle position and perturbed actin organization contribute to altered nephron morphogenesis in the absence of inversin. PMID:23515530

Proliferation assessment in breast carcinomas using digital image analysis based on virtual Ki67/cytokeratin double staining.

PubMed

Røge, Rasmus; Riber-Hansen, Rikke; Nielsen, Søren; Vyberg, Mogens

2016-07-01

Manual estimation of Ki67 Proliferation Index (PI) in breast carcinoma classification is labor intensive and prone to intra- and interobserver variation. Standard Digital Image Analysis (DIA) has limitations due to issues with tumor cell identification. Recently, a computer algorithm, DIA based on Virtual Double Staining (VDS), segmenting Ki67-positive and -negative tumor cells using digitally fused parallel cytokeratin (CK) and Ki67-stained slides has been introduced. In this study, we compare VDS with manual stereological counting of Ki67-positive and -negative cells and examine the impact of the physical distance of the parallel slides on the alignment of slides. TMAs, containing 140 cores of consecutively obtained breast carcinomas, were stained for CK and Ki67 using optimized staining protocols. By means of stereological principles, Ki67-positive and -negative cell profiles were counted in sampled areas and used for the estimation of PIs of the whole tissue core. The VDS principle was applied to both the same sampled areas and the whole tissue core. Additionally, five neighboring slides were stained for CK in order to examine the alignment algorithm. Correlation between manual counting and VDS in both sampled areas and whole core was almost perfect (correlation coefficients above 0.97). Bland-Altman plots did not reveal any skewness in any data ranges. There was a good agreement in alignment (>85 %) in neighboring slides, whereas agreement decreased in non-neighboring slides. VDS gave similar results compared with manual counting using stereological principles. Introduction of this method in clinical and research practice may improve accuracy and reproducibility of Ki67 PI.

Interleukin 4-producing CD4+ T cells in the skin of cats with allergic dermatitis.

PubMed

Roosje, P J; Dean, G A; Willemse, T; Rutten, V P M G; Thepen, T

2002-03-01

Lesional skin of cats with allergic dermatitis has a cellular infiltrate and a CD4/CD8 ratio comparable to that in humans with atopic dermatitis. CD4+ helper T cells and in particular cells belonging to the Th2 subset play an important role in disease pathogenesis in humans. We investigated the cytokine pattern of CD4+ T cells in situ, with special emphasis on the putative presence of cells producing interleukin 4 (IL4), in cats with allergic dermatitis. Immunohistochemical procedures were used to determine that CD4+ T cells in lesional and nonlesional skin of cats with allergic dermatitis can produce IL4, as occurs in humans. Lesional and nonlesional skin of cats with allergic dermatitis had significantly more IL4+ T cells (P = 0.001) than did skin of healthy control cats. Double staining indicated that all IL4+ cells were positive for pan-T or CD4 markers. Double labeling for mast cell chymase and IL4 stained primarily different cells. Western blotting demonstrated cross-reactivity between the antibody against human IL4 and a feline recombinant IL4. These results indicate that IL4 is primarily produced by CD4+ T cells and is also present in clinically uninvolved skin, indicating a role in the pathogenesis of allergic dermatitis in cats.

Temporal and spatial changes of cells positive for stem-like markers in different compartments and stages of human colorectal adenoma-carcinoma sequence

PubMed Central

Cui, Guanglin; Xu, Gang; Zhu, Li; Pang, Zhigang; Zheng, Wei; Li, Zhenfeng; Yuan, Aping

2017-01-01

Considerable evidence supports the idea that stem-like cells may play an essential role during the development of colorectal cancer (CRC). To accomplish this aim, we use immunohistochemistry (IHC) and double IHC with different potential stem-like markers, anti-musashi (Msi), anti-CD133, anti- LGR5 and anti-ALDH1 to examine the presentation of stem-like cells in different compartments including adenoma/CRC epithelium, transitional crypts and tumor stroma in colorectal adenoma and CRC. The results showed that cells positive for stem-like markers were remarkably increased in number and frequently observed in the adenoma/CRC epithelium, transitional crypts and tumor stroma. Notably, the population of cells positive for stem-liker markers was expanded from the base to the middle part of the transitional crypt in both adenoma and CRC tissues, reflecting that stem-like cells are likely involved in the process of colorectal tumorigenesis. Counting results showed that the grading scores of cells positive for LGR5 and ALDH1 in the adenoma/CRC epithelium were significantly increased relative with the control epithelium, and associated with the degree of dysplasia in the adenoma and node involvement in the CRC (all P < 0.05). In addition, the density of cells positive for stem–like markers in the adenomatous/cancerous stroma was also increased and paralleled an increase in the density of proliferative stromal cells labeled by PCNA, which were primarily identified as vimentin positive fibroblasts. Our results have revealed a changed temporal and spatial presentation of stem-like markers in different stages of human colorectal adenoma-carcinoma sequence, which might be a hallmark of the adenoma-carcinoma transition. PMID:28484082

A 10-aa-long sequence in SLP-76 upstream of the Gads binding site is essential for T cell development and function.

PubMed

Kumar, Lalit; Feske, Stefan; Rao, Anjana; Geha, Raif S

2005-12-27

The adapter SLP-76 is essential for T cell development and function. SLP-76 binds to the src homology 3 domain of Lck in vitro. This interaction depends on amino acids 185-194 of SLP-76. To examine the role of the Lck-binding region of SLP-76 in T cell development and function, SLP-76(-/-) mice were reconstituted with an SLP-76 mutant that lacks amino acids 185-194. Double and single positive thymocytes from reconstituted mice were severely reduced in numbers and exhibited impaired positive selection and increased apoptosis. Peripheral T cells were also reduced in numbers, exhibited impaired phospholipase C-gamma1 and Erk phosphorylation, and failed to flux calcium, secrete IL-2, and proliferate in response to T cell antigen receptor ligation. Delayed cutaneous hypersensitivity responses and Ab responses to T cell-dependent antigen were severely impaired. These results indicate that the Lck binding region of SLP-76 is essential for T cell antigen receptor signaling and normal T cell development and function.

Thymic Stromal-Cell Abnormalities and Dysregulated T-Cell Development in IL-2-Deficient Mice

PubMed Central

Reya, Tannishtha; Bassiri, Hamid; Biancaniello, Renée

1998-01-01

The role that interleukin-2 (IL-2) plays in T-cell development is not known. To address this issue, we have investigated the nature of the abnormal thymic development and autoimmune disorders that occurs in IL-2-deficient (IL-2–/–) mice. After 4 to 5 weeks of birth, IL-2–/– mice progressively develop a thymic disorder resulting in the disruption of thymocyte maturation. This disorder is characterized by a dramatic reduction in cellularity, the selective loss of immature CD4-8- (double negative; DN) and CD4+8+ (double positive; DP) thymocytes and defects in the thymic stromal-cell compartment. Immunohistochemical staining of sections of thymuses from specific pathogen-free and germ-free IL-2–/– mice of various ages showed a progressive ,loss of cortical epithelial cells, MHC class II-expressing cells, monocytes, and macrophages. Reduced numbers of macrophages were apparent as early as week after birth. Since IL-2–/– thymocyte progenitor populations could mature normally on transfer into a normal thymus, the thymic defect in IL-2–/– mice appears to be due to abnormalities among thymic stromal cells. These results underscore the role of IL-2 in maintaining functional microenvironments that are necessary to support thymocyte growth, development, and selection. PMID:9814585

A dual positive and negative regulation of monocyte activation by leukocyte Ig-like receptor B4 depends on the position of the tyrosine residues in its ITIMs.

PubMed

Park, Mijeong; Liu, Robert W; An, Hongyan; Geczy, Carolyn L; Thomas, Paul S; Tedla, Nicodemus

2017-05-01

The leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory cell surface receptor, primarily expressed on mono-myeloid cells. It contains 2 C-type Ig-like extracellular domains and a long cytoplasmic domain that contains three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Data suggest that LILRB4 suppresses Fc receptor-dependent monocyte functions via its ITIMs, but relative contributions of the three ITIMs are not characterised. To address this, tyrosine (Tyr) residues at positions 337, 389 and 419 were single, double or triple mutated to phenylalanine and stably transfected into a human monocytic cell line, THP-1. Intact Tyr 389 was sufficient to maximally inhibit FcγRI-mediated TNF-α production in THP-1 cells, but, paradoxically, Tyr 337 significantly enhanced TNF-α production. In contrast, bactericidal activity was significantly enhanced in mutants containing Tyr 419 , while Tyr 337 markedly inhibited bacteria killing. Taken together, these results indicate that LILRB4 might have dual inhibitory and activating functions, depending on the position of the functional tyrosine residues in its ITIMs and/or the nature of the stimuli.

Push back to respond better: regulatory inhibition of the DNA double-strand break response.

PubMed

Panier, Stephanie; Durocher, Daniel

2013-10-01

Single DNA lesions such as DNA double-strand breaks (DSBs) can cause cell death or trigger genome rearrangements that have oncogenic potential, and so the pathways that mend and signal DNA damage must be highly sensitive but, at the same time, selective and reversible. When initiated, boundaries must be set to restrict the DSB response to the site of the lesion. The integration of positive and, crucially, negative control points involving post-translational modifications such as phosphorylation, ubiquitylation and acetylation is key for building fast, effective responses to DNA damage and for mitigating the impact of DNA lesions on genome integrity.

[Aortic stenosis and mitral regurgitation complicated by hemolytic anemia and positive Direct Coombs test: a case report].

PubMed

Tamura, Shinjiro; Kitaoka, Hiroaki; Yamasaki, Naohito; Okawa, Makoto; Kubo, Toru; Matsumura, Yoshihisa; Furuno, Takashi; Takata, Jun; Nishinaga, Masanori; Sasaguri, Shiro; Doi, Yoshinori

2005-09-01

A 83-year-old man was admitted because of heart failure due to severe aortic stenosis and mitral regurgitation secondary to chordal rupture of the anterior leaflet. Mild anemia and elevated serum lactate dehydrogenase were present with reticulocytosis and haptoglobinemia. Direct Coombs test was positive. Coexistence of autoimmune hemolytic anemia was identified, but the main cause of his hemolysis was thought to be mechanical hemolysis due to stenotic valve and/or ruptured chordae because of the presence of red cell fragmentation. The patient successfully underwent double valve replacement. Improvement of anemia was coupled with reduction of the serum lactate dehydrogenase level. Valvular shear stress on the red cells and reduction of red cell deformability secondary to autoimmune hemolytic anemia were thought to be responsible for his hemolysis.

Variations in the Processing of DNA Double-Strand Breaks Along 60-MeV Therapeutic Proton Beams

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

Chaudhary, Pankaj; Marshall, Thomas I.; Currell, Frederick J.

Purpose: To investigate the variations in induction and repair of DNA damage along the proton path, after a previous report on the increasing biological effectiveness along clinically modulated 60-MeV proton beams. Methods and Materials: Human skin fibroblast (AG01522) cells were irradiated along a monoenergetic and a modulated spread-out Bragg peak (SOBP) proton beam used for treating ocular melanoma at the Douglas Cyclotron, Clatterbridge Centre for Oncology, Wirral, Liverpool, United Kingdom. The DNA damage response was studied using the 53BP1 foci formation assay. The linear energy transfer (LET) dependence was studied by irradiating the cells at depths corresponding to entrance, proximal, middle, andmore » distal positions of SOBP and the entrance and peak position for the pristine beam. Results: A significant amount of persistent foci was observed at the distal end of the SOBP, suggesting complex residual DNA double-strand break damage induction corresponding to the highest LET values achievable by modulated proton beams. Unlike the directly irradiated, medium-sharing bystander cells did not show any significant increase in residual foci. Conclusions: The DNA damage response along the proton beam path was similar to the response of X rays, confirming the low-LET quality of the proton exposure. However, at the distal end of SOBP our data indicate an increased complexity of DNA lesions and slower repair kinetics. A lack of significant induction of 53BP1 foci in the bystander cells suggests a minor role of cell signaling for DNA damage under these conditions.« less

Meta-analysis of Microbial Fuel Cells Using Waste Substrates.

PubMed

Dowdy, F Ryan; Kawakita, Ryan; Lange, Matthew; Simmons, Christopher W

2018-05-01

Microbial fuel cell experimentation using waste streams is an increasingly popular field of study. One obstacle to comparing studies has been the lack of consistent conventions for reporting results such that meta-analysis can be used for large groups of experiments. Here, 134 unique microbial fuel cell experiments using waste substrates were compiled for analysis. Findings include that coulombic efficiency correlates positively with volumetric power density (p < 0.001), negatively with working volume (p < 0.05), and positively with percentage removal of chemical oxygen demand (p < 0.005). Power density in mW/m 2 correlates positively with chemical oxygen demand loading (p < 0.005), and positively with maximum open-circuit voltage (p < 0.05). Finally, single-chamber versus double-chamber reactor configurations differ significantly in maximum open-circuit voltage (p < 0.005). Multiple linear regression to predict either power density or maximum open-circuit voltage produced no significant models due to the amount of multicollinearity between predictor variables. Results indicate that statistically relevant conclusions can be drawn from large microbial fuel cell datasets. Recommendations for future consistency in reporting results following a MIAMFCE convention (Minimum Information About a Microbial Fuel Cell Experiment) are included.

Antimycobacterial, antimicrobial, and biocompatibility properties of para-aminosalicylic acid with zinc layered hydroxide and Zn/Al layered double hydroxide nanocomposites

PubMed Central

Saifullah, Bullo; El Zowalaty, Mohamed E; Arulselvan, Palanisamy; Fakurazi, Sharida; Webster, Thomas J; Geilich, Benjamin M; Hussein, Mohd Zobir

2014-01-01

The treatment of tuberculosis by chemotherapy is complicated due to multiple drug prescriptions, long treatment duration, and adverse side effects. We report here for the first time an in vitro therapeutic effect of nanocomposites based on para-aminosalicylic acid with zinc layered hydroxide (PAS-ZLH) and zinc-aluminum layered double hydroxides (PAS-Zn/Al LDH), against mycobacteria, Gram-positive bacteria, and Gram-negative bacteria. The nanocomposites demonstrated good antimycobacterial activity and were found to be effective in killing Gram-positive and Gram-negative bacteria. A biocompatibility study revealed good biocompatibility of the PAS-ZLH nanocomposites against normal human MRC-5 lung cells. The para-aminosalicylic acid loading was quantified with high-performance liquid chromatography analysis. In summary, the present preliminary in vitro studies are highly encouraging for further in vivo studies of PAS-ZLH and PAS-Zn/Al LDH nanocomposites to treat tuberculosis. PMID:25114509

An assessment of mast cells and myofibroblasts in denture-induced fibrous hyperplasia.

PubMed

Kiuchi, Misa; Yamamura, Takashi; Okudera, Michisato; Souksavanh, Vongsa; Ishigami, Tomohiko; Iwase, Takashi; Warnakulasuriya, Saman; Komiyama, Kazuo

2014-01-01

The pathogenesis of denture-induced fibrous hyperplasias has not been examined in detail to explain how tissue injury results in fibrous hyperplasia of the oral mucosa. We examined the presence of mast cells and myofibroblasts in 33 denture-induced fibrous hyperplasias (DIFH) compared with 10 healthy gingival tissues. The parameters examined included mast cell numbers, tissue distribution, degranulation, and cell subtypes using immunohistochemistry. The presence of myofibroblasts and their likely origin was also examined by double immunofluorescense staining. Furthermore, we investigated the synthesis of osteopontin and TGF-β, considered to be involved in the transformation of a fibroblast to a myofibroblast. The results demonstrated that the mast cell numbers are significantly increased in the DIFH compared with non-disease controls. The mast cell localization in lesions was higher in the superficial areas with inflammatory cell infiltration compared with the deep fibrotic area (P < 0.01). The number of tryptase-positive mast cells was significantly higher compared with chymase-positive ones. The TGF-β- or osteopontin-positive cell infiltration into the lesion was found in high numbers. The presence of myofibroblasts was identified in 14 of 33 cases (42%), and some of these cells showed apoptosis when assessed by the TUNEL assay. On the survey of the origin of myofibroblasts, results showed αSMA and vimentin positivity indicating these transformed from fibroblasts. These results are the first to show that mast cells and myofibroblasts can be detected in DIFH, indicating important roles of these cells in the pathogenesis of this lesion. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Difference in the relative biological effectiveness and DNA damage repair processes in response to proton beam therapy according to the positions of the spread out Bragg peak.

PubMed

Hojo, Hidehiro; Dohmae, Takeshi; Hotta, Kenji; Kohno, Ryosuke; Motegi, Atsushi; Yagishita, Atsushi; Makinoshima, Hideki; Tsuchihara, Katsuya; Akimoto, Tetsuo

2017-07-03

Cellular responses to proton beam irradiation are not yet clearly understood, especially differences in the relative biological effectiveness (RBE) of high-energy proton beams depending on the position on the Spread-Out Bragg Peak (SOBP). Towards this end, we investigated the differences in the biological effect of a high-energy proton beam on the target cells placed at different positions on the SOBP, using two human esophageal cancer cell lines with differing radiosensitivities. Two human esophageal cancer cell lines (OE21, KYSE450) with different radiosensitivities were irradiated with a 235-MeV proton beam at 4 different positions on the SOBP (position #1: At entry; position #2: At the proximal end of the SOBP; position #3: Center of the SOBP; position #4: At the distal end of the SOBP), and the cell survivals were assessed by the clonogenic assay. The RBE 10 for each position of the target cell lines on the SOBP was determined based on the results of the cell survival assay conducted after photon beam irradiation. In addition, the number of DNA double-strand breaks was estimated by quantitating the number of phospho-histone H2AX (γH2AX) foci formed in the nuclei by immunofluorescence analysis. In regard to differences in the RBE of a proton beam according to the position on the SOBP, the RBE value tended to increase as the position on the SOBP moved distally. Comparison of the residual number of γH2AX foci at the end 24 h after the irradiation revealed, for both cell lines, a higher number of foci in the cells irradiated at the distal end of the SOPB than in those irradiated at the proximal end or center of the SOBP. The results of this study demonstrate that the RBE of a high-energy proton beam and the cellular responses, including the DNA damage repair processes, to high-energy proton beam irradiation, differ according to the position on the SOBP, irrespective of the radiosensitivity levels of the cell lines.

Intra-species bacterial quorum sensing studied at single cell level in a double droplet trapping system.

PubMed

Bai, Yunpeng; Patil, Santoshkumar N; Bowden, Steven D; Poulter, Simon; Pan, Jie; Salmond, George P C; Welch, Martin; Huck, Wilhelm T S; Abell, Chris

2013-05-21

In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP) from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

Evolution of Ionospheric Convection during a Double Transpolar Arc Phenomenon on February 11, 1999

NASA Technical Reports Server (NTRS)

Narita, Y.; Maezawa, K.; Spann, J. F.; Parks, G. K.; Marklund, G. T.; Kullen, A.; Ivchenko, N.; Greenwald, R. A.; Sato, N.; Yamagishi, H.;

Reliable method for generating double-stranded DNA vectors containing site-specific base modifications.

PubMed

Brégeon, Damien; Doetsch, Paul W

2004-11-01

Cells of all living organisms are continuously exposed to physical and chemical agents that damage DNA and alter the integrity of their genomes. Despite the relatively high efficiency of the different repair pathways, some lesions remain in DNA when it is replicated or transcribed. Lesion bypass by DNA and RNA polymerases has been the subject of numerous investigations. However, knowledge of the in vivo mechanism of transcription lesion bypass is very limited because no robust methodology is available. Here we describe a protocol based on the synthesis of a complementary strand of a circular, single-stranded DNA molecule, which allows for the production of large amounts of double-stranded DNA containing a lesion at a specific position in a transcribed sequence. Such constructs can subsequently be used for lesion bypass studies in vivo by RNA polymerase and to ascertain how these events can be affected by the genetic background of the cells.

Design, fabrication and characterization of a double layer solid oxide fuel cell (DLFC)

NASA Astrophysics Data System (ADS)

Wang, Guangjun; Wu, Xiangying; Cai, Yixiao; Ji, Yuan; Yaqub, Azra; Zhu, Bin

2016-11-01

A double layer solid oxide fuel cell (DLSOFC) without using the electrolyte (layer) has been designed by integrating advantages of positive electrode material of lithium ion battery(LiNi0.8Co0.15Al0.05O2) and oxygen-permeable membranes material (trace amount cobalt incorporated terbium doped ceria, TDC + Co) based on the semiconductor physics principle. Instead of using an electrolyte layer, the depletion layer between the anode and cathode served as an electronic insulator to block the electrons but to maintain the electrolyte function for ionic transport. Thus the device with two layers can realize the function of SOFC and at the same time avoids the electronic short circuiting problem. Such novel DLFC showed good performance at low temperatures, for instance, a maximum power density of 230 mWcm-2 was achieved at 500 °C. The working principle of the new device is presented.

Double-layered cell transfer technology for bone regeneration

PubMed Central

Akazawa, Keiko; Iwasaki, Kengo; Nagata, Mizuki; Yokoyama, Naoki; Ayame, Hirohito; Yamaki, Kazumasa; Tanaka, Yuichi; Honda, Izumi; Morioka, Chikako; Kimura, Tsuyoshi; Komaki, Motohiro; Kishida, Akio; Izumi, Yuichi; Morita, Ikuo

2016-01-01

For cell-based medicine, to mimic in vivo cellular localization, various tissue engineering approaches have been studied to obtain a desirable arrangement of cells on scaffold materials. We have developed a novel method of cell manipulation called “cell transfer technology”, enabling the transfer of cultured cells onto scaffold materials, and controlling cell topology. Here we show that using this technique, two different cell types can be transferred onto a scaffold surface as stable double layers or in patterned arrangements. Various combinations of adherent cells were transferred to a scaffold, amniotic membrane, in overlapping bilayers (double-layered cell transfer), and transferred cells showed stability upon deformations of the material including folding and trimming. Transplantation of mesenchymal stem cells from periodontal ligaments (PDLSC) and osteoblasts, using double-layered cell transfer significantly enhanced bone formation, when compared to single cell type transplantation. Our findings suggest that this double-layer cell transfer is useful to produce a cell transplantation material that can bear two cell layers. Moreover, the transplantation of an amniotic membrane with PDLSCs/osteoblasts by cell transfer technology has therapeutic potential for bone defects. We conclude that cell transfer technology provides a novel and unique cell transplantation method for bone regeneration. PMID:27624174

Activation of cholecystokinin neurons in the dorsal pallium of the telencephalon is indispensable for the acquisition of chick imprinting behavior.

PubMed

Maekawa, Fumihiko; Nakamori, Tomoharu; Uchimura, Motoaki; Fujiwara, Ken; Yada, Toshihiko; Tsukahara, Shinji; Kanamatsu, Tomoyuki; Tanaka, Kohichi; Ohki-Hamazaki, Hiroko

2007-09-01

Chick imprinting behavior is a good model for the study of learning and memory. Imprinting object is recognized and processed in the visual wulst, and the memory is stored in the intermediate medial mesopallium in the dorsal pallium of the telencephalon. We identified chicken cholecystokinin (CCK)-expressing cells localized in these area. The number of CCK mRNA-positive cells increased in chicks underwent imprinting training, and these cells expressed nuclear Fos immunoreactivity at high frequency in these regions. Most of these CCK-positive cells were glutamatergic and negative for parvalbumin immunoreactivity. Semi-quantitative PCR analysis revealed that the CCK mRNA levels were significantly increased in the trained chicks compared with untrained chicks. In contrast, the increase in CCK- and c-Fos-double-positive cells associated with the training was not observed after closure of the critical period. These results indicate that CCK cells in the dorsal pallium are activated acutely by visual training that can elicit imprinting. In addition, the CCK receptor antagonist significantly suppressed the acquisition of memory. These results suggest that the activation of CCK cells in the visual wulst as well as in the intermediate medial mesopallium by visual stimuli is indispensable for the acquisition of visual imprinting.

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

Yuan, Aping; Research Group of Gastrointestinal Diseases, The Second Affiliated Hospital of Zhengzhou University, Henan; Yang, Hang

Diverse T help (Th) cells play a crucial role in the processing and maintaining of chronic inflammation as seen in ulcerative colitis (UC). Th9, a novel subset of Th cells that primarily produces interleukin (IL)-9, has recently been associated with the development of inflammatory diseases. In this study, we evaluated the presentation of Th9 cells in inflamed tissues of human and experimental mouse UC, and examined the therapeutic efficiency of anti Th9 cytokine IL-9 in the experimental mouse UC. Using immunohistochemistry (IHC), we evaluated the presentation of Th9 cells labelled by transcriptional factor PU.1 in both human and dextran sulfatemore » sodium (DSS) induced mouse colitis biopsies. The results showed that increased PU.1 positive Th9 cells were mainly located in the lamina propria in relative with the controls, intraepithelial Th9 cells can also be observed but at low density. Double IHCs revealed that most of PU.1 positive cells were CD3 positive lymphocytes in human UC specimens. Anti-IL-9 antibody injection for 2 weeks reduced the severity of inflammation in DSS induced colitis mice. Our results suggest that The Th9/IL-9 is involved in the pathogenesis of UC. - Highlights: • The density of novel PU.1 positive Th9 cells is significantly increased in both human and mouse colitis tissues. • PU.1 positive Th9 cells are predominately located in the inflamed lamina propria in both human and mouse colitis tissues. • Blocking of Th9 cytokine IL-9 by antibody injection suppresses the severity of inflammation in the bowel in colitis mice. • Novel Th9 cells contribute to the pathogenesis of UC.« less

A global interaction network maps a wiring diagram of cellular function

PubMed Central

Costanzo, Michael; VanderSluis, Benjamin; Koch, Elizabeth N.; Baryshnikova, Anastasia; Pons, Carles; Tan, Guihong; Wang, Wen; Usaj, Matej; Hanchard, Julia; Lee, Susan D.; Pelechano, Vicent; Styles, Erin B.; Billmann, Maximilian; van Leeuwen, Jolanda; van Dyk, Nydia; Lin, Zhen-Yuan; Kuzmin, Elena; Nelson, Justin; Piotrowski, Jeff S.; Srikumar, Tharan; Bahr, Sondra; Chen, Yiqun; Deshpande, Raamesh; Kurat, Christoph F.; Li, Sheena C.; Li, Zhijian; Usaj, Mojca Mattiazzi; Okada, Hiroki; Pascoe, Natasha; Luis, Bryan-Joseph San; Sharifpoor, Sara; Shuteriqi, Emira; Simpkins, Scott W.; Snider, Jamie; Suresh, Harsha Garadi; Tan, Yizhao; Zhu, Hongwei; Malod-Dognin, Noel; Janjic, Vuk; Przulj, Natasa; Troyanskaya, Olga G.; Stagljar, Igor; Xia, Tian; Ohya, Yoshikazu; Gingras, Anne-Claude; Raught, Brian; Boutros, Michael; Steinmetz, Lars M.; Moore, Claire L.; Rosebrock, Adam P.; Caudy, Amy A.; Myers, Chad L.; Andrews, Brenda; Boone, Charles

2017-01-01

We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing over 23 million double mutants, identifying ~550,000 negative and ~350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell. PMID:27708008

Hybrid capacitor with activated carbon electrode, Ni(OH) 2 electrode and polymer hydrogel electrolyte

NASA Astrophysics Data System (ADS)

Nohara, Shinji; Asahina, Toshihide; Wada, Hajime; Furukawa, Naoji; Inoue, Hiroshi; Sugoh, Nozomu; Iwasaki, Hideharu; Iwakura, Chiaki

A new hybrid capacitor (HC) cell was assembled using an activated carbon (AC) negative electrode, an Ni(OH) 2 positive electrode and a polymer hydrogel electrolyte prepared from crosslinked potassium poly(acrylate) (PAAK) and KOH aqueous solution. The HC cell was characterized compared with an electric double layer capacitor (EDLC) using two AC electrodes and the polymer hydrogel electrolyte. It was found that the HC cell successfully worked in the larger voltage range and exhibited ca. 2.4 times higher capacitance than the EDLC cell. High-rate dischargeability of the HC cell was also superior to that of the EDLC cell. These improved characteristics strongly suggest that the HC cell can be a promising system of capacitors with high energy and power densities.

Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

DOEpatents

Stampfer, Martha R.; Garbe, James C.

2016-06-28

Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

DOEpatents

Stampfer, Martha R; Garbe, James C

2015-02-24

Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

Phospho-eNOS Ser-1176 is associated with the nucleoli and the Golgi complex in C6 rat glioma cells.

PubMed

Klinz, Franz-Josef; Herberg, Natalie; Arnhold, Stefan; Addicks, Klaus; Bloch, Wilhelm

2007-06-29

Enzymatic activity of endothelial nitric oxide synthase (eNOS) is controlled by posttranslational modifications, protein-protein interactions, and subcellular localization. For example, N-terminal fatty acid modifications target eNOS to the Golgi complex where it becomes phosphorylated. We show here by immunofluorescence analysis that phospho-eNOS Ser-1176 is enriched in the perinuclear region of interphase C6 rat glioma cells. Confocal double immunofluorescence microscopy with the Golgi marker protein 58K revealed that phospho-eNOS Ser-1176 is associated with the Golgi complex. Surprisingly, we observed several spots in the nucleus of C6 cells that were positive for phospho-eNOS Ser-1176. Confocal double immunofluorescence analysis with the nucleolus marker protein fibrillarin revealed that within the nucleus phospho-eNOS Ser-1176 is exclusively associated with the nucleoli. It is known that in mitotic cells nucleoli are lost during prophase and rebuild during telophase. In agreement with this, we find no nucleoli-like distribution of phospho-eNOS Ser-1176 in metaphase and anaphase C6 glioma cells. Our finding that phospho-eNOS Ser-1176 is selectively associated with the nucleoli points to a so far unknown role for eNOS in interphase glioma cells.

Quantum dots-based double imaging combined with organic dye imaging to establish an automatic computerized method for cancer Ki67 measurement.

PubMed

Wang, Lin-Wei; Qu, Ai-Ping; Liu, Wen-Lou; Chen, Jia-Mei; Yuan, Jing-Ping; Wu, Han; Li, Yan; Liu, Juan

2016-02-03

As a widely used proliferative marker, Ki67 has important impacts on cancer prognosis, especially for breast cancer (BC). However, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study is to establish quantum dots (QDs)-based double imaging of nuclear Ki67 as red signal by QDs-655, cytoplasmic cytokeratin (CK) as yellow signal by QDs-585, and organic dye imaging of cell nucleus as blue signal by 4',6-diamidino-2-phenylindole (DAPI), and to develop a computer-aided automatic method for Ki67 index measurement. The newly developed automatic computerized Ki67 measurement could efficiently recognize and count Ki67-positive cancer cell nuclei with red signals and cancer cell nuclei with blue signals within cancer cell cytoplasmic with yellow signals. Comparisons of computerized Ki67 index, visual Ki67 index, and marked Ki67 index for 30 patients of 90 images with Ki67 ≤ 10% (low grade), 10% < Ki67 < 50% (moderate grade), and Ki67 ≥ 50% (high grade) showed computerized Ki67 counting is better than visual Ki67 counting, especially for Ki67 low and moderate grades. Based on QDs-based double imaging and organic dye imaging on BC tissues, this study successfully developed an automatic computerized Ki67 counting method to measure Ki67 index.

Decreased interleukin 35 and CD4+EBI3+ T cells in patients with active systemic lupus erythematosus.

PubMed

Ouyang, Han; Shi, Yong-Bing; Liu, Zhi-Chun; Wang, Zhi; Feng, Sheng; Kong, Shu-Min; Lu, Ying

2014-08-01

Interleukin 35 (IL-35) is likely to contribute to the development of autoimmune diseases, as the Epstein-Barr virus-induced gene protein 3 (EBI3) is the specificity subunit of IL-35. Nevertheless, until recently, no studies have evaluated its role in systemic lupus erythematosus (SLE) in humans. The objective of this study was to investigate the serum IL-35 level and the percentage of CD4EBI3 T cells in the peripheral blood of patients with SLE and explore the roles of double-positive T cells and IL-35 in the pathogenesis of SLE and the effects of glucocorticoid on these roles. Fifty-five hospitalized patients with SLE were recruited, and 20 volunteers were enrolled as healthy controls. Serum IL-35 levels were measured by enzyme-linked immunosorbent assay, and the percentage of CD4EBI3 T cells was analyzed by flow cytometry. The serum IL-35 level and the percentage of CD4EBI3 T cells were significantly decreased in patients with active SLE compared with healthy controls and patients with inactive SLE. The serum IL-35 level and the percentage of CD4EBI3 T cells were negatively correlated with the SLE disease activity index. The percentages of CD4EBI3 T cells and serum IL-35 levels in 10 untreated patients with active SLE were increased at days l, 3, and 7 after the treatment with methylprednisolone (0.8 mg·kg·d) compared with the percentages before the treatment. These results demonstrate that abnormalities in IL-35 and CD4EBI3 T cells may play important roles in the pathogenesis of SLE; the percentage of double-positive T cells and the level of IL-35 are parameters for the evaluation of SLE activity and severity.

Peroxisome proliferator-activated receptor (PPAR)gamma is highly expressed in normal human pituitary gland.

PubMed

Bogazzi, F; Russo, D; Locci, M T; Chifenti, B; Ultimieri, F; Raggi, F; Viacava, P; Cecchetti, D; Cosci, C; Sardella, C; Acerbi, G; Gasperi, M; Martino, E

2005-11-01

Expression of peroxisome proliferator-activated receptor (PPAR)gamma in normal pituitary seems to be restricted to ACTH-secreting cells. The aim of the study was to evaluate the expression of PPARgamma in normal human pituitary tissue and to study its localization in the pituitary secreting cells. Normal pituitary tissue samples were obtained form 11 patients with non-secreting adenoma who underwent surgical excision of the tumor. Expression of PPARgamma was evaluated by immunostaining and western blotting; localization of PPARgamma in each pituitary secreting cell lineage was evaluated by double immunofluorescence using confocal microscopy. Pituitary non-functioning adenomas served as Controls. PPARgamma was highly expressed in all pituitary samples with a (mean +/- SD) 81 +/- 6.5% of stained cells; expression of PPARgamma was confirmed by western blotting. Non-functioning pituitary adenomas had 74 +/- 11% PPARgamma positive cells. Expression of PPARy was either in cytoplasm or nuclei. In addition, treatment of GH3 cells, with a PPARgamma ligand was associated with traslocation of the receptor from cytoplasm into the nucleus. Double immunostaining revealed that every pituitary secreting cell (GH, TSH, LH, FSH, PRL and ACTH) had PPARgamma expressed. The present study demonstrated that PPARgamma is highly expressed in every normal pituitary secreting cell lineage. It can translocate into the nucleus by ligand binding; however, its role in pituitary hormone regulation remains to be elucidated.

TTF-1 and napsin A on cell blocks and supernatants of pleural fluids for labeling malignant effusions.

PubMed

Porcel, José M; Palma, Rosa; Bielsa, Silvia; Esquerda, Aureli; Gatius, Sonia; Matias-Guiu, Xavier; Salud, Antonieta

2015-07-01

In this retrospective study of 80 pleural effusions, the combination of thyroid transcription factor 1 (TTF-1) and napsin A immunostaining on fluid cell blocks was positive in 80% of lung adenocarcinomas. Although measuring TTF-1 pleural fluid concentrations was of no value, quantification of napsin A levels allowed the identification of one third of the double-negative stained lung adenocarcinomas, with an overall accuracy similar to classical tumour markers for malignant-benign discrimination (sensitivity 40%, specificity 100%). © 2015 Asian Pacific Society of Respirology.

Establishment and characterization of outer root sheath (ORS) cell line from Jining grey goat.

PubMed

Cui, Zhifeng; Hu, Yanxia; Wang, Hui; Zeng, Yongqing; Dong, Bin; Zhu, Houshun; Dong, Zhongdian; Liu, Zhiyuan

2012-03-01

A new line of outer root sheath (ORS) cells was established from hair follicles of Jining grey goat by using a mechanical separation combined with enzyme digestion. Cell morphology is described at different phases. The chromosome analysis of ORS cells, identification of the ORS cells and morphological reversion test were detected at the 4th and 40th passages. The ORS cells were healthy and the growth characteristics were stable with a population doubling time of 52 h. Chromosome analysis showed that >58% of cells were diploid. Test for ORS cell line CK19 expression was positive. This newly established ORS cell line not only lays the foundation for further studying on the growth, regeneration, development law of goat hair follicle but also provides a mirror for the research of human hair in medical field.

In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)

USGS Publications Warehouse

Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Schumacher, Joanne; Lewis, Teresa D.; Leong, Jo-Ann C.; Casey, Rufina N.; Casey, James W.

2009-01-01

Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.

Intracerebral Mycobacterium bovis bacilli Calmette-Guerin infection-induced immune responses in the CNS 1

PubMed Central

Lee, JangEun; Ling, Changying; Kosmalski, Michelle M.; Hulseberg, Paul; Schreiber, Heidi A.; Sandor, Matyas; Fabry, Zsuzsanna

2010-01-01

To study whether cerebral mycobacterial infection induces granuloma and protective immunity similar to systemic infection, we intracerebrally infected mice with Mycobacterium bovis bacilli Calmette-Guerin. Granuloma and IFN-γ+CD4+ T cell responses are induced in the central nervous system (CNS) similar to periphery, but the presence of IFN-γIL-17 double-positive CD4+ T cells is unique to the CNS. The major CNS source of TNF-α is microglia, with modest production by CD4+ T cells and macrophage. Protective immunity is accompanied by accumulation of Foxp3+CD4+ T cells and PD-L2+ dendritic cells, suggesting that both inflammatory and anti-inflammatory responses develop in the CNS following mycobacterial infection. PMID:19535154

A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

PubMed

Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

2015-08-01

At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

[The expression of Cyclin D1 modulated by somatotropin on human pancreas cancer cell lines Bxpc-3].

PubMed

Li, Fei; Liu, Da-chuan; Sun, Jia-bang

2004-04-07

To observe the growth effect of somatostapin on human pancreas cancer lines Bxpc-3. The Bxpc-3 pancreas cancer cells were treated with Somatotropin. The cells hyperplasia were detected by MTT and were observed apoptosis cells determinated quantitatively by TUNEL, quantify immune fluoresence double marked the proliferation cells and apoptosis cells, the expression of Cyclin D1 detected by immunohistochemical. The growth effect of pancrea cancer cells were limited by 10(-7) M, 10(-8) M, 10(-9) M Somatotropin on 2 day. The limited effect was decreased from 3 day. The cells proliferation were increased by somotostapin on 4day to 5day. The relationship between the expression of Cyclin D1 and apoptosis was negative correlation and the cells hyperplasia was positive correlation in Bxpc-3 cell line. From the cell study we knew the expression of Cyclin D1 reflected the prolefiration of pancreas cancer cells.

Patients double-seropositive for ANCA and anti-GBM antibodies have varied renal survival, frequency of relapse, and outcomes compared to single-seropositive patients.

PubMed

McAdoo, Stephen P; Tanna, Anisha; Hrušková, Zdenka; Holm, Lisa; Weiner, Maria; Arulkumaran, Nishkantha; Kang, Amy; Satrapová, Veronika; Levy, Jeremy; Ohlsson, Sophie; Tesar, Vladimir; Segelmark, Mårten; Pusey, Charles D

2017-09-01

Co-presentation with both ANCA and anti-GBM antibodies is thought to be relatively rare. Current studies of such 'double-positive' cases report small numbers and variable outcomes. To study this further we retrospectively analyzed clinical features and long-term outcomes of a large cohort of 568 contemporary patients with ANCA-associated vasculitis, 41 patients with anti-GBM disease, and 37 double-positive patients with ANCA and anti-GBM disease from four European centers. Double-positive patients shared characteristics of ANCA-associated vasculitis (AAV), such as older age distribution and longer symptom duration before diagnosis, and features of anti-GBM disease, such as severe renal disease and high frequency of lung hemorrhage at presentation. Despite having more evidence of chronic injury on renal biopsy compared to patients with anti-GBM disease, double-positive patients had a greater tendency to recover from being dialysis-dependent after treatment and had intermediate long-term renal survival compared to the single-positive patients. However, overall patient survival was similar in all three groups. Predictors of poor patient survival included advanced age, severe renal failure, and lung hemorrhage at presentation. No single-positive anti-GBM patients experienced disease relapse, whereas approximately half of surviving patients with AAV and double-positive patients had recurrent disease during a median follow-up of 4.8 years. Thus, double-positive patients have a truly hybrid disease phenotype, requiring aggressive early treatment for anti-GBM disease, and careful long-term follow-up and consideration for maintenance immunosuppression for AAV. Since double-positivity appears common, further work is required to define the underlying mechanisms of this association and define optimum treatment strategies. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Double Negative Materials (DNM), Phenomena and Applications

DTIC Science & Technology

2009-07-01

Nanoparticles Formed by Pairs Of Concentric Double-Negative (DNG), Single-Negative ( SNG ) and/or Double-Positive (DPS) Metamaterial Layers.” J. Appl...material RRL Rapid Research Letters SHG second-harmonic generation SNG single-negative SSR split-ring resonator A-1 Appendix A. October 2008...Pairs of Concentric Double-Negative (DNG), Single-Negative ( SNG ), and/or Double-Positive (DPS) Metamaterial Layers.” J. Appl. Phys. 97, no. 9 (May

Depletion of pro-inflammatory CD161(+) double negative (CD3(+)CD4(-)CD8(-)) T cells in AIDS patients is ameliorated by expansion of the γδ T cell population.

PubMed

Singleterry, Will L; Henderson, Harold; Cruse, Julius M

2012-02-01

In this present investigation, flow cytometry was utilized to evaluate 13 healthy controls and 31 HIV-1 infected patients who had advanced to the AIDS stage of infection (CD4 count below 200 cells/mm(3)), for the expression of CD161 on CD3(+) double negative (DN) (CD3(+)CD4(-)CD8(-)) T cells, CD4(+) T cells, CD8(+) T cells and γδ T cells. The observed depletion of CD161(+) T cells from peripheral circulation was due primarily to the loss of CD4(+)CD161(+) T cells; as these cells represented 8.67±0.74% of the total healthy control peripheral T cell population, while the CD4(+)CD161(+) T cells of the AIDS group represented only 3.35±0.41% (p=<0.0001) of the total peripheral T cell population. We have also shown here that the DN T cell population was more than doubled in the AIDS group, with the DN T cell population expanding from 3.29±0.45% of the healthy control peripheral T cell population to 8.64±1.16% (p=0.0001) of the AIDS group peripheral T cell population. By evaluating the expression of CD161 on the surface of the DN T cells we showed that within the healthy control group, 47.4±4.99% of the DN T cells were positive for the expression of CD161, while only 26.4±3.54% (p=0.002) of the AIDS group's DN T cells expressed CD161. Despite CD161 expression being halved on the DN T cells of the AIDS group, when we compared the total peripheral T cell percentage of CD161(+) DN T cells between the healthy control group and the AIDS group, there was no statistical difference. Even though only 26.4% DN T cells within the AIDS group were positive for CD161(+), the overall DN T cell population had expanded to such an extent that there was no statistical difference between the groups with regard to CD161(+) DN T cells as a percentage of the total peripheral T cell population. Furthermore, we showed that within the DN T cell population, there was an approximate 2:1 ratio of γδ to αβ T cells, and this ratio was maintained in both the healthy control group and the AIDS group. While evaluating γδ T cells we also discovered that CD8(+) γδ T cells were expanded from 0.62±.09% of the healthy control peripheral T cell population to 5.01±.88% (p=<0.0001) of the peripheral T cell population of the AIDS group; and that this population of CD8(+) γδ T cells underwent the same reduction in percentage of cells expressing CD161(+), further demonstrated that the phenomenon of CD161(+) percentage reduction and compensatory increase in total cell population was affecting the entire circulating γδ T cell population. Copyright © 2011 Elsevier Inc. All rights reserved.

[Neuroprotective effect of curcumin to Aβ of double transgenic mice with Alzheimer's disease].

PubMed

Feng, Hui-Li; Fan, Hui; Dang, Hui-Zi; Chen, Xiao-Pei; Ren, Ying; Yang, Jin-Duo; Wang, Peng-Wen

2014-10-01

To observe the changes in Aβ40, Aβ42 and ADDLs in brains of 3 month-old APPswe/PS1dE9 double transgenic mice after six-month intervention with curcumin, in order to discuss the neuroprotective effect of curcumin. APPswe/PS1dE9dtg mice were randomly divided into the model group, the Rosiglitazone group (10 mg x kg(-1) x d(-1)) and curcumin high (400 mg x kg9-1) x d(-1)), medium (200 mg x kg(-1) x d(-1)) and low (100 mg x kg(-1) x d(-1)) dosage groups, with C57/BL6J mice of the same age and the same background in the normal control group. After 6 months, the immunohistochemical staining (IHC) and the Western blot method were used to observe the changes in positive cell of Aβ40, Aβ42 and ADDLs in hippocampal CA1 area, their distribution and protein expressions. Both of the immunohistochemical staining and the Western blot method showed more positive cell of Aβ40, Aβ42 and ADDLs in hippocampal CA1 area and higher protein expressions in the model group than the normal group (P < 0.01). IHC showed a lower result in the Rosiglitazone group than the model group (P < 0.05), while Western blot showed a much lower result (P < 0.01). The number of Aβ40, Aβ42 and ADDLs positive cells and the protein expressions decreased in the curcumin high group, the medium group showed a significant decrease (P < 0.01), and the low dose group also showed reductions in the protein expressions of Aβ40 and Aβ42. The six-month intervention with curcumin can significantly reduce the expressions of hippocampal Aβ40, Aβ42 and ADDLs in brains of APPswe/PS1dE9 double transgenic mice. Whether curcumin can impact Aβ cascade reaction by down-regulating expressions of Aβ40, Aβ42 and ADDLs and show the neuroprotective effect needs further studies.

Pasteurella multocida Toxin Manipulates T Cell Differentiation

PubMed Central

Hildebrand, Dagmar; Heeg, Klaus; Kubatzky, Katharina F.

2015-01-01

Pasteurella multocida causes various diseases in a broad range of wild and domestic animals. Toxigenic strains of the serotypes A and D produce an AB protein toxin named Pasteurella multocida toxin (PMT). PMT constitutively activates the heterotrimeric G protein subunits Gαq, Gα13, and Gαi through deamidation of a glutamine residue, which results in cytoskeletal rearrangements as well as increased proliferation and survival of the host cell. In human monocytes, PMT alters the lipopolysaccharide (LPS)-induced activation toward a phenotype that suppresses T cell activation. Here we describe that the toxin also modulates CD4-positive T helper (Th) cells directly. PMT amplifies the expansion of Th cells through enhanced cell cycle progression and suppression of apoptosis and manipulates the differentiation of Th subclasses through activation of Signal Transducers and Activators of Transcription (STAT) family members and induction of subtype-specific master transcription factors. A large population of toxin-treated T cells is double-positive for Foxp3 and RORγt, the transcription factors expressed by Treg and Th17 cells, respectively. This suggests that these cells could have the potential to turn into Th17 cells or suppressive Treg cells. However, in terms of function, the PMT-differentiated cells behave as inflammatory Th17 cells that produce IL-17 and trigger T cell proliferation. PMID:26635744

Functionalized carbon nanotube based hybrid electrochemical capacitors using neutral bromide redox-active electrolyte for enhancing energy density

NASA Astrophysics Data System (ADS)

Tang, Xiaohui; Lui, Yu Hui; Chen, Bolin; Hu, Shan

2017-06-01

A hybrid electrochemical capacitor (EC) with enhanced energy density is realized by integrating functionalized carbon nanotube (FCNT) electrodes with redox-active electrolyte that has a neutral pH value (1 M Na2SO4 and 0.5 M KBr mixed aqueous solution). The negative electrode shows an electric double layer capacitor-type behavior. On the positive electrode, highly reversible Br-/Br3- redox reactions take place, presenting a battery-type behavior, which contributes to increase the capacitance of the hybrid cell. The voltage window of the whole cell is extended up to 1.5 V because of the high over-potentials of oxygen and hydrogen evolution reactions in the neutral electrolyte. Compared with raw CNT, the FCNT has better wettability in the aqueous electrolyte and contributes to increase the electric double layer capacitance of the cell. As a result, the maximum energy density of 28.3 Wh kg-1 is obtained from the hybrid EC at 0.5 A g-1 without sacrificing its power density, which is around 4 times larger than that of the electrical double layer capacitor constructed by FCNT electrodes and 1 M Na2SO4 electrolyte. Moreover, the discharge capacity retained 86.3% of its initial performance after 10000 cycles of galvanostatic charge and discharge test (10 A/g), suggesting its long life cycle even at high current loading.

Facile synthesis of mercaptosuccinic acid-capped CdTe/CdS/ZnS core/double shell quantum dots with improved cell viability on different cancer cells and normal cells

NASA Astrophysics Data System (ADS)

Parani, Sundararajan; Bupesh, Giridharan; Manikandan, Elayaperumal; Pandian, Kannaiyan; Oluwafemi, Oluwatobi Samuel

2016-11-01

Water-soluble, mercaptosuccinic acid (MSA)-capped CdTe/CdS/ZnS core/double shell quantum dots (QDs) were prepared by successive growth of CdS and ZnS shells on the as-synthesized CdTe/CdSthin core/shell quantum dots. The formation of core/double shell structured QDs was investigated by ultraviolet-visible (UV-Vis) absorption and photoluminescence (PL) spectroscopy, PL decay studies, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The core/double shell QDs exhibited good photoluminescence quantum yield (PLQY) which is 70% higher than that of the parent core/shell QDs, and they are stable for months. The average particle size of the core/double shell QDs was ˜3 nm as calculated from the transmission electron microscope (TEM) images. The cytotoxicity of the QDs was evaluated on a variety of cancer cells such as HeLa, MCF-7, A549, and normal Vero cells by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assay. The results showed that core/double shell QDs were less toxic to the cells when compared to the parent core/shell QDs. MCF-7 cells showed proliferation on incubation with QDs, and this is attributed to the metalloestrogenic activity of cadmium ions released from QDs. The core/double shell CdTe/CdS/ZnS (CSS) QDs were conjugated with transferrin and successfully employed for the biolabeling and fluorescent imaging of HeLa cells. These core/double shell QDs are highly promising fluorescent probe for cancer cell labeling and imaging applications.

Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras.

PubMed

Papapetrou, Eirini P; Kovalovsky, Damian; Beloeil, Laurent; Sant'angelo, Derek; Sadelain, Michel

2009-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy.

Leg tendon glands in male bumblebees ( Bombus terrestris): structure, secretion chemistry, and possible functions

NASA Astrophysics Data System (ADS)

Jarau, Stefan; Žáček, Petr; Šobotník, Jan; Vrkoslav, Vladimír; Hadravová, Romana; Coppée, Audrey; Vašíčková, Soňa; Jiroš, Pavel; Valterová, Irena

2012-12-01

Among the large number of exocrine glands described in bees, the tarsal glands were thought to be the source of footprint scent marks. However, recent studies showed that the compounds used for marking by stingless bees are secreted by leg tendon instead of tarsal glands. Here, we report on the structure of leg tendon glands in males of Bombus terrestris, together with a description of the chemical composition of their secretions and respective changes of both during the males' lives. The ultrastructure of leg tendon glands shows that the secretory cells are located in three independent regions, separated from each other by unmodified epidermal cells: in the femur, tibia, and basitarsus. Due to the common site of secretion release, the organ is considered a single secretory gland. The secretion of the leg tendon glands of B. terrestris males differs in its composition from those of workers and queens, in particular by (1) having larger proportions of compounds with longer chain lengths, which we identified as wax esters; and (2) by the lack of certain hydrocarbons (especially long chain dienes). Other differences consist in the distribution of double bond positions in the unsaturated hydrocarbons that are predominantly located at position 9 in males but distributed at seven to nine different positions in the female castes. Double bond positions may change chemical and physical properties of a molecule, which can be recognized by the insects and, thus, may serve to convey specific information. The function of male-specific compounds identified from their tendon glands remains elusive, but several possibilities are discussed.

Expression of GDNF and GFR alpha 1 in mouse taste bud cells.

PubMed

Takeda, Masako; Suzuki, Yuko; Obara, Nobuko; Uchida, Nobuhiko; Kawakoshi, Kentaro

2004-11-01

GDNF (glial cell line-derived neurotrophic factor) affects the survival and maintenance of central and peripheral neurons. Using an immunocytochemical method, we examined whether the taste bud cells in the circumvallate papillae of normal mice expressed GDNF and its GFR alpha 1 receptor. Using double immunostaining for either of them and NCAM, PGP 9.5, or alpha-gustducin, we additionally sought to determine what type of taste bud cells expressed GDNF or GFR alpha 1, because NCAM is reported to be expressed in type-III cells, PGP 9.5, in type-III and some type-II cells, and alpha-gustducin, in some type-II cells. Normal taste bud cells expressed both GDNF and GFR alpha 1. The percentage of GDNF-immunoreactive cells among all taste bud cells was 31.63%, and that of GFR alpha 1-immunoreactive cells, 83.21%. Confocal laser scanning microscopic observations after double immunostaining showed that almost none of the GDNF-immunoreactive cells in the taste buds were reactive with anti-NCAM or anti-PGP 9.5 antibody, but could be stained with anti-alpha-gustducin antibody. On the other hand, almost all anti-PGP 9.5- or anti-alpha-gustducin-immunoreactive cells were positive for GFR alpha 1. Thus, GDNF-immunoreactive cells did not include type-III cells, but type-II cells, which are alpha-gustducin-immunoreactive; on the other hand, GFR alpha 1-immunoreactive cells included type-II and -III cells, and perhaps type-I cells. We conclude that GDNF in the type-II cells may exert trophic actions on type-I, -II, and -III taste bud cells by binding to their GFR alpha 1 receptors.

LAM-1 and FAT Genes Control Development of the Leaf Blade in Nicotiana sylvestris.

PubMed Central

McHale, NA

1993-01-01

Leaf primordia of the lam-1 mutant of Nicotiana sylvestris grow normally in length but remain bladeless throughout development. The blade initiation site is established at the normal time and position in lam-1 primordia. Anticlinal divisions proceed normally in the outer L1 and L2 layers, but the inner L3 cells fail to establish the periclinal divisions that normally generate the middle mesophyll core. The lam-1 mutation also blocks formation of blade mesophyll from distal L2 cells. This suggests that LAM-1 controls a common step in initiation of blade tissue from the L2 and L3 lineage of the primordium. Another recessive mutation (fat) was isolated in N. sylvestris that induces abnormal periclinal divisions in the mesophyll during blade initiation and expansion. This generates a blade approximately twice its normal thickness by doubling the number of mesophyll cell layers from four to approximately eight. Presumably, the fat mutation defines a negative regulator involved in repression of periclinal divisions in the blade. The lam-1 fat double mutant shows radial proliferation of mesophyll cells at the blade initiation site. This produces a highly disorganized, club-shaped blade that appears to represent an additive effect of the lam-1 and fat mutations on blade founder cells. PMID:12271096

Circulating CD4+CD28null and extra-thymic CD4+CD8+ double positive T cells are independently associated with disease damage in systemic lupus erythematosus patients.

PubMed

Ugarte-Gil, M F; Sánchez-Zúñiga, C; Gamboa-Cárdenas, R V; Aliaga-Zamudio, M; Zevallos, F; Tineo-Pozo, G; Cucho-Venegas, J M; Mosqueira-Riveros, A; Medina, M; Perich-Campos, R A; Alfaro-Lozano, J L; Rodriguez-Bellido, Z; Alarcón, G S; Pastor-Asurza, C A

2016-03-01

To determine whether circulating CD4+CD28null and extra-thymic CD4+CD8+ double positive (DP) T cells are independently associated with damage accrual in systemic lupus erythematosus (SLE) patients. This cross-sectional study was conducted between September 2013 and April 2014 in consecutive SLE patients from our Rheumatology Department. CD4+CD28null and CD4+CD8+ DP T-cell frequencies were analyzed by flow-cytometry. The association of damage (SLICC/ACR Damage Index, SDI) and CD4+CD28null and CD4+CD8+ DP T cells was examined by univariable and multivariable Poisson regression models, adjusting for possible confounders. All analyses were performed using SPSS 21.0. Patients' (n = 133) mean (SD) age at diagnosis was 35.5 (16.8) years, 124 (93.2%) were female; all were mestizo (mixed Caucasian and Amerindian ancestry). Disease duration was 7.4 (6.8) years. The SLE Disease Activity Index was 5.5 (4.2), and the SDI 0.9 (1.2). The percentages of CD4+CD28null and CD4+CD8+ DP T cells were 17.1 (14.4) and 0.4 (1.4), respectively. The percentage of CD4+CD28null and CD4+CD8+ DP T cells were positively associated with a higher SDI in both univariable (rate ratio (RR) 1.02, 95% confidence interval (CI): 1.01-1.03 and 1.17, 95% CI: 1.07-1.27, respectively; p < 0.001 for both) and multivariable analyses RR 1.02, 95% CI: 1.01-1.03, p = 0.001 for CD4+CD28null T cells and 1.28, 95% CI: 1.13-1.44, p < 0.001 for CD4+CD8+ DP T cells). Only the renal domain remained associated with CD4+CD28null in multivariable analyses (RR 1.023 (1.002-1.045); p = 0.034). In SLE patients, CD4+CD28null and CD4+CD8+ DP T cells are independently associated with disease damage. Longitudinal studies are warranted to determine the predictive value of these associations. © The Author(s) 2015.

Digestion of peptidoglycan near the cross-link is necessary for the growth of Bacillus subtilis.

PubMed

Hashimoto, Masayuki; Matsushima, Hiroaki; Suparthana, I Putu; Ogasawara, Hiroshi; Yamamoto, Hiroki; Teng, ChingHao; Sekiguchi, Junichi

2018-03-01

Bacterial cells are covered with peptidoglycan (PG) layer(s), serving as the cellular exoskeleton. The PG sacculus changes its shape during cell growth, and thus both the synthesis and disassembly of PG are important for cell proliferation. In Bacillus subtilis, four dl-endopeptidases (DLEPases; LytE, LytF, CwlO and CwlS) are involved in the maintenance of cell morphology. The lytE cwlO double mutant exhibits synthetic lethality and defective cell elongation, while the lytE lytF cwlS triple mutant exhibits defective cell separation, albeit with septum formation. LytE is involved in both cell separation and elongation. We propose that DLEPases have varied roles in cell separation and elongation. To determine these roles, the catalytic domain of LytE was substituted with another catalytic domain that digests the other bonds in PG. By using the chimeric enzymes, we assessed the suppression of the synthetic lethality by the cell elongation defect and the disruption of chain morphology by the cell separation defect. All the constructed chimeric enzymes suppressed the cell separation defect, restoring the chain morphology. Digestion at any position of PG broke the linkage between two daughter cells, releasing them from each other. However, only d,d-endopeptidases suppressed the lack of DLEPase in the lytE cwlO double mutant. This indicated that the release of tension on the expanding PG sacculus is not the sole essential function of DLEPases. Considering that the structure of the digested PG is important for cell elongation, the digested product might be reused in the growth process in some way.

Autophagy Induced by Intracellular Infection of Propionibacterium acnes

PubMed Central

Nakamura, Teruko; Furukawa, Asuka; Uchida, Keisuke; Ogawa, Tomohisa; Tamura, Tomoki; Sakonishi, Daisuke; Wada, Yuriko; Suzuki, Yoshimi; Ishige, Yuki; Minami, Junko; Akashi, Takumi

2016-01-01

Background Sarcoidosis is caused by Th1-type immune responses to unknown agents, and is linked to the infectious agent Propionibacterium acnes. Many strains of P. acnes isolated from sarcoid lesions cause intracellular infection and autophagy may contribute to the pathogenesis of sarcoidosis. We examined whether P. acnes induces autophagy. Methods Three cell lines from macrophages (Raw264.7), mesenchymal cells (MEF), and epithelial cells (HeLa) were infected by viable or heat-killed P. acnes (clinical isolate from sarcoid lymph node) at a multiplicity of infection (MOI) of 100 or 1000 for 1 h. Extracellular bacteria were killed by washing and culturing infected cells with antibiotics. Samples were examined by colony assay, electron-microscopy, and fluorescence-microscopy with anti-LC3 and anti-LAMP1 antibodies. Autophagy-deficient (Atg5-/-) MEF cells were also used. Results Small and large (≥5 μm in diameter) LC3-positive vacuoles containing few or many P. acnes cells (LC3-positive P. acnes) were frequently found in the three cell lines when infected by viable P. acnes at MOI 1000. LC3-positive large vacuoles were mostly LAMP1-positive. A few small LC3-positive/LAMP1-negative vacuoles were consistently observed in some infected cells for 24 h postinfection. The number of LC3-positive P. acnes was decreased at MOI 100 and completely abolished when heat-killed P. acnes was used. LC3-positive P. acnes was not found in autophagy-deficient Atg5-/- cells where the rate of infection was 25.3 and 17.6 times greater than that in wild-type Atg5+/+ cells at 48 h postinfection at MOI 100 and 1000, respectively. Electron-microscopic examination revealed bacterial cells surrounded mostly by a single-membrane including the large vacuoles and sometimes a double or multi-layered membrane, with occasional undigested bacterial cells in ruptured late endosomes or in the cytoplasm. Conclusion Autophagy was induced by intracellular P. acnes infection and contributed to intracellular bacterial killing as an additional host defense mechanism to endocytosis or phagocytosis. PMID:27219015

Activation of the Wnt/{beta}-catenin signaling pathway is associated with glial proliferation in the adult spinal cord of ALS transgenic mice

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

Chen, Yanchun; Department of Histology and Embryology, Shandong University School of Medicine, Jinan, Shandong; Guan, Yingjun, E-mail: guanyj@wfmc.edu.cn

Highlights: Black-Right-Pointing-Pointer Wnt3a and Cyclin D1 were upregulated in the spinal cord of the ALS mice. Black-Right-Pointing-Pointer {beta}-catenin translocated from the cell membrane to the nucleus in the ALS mice. Black-Right-Pointing-Pointer Wnt3a, {beta}-catenin and Cyclin D1 co-localized for astrocytes were all increased. Black-Right-Pointing-Pointer BrdU/Cyclin D1 double-positive cells were increased in the spinal cord of ALS mice. Black-Right-Pointing-Pointer BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. -- Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive and fatal loss of motor neurons. In ALS, there is a significant cell proliferation in response to neurodegeneration; however,more » the exact molecular mechanisms of cell proliferation and differentiation are unclear. The Wnt signaling pathway has been shown to be involved in neurodegenerative processes. Wnt3a, {beta}-catenin, and Cyclin D1 are three key signaling molecules of the Wnt/{beta}-catenin signaling pathway. We determined the expression of Wnt3a, {beta}-catenin, and Cyclin D1 in the adult spinal cord of SOD1{sup G93A} ALS transgenic mice at different stages by RT-PCR, Western blot, and immunofluorescence labeling techniques. We found that the mRNA and protein of Wnt3a and Cyclin D1 in the spinal cord of the ALS mice were upregulated compared to those in wild-type mice. In addition, {beta}-catenin translocated from the cell membrane to the nucleus and subsequently activated transcription of the target gene, Cyclin D1. BrdU and Cyclin D1 double-positive cells were increased in the spinal cord of these mice. Moreover, Wnt3a, {beta}-catenin, and Cyclin D1 were also expressed in both neurons and astrocytes. The expression of Wnt3a, {beta}-catenin or Cyclin D1 in mature GFAP{sup +} astrocytes increased. Moreover, BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. Our findings suggest that neurodegeneration activates the Wnt/{beta}-catenin signaling pathway, which is associated with glial proliferation in the adult spinal cord of ALS transgenic mice. This mechanism may be significant in clinical gene therapy.« less

CD95 (Fas) ligand expression of Epstein-Barr virus (EBV)-infected lymphocytes: a possible mechanism of immune evasion in chronic active EBV infection.

PubMed

Ohshima, K; Suzumiya, J; Sugihara, M; Nagafuchi, S; Ohga, S; Kikuchi, M

1999-01-01

The Epstein-Barr virus (EBV) induces infectious mononucleosis (IM) and can be associated with chronic active EBV infection (CAEBV). Cytotoxic T lymphocytes (CTL) play an important role in excluding EBV-infected cells. Two cytotoxic mechanisms of CTL have been demonstrated: one perforin/granzyme-based and the other Fas (CD95)/Fas ligand (FasL)-based. To clarify these two pathways in CAEBV, we analyzed six patients with CAEBV and four patients with IM using immunohistochemical staining of the lymph nodes. In both CAEBV and IM, CD8+ T-cells increased in number, but CD56+ natural killer cells were rare. In four of six cases with CAEBV, approximately half the lymphocytes were positive for T cell-restricted intracellular antigens (TIA-1), which were recognized by the cytolytic granules of CTL. In IM, the number of TIA-1 positive cells was smaller than that in CAEBV. Fas-positive lymphocytes were frequently encountered in both CAEBV and IM. However, FasL-positive lymphocytes increased in three of six patients with CAEBV, but not in patients with IM. Except for one case with CAEBV, the number of perforin- and/or granzyme-positive cells was small in number in both CAEBV and IM cases. In double-staining FasL and EBV in situ hybridization, FasL-positive EBV-infected lymphocytes were detected in CAEBV but not in IM. In CAEBV, the Fas/FasL pathway and not perforin pathways appears to play an important role in the pathogenesis. The data suggest that EBV-infected lymphocytes may evade immune attack through the expression of FasL.

Double-double bend achromat cell upgrade at the Diamond Light Source: From design to commissioning

NASA Astrophysics Data System (ADS)

Bartolini, R.; Abraham, C.; Apollonio, M.; Bailey, C. P.; Cox, M. P.; Day, A.; Fielder, R. T.; Hammond, N. P.; Heron, M. T.; Holdsworth, R.; Kay, J.; Martin, I. P. S.; Mhaskar, S.; Miller, A.; Pulampong, T.; Rehm, G.; Rial, E. C. M.; Rose, A.; Shahveh, A.; Singh, B.; Thomson, A.; Walker, R. P.

2018-05-01

Diamond has recently successfully commissioned a major change in the lattice consisting of the substitution of a standard double-bend achromat (DBA) cell with a modified four-bend achromat (4BA) cell called "double-double bend achromat" (DDBA). This work stems from the original studies initiated in 2012 towards a Diamond upgrade and provides the benefit of an additional straight section in the ring available for insertion devices. This paper reviews the DDBA design and layout, the implications for technical subsystems, the associated engineering challenges and the main results of the commissioning completed in April 2017.

From coin cells to 400 mAh pouch cells: Enhancing performance of high-capacity lithium-ion cells via modifications in electrode constitution and fabrication

NASA Astrophysics Data System (ADS)

Trask, Stephen E.; Li, Yan; Kubal, Joseph J.; Bettge, Martin; Polzin, Bryant J.; Zhu, Ye; Jansen, Andrew N.; Abraham, Daniel P.

2014-08-01

In this article we describe efforts to improve performance and cycle life of cells containing Li1.2Ni0.15Mn0.55Co0.1O2-based positive and graphite-based negative electrodes. Initial work to identify high-performing materials, compositions, fabrication variables, and cycling conditions is conducted in coin cells. The resulting information is then used for the preparation of double-sided electrodes, assembly of pouch cells, and electrochemical testing. We report the cycling performance of cells with electrodes prepared under various conditions. Our data indicate that cells with positive electrodes containing 92 wt.% Li1.2Ni0.15Mn0.55Co0.1O2, 4 wt.% carbons (no graphite), and 4 wt.% PVdF (92-4-4) show ∼20% capacity fade after 1000 cycles in the 2.5-4.4 V range, significantly better than our baseline cells that show the same fade after only 450 cycles. Our analyses indicate that the major contributors to cell energy fade are capacity loss and impedance rise. Therefore incorporating approaches that minimize capacity fade and impedance rise, such as electrode coatings and electrolyte additives, can significantly enhance calendar and cycle life of this promising cell chemistry.

From coin cells to 400 mAh pouch cells: Enhancing performance of high-capacity lithium-ion cells via modifications in electrode constitution and fabrication

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

Trask, Stephen E.; Li, Yan; Kubal, Joseph J.

2014-08-01

In this article we describe efforts to improve performance and cycle life of cells containing Li1.2Ni0.15Mn0.55Co0.1O2-based positive and graphite-based negative electrodes. Initial work to identify high-performing materials, compositions, fabrication variables, and cycling conditions is conducted in coin cells. The resulting information is then used for the preparation of double-sided electrodes, assembly of pouch cells, and electrochemical testing. We report the cycling performance of cells with electrodes prepared under various conditions. Our data indicate that cells with positive electrodes containing 92 wt% Li1.2Ni0.15Mn0.55Co0.1O2, 4 wt% carbons (no graphite), and 4 wt% PVdF (92-4-4) show ~20% capacity fade after 1000 cycles inmore » the 2.5-4.4V range, significantly better than our baseline cells that show the same fade after only 450 cycles. Our analyses indicate that the major contributors to cell energy fade are capacity loss and impedance rise. Therefore incorporating approaches that minimize capacity fade and impedance rise, such as electrode coatings and electrolyte additives, can significantly enhance calendar and cycle life of this promising cell chemistry.« less

Uneven colonization of the lymphoid periphery by T cells that undergo early TCR{alpha} rearrangements.

PubMed

Hendricks, Deborah W; Fink, Pamela J

2009-04-01

A sparse population of thymocytes undergoes TCRalpha gene rearrangement early in development, before the double-positive stage. The potential of these cells to contribute to the peripheral T cell pool is unknown. To examine the peripheral T cell compartment expressing a repertoire biased to early TCR gene rearrangements, we developed a mouse model in which TCRalpha rearrangements are restricted to the double-negative stage of thymocyte development. These mice carry floxed RAG2 alleles and a Cre transgene driven by the CD4 promoter. As expected, conventional T cell development is compromised in such Cre(+) RAG2(fl/fl) mice, and the TCRalphabeta(+) T cells that develop are limited in their TCRalpha repertoire, preferentially using early rearranging Valpha genes. In the gut, the Thy-1(+)TCRalphabeta(+) intraepithelial lymphocyte (IEL) compartment is surprisingly intact, whereas the Thy-1(-)TCRalphabeta(+) subset is almost completely absent. Thus, T cells expressing a TCRalpha repertoire that is the product of early gene rearrangements can preferentially populate distinct IEL compartments. Despite this capacity, Cre(+) RAG2(fl/fl) T cell progenitors cannot compete with wild-type T cell progenitors in mixed bone marrow chimeras, suggesting that in normal mice, there is only a small contribution to the peripheral T cell pool by cells that have undergone early TCRalpha rearrangements. In the absence of wild-type competitors, aggressive homeostatic proliferation in the IEL compartment can promote a relatively normal Thy-1(+) TCRalphabeta(+) T cell pool from the limited population derived from Cre(+) RAG2(fl/fl) progenitors.

Uneven colonization of the lymphoid periphery by T cells that undergo early TCRα rearrangements1

PubMed Central

Hendricks, Deborah W.; Fink, Pamela J.

2009-01-01

A sparse population of thymocytes undergoes TCRα gene rearrangement early in development, before the double positive stage. The potential of these cells to contribute to the peripheral T cell pool is unknown. To examine the peripheral T cell compartment expressing a repertoire biased to early TCR gene rearrangements, we developed a mouse model in which TCRα rearrangements are restricted to the double negative stage of thymocyte development. These mice carry floxed RAG2 alleles and a Cre transgene driven by the CD4 promoter. As expected, conventional T cell development is compromised in such Cre(+) RAG2fl/fl mice, and the TCRαβ+ T cells that develop are limited in their TCRα repertoire, preferentially utilizing early-rearranging Vα genes. In the gut, the Thy-1+TCRαβ+ intraepithelial lymphocyte (IEL) compartment is surprisingly intact, while the Thy-1−TCRαβ+ subset is almost completely absent. Thus, T cells expressing a TCRα repertoire that is the product of early gene rearrangements can preferentially populate distinct IEL compartments. Despite this capacity, Cre(+) RAG2fl/fl T cell progenitors cannot compete with wild-type (WT) T cell progenitors in mixed bone marrow chimeras, suggesting that in normal mice, there is only a small contribution to the peripheral T cell pool by cells that have undergone early TCRα rearrangements. In the absence of WT competitors, aggressive homeostatic proliferation in the IEL compartment can promote a relatively normal Thy-1+ TCRαβ+ T cell pool from the limited population derived from Cre(+) RAG2fl/fl progenitors. PMID:19299725

In vivo responses of macrophages and perisinusoidal cells to cholestatic liver injury.

PubMed Central

Hines, J. E.; Johnson, S. J.; Burt, A. D.

1993-01-01

We investigated the response of macrophages and perisinusoidal (Ito) cells (PSCs) during the development of secondary biliary cirrhosis after ligation and division of the common bile duct. Liver tissue was obtained from three groups of male Wistar rats: 1) untreated controls (n = 3); 2) common bile duct-ligated (CBDL) animals (n = 15); and 3) sham-operated controls (n = 15). Material from animal groups 2 and 3 was obtained on days 3, 7, 14, 21, and 28 after operation; in all animals 5-bromo-2-deoxyuridine was administered intraperitoneally before death. Monocytes and macrophages were detected using the monoclonal antibody ED1 and tissue macrophages using the antibody ED2. Cell proliferation within the macrophage population was demonstrated by double labeling for ED2 and incorporated 5-bromo-2-deoxyuridine. PSCs were demonstrated in tissue sections by immunolocalization of desmin; proliferating PSCs were identified by double labeling for desmin and incorporated 5-bromo-2-deoxyuridine. Evidence of phenotypic modulation of PSCs was sought using anti-alpha-smooth muscle actin (alpha-SMA) antibody. Increased numbers of ED1- and ED2-positive cells were seen in CBDL animals at all time points. Local proliferation of macrophages could be identified and reached a peak at day 3, thereafter falling toward control values. Compared with those of controls, livers of CBDL animals showed increased numbers of desmin-positive PSCs in periportal zones from day 3 on, reaching a peak at day 14 (127.8 +/- 10.99 cells/0.635 mm2) and followed by a plateau. PSC proliferation peaked at days 3 and 7 (labeling indices 11.2% and 11.2%, respectively) and thereafter fell toward control values; no expansion of the PSC population was seen in sham-operated rats. Increased alpha-SMA-positive cells were also noted from day 3, with a peak at day 21 (231.1 +/- 11.52 cells/0.635 mm2) and followed by a plateau. En face labeling experiments in days 14, 21, and 28 CBDL animals showed cells co-expressing alpha-SMA and desmin and cells expressing alpha-SMA alone. These results indicate that in response to chronic cholestatic liver injury, PSCs proliferate and undergo phenotypic modulation toward "myofibroblast-like" cells. The kinetics of the response are similar to those of the ED2-positive cell population in keeping with a hypothesis that PSC proliferation and activation may be mediated by factors released by macrophages in response to various forms of liver injury. We conclude that the responses of macrophages and PSCs to cholestatic injury are similar to those after toxin-induced hepatocyte necrosis. Images Figure 2 Figure 4 Figure 5 Figure 6 PMID:8434646

The synergistic effects of saxagliptin and metformin on CD34+ endothelial progenitor cells in early type 2 diabetes patients: a randomized clinical trial.

PubMed

Dore, Fiona J; Domingues, Cleyton C; Ahmadi, Neeki; Kundu, Nabanita; Kropotova, Yana; Houston, Sara; Rouphael, Carol; Mammadova, Aytan; Witkin, Linda; Khiyami, Anamil; Amdur, Richard L; Sen, Sabyasachi

2018-05-03

Type 2 diabetes is associated with endothelial dysfunction leading to cardiovascular disease. CD34+ endothelial Progenitor Cells (EPCs) are responsible for endothelial repair and neo-angiogenesis and can be used as a cardiovascular disease risk biomarker. This study investigated whether the addition of saxagliptin, a DPP-IV inhibitor, to metformin, may reduce cardiovascular disease risk in addition to improving glycemic control in Type 2 diabetes patients. In 12 week, double-blind, randomized placebo-controlled trial, 42 subjects already taking metformin 1-2 grams/day were randomized to placebo or saxagliptin 5 mg. Subjects aged 40-70 years with diabetes for < 10 years, with no known cardiovascular disease, BMI 25-39.9, HbA1C 6-9% were included. We evaluated EPCs number, function, surface markers and gene expression, in addition to arterial stiffness, blood biochemistries, resting energy expenditure, and body composition parameters. A mixed model regression to examine saxagliptin vs placebo, accounting for within-subject autocorrelation, was done with SAS (p < 0.05). Although there was no significant increase in CD34+ cell number, CD31+ cells percentage increased. Saxagliptin increased migration (in response to SDF1α) with a trend of higher colony formation count. MNCs cytometry showed higher percentage of CXCR4 double positivity for both CD34 and CD31 positive cells, indicating a functional improvement. Gene expression analysis showed an upregulation in CD34+ cells for antioxidant SOD1 (p < 0.05) and a downregulation in CD34- cells for IL-6 (p < 0.01). For arterial stiffness, both augmentation index and systolic blood pressure measures went down in saxagliptin subjects (p < 0.05). Saxagliptin, in combination with metformin, can help improve endothelial dysfunction in early diabetes before macrovascular complications appear. Trial registration Trial is registered under clinicaltrials.gov, NCT02024477.

Beneficial immunostimulatory effect of short-term Chlorella supplementation: enhancement of Natural Killer cell activity and early inflammatory response (Randomized, double-blinded, placebo-controlled trial)

PubMed Central

2012-01-01

Background In vitro and animal studies have demonstrated that Chlorella is a potent biological response modifier on immunity. However, there were no direct evidences for the effect of Chlorella supplementation on immune/inflammation response in healthy humans. Methods This study was designed for an 8-week randomized, double-blinded, placebo-controlled trial: 5g of Chlorella (n=23) or Placebo (n=28) as form of tablets. Mainly, cytotoxic activities of Natural killer (NK) cells and serum concentrations of interferon-γ, interleukin-1β and interleukin-12 were measured. Results After the 8-week, serum concentrations of interferon-γ (p<0.05) and interleukin-1β (p<0.001) significantly increased and that of interleukin-12 (p<0.1) tended to increase in the Chlorella group. The increments of these cytokines after the intervention were significantly bigger in the Chlorella group than those in the placebo group. In addition, NK cell activities (%) were significantly increased in Chlorella group, but not in Placebo group. The increments of NK cell activities (%) were also significantly bigger in the Chlorella group than the placebo group. Additionally, changed levels of NK cell activity were positively correlated with those of serum interleukin-1β (r=0.280, p=0.047) and interferon-γ (r=0.271, p<0.005). Signficantly positive correlations were also observed among the changed levels of serum cytokines; between interferon-γ and interleukin-1β (r=0.448, p<0.001), between interleukin-12 and interleukin-1β (r=0.416, p=0.003) and between interleukin-12 and interferon-γ (r=0.570, p<001). Conclusion These results may suggest a beneficial immunostimulatory effect of short-term Chlorella supplementation which enhances the NK cell activity and produces interferon-γ and interleukin-12 as well as interleukin-1β, the Th-1 cell-induced cytokines in healthy people. PMID:22849818

The cis/trans isomerization of the double bond of a fatty acid as a strategy for adaptation to changes in ambient temperature in the psychrophilic bacterium, Vibrio sp. strain ABE-1.

PubMed

Okuyama, H; Okajima, N; Sasaki, S; Higashi, S; Murata, N

1991-06-19

The major phospholipid, phosphatidylethanolamine (PE), of Vibrio sp. strain ABE-1 contains a unique trans-unsaturated fatty acid, 9-trans-hexadecenoic acid (16:1(9t], at the sn-2 position of the glycerol moiety. The major molecular species of PE that contain 16:1(9t) at the sn-2 position have either 9-cis-hexadecenoic acid (16:1(9c] or hexadecanoic acid (16:0) at the sn-1 position. The transition temperatures of the liquid-crystal to the gel phase of 16:1(9c)/16:1(9t)-PE and 16:0/16:1(9t)-PE were -3 degrees C and 38 degrees C, respectively, temperatures that were 31 degrees C and 18 degrees C higher than the corresponding temperatures for 16:1(9c)/16:1(9c)-PE and 16:0/16:1(9c)-PE. The proportion of 16:1(9c)/16:1(9t)-PE and 16:0/16:1(9t)-PE increased significantly in cells grown at 20 degrees C over that in cells grown at 5 degrees C. When cells grown at 5 degrees C were incubated at 20 degrees C in the presence of cerulenin, an inhibitor of the synthesis de novo of fatty acids, the level of 16:1(9t) increased almost in parallel with a concomitant decrease in the level of 16:1(9c) at the sn-2 position. These results suggest that 16:1(9c) is converted to 16:1(9t) by the cis/trans isomerization of the double bond in the fatty acid. This conversion is discussed as a possible strategy for adaptation by bacteria to changes in temperature.

K-RasG12D–induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to γ-secretase inhibitors

PubMed Central

Cornejo, Melanie G.; Scholl, Claudia; Liu, Jianing; Leeman, Dena S.; Haydu, J. Erika; Fröhling, Stefan; Lee, Benjamin H.; Gilliland, D. Gary

2008-01-01

To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-RasG12D murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to γ-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways. PMID:18663146

Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas.

PubMed

Lee, Hyung-Ok; He, Xiao; Mookerjee-Basu, Jayati; Zhongping, Dai; Hua, Xiang; Nicolas, Emmanuelle; Sulis, Maria Luisa; Ferrando, Adolfo A; Testa, Joseph R; Kappes, Dietmar J

2015-06-23

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell-specific ThPOK transgene (ThPOK(const) mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αβTCR antibody into ThPOK(const) RAG-deficient mice, which promotes development to the CD4(+)8(+) (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

Multiple-reflection optical gas cell

DOEpatents

Matthews, Thomas G.

1983-01-01

A multiple-reflection optical cell for Raman or fluorescence gas analysis consists of two spherical mirrors positioned transverse to a multiple-pass laser cell in a confronting plane-parallel alignment. The two mirrors are of equal diameter but possess different radii of curvature. The spacing between the mirrors is uniform and less than half of the radius of curvature of either mirror. The mirror of greater curvature possesses a small circular portal in its center which is the effective point source for conventional F1 double lens collection optics of a monochromator-detection system. Gas to be analyzed is flowed into the cell and irradiated by a multiply-reflected composite laser beam centered between the mirrors of the cell. Raman or fluorescence radiation originating from a large volume within the cell is (1) collected via multiple reflections with the cell mirrors, (2) partially collimated and (3) directed through the cell portal in a geometric array compatible with F1 collection optics.

Thymic hormone-containing cells. Characterization and localization of serum thymic factor in young mouse thymus studied by monoclonal antibodies

PubMed Central

1982-01-01

The characterization and distribution of cells containing the serum thymic factor (FTS) in the thymus of young mice was studied by immunofluorescence using monoclonal anti-FTS antibodies. FTS+ cells were distributed throughout the thymic parenchyma but were more frequent in the medullary region than in the cortex. FTS-containing cells presented a stellate or globular aspect, and some of them exhibited fluorescent cytoplasmic granules. The epithelial nature of FTS+ cells was confirmed by double-labeling experiments using an anti- keratin antiserum (as an epithelial cell marker). Nevertheless, only a minority of keratin-positive epithelial reticular cells contained FTS. All controls, including the incubation of sections from nonthymic tissues with the anti-FTS antibodies, were negative. Taken together, these results confirm the exclusive localization of FTS-containing cells within the mouse thymus. PMID:7047671

Circulating anti-double-stranded DNA antibody-secreting cells in patients with systemic lupus erythematosus: a novel biomarker for disease activity.

PubMed

Hanaoka, H; Okazaki, Y; Satoh, T; Kaneko, Y; Yasuoka, H; Seta, N; Kuwana, M

2012-10-01

Antibodies against double-stranded DNA (dsDNA) are widely used to diagnose systemic lupus erythematosus (SLE) and evaluate its activity in patients. This study was undertaken to examine the clinical utility of circulating anti-dsDNA antibody-secreting cells for evaluating SLE patients. Anti-dsDNA antibody-secreting cells quantified using an enzyme-linked immunospot assay were detected in the spleen, bone marrow and peripheral blood from MRL/lpr but not in control BALB/c mice. Circulating anti-dsDNA antibody-secreting cells were detected in 29 (22%) of 130 patients with SLE, but in none of 49 with non-SLE connective-tissue disease or 18 healthy controls. The presence of circulating anti-dsDNA antibody-secreting cells was associated with persistent proteinuria, high SLE disease activity index and systemic lupus activity measures, and a high serum anti-dsDNA antibody titre measured with an enzyme-linked immunosorbent assay. The positive predictive value for active disease was 48% for circulating anti-dsDNA antibody-secreting cells versus 17% for serum anti-dsDNA antibodies. A prospective cohort of patients with circulating anti-dsDNA antibodies and inactive SLE showed that the cumulative disease flare-free rate was significantly lower in patients with than without circulating anti-dsDNA antibody-secreting cells (p < 0.001). Circulating anti-dsDNA antibody-secreting cells are a useful biomarker for assessing disease activity in SLE patients.

Radioimmunotherapy of pancreatic cancer xenografts in nude mice using 90Y-labeled anti-α6β4 integrin antibody

PubMed Central

Aung, Winn; Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Ukai, Yoshinori; Kouda, Katsushi; Kurosawa, Yoshikazu; Furukawa, Takako; Saga, Tsuneo

2016-01-01

The contribution of integrin α6β4 (α6β4) overexpression to the pancreatic cancer invasion and metastasis has been previously shown. We have reported immunotargeting of α6β4 for radionuclide-based and near-infrared fluorescence imaging in a pancreatic cancer model. In this study, we prepared yttrium-90 labeled anti-α6β4 antibody (90Y-ITGA6B4) and evaluated its radioimmunotherapeutic efficacy against pancreatic cancer xenografts in nude mice. Mice bearing xenograft tumors were randomly divided into 5 groups: (1) single administration of 90Y-ITGA6B4 (3.7MBq), (2) double administrations of 90Y-ITGA6B4 with once-weekly schedule (3.7MBq × 2), (3) single administration of unlabeled ITGA6B4, (4) double administrations of unlabeled ITGA6B4 with once-weekly schedule and (5) the untreated control. Biweekly tumor volume measurements and immunohistochemical analyses of tumors at 2 days post-administration were performed to monitor the response to treatments. To assess the toxicity, body weight was measured biweekly. Additionally, at 27 days post-administration, blood samples were collected through cardiac puncture, and hematological parameters, hepatic and renal functions were analyzed. Both 90Y-ITGA6B4 treatment groups showed reduction in tumor volumes (P < 0.04), decreased cell proliferation marker Ki-67-positive cells and increased DNA damage marker p-H2AX-positive cells, compared with the other groups. Mice treated with double administrations of 90Y-ITGA6B4, exhibited myelosuppression. There were no significant differences in hepatic and renal functions between the 2 treatment groups and the other groups. Our results suggest that 90Y-ITGA6B4 is a promising radioimmunotherapeutic agent against α6β4 overexpressing tumors. In the future studies, dose adjustment for fractionated RIT should be considered carefully in order to get the optimal effect while avoiding myelotoxicity. PMID:27246980

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

Neft, R.E.; Tierney, L.A.; Belinsky, S.A.

Molecular and immunological techniques may enhance the usefulness of sputum cytology as a screening tool for lung cancer. These techniques may also be useful in detecting and following the early progression of disease from metaplasia to dysplasia, carcinoma in situ, and finally to invasive carcinoma. Longitudinal information on the evolution of these malignant changes in the respiratory epithelium can be gained by prospective study of populations at high risk for lung cancer. This work is significant because double-labeling of cells in sputum with p53 and cytokeratin antibodies facilitates rapid screening of p53 positive neoplastic and preneoplastic lung cells by brightfieldmore » and fluorescence microscopy.« less

Lymphatic and blood vessels in male breast cancer.

PubMed

Niemiec, Joanna; Sas-Korczynska, Beata; Harazin-Lechowska, Agnieszka; Martynow, Dariusz; Adamczyk, Agnieszka

2015-02-01

It is assumed that lymphatic vessels are responsible for breast cancer dissemination. In 32 male breast carcinomas we evaluated the correlation between: (i) lymphatic vessel density (LVD), distribution of podoplanin-immunostained vessels (DPV), blood vessel density (BVD), infiltration of immune cells and (ii) known clinicopathological parameters. Lymphatic and blood vessels were found in 77.8% and 100% of breast carcinomas, respectively. Double-negative estrogen and progesterone receptor tumors (ER-/PR-) presented significantly higher LVD than ER/PR positive cases, while high-grade tumors exhibited significantly higher DPV than low-grade carcinomas. We detected significantly higher frequency of vascular invasion in high-grade and double-negative carcinomas than in low-grade and ER/PR-positive ones, respectively. The relationship between high number of lymphatic vessels and high tumor grade or steroid receptor negativity might confirm the hypothesis regarding the influence of lymphangiogenesis on the formation of a more aggressive phenotype in male breast cancer. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Phenotypes and Karyotypes of Human Malignant Mesothelioma Cell Lines

PubMed Central

Relan, Vandana; Morrison, Leanne; Parsonson, Kylie; Clarke, Belinda E.; Duhig, Edwina E.; Windsor, Morgan N.; Matar, Kevin S.; Naidoo, Rishendran; Passmore, Linda; McCaul, Elizabeth; Courtney, Deborah; Yang, Ian A.; Fong, Kwun M.; Bowman, Rayleen V.

2013-01-01

Background Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. Methods Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM) and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. Results Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30–72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5–17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. Conclusion These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of mesothelioma during maintenance in artificial culture systems. These characteristics support their potential as in vitro model systems for studying cellular, molecular and genetic aspects of mesothelioma. PMID:23516439

Chromosome territories reposition during DNA damage-repair response

PubMed Central

2013-01-01

Background Local higher-order chromatin structure, dynamics and composition of the DNA are known to determine double-strand break frequencies and the efficiency of repair. However, how DNA damage response affects the spatial organization of chromosome territories is still unexplored. Results Our report investigates the effect of DNA damage on the spatial organization of chromosome territories within interphase nuclei of human cells. We show that DNA damage induces a large-scale spatial repositioning of chromosome territories that are relatively gene dense. This response is dose dependent, and involves territories moving from the nuclear interior to the periphery and vice versa. Furthermore, we have found that chromosome territory repositioning is contingent upon double-strand break recognition and damage sensing. Importantly, our results suggest that this is a reversible process where, following repair, chromosome territories re-occupy positions similar to those in undamaged control cells. Conclusions Thus, our report for the first time highlights DNA damage-dependent spatial reorganization of whole chromosomes, which might be an integral aspect of cellular damage response. PMID:24330859

Multihormonal pituitary adenoma concomitant with Pit-1 and Tpit lineage cells causing acromegaly associated with subclinical Cushing's disease: a case report.

PubMed

Takiguchi, Tomoko; Koide, Hisashi; Nagano, Hidekazu; Nakayama, Akitoshi; Fujimoto, Masanori; Tamura, Ai; Komai, Eri; Shiga, Akina; Kono, Takashi; Higuchi, Seiichiro; Sakuma, Ikki; Hashimoto, Naoko; Suzuki, Sawako; Miyabayashi, Yui; Ishiwatari, Norio; Horiguchi, Kentaro; Nakatani, Yukio; Yokote, Koutaro; Tanaka, Tomoaki

2017-09-02

A functional pituitary adenoma can produce multiple anterior-pituitary hormones, such as growth hormone (GH) -producing adenomas (GHoma) with prolactin or thyrotropin stimulating hormone production in the same lineage. However, it is very rare that acromegaly shows subclinical Cushing's disease (SCD) beyond the lineage. Here we describe the involvement of intratumoral coexistence with 2 types of hormone-producing cells associated with different lineage in acromegaly concomitant with SCD. In our study, we performed clinical evaluation of the patient showing acromegaly with SCD. To elucidate the mechanisms of this pathology, we analyzed immunohistochemistry and gene expression of anterior-pituitary hormones and transcriptional factors in the resected pituitary tumor. On immunohistochemical staining, most of the tumor cells were strongly stained for GH antibody, while some cells were strongly positive for adrenocorticotropic hormone (ACTH). Gene expression analysis of a transsphenoidal surgery sample of the pituitary gland revealed that ACTH-related genes, such as POMC, Tpit, and NeuroD1 mRNA, had higher expression in the tumor tissue than the nonfunctional adenoma but lower expression compared to an adenoma of typical Cushing's disease. Further, double-labeling detection methods with a fluorescent stain for ACTH and GH demonstrated the coexistence of ACTH-positive cells (GH-negative) among the GH-positive cells in the tumor. Additionally, Pit-1 expression was reduced in the ACTH-positive cells from tumor tissue primary culture. Here we described a case of a pituitary tumor diagnosed with acromegaly associated with SCD. We performed quantitative-expression analyses of transcriptional factors of the tumor tissue and immunohistochemistry analysis of tumor-derived primary culture cells, which suggested that the multihormonal pituitary adenoma concomitant with Pit-1 and Tpit lineage cells caused acromegaly associated with SCD.

Clinical Significance of "Double-hit" and "Double-protein" expression in Primary Gastric B-cell Lymphomas.

PubMed

He, Miaoxia; Chen, Keting; Li, Suhong; Zhang, Shimin; Zheng, Jianming; Hu, Xiaoxia; Gao, Lei; Chen, Jie; Song, Xianmin; Zhang, Weiping; Wang, Jianmin; Yang, Jianmin

2016-01-01

Primary gastric B-cell lymphoma is the second most common malignancy of the stomach. There are many controversial issues about its diagnosis, treatment and clinical management. "Double-hit" and "double-protein" involving gene rearrangement and protein expression of c-Myc and bcl2/bcl6 are the most used terms to describe DLBCL poor prognostic factors in recent years. However, very little is known about the role of these prognostic factors in primary gastric B-cell lymphomas. This study aims to obtain a molecular pathology prognostic model of gastric B-cell lymphoma for clinical stratified management by evaluating how the "double-hit" and "double-protein" in tumor cells as well as microenvironmental reaction of tumor stromal tissue affect clinical outcome in primary gastric B-cell lymphomas. Data and tissues of 188 cases diagnosed with gastric B-cell lymphomas were used in this study. Tumor tissue microarray (TMA) of formalin fixed and paraffin embedded (FFPE) tissues was constructed for fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analysis with a serial of biomarkers containing MYC, BCL2, BCL6, CD31, SPARC, CD10, MUM1 and Ki-67. Modeled period analysis was used to estimate 3-year and 5-year overall survival (OS) and disease-free survival (DFS) distributions. There was no definite "double-hit" case though the gene rearrangement of c-Myc (5.9%), bcl2 (0.1%) and bcl6 (7.4%) was found in gastric B-cell lymphomas. The gene amplification or copy gains of c-Myc (10.1%), bcl-2 (17.0%) and bcl-6 (0.9%) were present in these lymphomas. There were 12 cases of the lymphomas with the "double-protein" expression of MYC and BCL2/BCL6. All patients with "double-protein" gastric B-cell lymphomas had poor outcome compared with those without. More importantly, "MYC-BCL2-BCL6" negative group of gastric B-cell lymphoma patients had favorable clinical outcome regardless clinical stage, pathological types and therapeutic modalities. And the similar better prognosis was found in the cases with low microvessel density (MVD) in tumor tissue and high expression of SPARC (SPARC≥5%) in stromal cells. "Double-hit" lymphoma was rare among primary gastric lymphoma, while patients with multiple gene amplification and/or copy gains of c-Myc, bcl2 and bcl6, and "double-protein" gastric B-cell lymphomas had a poor clinical outcome. In addition, patients with MYC, BCL2 and BCL6 expression negative or low MVD in tumor tissue with high expression of SPARC in stromal cells could have better prognosis than other gastric B-cell lymphomas regardless of their clinical stage and pathological types. These results would be of very importance for clinical stratified management and precision medicine of gastric B-cell lymphomas.

Espin cytoskeletal proteins in the sensory cells of rodent taste buds

PubMed Central

Sekerková, Gabriella; Freeman, David; Mugnaini, Enrico; Bartles, James R.

2010-01-01

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste bud cells contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of rat circumvallate taste buds. In confocal images, we counted 21.5±0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7±1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP3R3),α-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP3R3, PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP3R3, α-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of ~3 μm-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10-12 μm into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells. PMID:16841162

BI-31ANALYSIS AND QUANTIFICATION OF MULTIPLE ANTIGEN EXPRESSION IN GLIOBLASTOMA

PubMed Central

Weng, Lihong; Zhai, Yubo; D'Apuzzo, Massimo; Badie, Behnam; Forman, Stephen J.; Barish, Michael; Brown, Christine E.

2014-01-01

Glioblastoma (GBM), one of the most common and fatal types of brain tumor, is marked by significant antigenic heterogeneity. Identification and quantification of tumor related antigens in the context of GBM tissue is an essential step towards developing antigen- targeted therapies. Immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) clinical specimens is a valuable technique for evaluating antigen expression in large study cohorts. To overcome the limitations of manual semi-quantitative scoring and subjectivity in the evaluation of IHC staining, we analyzed and quantified multiple antigens across entire tumor sections using Image Pro Premier v9.1 (DAB plug-in). For each slide, dense tumor regions (DTRs, tumor cells >60%), tumor infiltration regions (TIRs, tumor cells <50%) and pseudopalisading necrosis regions (PPNs) were defined from HE section by a neuropathologist. We quantified the expression of tumor-associated antigens IL13Rα2, HER2, EGFR and the proliferation marker Ki67 within these defined regions for 44 brain tumor samples (35 stage IV and 9 stage III). Our results demonstrate the heterogeneous expression patterns of IL13Rα2, HER2 and EGFR in GBMs. For example, in dense tumor regions 52%, 61% and 77% of samples showed IL13Rα2, HER2 or EGFR positivity, respectively. In correlation studies, 25% of samples were triple positive, 11% of samples showed double positivity for IL13Rα2 and HER2 or IL13Rα2 and EGFR, and 25% of samples were double positive of EGFR and HER2. Less than 7% of samples were negative for all three antigens. Interestingly, a higher percentage of samples showed triple positive expression in PPN regions (43%) versus the DTR (25%) and TIR (25%) regions. Also, Ki67 positivity was higher in PPN and DTR regions. In this study we developed methods for combining pathological annotations with DAB-capturing software, which allowed us to quantify protein expression in a more precise, consistent and efficient manner.

Double-hit lymphomas constitute a highly aggressive subgroup in diffuse large B-cell lymphomas in the era of rituximab.

PubMed

Kobayashi, Tsutomu; Tsutsumi, Yasuhiko; Sakamoto, Natsumi; Nagoshi, Hisao; Yamamoto-Sugitani, Mio; Shimura, Yuji; Mizutani, Shinsuke; Matsumoto, Yosuke; Nishida, Kazuhiro; Horiike, Shigeo; Asano, Naoko; Nakamura, Shigeo; Kuroda, Junya; Taniwaki, Masafumi

2012-11-01

The incorporation of rituximab in immunochemotherapy has improved treatment outcomes for diffuse large B-cell lymphoma, but the prognosis for some diffuse large B-cell lymphomas remains dismal. Identification of adverse prognostic subgroups is essential for the choice of appropriate therapeutic strategy. We retrospectively investigated the impact of so-called 'double-hit' cytogenetic abnormalities, i.e. cytogenetic abnormalities involving c-MYC co-existing with other poor prognostic cytogenetic abnormalities involving BCL2, BCL6 or BACH2, on treatment outcomes for 93 consecutive diffuse large B-cell lymphoma patients. According to the revised international prognostic index, no patients were cytogenetically diagnosed with double-hit lymphomas in the 'very good' risk group or in the 'good' risk group, while 5 of 33 patients had double-hit lymphomas in the 'poor' risk group. All the double-hit lymphoma patients possessed both nodal and extranodal involvement. The overall complete response rate was 89.3%, overall survival 87.1% and progression-free survival 75.8% over 2 years (median observation period: 644 days). The complete response rates were 93.2% for the non-double-hit lymphoma patients and 40.0% for the double-hit lymphoma patients. Significantly longer progression-free survival and overall survival were observed for the 'very good' and the 'good' risk patients than for the 'poor' risk patients. Moreover, the progression-free survival of double-hit lymphoma was significantly shorter than that of the non-double-hit lymphoma 'poor' risk patients (P = 0.016). In addition, the overall survival of the double-hit lymphoma patients also tended to be shorter than that of the non-double-hit lymphoma 'poor' risk group. The diagnosis of double-hit lymphoma can help discriminate a subgroup of highly aggressive diffuse large B-cell lymphomas and indicate the need for the development of novel therapeutic strategies for double-hit lymphoma.

Discovery of melanocortin ligands via a double simultaneous substitution strategy based on the Ac-His-DPhe-Arg-Trp-NH2 template.

PubMed

Todorovic, Aleksandar; Lensing, Cody J; Holder, Jerry Ryan; Scott, Joseph W; Sorensen, Nicholas B; Haskell-Luevano, Carrie

2018-05-21

The melanocortin system regulates an array of diverse physiological functions including pigmentation, feeding behavior, energy homeostasis, cardiovascular regulation, sexual function, and steroidogenesis. Endogenous melanocortin agonist ligands all possess the minimal messaging tetrapeptide sequence His-Phe-Arg-Trp. Based on this endogenous sequence, the Ac-His1-DPhe2-Arg3-Trp4-NH 2 tetrapeptide has previously been shown to be a useful scaffold when utilizing traditional positional scanning approaches to modify activity at the various melanocortin receptors (MC1-5R). The study reported herein was undertaken to evaluate a double simultaneous substitution strategy as an approach to further diversify the Ac-His1-DPhe2-Arg3-Trp4-NH 2 tetrapeptide with concurrent introduction of natural and unnatural amino acids at positions 1, 2, or 4 as well as an octanoyl residue at the N-terminus. The designed library includes the following combinations: (A) double simultaneous substitution at capping group position (Ac) together with position 1, 2, or 4, (B) double simultaneous substitution at position 1 and 2, (C) double simultaneous substitution at position 1 and 4, and (D) double simultaneous substitution at position 2 and 4. Several lead ligands with unique pharmacologies were discovered in the current study including antagonists targeting the neuronal mMC3R with minimal agonist activity and ligands with selective profiles for the various melanocortin subtypes. The results suggest that the double simultaneous substitution strategy is a suitable approach in altering melanocortin receptor potency, selectivity, or converting agonists into antagonists and vice versa.

CD22 x Siglec-G double-deficient mice have massively increased B1 cell numbers and develop systemic autoimmunity.

PubMed

Jellusova, Julia; Wellmann, Ute; Amann, Kerstin; Winkler, Thomas H; Nitschke, Lars

2010-04-01

CD22 and Siglec-G are inhibitory coreceptors for BCR-mediated signaling. Although CD22-deficient mice show increased calcium signaling in their conventional B2 cells and a quite normal B cell maturation, Siglec-G-deficient mice have increased calcium mobilization just in B1 cells and show a large expansion of the B1 cell population. Neither CD22-deficient, nor Siglec-G-deficient mice on a pure C57BL/6 or BALB/c background, respectively, develop autoimmunity. Using Siglec-G x CD22 double-deficient mice, we addressed whether Siglec-G and CD22 have redundant functions. Siglec-G x CD22 double-deficient mice show elevated calcium responses in both B1 cells and B2 cells, increased serum IgM levels and an enlarged population of B1 cells. The enlargement of B1 cell numbers is even higher than in Siglecg(-/-) mice. This expansion seems to happen at the expense of B2 cells, which are reduced in absolute cell numbers, but show an activated phenotype. Furthermore, Siglec-G x CD22 double-deficient mice show a diminished immune response to both thymus-dependent and thymus-independent type II Ags. In contrast, B cells from Siglec-G x CD22 double-deficient mice exhibit a hyperproliferative response to stimulation with several TLR ligands. Aged Siglec-G x CD22 double-deficient mice spontaneously develop anti-DNA and antinuclear autoantibodies. These resulted in a moderate form of immune complex glomerulonephritis. These results show that Siglec-G and CD22 have partly compensatory functions and together are crucial in maintaining the B cell tolerance.

The double universal joint wrist on a manipulator: Solution of inverse position kinematics and singularity analysis

NASA Technical Reports Server (NTRS)

Williams, Robert L., III

1992-01-01

This paper presents three methods to solve the inverse position kinematics position problem of the double universal joint attached to a manipulator: (1) an analytical solution for two specific cases; (2) an approximate closed form solution based on ignoring the wrist offset; and (3) an iterative method which repeats closed form position and orientation calculations until the solution is achieved. Several manipulators are used to demonstrate the solution methods: cartesian, cylindrical, spherical, and an anthropomorphic articulated arm, based on the Flight Telerobotic Servicer (FTS) arm. A singularity analysis is presented for the double universal joint wrist attached to the above manipulator arms. While the double universal joint wrist standing alone is singularity-free in orientation, the singularity analysis indicates the presence of coupled position/orientation singularities of the spherical and articulated manipulators with the wrist. The cartesian and cylindrical manipulators with the double universal joint wrist were found to be singularity-free. The methods of this paper can be implemented in a real-time controller for manipulators with the double universal joint wrist. Such mechanically dextrous systems could be used in telerobotic and industrial applications, but further work is required to avoid the singularities.

Thymus-directed immunotoxicity of airborne dust particles from Upper Silesia (Poland) under acute extrapulmonary studies in mice

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

Kozlowska, E.; Krzystniak, K.; Drela, N.

1996-12-27

Industrial air pollutants from Upper Silesia, Poland, contain over 250 polycyclic and heterocyclic aromatic hydrocarbons and heavy metals, including mutagenic and carcinogenic chemicals that have been shown to from DNA adducts. Over 4 million habitants of Silesia are permanently exposed to the industrial pollution by pulmonary and dermal routes and by contaminated food and water. These chemicals, when examined separately in animals models, were proven immunotoxic. We studied the extrapulmonary immunotoxic potential of a typical mixture of Silesian filter-suspended matter from a selected area, over a specific season and time period. Early changes in the immune system were analyzed inmore » BALB/c mice exposed ip to acute doses of 20-330 mg dust mixture/kg body weight (0.06-1.0 LD50). No major changes were noted for weight and the cellularity of spleen, liver and kidneys. However, dramatic decrease in thymus weight index and thymocyte cell count were noted as early as 24-72 h postexposure, which correlated with almost complete depletion of immature, double-positive CD4{sup +}CD8{sup +} thymocytes. Changes in spleen were less profound; however, increased depletion of B cells over T cells was noted at high doses of the suspended matter. Exposure to the airborne dust also decreased cytokine production by spleen cells, such as interferon-{gamma} (IFN-{gamma}) and tumor necrosis factor-{alpha} (TNF-{alpha}). Overall, a single exposure to Silesian dust, even at the relatively low 0.06 LD50 dose, affected lymphokine production, suppressed B-cell proliferative response, and depleted thymuses of immature, double-positive CD4{sup +}CD8{sup +} cells. A chemical synergism is suspected. To our knowledge, none of the known components of Silesian suspended matter, when examined as a single chemical, was shown to exert such a profound biological effect. 32 refs., 5 figs.« less

Environmental factor and inflammation-driven alteration of the total peripheral T-cell compartment in granulomatosis with polyangiitis.

PubMed

Kerstein, Anja; Schüler, Silke; Cabral-Marques, Otávio; Fazio, Juliane; Häsler, Robert; Müller, Antje; Pitann, Silke; Moosig, Frank; Klapa, Sebastian; Haas, Christian; Kabelitz, Dieter; Riemekasten, Gabriela; Wolters, Steffen; Lamprecht, Peter

2017-03-01

Autoimmune diseases are initiated by a combination of predisposing genetic and environmental factors resulting in self-perpetuating chronic inflammation and tissue damage. Autoantibody production and an imbalance of effector and regulatory T-cells are hallmarks of autoimmune dysregulation. While expansion of circulating effector memory T-cells is linked to disease pathogenesis and progression, the causes driving alterations of the peripheral T-cell compartment have remained poorly understood so far. In granulomatosis with polyangiitis (GPA), a prototypical autoimmune disorder of unknown aetiology, we performed for the first time a combined approach using phenotyping, transcriptome and functional analyses of T-cell populations to evaluate triggers of memory T-cell expansion. In more detail, we found increased percentages of circulating CD4+CD28-, CD8+CD28- and CD4+CD161+ single-positive and CD4+CD8+ double-positive T-cells in GPA. Transcriptomic profiling of sorted T-cell populations showed major differences between GPA and healthy controls reflecting antigen- (bacteria, viruses, fungi) and cytokine-driven impact on T-cell populations in GPA. Concomitant cytomegalovirus (CMV) and Epstein-Barr virus (EBV) - positivity was associated with a significant increase in the percentage of CD28- T-cells in GPA-patients compared to sole CMV- or EBV-positivity or CMV- and EBV-negativity. T-cells specific for other viruses (influenza A virus, metapneumovirus, respiratory syncytial virus) and the autoantigen proteinase 3 (PR3) were infrequently detected in GPA. Antigen-specific T-cells were not specifically enriched in any of the T-cell subsets. Altogether, on a genetic and cellular basis, here we show that alterations of the peripheral T-cell compartment are driven by inflammation and various environmental factors including concomitant CMV and EBV infection. Our study provides novel insights into mechanisms driving autoimmune disease and on potential therapeutic targets. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

[First attempts of detecting fetal cells in the maternal circulation].

PubMed

Nagy, Gyula Richárd; Bán, Zoltán; Sipos, Ferenc; Fent, János; Oroszné Nagy, Judit; Beke, Artúr; Furész, József; Papp, Zoltán

2004-10-31

In prenatal diagnosis there is great interest for noninvasive diagnostic methods. Authors report their first results in detecting fetal cells in the maternal circulation during pregnancy. The aim of the study was to detect fetal gender from maternal peripheral blood samples during pregnancy. Authors have analysed fetal nucleated red blood cells. In 12 cases after a double density Percoll gradient separation they labelled the surface antigens of the cells with anti-glycophorin-A and anti-CD45 fluorescent antibodies, did an intracellular staining of the epsilon haemoglobin chain, and analysed the cells with flow cytometry. The CD45 negative/glycophorin-A positive/epsilon-haemoglobin chain positive cells were considered as fetal cells. Having the results, in another 13 cases magnetic activated cell sorting with CD71 antibody were used as an enrichment step. Authors made an intracellular staining of the epsilon haemoglobin chain, the positive cells were isolated by micromanipulation, and analysed by single cell fluorescent polymerase chain reaction. Primers for the amelogenin gene were used to detect fetal gender. Only the Percoll enrichment step itself is not enough for using the samples for diagnostic molecular-biologic examinations, a following enrichment step is needed. For this the authors used magnetic activated cell sorting with CD71 antibody. With the help of this enrichment step, after the intracellular staining of the epsilon haemoglobin chain the direct micromanipulator isolation of the epsilon haemoglobin chain positive cells could be done. After analysing single cells by fluorescent polymerase chain reaction, in 8 out of the 11 comparable cases the results were similar to those, what was found during the genetic amniocentesis. In 2 cases from this 8, genetic amniocentesis proved Klinefelter syndrome, which they could also confirm with the examination of fetal cells in the maternal circulation. The results of the study suggest that the method described above can be useful in prenatal genetic diagnosis, and improving it could be useful to detect other genetic abnormalities (chromosomal abnormalities, single gene disorders) as well.

Localization of acyl ghrelin- and des-acyl ghrelin-immunoreactive cells in the rat stomach and their responses to intragastric pH.

PubMed

Mizutani, Makoto; Atsuchi, Kaori; Asakawa, Akihiro; Matsuda, Norifumi; Fujimura, Masaki; Inui, Akio; Kato, Ikuo; Fujimiya, Mineko

2009-11-01

Acyl ghrelin has a 28-amino acid sequence with O-n-octanoyl acid modification at the serine 3 position, whereas des-acyl ghrelin has no octanoyl acid modification. Although these peptides exert different physiological functions, no previous studies have shown the different localization of acyl ghrelin and des-acyl ghrelin in the stomach. Here we have developed an antibody specific for des-acyl ghrelin that does not crossreact with acyl ghrelin. Both acyl ghrelin- and des-acyl ghrelin-immunoreactive cells were distributed in the oxyntic and antral mucosa of the rat stomach, with higher density in the antral mucosa than oxyntic mucosa. Immunofluorescence double staining showed that acyl ghrelin- and des-acyl ghrelin-positive reactions overlapped in closed-type round cells, whereas des-acyl ghrelin-positive reaction was found in open-type cells in which acyl ghrelin was negative. Acyl ghrelin-/des-acyl ghrelin-positive closed-type cells contain obestatin; on the other hand, des-acyl ghrelin-positive open-type cells contain somatostatin. We measured the release of acyl ghrelin and des-acyl ghrelin in vascularly perfused rat stomach by ELISA, and the effects of different intragastric pH levels on the release of each peptide were examined. The release of des-acyl ghrelin from the perfused stomach was greater at pH 2 than at pH 4; however, the release of acyl ghrelin was not affected by intragastric pH. The present study demonstrated the differential localization of acyl ghrelin and des-acyl ghrelin in the rat stomach and their different responses to the intragastric pH.

Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites or axon terminals in the hippocampus.

PubMed

Hájos, N; Papp, E C; Acsády, L; Levey, A I; Freund, T F

1998-01-01

In previous studies m2 muscarinic acetylcholine receptor-immunoreactive interneurons and various types of m2-positive axon terminals have been described in the hippocampal formation. The aim of the present study was to identify the types of interneurons expressing m2 receptor and to examine whether the somadendritic and axonal m2 immunostaining labels the same or distinct cell populations. In the CA1 subfield, neurons immunoreactive for m2 have horizontal dendrites, they are located at the stratum oriens/alveus border and have an axon that project to the dendritic region of pyramidal cells. In the CA3 subfield and the hilus, m2-positive neurons are multipolar and are scattered in all layers except stratum lacunosum-moleculare. In stratum pyramidale of the CA1 and CA3 regions, striking axon terminal staining for m2 was observed, surrounding the somata and axon initial segments of pyramidal cells in a basket-like manner. The co-localization of m2 with neurochemical markers and GABA was studied using the "mirror" technique and fluorescent double-immunostaining at the light microscopic level and with double-labelling using colloidal gold-conjugated antisera and immunoperoxidase reaction (diaminobenzidine) at the electron microscopic level. GABA was shown to be present in the somata of most m2-immunoreactive interneurons, as well as in the majority of m2-positive terminals in all layers. The calcium-binding protein parvalbumin was absent from practically all m2-immunoreactive cell bodies and dendrites. In contrast, many of the terminals synapsing on pyramidal cell somata and axon initial segments co-localized parvalbumin and m2, suggesting a differential distribution of m2 receptor immunoreactivity on the axonal and somadendritic membrane of parvalbumin-containing basket and axo-axonic cells. The co-existence of m2 receptors with the calcium-binding protein calbindin and the neuropeptides cholecystokinin and vasoactive intestinal polypeptide was rare throughout the hippocampal formation. Only calretinin and somatostatin showed an appreciable degree of co-localization with m2 (20% and 15%, respectively). Using retrograde tracing, some of the m2-positive cells in stratum oriens were shown to project to the medial septum, accouting for 38% of all projection neurons. The present results demonstrate that there is a differential distribution of m2 receptor immunoreactivity on the axonal vs the somadendritic membranes of distinct interneuron types and suggest that acetylcholine via m2 receptors may reduce GABA release presynaptically from the terminals of perisomatic inhibitory cells, while it may act to increase the activity of another class of interneuron, which innervates the dendritic region of pyramidal cells.

Cell Size Regulation in Bacteria

NASA Astrophysics Data System (ADS)

Amir, Ariel

2014-05-01

Various bacteria such as the canonical gram negative Escherichia coli or the well-studied gram positive Bacillus subtilis divide symmetrically after they approximately double their volume. Their size at division is not constant, but is typically distributed over a narrow range. Here, we propose an analytically tractable model for cell size control, and calculate the cell size and interdivision time distributions, as well as the correlations between these variables. We suggest ways of extracting the model parameters from experimental data, and show that existing data for E. coli supports partial size control, and a particular explanation: a cell attempts to add a constant volume from the time of initiation of DNA replication to the next initiation event. This hypothesis accounts for the experimentally observed correlations between mother and daughter cells as well as the exponential dependence of size on growth rate.

Triterpene esters from Uncaria rhynchophylla drive potent IL-12-dependent Th1 polarization.

PubMed

Umeyama, Akemi; Yahisa, Yoshinori; Okada, Minori; Okayama, Eriko; Uda, Ayaka; Shoji, Noboru; Lee, Je-Jung; Takei, Masao; Hashimoto, Toshihiro

2010-10-01

Dendritic cells (DC) are key antigen-presenting cells that link innate and adaptive immunity and ultimately activate antigen-specific T cells. In the current study, we demonstrated that two triterpene esters, uncarinic acid C (1) and uncarinic acid D (2), which are isolated from the hooks of Uncaria rhynchophylla, activate phenotypic and cytokine production alterations in DC. We also show that 1 and 2 modulate human DC function in a fashion that favors Th1 cell polarization. The effect of 1 (E configuration at the 2' position) was approximately 20 times more potent than that of 2 (Z configuration at 2'). These results indicated that the configuration of the 2' double bond greatly effects activity. Thus, 1 and 2 may prove useful as DC-based vaccines for cancer immunotherapy.

The organophosphate insecticide chlorpyrifos confers its genotoxic effects by inducing DNA damage and cell apoptosis.

PubMed

Li, Diqiu; Huang, Qingchun; Lu, Miaoqing; Zhang, Lei; Yang, Zhichuan; Zong, Mimi; Tao, Liming

2015-09-01

The organophosphate insecticide chlorpyrifos (CPF) is known to induce neurological effects, malformation and micronucleus formation, persistent developmental disorders, and maternal toxicity in rats and mice. The binding of chlorpyrifos with DNA to produce DNA adducts leads to an increasing social concern about the genotoxic risk of CPF in human, but CPF-induced cytotoxicity through DNA damage and cell apoptosis is not well understood. Here, we quantified the cytotoxicity and potential genotoxicity of CPF using the alkaline comet assay, γH2AX foci formation, and the DNA laddering assay in order to detect DNA damage and apoptosis in human HeLa and HEK293 cells in vitro. Drosophila S2 cells were used as a positive control. The alkaline comet assay showed that sublethal concentrations of CPF induced significant concentration-dependent increases in single-strand DNA breaks in the treated cells compared with the control. The percentage of γH2AX-positive HeLa cells revealed that CPF also causes DNA double-strand breaks in a time-dependent manner. Moreover, DNA fragmentation analysis demonstrated that exposure to CPF induced a significant concentration- and time-dependent increase in cell apoptosis. We conclude that CPF is a strongly genotoxic agent that induces DNA damage and cell apoptosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

The cellular lesion of humoral rejection: predominant recruitment of monocytes to peritubular and glomerular capillaries.

PubMed

Fahim, T; Böhmig, G A; Exner, M; Huttary, N; Kerschner, H; Kandutsch, S; Kerjaschki, D; Bramböck, A; Nagy-Bojarszky, K; Regele, H

2007-02-01

Accumulation of inflammatory cells within capillaries is a common morphologic feature of humoral renal allograft rejection and is most easily appreciated if it occurs in glomeruli. The aim of our study was to determine the amount and composition of immune cells within glomeruli and peritubular capillaries (PTC) in cellular and humoral allograft rejection. Immunofluorescent double-labeling for CD31 and CD3 or CD68 was used for phenotyping and enumerating immune cells within glomeruli and PTC. The major findings are: (1) accumulation of immune cells in PTC is far more common than it would be anticipated based on the assessment by conventional histology; (2) it is not the absolute number of immune cells accumulating within capillaries, but rather the composition of the intracapillary cell population that distinguishes humoral rejection from cellular rejection and (3) in C4d positive biopsies a predominantly monocytic cell population accumulates not only within glomeruli but also within PTC. The median value of monocyte/T-cell ratio within PTC was 2.3 in C4d positive biopsies but only 1 (p = 0.0008) in C4d negative biopsies. Given their prominent presence within capillaries and their extensive biological versatility monocytes might contribute to the capillary damage observed in acute and chronic allograft rejection.

[Construction of fetal mesenchymal stem cell cDNA subtractive library].

PubMed

Yang, Li; Wang, Dong-Mei; Li, Liang; Bai, Ci-Xian; Cao, Hua; Li, Ting-Yu; Pei, Xue-Tao

2002-04-01

To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp. SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

A combination of biomolecules enhances expression of E-cadherin and peroxisome proliferator-activated receptor gene leading to increased cell proliferation in primary human meniscal cells: an in vitro study.

PubMed

Pillai, Mamatha M; Elakkiya, V; Gopinathan, J; Sabarinath, C; Shanthakumari, S; Sahanand, K Santosh; Dinakar Rai, B K; Bhattacharyya, Amitava; Selvakumar, R

2016-10-01

The present study investigates the impact of biomolecules (biotin, glucose, chondroitin sulphate, proline) as supplement, (individual and in combination) on primary human meniscus cell proliferation. Primary human meniscus cells isolated from patients undergoing meniscectomy were maintained in Dulbecco's Modified Eagle's Medium (DMEM). The isolated cells were treated with above mentioned biomolecules as individual (0-100 µg/ml) and in combinations, as a supplement to DMEM. Based on the individual biomolecule study, a unique combination of biomolecules (UCM) was finalized using one way ANOVA analysis. With the addition of UCM as supplement to DMEM, meniscal cells reached 100 % confluency within 4 days in 60 mm culture plate; whereas the cells in medium devoid of UCM, required 36 days for reaching confluency. The impact of UCM on cell viability, doubling time, histology, gene expression, biomarkers expression, extra cellular matrix synthesis, meniscus cell proliferation with respect to passages and donor's age were investigated. The gene expression studies for E-cadherin and peroxisome proliferator-activated receptor (PPAR∆) using RT-qPCR and immunohistochemical analysis for Ki67, CD34 and Vimentin confirmed that UCM has significant impact on cell proliferation. The extracellular collagen and glycosaminoglycan secretion in cells supplemented with UCM were found to increase by 31 and 37 fold respectively, when compared to control on the 4th day. The cell doubling time was reduced significantly when supplemented with UCM. The addition of UCM showed positive influence on different passages and age groups. Hence, this optimized UCM can be used as an effective supplement for meniscal tissue engineering.

3D single-molecule super-resolution microscopy with a tilted light sheet.

PubMed

Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

2018-01-09

Tilted light sheet microscopy with 3D point spread functions (TILT3D) combines a novel, tilted light sheet illumination strategy with long axial range point spread functions (PSFs) for low-background, 3D super-localization of single molecules as well as 3D super-resolution imaging in thick cells. Because the axial positions of the single emitters are encoded in the shape of each single-molecule image rather than in the position or thickness of the light sheet, the light sheet need not be extremely thin. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The result is simple and flexible 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validate TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed tetrapod PSFs for fiducial bead tracking and live axial drift correction.

Accelerated cell-sheet recovery from a surface successively grafted with polyacrylamide and poly(N-isopropylacrylamide).

PubMed

Akiyama, Yoshikatsu; Kikuchi, Akihiko; Yamato, Masayuki; Okano, Teruo

2014-08-01

A double polymeric nanolayer consisting of poly(N-isopropylacrylamide) (PIPAAm) and hydrophilic polyacrylamide (PAAm) was deposited on tissue culture polystyrene (TCPS) surfaces using electron beam irradiation to form a new temperature-responsive cell culture surface in which the basal hydrophilic PAAm component in the double polymeric layer promotes the hydration of the upper PIPAAm layer and induces rapid cell detachment compared to a conventional temperature-responsive cell culture surface, PIPAAm-grafted TCPS (PIPAAm-TCPS). Take-off angle-dependent X-ray photoelectron spectroscopy spectral analysis demonstrated that the grafted PIPAAm and PAAm components were located in the upper and basal regions of the double polymeric layer, respectively, suggesting that the double polymeric layer forms an inter-penetrating-network-like structure with PAAm at the basal portion of the PIPAAm grafted chains. The wettability of the temperature-responsive cell culture surfaces with the double polymeric layer tended to be more hydrophilic, with an increase in the basal PAAm graft density at a constant PIPAAm graft density. However, when the graft densities of the upper PIPAAm and basal PAAm were optimized, the resulting temperature-responsive cell culture surface with the double polymeric layer exhibited rapid cell detachment while maintaining cell adhesive character comparable to that of PIPAAm-TCPS. The cell adhesive character was altered from cell-adhesive to cell-repellent with increasing PAAm or PIPAAm graft density. The cell adhesive character of the temperature-responsive cell culture surfaces was relatively consistent with their contact angles. These results strongly suggest that the basal PAAm surface properties affect the degree of hydration and dehydration of the subsequently grafted PIPAAm. In addition, the roles of the hydrophilic component in accelerating cell detachment are further discussed in terms of the mobility of the grafted PIPAAm chains. Applications of this insight might be useful for designing temperature-responsive cell culture surfaces for achieving efficient cell culture and quick target cell detachment. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Double versus single reading of mammograms in a breast cancer screening programme: a cost-consequence analysis.

PubMed

Posso, Margarita C; Puig, Teresa; Quintana, Ma Jesus; Solà-Roca, Judit; Bonfill, Xavier

2016-09-01

To assess the costs and health-related outcomes of double versus single reading of digital mammograms in a breast cancer screening programme. Based on data from 57,157 digital screening mammograms from women aged 50-69 years, we compared costs, false-positive results, positive predictive value and cancer detection rate using four reading strategies: double reading with and without consensus and arbitration, and single reading with first reader only and second reader only. Four highly trained radiologists read the mammograms. Double reading with consensus and arbitration was 15 % (Euro 334,341) more expensive than single reading with first reader only. False-positive results were more frequent at double reading with consensus and arbitration than at single reading with first reader only (4.5 % and 4.2 %, respectively; p < 0.001). The positive predictive value (9.3 % and 9.1 %; p = 0.812) and cancer detection rate were similar for both reading strategies (4.6 and 4.2 per 1000 screens; p = 0.283). Our results suggest that changing to single reading of mammograms could produce savings in breast cancer screening. Single reading could reduce the frequency of false-positive results without changing the cancer detection rate. These results are not conclusive and cannot be generalized to other contexts with less trained radiologists. • Double reading of digital mammograms is more expensive than single reading. • Compared to single reading, double reading yields a higher proportion of false-positive results. • The cancer detection rate was similar for double and single readings. • Single reading may be a cost-effective strategy in breast cancer screening programmes.

Pseudocapacitance of MXene nanosheets for high-power sodium-ion hybrid capacitors

PubMed Central

Wang, Xianfen; Kajiyama, Satoshi; Iinuma, Hiroki; Hosono, Eiji; Oro, Shinji; Moriguchi, Isamu; Okubo, Masashi; Yamada, Atsuo

2015-01-01

High-power Na-ion batteries have tremendous potential in various large-scale applications. However, conventional charge storage through ion intercalation or double-layer formation cannot satisfy the requirements of such applications owing to the slow kinetics of ion intercalation and the small capacitance of the double layer. The present work demonstrates that the pseudocapacitance of the nanosheet compound MXene Ti2C achieves a higher specific capacity relative to double-layer capacitor electrodes and a higher rate capability relative to ion intercalation electrodes. By utilizing the pseudocapacitance as a negative electrode, the prototype Na-ion full cell consisting of an alluaudite Na2Fe2(SO4)3 positive electrode and an MXene Ti2C negative electrode operates at a relatively high voltage of 2.4 V and delivers 90 and 40 mAh g−1 at 1.0 and 5.0 A g−1 (based on the weight of the negative electrode), respectively, which are not attainable by conventional electrochemical energy storage systems. PMID:25832913

Perinatal asphyxia induces neurogenesis in hippocampus: an organotypic culture study.

PubMed

Morales, P; Huaiquín, P; Bustamante, D; Fiedler, J; Herrera-Marschitz, M

2007-07-01

There is clinical and experimental evidence indicating that neurocircuitries of the hippocampus are vulnerable to hypoxia/ischemia occurring at birth, inducing, upon re-oxygenation/re-circulation, delayed neuronal death, but also compensatory mechanisms, including neurogenesis. In the present report, perinatal asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath at 37 degrees C for 20 min. Some pups were delivered immediately after the hysterectomy to be used as non-asphyxiated caesarean-delivered controls. The pups were sacrificed after seven days for preparing organotypic hippocampal cultures. The cultures were grown on a coverslip in a medium-containing culture tube inserted in a hole of a roller device standing on the internal area of a cell incubator at 35 degrees C, 10% CO2. At days in vitro (DIV) 25-27, cultures were fixed for assaying cell proliferation and neuronal phenotype with antibodies against 5-bromo-2'deoxyuridine (BrdU) and microtubule associated protein-2 (MAP-2), respectively. Confocal microscopy revealed that there was a 2-fold increase of BrdU-positive, but a 40% decrease of MAP-2-positive cells/mm3 in cultures from asphyxia-exposed, compared to that from control animals. Approximately 30% of BrdU-positive cells were also positive for MAP-2 (approximately 4800 cells), mainly seen in the dentate gyrus of the hippocampus, demonstrating a 3-fold increase of postnatal neurogenesis, when the total amount of double-labelled cells seen in cultures from asphyxia-exposed animals is compared to that from control animals.

Restoring Cytokine Balance in HIV-Positive Individuals with Low CD4 T Cell Counts

PubMed Central

Valdivia, Anddre; Ly, Judy; Gonzalez, Leslie; Hussain, Parveen; Saing, Tommy; Islamoglu, Hicret; Pearce, Daniel; Ochoa, Cesar

2017-01-01

Abstract HIV infects and destroys CD4+ T cells leading to a compromised immune system. In a double-blinded study, a group of HIV-infected individuals with CD4+ T cell counts below 350 cells/mm3 were given either an empty liposomal supplement or a liposomal glutathione (L-GSH) supplement to take over a 3-month period. Baseline measurements in HIV-positive subjects show a significant decrease in levels of interleukin (IL)-12, IL-2, and interferon (IFN)-γ, along with a substantial increase in the levels of IL-6, IL-10, transforming growth factor (TGF)-β, and free radicals, compared to healthy individuals. Supplementation of HIV-positive subjects with L-GSH for 3 months resulted in a notable increase in the levels of IL-12, IL-2, and IFN-γ, with a concomitant decrease in the levels of IL-6, IL-10, and free radicals, and stabilization in the levels of TGF-β, IL-1, and IL-17, compared to their placebo counterparts. Levels of free radicals in CD4+ T cells stabilized, while GSH levels increased in the treatment group. Those in the placebo group showed no significant difference throughout the study. In summary, supplementation with L-GSH in HIV-infected individuals with CD4+ T cell counts below 350 cells/mm3 can help restore redox homeostasis and cytokine balance, therefore aiding the immune system to control opportunistic infections. PMID:28398068

Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells.

PubMed

Wu, Wei; Liu, Juli; Su, Zhenghui; Li, Zhonghao; Ma, Ning; Huang, Ke; Zhou, Tiancheng; Wang, Linli

2018-08-25

Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6 + neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6 WT/EGFP H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6 WT/EGFP NPCs could be purified, expanded and specified to post-mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512-3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application. Copyright © 2018 Elsevier Inc. All rights reserved.

Influence of LTB4 on CD4-, CD8- thymocytes. Evidence that LTB4 plus IL-2 generate CD8+ suppressor thymocytes involved in tolerance to self. Effect of LTB4 and IL-2 on double negative thymocytes.

PubMed

Gualde, N; Cogny van Weydevelt, F; Buffière, F; Jauberteau, M O; Daculsi, R; Vaillier, D

1991-09-01

Leukotriene B4 (LTB4) is produced by a large variety of cells involved in immune response and it has been demonstrated that this arachidonic acid metabolite acts as an immunomodulator. Because LTB4 and IL-2 both influence the physiology of immature cells we studied the effects of the leukotriene on double negative thymocytes. For that purpose C57 Bl/6 double negative thymocytes were treated by LTB4 plus IL-2 in the presence of either autologous or allogenic red blood cells (RBC). Then, preincubated CD4- CD8- thymocytes were cocultured with red blood cells stimulated fresh splenocytes. We observed that fresh splenocytes responding to autologous RBC were CD4+ cells and that the proliferative response of spleen lymphocytes driven by RBC was inhibited by preincubated double negative thymocytes. On the other hand a majority of double negative thymocytes overnight preincubated in vitro in the presence of both IL-2 and LTB4 give rise to CD8+ CD4- cells. Therefore we speculate that LTB4 plus IL-2 generate CD8+ suppressor thymocytes among double negative thymocytes and that these suppressive T cells are involved in tolerance to self.

Hybrid capacitors utilizing halogen-based redox reactions at interface between carbon positive electrode and aqueous electrolytes

NASA Astrophysics Data System (ADS)

Yamazaki, Shigeaki; Ito, Tatsuya; Murakumo, Yuka; Naitou, Masashi; Shimooka, Toshiharu; Yamagata, Masaki; Ishikawa, Masashi

2016-09-01

We propose novel hybrid capacitors (HCs) with electrolyte-involved redox reactions of bromide or iodide species by pretreatment of an activated carbon positive electrode. The treatment is simple; impregnation of pores at an activated carbon fiber cloth (ACFC) as a positive electrode with bromine- or iodine-containing water before cell assembly. The treated positive electrode is applied to a HC cell with a non-treated negative electrode of ACFC and its electrochemical performance is investigated by galvanostatic cycling and leakage current tests. Few studies on such "electrolytic" charge storage systems have provided acceptable capacitor performance because of inevitable self-discharge caused by diffusion of charged species form an electrode to the other one through an electrolyte. Nevertheless, our electrolyte-redox-based HCs show excellent performance without undesirable diffusion of charged species. Moreover, the present HC utilizing a bromide redox system fulfills a practical cell voltage of 1.8 V in spite of an aqueous electrolyte system. This high voltage provides excellent energy density, which is 5 times higher than that in a conventional aqueous electric double-layer capacitor (EDLC), and 1.2 times higher even than that in a 2.7 V-class non-aqueous EDLC, while keeping high charge-discharge rate capability.

Upregulation of the axonal growth and the expression of substance P and its NK1 receptor in human allergic contact dermatitis.

PubMed

El-Nour, H; Lundeberg, L; Al-Tawil, R; Granlund, A; Lonne-Rahm, S-B; Nordlind, K

2006-01-01

Nerve fibers and sensory neuropeptides substance P and calcitonin gene-related peptide (CGRP) have been reported to be involved in allergic contact dermatitis (ACD). In the present study, we investigated the general innervation (using antibody against protein gene product 9.5, PGP 9.5), axonal growth (using antibody against growth associated protein, GAP-43), CGRP, and substance P with its receptor neurokinin 1 (NK1), in positive epicutaneous reactions to nickel sulphate from nickel-allergic patients, at the peak of inflammation, 72 hr after challenge with the antigen. There was an increased (p < 0.01) number of GAP-43 positive fibers in the eczematous compared with control skin, indicating an increased axonal growth already at 72 hr postchallenge. Double staining revealed a coexpression of CGRP and GAP-43 on dermal nerve fibers. There was no difference in the number of substance P and CGRP positive nerve fibers between eczematous and control skin. However, semiquantification analyses showed an increased expression of substance P positive inflammatory cells, being CD3, CD4, or CD8 positive, and NK1R positive inflammatory cells, being tryptase or CD3 positive. These results indicate a contribution of regenerating nerve fibers and substance P to the contact allergic reaction.

Student Observations of Double Star Delta Orionis (STFA 14 AC)

NASA Astrophysics Data System (ADS)

Estrada, Reed; Aguilera, Sophia; Bowden, Sam; Gillette, Travis; Givens, Jalynn; Reder, Gabriel; Rhoades, Breauna; Sharpe, Scott; Shattles, Jenna; Cha, Brendon; Do, Vicky; Ewing, Malachi; Kiamco, Alex Junior; Nelms, Brenda; Peña, Emilie; Maricarmen, Richard; Thielen, Austin

2018-01-01

A group of eight eighth graders and eight high schoolers studied the double star STFA 14 AC. They used the procedure from Argyle's book to get the separation and position angle for the double star. The students used a Celestron C8 Schmidt-Cassegrain telescope with a Baader Planetarium microguide eyepiece with similar markings to a Celestron Eyepiece. The students determined the separation to be 56 arcseconds and the position angle to be 4.19°. They compared their results to the Washington Double Star Catalog and found that they had a 2.88 arcseconds difference in separation and a 2.19° in position angle.

The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath)

NASA Technical Reports Server (NTRS)

Jahnke, L. L.

1992-01-01

Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 degrees C resulted in changes to the whole cell lipid constituents. As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the delta 9, delta 10 and delta 11 C16:1 double bond positional isomers changed. Methyl sterol content also increased as the growth temperature was lowered. The highest amounts of methyl sterol were found in 30 degrees C cells and the lowest in 50 degrees C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively). The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.

The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath)

NASA Technical Reports Server (NTRS)

Jahnke, Linda L.

1992-01-01

Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 C resulted in changes to the whole cell lipid constituents. As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the Delta9, Delta10, and Delta11 C16:1 double bond positional isomers changed. Methyl sterol content also increased as the growth temperature was lowered. The highest amounts of methyl sterol were found in 30 C cells and the lowest in 50 C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively). The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.

[Effect of electroacupuncture on differentiation and proliferation of hippocampal nerve stem cells in splenic asthenia pedo-rats].

PubMed

Zhuo, Yuan-yuan; Yang, Zhuo-xin; Wu, Jia-man

2011-10-01

To observe the effect of electroacupuncture (EA) on the differentiation and proliferation of nerve stem cells in the hippocampal dentate gyrus (DG) in splenic asthenia pedo-rats so as to study its central mechanism. A total of 72 SD male rats were randomly assigned to normal control group (n=24), model group (n=24) and EA group (n=24) which were further divided into 7 d, 14 d, 28 d and 49 d time-points (n=6). Splenic asthenia model was established by intraperitoneal injection of reserpine and gavage of Dahuang (Radix et Rhizoma Rhei) fluid. EA was applied to bilateral "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) for 20 min, once daily for 7, 14, 28 and 49 days respectively. Brdu, Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE) expression in the DG of hippocampus were detected by immunohistochemistry double staining. Compared with the normal control group, the numbers of Brdu, Brdu/GFAP, Brdu/NSE Immunoreactive (IR) positive cells in the DG of hippocampus on day 7 and 14, and that of Brdu/Nestin IR-positive cells on day 7 were decreased considerably in the model group (P < 0.05). Compared to the model group, the numbers of hippocampal Brdu IR-positive cells at the 4 time-points, Brdu/Nestin and Brdu/GFAP on day 7, 14 and 49, and Brdu/NSE on day 7, 14 and 28 were increased significantly in the EA group (P < 0.05). No significant differences were found between the model and control groups in the numbers of hippocampal Brdu and Brdu/NSE IR-positive cells on day 28 and 49, in the number of Brdu/Nestin IR-positive cells on day 14 and 49, in the number of Brdu/GFAP IR-positive cells on day 28; and between the EA and model groups in the numbers of Brdu/Nestin and Brdu/GFAP IR-positive cells on day 28, and in the number of Brdu/NSE IR-positive cells on day 49 (P > 0.05). EA of ST 36 and SP 6 can effectively suppress splenic asthenia syndrome-induced decrease of the numbers of Brdu, Brdu/GFAP, Brdu/Nestin and Brdu/NSE IR-positive cells in the DG of hippocampus at the early stage in the splenic asthenia rats, which may contribute to its effect in improving splenic asthenia symptoms in clinic by promoting the proliferation and differentiation of some nerve stem cells in the hippocampus.

Establishment of ultra long-lived cell lines by transfection of TERT into normal human fibroblast TIG-1 and their characterization.

PubMed

Kamada, Mizuna; Kumazaki, Tsutomu; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

2012-06-01

To establish useful human normal cell lines, TERT (telomerase reverse transcriptase) cDNA was transfected into normal female lung fibroblast, TIG-1. After long-term-sub-cultivation of 74 individual clones selected for resistance to G418, we obtained 55 cultures with normal range of life span [75 PDL (population doubling level)], 16 cultures with extended life span (75-140 PDL). In addition, 3 immortal cell strains and unexpectedly, one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of the immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and an extremely low level of p16INK4A. They also showed moderate p53 and p21CIP1 expression, keeping vigorous growth rate even at 450 PDL. High level of fibronectin and collagen 1α expression confirmed IMT-1 as normal fibroblasts, although one X chromosome had been lost. ULT-1, however, kept a near normal karyotypes and had shortening of telomere length, high expression of p16INK4A, moderate levels of senescence associated-β-galactosidase positive cells and decreased growth rate only after 150 PDs (population doublings), and finally reached senescence at 166 PDL with morphology of normal senescent fibroblasts. As resources of standard normal human cell, abundant vials of early and middle passages of ULT-1 have been stocked. The use of the cell line is discussed, focusing on isograft of artificial skin and screening of anti-aging or safe chemical agents.

Perturbed thymopoiesis in vitro in the absence of Suppressor of Cytokine Signalling 1 and 3

PubMed Central

Croom, Hayley A.; Izon, David J.; Chong, Mark M.; Curtis, David J.; Roberts, Andrew W.; Kay, Thomas W.H.; Hilton, Douglas J.; Alexander, Warren S.; Starr, Robyn

2014-01-01

Cytokine signals are central to the differentiation of thymocytes and their stepwise progression through defined developmental stages. The intensity and duration of cytokine signals are regulated by the suppressor of cytokine signalling (SOCS) proteins. A clear role for SOCS1 during the later stages of thymopoiesis has been established, but little is known about its role during early thymopoiesis, nor the function of its closest relative, SOCS3. Here, we find that both SOCS1 and SOCS3 are expressed during early thymopoiesis, with expression coincident during the double negative (DN)2 and DN3 stages. We examined thymocyte differentiation in vitro by co-culture of SOCS-deficient bone marrow cells with OP9 cells expressing the Notch ligand Delta-like1 (OP9-DL1). Cells lacking SOCS1 were retarded at the DN3:DN4 transition and appeared unable to differentiate into double positive (DP) thymocytes. Cells lacking both SOCS1 and SOCS3 were more severely affected, and displayed an earlier block in T cell differentiation at DN2, the stage at which expression of SOCS1 and SOCS3 coincides. This indicates that, in addition to their specific roles, SOCS1 and SOCS3 share overlapping roles during thymopoiesis. This is the first demonstration of functional redundancy within the SOCS family, and has uncovered a vital role for SOCS1 and SOCS3 during two important checkpoints in early T cell development. PMID:18321577

Functional characteristics of a double positive feedback loop coupled with autorepression

NASA Astrophysics Data System (ADS)

Banerjee, Subhasis; Bose, Indrani

2008-12-01

We study the functional characteristics of a two-gene motif consisting of a double positive feedback loop and an autoregulatory negative feedback loop. The motif appears in the gene regulatory network controlling the functional activity of pancreatic β-cells. The model exhibits bistability and hysteresis in appropriate parameter regions. The two stable steady states correspond to low (OFF state) and high (ON state) protein levels, respectively. Using a deterministic approach, we show that the region of bistability increases in extent when the copy number of one of the genes is reduced from 2 to 1. The negative feedback loop has the effect of reducing the size of the bistable region. Loss of a gene copy, brought about by mutations, hampers the normal functioning of the β-cells giving rise to the genetic disorder, maturity-onset diabetes of the young (MODY). The diabetic phenotype makes its appearance when a sizable fraction of the β-cells is in the OFF state. Using stochastic simulation techniques we show that, on reduction of the gene copy number, there is a transition from the monostable ON to the ON state in the bistable region of the parameter space. Fluctuations in the protein levels, arising due to the stochastic nature of gene expression, can give rise to transitions between the ON and OFF states. We show that as the strength of autorepression increases, the ON → OFF state transitions become less probable whereas the reverse transitions are more probable. The implications of the results in the context of the occurrence of MODY are pointed out.

Virus Assembly and Maturation

NASA Astrophysics Data System (ADS)

Johnson, John E.

2004-03-01

We use two techniques to look at three-dimensional virus structure: electron cryomicroscopy (cryoEM) and X-ray crystallography. Figure 1 is a gallery of virus particles whose structures Timothy Baker, one of my former colleagues at Purdue University, used cryoEM to determine. It illustrates the variety of sizes of icosahedral virus particles. The largest virus particle on this slide is the Herpes simplex virus, around 1200Å in diameter; the smallest we examined was around 250Å in diameter. Viruses bear their genomic information either as positive-sense DNA and RNA, double-strand DNA, double-strand RNA, or negative-strand RNA. Viruses utilize the various structure and function "tactics" seen throughout cell biology to replicate at high levels. Many of the biological principles that we consider general were in fact discovered in the context of viruses ...

The cell biology of aging.

PubMed

Hayflick, L

1985-02-01

It is only within the past ten years that biogerontology has become attractive to a sufficient number of biologists so that the field can be regarded as a seriously studied discipline. Cytogerontology, or the study of aging at the cellular level, had its genesis about 20 years ago when the dogma that maintained that cultured normal cells could replicate forever was overturned. Normal human and animal cells have a finite capacity to replicate and function whether they are cultured in vitro or transplanted as grafts in vivo. This phenomenon has been interpreted to be aging at the cellular level. Only abnormal somatic cells are capable of immortality. In recent years it has been found that the number of population doublings of which cultured normal cells are capable is inversely proportional to donor age. There is also good evidence that the number of population doublings of cultured normal fibroblasts is directly proportional to the maximum lifespan of ten species that have been studied. Cultures prepared from patients with accelerated aging syndromes (progeria and Werner's syndrome) undergo far fewer doublings than do those of age-matched controls. The normal human fibroblast cell strain WI-38 was established in 1962 from fetal lung, and several hundred ampules of these cells were frozen in liquid nitrogen at that time. These ampules have been reconstituted periodically and shown to be capable of replication. This represents the longest period of time that a normal human cell has ever been frozen. Normal human fetal cell strains such as WI-38 have the capacity to double only about 50 times. If cultures are frozen at various population doublings, the number of doublings remaining after reconstitution is equal to 50 minus the number of doublings that occurred prior to freezing. The memory of the cells has been found to be accurate after 23 years of preservation in liquid nitrogen. Normal human cells incur many physiologic decrements that herald the approach of their failure to divide. Many of these functional decrements are identical to decrements found in humans as they age. Thus it is likely that these decrements are also the precursors of age changes in vivo. The finite replicative capacity of normal cells is never seen to occur in vivo because aging and death of the individual occurs well before the doubling limit is reached.

Immunohistochemical detection of autophagy-related microtubule-associated protein 1 light chain 3 (LC3) in the cerebellums of dogs naturally infected with canine distemper virus.

PubMed

Kabak, Y B; Sozmen, M; Yarim, M; Guvenc, T; Karayigit, M O; Gulbahar, M Y

2015-01-01

We investigated the expression of microtubule-associated protein 1 light chain 3 (LC3) protein in the cerebellums of dogs infected with canine distemper virus (CDV) using immunohistochemistry to detect autophagy. The cerebellums of 20 dogs infected with CDV were used. Specimens showing demyelination of white matter were considered to have an acute infection, whereas specimens showing signs of severe perivascular cuffing and demyelination of white matter were classified as having chronic CDV. Cerebellar sections were immunostained with CDV and LC3 antibodies. The cytoplasm of Purkinje cells, granular layer cells, motor neurons in large cerebellar ganglia and some neurons in white matter were positive for the LC3 antibody in both the control and CDV-infected dogs. In the infected cerebellums, however, white matter was immunostained more intensely, particularly the neurons and gemistocytic astrocytes in the demyelinated areas, compared to controls. Autophagy also was demonstrated in CDV-positive cells using double immunofluorescence staining. Our findings indicate that increased autophagy in the cerebellum of dogs naturally infected with CDV may play a role in transferring the virus from cell to cell.

A threshold level of NFATc1 activity facilitates thymocyte differentiation and opposes notch-driven leukaemia development

PubMed Central

Klein-Hessling, Stefan; Rudolf, Ronald; Muhammad, Khalid; Knobeloch, Klaus-Peter; Maqbool, Muhammad Ahmad; Cauchy, Pierre; Andrau, Jean-Christophe; Avots, Andris; Talora, Claudio; Ellenrieder, Volker; Screpanti, Isabella; Serfling, Edgar; Patra, Amiya Kumar

2016-01-01

NFATc1 plays a critical role in double-negative thymocyte survival and differentiation. However, the signals that regulate Nfatc1 expression are incompletely characterized. Here we show a developmental stage-specific differential expression pattern of Nfatc1 driven by the distal (P1) or proximal (P2) promoters in thymocytes. Whereas, preTCR-negative thymocytes exhibit only P2 promoter-derived Nfatc1β expression, preTCR-positive thymocytes express both Nfatc1β and P1 promoter-derived Nfatc1α transcripts. Inducing NFATc1α activity from P1 promoter in preTCR-negative thymocytes, in addition to the NFATc1β from P2 promoter impairs thymocyte development resulting in severe T-cell lymphopenia. In addition, we show that NFATc1 activity suppresses the B-lineage potential of immature thymocytes, and consolidates their differentiation to T cells. Further, in the pTCR-positive DN3 cells, a threshold level of NFATc1 activity is vital in facilitating T-cell differentiation and to prevent Notch3-induced T-acute lymphoblastic leukaemia. Altogether, our results show NFATc1 activity is crucial in determining the T-cell fate of thymocytes. PMID:27312418

Effect of concentration boundary layers on passive solute flows in a system of two polymeric membranes positioned in vertical planes.

PubMed

Slezak, Andrzej; Jasik-Slezak, Jolanta; Dworecki, Kazimierz

2003-01-01

The results of studies of influence of concentration boundary layers on passive diffusive transport in a double-membrane osmo-diffusive cell, containing a series of two (Ml and M(r)) vertically positioned, flat, microporous and symmetric polymer membranes (Nephrophane and Cellulose IMP-1) are presented in this paper. The membranes separated three compartments (l, m, r) containing binary, heterogeneous and non-ionic solutions (aqueous solutions of glucose or ethanol) or ternary non-electrolyte solutions (glucose solutions in 0.75 mol.l-1 solution of ethanol or ethanol solutions in 0.1 mol.l-1 aqueous solution of glucose). Solution concentrations fulfilled the condition C(k)l > C(k)m > C(k)r. The intermembrane compartment (m) was an infinitesimal solution layer. The volume of the m compartment and the volumes of the external (l and r) compartments fulfilled the condition Vl = Vr approximately 170 Vm. The tests were performed for configurations A and B of a double-membrane osmo-diffusive cell. In configuration A, the solution was located behind the M(r) membrane, and water was placed behind the Ml membrane, while in configuration B this sequence was reversed. The results obtained during experiment were interpreted in the categories of convective instability, which increased the value of diffusive permeability coefficient of the system: concentration boundary layer/membrane/concentration boundary layer.

The cell biology of aging.

PubMed

Hayflick, L

1979-07-01

Cultured normal human and animal cells are predestinued to undergo irreversible functional decrements that mimick age changes in the whole organism. When normal human embryonic fibroblasts are cultured in vitro, 50 +/- 10 population doublings occur. This maximum potential is diminished in cells derived from older donors and appears to be inversely proportional to their age. The 50 population doubling limit can account for all cells produced during a lifetime. The limitation on doubling potential of cultured normal cells is also expressed in vivo when serial transplants are made. There may be a direct correlation between the mean maximum life spans of several species and the population doubling potential of their cultured cells. A plethora of functional decrements occur in cultured normal cells as they approach their maximum division capability. Many of these decrements are similar to those occurring in intact animals as they age. We have concluded that these functional decrements expressed in vitro, rather than cessation of cell division, are the essential contributors to age changes in intact animals. Thus, the study of events leading to functional losses in cultured normal cells may provide useful insights into the biology of aging.

Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

PubMed Central

Alonso, Jose L.; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A.; Hernández, Javier

2002-01-01

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells. PMID:12324366

Bach2 Controls Homeostasis of Eosinophils by Restricting the Type-2 Helper Function of T Cells.

PubMed

Sato, Yuki; Kato, Hiroki; Ebina-Shibuya, Risa; Itoh-Nakadai, Ari; Okuyama, Ryuhei; Igarashi, Kazuhiko

2017-03-01

Bach2 is a transcription factor which represses its target genes and plays important roles in the differentiation of B and T lymphoid cells. Bach2-deficient (KO) mice develop severe pulmonary alveolar proteinosis, which is associated with increased numbers of granulocytes and T cells. Bach2 is essential for the regulation of T cells, but its role in the regulation of granulocytes is not clear. Here, we observed increased numbers of eosinophils but not neutrophils in the bone marrow, spleen, peripheral blood, and bronchoalveolar lavage fluids of Bach2 KO mice compared with those of wild-type (WT) mice. Upon co-transplantation of the bone marrow cells from CD45.2 Bach2 KO and CD45.1/CD45.2 double-positive WT mice to irradiated WT CD45.1/CD45.2 mice, the reconstituted numbers of eosinophils were similar between Bach2 KO and WT cells. These results showed that the deficiency of Bach2 in eosinophils did not directly drive the differentiation of eosinophils. To investigate the effect of Bach2 KO CD4 + T cells upon eosinophils, we analyzed Rag2/Bach2-double deficient (dKO) mice which lack lymphocytes including CD4 + T cells. Rag2/Bach2 dKO mice did not show any increase in the numbers of eosinophils. Importantly, Bach2 KO mice showed an increase of interleukin-5 (Il-5) in the sera compared with WT mice. These results suggest that up-regulated functions of CD4 + T cells including secretion of Il-5 resulted in proliferation and/or migration to peripheral tissues of eosinophils in Bach2 KO mice. We propose that Bach2 controls homeostasis of eosinophils via restricting the production of Il-5 in CD4 + T cells.

Immunocytochemical detection of the microsomal glucose-6-phosphatase in human brain astrocytes.

PubMed

Bell, J E; Hume, R; Busuttil, A; Burchell, A

1993-10-01

Using an antibody raised against the catalytic subunit of glucose-6-phosphatase, this enzyme was immunolocalized in many astrocytes in 20 normal human brains. Double immunofluorescence studies showed co-localization of glial fibrillary acidic protein (GFAP) with glucose-6-phosphatase in astrocytes. However, not all GFAP-positive cells were also glucose-6-phosphatase positive, indicating that some astrocytes do not contain demonstrable expression of this enzyme. Reactive astrocytes in a variety of abnormal brains were strongly glucose-6-phosphatase positive, but neoplastic astrocytes were often only weakly positive. Expression of the enzyme could not be demonstrated in radial glia, neurons or oligodendroglia. Astrocytes normally contain glycogen and the demonstration that some astrocytes also contain glucose-6-phosphatase indicates that they are competent for both glycogenolysis and gluconeogenesis, which may be critical for neuronal welfare.

Programmed death-1 ligands 1 and 2 expression in cutaneous squamous cell carcinoma and their relationship with tumour- infiltrating dendritic cells.

PubMed

Jiao, Q; Liu, C; Li, W; Li, W; Fang, F; Qian, Q; Zhang, X

2017-06-01

The programmed death-1 (PD-1) receptor ligands, PD-L1 and PD-L2, are co-stimulatory molecules that contribute to the negative regulation of T lymphocyte activation. It is still unclear whether there is correlation between PD-L1 or PD-L2 and tumour-infiltrating dendritic cells (TIDCs) in cutaneous squamous cell carcinoma (CSCC). The aim of this study was to analyse PD-L1 and PD-L2 expression and dendritic cells infiltration in tumour tissue of CSCC patients and investigate their clinical significance. Immunohistochemical analysis was used to evaluate the expression of PD-L1, PD-L2, CD1a and CD83 in 61 CSCC tissues. The immunofluoresence double-labelling technique was performed to detect the co-expression of PD-L1 or PD-L2 and CD1a or CD83 in tumour tissues. We found that 25 of 61 cases CSCC (40·98%) exhibited positivity for PD-L1, whereas 37 of 61 cases CSCC (60·66%) exhibited positivity for PD-L2. A higher percentage of CD1a-positive cases were observed on both PD-L1-positive and PD-L2-positive specimens compared with that of CD83-positive cases (92·29% versus 37·60%, 83·20% versus 33·16%). The expression of PD-L1 and PD-L2 on CD1a + cells was significantly higher than that on CD83 + cells in tumour tissues of CSCC patients. Furthermore, the expression rate of PD-L1 was associated with UICC stage, and the expression rate of PD-L2 was associated with predominant differentiation and tumour size in CSCC. Our results indicated that higher expression of PD-L1 and PD-L2 on CD1a + cells than that on CD83 + cells in CSCC tumour tissues may contribute to negative regulation in anti-tumour immune responses. © 2017 British Society for Immunology.

Meiotic synapsis of homogeneously staining regions (HSRs) in chromosome 1 of Mus musculus.

PubMed

Winking, H; Reuter, C; Traut, W

1993-05-01

About 50 copies of a long-range repeat DNA family with a repeat size of roughly 100 kb and with sequence homology to mRNAs are clustered in the G-light band D of chromosome 1 of the house mouse, Mus musculus. We studied amplified versions of the cluster which are found in many wild populations of M. musculus. They are cytogenetically conspicuous as one or two C-band positive homogeneously staining regions (single- and double band HSRs) which increase the mitotic length of chromosome 1. The double band HSR was phylogenetically derived from a single band HSR by a paracentric inversion. In homozygous condition, such HSRs contribute, albeit not as much as expected from their mitotic length, to the synaptonemal complex (SC) length of chromosome 1. In HSR heterozygous animals an elongation of the SCs was not noticeable. In single band HSR heterozygous males, synapsis proceeds regularly and continuously from the distal telomere towards the centromeric end without forming buckles. Thus, the single band HSR has no adverse effect on pairing. The same straight pairing behaviour was found in the majority of double band HSR heterozygous spermatocytes. This shows that extensive nonhomologous pairing can take place in the earliest phase of synapsis. Synapsis was discontinuous, leaving the central part of the bivalent 1 asynapsed, in only 14.3% of double band HSR heterozygous cells. In such cells the chromosome 1 SC is completed at a later stage of meiosis. The delay is presumably an effect of the inversion that includes one HSR band and the segment between the two HSR bands.

Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.

PubMed

Novak, K D; Peterson, M D; Reedy, M C; Titus, M A

1995-12-01

The functional relationship between three Dictyostelium myosin Is, myoA, myoB, and myoC, has been examined through the creation of double mutants. Two double mutants, myoA-/B- and myoB-/C-, exhibit similar conditional defects in fluid-phase pinocytosis. Double mutants grown in suspension culture are significantly impaired in their ability to take in nutrients from the medium, whereas they are almost indistinguishable from wild-type and single mutant strains when grown on a surface. The double mutants are also found to internalize gp126, a 116-kD membrane protein, at a slower rate than either the wild-type or single mutant cells. Ultrastructural analysis reveals that both double mutants possess numerous small vesicles, in contrast to the wild-type or myosin I single mutants that exhibit several large, clear vacuoles. The alterations in fluid and membrane internalization in the suspension-grown double mutants, coupled with the altered vesicular profile, suggest that these cells may be compromised during the early stages of pinocytosis, a process that has been proposed to occur via actin-based cytoskeletal rearrangements. Scanning electron microscopy and rhodamine-phalloidin staining indicates that the myosin I double mutants appear to extend a larger number of actin-filled structures, such as filopodia and crowns, than wild-type cells. Rhodamine-phalloidin staining of the F-actin cytoskeleton of these suspension-grown cells also reveals that the double mutant cells are delayed in the rearrangement of cortical actin-rich structures upon adhesion to a substrate. We propose that myoA, myoB, and myoC play roles in controlling F-actin filled membrane projections that are required for pinosome internalization in suspension.

Morphological and immunohistochemical characterization of isolated tumor cells by p53 status in gastrointestinal tumors.

PubMed

Milsmann, C; Füzesi, L; Heinmöller, E; Krause, P; Werner, C; Becker, H; Horstmann, O

2008-01-01

Isolated tumor cells (ITCs) in cancer patients are retrieved mostly using immunohistochemistry with antibodies directed against antiepithelial antigens (for example Ber-EP4), which are supposed not to be present in metastatic-free tissue. To date, there has been ongoing controversy whether those cells have biologic significance and are linked with tumor progression and impaired patient's prognosis. Therefore, the aim of this study was to further characterize Ber-EP4-positive cells in various tissues, with special emphasis on their tumorigenic origin. The frequency and prognostic impact of ITCs in lymph nodes displayed by means of monoclonal antibody Ber-EP4 were evaluated in retrospective (n = 292) and prospective (n = 100) collectives of various gastrointestinal carcinomas free of metastatic disease in conventional histopathology (pN0). Furthermore, the frequency of ITCs in the peritoneal cavity and bone marrow was analyzed in case of absence of overt distant metastasis (pM0) in the prospective collective. Ber-EP4-immunoreactive cells were further characterized for tumorigenic origin using morphological criteria and immunohistochemical double staining for Ber-EP4 and p53. Ber-EP4-positive cells could be revealed in lymph nodes in 44.3% of pN0-gastrointestinal carcinomas, in the peritoneal cavity in 19%, and in the bone marrow in 10%. In lymph nodes, BerEP4-immunoreactive cells exhibited a metastatic-atypical morphology in 59%; however, it was always typical for true tumor cells in the peritoneal cavity or bone marrow. The cumulative 5-year survival rate was adversely affected by Ber-EP4-immunoreactive cells in uni- and multivariate analysis, irrespective of the underlying cell morphology (68% for Ber-EP4 negative, 41% for Ber-EP4 positive with atypical and typical morphology each). In the case of a p53-positive primary tumor, 70% of the corresponding ITCs also overexpressed p53, while the remainder was deemed p53 negative (p = 0.002). ITCs detected by the antiepithelial antibody Ber-EP4 are present in a substantial proportion of apparently tumor-free lymph nodes. These cells impair patients' prognoses, irrespective of the underlying cell morphology. As approximately one third of Ber-EP4-positive cells in p53-positive primary tumors do not overexpress p53; their true tumorigenic origin needs to be further investigated.

Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis.

PubMed

Zhang, Yanjun; Clausmeyer, Jan; Babakinejad, Babak; Córdoba, Ainara López; Ali, Tayyibah; Shevchuk, Andrew; Takahashi, Yasufumi; Novak, Pavel; Edwards, Christopher; Lab, Max; Gopal, Sahana; Chiappini, Ciro; Anand, Uma; Magnani, Luca; Coombes, R Charles; Gorelik, Julia; Matsue, Tomokazu; Schuhmann, Wolfgang; Klenerman, David; Sviderskaya, Elena V; Korchev, Yuri

2016-03-22

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.

Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis

PubMed Central

Córdoba, Ainara López; Ali, Tayyibah; Shevchuk, Andrew; Takahashi, Yasufumi; Novak, Pavel; Edwards, Christopher; Lab, Max; Gopal, Sahana; Chiappini, Ciro; Anand, Uma; Magnani, Luca; Coombes, R. Charles; Gorelik, Julia; Matsue, Tomokazu; Schuhmann, Wolfgang; Klenerman, David; Sviderskaya, Elena V.; Korchev, Yuri

2016-01-01

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements. PMID:26816294

Oncogenes: The Passport for Viral Oncolysis Through PKR Inhibition.

PubMed

Fernandes, Janaina

2016-01-01

The transforming properties of oncogenes are derived from gain-of-function mutations, shifting cell signaling from highly regulated homeostatic to an uncontrolled oncogenic state, with the contribution of the inactivating mutations in tumor suppressor genes P53 and RB, leading to tumor resistance to conventional and target-directed therapy. On the other hand, this scenario fulfills two requirements for oncolytic virus infection in tumor cells: inactivation of tumor suppressors and presence of oncoproteins, also the requirements to engage malignancy. Several of these oncogenes have a negative impact on the main interferon antiviral defense, the double-stranded RNA-activated protein kinase (PKR), which helps viruses to spontaneously target tumor cells instead of normal cells. This review is focused on the negative impact of overexpression of oncogenes on conventional and targeted therapy and their positive impact on viral oncolysis due to their ability to inhibit PKR-induced translation blockage, allowing virion release and cell death.

Mutations in MYB3R1 and MYB3R4 Cause Pleiotropic Developmental Defects and Preferential Down-Regulation of Multiple G2/M-Specific Genes in Arabidopsis1[C][W

PubMed Central

Haga, Nozomi; Kobayashi, Kosuke; Suzuki, Takamasa; Maeo, Kenichiro; Kubo, Minoru; Ohtani, Misato; Mitsuda, Nobutaka; Demura, Taku; Nakamura, Kenzo; Jürgens, Gerd; Ito, Masaki

2011-01-01

R1R2R3-Myb proteins represent an evolutionarily conserved class of Myb family proteins important for cell cycle regulation and differentiation in eukaryotic cells. In plants, this class of Myb proteins are believed to regulate the transcription of G2/M phase-specific genes by binding to common cis-elements, called mitosis-specific activator (MSA) elements. In Arabidopsis (Arabidopsis thaliana), MYB3R1 and MYB3R4 act as transcriptional activators and positively regulate cytokinesis by activating the transcription of KNOLLE, which encodes a cytokinesis-specific syntaxin. Here, we show that the double mutation myb3r1 myb3r4 causes pleiotropic developmental defects, some of which are due to deficiency of KNOLLE whereas other are not, suggesting that multiple target genes are involved. Consistently, microarray analysis of the double mutant revealed altered expression of many genes, among which G2/M-specific genes showed significant overrepresentation of the MSA motif and a strong tendency to be down-regulated by the double mutation. Our results demonstrate, on a genome-wide level, the importance of the MYB3R-MSA pathway for regulating G2/M-specific transcription. In addition, MYB3R1 and MYB3R4 may have diverse roles during plant development by regulating G2/M-specific genes with various functions as well as genes possibly unrelated to the cell cycle. PMID:21862669

Alternating air-medium exposure in rotating bioreactors optimizes cell metabolism in 3D novel tubular scaffold polyurethane foams.

PubMed

Tresoldi, Claudia; Stefani, Ilaria; Ferracci, Gaia; Bertoldi, Serena; Pellegata, Alessandro F; Farè, Silvia; Mantero, Sara

2017-04-26

In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications.

6,6″-Dimethyl-2,2':6',2″-terpyridine revisited: new fluorescent silver(I) helicates with in vitro antiproliferative activity via selective nucleoli targeting.

PubMed

Fik, Marta A; Gorczyński, Adam; Kubicki, Maciej; Hnatejko, Zbigniew; Fedoruk-Wyszomirska, Agnieszka; Wyszko, Eliza; Giel-Pietraszuk, Małgorzata; Patroniak, Violetta

2014-10-30

6,6″-Dimethyl-2,2':6',2″-terpyridine ligand (L) reacts in equimolar ratio with Ag(I) ions what results in formation of dinuclear double helicates, which differ in terms of framework and complexity in accordance to counterions and solvent applied. Obtained complexes were thoroughly studied in terms of their biological activity, with the positive antiproliferative outcome on three human cancer cell lines: human breast cancer (T47D), human cervical carcinoma (HeLa) and human lung cancer (A-549). Performed DNA binding experiments showed that given Ag(I) species specifically interact with DNA double helix via intercalation and were visualized by confocal microscopy to specifically bind to the nuclei. All newly synthesized helical systems exhibit promising antimicrobial activity against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacterial strains. Spectrophotometric properties were described as fulfilment of structural studies of newly presented complexes confirming their helical structure in solution. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting

NASA Technical Reports Server (NTRS)

Allen, D. L.; Linderman, J. K.; Roy, R. R.; Bigbee, A. J.; Grindeland, R. E.; Mukku, V.; Edgerton, V. R.

1997-01-01

The role of apoptosis in the elimination of myonuclei during hindlimb unloading-induced atrophy and the inhibition of apoptosis in the prevention of muscle atrophy were examined. The number of nuclei demonstrating double-stranded DNA fragmentation seen by terminal deoxynucleotidyl transferase (TDT) histochemical staining, an indicator of apoptosis, was significantly increased after 14 days of suspension. Double staining with TDT and antilaminin immunohistochemistry revealed that some TDT-positive nuclei were within the fiber lamina and were most likely myonuclei. The number of fibers containing morphologically abnormal nuclei was also significantly greater in suspended compared with control rats. Combined treatment with growth hormone and insulin-like growth factor I (GH/ IGF-I) and resistance exercise attenuated the increase in TDT-positive nuclei (approximately 26%, P > 0.05) and significantly decreased the number of fibers with morphologically abnormal nuclei. The data suggest that 1) "programmed nuclear death" contributes to the elimination of myonuclei and/or satellite cells from atrophying fibers, and 2) GH/IGF-I administration plus muscle loading ameliorates the apoptosis associated with hindlimb unloading.

Brickworx builds recurrent RNA and DNA structural motifs into medium- and low-resolution electron-density maps

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

Chojnowski, Grzegorz, E-mail: gchojnowski@genesilico.pl; Waleń, Tomasz; University of Warsaw, Banacha 2, 02-097 Warsaw

2015-03-01

A computer program that builds crystal structure models of nucleic acid molecules is presented. Brickworx is a computer program that builds crystal structure models of nucleic acid molecules using recurrent motifs including double-stranded helices. In a first step, the program searches for electron-density peaks that may correspond to phosphate groups; it may also take into account phosphate-group positions provided by the user. Subsequently, comparing the three-dimensional patterns of the P atoms with a database of nucleic acid fragments, it finds the matching positions of the double-stranded helical motifs (A-RNA or B-DNA) in the unit cell. If the target structure ismore » RNA, the helical fragments are further extended with recurrent RNA motifs from a fragment library that contains single-stranded segments. Finally, the matched motifs are merged and refined in real space to find the most likely conformations, including a fit of the sequence to the electron-density map. The Brickworx program is available for download and as a web server at http://iimcb.genesilico.pl/brickworx.« less

Periodontal treatment with the frequency-doubled Alexandrite laser in dogs

NASA Astrophysics Data System (ADS)

Rechmann, Peter; Hennig, Thomas; Reichart, Peter

2000-03-01

While earlier periodontal investigations have proved the frequency doubled Alexandrite laser to eliminate efficiently and selectively dental calculus as well as bacteria the aim of this study was to demonstrate the safety of this laser for removal of dental calculus with respect to the dental pulp. Four adult Labrador dogs were treated with a frequency doubled Alexandrite laser (laboratory prototype, q-switched, fiber guided, wavelength 377 nm, pulse duration 1 microsecond, pulse repetition rate 70 Hz, water cooling) to remove dental calculus. After performing a modified Widman flap procedure the buccal surface of nine teeth in the lower and upper right jaw were irradiated for four minutes per tooth. Three different laser fluences up to four times higher than the fluence required for calculus removal were used (1.5, 3 and 6 J/cm2). At three other sites of the right jaw deep cavities were prepared with a dental drill and filled with compomere material (DyractR, Dentsply, Germany) to serve as a positive control with regard to possible pulpal reactions. The corresponding teeth of the lower and upper left jaw served as controls. Animals were sacrificed one day, one week, four weeks and six weeks after treatment. Teeth were separated, fixed in formalin and decalcified. After embedding and sectioning the histological sections were stained and investigated by a totally blinded investigator (P.A.R). Histological investigations revealed that irradiation with the frequency doubled Alexandrite laser for periodontal treatment with fluences of 1.5 J/cm2 -- those fluences necessary for the selective removal of dental calculus and microbial plaque -- had no adverse side effects to the pulpal tissues. Moreover this pulpal safety study demonstrated that even applying fluences two or four times higher than those suggested for calculus removal do not lead to observable changes or alterations in the odontoblast cell layer or the pulpal tissues. No inflammatory reactions and no differences between irradiated and control teeth occurred, while the positive controls showed reactions in the odontoblast cell layer and the connective pulpal tissue.

The Dawn of Lead-Free Perovskite Solar Cell: Highly Stable Double Perovskite Cs2AgBiBr6 Film.

PubMed

Wu, Cuncun; Zhang, Qiaohui; Liu, Yang; Luo, Wei; Guo, Xuan; Huang, Ziru; Ting, Hungkit; Sun, Weihai; Zhong, Xinrui; Wei, Shiyuan; Wang, Shufeng; Chen, Zhijian; Xiao, Lixin

2018-03-01

Recently, lead-free double perovskites have emerged as a promising environmentally friendly photovoltaic material for their intrinsic thermodynamic stability, appropriate bandgaps, small carrier effective masses, and low exciton binding energies. However, currently no solar cell based on these double perovskites has been reported, due to the challenge in film processing. Herein, a first lead-free double perovskite planar heterojunction solar cell with a high quality Cs 2 AgBiBr 6 film, fabricated by low-pressure assisted solution processing under ambient conditions, is reported. The device presents a best power conversion efficiency of 1.44%. The preliminary efficiency and the high stability under ambient condition without encapsulation, together with the high film quality with simple processing, demonstrate promise for lead-free perovskite solar cells.

A leukocyte activation test identifies food items which induce release of DNA by innate immune peripheral blood leucocytes.

PubMed

Garcia-Martinez, Irma; Weiss, Theresa R; Yousaf, Muhammad N; Ali, Ather; Mehal, Wajahat Z

2018-01-01

Leukocyte activation (LA) testing identifies food items that induce a patient specific cellular response in the immune system, and has recently been shown in a randomized double blinded prospective study to reduce symptoms in patients with irritable bowel syndrome (IBS). We hypothesized that test reactivity to particular food items, and the systemic immune response initiated by these food items, is due to the release of cellular DNA from blood immune cells. We tested this by quantifying total DNA concentration in the cellular supernatant of immune cells exposed to positive and negative foods from 20 healthy volunteers. To establish if the DNA release by positive samples is a specific phenomenon, we quantified myeloperoxidase (MPO) in cellular supernatants. We further assessed if a particular immune cell population (neutrophils, eosinophils, and basophils) was activated by the positive food items by flow cytometry analysis. To identify the signaling pathways that are required for DNA release we tested if specific inhibitors of key signaling pathways could block DNA release. Foods with a positive LA test result gave a higher supernatant DNA content when compared to foods with a negative result. This was specific as MPO levels were not increased by foods with a positive LA test. Protein kinase C (PKC) inhibitors resulted in inhibition of positive food stimulated DNA release. Positive foods resulted in CD63 levels greater than negative foods in eosinophils in 76.5% of tests. LA test identifies food items that result in release of DNA and activation of peripheral blood innate immune cells in a PKC dependent manner, suggesting that this LA test identifies food items that result in release of inflammatory markers and activation of innate immune cells. This may be the basis for the improvement in symptoms in IBS patients who followed an LA test guided diet.

CINtec PLUS immunocytochemistry as a tool for the cytologic diagnosis of glandular lesions of the cervix uteri.

PubMed

Ravarino, Alberto; Nemolato, Sonia; Macciocu, Elena; Fraschini, Matteo; Senes, Giancarlo; Faa, Gavino; Negri, Giovanni

2012-11-01

Cytologic findings of glandular lesions of the cervix uteri are often difficult to evaluate. We studied the usefulness of CINtec PLUS p16/Ki-67 double stain (mtm laboratories, Heidelberg, Germany) for the diagnosis of glandular lesions. The study included 47 abnormal results on liquid-based cytologic tests with a subsequent histologic diagnosis of adenocarcinoma in situ or with early invasion, and 16 samples with negative results on follow-up. All samples were stained with CINtec PLUS p16/Ki-67 double stain. Of the neoplastic samples, 7 were excluded because of insufficient residual cellularity or loss of neoplastic cells. Of the samples that were adequate, 92.5% were stained with CINtec PLUS, whereas 7.5% were judged inconclusive. All inconclusive cases were at least 3 years old. Of the 16 negative samples, 15 (93.8%) stained negative and only 1 (6.2%) showed several positive clusters of cells. Our study shows that CINtec PLUS is a robust and useful tool for the diagnosis of glandular lesions of the cervix uteri.

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

PubMed Central

2014-01-01

Background Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. Results The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. Conclusion There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system. PMID:24731213

Risk of Digital Vascular Events in Scleroderma Patients Who Have Both Anticentromere and Anti-Interferon-Inducible Protein 16 Antibodies.

PubMed

McMahan, Zsuzsanna H; Wigley, Frederick M; Casciola-Rosen, Livia

2017-06-01

To evaluate whether scleroderma patients who are double-positive for anti-interferon-inducible protein 16 (anti-IFI-16) antibodies and anticentromere (anti-CENP) antibodies are at increased risk for significant digital vascular events relative to patients positive for anti-CENP antibodies alone. Sera from 165 scleroderma patients who tested positive for anti-CENP antibodies upon clinical evaluation were reassayed for both anti-CENP and anti-IFI-16 antibodies using enzyme-linked immunosorbent assay testing. Patients who were positive for anti-CENP antibodies alone were then compared to patients who were double-positive for both anti-IFI-16 and anti-CENP antibodies. The association between a history of significant digital vascular events (digital pits, ischemic digital ulcers, and/or gangrene) and double-positive antibody status was examined using chi-square tests. After completion of univariate analysis, multivariable analyses were done to adjust for clinically relevant covariates. Of the 165 anti-CENP antibody positive patients, 21 (12.7%) also had anti-IFI-16 antibodies. Patients who were double-positive for anti-CENP and anti-IFI-16 antibodies were more likely to have had digital pits, ischemic digital ulcers, and/or gangrene (P = 0.03). After adjustment for clinically relevant covariates (age, cutaneous subtype, disease duration, and smoking), double-positive patients remained at significantly higher odds of having severe Raynaud's phenomenon (odds ratio 3.5 [95% confidence interval 1.1-11.1]; P = 0.03). Scleroderma patients who are double-positive for antibodies recognizing CENP and IFI-16 are significantly more likely to have significant digital vascular events during the course of their disease. This study provides further evidence that anti-CENP and anti-IFI-16 antibodies are disease biomarkers that may be used for risk stratification of vascular events in scleroderma. © 2016, American College of Rheumatology.

Subcellular Spatial Correlation of Particle Traversal and Biological Response in Clinical Ion Beams

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

Niklas, Martin, E-mail: m.niklas@dkfz.de; German Cancer Consortium, National Center for Radiation Research in Oncology, Heidelberg Institute of Radiation Oncology, Heidelberg; Abdollahi, Amir

2013-12-01

Purpose: To report on the spatial correlation of physical track information (fluorescent nuclear track detectors, FNTDs) and cellular DNA damage response by using a novel hybrid detector (Cell-Fit-HD). Methods and Materials: The FNTDs were coated with a monolayer of human non-small cell lung carcinoma (A549) cells and irradiated with carbon ions (270.55 MeV u{sup −1}, rising flank of the Bragg peak). Phosphorylated histone variant H2AX accumulating at the irradiation-induced double-strand break site was labeled (RIF). The position and direction of ion tracks in the FNTD were registered with the location of the RIF sequence as an ion track surrogate inmore » the cell layer. Results: All RIF sequences could be related to their corresponding ion tracks, with mean deviations of 1.09 μm and −1.72 μm in position and of 2.38° in slope. The mean perpendicular between ion track and RIF sequence was 1.58 μm. The mean spacing of neighboring RIFs exhibited a regular rather than random spacing. Conclusions: Cell-Fit-HD allows for unambiguous spatial correlation studies of cell damage with respect to the intracellular ion traversal under therapeutic beam conditions.« less

Zinc Chromate Induces Chromosome Instability and DNA Double Strand Breaks in Human Lung Cells

PubMed Central

Xie, Hong; Holmes, Amie L.; Young, Jamie L.; Qin, Qin; Joyce, Kellie; Pelsue, Stephen C.; Peng, Cheng; Wise, Sandra S.; Jeevarajan, Antony S.; Wallace, William T.; Hammond, Dianne; Wise, John Pierce

2014-01-01

Hexavalent chromium Cr(VI) is a respiratory toxicant and carcinogen, with solubility playing an important role in its carcinogenic potential. Zinc chromate, a water insoluble or ‘particulate’ Cr(VI) compound, has been shown to be carcinogenic in epidemiology studies and to induce tumors in experimental animals, but its genotoxicity is poorly understood. Our study shows that zinc chromate induced concentration-dependent increases in cytotoxicity, chromosome damage and DNA double strand breaks in human lung cells. In response to zinc chromate-induced breaks, MRE11 expression was increased and ATM and ATR were phosphorylated, indicating that the DNA double strand break repair system was initiated in the cells. In addition, our data show that zinc chromate-induced double strand breaks were only observed in the G2/M phase population, with no significant amount of double strand breaks observed in G1 and S phase cells. These data will aid in understanding the mechanisms of zinc chromate toxicity and carcinogenesis. PMID:19027772

Coexpression patterns of vimentin and glial filament protein with cytokeratins in the normal, hyperplastic, and neoplastic breast.

PubMed Central

Gould, V. E.; Koukoulis, G. K.; Jansson, D. S.; Nagle, R. B.; Franke, W. W.; Moll, R.

1990-01-01

The authors studied by immunohistochemistry the intermediate filament (IF) protein profile of 66 frozen samples of breast tissue, including normal parenchyma, all variants of fibrocystic disease (FCD), fibroadenomas, cystosarcoma phylloides, and ductal and lobular carcinomas. Monoclonal antibodies (MAbs) to cytokeratins included MAb KA 1, which binds to polypeptide 5 in a complex with polypeptide 14 and recognizes preferentially myoepithelial cells; MAb KA4, which binds to polypeptides 14, 15, 16 and 19; individual MAbs to polypeptides 7, 13, and 16, 17, 18, and 19, and the MAb mixture AE1/AE3. The authors also applied three MAbs to vimentin (Vim), and three MAbs to glial filament protein (GFP). Selected samples were studied by double-label immunofluorescence microscopy and by staining sequential sections with some of the said MAbs, an MAb to alpha-smooth muscle actin, and well-characterized polyclonal antibodies for the possible coexpression of diverse types of cytoskeletal proteins. Gel electrophoresis and immunoblot analysis also were performed. All samples reacted for cytokeratins with MAbs AE1/AE3, although the reaction did not involve all cells. Monoclonal antibody KA4 stained preferentially the luminal-secretory cells in the normal breast and in FCD, whereas it stained the vast majority of cells in all carcinomas. Monoclonal antibody KA1 stained preferentially the basal-myoepithelial cells of the normal breast and FCD while staining tumor cell subpopulations in 4 of 31 carcinomas. Vimentin-positive cells were found in 8 of 12 normal breasts and in 12 of 20 FCD; in most cases, Vim-reactive cells appeared to be myoepithelial, but occasional luminal cells were also stained. Variable subpopulations of Vim-positive cells were noted in 9 of 20 ductal and in 1 of 7 lobular carcinomas. Glial filament protein-reactive cells were found in normal breast lobules and ducts and in 15 of 20 cases of FCD; with rare exceptions, GFP-reactivity was restricted to basally located, myoepithelial-appearing cells. Occasional GFP-reactive cells were found in 3 of 31 carcinomas. Evaluation of sequential sections and double-label immunofluorescence microscopy showed the coexpression of certain cytokeratins (possibly including polypeptides 14 and 17) with vimentin and alpha-smooth muscle actin together with GFP in some myoepithelial cells. The presence of GFP in myoepithelial cells was confirmed by gel electrophoresis and immunoblotting. Our results indicate that coexpression of cytokeratin with vimentin and/or GFP is comparatively frequent in normal basal-myoepithelial cells of the breast.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1700618

High-rate lithium/manganese dioxide batteries; the double cell concept

NASA Astrophysics Data System (ADS)

Drews, Jürgen; Wolf, Rüdiger; Fehrmann, Gerd; Staub, Roland

An implantable defibrillator battery has to provide pulse-power capabilities as well as high energy density. Low self-discharge rates are mandatory and an ability to check the state of charge is required. To accomplish these requirements, a lithium/manganese dioxide battery with a modified active cathode mass has been developed. Usage of a double cell design increases significantly the battery performance within an implantable defibrillator. The design features of a high-rate, pulse-power, manganese dioxide double cell are described.

Holographic study of non-affine deformation in copper foam with a negative Poisson's ratio of -0.8

NASA Technical Reports Server (NTRS)

Chen, C. P.; Lakes, R. S.

1993-01-01

While conventional foams have positive Poisson's ratios (become smaller in cross-section when stretched and larger when compressed), foam materials have recently been defined which possess 'reentrant' cellular architectures; in these, inwardly-protruding cell ribs are responsible for negative Poisson's ratio behavior, yielding greater resilience than conventional foams. Double-exposure holographic interferometry is presently used to examine the microdeformation of a reentrant copper foam. Attention is given to the nonaffine (inhomogeneous) deformation of this foam.

A rational approach for cancer stem-like cell isolation and characterization using CD44 and prominin-1(CD133) as selection markers

PubMed Central

Lee, Yi-Jen; Wu, Chang-Cheng; Li, Jhy-Wei; Ou, Chien-Chih; Hsu, Shih-Chung; Tseng, Hsiu-Hsueh; Kao, Ming-Ching; Liu, Jah-Yao

2016-01-01

The availability of adequate cancer stem cells or cancer stem-like cell (CSC) is important in cancer study. From ovarian cancer cell lines, SKOV3 and OVCAR3, we induced peritoneal ascites tumors in immunodeficient mice. Among the cells (SKOV3.PX1 and OVCAR3.PX1) from those tumors, we sorted both CD44 and CD133 positive cells (SKOV3.PX1_133+44+, OVCAR3.PX1_133+44+), which manifest the characteristics of self-renewal, multi-lineage differentiation, chemoresistance and tumorigenicity, those of cancer stem-like cells (CSLC). Intraperitoneal transplantation of these CD44 and CD133 positive cells resulted in poorer survival in the engrafted animals. Clinically, increased CD133 expression was found in moderately and poorly differentiated (grade II and III) ovarian serous cystadenocarcinomas. The ascites tumor cells from human ovarian cancers demonstrated more CD133 and CD44 expressions than those from primary ovarian or metastatic tumors and confer tumorigenicity in immunodeficient mice. Compared to their parental cells, the SKOV3.PX1_133+44+ and OVCAR3.PX1_133+44+ cells uniquely expressed 5 CD markers (CD97, CD104, CD107a, CD121a, and CD125). Among these markers, CD97, CD104, CD107a, and CD121a are significantly more expressed in the CD133+ and CD44+ double positive cells of human ovarian ascites tumor cells (Ascites_133+44+) than those from primary ovarian or metastatic tumors. The cancer stem-like cells were enriched from 3% to more than 70% after this manipulation. This intraperitoneal enrichment of cancer stem-like cells, from ovarian cancer cell lines or primary ovarian tumor, potentially provides an adequate amount of ovarian cancer stem-like cells for the ovarian cancer study and possibly benefits cancer therapy. PMID:27655682

Recent advances in the cell biology of aging.

PubMed

Hayflick, L

1980-01-01

Cultured normal human and animal cells are predestined to undergo irreversible functional decrements that mimic age changes in the whole organism. When normal human embryonic fibroblasts are cultured in vitro, 50 +/- 10 population doublings occur. This maximum potential is diminished in cells derived from older donors and appears to be inversely proportional to their age. The 50 population doubling limit can account for all cells produced during a lifetime. The limitation on doubling potential of cultured normal cells is also expressed in vivo when serial transplants are made. There may be a direct correlation between the mean maximum life spans of several species and the population doubling potential of their cultured cells. A plethora of functional decrements occurs in cultured normal cells as they approach their maximum division capability. Many of these decrements are similar to those occurring in intact animals as they age. We have concluded that these functional decrements expressed in vitro, rather than cessation of cell division, are the essential contributors to age changes in intact animals. Thus, the study of events leading to functional losses in cultured normal cells may provide useful insights into the biology of aging.

Involvement of hypothalamus autoimmunity in patients with autoimmune hypopituitarism: role of antibodies to hypothalamic cells.

PubMed

De Bellis, A; Sinisi, A A; Pane, E; Dello Iacovo, A; Bellastella, G; Di Scala, G; Falorni, A; Giavoli, C; Gasco, V; Giordano, R; Ambrosio, M R; Colao, A; Bizzarro, A; Bellastella, A

2012-10-01

Antipituitary antibodies (APA) but not antihypothalamus antibodies (AHA) are usually searched for in autoimmune hypopituitarism. Our objective was to search for AHA and characterize their hypothalamic target in patients with autoimmune hypopituitarism to clarify, on the basis of the cells stained by these antibodies, the occurrence of autoimmune subclinical/clinical central diabetes insipidus (CDI) and/or possible joint hypothalamic contribution to their hypopituitarism. We conducted a cross-sectional cohort study. Ninety-five APA-positive patients with autoimmune hypopituitarism, 60 without (group 1) and 35 with (group 2) lymphocytic hypophysitis, were studied in comparison with 20 patients with postsurgical hypopituitarism and 50 normal subjects. AHA by immunofluorescence and posterior pituitary function were evaluated; then AHA-positive sera were retested by double immunofluorescence to identify the hypothalamic cells targeted by AHA. AHA were detected at high titer in 12 patients in group 1 and in eight patients in group 2. They immunostained arginine vasopressin (AVP)-secreting cells in nine of 12 in group 1 and in four of eight in group 2. All AVP cell antibody-positive patients presented with subclinical/clinical CDI; in contrast, four patients with GH/ACTH deficiency but with APA staining only GH-secreting cells showed AHA targeting CRH- secreting cells. The occurrence of CDI in patients with lymphocytic hypophysitis seems due to an autoimmune hypothalamic involvement rather than an expansion of the pituitary inflammatory process. To search for AVP antibody in these patients may help to identify those of them prone to develop an autoimmune CDI. The detection of AHA targeting CRH-secreting cells in some patients with GH/ACTH deficiency but with APA targeting only GH-secreting cells indicates that an autoimmune aggression to hypothalamus is jointly responsible for their hypopituitarism.

Constant Flux of Spatial Niche Partitioning through High-Resolution Sampling of Magnetotactic Bacteria.

PubMed

He, Kuang; Gilder, Stuart A; Orsi, William D; Zhao, Xiangyu; Petersen, Nikolai

2017-10-15

Magnetotactic bacteria (MTB) swim along magnetic field lines in water. They are found in aquatic habitats throughout the world, yet knowledge of their spatial and temporal distribution remains limited. To help remedy this, we took MTB-bearing sediment from a natural pond, mixed the thoroughly homogenized sediment into two replicate aquaria, and then counted three dominant MTB morphotypes (coccus, spirillum, and rod-shaped MTB cells) at a high spatiotemporal sampling resolution: 36 discrete points in replicate aquaria were sampled every ∼30 days over 198 days. Population centers of the MTB coccus and MTB spirillum morphotypes moved in continual flux, yet they consistently inhabited separate locations, displaying significant anticorrelation. Rod-shaped MTB were initially concentrated toward the northern end of the aquaria, but at the end of the experiment, they were most densely populated toward the south. The finding that the total number of MTB cells increased over time during the experiment argues that population reorganization arose from relative changes in cell division and death and not from migration. The maximum net growth rates were 10, 3, and 1 doublings day -1 and average net growth rates were 0.24, 0.11, and 0.02 doublings day -1 for MTB cocci, MTB spirilla, and rod-shaped MTB, respectively; minimum growth rates for all three morphotypes were -0.03 doublings day -1 Our results suggest that MTB cocci and MTB spirilla occupy distinctly different niches: their horizontal positioning in sediment is anticorrelated and under constant flux. IMPORTANCE Little is known about the horizontal distribution of magnetotactic bacteria in sediment or how the distribution changes over time. We therefore measured three dominant magnetotactic bacterium morphotypes at 36 places in two replicate aquaria each month for 7 months. We found that the spatial positioning of population centers changed over time and that the two most abundant morphotypes (MTB cocci and MTB spirilla) occupied distinctly different niches in the aquaria. Maximum and average growth and death rates were quantified for each of the three morphotypes based on 72 sites that were measured six times. The findings provided novel insight into the differential behavior of noncultured magnetotactic bacteria. Copyright © 2017 American Society for Microbiology.

Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions.

PubMed

Al-Mulla, Hawaa M N; Turrell, Lauren; Smith, Nicola M; Payne, Luke; Baliji, Surendranath; Züst, Roland; Thiel, Volker; Baker, Susan C; Siddell, Stuart G; Neuman, Benjamin W

2014-04-01

Positive-stranded viruses synthesize their RNA in membrane-bound organelles, but it is not clear how this benefits the virus or the host. For coronaviruses, these organelles take the form of double-membrane vesicles (DMVs) interconnected by a convoluted membrane network. We used electron microscopy to identify murine coronaviruses with mutations in nsp3 and nsp14 that replicated normally while producing only half the normal amount of DMVs under low-temperature growth conditions. Viruses with mutations in nsp5 and nsp16 produced small DMVs but also replicated normally. Quantitative reverse transcriptase PCR (RT-PCR) confirmed that the most strongly affected of these, the nsp3 mutant, produced more viral RNA than wild-type virus. Competitive growth assays were carried out in both continuous and primary cells to better understand the contribution of DMVs to viral fitness. Surprisingly, several viruses that produced fewer or smaller DMVs showed a higher fitness than wild-type virus at the reduced temperature, suggesting that larger and more numerous DMVs do not necessarily confer a competitive advantage in primary or continuous cell culture. For the first time, this directly demonstrates that replication and organelle formation may be, at least in part, studied separately during infection with positive-stranded RNA virus. IMPORTANCE The viruses that cause severe acute respiratory syndrome (SARS), poliomyelitis, and hepatitis C all replicate in double-membrane vesicles (DMVs). The big question about DMVs is why they exist in the first place. In this study, we looked at thousands of infected cells and identified two coronavirus mutants that made half as many organelles as normal and two others that made typical numbers but smaller organelles. Despite differences in DMV size and number, all four mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelles, we carried out competitive fitness experiments. None of these viruses was found to be significantly less fit than wild-type, and two were actually fitter in tests in two kinds of cells. This suggests that viruses have evolved to have tremendous plasticity in the ability to form membrane-associated replication complexes and that large and numerous DMVs are not exclusively associated with efficient coronavirus replication.

Diphosphates at the 5' end of the positive strand of yeast L-A double-stranded RNA virus as a molecular self-identity tag.

PubMed

Fujimura, Tsutomu; Esteban, Rosa

2016-10-01

The 5'end of RNA conveys important information on self-identity. In mammalian cells, double-stranded RNA (dsRNA) with 5'di- or triphosphates generated during virus infection is recognized as foreign and elicits the host innate immune response. Here, we analyze the 5' ends of the dsRNA genome of the yeast L-A virus. The positive strand has largely diphosphates with a minor amount of triphosphates, while the negative strand has only diphosphates. Although the virus can produce capped transcripts by cap snatching, neither strand carried a cap structure, suggesting that only non-capped transcripts serve as genomic RNA for encapsidation. We also found that the 5' diphosphates of the positive but not the negative strand within the dsRNA genome are crucial for transcription in vitro. Furthermore, the presence of a cap structure in the dsRNA abrogated its template activity. Given that the 5' diphosphates of the transcripts are also essential for cap acquisition and that host cytosolic RNAs (mRNA, rRNA, and tRNA) are uniformly devoid of 5' pp-structures, the L-A virus takes advantage of its 5' terminal diphosphates, using them as a self-identity tag to propagate in the host cytoplasm. © 2016 John Wiley & Sons Ltd.

Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases.

PubMed

Nerys-Junior, Arildo; Costa, Lendel C; Braga-Dias, Luciene P; Oliveira, Márcia; Rossi, Atila D; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S; Tanuri, Amilcar

2014-03-01

Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

100 positive double-blind studies: enough or too little?

NASA Astrophysics Data System (ADS)

Tuner, Jan; Hode, Lars

2000-06-01

A major argument among the opponents of laser therapy has been the absence of scientific documentation. This was a valid position in the 80s and partly in the 90s. But today, is this still a sound argument. There are more than 2,000 published studies in the field, including meeting abstracts and anecdotal reports. The vast majority of these papers reports positive effects of LLLT in vitro and in vivo. It is fair to argue that negative results are less prone to be published, but certainly more than 80 percent of the published studies are positive. In the field of dentistry, for instance, the positive percentage is well above 90 percent. The present literature study will look at the heart of the positive documentation: the positive double blind studies. It may come as a surprise to many critics that there are more than 100 positive double blind studies in the field laser therapy. This is a god base for a further understanding of the effects of low level laser in the clinical setting. We must, however, be as critical as the sceptics themselves in order to obtain a constructive dialogue between 'attorneys' and sceptics. In this paper, a critical review of 100 positive double blind studies will be presented.

Synergetic effect of double-step blocking layer for the perovskite solar cell

NASA Astrophysics Data System (ADS)

Kim, Jinhyun; Hwang, Taehyun; Lee, Sangheon; Lee, Byungho; Kim, Jaewon; Kim, Jaewook; Gil, Bumjin; Park, Byungwoo

2017-10-01

In an organometallic CH3NH3PbI3 (MAPbI3) perovskite solar cell, we have demonstrated a vastly compact TiO2 layer synthesized by double-step deposition, through a combination of sputter and solution deposition to minimize the electron-hole recombination and boost the power conversion efficiency. As a result, the double-step strategy allowed outstanding transmittance of blocking layer. Additionally, crystallinity and morphology of the perovskite film were significantly modified, provoking enhanced photon absorption and solar cell performance with the reduced recombination rate. Thereby, this straightforward double-step strategy for the blocking layer exhibited 12.31% conversion efficiency through morphological improvements of each layer.

High sensitive position-dependent photodetection observed in Cu-covered Si nanopyramids

NASA Astrophysics Data System (ADS)

Mei, Chunlian; Zou, Jiaren; Huang, Xu; Zou, Bugao; Zhou, Peiqi; Gan, Zhikai; Hu, Jieqiong; Zhang, Qian; Wang, Hui

2018-05-01

Silicon nanopyramids with the excellent ability of light absorption have been mostly reported in solar cells. Here, we report an obviously enhanced lateral photovoltaic effect (LPE) in copper-nanoparticle-covered random Si nanopyramids (Cu@Si-pyramid). Remarkable photoelectric responses are achieved in broadband from 405 to 780 nm. Furthermore, a prominent LPE is double-enhanced from 74.0 to 157.9 mV mm‑1 when the linear region decreases from 3 to 1 mm. Finite-difference time-domain simulation is applied to investigate the origin of the exceptional results. This work declares a position-sensitive property of Si-nanopyramid systems and proposes promising applications to photodetections based on LPE.

High sensitive position-dependent photodetection observed in Cu-covered Si nanopyramids.

PubMed

Mei, Chunlian; Zou, Jiaren; Huang, Xu; Zou, Bugao; Zhou, Peiqi; Gan, Zhikai; Hu, Jieqiong; Zhang, Qian; Wang, Hui

2018-05-18

Silicon nanopyramids with the excellent ability of light absorption have been mostly reported in solar cells. Here, we report an obviously enhanced lateral photovoltaic effect (LPE) in copper-nanoparticle-covered random Si nanopyramids (Cu@Si-pyramid). Remarkable photoelectric responses are achieved in broadband from 405 to 780 nm. Furthermore, a prominent LPE is double-enhanced from 74.0 to 157.9 mV mm -1 when the linear region decreases from 3 to 1 mm. Finite-difference time-domain simulation is applied to investigate the origin of the exceptional results. This work declares a position-sensitive property of Si-nanopyramid systems and proposes promising applications to photodetections based on LPE.

Myosins XI-K, XI-1, and XI-2 are required for development of pavement cells, trichomes, and stigmatic papillae in Arabidopsis

PubMed Central

2012-01-01

Background The positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport. Results Simultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils. Conclusions We conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes. PMID:22672737

Cooperative function of Pdx1 and Oc1 in multipotent pancreatic progenitors impacts postnatal islet maturation and adaptability.

PubMed

Kropp, Peter A; Dunn, Jennifer C; Carboneau, Bethany A; Stoffers, Doris A; Gannon, Maureen

2018-04-01

The transcription factors pancreatic and duodenal homeobox 1 (Pdx1) and onecut1 (Oc1) are coexpressed in multipotent pancreatic progenitors (MPCs), but their expression patterns diverge in hormone-expressing cells, with Oc1 expression being extinguished in the endocrine lineage and Pdx1 being maintained at high levels in β-cells. We previously demonstrated that cooperative function of these two factors in MPCs is necessary for proper specification and differentiation of pancreatic endocrine cells. In those studies, we observed a persistent decrease in expression of the β-cell maturity factor MafA. We therefore hypothesized that Pdx1 and Oc1 cooperativity in MPCs impacts postnatal β-cell maturation and function. Here our model of Pdx1-Oc1 double heterozygosity was used to investigate the impact of haploinsufficiency for both of these factors on postnatal β-cell maturation, function, and adaptability. Examining mice at postnatal day (P) 14, we observed alterations in pancreatic insulin content in both Pdx1 heterozygotes and double heterozygotes. Gene expression analysis at this age revealed significantly decreased expression of many genes important for glucose-stimulated insulin secretion (e.g., Glut2, Pcsk1/2, Abcc8) exclusively in double heterozygotes. Analysis of P14 islets revealed an increase in the number of mixed islets in double heterozygotes. We predicted that double-heterozygous β-cells would have an impaired ability to respond to stress. Indeed, we observed that β-cell proliferation fails to increase in double heterozygotes in response to either high-fat diet or placental lactogen. We thus report here the importance of cooperation between regulatory factors early in development for postnatal islet maturation and adaptability.

[Simultaneous staining with fluorescein diacetate-propidium iodide to determine isolated cochlear outer hair cell viability of guinea pig].

PubMed

Yu, Q; Shi, H; Wang, J

1995-01-01

A simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is discribed for use in the determination of isolated cochlear outer hair cell viability. With exciter light, viable cells fluoresce bright green, while nonviable cells are bright red. In cell culture and cytotoxicity studies, double-staining with FDA-PI is a accurate method to discriminate between live and nonviable cells.

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

PubMed

Son, Kyung-No; Liang, Zhiguo; Lipton, Howard L

2015-09-01

Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Dawn of Lead‐Free Perovskite Solar Cell: Highly Stable Double Perovskite Cs2AgBiBr6 Film

PubMed Central

Wu, Cuncun; Zhang, Qiaohui; Liu, Yang; Luo, Wei; Guo, Xuan; Huang, Ziru; Ting, Hungkit; Sun, Weihai; Zhong, Xinrui; Wei, Shiyuan

2017-01-01

Abstract Recently, lead‐free double perovskites have emerged as a promising environmentally friendly photovoltaic material for their intrinsic thermodynamic stability, appropriate bandgaps, small carrier effective masses, and low exciton binding energies. However, currently no solar cell based on these double perovskites has been reported, due to the challenge in film processing. Herein, a first lead‐free double perovskite planar heterojunction solar cell with a high quality Cs2AgBiBr6 film, fabricated by low‐pressure assisted solution processing under ambient conditions, is reported. The device presents a best power conversion efficiency of 1.44%. The preliminary efficiency and the high stability under ambient condition without encapsulation, together with the high film quality with simple processing, demonstrate promise for lead‐free perovskite solar cells. PMID:29593974

Cisplatin selects for stem-like cells in osteosarcoma by activating Notch signaling

PubMed Central

Yang, Jian; Gao, Tian; Simões, Bruno M.; Eyre, Rachel; Guo, Weichun; Clarke, Robert B.

2016-01-01

Notch signaling regulates normal stem cells and is also thought to regulate cancer stem cells (CSCs). Recent data indicate that Notch signaling plays a role in the development and progression of osteosarcoma, however the regulation of Notch in chemo-resistant stem-like cells has not yet been fully elucidated. In this study we generated cisplatin-resistant osteosarcoma cells by treating them with sub-lethal dose of cisplatin, sufficient to induce DNA damage responses. Cisplatin-resistant osteosarcoma cells exhibited lower proliferation, enhanced spheroid formation and more mesenchymal characteristics than cisplatin-sensitive cells, were enriched for Stro-1+/CD117+ cells and showed increased expression of stem cell-related genes. A similar effect was observed in vivo, and in addition in vivo tumorigenicity was enhanced during serial transplantation. Using several publicly available datasets, we identified that Notch expression was closely associated with osteosarcoma stem cells and chemotherapy resistance. We confirmed that cisplatin-induced enrichment of osteosarcoma stem cells was mediated through Notch signaling in vitro, and immunohistochemistry showed that cleaved Notch1 (NICD1) positive cells were significantly increased in a relapsed xenograft which had received cisplatin treatment. Furthermore, pretreatment with a γ-secretase inhibitor (GSI) to prevent Notch signalling inhibited cisplatin-enriched osteosarcoma stem cell activity in vitro, including Stro-1+/CD117+ double positive cells and spheroid formation capacity. The Notch inhibitor DAPT also prevented tumor recurrence in resistant xenograft tumors. Overall, our results show that cisplatin induces the enrichment of osteosarcoma stem-like cells through Notch signaling, and targeted inactivation of Notch may be useful for the elimination of CSCs and overcoming drug resistance. PMID:27102300

Abnormal thymic maturation and lymphoproliferation in MRL-Fas lpr/lpr mice can be partially reversed by synthetic oligonucleotides: implications for systemic lupus erythematosus and autoimmune lymphoproliferative syndrome.

PubMed

Ashman, R F; Singh, N; Lenert, P S

2017-06-01

MRL-Fas lpr/lpr mice represent an excellent animal model for studying non-malignant lymphoproliferation, regeneration and systemic autoimmunity. Retro-transposon insertion into the second intron of the pro-apoptotic Fas gene appears to be responsible for both lymphoproliferation and autoimmunity, while other genes are more likely to contribute to the regenerative healing characteristic of this mouse strain. Previous studies have shown that neonatal thymectomy can halt the development of abnormal lymphoproliferation. Whereas at four weeks of age primary and secondary lymphoid organs appear to be grossly intact, vigorous lymphoproliferation and autoantibody production subsequently ensues. This is first noticeable at six weeks of age, at which time lymph nodes, spleens and thymuses, but not the bone marrow, become infiltrated with abnormal B220 + CD3 + CD4 - CD8 - T cells. Around the same time, thymuses show a significant drop in CD4 + CD8 + double-positive T cells generating an abnormal ratio between double-positive and single-positive thymocytes. The objective of current study was to evaluate the effect of synthetic oligonucleotides-toll-like receptor antagonists on early lymphoid development in this strain of mice. Herein, we demonstrate the ability of synthetic oligonucleotides made with the nuclease-resistant phosphorothioate backbone to partially reverse abnormal lymphoproliferation and thymic involution in pre-diseased MRL-Fas lpr/lpr mice when administered intraperitoneally starting from week four of age. This curative effect of oligonucleotides was primary sequence/secondary oligonucleotide structure-independent, suggesting an effect through the toll-like receptor 7. A similar approach may potentially benefit patients with autoimmune lymphoproliferative syndrome who, like MRL-Fas lpr/lpr mice, carry a mutation in the Fas gene.

Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions

PubMed Central

Rimkute, Lina; Kraujalis, Tadas; Snipas, Mindaugas; Palacios-Prado, Nicolas; Jotautis, Vaidas; Skeberdis, Vytenis A.; Bukauskas, Feliksas F.

2018-01-01

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pHi) and cytosolic magnesium ion concentration ([Mg2+]i), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pHi and [Mg2+]i affect junctional conductance (gj) in an interdependent manner; in other words, intracellular acidification cause increase or decay in gj depending on whether [Mg2+]i is high or low, respectively, and intracellular alkalization cause reduction in gj independently of [Mg2+]i. Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pHi and [Mg2+]i. Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pHi and [Mg2+]i. Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg2+]i, while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36*E8Q lost the initial increase of gj at low [Mg2+]i and double mutation lost the sensitivity to high [Mg2+]i. These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg2+ and H+ ions. PMID:29706896

Pilot, double-blind, randomized, placebo-controlled clinical trial of the supplement food Nyaditum resae® in adults with or without latent TB infection: Safety and immunogenicity

PubMed Central

Montané, Eva; Barriocanal, Ana Maria; Arellano, Ana Lucía; Valderrama, Angelica; Sanz, Yolanda; Perez-Alvarez, Nuria; Cardona, Paula; Vilaplana, Cristina

2017-01-01

Background Nyaditum resae® (NR) is a galenic preparation of heat-killed Mycobacterium manresensis, a new species of the fortuitum complex, that is found in drinkable water, and that has demonstrated to protect against the development of active TB in a murine experimental model that develop human-like lesions. Methods Double-blind, randomized, placebo-controlled Clinical Trial (51 volunteers included). Two different doses of NR and a placebo were tested, the randomization was stratified by Latent Tuberculosis Infection (LTBI)-positive (n = 21) and LTBI-negative subjects (n = 30). Each subject received 14 drinkable daily doses for 2 weeks. Results All patients completed the study. The 46.3% of the overall reported adverse events (AE) were considered related to the investigational treatment. None of them were severe (94% were mild and 6% moderate). No statistical differences were found when comparing the median number of AE between the placebo group and both treatment groups. The most common AE reported were gastrointestinal events, most frequently mild abdominal pain and increase in stool frequency. Regarding the immunogenic response, both LTBI-negative and LTBI-positive volunteers treated with NR experienced a global increase on the Treg response, showed both in the population of CD25+CD39-, mainly effector Treg cells, or CD25+CD39+ memory PPD-specific Treg cells. Conclusion This clinical trial demonstrates an excellent tolerability profile of NR linked to a significant increase in the population of specific effector and memory Tregs in the groups treated with NR in both LTBI-positive and negative subjects. NR shows a promising profile to be used to reduce the risk of active TB. PMID:28182700

Pilot, double-blind, randomized, placebo-controlled clinical trial of the supplement food Nyaditum resae® in adults with or without latent TB infection: Safety and immunogenicity.

PubMed

Montané, Eva; Barriocanal, Ana Maria; Arellano, Ana Lucía; Valderrama, Angelica; Sanz, Yolanda; Perez-Alvarez, Nuria; Cardona, Paula; Vilaplana, Cristina; Cardona, Pere-Joan

2017-01-01

Nyaditum resae® (NR) is a galenic preparation of heat-killed Mycobacterium manresensis, a new species of the fortuitum complex, that is found in drinkable water, and that has demonstrated to protect against the development of active TB in a murine experimental model that develop human-like lesions. Double-blind, randomized, placebo-controlled Clinical Trial (51 volunteers included). Two different doses of NR and a placebo were tested, the randomization was stratified by Latent Tuberculosis Infection (LTBI)-positive (n = 21) and LTBI-negative subjects (n = 30). Each subject received 14 drinkable daily doses for 2 weeks. All patients completed the study. The 46.3% of the overall reported adverse events (AE) were considered related to the investigational treatment. None of them were severe (94% were mild and 6% moderate). No statistical differences were found when comparing the median number of AE between the placebo group and both treatment groups. The most common AE reported were gastrointestinal events, most frequently mild abdominal pain and increase in stool frequency. Regarding the immunogenic response, both LTBI-negative and LTBI-positive volunteers treated with NR experienced a global increase on the Treg response, showed both in the population of CD25+CD39-, mainly effector Treg cells, or CD25+CD39+ memory PPD-specific Treg cells. This clinical trial demonstrates an excellent tolerability profile of NR linked to a significant increase in the population of specific effector and memory Tregs in the groups treated with NR in both LTBI-positive and negative subjects. NR shows a promising profile to be used to reduce the risk of active TB.

Pneumocystis jirovecii colonisation in HIV-positive and HIV-negative subjects in Cameroon.

PubMed

Riebold, D; Enoh, D O; Kinge, T N; Akam, W; Bumah, M K; Russow, K; Klammt, S; Loebermann, M; Fritzsche, C; Eyong, J E; Eppel, G; Kundt, G; Hemmer, C J; Reisinger, E C

2014-06-01

To determine the prevalence of Pneumocystis pneumonia (PCP), a major opportunistic infection in AIDS patients in Europe and the USA, in Cameroon. Induced sputum samples from 237 patients without pulmonary symptoms (126 HIV-positive and 111 HIV-negative outpatients) treated at a regional hospital in Cameroon were examined for the prevalence of Pneumocystis jirovecii by specific nested polymerase chain reaction (nPCR) and staining methods. CD 4 counts and the history of antiretroviral therapy of the subjects were obtained through the ESOPE database system. Seventy-five of 237 study participants (31.6%) were colonised with Pneumocystis, but none showed active PCP. The Pneumocystis colonisation rate in HIV-positive subjects was more than double that of HIV-negative subjects (42.9% vs. 18.9%, P < 0.001). In the HIV-positive group, the colonisation rate corresponds to the reduction in the CD 4 lymphocyte counts. Subjects with CD 4 counts >500 cells/μl were colonised at a rate of 20.0%, subjects with CD 4 counts between 200 and 500 cells/μl of 42.5%, and subjects with CD 4 counts <200 cells/μl of 57.1%. Colonisation with Pneumocystis in Cameroon seems to be comparable to rates found in Western Europe. Prophylactic and therapeutic measures against Pneumocystis should be taken into account in HIV care in western Africa. © 2014 John Wiley & Sons Ltd.

Temporal Expression of Bim Limits the Development of Agonist-Selected Thymocytes and Skews Their TCRβ Repertoire

PubMed Central

Li, Kun-Po; Fahnrich, Anke; Roy, Eron; Cuda, Carla M.; Grimes, H. Leighton; Perlman, Harris R.; Kalies, Kathrin; Hildeman, David A.

2017-01-01

CD8αα TCRαβ+ intestinal intraepithelial lymphocytes play a critical role in promoting intestinal homeostasis, although mechanisms controlling their development and peripheral homeostasis remain unclear. In this study, we examined the spatiotemporal role of Bim in the thymic selection of CD8αα precursors and the fate of these cells in the periphery. We found that T cell–specific expression of Bim during early/cortical, but not late/medullary, thymic development controls the agonist selection of CD8αα precursors and limits their private TCRβ repertoire. During this process, agonist-selected double-positive cells lose CD4/8 coreceptor expression and masquerade as double-negative (DN) TCRαβhi thymocytes. Although these DN thymocytes fail to re-express coreceptors after OP9-DL1 culture, they eventually mature and accumulate in the spleen where TCR and IL-15/STAT5 signaling promotes their conversion to CD8αα cells and their expression of gut-homing receptors. Adoptive transfer of splenic DN cells gives rise to CD8αα cells in the gut, establishing their precursor relationship in vivo. Interestingly, Bim does not restrict the IL-15–driven maturation of CD8αα cells that is critical for intestinal homeostasis. Thus, we found a temporal and tissue-specific role for Bim in limiting thymic agonist selection of CD8αα precursors and their TCRβ repertoire, but not in the maintenance of CD8αα intraepithelial lymphocytes in the intestine. PMID:27852740

Anti-tumor function of double-promoter regulated adenovirus carrying SEA gene, in the treatment of bladder cancer.

PubMed

Hu, Jianpeng; Xuan, Xujun; Han, Conghui; Hao, Lin; Zhang, Peiying; Chen, Meng; He, Houguang; Fan, Tao; Dong, Binzheng

2012-03-01

To construct an adenovirus carrying SEA gene and regulated by telomerase reverse transcriptase (hTERT) and hypoxia-inducible factor (HIF) promoters and investigate its anti-tumor function in vitro, as well as its role in lymphocyte production. hTERT and HIF genes were cloned into adenovirus E1A and E1B shuttle plasmids. The control vector for SEA gene expression is under the regulation of CMV and SV40 promoters. Double regulation was obtained through homologous recombination. The positive clones of replicable adenovirus H2-SEA-Ad were selected by plaque assay. The adenovirus was purified, titrated, and DNA was verified by PCR. The obtained virus was used to infect EJ bladder tumor cells and the SEA Mrna, and protein expression was measured by RT-PCR, western blot, and immunofluorescence microscopy, respectively. Co-culture of lymphocytes and tumor cells was observed dynamically under microscope. The construction of shuttle plasmid p315-CSS-SEA was confirmed by PCR and DNA sequencing. Insertion of superantigen SEA gene in adenovirus (H2-SEA-Ad.SEA) was obtained by homologous recombination. In lymphocytes and tumor cell co-culture, the number of viable tumor cells in test groups was significantly lower than that in control group after 12, 24, and 48 h of treatment. Production of interleukin-2, interleukin-4, and tumor necrosis factor were higher in test groups than in control group. Expression of SEA gene in bladder tumor cells by adenoviral vector caused reduced tumor cell proliferation, as well as stimulation of inflammatory cytokine productions in co-cultures with lymphocytes.

Mode Transition of RNA Trap by Electric and Hydraulic Force Field in Microfluidic Taper Shape Channel

NASA Astrophysics Data System (ADS)

Takamura, Yuzuru; Ueno, Kunimitsu; Nagasaka, Wako; Tomizawa, Yuichi; Tamiya, Eiichi

2007-03-01

We have discovered a phenomenon of accumulation of DNA near the constricted position of a microfluidic chip with taper shaped channel when both hydro pressure and electric field are applied in opposite directions. However, RNA has not been able to trap so far, unlike huge and uniformly double stranded DNA molecules, RNAs are smaller in size and single stranded with complicated conformation like blocks in lysed cell solution. In this paper, we will report not only large but also small RNA (100˜10b) are successfully trapped in relatively large microfluidic taper shape channel (width >10um). RNA are trapped in circular motion near the constricted position of taper shape channel, and the position and shape of the trapped RNA are controlled and make mode transition by changing the hydraulic and the electric force. Using this technique, smaller size molecule can be trapped in larger micro fluidic structure compared to the trap using dielectrophoresis. This technique is expected to establish easy and practical device as a direct total RNA extraction tool from living cells or tissues.

Positive zeta potential of a negatively charged semi-permeable plasma membrane

NASA Astrophysics Data System (ADS)

Sinha, Shayandev; Jing, Haoyuan; Das, Siddhartha

2017-08-01

The negative charge of the plasma membrane (PM) severely affects the nature of moieties that may enter or leave the cells and controls a large number of ion-interaction-mediated intracellular and extracellular events. In this letter, we report our discovery of a most fascinating scenario, where one interface (e.g., membrane-cytosol interface) of the negatively charged PM shows a positive surface (or ζ) potential, while the other interface (e.g., membrane-electrolyte interface) still shows a negative ζ potential. Therefore, we encounter a completely unexpected situation where an interface (e.g., membrane-cytosol interface) that has a negative surface charge density demonstrates a positive ζ potential. We establish that the attainment of such a property by the membrane can be ascribed to an interplay of the nature of the membrane semi-permeability and the electrostatics of the electric double layer established on either side of the charged membrane. We anticipate that such a membrane property can lead to such capabilities of the cell (in terms of accepting or releasing certain kinds of moieties as well regulating cellular signaling) that was hitherto inconceivable.

How do culture media influence in vitro perivascular cell behavior?

PubMed

Huber, Birgit; Volz, Ann-Cathrin; Kluger, Petra Juliane

2015-12-01

Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries. © 2015 International Federation for Cell Biology.

Sol-gel-Derived nano-sized double layer anti-reflection coatings (SiO2/TiO2) for low-cost solar cell fabrication.

PubMed

Lee, Seung Jun; Hur, Man Gyu; Yoon, Dae Ho

2013-11-01

We investigate nano-sized double layer anti-reflection coatings (ARCs) using a TiO2 and SiO2 sol-gel solution process for mono-crystalline silicon solar cells. The process can be easily adapted for spraying sol-gel coatings to reduce manufacturing cost. The spray-coated SiO2/TiO2 nano-sized double layer ARCs were deposited on mono-crystalline silicon solar cells, and they showed good optical properties. The spray coating process is a lower-cost fabrication process for large-scale coating than vacuum deposition processes such as PECVD. The measured average optical reflectance (300-1200 nm) was about approximately 8% for SiO2/TiO2 nano-sized double layer ARCs. The electrical parameters of a mono-crystalline silicon solar cell and reflection losses show that the SiO2/TiO2 stacks can improve cell efficiency by 0.2% compared to a non-coated mono-crystalline silicon solar cell. In the results, good correlation between theoretical and experimental data was obtained. We expect that the sol-gel spray-coated mono-crystalline silicon solar cells have high potential for low-cost solar cell fabrication.

Irradiation inhibits vascular anastomotic stenosis in a canine model.

PubMed

Saito, Takeshi; Iguchi, Atsushi; Tabayashi, Koichi

2009-08-01

The graft patency rate after coronary artery bypass grafting (CABG) correlates with anastomotic stenosis. Intracoronary radiation therapy is effective for preventing restenosis after percutaneous coronary intervention (PCI). We postulated that intracoronary radiation therapy could prevent anastomotic stenosis and tested this hypothesis in an animal model. Femoral arteries and veins of beagle dogs were harvested, and composite arterioarterial and arteriovenous grafts were prepared. After external irradiation of the anastomotic sites, these composite grafts were transplanted into femoral arteries. Histomorphometric and immunohistological analyses of the anastomotic sites were performed. The study groups consisted of controls and animals exposed to 10 Gy, 20 Gy, and 30 Gy (n = 5, in each group). In the artery graft model, the ratio of negative remodeling was significantly increased in all groups exposed to >or=10 Gy. The ratio of neointimal hyperplasia was significantly decreased in all groups exposed to >or=10 Gy. Cell density of anti-alpha-actin antibody-positive cells and anti-proliferating cell nuclear antigen (PCNA) antibody-positive cells was highest in the adventitial layer, and the density decreased as the dosage increased. Experimental results were almost the same in the vein graft models as in the artery graft models. With double immunohistostaining, the anti-PCNA antibody-positive cells expressed alpha-actin. Irradiation can inhibit anastomotic stenosis in a canine model. Adventitia is a factor in the creation of stenosis, and irradiation appears to target the adventitia. We speculate that there might be a possible role for intracoronary irradiation in the future to prevent anastomotic stenosis.

Double-layered microstrip metamaterial beam scanning leaky wave antenna with consistent gain and low cross-polarization

NASA Astrophysics Data System (ADS)

An, Yong-li; Tan, Yi-li; Zhang, Hong-bo; Wu, Guo-cheng

2017-12-01

In this paper, a novel double-layered microstrip metamaterial beam scanning leaky wave antenna (LWA) is proposed and investigated to achieve consistent gain and low cross-polarization. Thanks to the continuous phase constant changing from negative to positive values over the passband of the double-layered microstrip metamaterial, the proposed LWA, which consists of 20 identical microstrip metamaterial unit cells, can obtain a continuous beam scanning property from backward to forward directions. The proposed LWA is fabricated and measured. The measured results show that the fabricated antenna obtains a continuous beam scanning angle of 140° over the operating frequency band of 3.80-5.25 GHz (32%), the measured 3 dB gain bandwidth is 30.17% with maximum gain of 11.7 dB. Besides, the measured cross-polarization of the fabricated antenna keeps at a level of at least 30 dB below the co-polarization across the entire radiation region. Moreover, the measured and simulated results are in good agreement with each other, indicating the significance and effectiveness of this method.

Fluorescence-based recombination assay for sensitive and specific detection of genotoxic carcinogens in human cells.

PubMed

Ireno, Ivanildce C; Baumann, Cindy; Stöber, Regina; Hengstler, Jan G; Wiesmüller, Lisa

2014-05-01

In vitro genotoxicity tests are known to suffer from several shortcomings, mammalian cell-based assays, in particular, from low specificities. Following a novel concept of genotoxicity detection, we developed a fluorescence-based method in living human cells. The assay quantifies DNA recombination events triggered by DNA double-strand breaks and damage-induced replication fork stalling predicted to detect a broad spectrum of genotoxic modes of action. To maximize sensitivities, we engineered a DNA substrate encompassing a chemoresponsive element from the human genome. Using this substrate, we screened various human tumor and non-transformed cell types differing in the DNA damage response, which revealed that detection of genotoxic carcinogens was independent of the p53 status but abrogated by apoptosis. Cell types enabling robust and sensitive genotoxicity detection were selected for the generation of reporter clones with chromosomally integrated DNA recombination substrate. Reporter cell lines were scrutinized with 21 compounds, stratified into five sets according to the established categories for identification of carcinogenic compounds: genotoxic carcinogens ("true positives"), non-genotoxic carcinogens, compounds without genotoxic or carcinogenic effect ("true negatives") and non-carcinogenic compounds, which have been reported to induce chromosomal aberrations or mutations in mammalian cell-based assays ("false positives"). Our results document detection of genotoxic carcinogens in independent cell clones and at levels of cellular toxicities <60 % with a sensitivity of >85 %, specificity of ≥90 % and detection of false-positive compounds <17 %. Importantly, through testing cyclophosphamide in combination with primary hepatocyte cultures, we additionally provide proof-of-concept for the identification of carcinogens requiring metabolic activation using this novel assay system.

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

PubMed

Koo, Ok Jae; Park, Sol Ji; Lee, Choongil; Kang, Jung Taek; Kim, Sujin; Moon, Joon Ho; Choi, Ji Yei; Kim, Hyojin; Jang, Goo; Kim, Jin-Soo; Kim, Seokjoong; Lee, Byeong-Chun

2014-03-01

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP(+)/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Neuro-immune interactions in the dove brain.

PubMed

Wilhelm, Marta

2011-05-15

Mast cells (MC) are of hematopoetic origin. Connective tissue type MCs are able to function in IgE dependent and independent fashion, change their phenotype according to the tissue environment. They are able to enter the brain under normal physiological conditions, and move into this compact tissue made of neurons. In doves MCs are found only in the medial habenula (MH) and their number is changing according to the amount of sex steroids in the body. MCs are able to synthesize and store a great variety of biologically active compounds, like transmitters, neuromodulators and hormones. They are able to secrete GnRH. With the aid of electron microscopy we were able to describe MC-neuron interactions between GnRH-positive MCs and neurons. Piecemeal degranulation (secretory vesicles budding off swollen and active granules) seems to be a very efficient type of communication between MCs and surrounding neurons. Different types of granular and vesicular transports are seen between GnRH-immunoreactive MCs and neurons in the MH of doves. Sometimes whole granules are visible in the neuronal cytoplasm, in other cases exocytotic vesicles empty materials of MC origin. Thus MCs might modulate neuronal functions. Double staining experiments with IP3-receptor (IP3R), Ryanodine-receptor (RyR) and serotonin antibodies showed active MC population in the habenula. Light IP3R-labeling was present in 64-97% of the cells, few granules were labeled in 7-10% of MCs, while strong immunoreactivity was visible in 1-2% of TB stained cells. No immunoreactivity was visible in 28-73% of MCs. According to cell counts, light RyR-positivity appeared in 27-52%, few granules were immunoreactive in 4-19%, while strong immunopositivity was found only in one animal. In this case 22% of MCs were strongly RyR-positive. No staining was registered in 44-73% of MCs. Double staining with 5HT and these receptor markers proved that indeed only a part of MCs is actively secreting. Resting cells with only 5HT-immunopositivity are often visible. The activational state of MCs is changing at higher estrogen/testosterone level, thus with the secretion of neuromodulators they might alter sexual and parental behavior of the animals. Copyright © 2011 Elsevier Inc. All rights reserved.

Progesterone biotransformation by plant cell suspension cultures.

PubMed Central

Yagen, B; Gallili, G E; Mateles, R I

1978-01-01

Progesterone was converted to 5alpha-pregnane-3alpha-ol-20-one, delta4-pregnene-20alpha-ol-3-one, delta4-pregnene-14alpha-ol-3,20-dione, delta4-pregnene-7beta,14alpha-diol-3,20-dione, and delta4-pregnene-6beta,11alpha-diol-3,20-dione by cell cultures of Lycopersicon esculentum. Cell cultures of Capsicum frutescens (green) metabolized progesterone to delta4-pregnene-20alpha-ol-3-one in very high yield, and Vinca rosea yielded delta4-pregnene-20beta-ol-3-one and delta4-pregnene-14alpha-ol-3,20-dione. A stereospecific reduction of the keto groups and a double bond and stereospecific introduction of hydroxyl groups at the 6, 11, and 14 positions have been observed. The mono- and dihydroxylated progesterones have not previously been reported as metabolic products of progesterone by plant cell systems and represent de novo hydroxylation of a nonglycosylated steroid. PMID:697360

A New Case of Syringocystadenocarcinoma Papilliferum: A Rare Pathology for a Wide-Ranging Comprehension

PubMed Central

Paradiso, Beatrice; Bianchini, Enzo; Cifelli, Pierangelo; Cavazzini, Luigi; Lanza, Giovanni

2014-01-01

We report a new case of p63/cytokeratin 7 (CK7) positive syringocystadenocarcinoma papilliferum (SCACP), on the shoulder of an 88-year-old man, with superficial dermal infiltration and squamoid differentiation. We describe the 24th case of SCACP, the malignant counterpart of syringocystadenoma papilliferum (SCAP). At the present, we do not know whether SCACP arises from eccrine or apocrine glands because of the contrasting opinions in the literature. Only few histochemical and ultrastructural studies have previously advised that SCACP could arise from pluripotent stem cells. Through our case, we wish to suggest the stem cell-like properties of the syringocystadenocarcinoma papilliferum. This rare neoplasm shows two different patterns of stem cell marker expression in the glandular and squamous components, respectively. For the double phenotype of SCACP, we propose it like an intriguing model to study histogenesis and stem cell properties for more wide-ranging epithelial tumors. PMID:24959179

Safety and immunogenicity of the HPV-16/18 AS04-adjuvanted vaccine in HIV-positive women in South Africa: a partially-blind randomised placebo-controlled study.

PubMed

Denny, Lynette; Hendricks, Bronwyn; Gordon, Chivaugn; Thomas, Florence; Hezareh, Marjan; Dobbelaere, Kurt; Durand, Christelle; Hervé, Caroline; Descamps, Dominique

2013-11-19

In developing countries, risk of human papillomavirus (HPV) infection may be increased by the high prevalence of human immunodeficiency virus (HIV) infection. We evaluated the safety and immunogenicity of the HPV-16/18 AS04-adjuvanted vaccine in HIV-infected women in South Africa. Asymptomatic HIV-positive women aged 18-25 years (N=120) were stratified by CD4⁺ T-cell count and randomised (1:1) to receive HPV-16/18 vaccine (Cervarix®; GlaxoSmithKline Vaccines) or placebo (Al[OH]3) at 0, 1 and 6 months (double-blind). HIV-negative women (N=30) received HPV-16/18 vaccine (open label). Anti-HPV-16/18 antibody and CD4⁺ T-cell responses, CD4⁺ T-cell count, HIV viral load, HIV clinical stage and safety were evaluated for 12 months. The safety and reactogenicity profile of the HPV-16/18 vaccine was comparable in HIV-positive and HIV-negative women. Irrespective of baseline HPV status, all HIV-positive and HIV-negative women who received the HPV-16/18 vaccine were seropositive for both HPV-16 and HPV-18 after the second vaccine dose (month 2) and remained seropositive for both antigens at month 12. Anti-HPV-16/18 antibody titres at month 12 remained substantially above levels associated with natural infection. The HPV-16/18 vaccine induced sustained anti-HPV-16/18 CD4⁺ T-cell responses in both HIV-positive and HIV-negative women. No impact of baseline CD4⁺ T-cell count or HIV viral load was observed on the magnitude of the immune response in HIV-positive women. In HIV-positive women, CD4⁺ T-cell count, HIV viral load and HIV clinical stage were unaffected by HPV-16/18 vaccine administration. In conclusion, the HPV-16/18 AS04-adjuvanted vaccine appears immunogenic and well-tolerated in women with HIV infection. Study ID: 107863/NCT00586339. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cartilage formation in the CELLS 'double bubble' hardware

NASA Technical Reports Server (NTRS)

Duke, P. J.; Arizpe, Jorge; Montufar-Solis, Dina

1991-01-01

The CELLS experiment scheduled to be flown on the first International Microgravity Laboratory is designed to study the effect of microgravity on the cartilage formation, by measuring parameters of growth in a differentiating cartilage cell culture. This paper investigates the conditions for this experiment by studying cartilage differentiation in the 'bubble exchange' hardware with the 'double bubble' design in which the bubbles are joined by a flange which also overlays the gasket. Four types of double bubbles (or double gas permeable membranes) were tested: injection-molded bubbles 0.01- and 0.005-in. thick, and compression molded bubbles 0.015- and 0.01-in. thick. It was found that double bubble membranes of 0.005- and 0.010-in. thickness supported cartilage differentiation, while the 0.015-in. bubbles did not. It was also found that nodule count, used in this study as a parameter, is not the best measure of the amount of cartilage differentiation.

Stalk cell differentiation without polyketides in the cellular slime mold.

PubMed

Sato, Yukie G; Suarez, Teresa; Saito, Tamao

2016-07-01

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells-the major part of the prestalk cells-are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.

CD146 positive human dental pulp stem cells promote regeneration of dentin/pulp-like structures.

PubMed

Matsui, Mikiko; Kobayashi, Tomoko; Tsutsui, Takeo W

2018-04-01

CD146 and STRO-1 are endothelial biomarkers that are co-expressed on the cellular membranes of blood vessels within human dental pulp tissue. This study characterized the percentage of dentin-like structures produced by CD146-positive (CD146 + ) human dental pulp stem cells (DPSCs), compared with their CD146-negative (CD146 - ) counterparts. DPSC populations were enriched using magnetic-activated cell sorting (MACS), yielding CD146 + and CD146 - cells, as well as mixtures composed of 25% CD146 + cells and 75% CD146 - cells (CD146 +/- ). Cell growth assays indicated that CD146 + cells exhibit an approximate 3-4 h difference in doubling time, compared with CD146 - cells. Cell cycle distributions were determined by flow cytometry analysis. The low percentage of CD146 + cells' DNA content in G 0 /G 1 phase were compared with CD146 - and non-separated cells. In contrast to CD146 - and non-separated cells, prompt mineralization was observed in CD146 + cells. Subsequently, qRT-PCR revealed high mRNA expression of CD146 and Alkaline phosphatase in mineralization-induced CD146 + cells. CD146 + cells were also observed high adipogenic ability by Oil red O staining. Histological examinations revealed an increased area of dentin/pulp-like structures in transplanted CD146 + cells, compared with CD146 - and CD146 +/- cells. Immunohistochemical studies detected dentin matrix protein-1 (DMP1) and dentin sialophosphoprotein (DSPP), as well as human mitochondria, in transplanted DPSCs. Co-expression of CD146 and GFP indicated that CD146 was expressed in transplanted CD146 + cells. CD146 + cells may promote mineralization and generate dentin/pulp-like structures, suggesting a role in self-renewal of stem cells and dental pulp regenerative therapy.

Synthesis and biological evaluation of novel N-phenyl ureidobenzenesulfonate derivatives as potential anticancer agents. Part 2. Modulation of the ring B.

PubMed

Gagné-Boulet, Mathieu; Moussa, Hanane; Lacroix, Jacques; Côté, Marie-France; Masson, Jean-Yves; Fortin, Sébastien

2015-10-20

DNA double strand-breaks (DSBs) are the most deleterious lesions that can affect the genome of living beings and are lethal if not quickly and properly repaired. Recently, we discovered a new family of anticancer agents designated as N-phenyl ureidobenzenesulfonates (PUB-SOs) that are blocking the cells cycle progression in S-phase and inducing DNA DSBs. Previously, we have studied the effect of several modifications on the molecular scaffold of PUB-SOs on their cytocidal properties. However, the effect of the nature and the position of substituents on the aromatic ring B is still poorly studied. In this study, we report the preparation and the biological evaluation of 45 new PUB-SO derivatives substituted by alkyl, alkoxy, halogen and nitro groups at different positions on the aromatic ring B. All PUB-SOs were active in the submicromolar to low micromolar range (0.24-20 μM). The cell cycle progression analysis showed that PUB-SOs substituted at position 2 by alkyl, halogen or nitro groups or substituted at position 4 by a hydroxyl group arrest the cell cycle progression in S-phase. Interestingly, all others PUB-SOs substituted at positions 3 and 4 arrested the cell cycle in G2/M-phase. PUB-SOs arresting the cell cycle progression in S-phase also induced the phosphorylation of H2AX (γH2AX) which is indicating the generation of DNA DSBs. We evidenced that few modifications on the ring B of PUB-SOs scaffold lead to cytocidal derivatives arresting the cell cycle in S-phase and inducing γH2AX and DSBs. In addition, this study shows that these new anticancer agents are promising and could be used as alternative to circumvent some of the biopharmaceutical complications that might be encountered during the development of PUB-SOs. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Role of nucleocapsid protein of hantaviruses in intracellular traffic of viral glycoproteins.

PubMed

Shimizu, Kenta; Yoshimatsu, Kumiko; Koma, Takaaki; Yasuda, Shumpei P; Arikawa, Jiro

2013-12-26

To understand the role of nucleocapsid protein (NP) of hantaviruses in viral assembly, the effect of NP on intracellular traffic of viral glycoproteins Gn and Gc was investigated. Double staining of viral and host proteins in Hantaan virus (HTNV)-infected Vero E6 cells showed that Gn and Gc were localized to cis-Golgi, in which virus particles are thought to be formed. When HTNV Gn and Gc were expressed by a plasmid encoding glycoprotein precursor (GPC), which is posttranslationally cleaved into Gn and Gc, Gn was localized to cis-Golgi, whereas Gc showed diffuse distribution in the cytoplasm in 32.9% of Gc-positive cells. The ratio of the diffused Gc-positive cells was significantly decreased to 15.0% by co-expression of HTNV NP. Co-expression of HTNV GPC with NPs of other hantaviruses, such as Seoul virus, Puumala virus and Sin Nombre virus, also reduced the ratios of diffused Gc-positive cells to 13.5%, 25.2%, and 11.6%, respectively. Among amino- and carboxyl-terminally truncated HTNV NPs, NP75-429, NP116-429, NP1-333, NP1-233, and NP1-155 possessed activity to reduce the ratio of diffused Gc-positive cells, while NP155-429 and NP1-116 did not. NP30-429 has partial activity. These results indicate that amino acid region 116-155 of NP is important for the activity, although amino acid region 1-30 is partially related. Truncation of the HTNV Gc cytoplasmic tail caused an increase in diffused Gc-positive cells. In addition, the effect of coexpression of HTNV NP was weakened. These results suggest that HTNV NP has a role to promote Golgi localization of Gc through a mechanism possibly mediated by the Gc cytoplasmic tail. Copyright © 2013 Elsevier B.V. All rights reserved.

Multiparameter double hole contrast detail phantom: Ability to detect image displacement due to off position anode stem

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

Pauzi, Nur Farahana; Majid, Zafri Azran Abdul; Sapuan, Abdul Halim

Contrast Detail phantom is a quality control tool to analyze the performance of imaging devices. Currently, its function is solely to evaluate the contrast detail characteristic of imaging system. It consists of drilled hole which gives effect to the penetration of x-ray beam divergence to pass through the base of each hole. This effect will lead to false appearance of image from its original location but it does not being visualized in the radiograph. In this study, a new design of Contrast Detail phantom’s hole which consists of double hole construction has been developed. It can detect the image displacementmore » which is due to off position of anode stem from its original location. The double hole differs from previous milled hole, whereby it consists of combination of different hole diameters. Small hole diameter (3 mm) is positioned on top of larger hole diameter (10 mm). The thickness of double hole acrylic blocks is 13 mm. Result revealed that Multiparameter Double Hole Contrast Detail phantom can visualize the shifted flaw image quality produced by x-ray machine due to improper position of the anode stem which is attached to rotor and stator. The effective focal spot of x-ray beam also has been shifted from the center of collimator as a result of off-position anode stem. As a conclusion, the new design of double hole Contrast Detail phantom able to measure those parameters in a well manner.« less

The scaling of relativistic double-year widths - Poisson-Vlasov solutions and particle-in-cell simulations

NASA Technical Reports Server (NTRS)

Sulkanen, Martin E.; Borovsky, Joseph E.

1992-01-01

The study of relativistic plasma double layers is described through the solution of the one-dimensional, unmagnetized, steady-state Poisson-Vlasov equations and by means of one-dimensional, unmagnetized, particle-in-cell simulations. The thickness vs potential-drop scaling law is extended to relativistic potential drops and relativistic plasma temperatures. The transition in the scaling law for 'strong' double layers suggested by analytical two-beam models by Carlqvist (1982) is confirmed, and causality problems of standard double-layer simulation techniques applied to relativistic plasma systems are discussed.

HER2 expression identifies dynamic functional states within circulating breast cancer cells.

PubMed

Jordan, Nicole Vincent; Bardia, Aditya; Wittner, Ben S; Benes, Cyril; Ligorio, Matteo; Zheng, Yu; Yu, Min; Sundaresan, Tilak K; Licausi, Joseph A; Desai, Rushil; O'Keefe, Ryan M; Ebright, Richard Y; Boukhali, Myriam; Sil, Srinjoy; Onozato, Maristela L; Iafrate, Anthony J; Kapur, Ravi; Sgroi, Dennis; Ting, David T; Toner, Mehmet; Ramaswamy, Sridhar; Haas, Wilhelm; Maheswaran, Shyamala; Haber, Daniel A

2016-09-01

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER + /HER2 - primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2 + and HER2 - subpopulations: HER2 + circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2 - circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2 + and HER2 - circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2 + and HER2 - circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2 + state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2 - phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.

Reversible conversion of immortal human cells from telomerase-positive to telomerase-negative cells.

PubMed

Kumakura, Shin-ichi; Tsutsui, Takeo W; Yagisawa, Junko; Barrett, J Carl; Tsutsui, Takeki

2005-04-01

Immortal cell lines and tumors maintain their telomeres via the telomerase pathway or via a telomerase-independent pathway, referred to as alternative lengthening of telomeres (ALT). Here, we show the reversible conversion of the human papillomavirus type 16 E6-induced immortal human fibroblasts E6 Cl 6 from telomerase-positive (Tel(+)) to telomerase-negative (Tel(-)) cells. Tel(+) cells converted spontaneously to Tel(-) cells that reverted to Tel(+) cells following treatment with trichostatin A (TSA) and/or 5-aza-2'-deoxycytidine (5-AZC), which induced the reversion from complete to partial methylation of the CpG islands of the human telomerase reverse transcriptase (hTERT) promoter in Tel(-) E6 Cl 6 cells. Tel(-) E6 Cl 6 cells lacked the phenotypes characteristic of ALT cell lines such as very long and heterogenous telomeres and ALT-associated promyelocytic leukemia nuclear bodies (APB) but grew for >240 population doublings (PD) after they became telomerase negative. The ratios of histone H3 (H3) lysine (K) 9 methylation to each of H3-K4 methylation, H3-K9 acetylation, and H3-K14 acetylation of the chromatin containing the hTERT promoter in Tel(-) E6 Cl 6 cells and ALT cell lines were greater than those in Tel(+) cells and decreased following treatment with TSA and/or 5-AZC, inversely corresponding to telomerase activity. Our findings suggest the possibility that human tumors may be able to reversibly interconvert their telomere maintenance phenotypes by chromatin structure-mediated regulation of hTERT expression.

31P NMR spectroscopy studies of phospholipid metabolism in human melanoma xenograft lines differing in rate of tumour cell proliferation.

PubMed

Lyng, H; Olsen, D R; Petersen, S B; Rofstad, E K

1995-04-01

The concentration of phospholipid metabolites in tumours has been hypothesized to be related to rate of cell membrane turnover and may reflect rate of cell proliferation. The purpose of the study reported here was to investigate whether 31P NMR resonance ratios involving the phosphomonoester (PME) or phosphodiester (PDE) resonance are correlated to fraction of cells in S-phase or volume-doubling time in experimental tumours. Four human melanoma xenograft lines (BEX-t, HUX-t, SAX-t, WIX-t) were included in the study. The tumours were grown subcutaneously in male BALB/c-nu/nu mice. 31P NMR spectroscopy was performed at a magnetic field strength of 4.7 T. Fraction of cells in S-phase was measured by flow cytometry. Tumour volume-doubling time was determined by Gompertzian analysis of volumetric growth data. BEX-t and SAX-t tumours differed in fraction of cells in S-phase and volume-doubling time, but showed similar 31P NMR resonance ratios. BEX-t and WIX-t tumours showed significantly different 31P NMR resonance ratios but similar fractions of cells in S-phase. The 31P NMR resonance ratios were significantly different for small and large HUX-t tumours even though fraction of cells in S-phase and volume-doubling time did not differ with tumour volume. None of the 31P NMR resonance ratios showed significant increase with increasing fraction of cells in S-phase or significant decrease with increasing tumour volume-doubling time across the four xenograft lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence of antibodies to soluble antigen of Epstein-Barr virus-producing cells (P3HR-1) in the sera of hamadryas baboons of the high lymphoma incidence stock.

PubMed

Voevodin, A F; Lapin, B A; Yakovleva, L A; Ponomarjova, T I; Agrba, V Z

1979-01-01

Soluble antigen of P3HR-1 cells (SA-P3HR-1) was identified in indirect double immunodiffusion enhanced with tannic acid using serum of a nasopharyngeal carcinoma patient containing high-titer antibodies to Epstein-Barr virus (EBV) antigens. SA-P3HR-1 was nonidentical to soluble antigen of Raji cells. Human and baboon sera containing antibodies to all the known antigen of EBV and HVP respectively were anti-SA-3HR-1-positive. Human and baboon sera containing antibodies to all the known antigens of EBV and herpesvirus Papio (HVP) were also anti-SA-P3HR-1-negative. Prevalence of anti-SA-P3HR-1 was very high in two groups of the high-lymphoma incidence stock of hamadryas baboons of the Sukhumi monkey colony. 54% (15 of 28) and 38% (13 of 34) of clinically lymphomatous and clinical normal monkeys, respectively, were anti-SA-P3HR-1-positive.. Only 1 of 30 normal baboons studied, living in the forest and having no contacts with the baboons in the main stock of the Sukhumi monkey colony, was anti-SA-P3HR-1-positive (3%).

2-Aminoanthracene, 5-fluorouracil, colchicine, benzo[a]pyrene, cadmium chloride and cytosine arabinoside tested in the in vitro mammalian cell micronucleus test (MNvit) in Chinese hamster ovary (CHO) cells at Covance Laboratories, Harrogate UK in support of OECD draft Test Guideline 487.

PubMed

Whitwell, James; Fowler, Paul; Allars, Sarah; Jenner, Karen; Lloyd, Melvyn; Wood, Debbie; Smith, Katie; Young, Jamie; Jeffrey, Laura; Kirkland, David

2010-10-29

The reference genotoxic agents 2-aminoanthracene (a metabolism dependent weak clastogen), 5-fluorouracil (a nucleoside analogue, characterised by a steep dose response profile), colchicine (an aneugen that inhibits tubulin polymerisation), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation), cadmium chloride (an inorganic carcinogen), and cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair) were tested in the in vitro micronucleus assay using the Chinese hamster ovary (CHO) cell line at Covance Laboratories, Harrogate, UK. All chemicals were treated in the absence and presence of cytokinesis block (via addition of cytochalasin B) with this work forming part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 on the In vitro Mammalian Cell Micronucleus Test (MNvit). The toxicity measures used, detecting a possible combination of both cytostasis and cell death (though not cell death directly), were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index in the presence of cytokinesis block. All of the chemicals tested either gave marked positive increases in the percentage of micronucleated cells with and without cytokinesis block, or did not induce micronuclei at concentrations giving approximately 50-60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcome from this series of tests supports the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay. Copyright © 2010 Elsevier B.V. All rights reserved.

Nuclear ARP2/3 drives DNA break clustering for homology-directed repair.

PubMed

Schrank, Benjamin R; Aparicio, Tomas; Li, Yinyin; Chang, Wakam; Chait, Brian T; Gundersen, Gregg G; Gottesman, Max E; Gautier, Jean

2018-06-20

DNA double-strand breaks repaired by non-homologous end joining display limited DNA end-processing and chromosomal mobility. By contrast, double-strand breaks undergoing homology-directed repair exhibit extensive processing and enhanced motion. The molecular basis of this movement is unknown. Here, using Xenopus laevis cell-free extracts and mammalian cells, we establish that nuclear actin, WASP, and the actin-nucleating ARP2/3 complex are recruited to damaged chromatin undergoing homology-directed repair. We demonstrate that nuclear actin polymerization is required for the migration of a subset of double-strand breaks into discrete sub-nuclear clusters. Actin-driven movements specifically affect double-strand breaks repaired by homology-directed repair in G2 cell cycle phase; inhibition of actin nucleation impairs DNA end-processing and homology-directed repair. By contrast, ARP2/3 is not enriched at double-strand breaks repaired by non-homologous end joining and does not regulate non-homologous end joining. Our findings establish that nuclear actin-based mobility shapes chromatin organization by generating repair domains that are essential for homology-directed repair in eukaryotic cells.

Fetal progenitor cell transplantation treats methylmalonic aciduria in a mouse model

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

Buck, Nicole E., E-mail: nicole.buck@mcri.edu.au; Pennell, Samuel D.; Wood, Leonie R.

Highlights: Black-Right-Pointing-Pointer Fetal cells were transplanted into a methylmalonic acid mouse model. Black-Right-Pointing-Pointer Cell engraftment was detected in liver, spleen and bone marrow. Black-Right-Pointing-Pointer Biochemical disease correction was measured in blood samples. Black-Right-Pointing-Pointer A double dose of 5 million cells (1 week apart) proved more effective. Black-Right-Pointing-Pointer Higher levels of engraftment may be required for greater disease correction. -- Abstract: Methylmalonic aciduria is a rare disorder caused by an inborn error of organic acid metabolism. Current treatment options are limited and generally focus on disease management. We aimed to investigate the use of fetal progenitor cells to treat this disordermore » using a mouse model with an intermediate form of methylmalonic aciduria. Fetal liver cells were isolated from healthy fetuses at embryonic day 15-17 and intravenously transplanted into sub-lethally irradiated mice. Liver donor cell engraftment was determined by PCR. Disease correction was monitored by urine and blood methylmalonic acid concentration and weight change. Initial studies indicated that pre-transplantation sub-lethal irradiation followed by transplantation with 5 million cells were suitable. We found that a double dose of 5 million cells (1 week apart) provided a more effective treatment. Donor cell liver engraftment of up to 5% was measured. Disease correction, as defined by a decrease in blood methylmalonic acid concentration, was effected in methylmalonic acid mice transplanted with a double dose of cells and who showed donor cell liver engraftment. Mean plasma methylmalonic acid concentration decreased from 810 {+-} 156 (sham transplanted) to 338 {+-} 157 {mu}mol/L (double dose of 5 million cells) while mean blood C3 carnitine concentration decreased from 20.5 {+-} 4 (sham transplanted) to 5.3 {+-} 1.9 {mu}mol/L (double dose of 5 million cells). In conclusion, higher levels of engraftment may be required for greater disease correction; however these studies show promising results for cell transplantation biochemical correction of a metabolic disorder.« less

In Vitro Targeted Photodynamic Therapy with a Pyropheophorbide-a Conjugated Inhibitor of Prostate Specific Membrane Antigen

PubMed Central

Liu, Tiancheng; Wu, Lisa Y.; Choi, Joseph K.; Berkman, Clifford E.

2009-01-01

BACKROUND The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on photosensitizer-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. METHODS Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. RESULTS Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 h by HOE33342/PI double-staining, becoming more intense by 4 h. Evidence for the apoptotic caspase cascade being activated was based on the appearance of PARP p85 fragment. TUNEL assay detected DNA fragmentation 16 h post-PDT, confirming apoptotic events. CONCLUSIONS Cell permeability by HOE33342/PI double-staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. PMID:19142895

In vitro targeted photodynamic therapy with a pyropheophorbide--a conjugated inhibitor of prostate-specific membrane antigen.

PubMed

Liu, Tiancheng; Wu, Lisa Y; Choi, Joseph K; Berkman, Clifford E

2009-05-01

The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on PS-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 hr by HOE33342/PI double staining, becoming more intense by 4 hr. Evidence for the apoptotic caspase cascade being activated was based on the appearance of poly-ADP-ribose polymerase (PARP) p85 fragment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detected DNA fragmentation 16 hr post-PDT, confirming apoptotic events. Cell permeability by HOE33342/PI double staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. (c) 2009 Wiley-Liss, Inc.

The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma.

PubMed

Lowery, Caitlin D; VanWye, Alle B; Dowless, Michele; Blosser, Wayne; Falcon, Beverly L; Stewart, Julie; Stephens, Jennifer; Beckmann, Richard P; Bence Lin, Aimee; Stancato, Louis F

2017-08-01

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G 2 -M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma. Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma. Results: Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature. Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354-63. ©2017 AACR . ©2017 American Association for Cancer Research.

Efficient repair of DNA double-strand breaks in malignant cells with structural instability

PubMed Central

Cheng, Yue; Zhang, Zhenhua; Keenan, Bridget; Roschke, Anna V.; Nakahara, Kenneth; Aplan, Peter D.

2009-01-01

Aberrant repair of DNA double strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1α) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1α promoter from the TK gene, or deletion of either the EF1α promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR. PMID:19909760

Efficient repair of DNA double-strand breaks in malignant cells with structural instability.

PubMed

Cheng, Yue; Zhang, Zhenhua; Keenan, Bridget; Roschke, Anna V; Nakahara, Kenneth; Aplan, Peter D

2010-01-05

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1alpha) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1alpha promoter from the TK gene, or deletion of either the EF1alpha promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.

P63 EXPRESSION LEVELS IN SIDE POPULATION AND LOW LIGHT SCATTERING OCULAR SURFACE EPITHELIAL CELLS

PubMed Central

Epstein, Seth P; Wolosin, J. Mario; Asbell, Penny A

2005-01-01

Purpose Because stem cells exhibit high self-renewal capacity, slow cycling, and high proliferative potential, and one of many markers postulated for epithelial stem cells, p63, is challenged by widespread expression within stem cell–free regions, we examined p63 expression in these stem cell–associated cohorts compared with their controls. Methods Rabbit limbocorneal cryosections, cytospun cell-sorted (by fluorescence-activated cell sorter) side population (SP) and low side scatter (LSSC) cells, and limbal epithelial cells over feeders were stained for p63 by indirect immunofluorescence. Clones were fixed and stained daily for 7 days. Image analysis measured p63 intensity, plotting it against colony size. Results All basal limbal cells were positive for p63, yet only 5% to 7% expressed high p63 intensities, 40% intermediate, and the majority low. Side population cells were less than 1% of total cells. The average intensity of SP staining was three times that of controls. Subpopulations displaying stemlike features exhibited highest p63 expression. Replication rates of isolated cells differed. Day 5 colonies contained 256 (16 hours/cycle) to two (96 hours/cycle) cells. Whereas all cells were positive for p63, intensity in slow-cycling cells was three to four times that in rapidly proliferating congeners. Increased cell doublings did not decrease fluorescence. Conclusions Results suggest that p63 concentration is maximal in stem cells and decreases with differentiation. High p63 levels seem to correlate with cells of the SP and LSSC phenotypes, indicating high cell stemness. With identification of stem cells, further studies can elucidate their use in supporting ocular surface health. PMID:17057802

The carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule.

PubMed Central

Hufnagle, W O; Tremaine, M T; Betley, M J

1991-01-01

Staphylococcal enterotoxin type A (SEA) gene (sea+) mutations were constructed by exonuclease III digestion or cassette mutagenesis. Five different sea mutations that had 1, 3, 7, 39, and 65 codons deleted from the 3' end of sea+ were identified and confirmed by restriction enzyme and nucleotide sequence analyses. Each of these sea mutations was constructed in Escherichia coli and transferred to Staphylococcus aureus by using the plasmid vector pC194. Culture supernatants from the parent S. aureus strain that lacked an enterotoxin gene (negative controls) and from derivatives that contained either sea+ (positive control) or a sea mutation were examined for in vitro sensitivity to degradation by monkey stomach lavage fluid, the ability to cause emesis when administered by an intragastric route to rhesus monkeys, and the ability to induce T-cell proliferation and by Western immunoblot analysis and a gel double-diffusion assay with polyclonal antibodies prepared against SEA. Altered SEAs corresponding to the predicted sizes were visualized by Western blot analysis of culture supernatants for each of the staphylococcal derivatives that contained a sea mutation. The altered SEA that lacked the C-terminal amino acid residue behaved like SEA in all of the assays performed. The altered SEA that lacked the three C-terminal residues of SEA caused T-cell proliferation but was not emetic; this altered SEA was degraded in vitro by monkey stomach lavage fluid and did not reach in the gel double diffusion assay. Altered SEAs that lacked 7, 39, or 65 carboxyl-terminal residues were degraded by stomach lavage fluid in vitro, did not produce an emetic response, and did not induce T-cell proliferation or form a visible reaction in the gel double-diffusion assay. Images PMID:1903773

Coexistence of immune-neuro-endocrine substances in the rat central neurons.

PubMed

Zhu, C; Liu, Q; Wei, Y; Ma, C; Hao, J; Yan, P

1999-01-01

To investigate the expression of interleukin-2 (IL-2), metabotropic glutamate receptor subunit 1 (mGluR1) and estrogen receptor (ER) in neurons of the rat central nervous system (CNS) and identify the coexistence possibility of these immune-neuro-endocrine substances in the central neurons, the tri-labeling immunocytochemical technique with different species-specific primary antibodies (goat anti-IL-2 antibody, rabbit anti-mGluR1 antibody and mouse anti-ER antibody) were used to incubate two serial neighbor sections (one for demonstrating IL-2, another for mGluR1 and ER) of the cerebral cortex, medulla oblongata and spinal cord. There were IL-2-, mGluR1- and ER-immunoreactivity (IR)-positive labeled neurons in the above-mentioned central areas. The IL-2-IR production showed brown color, located in the cytoplasm; In the neighbor serial section, the mGluR1-IR, production showed blue-black color, located on the cell membrane; the ER-IR production also showed brown color, located in the cytoplasm and nuclei. There were mGluR1/ER double-labeled cells in the same section, which accounted for about 50%-60% of the total single and double labeled neurons. It was identified by projection check of serial neighbor sections that had mGluR1/ER/IL-2 tri-labeled cells, which accounted for about 30% of total mGluR1/ER double-labeled neurons. The results indicate that mGluR1, ER and Il-2 can coexist in the same rat central neurons, therefore, providing morphological basis for the theory about immune-neuro-endocrine network at the cellular level for the first time.

Glial-derived neurotropic factor and RET gene expression in normal human anterior pituitary cell types and in pituitary tumors.

PubMed

Japón, Miguel A; Urbano, Angel G; Sáez, Carmen; Segura, Dolores I; Cerro, Alfonso Leal; Diéguez, Carlos; Alvarez, Clara V

2002-04-01

Glial-derived neurotropic factor (GDNF) signaling is mediated through a 2-component system consisting of the so-called GDNF receptor-alpha (GFRalpha1), which binds to GDNF. This complex activates the tyrosine kinase receptor RET. In this paper we demonstrate GDNF, GFRalpha1, and RET mRNA and protein expression in the human anterior pituitary gland. Double immunohistochemistry of anterior pituitary sections showed GDNF immunoreactivity in more than 95% of somatotrophs and to a lesser extent in corticotrophs (20%); it was almost absent in the remaining cell types. Also, although more than 95% of somatotrophs were stained for RET, no positive immunostaining could be detected in other cell types. Furthermore, we have looked for GDNF and RET in human pituitary adenomas of various hormonal phenotypes. Strong positive immunostaining was found for c-RET in all of the GH-secreting adenomas screened as well as in 50% of ACTH-producing adenomas. Positive immunostaining for GDNF was found in all of the GH-secreting adenomas and in 10% of the corticotropinomas. Lastly, we found strong positive immunostaining for GFRalpha1 in 90% of the somatotropinomas and 50% of the corticotropinomas as well as in 1 of 8 prolactinomas and 1 of 13 nonfunctioning adenomas. All of the remaining pituitary tumors screened were negative for RET, GDNF, and GFRalpha1. This study indicates that GDNF may well be acting in the regulation of somatotroph cell growth and/or cell function in the normal human anterior pituitary gland. The expression of RET in all of the somatotropinomas and in 50% of the ACTH-producing tumors implies that GDNF and RET could be involved in the pathogenesis of pituitary tumors.

Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

NASA Astrophysics Data System (ADS)

Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

1983-11-01

Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

PubMed Central

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies. PMID:25460012

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

PubMed

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

β-MSCs: successful fusion of MSCs with β-cells results in a β-cell like phenotype.

PubMed

Azizi, Zahra; Lange, Claudia; Paroni, Federico; Ardestani, Amin; Meyer, Anke; Wu, Yonghua; Zander, Axel R; Westenfelder, Christof; Maedler, Kathrin

2016-08-02

Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, anti-apoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow-derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes.Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherry-expressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment.With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP.After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of beta-cell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes.

OPC-12759 increases proliferation of cultured rat conjunctival goblet cells.

PubMed

Ríos, José D; Shatos, Marie; Urashima, Hiroki; Tran, Hao; Dartt, Darlene A

2006-06-01

To determine if the gastroprotective drug OPC-12759 increased proliferation of rat conjunctival goblet cells in culture. Cultured goblet cells were incubated with 10(-12) to 10(-8) M OPC-12759 for 1 to 7 days. Fetal bovine serum (FBS) was used as a positive control. Cell proliferation was determined by a MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] colorimetric assay and by immunohistochemical staining with anti-Ki-67, a marker of cell division. Goblet cells were identified by double-labeling with anti-Ki-67, a marker of cell division, and Ulex europaeus agglutinin I lectin, anti-MUC5AC and anticytokeratin 7. Stratified squamous cells were identified by using Griffonia (Bandeiraea) simplicifolia lectin and anticytokeratin 4 antibody. As determined by MTT conversion to formazan, OPC-12579 at 10(-11) M induced an almost 2-fold increase in goblet cell proliferation on Days 1 and 3 of incubation but not on Days 5 and 7. The FBS at 10% increased cell proliferation by 2- to 3-fold at each time point. Daily replenishment of OPC-12579 for 3 consecutive days induced cell proliferation at all concentrations. Proliferation as determined by the number of Ki-67 positive cells increased by 4- and 3-fold at Days 1 and 3, respectively with addition of 10(-11) M OPC-12579. The FBS at 10% induced a 10-fold increase in goblet cell proliferation on Days 1, 3, and 5. Colocalization of Ulex europaeus agglutinin I, MUC5AC and anticytokeratin 7 with Ki-67 indicated that proliferating cells were goblet cells. Proliferating cells were negative for the nongoblet cell markers Bandeiraea lectin and anticytokeratin 4. The OPC-12759 stimulates proliferation of conjunctival goblet cells in primary culture.

Replication of tobacco mosaic virus RNA.

PubMed Central

Buck, K W

1999-01-01

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA. PMID:10212941

Protein expression differs between neural progenitor cells from the adult rat brain subventricular zone and olfactory bulb.

PubMed

Maurer, Martin H; Feldmann, Robert E; Bürgers, Heinrich F; Kuschinsky, Wolfgang

2008-01-16

Neural progenitor cells can be isolated from various regions of the adult mammalian brain, including the forebrain structures of the subventricular zone and the olfactory bulb. Currently it is unknown whether functional differences in these progenitor cell populations can already be found on the molecular level. Therefore, we compared protein expression profiles between progenitor cells isolated from the subventricular zone and the olfactory bulb using a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry. The subventricular zone and the olfactory bulb are connected by the Rostral Migratory Stream (RMS), in which glial fibrillary acidic protein (GFAP)-positive cells guide neuroblasts. Recent literature suggested that these GFAP-positive cells possess neurogenic potential themselves. In the current study, we therefore compared the cultured neurospheres for the fraction of GFAP-positive cells and their morphology of over a prolonged period of time. We found significant differences in the protein expression patterns between subventricular zone and olfactory bulb neural progenitor cells. Of the differentially expressed protein spots, 105 were exclusively expressed in the subventricular zone, 23 showed a lower expression and 51 a higher expression in the olfactory bulb. The proteomic data showed that more proteins are differentially expressed in olfactory bulb progenitors with regard to proteins involved in differentiation and microenvironmental integration, as compared to the subventricular zone progenitors. Compared to 94% of all progenitors of the subventricular zone expressed GFAP, nearly none in the olfactory bulb cultures expressed GFAP. Both GFAP-positive subpopulations differed also in morphology, with the olfactory bulb cells showing more branching. No differences in growth characteristics such as doubling time, and passage lengths could be found over 26 consecutive passages in the two cultures. In this study, we describe differences in protein expression of neural progenitor populations isolated from two forebrain regions, the subventricular zone and the olfactory bulb. These subpopulations can be characterized by differential expression of marker proteins. We isolated fractions of progenitor cells with GFAP expression from both regions, but the GFAP-positive cells differed in number and morphology. Whereas in vitro growth characteristics of neural progenitors are preserved in both regions, our proteomic and immunohistochemical data suggest that progenitor cells from the two regions differ in morphology and functionality, but not in their proliferative capacity.

Single versus double blade technique for skin incision and deep dissection in surgery for closed fracture: a prospective randomised control study.

PubMed

Trikha, V; Saini, P; Mathur, P; Agarwal, A; Kumar, S V; Choudhary, B

2016-04-01

To compare blade cultures in surgery for closed fracture using a single or double blade technique to determine whether the current practice of double blade technique is justified. 155 men and 29 women aged 20 to 60 (mean, 35) years who underwent surgery for closed fracture with healthy skin at the incision site were included. Patients were block randomised to the single (n=92) or double (n=92) blade technique. Blades were sent for bacteriological analysis. Outcome measures were early surgical site infection (SSI) within 30 days and cultures from the blades. The 2 groups were comparable in baseline characteristics. In the single blade group, 6 surgical blades and 2 control blades showed positive cultures; 4 patients developed SSI, but only one had a positive culture from the surgical blade (with different organism isolated from the wound culture). In the double blade group, 6 skin blades, 7 deep blades, and 0 control blade showed positive culture; only 2 patients had the same bacteria grown from both skin and deep blade. Five patients developed SSI, but only one patient had a positive culture from the deep blade (with different organism isolated from the wound culture). The difference in incidence of culture-positive blade or SSI between the 2 groups was not significant. The relative risk of SSI in the single blade group was 0.8. Positive blade culture was not associated with SSI in the single or double blade group. The practice of changing blade following skin incision has no effect on reducing early SSI in surgery for closed fracture in healthy patients with healthy skin.

Quantitative, non-invasive imaging of radiation-induced DNA double strand breaks in vivo

PubMed Central

Li, Wenrong; Li, Fang; Huang, Qian; Shen, Jingping; Wolf, Frank; He, Yujun; Liu, Xinjian; Hu, Y. Angela; Bedford, Joel. S.; Li, Chuan-Yuan

2011-01-01

DNA double strand breaks is a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA double strand breaks is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here we report a novel approach to image and quantify DNA double strand breaks in live mammalian cells through bi-fragment luciferase reconstitution. N- and C- terminal fragments of firefly luciferase gene were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DNA double strand breaks, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C- luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, non-invasive quantification of DNA double strand breaks in cells irradiated with x-rays and 56Fe ions. Furthermore, it allowed for the evaluation of DNA double strand breaks (DSBs) non-invasively in vivo in irradiated tumors over two weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DNA double strand break repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time. PMID:21527553

Clinicopathological study of severe chronic active Epstein-Barr virus infection that developed in association with lymphoproliferative disorder and/or hemophagocytic syndrome.

PubMed

Ohshima, K; Suzumiya, J; Sugihara, M; Nagafuchi, S; Ohga, S; Kikuchi, M

1998-12-01

Chronic active Epstein-Barr virus (CAEBV) infection has been previously reported to be sometimes associated with an aggressive clinical course. However, the role of EBV in the CAEBV is not well clarified. A retrospective study was performed on nine adult and five child patients (eight males and six females). Histologically, at first admission, the presence of neoplastic lesions could not be confirmed. The lymph nodes in half of all cases revealed paracortical hyperplasia with transformed lymphocytes (hyperplastic type). Half of the cases showed non-suppurative necrosis and an increased number of histiocytes with phagocytosis (histiocytic type). Activated histiocytes with lymphokine positivity were frequently detected in the histiocytic type. In the phenotypical study, 10 of the examined 11 cases showed increased numbers of natural killer (NK) cells and/or CD8-positive T lymphocytes. In situ hybridization (ISH) showed EBV-infected lymphoid cells, but the number of EBV-infected cells varied. Double-labeling immunochemistry/ISH demonstrated EBV-infected T cells, including NK cells, but not B cells. In addition, three cases showed a monoclonal dissemination of EBV terminal repetitive sequence (TR), and two cases showed oligoclonal dissemination. From those findings, monoclonal, oligoclonal and polyclonal populations of EBV-infected T or NK cells were considered to be present in CAEBV states. During the clinical course, 12 of the 14 cases died within 5 years. Six cases died from EBV-associated hematopoietic tumors (histiocytic tumor, T cell lymphoma, B cell lymphoma, plasmacytoma, and NK cell leukemia); one from non-EBV-associated acute myelogenous leukemia, and five due to hemophagocytic syndrome. The examined EBV-associated hematopoietic tumors showed monoclonal EBV terminal repetitive sequences. There is a possibility that the monoclonal dissemination of EBV-infected cells develops from oligoclonal or polyclonal EBV-infected cells. And active histiocytes with lymphokine positivity were frequently detected in the cases with histologically histiocytic type. These findings seem to be related with the causes of death due to hemophagocytic syndrome.

CD30 Expression by B and T Cells: A Frequent Finding in Angioimmunoblastic T-Cell Lymphoma and Peripheral T-Cell Lymphoma-Not Otherwise Specified.

PubMed

Onaindia, Arantza; Martínez, Nerea; Montes-Moreno, Santiago; Almaraz, Carmen; Rodríguez-Pinilla, Socorro M; Cereceda, Laura; Revert, Jose B; Ortega, César; Tardio, Antoni; González, Lucía; García, Sonia; Camacho, Francisca I; González-Vela, Carmen; Piris, Miguel A

2016-03-01

CD30 expression in peripheral T-cell lymphoma (PTCL) and angioimmunoblastic T-cell lymphoma (AITL) is currently of great interest because therapy targeting CD30 is of clinical benefit, but the clinical and therapeutic relevance of CD30 expression in these neoplasms still remains uncertain. The aim of this study was to better quantify CD30 expression in AITL and PTCL-not otherwise specified (NOS). The secondary objective was to determine whether CD30 cells exhibit a B-cell or a T-cell phenotype. Gene expression profiling was studied in a series of 37 PTCL cases demonstrating a continuous spectrum of TNFRSF8 expression. This prompted us to study CD30 immunohistochemical (IHC) expression and mRNA levels by reverse transcription polymerase chain reaction (RT-PCR) in a different series of 51 cases (43 AITLs and 8 PTCL-NOSs) in routine samples. Double stainings with PAX5/CD30, CD3/CD30, and LEF1/CD30 were performed to study the phenotype of CD30 cells. Most (90%) of the cases showed some level of CD30 expression by IHC (1% to 95%); these levels were high (>50% of tumoral cells) in 14% of cases. CD30 expression was not detected in 10% of the cases. Quantitative RT-PCR results largely confirmed these findings, demonstrating a moderately strong correlation between global CD30 IHC and mRNA levels (r=0.65, P=1.75e-7). Forty-four of the positive cases (98%) contained CD30-positive B cells (PAX5), whereas atypical CD30-positive T cells were detected in 42 cases (93%). In conclusion, our data show that most AITL and PTCL-NOS cases express CD30, exhibiting very variable levels of CD30 expression that may be measured by IHC or RT-PCR techniques.

Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice.

PubMed

Li, Yafan; Wheeler, Deric L; Ananthaswamy, Honnavara N; Verma, Ajit K; Oberley, Terry D

2007-12-01

Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

[Cytosine deaminase and thymidine kinase double suicide gene system driven by carcinoembryonic antigen promoter for the treatment of colorectal carcinoma xenograft in nude mice].

PubMed

Huang, Zheng-jie; Feng, Qing-zhao; You, Jun; Xu, Lin; Luo, Wei-yuan; Yi, Wen-cheng; Zeng, Yue-yue; Luo, Qi

2013-07-23

To explore the therapeutic efficacy of double suicide gene system driven by carcinoembryonic antigen (CEA) promoter (Cp-CDglyTK) on colorectal carcinoma xenograft in nude mice. The plasmid pcDNA3.1(-)Cp-CDglyTK was transfected into the CEA-positive SW480 and CEA-negative HeLa cells respectively. The expression of suicide gene was detected by RT-PCR. And the transfected cells were treated with 5-fluorocytosine (5-FC) and ganciclovir (GCV) at different concentrations and the cell-killing and bystander effects assayed by methyl thiazolyl tetrazolium (MTT). By a transplantation of cultivated cells, SW480 or HeLa cell lines were injected subcutaneously into right axillary of nude mice to establish 96 SW480 and 72 HeLa tumor animal models. Nude mice were completely randomized with statistical software according to tumor volume. For prodrug therapy, 48 SW480-bearing mice were divided equally into 4 groups of I-IV. At the same time, 48 HeLa-bearing mice were divided equally into 4 groups of V-VIII. Groups I & V received an intratumoral injection of PBS, groups II & VIGCV and 5-FC intratumorally, groups III & VII PBS intraperitoneally and groups IV & VIII GCV and 5-FC intraperitoneally. Forty-eight SW480-bearing mice were divided equally into 4 groups of IX∼XII and 24 Hela-bearing ones into groups of & in therapy experiment by suicide gene plus prodrug. Six groups received an intratumoral injection of liposome Lipofectamine and plasmid CP-CDglyTK and then an intraperitoneal injection of drug. The groups of IX and received an injection of PBS, group X GCV, group XI 5-FC and groups XII & GCV and 5-FC. The observation parameters included tumor bulk, tumor weight, survival time and treatment effect in each group. SW480 cells transfected by plasmid pcDNA3.1(-)Cp- CDglyTK expressed CDglyTK gene. The inhibition rates of GCV and 5-FC were significantly higher than those of HeLa cells (59.87% ± 0.21% vs 9.90% ± 0.09%, P < 0.01). And higher inhibition rates and stronger bystander effect existed in double versus single produg (all P < 0.05). Tumor size, final tumor weight and survival time of nude mice in groups ofII, IV, VI & VIII had no significant difference with groups ofI, III, V & VII (all P < 0.05). Final tumor size and weight of group XII was significantly smaller than those of groups of IX, X and XI ((150.0 ± 3.2) vs (522.5 ± 1.9) and (256.8 ± 10.4) and (260.7 ± 2.2) mm(3), (54.1 ± 10.4) vs (682.0 ± 12.0) and (251.8 ± 15.1) and (271.6 ± 17.7) mg, all P < 0.05). Meanwhile, the tumor inhibition rate and survival time of group XII(92.1% and (25.7 ± 0.8)d) were significant higher and longer than group X (63.1% and (21.8 ± 0.5) d) and group XI (60.2% and (18.0 ± 0.9) d) (all P < 0.05). However, no significant difference existed in tumor size, final tumor weight and survival time between groups and (all P > 0.05). The inhibition rate of group was merely 0.9%. CDglyTK double suicide gene system driven by CEA promoter may inhibit CEA positive colorectal cancer xenograft in prodrug-treated nude mice.

Electromechanical and Elastic Probing of Bacteria in Cell Culture Medium

PubMed Central

Thompson, G.L.; Reukov, V.V.; Nikiforov, M.P.; Jesse, S.; Kalinin, S.V.; Vertegel, A.A.

2012-01-01

Rapid phenotype characterization and identification of cultured cells, which is needed for progress in tissue engineering and drug testing, requires an experimental technique that measures physical properties of cells with sub-micron resolution. Recently, band excitation piezoresponse force microscopy (BEPFM) has been proven useful for recognition and imaging of different types of bacteria in pure water. Here, the BEPFM method is performed for the first time in physiologically-relevant electrolyte media, such as Dulbecco’s phosphate-buffered saline (DPBS) and Dulbecco’s modified Eagle’s medium (DMEM). Distinct electromechanical responses for Micrococcus lysodeikticus (Gram-positive) and Pseudomonas fluorescens (Gram-negative) bacteria are demonstrated in DPBS. The results suggest that mechanical properties of the outer surface coating each bacterium, as well as the electrical double layer around them, are responsible for the BEPFM image formation mechanism in electrolyte media. PMID:22641388

Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

PubMed Central

Nerys-Junior, Arildo; Costa, Lendel C.; Braga-Dias, Luciene P.; Oliveira, Márcia; Rossi, Átila D.; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S.; Tanuri, Amilcar

2014-01-01

Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity. PMID:24688299

Structural investigation of cooperite (PtS) crystals

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

Rozhdestvina, V. I., E-mail: veronika@ascnet.ru; Udovenko, A. A.; Rubanov, S. V.

2016-03-15

The single-crystal structure of cooperite, a natural platinum sulfide PtS, is studied by X-ray diffraction supported by high-resolution scanning transmission electron microscopy and X-ray spectrum microanalysis. It is found that, in addition to the main reflections corresponding to the known tetragonal cell (a = 3.47 and c = 6.11 Å; space group P4{sub 2}/mmc), many weak reflections with intensities I ≤ 60σ(I) are clearly observed. These reflections fit the tetragonal cell (space group I4/mmm) with doubled parameters. In structures with small (P4{sub 2}/mmc) and large (I4/mmm) cells, the S atoms occupy statistically two special positions. It is shown that themore » chemical composition of the cooperite crystals deviates from the stoichiometric composition: sulfur-deficient specimens predominate.« less

Electromechanical and elastic probing of bacteria in a cell culture medium

NASA Astrophysics Data System (ADS)

Thompson, G. L.; Reukov, V. V.; Nikiforov, M. P.; Jesse, S.; Kalinin, S. V.; Vertegel, A. A.

2012-06-01

Rapid phenotype characterization and identification of cultured cells, which is needed for progress in tissue engineering and drug testing, requires an experimental technique that measures physical properties of cells with sub-micron resolution. Recently, band excitation piezoresponse force microscopy (BEPFM) has been proven useful for recognition and imaging of bacteria of different types in pure water. Here, the BEPFM method is performed for the first time on physiologically relevant electrolyte media, such as Dulbecco’s phosphate-buffered saline (DPBS) and Dulbecco’s modified Eagle’s medium (DMEM). Distinct electromechanical responses for Micrococcus lysodeikticus (Gram-positive) and Pseudomonas fluorescens (Gram-negative) bacteria in DPBS are demonstrated. The results suggest that mechanical properties of the outer surface coating each bacterium, as well as the electrical double layer around them, are responsible for the BEPFM image formation mechanism in electrolyte media.

Forkhead box-P3+ regulatory T cells and toll-like receptor 2 co-expression in oral squamous cell carcinoma.

PubMed

Hussaini, H M; Parachuru, V P B; Seymour, G J; Rich, A M

2017-04-01

The function of forkhead box-P3 (FoxP3) regulatory T cells (Treg) and toll-like receptor (TLR)2 protein in the oral cancer microenvironment is not fully understood, but evidence from other malignancies suggests it is likely they are involved with tumour development and progression. The aim of this study was to investigate the distribution of FoxP3 + cells, TLR2 + cells and double-labelled FoxP3 + TLR2 + immune cells in oral squamous cell carcinoma (OSCC), using immunohistochemistry (IHC) and immunofluorescence (IF). 25 archival cases of OSCC were immunostained with anti-FoxP3 and anti-TLR2 antibodies. Inflamed hyperplastic oral mucosal tissues were used as controls. The proportion of single-labelled, double-labelled and negative cells was determined. A higher frequency of double-labelled FoxP3 + TLR2 + Tregs was observed within the immune cells of OSCC compared to inflamed controls using IHC (p<0.05). Cell-to-cell contact between single-stained TLR2 + cells and FoxP3 + cells was noted. Double IF studies validated demonstration of co-expression of FoxP3 + /TLR2 + immune cells in OSCC. The presence of FoxP3 + TLR2 + cells within the OSCC microenvironment may represent a dendritic cell-dependent pathway capable of inhibiting Treg suppressive activity, potentially enhancing the anti-tumour response. Modulation of TLR2-Treg interactions should be further explored to determine if they have a role in the therapeutic management of OSCC. Copyright © 2017 Elsevier GmbH. All rights reserved.

Subcellular distribution of Lck during CD4 T-cell maturation in the thymic medulla regulates the T-cell activation threshold.

PubMed

Stephen, Tom Li; Wilson, Bridget S; Laufer, Terri M

2012-05-08

Mature peripheral T cells respond to foreign but not to self-antigens. During development in the thymus, deletion of high-affinity self-reactive immature thymocytes contributes to tolerance of mature T cells. However, double-positive thymocytes are positively selected to survive if they respond to self-peptide-MHC complexes; thus, there must be mechanisms to prevent overt reactivity to those same complexes in the periphery. "Developmental tuning" is the active process through which T-cell receptor (TCR)-associated signaling pathways of single-positive (SP) thymocytes are attenuated to respond appropriately to self-peptide-MHC complexes in the periphery. We previously showed that MHC class II expression in the thymic medulla was necessary to tune CD4(+) SP (CD4 SP) thymocytes. CD4 SP thymocytes from mice lacking medullary MHC class II expression had inappropriately enhanced proximal TCR signaling to low-affinity self-ligands that was associated with altered cellular distribution of the tyrosine kinase Lck. Now, we report that activation of both tuned and untuned CD4 SP thymocytes is Lck-dependent. Untuned CD4 SP cells contain a pool of Lck with increased basal phosphorylation that is not associated with the CD4 coreceptor. Phosphorylation of this pool of Lck decreases with tuning. Immunogold transmission electron microscopy of membrane sheets permitted direct visualization of Lck. In the absence of tuning, a significant proportion of Lck and the TCR subunit CD3ζ are expressed on the same protein island; this close association of Lck and the TCR probably explains the enhanced activation of untuned CD4 SP cells. Thus, changes in membrane topography during thymic maturation determine the set point for TCR responsiveness.

Wind-Tunnel Investigation of an NACA 23021 Airfoil with a 0.32-Airfoil-Chord Double Slotted Flap

NASA Technical Reports Server (NTRS)

Fischel, Jack; Riebe, John M

1944-01-01

An investigation was made in the LMAL 7- by 10-foot wind tunnel of a NACA 23021 airfoil with a double slotted flap having a chord 32 percent of the airfoil chord (0.32c) to determine the aerodynamic section characteristics with the flaps deflected at various positions. The effects of moving the fore flap and rear flap as a unit and of deflecting or removing the lower lip of the slot were also determined. Three positions were selected for the fore flap and at each position the maximum lift of the airfoil was obtained with the rear flap at the maximum deflection used at that fore-flap position. The section lift of the airfoil increased as the fore flap was extended and maximum lift was obtained with the fore flap deflected 30 deg in the most extended position. This arrangement provided a maximum section lift coefficient of 3.31, which was higher than the value obtained with either a 0.2566c or a 0.40c single-slotted-flap arrangement and 0.25 less than the value obtained with a 0.4c double-slotted-flap arrangement on the same airfoil. The values of the profile-drag coefficient obtained with the 0.32c double slotted flap were larger than those for the 0.2566c or 0.40c single slotted flaps for section lift coefficients between 1.0 and approximately 2.7. At all values of the section lift coefficient above 1.0, the 0.40c double slotted flap had a lower profile drag than the 0.32c double slotted flap. At various values of the maximum section lift coefficient produced by various flap defections, the 0.32c double slotted flap gave negative section pitching-moment coefficients that were higher than those of other slotted flaps on the same airfoil. The 0.32c double slotted flap gave approximately the same maximum section lift coefficient as, but higher profile-drag coefficients over the entire lift range than, a similar arrangement of a 0.30c double slotted flap on an NACA 23012 airfoil.

A hybrid of B and T lymphoblastic cell line could potentially substitute dendritic cells to efficiently expand out Her-2/neu-specific cytotoxic T lymphocytes from advanced breast cancer patients in vitro.

PubMed

Chen, Sheng; Gu, Feifei; Li, Kang; Zhang, Kai; Liu, Yangyang; Liang, Jinyan; Gao, Wei; Wu, Gang; Liu, Li

2017-02-28

Adoptive transfer of cytotoxic T lymphocytes (CTLs) holds promises to cure cancer. However, this treatment is hindered by lacking a robust way to specifically expand out CTLs. Here, we developed a hybrid of B lymphoblastic cell line and T lymphoblastic cell line (T2 cells) as a substitute of dendritic cells, together with irradiated autologous peripheral blood mononuclear cell (PBMC) as feeder cells and rhIL-2, to activate and expand Her-2/neu-specific CD8 + T cells from human epidermal growth factor receptor 2 (Her-2/neu) and human leukocyte antigen (HLA)-A2 double positive advanced breast cancer patients in vitro. These Her-2/neu-loaded T2 cells reproducibly activated and expanded out Her-2/neu-specific CD8 + T cells to 10 7 in 8 weeks. Furthermore, these Her-2/neu-specific CD8 + T cells had good sensitivity of recognition and killing Her-2/neu-overexpressed breast cancer cell line SK.BR.3. This technique gives us another insight on how to rapidly obtain sufficient CTLs for adoptive cancer immunotherapy.

Graphene-Iodine Nanocomposites: Highly Potent Bacterial Inhibitors that are Bio-compatible with Human Cells

PubMed Central

Some, Surajit; Sohn, Ji Soo; Kim, Junmoo; Lee, Su-Hyun; Lee, Su Chan; Lee, Jungpyo; Shackery, Iman; Kim, Sang Kyum; Kim, So Hyun; Choi, Nakwon; Cho, Il-Joo; Jung, Hyo-Il; Kang, Shinill; Jun, Seong Chan

2016-01-01

Graphene-composites, capable of inhibiting bacterial growth which is also bio-compatible with human cells have been highly sought after. Here we report for the first time the preparation of new graphene-iodine nano-composites via electrostatic interactions between positively charged graphene derivatives and triiodide anions. The resulting composites were characterized by X-ray photoemission spectroscopy, UV-spectroscopy, Raman spectroscopy and Scanning electron microscopy. The antibacterial potential of these graphene-iodine composites against Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirobilis, Staphylococcus aureus, and E. coli was investigated. In addition, the cytotoxicity of the nanocomposite with human cells [human white blood cells (WBC), HeLa, MDA-MB-231, Fibroblast (primary human keratinocyte) and Keratinocyte (immortalized fibroblast)], was assessed. DGO (Double-oxidizes graphene oxide) was prepared by the additional oxidation of GO (graphene oxide). This generates more oxygen containing functional groups that can readily trap more H+, thus generating a positively charged surface area under highly acidic conditions. This step allowed bonding with a greater number of anionic triiodides and generated the most potent antibacterial agent among graphene-iodine and as-made povidone-iodine (PVP-I) composites also exhibited nontoxic to human cells culture. Thus, these nano-composites can be used to inhibit the growth of various bacterial species. Importantly, they are also very low-cytotoxic to human cells culture. PMID:26843066

MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

PubMed

Constantin, Lena; Wainwright, Brandon J

2015-12-01

MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

Peroxisome proliferator-activated receptor-γ is expressed in eosinophils in nasal polyps.

PubMed

Asaka, Chikara; Honda, Kohei; Ito, Eiko; Fukui, Naoko; Chihara, Junichi; Ishikawa, Kazuo

2011-01-01

Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptors, which regulate fatty acid metabolites. One of the natural ligands for PPARγ is 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a major metabolite of prostaglandin D(2) (PGD(2)). Recently, PPARγ has been shown to play an important role in anti-inflammatory reactions, including within-airway allergic diseases, in a mouse model. Our aim was to clarify the expression and localization of PPARγ and PGD(2) synthase, which produces ligands of PPARγ, in nasal polyps by immunohistochemical analysis. Nasal polyps of chronic rhinosinusitis patients (6 asthmatic patients and 6 nonasthmatic patients) were obtained during surgery. May-Grünwald-Giemsa staining was performed to evaluate the eosinophil infiltration of the polyps. To identify the cells expressing PPARγ protein and PGD(2) synthase, double immunostaining was performed using anti-PPARγ antibody, monoclonal antileukocyte antibodies, and PGD(2) synthase antibody. The number of eosinophils and the number of PPARγ-positive cells in the nasal polyps of the asthmatic patients were significantly higher than those in the nonasthmatic patients. PPARγ was expressed on eosinophils and T cells of the infiltrating cells in the nasal polyps. PGD(2) synthase was also expressed on PPARγ-positive cells. PPARγ is involved in nasal polyposis pathogenesis, acting on eosinophils and T cells. Copyright © 2011 S. Karger AG, Basel.

Auditory hair cell centrioles undergo confined Brownian motion throughout the developmental migration of the kinocilium.

PubMed

Lepelletier, Léa; de Monvel, Jacques Boutet; Buisson, Johanna; Desdouets, Chantal; Petit, Christine

2013-07-02

Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements of the kinocilium basal body (mother centriole) and its daughter centriole, which we analyzed using particle tracking and modeling. We found that both hair cell centrioles undergo confined Brownian movements around their equilibrium positions, under the apparent constraint of a radial restoring force of ∼0.1 pN. This magnitude depended little on centriole position, suggesting nonlinear interactions with constraining, presumably cytoskeletal elements. The only dynamic change observed during the period of kinocilium migration was a doubling of the centrioles' confinement area taking place early in the process. It emerges from these static and dynamic observations that kinocilia migrate gradually in parallel with the organization of hair cells into rows during cochlear neuroepithelium extension. Analysis of the confined motion of hair cell centrioles under normal and pathological conditions should help determine which structures contribute to the restoring force exerting on them. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Auditory Hair Cell Centrioles Undergo Confined Brownian Motion Throughout the Developmental Migration of the Kinocilium

PubMed Central

Lepelletier, Léa; de Monvel, Jacques Boutet; Buisson, Johanna; Desdouets, Chantal; Petit, Christine

2013-01-01

Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements of the kinocilium basal body (mother centriole) and its daughter centriole, which we analyzed using particle tracking and modeling. We found that both hair cell centrioles undergo confined Brownian movements around their equilibrium positions, under the apparent constraint of a radial restoring force of ∼0.1 pN. This magnitude depended little on centriole position, suggesting nonlinear interactions with constraining, presumably cytoskeletal elements. The only dynamic change observed during the period of kinocilium migration was a doubling of the centrioles’ confinement area taking place early in the process. It emerges from these static and dynamic observations that kinocilia migrate gradually in parallel with the organization of hair cells into rows during cochlear neuroepithelium extension. Analysis of the confined motion of hair cell centrioles under normal and pathological conditions should help determine which structures contribute to the restoring force exerting on them. PMID:23823223

Tilted light sheet microscopy with 3D point spread functions for single-molecule super-resolution imaging in mammalian cells

NASA Astrophysics Data System (ADS)

Gustavsson, Anna-Karin; Petrov, Petar N.; Lee, Maurice Y.; Shechtman, Yoav; Moerner, W. E.

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

"Double-Cable" Conjugated Polymers with Linear Backbone toward High Quantum Efficiencies in Single-Component Polymer Solar Cells.

PubMed

Feng, Guitao; Li, Junyu; Colberts, Fallon J M; Li, Mengmeng; Zhang, Jianqi; Yang, Fan; Jin, Yingzhi; Zhang, Fengling; Janssen, René A J; Li, Cheng; Li, Weiwei

2017-12-27

A series of "double-cable" conjugated polymers were developed for application in efficient single-component polymer solar cells, in which high quantum efficiencies could be achieved due to the optimized nanophase separation between donor and acceptor parts. The new double-cable polymers contain electron-donating poly(benzodithiophene) (BDT) as linear conjugated backbone for hole transport and pendant electron-deficient perylene bisimide (PBI) units for electron transport, connected via a dodecyl linker. Sulfur and fluorine substituents were introduced to tune the energy levels and crystallinity of the conjugated polymers. The double-cable polymers adopt a "face-on" orientation in which the conjugated BDT backbone and the pendant PBI units have a preferential π-π stacking direction perpendicular to the substrate, favorable for interchain charge transport normal to the plane. The linear conjugated backbone acts as a scaffold for the crystallization of the PBI groups, to provide a double-cable nanophase separation of donor and acceptor phases. The optimized nanophase separation enables efficient exciton dissociation as well as charge transport as evidenced from the high-up to 80%-internal quantum efficiency for photon-to-electron conversion. In single-component organic solar cells, the double-cable polymers provide power conversion efficiency up to 4.18%. This is one of the highest performances in single-component organic solar cells. The nanophase-separated design can likely be used to achieve high-performance single-component organic solar cells.

Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

PubMed Central

Echevarria-Lima, Juliana; Kyle-Cezar, Fernanda; Leite, Daniela F P; Capella, Luiz; Capella, Márcia A M; Rumjanek, Vivian M

2005-01-01

Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2′-7′-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation. PMID:15804283

Automatic alternative phase-shift mask CAD layout tool for gate shrinkage of embedded DRAM in logic below 0.18 μm

NASA Astrophysics Data System (ADS)

Ohnuma, Hidetoshi; Kawahira, Hiroichi

1998-09-01

An automatic alternative phase shift mask (PSM) pattern layout tool has been newly developed. This tool is dedicated for embedded DRAM in logic device to shrink gate line width with improving line width controllability in lithography process with a design rule below 0.18 micrometers by the KrF excimer laser exposure. The tool can crete Levenson type PSM used being coupled with a binary mask adopting a double exposure method for positive photo resist. By using graphs, this tool automatically creates alternative PSM patterns. Moreover, it does not give any phase conflicts. By adopting it to actual embedded DRAM in logic cells, we have provided 0.16 micrometers gate resist patterns at both random logic and DRAM areas. The patterns were fabricated using two masks with the double exposure method. Gate line width has been well controlled under a practical exposure-focus window.

Measures of precision for dissimilarity-based multivariate analysis of ecological communities

PubMed Central

Anderson, Marti J; Santana-Garcon, Julia

2015-01-01

Ecological studies require key decisions regarding the appropriate size and number of sampling units. No methods currently exist to measure precision for multivariate assemblage data when dissimilarity-based analyses are intended to follow. Here, we propose a pseudo multivariate dissimilarity-based standard error (MultSE) as a useful quantity for assessing sample-size adequacy in studies of ecological communities. Based on sums of squared dissimilarities, MultSE measures variability in the position of the centroid in the space of a chosen dissimilarity measure under repeated sampling for a given sample size. We describe a novel double resampling method to quantify uncertainty in MultSE values with increasing sample size. For more complex designs, values of MultSE can be calculated from the pseudo residual mean square of a permanova model, with the double resampling done within appropriate cells in the design. R code functions for implementing these techniques, along with ecological examples, are provided. PMID:25438826

Lack of clinical AIDS in SIV-infected sooty mangabeys with significant CD4+ T cell loss is associated with double-negative T cells.

PubMed

Milush, Jeffrey M; Mir, Kiran D; Sundaravaradan, Vasudha; Gordon, Shari N; Engram, Jessica; Cano, Christopher A; Reeves, Jacqueline D; Anton, Elizabeth; O'Neill, Eduardo; Butler, Eboneé; Hancock, Kathy; Cole, Kelly S; Brenchley, Jason M; Else, James G; Silvestri, Guido; Sodora, Donald L

2011-03-01

SIV infection of natural host species such as sooty mangabeys results in high viral replication without clinical signs of simian AIDS. Studying such infections is useful for identifying immunologic parameters that lead to AIDS in HIV-infected patients. Here we have demonstrated that acute, SIV-induced CD4(+) T cell depletion in sooty mangabeys does not result in immune dysfunction and progression to simian AIDS and that a population of CD3(+)CD4(-)CD8(-) T cells (double-negative T cells) partially compensates for CD4(+) T cell function in these animals. Passaging plasma from an SIV-infected sooty mangabey with very few CD4(+) T cells to SIV-negative animals resulted in rapid loss of CD4(+) T cells. Nonetheless, all sooty mangabeys generated SIV-specific antibody and T cell responses and maintained normal levels of plasma lipopolysaccharide. Moreover, all CD4-low sooty mangabeys elicited a de novo immune response following influenza vaccination. Such preserved immune responses as well as the low levels of immune activation observed in these animals were associated with the presence of double-negative T cells capable of producing Th1, Th2, and Th17 cytokines. These studies indicate that SIV-infected sooty mangabeys do not appear to rely entirely on CD4(+) T cells to maintain immunity and identify double-negative T cells as a potential subset of cells capable of performing CD4(+) T cell-like helper functions upon SIV-induced CD4(+) T cell depletion in this species.

The Bacillus subtilis Acyl Lipid Desaturase Is a Δ5 Desaturase

PubMed Central

Altabe, Silvia G.; Aguilar, Pablo; Caballero, Gerardo M.; de Mendoza, Diego

2003-01-01

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids. PMID:12730185

Does infection with Chlamydia pneumoniae and/or Helicobacter pylori increase the expression of endothelial cell adhesion molecules in humans?

PubMed

Schumacher, A; Seljeflot, I; Lerkerød, A B; Sommervoll, L; Otterstad, J E; Arnesen, H

2002-10-01

To investigate if Chlamydia pneumoniae and/or Helicobacter pylori seropositivity is associated with elevated levels of soluble endothelial cell adhesion molecules (sCAMs) as markers of atherosclerotic activity. Immunoglobulin A (IgA) and IgG antibodies to the two bacteria, soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and E-selectin were measured in coronary heart disease (CHD) patients (n = 193) and age- and sex-matched controls (n = 193). Two different serological methods were used for the detection of Chlamydia antibodies: Labsystems microimmunofluorescence to detect species-specific C. pneumoniae antibodies and Medac's recombinant enzyme-linked immunosorbent assay to detect genus-specific lipopolysaccharide antibodies. The concentrations of sICAM-1 and E-selectin were higher in CHD patients with positive vs. negative Chlamydia lipopolysaccharide IgA (P = 0.044 for both). H. pylori antibodies alone did not predict raised levels of sCAMs, but in CHD patients sICAM-1 was increased with IgA seropositivity to both bacteria compared to double seronegativity (P = 0.034). Concentrations of sVCAM-1 were elevated in CHD patients with double IgA seropositivity compared to those with Chlamydia lipopolysaccharide IgA seropositivity alone (P = 0.018). Our results may indicate that C. pneumoniae contributes to increased inflammation in CHD, and that this contribution is even more pronounced when present in combination with H. pylori IgA antibodies.

Expression Profile of NOTCH3 in Mouse Spermatogonia.

PubMed

Okada, Ryu; Fujimagari, Megumi; Koya, Eri; Hirose, Yoshikazu; Sato, Tomomi; Nishina, Yukio

2017-01-01

Stable and sustainable spermatogenesis is supported by the strict regulation of self-renewal and differentiation of spermatogonial stem cells (SSC), which are a rare population of undifferentiated spermatogonia. It has been revealed that some signaling factors regulate the self-renewal of SSC; however, the molecular mechanism of SSC maintenance is still not completely understood. Notch signaling is an evolutionarily conserved juxtacrine signaling that plays important roles in the cell fate determination of various tissue stem cells. Recently, analyses of loss- and gain-of-function suggested that Notch signaling was necessary for normal spermatogenesis. However, the expression of Notch signal components in spermatogonia is still unclear. Here, we analyzed the distribution of NOTCH3-expressing spermatogonia and the target genes. Double immunostaining with differentiation markers revealed that NOTCH3 was expressed in some undifferentiated and differentiated spermatogonia in mouse testes. To define the target gene of Notch3 signaling in spermatogonia, we analyzed the mRNA expression pattern of Hes and Hey family genes during testis development. Hes1 abundance was decreased during testis development, suggesting that spermatogonia may express Hes1. Immunohistochemical analysis showed that HES1 was expressed in prepubertal spermatogonia, whereas it was expressed predominantly in adult Sertoli cells and weakly in adult spermatogonia. Furthermore, NOTCH3-HES1 double-positive spermatogonia were in pup and adult testes. These results suggest that Notch3 signaling in spermatogonia could promote Hes1 expression. © 2017 S. Karger AG, Basel.

Photoassisted Oxygen Reduction Reaction in H2 -O2 Fuel Cells.

PubMed

Zhang, Bingqing; Wang, Shengyang; Fan, Wenjun; Ma, Weiguang; Liang, Zhenxing; Shi, Jingying; Liao, Shijun; Li, Can

2016-11-14

The oxygen reduction reaction (ORR) is a key step in H 2 -O 2 fuel cells, which, however, suffers from slow kinetics even for state-of-the-art catalysts. In this work, by making use of photocatalysis, the ORR was significantly accelerated with a polymer semiconductor (polyterthiophene). The onset potential underwent a positive shift from 0.66 to 1.34 V, and the current was enhanced by a factor of 44 at 0.6 V. The improvement was further confirmed in a proof-of-concept light-driven H 2 -O 2 fuel cell, in which the open circuit voltage (V oc ) increased from 0.64 to 1.18 V, and the short circuit current (J sc ) was doubled. This novel tandem structure combining a polymer solar cell and a fuel cell enables the simultaneous utilization of photo- and electrochemical energy, showing promising potential for applications in energy conversion and storage. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.

PubMed

Taniuchi, Ichiro; Osato, Motomi; Egawa, Takeshi; Sunshine, Mary Jean; Bae, Suk Chul; Komori, Toshihisa; Ito, Yoshiaki; Littman, Dan R

2002-11-27

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

Haloperidol and reduced haloperidol concentrations in plasma and red blood cells from chronic schizophrenic patients.

PubMed

Ko, G N; Korpi, E R; Kirch, D G

1989-06-01

In a double-blind, placebo-controlled study, 15 drug-free chronic schizophrenic inpatients were treated with a fixed dose of haloperidol for 6 weeks. Haloperidol and its metabolite, reduced haloperidol, were measured in plasma and red blood cells after 2, 4, and 6 weeks of treatment. Behavioral change was rated using the Brief Psychiatric Rating Scale (BPRS). Not only the raw concentrations, but also blood compartment sums and ratios of these four drug measurements were tested for their strength of association with behavioral improvement. Positive associations with some BPRS subscales at some time points emerged; however, no significant correlations were found to extend across all time points measured. There was a trend in this cohort for negative symptom improvement to be associated with the ratio of haloperidol to reduced haloperidol in red blood cells. The ratio of haloperidol to reduced haloperidol in plasma was always greater than that in the red blood cells for all patients, reflecting an accumulation of the metabolite in red blood cells.

Silicon wafer-based tandem cells: The ultimate photovoltaic solution?

NASA Astrophysics Data System (ADS)

Green, Martin A.

2014-03-01

Recent large price reductions with wafer-based cells have increased the difficulty of dislodging silicon solar cell technology from its dominant market position. With market leaders expected to be manufacturing modules above 16% efficiency at 0.36/Watt by 2017, even the cost per unit area (60-70/m2) will be difficult for any thin-film photovoltaic technology to significantly undercut. This may make dislodgement likely only by appreciably higher energy conversion efficiency approaches. A silicon wafer-based cell able to capitalize on on-going cost reductions within the mainstream industry, but with an appreciably higher than present efficiency, might therefore provide the ultimate PV solution. With average selling prices of 156 mm quasi-square monocrystalline Si photovoltaic wafers recently approaching 1 (per wafer), wafers now provide clean, low cost templates for overgrowth of thin, wider bandgap high performance cells, nearly doubling silicon's ultimate efficiency potential. The range of possible Si-based tandem approaches is reviewed together with recent results and ultimate prospects.

Chalcones: structural requirements for antioxidant, estrogenic and antiproliferative activities.

PubMed

Calliste, C A; Le Bail, J C; Trouillas, P; Pouget, C; Habrioux, G; Chulia, A J; Duroux, J L

2001-01-01

Flavonoids are largely studied for their biological properties and particularly for their scavenging and antioxidant activities. In the present study, we first evaluated the antioxidant and the estrogenic actions of chalcones, then we tested their effects on MCF-7 cell proliferation. Chalcones are unique in the flavonoids family in lacking a heterocyclic C ring. We tested substituted chalcones with different numbers and different positions of the hydroxy groups: 2'-hydroxychalcone, 4'-hydroxychalcone, 4-hydroxychalcone, 2',4-dihydroxychalcone, isoliquiritigenin, 2',4'-dihydroxychalcone, phloretin and naringenin chalcone. For the antioxidant tests we established the importance of the alpha-beta double bond and the 6'-hydroxy group. The establishment of the structure-activity relationship for the estrogenic properties showed a correlation between the antioxidant and the estrogenic properties. The importance of conformation and hydroxy group positions observed for chalcones, having antioxidant and estrogenic properties, was also observed on MCF-7 cell growth with the same structure-activity relationship. The role of electron and hydrogen transfer in the correlation between these three biological activities was discussed.

10 CFR Appendix H to Part 73 - Weapons Qualification Criteria

Code of Federal Regulations, 2014 CFR

2014-01-01

... position Shotgun 1 1 7 yards 2 Double 0 buck-shot 4 seconds At low ready position fire 2 rounds standing Minimum qualifying = 70%. 2 12 15 yards 4 Double 0 buck-shot 15 seconds At low ready position fire 2 rounds standing, reload and fire 2 rounds 3 12 25 yards 4 rifled slugs or 00 buck-shot 20 seconds On...

10 CFR Appendix H to Part 73 - Weapons Qualification Criteria

Code of Federal Regulations, 2011 CFR

2011-01-01

... position Shotgun 1 1 7 yards 2 Double 0 buck-shot 4 seconds At low ready position fire 2 rounds standing Minimum qualifying = 70%. 2 12 15 yards 4 Double 0 buck-shot 15 seconds At low ready position fire 2 rounds standing, reload and fire 2 rounds 3 12 25 yards 4 rifled slugs or 00 buck-shot 20 seconds On...

10 CFR Appendix H to Part 73 - Weapons Qualification Criteria

Code of Federal Regulations, 2012 CFR

2012-01-01

... position Shotgun 1 1 7 yards 2 Double 0 buck-shot 4 seconds At low ready position fire 2 rounds standing Minimum qualifying = 70%. 2 12 15 yards 4 Double 0 buck-shot 15 seconds At low ready position fire 2 rounds standing, reload and fire 2 rounds 3 12 25 yards 4 rifled slugs or 00 buck-shot 20 seconds On...

10 CFR Appendix H to Part 73 - Weapons Qualification Criteria

Code of Federal Regulations, 2010 CFR

2010-01-01

... position Shotgun 1 1 7 yards 2 Double 0 buck-shot 4 seconds At low ready position fire 2 rounds standing Minimum qualifying = 70%. 2 12 15 yards 4 Double 0 buck-shot 15 seconds At low ready position fire 2 rounds standing, reload and fire 2 rounds 3 12 25 yards 4 rifled slugs or 00 buck-shot 20 seconds On...

10 CFR Appendix H to Part 73 - Weapons Qualification Criteria

Code of Federal Regulations, 2013 CFR

2013-01-01

... position Shotgun 1 1 7 yards 2 Double 0 buck-shot 4 seconds At low ready position fire 2 rounds standing Minimum qualifying = 70%. 2 12 15 yards 4 Double 0 buck-shot 15 seconds At low ready position fire 2 rounds standing, reload and fire 2 rounds 3 12 25 yards 4 rifled slugs or 00 buck-shot 20 seconds On...

Inflammation-induced synthesis of proteoheparan sulfate: a novel acute-phase reactant in rat hepatocytes

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

Djovkar, A.; Gressner, A.M.

1987-03-01

The synthesis of proteoheparan sulfate in hepatocytes is positively regulated under acute-phase conditions produced either by turpentine or deep back incision. In both cases the incorporation of (/sup 35/S)sulfate and (/sup 14/C)glucosamine is doubled during a 4-h incubation period if compared with control rat hepatocytes. Neither the fractional secretion rate of heparan sulfate into the medium (less than 0.1 of cell-associated glycosaminoglycans) nor the composition of newly formed proteoglycans in hepatocytes are affected during acute phase reaction.

Unpaired 5' ppp-nucleotides, as found in arenavirus double-stranded RNA panhandles, are not recognized by RIG-I.

PubMed

Marq, Jean-Baptiste; Kolakofsky, Daniel; Garcin, Dominique

2010-06-11

Arenavirus and bunyavirus RNA genomes are unusual in that they are found in circular nucleocapsids, presumably due to the annealing of their complementary terminal sequences. Moreover, arenavirus genome synthesis initiates with GTP at position +2 of the template rather than at the precise 3' end (position +1). After formation of a dinucleotide, 5' pppGpC(OH) is then realigned on the template before this primer is extended. The net result of this "prime and realign" mechanism of genome initiation is that 5' pppG is found as an unpaired 5' nucleotide when the complementary genome ends anneal to form a double-stranded (dsRNA) panhandle. Using 5' pppRNA made in vitro and purified so that all dsRNA side products are absent, we have determined that both this 5' nucleotide overhang, as well as mismatches within the dsRNA (as found in some arenavirus genomes), clearly reduce the ability of these model dsRNAs to induce interferon upon transfection into cells. The presence of this unpaired 5' ppp-nucleotide is thus another way that some viruses appear to use to avoid detection by cytoplasmic pattern recognition receptors.

IL-12 and IL-15 induce the expression of CXCR6 and CD49a on peripheral natural killer cells.

PubMed

Hydes, Theresa; Noll, Angela; Salinas-Riester, Gabriela; Abuhilal, Mohammed; Armstrong, Thomas; Hamady, Zaed; Primrose, John; Takhar, Arjun; Walter, Lutz; Khakoo, Salim I

2018-03-01

Murine hepatic NK cells exhibit adaptive features, with liver-specific adhesion molecules CXCR6 and CD49a acting as surface markers. We investigated human liver-resident CXCR6+ and CD49a+ NK cells using RNA sequencing, flow cytometry, and functional analysis. We further assessed the role of cytokines in generating NK cells with these phenotypes from the peripheral blood. Hepatic CD49a+ NK cells could be induced using cytokines and produce high quantities of IFNγ and TNFα, in contrast to hepatic CXCR6+ NK cells. RNA sequencing of liver-resident CXCR6+ NK cells confirmed a tolerant immature phenotype with reduced expression of markers associated with maturity and cytotoxicity. Liver-resident double-positive CXCR6 + CD49a+ hepatic NK cells are immature but maintain high expression of Th1 cytokines as observed for single-positive CD49a+ NK cells. We show that stimulation with activating cytokines can readily induce upregulation of both CD49a and CXCR6 on NK cells in the peripheral blood. In particular, IL-12 and IL-15 can generate CXCR6 + CD49a+ NK cells in vitro from NK cells isolated from the peripheral blood, with comparable phenotypic and functional features to liver-resident CD49a+ NK cells, including enhanced IFNγ and NKG2C expression. IL-12 and IL-15 may be key for generating NK cells with a tissue-homing phenotype and strong Th1 cytokine profile in the blood, and links peripheral activation of NK cells with tissue-homing. These findings may have important therapeutic implications for immunotherapy of chronic liver disease. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Lack of clinical AIDS in SIV-infected sooty mangabeys with significant CD4+ T cell loss is associated with double-negative T cells

PubMed Central

Milush, Jeffrey M.; Mir, Kiran D.; Sundaravaradan, Vasudha; Gordon, Shari N.; Engram, Jessica; Cano, Christopher A.; Reeves, Jacqueline D.; Anton, Elizabeth; O’Neill, Eduardo; Butler, Eboneé; Hancock, Kathy; Cole, Kelly S.; Brenchley, Jason M.; Else, James G.; Silvestri, Guido; Sodora, Donald L.

2011-01-01

SIV infection of natural host species such as sooty mangabeys results in high viral replication without clinical signs of simian AIDS. Studying such infections is useful for identifying immunologic parameters that lead to AIDS in HIV-infected patients. Here we have demonstrated that acute, SIV-induced CD4+ T cell depletion in sooty mangabeys does not result in immune dysfunction and progression to simian AIDS and that a population of CD3+CD4–CD8– T cells (double-negative T cells) partially compensates for CD4+ T cell function in these animals. Passaging plasma from an SIV-infected sooty mangabey with very few CD4+ T cells to SIV-negative animals resulted in rapid loss of CD4+ T cells. Nonetheless, all sooty mangabeys generated SIV-specific antibody and T cell responses and maintained normal levels of plasma lipopolysaccharide. Moreover, all CD4-low sooty mangabeys elicited a de novo immune response following influenza vaccination. Such preserved immune responses as well as the low levels of immune activation observed in these animals were associated with the presence of double-negative T cells capable of producing Th1, Th2, and Th17 cytokines. These studies indicate that SIV-infected sooty mangabeys do not appear to rely entirely on CD4+ T cells to maintain immunity and identify double-negative T cells as a potential subset of cells capable of performing CD4+ T cell–like helper functions upon SIV-induced CD4+ T cell depletion in this species. PMID:21317533

DNA Polyplexes as Combinatory Drug Carriers of Doxorubicin and Cisplatin: An In Vitro Study

PubMed Central

Kang, Han Chang; Cho, Hana; Bae, You Han

2015-01-01

Double helix nucleic acids were used as a combination drug carrier for doxorubicin (DOX), which physically intercalates with DNA double helices, and cisplatin (CDDP), which binds to DNA without an alkylation reaction. DNA interacting with DOX, CDDP, or both was complexed with positively charged, endosomolytic polymers. Compared with the free drug, the polyplexes (100 ~ 170 nm in size) delivered more drug into the cytosol and the nucleus and demonstrated similar or superior (up to a 7-fold increase) in vitro cell-killing activity. Additionally, the gene expression activities of most of the chemical drug-loaded plasmid DNA (pDNA) polyplexes were not impaired by the physical interactions between the nucleic acid and DOX/CDDP. When a model reporter pDNA (luciferase) was employed, it expressed luciferase protein at 0.7- ~ 1.4-fold the amount expressed by the polyplex with no bound drugs (a control), which indicated the fast translocation of the intercalated or bound drugs from the “carrier DNA” to the “nuclear DNA” of target cells. The proposed concept may offer the possibility of versatile combination therapies of genetic materials and small molecule drugs that bind to nucleic acids to treat various diseases. PMID:26132975

First evidence of dengue infection in domestic dogs living in different ecological settings in Thailand.

PubMed

Thongyuan, Suporn; Kittayapong, Pattamaporn

2017-01-01

Dengue is a vector-borne disease transmitted by Aedes mosquitoes. It is considered an important public health problem in many countries worldwide. However, only a few studies have been conducted on primates and domestic animals that could potentially be a reservoir of dengue viruses. Since domestic dogs share both habitats and vectors with humans, this study aimed to investigate whether domestic dogs living in different ecological settings in dengue endemic areas in Thailand could be naturally infected with dengue viruses. Serum samples were collected from domestic dogs in three different ecological settings of Thailand: urban dengue endemic areas of Nakhon Sawan Province; rubber plantation areas of Rayong Province; and Koh Chang, an island tourist spot of Trat Province. These samples were screened for dengue viral genome by using semi-nested RT-PCR. Positive samples were then inoculated in mosquito and dog cell lines for virus isolation. Supernatant collected from cell culture was tested for the presence of dengue viral genome by semi-nested RT-PCR, then double-strand DNA products were double-pass custom-sequenced. Partial nucleotide sequences were aligned with the sequences already recorded in GenBank, and a phylogenetic tree was constructed. In the urban setting, 632 domestic dog serum samples were screened for dengue virus genome by RT-PCR, and six samples (0.95%) tested positive for dengue virus. Four out of six dengue viruses from positive samples were successfully isolated. Dengue virus serotype 2 and serotype 3 were found to have circulated in domestic dog populations. One of 153 samples (0.65%) collected from the rubber plantation area showed a PCR-positive result, and dengue serotype 3 was successfully isolated. Partial gene phylogeny revealed that the isolated dengue viruses were closely related to those strains circulating in human populations. None of the 71 samples collected from the island tourist spot showed a positive result. We concluded that domestic dogs can be infected with dengue virus strains circulating in dengue endemic areas. The role of domestic dogs in dengue transmission needs to be further investigated, i.e., whether they are potential reservoirs or incidental hosts of dengue viruses.

Functional Analysis of the Lactobacillus casei BL23 Sortases

PubMed Central

Muñoz-Provencio, Diego; Rodríguez-Díaz, Jesús; Collado, María Carmen; Langella, Philippe; Bermúdez-Humarán, Luis G.

2012-01-01

Sortases are a class of enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria. Lactobacillus casei BL23 harbors four sortase genes, two belonging to class A (srtA1 and srtA2) and two belonging to class C (srtC1 and srtC2). Class C sortases were clustered with genes encoding their putative substrates that were homologous to the SpaEFG and SpaCBA proteins that encode mucus adhesive pili in Lactobacillus rhamnosus GG. Twenty-three genes encoding putative sortase substrates were identified in the L. casei BL23 genome with unknown (35%), enzymatic (30%), or adhesion-related (35%) functions. Strains disrupted in srtA1, srtA2, srtC1, and srtC2 and an srtA1 srtA2 double mutant were constructed. The transcription of all four sortase encoding genes was detected, but only the mutation of srtA1 resulted in a decrease in bacterial surface hydrophobicity. The β-N-acetyl-glucosaminidase and cell wall proteinase activities of whole cells diminished in the srtA1 mutant and, to a greater extent, in the srtA1 srtA2 double mutant. Cell wall anchoring of the staphylococcal NucA reporter protein fused to a cell wall sorting sequence was also affected in the srtA mutants, and the percentages of adhesion to Caco-2 and HT-29 intestinal epithelial cells were reduced for the srtA1 srtA2 strain. Mutations in srtC1 or srtC2 result in an undetectable phenotype. Together, these results suggest that SrtA1 is the housekeeping sortase in L. casei BL23 and SrtA2 would carry out redundant or complementary functions that become evident when SrtA1 activity is absent. PMID:23042174

Redundant role of protein kinase C delta and epsilon during mouse embryonic development.

PubMed

Carracedo, Sergio; Sacher, Frank; Brandes, Gudrun; Braun, Ursula; Leitges, Michael

2014-01-01

Protein Kinase C delta and epsilon are mediators of important cellular events, such as cell proliferation, migration or apoptosis. The formation of blood vessels, i.e., vasculo- and angiogenesis, is a process where these isoforms have also been shown to participate. However, mice deficient in either Protein Kinase C delta or epsilon are viable and therefore their individual contribution to the formation of the vasculature appeared so far dispensable. In this study, we show that double null mutation of Protein Kinase C delta and epsilon causes embryonic lethality at approximately E9.5. At this stage, whole mount staining of the endothelial marker CD31 in double null embryos revealed defective blood vessel formation. Moreover, culture of double deficient mouse allantois showed impaired endothelial cell organization, and analyses of double deficient embryo sections showed dilated vessels, decreased endothelial-specific adherent junctions, and decreased contact of endothelial cells with mural cells. Protein kinase C delta and epsilon also appeared essential for vascular smooth muscle cell differentiation, since α-smooth muscle actin, a classical marker for vascular smooth muscle cells, was almost undetectable in double deficient embryonic aorta at E9.5. Subsequent qPCR analyses showed decreased VE-cadherin, Vegfr2, Cd31, Cdh2, Ets1, and Fli-1, among other angiogenesis related transcripts in double deficient embryos. Taken together, these data suggest for the first time an in vivo redundant role between members of the novel Protein Kinase C subfamily that allows for mutual compensation during mouse embryonic development, with vasculogenesis/angiogenesis as an obvious common function of these two Protein Kinase Cs. Protein Kinase C delta and epsilon might therefore be useful targets for inhibiting vasculo- and/or angiogenesis.

Immunohistochemical detection of vimentin in pancreatic islet β- and α-cells of macrosomic infants of diabetic and nondiabetic mothers.

PubMed

Krivova, Yuliya S; Proshchina, Alexandra E; Barabanov, Valeriy M; Barinova, Irina V; Saveliev, Sergey V

2018-02-01

Expression of the intermediate filament protein vimentin has been recently observed in the pancreatic islet β- and α-cells of humans with type 2 diabetes mellitus. It was suggested that the presence of vimentin in endocrine cells may indicate islet tissue renewal, or potentially represent the dedifferentiation of endocrine cells, which could contribute to the onset of type 2 diabetes or islet cell dysfunction. To analyze the expression of vimentin in pancreatic β- and α-cells of macrosomic infants of diabetic and nondiabetic mothers. Pancreatic samples of five macrosomic infants (gestational age 34-40weeks) from three diabetic and two nondiabetic mothers were compared to six control infants (32-40weeks, weight appropriate for gestational age) from normoglycemic mothers. Pancreatic autopsy samples were examined by double immunofluorescent labeling with antibodies against vimentin and either insulin or glucagon. Alterations in the endocrine pancreas were measured using morphometric methods, then data were statistically analyzed. In the pancreatic islets of macrosomic infants from diabetic and nondiabetic mothers, we observed vimentin-positive cells, some of which simultaneously contained insulin or glucagon. We also quantitatively showed that the presence of such cells was associated with hypertrophy and hyperplasia of the islets, and with an increase in β- and α-cell density. We speculate that the appearance of vimentin-positive islet cells may reflect induction of differentiation in response to the increased insulin demand, and vimentin may serve as an early marker of endocrine pancreas disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Cholinergic urethral brush cells are widespread throughout placental mammals.

PubMed

Deckmann, Klaus; Krasteva-Christ, Gabriela; Rafiq, Amir; Herden, Christine; Wichmann, Judy; Knauf, Sascha; Nassenstein, Christina; Grevelding, Christoph G; Dorresteijn, Adriaan; Chubanov, Vladimir; Gudermann, Thomas; Bschleipfer, Thomas; Kummer, Wolfgang

2015-11-01

We previously identified a population of cholinergic epithelial cells in murine, human and rat urethrae that exhibits a structural marker of brush cells (villin) and expresses components of the canonical taste transduction signaling cascade (α-gustducin, phospholipase Cβ2 (PLCβ2), transient receptor potential cation channel melanostatin 5 (TRPM5)). These cells serve as sentinels, monitoring the chemical composition of the luminal content for potentially hazardous compounds such as bacteria, and initiate protective reflexes counteracting further ingression. In order to elucidate cross-species conservation of the urethral chemosensory pathway we investigated the occurrence and molecular make-up of urethral brush cells in placental mammals. We screened 11 additional species, at least one in each of the five mammalian taxonomic units primates, carnivora, perissodactyla, artiodactyla and rodentia, for immunohistochemical labeling of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), villin, and taste cascade components (α-gustducin, PLCβ2, TRPM5). Corresponding to findings in previously investigated species, urethral epithelial cells with brush cell shape were immunolabeled in all 11 mammals. In 8 species, immunoreactivities against all marker proteins and ChAT were observed, and double-labeling immunofluorescence confirmed the cholinergic nature of villin-positive and chemosensory (TRPM5-positive) cells. In cat and horse, these cells were not labeled by the ChAT antiserum used in this study, and unspecific reactions of the secondary antiserum precluded conclusions about ChAT-expression in the bovine epithelium. These data indicate that urethral brush cells are widespread throughout the mammalian kingdom and evolved not later than about 64.5millionyears ago. Copyright © 2015 Elsevier B.V. All rights reserved.

[Effect of filial imprinting procedure on cell proliferation in the chick brain].

PubMed

Komissarova, N V; Anokhin, K V

2007-01-01

In the present study we tested the hypothesis that memory formation during visual imprinting might be related to generation of new cells in the brain of newborn domestic chicks. Cell proliferation was examined in the intermediate medial mesopallium (IMM), arcopallium intermedium (AI), medial part of nidopallium and mesopallium (MNM), nidopallium dorso-caudalis (Ndc), hippocampus (Hp) and area parahippocampalis (APH), as well as in corresponding ventricular zones. Number of new cells was measured by BrdU incorporation 24 h or 7 days after training, BrdU was injected before training. 24 h after imprinting the number of BrdU-positive cells increased significantly in IMM. 7 days after training no changes were observed in IMM, while the number of new cells decreased in MNM and Ndc in comparison to the control group. These data suggest that newly generated cells in the brain of young chicks are influenced by imprinting procedure, which has opposite short-term and long-term effects. A possible reason for such double action of imprinting in contrast to conventional learning can be its additional stimulation of development of predisposition for features of natural parents.

Long-term Persistence of Innate Lymphoid Cells in the Gut After Intestinal Transplantation.

PubMed

Weiner, Joshua; Zuber, Julien; Shonts, Brittany; Yang, Suxiao; Fu, Jianing; Martinez, Mercedes; Farber, Donna L; Kato, Tomoaki; Sykes, Megan

2017-10-01

Little is known about innate lymphoid cell (ILC) populations in the human gut, and the turnover of these cells and their subsets after transplantation has not been described. Intestinal samples were taken from 4 isolated intestine and 3 multivisceral transplant recipients at the time of any operative resection, such as stoma closure or revision. ILCs were isolated and analyzed by flow cytometry. The target population was defined as being negative for lineage markers and double-positive for CD45/CD127. Cells were further stained to define ILC subsets and a donor-specific or recipient-specific HLA marker to analyze chimerism. Donor-derived ILCs were found to persist greater than 8 years after transplantation. Additionally, the percentage of cells thought to be lymphoid tissue inducer cells among donor ILCs was far higher than that among recipient ILCs. Our findings demonstrate that donor-derived ILCs persist long-term after transplantation and support the notion that human lymphoid tissue inducer cells may form in the fetus and persist throughout life, as hypothesized in rodents. Correlation between chimerism and rejection, graft failure, and patient survival requires further study.

Mitomycin C-induced apoptosis in cultured human Tenon's capsule fibroblasts.

PubMed

Kim, J W; Kim, S K; Song, I H; Kim, I T

1999-06-01

To investigate the mitomycin C-induced apoptotic cell death of fibroblasts, the primarily cultured human Tenon's capsule fibroblasts were exposed to a clinically used dosage of 0.4 mg/ml of mitomycin C for 5 minutes. TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay and electron microscopic studies were performed to determine the extent of mitomycin C-induced apoptosis. A flow cytometric study was performed to quantify the apoptotic cell population over time. The TUNEL stains were positive and electron microscopy showed features of apoptotic cell death in some fibroblasts 3 and 5 days after treatment. Flow cytometric analysis using Annexin V-propidium iodide double staining detected apoptotic cells 3 days after treatment. These apoptotic cell populations increased at 4 days and were sustained for one week. This study revealed that the clinical effects of mitomycin C on fibroblasts may be mediated not only by antiproliferative but also apoptotic cell death to some degree. Therefore, the apoptotic cell death of fibroblasts induced by mitomycin C should be considered to properly understand the mechanism of wound healing after trabeculectomy with adjunctive mitomycin C.

Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

PubMed

Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

2014-01-01

Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

THP-1 macrophage lipid accumulation unaffected by fatty acid double bond geometric or positional configuration

USDA-ARS?s Scientific Manuscript database

Dietary fatty acid type alters atherosclerotic lesion progression and macrophage lipid accumulation. Incompletely elucidated are the mechanisms by which fatty acids differing in double-bond geometric or positional configuration alter arterial lipid accumulation. The objective of this study was to ev...

Double-hit or dual expression of MYC and BCL2 in primary cutaneous large B-cell lymphomas.

PubMed

Menguy, Sarah; Frison, Eric; Prochazkova-Carlotti, Martina; Dalle, Stephane; Dereure, Olivier; Boulinguez, Serge; Dalac, Sophie; Machet, Laurent; Ram-Wolff, Caroline; Verneuil, Laurence; Gros, Audrey; Vergier, Béatrice; Beylot-Barry, Marie; Merlio, Jean-Philippe; Pham-Ledard, Anne

2018-03-26

In nodal diffuse large B-cell lymphoma, the search for double-hit with MYC and BCL2 and/or BCL6 rearrangements or for dual expression of BCL2 and MYC defines subgroups of patients with altered prognosis that has not been evaluated in primary cutaneous large B-cell lymphoma. Our objectives were to assess the double-hit and dual expressor status in a cohort of 44 patients with primary cutaneous large B-cell lymphoma according to the histological subtype and to evaluate their prognosis relevance. The 44 cases defined by the presence of more than 80% of large B-cells in the dermis corresponded to 21 primary cutaneous follicle centre lymphoma with large cell morphology and 23 primary cutaneous diffuse large B-cell lymphoma, leg type. Thirty-one cases (70%) expressed BCL2 and 29 (66%) expressed MYC. Dual expressor profile was observed in 25 cases (57%) of either subtypes (n = 6 or n = 19, respectively). Only one primary cutaneous follicle centre lymphoma, large-cell case had a double-hit status (2%). Specific survival was significantly worse in primary cutaneous diffuse large B-cell lymphoma, leg type than in primary cutaneous follicle centre lymphoma, large cell (p = 0.021) and for the dual expressor primary cutaneous large B-cell lymphoma group (p = 0.030). Both overall survival and specific survival were worse for patients belonging to the dual expressor primary cutaneous diffuse large B-cell lymphoma, leg type subgroup (p = 0.001 and p = 0.046, respectively). Expression of either MYC and/or BCL2 negatively impacted overall survival (p = 0.017 and p = 0.018 respectively). As the differential diagnosis between primary cutaneous follicle centre lymphoma, large cell and primary cutaneous diffuse large B-cell lymphoma, leg type has a major impact on prognosis, dual-expression of BCL2 and MYC may represent a new diagnostic criterion for primary cutaneous diffuse large B-cell lymphoma, leg type subtype and further identifies patients with impaired survival. Finally, the double-hit assessment does not appear clinically relevant in primary cutaneous large B-cell lymphoma.

Abundance of the Organic Anion-transporting Polypeptide OATP4A1 in Early-Stage Colorectal Cancer Patients: Association With Disease Relapse.

PubMed

Buxhofer-Ausch, Veronika; Sheikh, Maidah; Ausch, Christoph; Zotter, Simone; Bauer, Heike; Mollik, Marina; Reiner, Angelika; Gleiss, Andreas; Jäger, Walter; Sebesta, Christian; Kriwanek, Stephan; Thalhammer, Theresia

2018-05-03

The abundance of OATP4A1 in colorectal cancer (CRC) might be related to tumor progression. This was studied by immunohistochemistry on paraffin-embedded samples obtained from 178 patients (43 patients with a relapse within 5 y) with early-stage CRC. Positivity for OATP4A1 in tumor cells and noncancerous mucosal cells was proved by double-immunofluorescence staining with antibodies against OATP4A1 and keratin 8, whereas antibodies against appropriate CD markers were used to identify immune cells. Automated microscopic image analysis was used to measure the percentage of OATP4A1-positive cells and OATP4A1 staining intensity in tumor, immune, and adjacent normal-looking mucosal cells separately, as well as in the mucosal and immune cells of 14 nonmalignant tissue samples. In CRC the percentage of OATP4A1-positive cells, but not staining intensity, was significantly higher in tumor and mucosal cells adjacent to the tumor compared to the mucosa of nonmalignant samples (P<0.001 each). No difference was registered between immune cells in malignant and nonmalignant samples. Importantly, high levels of OATP4A1 in immune (odds ratio, 0.73; confidence interval, 0.63-0.85; P<0.001), and tumor cells (odds ratio, 0.79; confidence interval, 0.69-0.91; P<0.001) are significantly associated with a low risk of recurrence and also significantly enhance the discriminative power of other clinical parameters [such as International Union Against Cancer (UICC), adjuvant therapy, localization of the primary tumor] of the risk of relapse (receiver operating characteristics analysis; P=0.002). Using an advanced digital microscopic quantification procedure, we showed that OATP4A1 abundance is negatively associated with tumor recurrence in early-stage CRC. This digital scoring procedure may serve as a novel tool for the assessment of potential prognostic markers in early-stage CRC.

Modelling the balance between quiescence and cell death in normal and tumour cell populations.

PubMed

Spinelli, Lorenzo; Torricelli, Alessandro; Ubezio, Paolo; Basse, Britta

2006-08-01

When considering either human adult tissues (in vivo) or cell cultures (in vitro), cell number is regulated by the relationship between quiescent cells, proliferating cells, cell death and other controls of cell cycle duration. By formulating a mathematical description we see that even small alterations of this relationship may cause a non-growing population to start growing with doubling times characteristic of human tumours. Our model consists of two age structured partial differential equations for the proliferating and quiescent cell compartments. Model parameters are death rates from and transition rates between these compartments. The partial differential equations can be solved for the steady-age distributions, giving the distribution of the cells through the cell cycle, dependent on specific model parameter values. Appropriate formulas can then be derived for various population characteristic quantities such as labelling index, proliferation fraction, doubling time and potential doubling time of the cell population. Such characteristic quantities can be estimated experimentally, although with decreasing precision from in vitro, to in vivo experimental systems and to the clinic. The model can be used to investigate the effects of a single alteration of either quiescence or cell death control on the growth of the whole population and the non-trivial dependence of the doubling time and other observable quantities on particular underlying cell cycle scenarios of death and quiescence. The model indicates that tumour evolution in vivo is a sequence of steady-states, each characterised by particular death and quiescence rate functions. We suggest that a key passage of carcinogenesis is a loss of the communication between quiescence, death and cell cycle machineries, causing a defect in their precise, cell cycle dependent relationship.

Expression of the Thomsen-Friedenreich (TF) tumor antigen in human abort placentas.

PubMed

Richter, D U; Jeschke, U; Bergemann, C; Makovitzky, J; Lüthen, F; Karsten, U; Briese, V

2005-01-01

The Thomsen-Friedenreich antigen (TF), or more precisely epitope, has been known as a pancarcinoma antigen. It consists of galactose-beta1-3-N-acetylgalactose. We have already described the expression of TF in the normal placenta. TF is expressed by the syncytium and by extravillous trophoblast cells. In this study, we investigated the expression of TF in the abort placenta. Frozen samples of human abort placentas (12 placentas), obtained from the first and second trimesters of pregnancy and, for comparison, samples of normal placentas (17 placentas) from the first, second and third trimesters of pregnancy, were used. Expression of TF was investigated by immunohistochemical methods. For identification of TF-positive cells in abort placentas, immunofluorescence methods were used. Evaluation of simple and double immunofluorescence was performed on a laser scanning microscope. Furthermore, we isolated trophoblast cells from first and third trimester placentas and evaluated cytokeratin 7 and Muc1 expression by immunofluorescence methods. We observed expression of TF antigen in the syncytiotrophoblasts layer of the placenta in all three trimesters of pregnancy in normal and abort placentas evaluated by immunohistochemical methods. There was no expression of TF antigen in the decidua of abort placentas. Immunofluorescence double staining of TF antigen and cytokeratin 7 showed reduced expression of both antigens in the abort decidua and co-expression of both antigens in the syncytiotrophoblast layer of normal and abort placentas. TF expression in the syncytiotrophoblast was reduced in abort placentas. In the isolated trophoblast cells, no TF expression was found, however, Muc1 expression was visualized. Expression of TF antigen was reduced in the first and second trimester abort decidua compared to the normal decidua during the same time of pregnancy. TF antigen was restricted to the syncytiotrophoblast and extravillous trophoblast cells in the decidua. Abort placentas expressed TF antigen on the syncytiotrophoblast layer, but with lower intensity compared to normal placentas. We found a significantly reduced co-expression of TF antigen and cytokeratin 7 in the decidua of abort placentas. These data suggested a reduction of extravillous trophoblast cells in the decidua of abort placentas. In addition, we found higher numbers of CD45-positive cells in the abort decidua compared to normal placentas.

Dynamics and control of the ERK signaling pathway: Sensitivity, bistability, and oscillations.

PubMed

Arkun, Yaman; Yasemi, Mohammadreza

2018-01-01

Cell signaling is the process by which extracellular information is transmitted into the cell to perform useful biological functions. The ERK (extracellular-signal-regulated kinase) signaling controls several cellular processes such as cell growth, proliferation, differentiation and apoptosis. The ERK signaling pathway considered in this work starts with an extracellular stimulus and ends with activated (double phosphorylated) ERK which gets translocated into the nucleus. We model and analyze this complex pathway by decomposing it into three functional subsystems. The first subsystem spans the initial part of the pathway from the extracellular growth factor to the formation of the SOS complex, ShC-Grb2-SOS. The second subsystem includes the activation of Ras which is mediated by the SOS complex. This is followed by the MAPK subsystem (or the Raf-MEK-ERK pathway) which produces the double phosphorylated ERK upon being activated by Ras. Although separate models exist in the literature at the subsystems level, a comprehensive model for the complete system including the important regulatory feedback loops is missing. Our dynamic model combines the existing subsystem models and studies their steady-state and dynamic interactions under feedback. We establish conditions under which bistability and oscillations exist for this important pathway. In particular, we show how the negative and positive feedback loops affect the dynamic characteristics that determine the cellular outcome.

The diagnosis of chronic endometritis in infertile asymptomatic women: a comparative study of histology, microbial cultures, hysteroscopy, and molecular microbiology.

PubMed

Moreno, Inmaculada; Cicinelli, Ettore; Garcia-Grau, Iolanda; Gonzalez-Monfort, Marta; Bau, Davide; Vilella, Felipe; De Ziegler, Dominique; Resta, Leonardo; Valbuena, Diana; Simon, Carlos

2018-06-01

Chronic endometritis is a persistent inflammation of the endometrial mucosa caused by bacterial pathogens such as Enterobacteriaceae, Enterococcus, Streptococcus, Staphylococcus, Mycoplasma, and Ureaplasma. Although chronic endometritis can be asymptomatic, it is found in up to 40% of infertile patients and is responsible for repeated implantation failure and recurrent miscarriage. Diagnosis of chronic endometritis is based on hysteroscopy of the uterine cavity, endometrial biopsy with plasma cells being identified histologically, while specific treatment is determined based on microbial culture. However, not all microorganisms implicated are easily or readily culturable needing a turnaround time of up to 1 week. We sought to develop a molecular diagnostic tool for chronic endometritis based on real-time polymerase chain reaction equivalent to using the 3 classic methods together, overcoming the bias of using any of them alone. Endometrial samples from patients assessed for chronic endometritis (n = 113) using at least 1 or several conventional diagnostic methods namely histology, hysteroscopy, and/or microbial culture, were blindly evaluated by real-time polymerase chain reaction for the presence of 9 chronic endometritis pathogens: Chlamydia trachomatis, Enterococcus, Escherichia coli, Gardnerella vaginalis, Klebsiella pneumoniae, Mycoplasma hominis, Neisseria gonorrhoeae, Staphylococcus, and Streptococcus. The sensitivity and specificity of the molecular analysis vs the classic diagnostic techniques were compared in the 65 patients assessed by all 3 recognized classic methods. The molecular method showed concordant results with histological diagnosis in 30 samples (14 double positive and 16 double negative) with a matching accuracy of 46.15%. Concordance of molecular and hysteroscopic diagnosis was observed in 38 samples (37 double positive and 1 double negative), with an accuracy of 58.46%. When the molecular method was compared to microbial culture, concordance was present in 37 samples (22 double positive and 15 double negative), a matching rate of 56.92%. When cases of potential contamination and/or noncultivable bacteria were considered, the accuracy increased to 66.15%. Of these 65 patients, only 27 patients had consistent histological + hysteroscopic diagnosis, revealing 58.64% of nonconcordant results. Only 13 of 65 patients (20%) had consistent histology + hysteroscopy + microbial culture results. In these cases, the molecular microbiology matched in 10 cases showing a diagnostic accuracy of 76.92%. Interestingly, the molecular microbiology confirmed over half of the isolated pathogens and provided additional detection of nonculturable microorganisms. These results were confirmed by the microbiome assessed by next-generation sequencing. In the endometrial samples with concordant histology + hysteroscopy + microbial culture results, the molecular microbiology diagnosis demonstrates 75% sensitivity, 100% specificity, 100% positive and 25% negative predictive values, and 0% false-positive and 25% false-negative rates. The molecular microbiology method describe herein is a fast and inexpensive diagnostic tool that allows for the identification of culturable and nonculturable endometrial pathogens associated with chronic endometritis. The results obtained were similar to all 3 classic diagnostic methods together with a degree of concordance of 76.92% providing an opportunity to improve the clinical management of infertile patients with a risk of experiencing this ghost endometrial pathology. Copyright © 2018 Elsevier Inc. All rights reserved.

Expression of small leucine-rich proteoglycans in rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Syaidah, Rahimi; Fujiwara, Ken; Tsukada, Takehiro; Ramadhani, Dini; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

2013-01-01

Proteoglycans are components of the extracellular matrix and comprise a specific core protein substituted with covalently linked glycosaminoglycan chains. Small leucine-rich proteoglycans (SLRPs) are a major family of proteoglycans and have key roles as potent effectors in cellular signaling pathways. Research during the last two decades has shown that SLRPs regulate biological functions in many tissues such as skin, tendon, kidney, liver, and heart. However, little is known of the expression of SLRPs, or the characteristics of the cells that produce them, in the anterior pituitary gland. Therefore, we have determined whether SLRPs are present in rat anterior pituitary gland. We have used real-time reverse transcription with the polymerase chain reaction to analyze the expression of SLRP genes and have identified the cells that produce SLRPs by using in situ hybridization with a digoxigenin-labeled cRNA probe. We have clearly detected the mRNA expression of SLRP genes, and cells expressing decorin, biglycan, fibromodulin, lumican, proline/arginine-rich end leucine-rich repeat protein (PRELP), and osteoglycin are located in the anterior pituitary gland. We have also investigated the possible double-staining of SLRP mRNA and pituitary hormones, S100 protein (a marker of folliculostellate cells), desmin (a marker of capillary pericytes), and isolectin B4 (a marker of endothelial cells). Decorin, biglycan, fibromodulin, lumican, PRELP, and osteoglycin mRNA have been identified in S100-protein-positive and desmin-positive cells. Thus, we conclude that folliculostellate cells and pericytes produce SLRPs in rat anterior pituitary gland.

Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology.

PubMed

Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

2016-03-01

Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct. Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system. Published by Elsevier B.V.

Abdominopelvic 1.5-T and 3.0-T MR Imaging in Healthy Volunteers: Relationship to Formation of DNA Double-Strand Breaks.

PubMed

Suntharalingam, Saravanabavaan; Mladenov, Emil; Sarabhai, Theresia; Wetter, Axel; Kraff, Oliver; Quick, Harald H; Forsting, Michael; Iliakis, Georg; Nassenstein, Kai

2018-05-01

Purpose To investigate the relationship between abdominopelvic magnetic resonance (MR) imaging and formation of DNA double-strand breaks (DSBs) in peripheral blood lymphocytes among a cohort of healthy volunteers. Materials and Methods Blood samples were obtained from 40 healthy volunteers (23 women and 17 men; mean age, 27.2 years [range, 21-37 years]) directly before and 5 and 30 minutes after abdominopelvic MR imaging performed at 1.5 T (n = 20) or 3.0 T (n = 20). The number of DNA DSBs in isolated blood lymphocytes was quantified after indirect immunofluorescent staining of a generally accepted DSB marker, γ-H2AX, by means of high-throughput automated microscopy. As a positive control of DSB induction, blood lymphocytes from six volunteers were irradiated in vitro with x-rays at a dose of 1 Gy (70-90 keV). Statistical analysis was performed by using a Friedman test. Results No significant alteration in the frequency of DNA DSB induction was observed after MR imaging (before imaging: 0.22 foci per cell, interquartile range [IQR] = 0.54 foci per cell; 5 minutes after MR imaging: 0.08 foci per cell, IQR = 0.39 foci per cell; 30 minutes after MR imaging: 0.09 foci per cell, IQR = 0.63 foci per cell; P = .057). In vitro radiation of lymphocytes with 1 Gy led to a significant increase in DSBs (0.22 vs 3.43 foci per cell; P = .0312). The frequency of DSBs did not differ between imaging at 1.5 T and at 3.0 T (5 minutes after MR imaging: 0.23 vs 0.06 foci per cell, respectively [P = .57]; 30 minutes after MR imaging: 0.12 vs 0.08 foci per cell [P = .76]). Conclusion Abdominopelvic MR imaging performed at 1.5 T or 3.0 T does not affect the formation of DNA DSBs in peripheral blood lymphocytes. © RSNA, 2018.

[The establishment of the immortalized mouse brain microvascular pericytes model and its preliminary application in screening of cerebrovascular toxicants].

PubMed

Zhao, H P; Gao, Y F; Xia, D; Zhao, Z Q; Wu, S; Wang, X H; Liu, H X; Xiao, C; Xing, X M; He, Y

2018-05-06

Objective: To establish the immortalized mouse brain microvascular pericytes model and to apply to the cerebrovascular toxicants screening study. Methods: Brain pericytes were isolated from 3 weeks of mice by tissue digestion. Immortalized pericyte cell line was constructed by infecting with LT retrovirus. Monoclone was selected to purify the immortalized pericyte cell line. The pericyte characteristics and purity were explored by immunocytochemistry. Cell proliferation was measured by using the Pomega MTS cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 0, 160, 320, 640, 1 280, 2 560 μmol/L lead acetate, 0, 5, 10, 20, 40, 80 μmol/L cadmium chloride and 0, 5, 10, 20, 40, 80 μmol/L sodium arsenite in 24 hours. Cell toxicity of each group was determined by MTS assay, median lethal dose (LD(50)) was calculated in linear regression. Results: Mouse brain pericytes were successfully isolated by tissue separation and enzyme digestion method. After immortalized by LT retroviruses, monoclone was selected and expanded to establish pericyte cell line. The brain pericytes exhibited typical long spindle morphology and positive staining for α-SMA and Vimentin. The proliferation of brain pericytes cell lines was very slowly, and the doubling time was about 48 hours. The proliferation of immortalized brain pericytes cell lines was very quickly, and the doubling time was about 24 hours. After lead acetate, cadmium chloride and sodium arsenite treatment for 24 hours respectively, gradual declines in cell viability were observed. The LD(50) of lead acetate was 2 025.0 μmol/L, the LD(50) of cadmium chloride was 36.6 μmol/L, and the LD(50) of sodium arsenite was 33.2 μmol/L. Conclusion: The immortalized mouse brain microvascular pericyte model is established successfully by infecting with LT retrovirus, and can be applied to screen cerebrovascular toxicants. The toxicity of these toxicants to immortalized mouse brain microvascular pericyte is in sequence: sodium arsenite,cadmium chloride, lead acetate.

Proliferating cellular nuclear antigen expression as a marker of perivascular macrophages in simian immunodeficiency virus encephalitis.

PubMed

Williams, Kenneth; Schwartz, Annette; Corey, Sarah; Orandle, Marlene; Kennedy, William; Thompson, Brendon; Alvarez, Xavier; Brown, Charlie; Gartner, Suzanne; Lackner, Andrew

2002-08-01

Brain perivascular macrophages are a major target of simian immunodeficiency virus (SIV) infection in rhesus macaques and HIV infection in humans. Perivascular macrophages are distinct from parenchymal microglia in their location, morphology, expression of myeloid markers, and turnover in the CNS. In contrast to parenchymal microglia, perivascular macrophages are continuously repopulated by blood monocytes, which undergo maturation to macrophages on entering the central nervous system (CNS). We studied differences in monocyte/macrophages in vivo that might account for preferential infection of perivascular macrophages by SIV. In situ hybridization for SIV and proliferating cellular nuclear antigen (PCNA) immunohistochemistry demonstrated that SIV-infected and PCNA-positive cells were predominantly found in perivascular cuffs of viremic animals and in histopathological lesions that characterize SIV encephalitis (SIVE) in animals with AIDS. Multilabel techniques including double-label immunohistochemistry and combined in situ hybridization and immunofluorescence confocal microscopy revealed numerous infected perivascular macrophages that were PCNA-positive. Outside the CNS, SIV-infected, PCNA-expressing macrophage subpopulations were found in the small intestine and lung of animals with AIDS. While PCNA is used as a marker of cell proliferation it is also strongly expressed in non-dividing cells undergoing DNA synthesis and repair. Therefore, more specific markers for cell proliferation including Ki-67, topoisomerase IIalpha, and bromodeoxyuridine (BrdU) incorporation were used which indicated that PCNA-positive cells within SIVE lesions were not proliferating. These observations are consistent with perivascular macrophages as terminally differentiated, non-dividing cells and underscores biological differences that could potentially define mechanisms of preferential, productive infection of perivascular macrophages in the rhesus macaque model of neuroAIDS. These studies suggest that within CNS and non-CNS tissues there exist subpopulations of macrophages that are SIV-infected and express PCNA.

Association of reticular cells with CD34+/Sca-1+ apoptotic cells in the hemopoietic organ of grasshopper, Euprepocnemis shirakii.

PubMed

Lim, Jong Yeon; Lee, Bong Hee; Kang, Seok Woo; Wago, Haruhisa; Han, Sung Sik

2004-07-01

Hemopoiesis in orthopteran insects occurs in a hemopoietic organ that is located bilaterally along the aorta. This organ is also known as a reticulo-hemopoietic organ because of the rich presence of reticular cells. This study was performed to further elucidate hemopoiesis in the reticulo-hemopoietic organ of an orthopteran, Euprepocnemis shirakii. We focused on the question why reticular cells are so abundant (35% of cells in hemopoietic organ). Interestingly, 21% of these reticular cells surrounded hemocytes with their reticular cytoplasm. The surrounded hemocytes were distinguished by their different size and darkly stained nucleus. These cells were characterized by immunostaining using antibodies against several types of hemocytes: 45% of the surrounded hemocytes were CD34+, and these positive cells were double stained (over 85%) when immunostained by another hemopoietic pluripotent cell marker, Sca-1. Transmission electron microscopic analysis showed that reticular cells surrounded hemocytes containing large nuclei and poorly developed cytoplasmic organelles. This strongly suggests that the reticular cells surround hemopoietic stem cells. Additionally, surrounded hemopoietic progenitor cells are undergoing apoptosis as indicated by the TUNEL assay. The enclosed apoptotic cells are engulfed and then phagocytosed by reticular cells. Our results suggest that reticular cells are related to the differentiation and apoptosis of hemopoietic stem cells.

Deletion of the TNFAIP3/A20 gene detected by FICTION analysis in classical Hodgkin lymphoma

PubMed Central

2012-01-01

Background The TNFAIP3 gene, which encodes a ubiquitin-modifying enzyme (A20) involved in the negative regulation of NF-κB signaling, is frequently inactivated by gene deletions/mutations in a variety of B-cell malignancies. However, the detection of this in primary Hodgkin lymphoma (HL) specimens is hampered by the scarcity of Hodgkin Reed-Sternberg (HR-S) cells even after enrichment by micro-dissection. Methods We used anti-CD30 immunofluorescence with fluorescence in-situ hybridization (FISH) to evaluate the relative number of TNFAIP3/CEP6 double-positive signals in CD30-positive cells. Results From a total of 47 primary classical Hodgkin lymphoma (cHL) specimens, 44 were evaluable. We found that the relative numbers of TNFAIP3/CD30 cells were distributed among three groups, corresponding to those having homozygous (11%), heterozygous (32%), and no (57%) deletions in TNFAIP3. This shows that TNFAIP3 deletions could be sensitively detected using our chosen methods. Conclusions Comparing the results with mutation analysis, TNFAIP3 inactivation was shown to have escaped detection in many samples with homozygous deletions. This suggests that TNFAIP3 inactivation in primary cHL specimens might be more frequent than previously reported. PMID:23039325

Nucleosome regulatory dynamics in response to TGFβ

PubMed Central

Enroth, Stefan; Andersson, Robin; Bysani, Madhusudhan; Wallerman, Ola; Termén, Stefan; Tuch, Brian B.; De La Vega, Francisco M.; Heldin, Carl-Henrik; Moustakas, Aristidis; Komorowski, Jan; Wadelius, Claes

2014-01-01

Nucleosomes play important roles in a cell beyond their basal functionality in chromatin compaction. Their placement affects all steps in transcriptional regulation, from transcription factor (TF) binding to messenger ribonucleic acid (mRNA) synthesis. Careful profiling of their locations and dynamics in response to stimuli is important to further our understanding of transcriptional regulation by the state of chromatin. We measured nucleosome occupancy in human hepatic cells before and after treatment with transforming growth factor beta 1 (TGFβ1), using massively parallel sequencing. With a newly developed method, SuMMIt, for precise positioning of nucleosomes we inferred dynamics of the nucleosomal landscape. Distinct nucleosome positioning has previously been described at transcription start site and flanking TF binding sites. We found that the average pattern is present at very few sites and, in case of TF binding, the double peak surrounding the sites is just an artifact of averaging over many loci. We systematically searched for depleted nucleosomes in stimulated cells compared to unstimulated cells and identified 24 318 loci. Depending on genomic annotation, 44–78% of them were over-represented in binding motifs for TFs. Changes in binding affinity were verified for HNF4α by qPCR. Strikingly many of these loci were associated with expression changes, as measured by RNA sequencing. PMID:24771338

[Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

PubMed

An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

2012-12-01

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

Production of Exocytic Vesicular Antigens by Primary Liver Cell Cultures

DTIC Science & Technology

1990-05-08

cells should be plated over the basement membrane proteins, and for optimal results, a second layer of protein should be precipitated over the cells...culture as two layer (two gelatin coated nylon sheets stapled together) and single layer carriers seeded with cells (Table 2). From the performance results...summarized in table 2, it can be seen that double sheets of 2% gelatin: 6% glutaraldehyde (carrier II) made the best carriers. A double layer of

Hilar granule cells of the mouse dentate gyrus: effects of age, septotemporal location, strain, and selective deletion of the proapoptotic gene BAX.

PubMed

Bermudez-Hernandez, Keria; Lu, Yi-Ling; Moretto, Jillian; Jain, Swati; LaFrancois, John J; Duffy, Aine M; Scharfman, Helen E

2017-09-01

The dentate gyrus (DG) principal cells are glutamatergic granule cells (GCs), and they are located in a compact cell layer. However, GCs are also present in the adjacent hilar region, but have been described in only a few studies. Therefore, we used the transcription factor prospero homeobox 1 (Prox1) to quantify GCs at postnatal day (PND) 16, 30, and 60 in a common mouse strain, C57BL/6J mice. At PND16, there was a large population of Prox1-immunoreactive (ir) hilar cells, with more in the septal than temporal hippocampus. At PND30 and 60, the size of the hilar Prox1-ir cell population was reduced. Similar numbers of hilar Prox1-expressing cells were observed in PND30 and 60 Swiss Webster mice. Prox1 is usually considered to be a marker of postmitotic GCs. However, many Prox1-ir hilar cells, especially at PND16, were not double-labeled with NeuN, a marker typically found in mature neurons. Most hilar Prox1-positive cells at PND16 co-expressed doublecortin (DCX) and calretinin, markers of immature GCs. Double-labeling with a marker of actively dividing cells, Ki67, was not detected. These results suggest that, surprisingly, a large population of cells in the hilus at PND16 are immature GCs (Type 2b and Type 3 cells). We also asked whether hilar Prox1-ir cell numbers are modifiable. To examine this issue, we conditionally deleted the proapoptotic gene BAX in Nestin-expressing cells at a time when there are numerous immature GCs in the hilus, PND2-8. When these mice were examined at PND60, the numbers of Prox1-ir hilar cells were significantly increased compared to control mice. However, deletion of BAX did not appear to change the proportion that co-expressed NeuN, suggesting that the size of the hilar Prox1-expressing population is modifiable. However, deleting BAX, a major developmental disruption, does not appear to change the proportion that ultimately becomes neurons.

In situ hybridization evidence for the coexistence of ASIC and TRPV1 within rat single sensory neurons.

PubMed

Ugawa, Shinya; Ueda, Takashi; Yamamura, Hisao; Shimada, Shoichi

2005-05-20

The activation of nociceptors by protons plays a crucial role in the initiation and maintenance of acidosis-linked pain. Acid-sensing ion channel (ASIC) and transient receptor potential/vanilloid receptor subtype-1 (TRPV1) encode proton-activated cation channels expressed by nociceptors and the opening of these channels results in nociceptor excitation. Histological relations among ASIC clones and the colocalization of each ASIC subunit and TRPV1 within single sensory neurons were examined on serial sections of rat dorsal root ganglia (DRG) using in situ hybridization histochemistry. ASIC1a transcripts were expressed in 20-25% of the DRG neurons, and most of the neurons had small (<30 microm)-diameter cell bodies. ASIC1b transcripts and ASIC3 transcripts were expressed in approximately 10% and 30-35% of the DRG neurons, respectively, and the greater part of each population was located in small-to-medium (30-50 microm)-diameter cells. The ASIC1a transcripts and ASIC1b transcripts were basically localized in the distinct populations of the DRG neurons, while approximately 20% of the ASIC1a-positive neurons and approximately 10% of the ASIC1b-positive neurons expressed ASIC3 transcripts. TRPV1 transcripts were expressed in 35-40% of the DRG neurons, and most of the TRPV1-positive neurons had small-diameter cell bodies. Intense expression signals for ASIC1a transcripts were detected in 40-45% of the TRPV1-positive neurons. Neurons expressing both ASIC1b and TRPV1 transcripts were barely detected in the DRG. Approximately 30% of the TRPV1-positive neurons expressed ASIC3 transcripts, and the double-labeled neurons were comprised of both small-diameter and medium-diameter cells. Approximately 13% of the TRPV1-positive neurons expressed both ASIC1a and ASIC3 transcripts.

Langmuir probe measurements of double-layers in a pulsed discharge

NASA Technical Reports Server (NTRS)

Levine, J. S.; Crawford, F. W.

1980-01-01

Langmuir probe measurements were carried out which confirm the occurrence of double-layers in an argon positive column. Pulsing the discharge current permitted probe measurements to be performed in the presence of the double-layer. Supplementary evidence, obtained from DC and pulsed discharges, indicated that the double-layers formed in the two modes of operation were similar. The double-layers observed were weak and stable; their relation to other classes of double-layers are discussed, and directions for future work are suggested.

14-3-3 eta isoform colocalizes TDP-43 on the coarse granules in the anterior horn cells of patients with sporadic amyotrophic lateral sclerosis.

PubMed

Umahara, Takahiko; Uchihara, Toshiki; Shibata, Noriyuki; Nakamura, Ayako; Hanyu, Haruo

2016-09-01

The immunolocalization of the 14-3-3 eta isoform in the anterior horn cells (AHCs) of patients with sporadic amyotrophic lateral sclerosis (ALS) and controls was examined. Compared with the immunolocalization of other 14-3-3 isoforms, the immunolocalization of the 14-3-3 eta isoform was either synaptic at the periphery of AHCs, spindle-shaped in neurites, or granular in the cytoplasm. By double labeling with phosphorylated (p-)TDP-43, the transactivation response DNA binding protein of 43kDa (TDP-43) demonstrated frequent colocalization of the 14-3-3 eta isoform in granular structures (90%) and spindle-shaped structures (85.4%), but not in p-TDP-43-positive round inclusions. It is speculated that the 14-3-3 eta isoform is associated with not only a synaptic pathology of ALS but also TDP-positive small lesions in the cytoplasm and neurites. The absence of eta-like immunoreactivity in p-TDP-43-positive large inclusions suggests the restricted relevance of the 14-3-3 eta isoform during ALS pathogenesis to some phases of the p-TDP pathology. Copyright © 2016 Elsevier B.V. All rights reserved.

Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

PubMed Central

Asai, Hiroaki; Fujiwara, Hiroshi; An, Jun; Ochi, Toshiki; Miyazaki, Yukihiro; Nagai, Kozo; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Inoue, Hirofumi; Yasukawa, Masaki

2013-01-01

Background and Purpose Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. Methodology/Principal Findings Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402+ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8+ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1235–243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3+ T cells both in vitro and in vivo. Double gene-modified CD3+ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modifiedCD3+ T cells. Conclusion/Significance Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer reactivity mediated by CD8+ T cells double gene-modified to express WT1-specific TCR and CCR2 not only via CCL2-tropic tumor trafficking, but also CCL2-enhanced WT1-responsiveness. PMID:23441216

Testing stem cell therapy in a rat model of inflammatory bowel disease: role of bone marrow stem cells and stem cell factor in mucosal regeneration.

PubMed

Qu, Bo; Xin, Guo-Rong; Zhao, Li-Xia; Xing, Hui; Lian, Li-Ying; Jiang, Hai-Yan; Tong, Jia-Zhao; Wang, Bei-Bei; Jin, Shi-Zhu

2014-01-01

The gastrointestinal (GI) mucosal cells turnover regularly under physiological conditions, which may be stimulated in various pathological situations including inflammation. Local epithelial stem cells appear to play a major role in such mucosal renewal or pathological regeneration. Less is clear about the involvement of multipotent stem cells from blood in GI repair. We attempted to explore a role of bone marrow mesenchymal stromal cells (BMMSCs) and soluble stem cell factor (SCF) in GI mucosa regeneration in a rat model of inflammatory bowel diseases (IBD). BMMSCs labelled with the fluorescent dye PKH26 from donor rats were transfused into rats suffering indomethacin-induced GI injury. Experimental effects by BMMSCs transplant and SCF were determined by morphometry of intestinal mucosa, double labeling of PKH26 positive BMMSCs with endogenous proliferative and intestinal cell markers, and western blot and PCR analyses of the above molecular markers in the recipient rats relative to controls. PKH26 positive BMMSCs were found in the recipient mucosa, partially colocalizing with the proliferating cell nuclear antigen (PCNA), Lgr5, Musashi-1 and ephrin-B3. mRNA and protein levels of PCNA, Lgr5, Musashi-1 and ephrin-B3 were elevated in the intestine in BMMSCs-treated rats, most prominent in the BMMSCs-SCF co-treatment group. The mucosal layer and the crypt layer of the small intestine were thicker in BMMSCs-treated rats, more evident in the BMMSCs-SCF co-treatment group. BMMSCs and SCF participate in but may play a synergistic role in mucosal cell regeneration following experimentally induced intestinal injury. Bone marrow stem cell therapy and SCF administration may be of therapeutic value in IBD.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

PubMed

Pucci, Angela; Mattioli, Claudia; Matteucci, Marco; Lorenzini, Daniele; Panvini, Francesca; Pacini, Simone; Ippolito, Chiara; Celiento, Michele; De Martino, Andrea; Dolfi, Amelio; Belgio, Beatrice; Bortolotti, Uberto; Basolo, Fulvio; Bartoloni, Giovanni

2018-05-22

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

Human recombinant Fab fragment from combinatorial libraries of a B-cell lymphoma patient recognizes core protein of chondroitin sulphate proteoglycan 4.

PubMed

Egami, Yoko; Narushima, Yuta; Ohshima, Motohiro; Yoshida, Akira; Yoneta, Naruki; Masaki, Yasufumi; Itoh, Kunihiko

2018-01-01

CD antigens are well known as therapeutic targets of B-cell lymphoma. To isolate therapeutic antibodies that recognize novel targets other than CD antigens, we constructed a phage display combinatorial antibody Fab library from bone marrow lymphocytes of B-cell lymphoma patient. To eliminate antibodies reactive with known B-cell lymphoma antigen, non-hematopoietic and patient's sera reactive HeLaS3 cells was selected as a target of whole cell panning. Five rounds of panning against live HeLaS3 cells retrieved single Fab clone, termed AHSA (Antibody to HeLa Surface Antigen). Using phage display random peptide library, LSYLEP was identified as an epitope sequence of AHSA. LC-MS/MS analysis of AHSA-precipitated HeLaS3 cell lysates detected several fragments corresponding to the sequence of chondroitin sulphate proteoglycan 4 (CSPG4) core protein. Since LSYLEP sequence was at the position of 313-318 of CSPG4, we considered that CSPG4 was AHSA-associated antigen. Double staining of CSPG4-postive MDA-MB-435S cells with AHSA and anti-CSPG4 rabbit antibody showed identical staining position, and reduced AHSA reactivity was observed in CSPG4-siRNA treated MDA-MB-435S cells. In conclusion, we retrieved a human Fab from antibody library of B-cell lymphoma patient, and identified CSPG4 as a recognizing antigen. AHSA may have potential benefits for development of CSPG4-targeting theranostics for B-cell lymphoma. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells.

PubMed

Fielitz, Kathrin; Althoff, Kristina; De Preter, Katleen; Nonnekens, Julie; Ohli, Jasmin; Elges, Sandra; Hartmann, Wolfgang; Klöppel, Günter; Knösel, Thomas; Schulte, Marc; Klein-Hitpass, Ludger; Beisser, Daniela; Reis, Henning; Eyking, Annette; Cario, Elke; Schulte, Johannes H; Schramm, Alexander; Schüller, Ulrich

2016-11-15

Amplification or overexpression of MYCN is involved in development and maintenance of multiple malignancies. A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma. In order to model the spectrum of MYCN-driven neoplasms in mice, we transgenically overexpressed MYCN under the control of the human GFAP-promoter that, among other targets, drives expression in neural progenitor cells. However, LSL-MYCN;hGFAP-Cre double transgenic mice did neither develop neural crest tumors nor tumors of the central nervous system, but presented with neuroendocrine tumors of the pancreas and, less frequently, the pituitary gland. Pituitary tumors expressed chromogranin A and closely resembled human pituitary adenomas. Pancreatic tumors strongly produced and secreted glucagon, suggesting that they derived from glucagon- and GFAP-positive islet cells. Interestingly, 3 out of 9 human pancreatic neuroendocrine tumors expressed MYCN, supporting the similarity of the mouse tumors to the human system. Serial transplantations of mouse tumor cells into immunocompromised mice confirmed their fully transformed phenotype. MYCN-directed treatment by AuroraA- or Brd4-inhibitors resulted in significantly decreased cell proliferation in vitro and reduced tumor growth in vivo. In summary, we provide a novel mouse model for neuroendocrine tumors of the pancreas and pituitary gland that is dependent on MYCN expression and that may help to evaluate MYCN-directed therapies.

Clinical magnification and residual refraction after implantation of a double intraocular lens system in patients with macular degeneration.

PubMed

Amselem, Luis; Diaz-Llopis, Manuel; Felipe, Adelina; Artigas, Jose M; Navea, Amparo; García-Delpech, Salvador

2008-09-01

To evaluate the efficacy of a standard double intraocular lens (IOL) system (IOL-Vip) in patients with low vision and central scotoma due to macular degeneration and assess the predictability of the residual refraction and magnification. Ophthalmology Department, Hospital General Universitario, Valencia, Spain. This interventional prospective noncomparative case series comprised 13 consecutive surgical procedures in 10 patients with central scotoma. Follow-up was 12 months. Evaluation included the difference between preoperative and postoperative best corrected visual acuity (BCVA), refraction, position of the IOLs, endothelial cell density, and occurrence of postoperative complications. Residual refraction and eye magnification were calculated using a theory developed in a previous study, and the values were compared with the clinical results. The mean BCVA was 1.37 logMAR preoperatively and 0.68 logMAR 1 year postoperatively. The mean best corrected clinical gain was 44%. There was no statistically significant difference between the clinically evaluated and theoretically calculated residual refractions (P = .17). No intraoperative or postoperative complications occurred. Implantation of the double IOL system improved BCVA in patients with low vision due to advanced maculopathy. The results were best in myopic patients (long eyes); patients with hyperopia (short eyes) had high residual refraction. The postoperative clinical gain and residual refraction were predictable, showing the feasibility of implanting a customized double IOL.

APLP2 regulates neuronal stem cell differentiation during cortical development.

PubMed

Shariati, S Ali M; Lau, Pierre; Hassan, Bassem A; Müller, Ulrike; Dotti, Carlos G; De Strooper, Bart; Gärtner, Annette

2013-03-01

Expression of amyloid precursor protein (APP) and its two paralogues, APLP1 and APLP2 during brain development coincides with key cellular events such as neuronal differentiation and migration. However, genetic knockout and shRNA studies have led to contradictory conclusions about their role during embryonic brain development. To address this issue, we analysed in depth the role of APLP2 during neurogenesis by silencing APLP2 in vivo in an APP/APLP1 double knockout mouse background. We find that under these conditions cortical progenitors remain in their undifferentiated state much longer, displaying a higher number of mitotic cells. In addition, we show that neuron-specific APLP2 downregulation does not impact the speed or position of migrating excitatory cortical neurons. In summary, our data reveal that APLP2 is specifically required for proper cell cycle exit of neuronal progenitors, and thus has a distinct role in priming cortical progenitors for neuronal differentiation.

Nucleolar Reorganization Upon Site-Specific Double-Strand Break Induction.

PubMed

Franek, Michal; Kovaříková, Alena; Bártová, Eva; Kozubek, Stanislav

2016-11-01

DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.

The discovery and early structural studies of arachidonic acid

PubMed Central

Martin, Sarah A.; Brash, Alan R.; Murphy, Robert C.

2016-01-01

Arachidonic acid and esterified arachidonate are ubiquitous components of every mammalian cell. This polyunsaturated fatty acid serves very important biochemical roles, including being the direct precursor of bioactive lipid mediators such as prostaglandin and leukotrienes. This 20 carbon fatty acid with four double bonds was first isolated and identified from mammalian tissues in 1909 by Percival Hartley. This was accomplished prior to the advent of chromatography or any spectroscopic methodology (MS, infrared, UV, or NMR). The name, arachidonic, was suggested in 1913 based on its relationship to the well-known arachidic acid (C20:0). It took until 1940 before the positions of the four double bonds were defined at 5,8,11,14 of the 20-carbon chain. Total synthesis was reported in 1961 and, finally, the configuration of the double bonds was confirmed as all-cis-5,8,11,14. By the 1930s, the relationship of arachidonic acid within the family of essential fatty acids helped cue an understanding of its structure and the biosynthetic pathway. Herein, we review the findings leading up to the discovery of arachidonic acid and the progress toward its complete structural elucidation. PMID:27142391

Bone morphogenetic protein 4 and bone morphogenetic protein receptor expression in the pituitary gland of adult dogs in healthy condition and with ACTH-secreting pituitary adenoma.

PubMed

Sato, A; Ochi, H; Harada, Y; Yogo, T; Kanno, N; Hara, Y

2017-01-01

The purpose of this study was to investigate the expression of bone morphogenetic protein 4 (BMP4) and its receptors, bone morphogenetic protein receptor I (BMPRI) and BMPRII, in the pituitary gland of healthy adult dogs and in those with ACTH-secreting pituitary adenoma. Quantitative polymerase chain reaction analysis showed that the BMP4 messenger RNA expression level in the ACTH-secreting pituitary adenoma samples was significantly lower than that in the normal pituitary gland samples (P = 0.03). However, there were no statistically significant differences between samples with respect to the messenger RNA expression levels of the receptors BMPRIA, BMPRIB, and BMPRII. Double-immunofluorescence analysis of the normal canine pituitary showed that BMP4 was localized in the thyrotroph (51.3 ± 7.3%) and not the corticotroph cells. By contrast, BMPRII was widely expressed in the thyrotroph (19.9 ± 5.2%) and somatotroph cells (94.7 ± 3.6%) but not in the corticotroph cells (P < 0.001, thyrotroph cells vs somatotroph cells). Similarly, in ACTH-secreting pituitary adenoma, BMP4 and BMPRII were not expressed in the corticotroph cells. Moreover, the percentage of BMP4-positive cells was also significantly reduced in the thyrotroph cells of the surrounding normal pituitary tissue obtained from the resected ACTH-secreting pituitary adenoma (8.3 ± 7.9%) compared with that in normal canine pituitary (P < 0.001). BMP4 has been reported to be expressed in corticotroph cells in the human pituitary gland. Therefore, the results of this study reveal a difference in the cellular pattern of BMP4-positive staining in the pituitary gland between humans and dogs and further revealed the pattern of BMPRII-positive staining in the dog pituitary gland. These species-specific differences regarding BMP4 should be considered when using dogs as an animal model for Cushing's disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Washington Double Star Catalog Cross Index (1950 position sort)

NASA Technical Reports Server (NTRS)

1993-01-01

A machine-readable version of the Washington Catalog of Visual Double Stars (WDS) was prepared in 1984 on the basis of a data file that was collected and maintained for more than a century by a succession of double-star observers. Although this catalog is being continually updated, a new copy for distribution is not expected to be available for a few years. The WDS contains DM numbers, but many of these are listed only in the notes, which makes it difficult to search for double-star information, except by position. Hence, a cross index that provides complete DM identifications is desirable, and it appears useful to add HD numbers for systems in that catalog. Aitken Double Star (ADS) numbers were retained from the WDS, but no attempt was made to correct these except for obvious errors.

Birth and death of human β-cells in pancreases from cadaver donors, autopsies, surgical specimens, and islets transplanted into mice.

PubMed

Caballero, Francisco; Siniakowicz, Karolina; Hollister-Lock, Jennifer; Duran, Luisa; Katsuta, Hitoshi; Yamada, Takatsugu; Lei, Ji; Deng, Shaoping; Westermark, Gunilla T; Markmann, James; Bonner-Weir, Susan; Weir, Gordon C

2014-02-01

There is great interest in the potential of the human endocrine pancreas for regeneration by β-cell replication or neogenesis. Our aim was to explore this potential in adult human pancreases and in both islet and exocrine tissue transplanted into mice. The design was to examine pancreases obtained from cadaver donors, autopsies, and fresh surgical specimens and compare these findings with those obtained from islet and duct tissue grafted into the kidney. Islets and exocrine tissue were transplanted into normoglycemic ICR-SCID mice and studied 4 and 14 weeks later. β-Cell replication, as assessed by double staining for insulin and Ki67, was 0.22 ± 0.03% at 4 weeks and 0.13 ± 0.03% at 14 weeks. In contrast, no evidence of β-cell replication could be found in 11 cadaver donor and 10 autopsy pancreases. However, Ki67 staining of β-cells in frozen sections obtained at surgery was comparable to that found in transplanted islets. Evidence for neogenesis in transplanted pancreatic exocrine tissue was supported by finding β-cells within the duct epithelium and the presence of cells double stained for insulin and cytokeratin 19 (CK19). However, β-cells within the ducts never constituted more than 1% of the CK19-positive cells. With confocal microscopy, 7 of 12 examined cells expressed both markers, consistent with a neogeneic process. Mice with grafts containing islet or exocrine tissue were treated with various combinations of exendin-4, gastrin, and epidermal growth factor; none increased β-cell replication or stimulated neogenesis. In summary, human β-cells replicate at a low level in islets transplanted into mice and in surgical pancreatic frozen sections, but rarely in cadaver donor or autopsy pancreases. The absence of β-cell replication in many adult cadaver or autopsy pancreases could, in part, be an artifact of the postmortem state. Thus, it appears that adult human β-cells maintain a low level of turnover through replication and neogenesis.

Birth and death of human β-cells in pancreas from cadaver donors, autopsies, surgical specimens, and islets transplanted into mice

PubMed Central

Caballero, Francisco; Siniakowicz, Karolina; Jennifer-Hollister-Lock; Duran, Luisa; Katsuta, Hitoshi; Yamada, Takatsugu; Lei, Ji; Deng, Shaoping; Westermark, Gunilla T.; Markmann, James; Bonner-Weir, Susan; Weir, Gordon C.

2013-01-01

There is great interest in the potential of the human endocrine pancreas for regeneration by β-cell replication or neogenesis. Our aim was to explore this potential in adult human pancreases and in both islet and exocrine tissue transplanted into mice. The design was to examine pancreases obtained from cadaver donors, autopsies and fresh surgical specimens and compare these findings with those obtained from islet and duct tissue grafted into the kidney. Islets and exocrine tissue were transplanted into normoglycemic ICR/SCID mice and studied 4 and 14 wk later. β-cell replication as assessed by double staining for insulin and Ki67 was 0.22 ± 0.03 % at 4 wk and 0.13 ± 0.03 % at 14 wk. In contrast, no evidence of β-cell replication could be found in 11 cadaver donor and 10 autopsy pancreases. However, Ki67 staining of β-cells in frozen sections obtained at surgery was comparable to that found in transplanted islets. Evidence for neogenesis in transplanted pancreatic exocrine tissue was supported by finding β-cells within the duct epithelium, and the presence of cells double stained for insulin and cytokeratin 19 (CK19). However, β-cells within the ducts never constituted more than 1% of the CK19 positive cells. With confocal microscopy, 7 of 12 examined cells expressed both markers, consistent with a neogeneic process. Mice with grafts containing islet or exocrine tissue were treated with various combinations exendin-4, gastrin and epidermal growth factor; none increased β-cell replication or stimulated neogenesis. In summary, human β-cells replicate at a low level in islets transplanted into mice and in surgical pancreatic frozen sections but rarely in cadaver donor or autopsy pancreases. The absence of β-cell replication in many adult cadaver or autopsy pancreases could, in part, be an artifact of the postmortem state. Thus, it appears that adult human β-cells maintain a low level of turnover through replication and neogenesis. PMID:23321263

Student Observation of HR 2282 (Furud)

NASA Astrophysics Data System (ADS)

Estrada, Reed; Estrada, Chris; Anker, Payton; Barrientos, Destiny; Colbert, Charlie; Dondelinger, Edward; Gillette, Lindsey; Goodrow, Jeremy; Izadi, Tara; Mayo, Colin; Milton, Jordan; Stuart, Sarah; Varela, Nick

2017-04-01

A selected team of 8th graders measured the separation and the position angle of double star HR 2282 also known as Furud. They used a 22- inch Newtonian Alt/Az telescope to determine the scale constant, separation, and the position angle. The separation angle was 169.6 arc seconds and the position angle was 339.7 degrees. The results were compared to the 1999 Washington Double Star Catalog and were found to be extremely close.

Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

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

Gambhir, Sanjiv; Pritha, Ray

Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

DOEpatents

Gambhir, Sanjiv; Pritha, Ray

2015-07-14

Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

Ontogeny of neuro-insular complexes and islets innervation in the human pancreas.

PubMed

Proshchina, Alexandra E; Krivova, Yulia S; Barabanov, Valeriy M; Saveliev, Sergey V

2014-01-01

The ontogeny of the neuro-insular complexes (NIC) and the islets innervation in human pancreas has not been studied in detail. Our aim was to describe the developmental dynamics and distribution of the nervous system structures in the endocrine part of human pancreas. We used double-staining with antibodies specific to pan-neural markers [neuron-specific enolase (NSE) and S100 protein] and to hormones of pancreatic endocrine cells. NSE and S100-positive nerves and ganglia were identified in the human fetal pancreas from gestation week (gw) 10 onward. Later the density of S100 and NSE-positive fibers increased. In adults, this network was sparse. The islets innervation started to form from gw 14. NSE-containing endocrine cells were identified from gw 12 onward. Additionally, S100-positive cells were detected both in the periphery and within some of the islets starting at gw 14. The analysis of islets innervation has shown that the fetal pancreas contained NIC and the number of these complexes was reduced in adults. The highest density of NIC is detected during middle and late fetal periods, when the mosaic islets, typical for adults, form. The close integration between the developing pancreatic islets and the nervous system structures may play an important role not only in the hormone secretion, but also in the islets morphogenesis.

Ontogeny of Neuro-Insular Complexes and Islets Innervation in the Human Pancreas

PubMed Central

Proshchina, Alexandra E.; Krivova, Yulia S.; Barabanov, Valeriy M.; Saveliev, Sergey V.

2014-01-01

The ontogeny of the neuro-insular complexes (NIC) and the islets innervation in human pancreas has not been studied in detail. Our aim was to describe the developmental dynamics and distribution of the nervous system structures in the endocrine part of human pancreas. We used double-staining with antibodies specific to pan-neural markers [neuron-specific enolase (NSE) and S100 protein] and to hormones of pancreatic endocrine cells. NSE and S100-positive nerves and ganglia were identified in the human fetal pancreas from gestation week (gw) 10 onward. Later the density of S100 and NSE-positive fibers increased. In adults, this network was sparse. The islets innervation started to form from gw 14. NSE-containing endocrine cells were identified from gw 12 onward. Additionally, S100-positive cells were detected both in the periphery and within some of the islets starting at gw 14. The analysis of islets innervation has shown that the fetal pancreas contained NIC and the number of these complexes was reduced in adults. The highest density of NIC is detected during middle and late fetal periods, when the mosaic islets, typical for adults, form. The close integration between the developing pancreatic islets and the nervous system structures may play an important role not only in the hormone secretion, but also in the islets morphogenesis. PMID:24795697

Neural metabolite changes in corpus striatum after rat multipotent mesenchymal stem cells transplanted in hemiparkinsonian rats by magnetic resonance spectroscopy.

PubMed

Fu, Wenyu; Zheng, Zhijuan; Zhuang, Wenxin; Chen, Dandan; Wang, Xiaocui; Sun, Xihe; Wang, Xin

2013-12-01

To investigate the biochemical changes in striatum after rat bone marrow mesenchymal stem cells (MSCs) were transplanted into hemiparkinsonian rats and to further confirm the therapeutic effects of rat MSCs for Parkinson's disease (PD). 5-bromo-2-deoxyuridine (BrdU)-labeled MSCs were transplanted into the corpus striatum of the 6-hydroxydopamine (6-OHDA)-injected side of six PD model rats. Before and 8 weeks after MSC transplantation, ethological changes in PD rats were assessed. The expression of tyrosine hydroxylase (TH) in substantia nigra (SN) and striatum were measured using immunohistochemical methods. The differentiation of MSCs was detected by double immunofluorescence techniques. The concentrations of neural metabolites of N-acetylaspartate (NAA), choline (Cho) and creatine (Cr) were measured by ¹H-magnetic resonance spectroscopy (MRS). Relative concentrations of NAA/Cr and Cho/Cr were calculated. The behavior of PD rats in rotarod tests improved, and there were statistical differences in TH-positive cells in SN and TH-positive terminals in striatum after the transplantation of BrdU-labeled MSCs. Transplanted MSCs differentiated into MAP-2-positive neurons. Especially compared with pre-MSC transplantation, the neural metabolite NAA/Cr ratio of the 6-OHDA-injected side of the striatum increased (P < 0.05) and the Cho/Cr ratio decreased (P < 0.05). MSCs transplantation apparently improves neuronal function in the striatum of PD rats.

Pathogenesis of Double-Dose Proton Pump Inhibitor-Resistant Non-Erosive Reflux Disease, and Mechanism of Reflux Symptoms and Gastric Acid Secretion-Suppressive Effect in the Presence or Absence of Helicobacter pylori Infection.

PubMed

Kawami, Noriyuki; Takenouchi, Nana; Umezawa, Mariko; Hoshino, Shintaro; Hanada, Yuriko; Hoshikawa, Yoshimasa; Sano, Hirohito; Hoshihara, Yoshio; Nomura, Tsutomu; Uchida, Eiji; Iwakiri, Katsuhiko

2017-01-01

Various mechanisms have been suggested to be responsible for contributing to the occurrence of proton pump inhibitor (PPI)-resistant non-erosive reflux disease (NERD). The aims of this study were to clarify the pathogenesis of PPI-resistant NERD. Fifty-three patients with NERD, who had persistent reflux symptoms despite taking double-dose PPI, were included in this study. After excluding eosinophilic esophagitis (EoE) and primary esophageal motility disorder, esophageal impedance-pH monitoring was carried out. In symptom index (SI)-positive patients, the mechanism of SI positivity and the percent time with intragastric pH >4 were investigated according to the presence or absence of Helicobacter pylori infection. One of the 53 patients had EoE, and 4 had primary esophageal motility disorder. Twenty-three and 2 patients were SI-positive for liquid and gas-only reflux respectively. Of 17 SI-positive, H. pylori-negative patients, 5 were SI-positive for acid reflux, whereas all of the H. pylori-positive patients were SI-positive for non-acid reflux. The percent time with intragastric pH >4 was significantly lower in the H. pylori-negative patients than in the H. pylori-positive patients. The pathogenesis of double-dose PPI-resistant NERD was identified in 57%. In some of H. pylori-negative patients, acid-related symptoms were observed. However, in H. pylori-positive patients, these symptoms were excluded by taking double-dose PPI. © 2017 S. Karger AG, Basel.

Student Measurements of Double Star STF 747AB

NASA Astrophysics Data System (ADS)

Bateman, Grace; Funk, Benjamin; Gillette, Travis; Rhoades, Breauna; Rhoades, Mark; Schlosser, Ruth; Sharpe, Scott; Thompson, Leone

2017-04-01

Data gathered from a 22-Inch Newtonian Alt/Az telescope and a Celestron Micro Guide eyepiece were used to measure the double star STF 747AB. Students from Apple Valley High School determined the separation to be 39.97 arc sec and the position angle to be 227.91 degrees. The students also used data from the digitized sky survey and determined a separation of 39.99 arc sec and a position angle of 225 degrees. The research was semi-independent from the Vanguard Double Star Workshop 2016 in Apple Valley, California.

[Survival of patients with primary central nervous system diffuse large B-cell lymphoma: impact of gene aberrations and protein overexpression of bcl-2 and C-MYC, and selection of chemotherapy regimens].

PubMed

Yin, W J; Zhu, X; Yang, H Y; Sun, W Y; Wu, M J

2018-01-08

Objective: To investigate the impact of clinicopathological features, gene rearrangements and protein expression of bcl-6, bcl-2, C-MYC and chemotherapy regime on the prognosis of patients with primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL). Methods: Thirty-three cases of PCNS-DLBCL diagnosed from January 2006 to December 2016 at Zhejiang Cancer Hospital were collected. The expression of CD10, bcl-6, bcl-2, MUM1 and MYC were detected by immunohistochemical staining (IHC). The presence of EB virus was detected by in situ hybridization(EBER). Copy number variation (ICN) and translocation status of bcl-6, bcl-2 and C-MYC genes were detected by fluorescence in situ hybridization (FISH). The relationship between the above indexes and the prognosis was analyzed by univariate, bivariate survival analysis and multiple Cox hazard regression analysis. Results: The study included 33 patients of PCNS-DLBCL, without evidence of primary or secondary immunodeficient disease. Male to female ratio was 1.36∶1.00, and the average age was 56 years. Twenty cases had single lesion while 13 had multiple lesions. Deep brain involvement was seen in 12 cases. All patients underwent partial or total tumor resection. Five patients received whole brain post-surgery radiotherapy, nine patients received high-dose methotrexate (HD-MTX) based chemotherapy, and 12 patients received whole-brain radiotherapy combined with HD-MTX based chemotherapy. Severn patients received no further treatment and rituximab was used in 8 patients. According to the Hans model, 27 cases were classified as non-GCB subtypes (81.8%). Bcl-2 was positive in 25 cases (75.8%, 25/33) and highly expressed in 8 (24.2%). MYC was positive in 12 cases (36.4%) and double expression of bcl-2 and MYC was seen in 6 cases. EBER positive rate was 10.0%(3/30), all of which had multiple lesions. Two bcl-6 gene translocations and 3 amplifications were found in 28 patients. Two translocations, 3 ICN or with both bcl-2 gene translocation and ICN were found in 30 patients. Four ICNs of C-MYC gene were found in 28 patients. Elevated protein in cerebrospinal fluid (CSF) was found in 13 patients. LDH increased in 10 cases. Follow-up period was 2-90 months with the average survival time of (23.0±3.7) months and two-year survival rate of 39.0%. Univariate survival analysis showed that overexpression of bcl-2 protein (≥70%) and MYC protein (≥40%), bcl-2 gene abnormality (including copy number increase and translocation), C-MYC gene copy number increased were adverse factors for survival. C-MYC/ bcl-2 gene double hit was seen in 2 cases. Bivariate survival analysis found that of bcl-2/MYC protein double expression and bcl-2 and C-MYC genes double aberration were significantly associated with adverse outcomes. Cox multivariate risk regression analysis found that gender, cerebrospinal fluid protein increasing, and ICN of C-MYC gene were independent poor prognostic factors. DH-MTX based comprehensive chemotherapy was associated with better prognosis. Conclusions: Double hit at genomic level (copy number variations and gene rearrangements) and double protein expression of bcl-2 and C-MYC in PCNS-DLBCL are significantly associated with an adverse outcome. DH-MTX based comprehensive treatment may prolong the patient survival.

Modeling and characterization of double resonant tunneling diodes for application as energy selective contacts in hot carrier solar cells

NASA Astrophysics Data System (ADS)

Jehl, Zacharie; Suchet, Daniel; Julian, Anatole; Bernard, Cyril; Miyashita, Naoya; Gibelli, Francois; Okada, Yoshitaka; Guillemolles, Jean-Francois

2017-02-01

Double resonant tunneling barriers are considered for an application as energy selective contacts in hot carrier solar cells. Experimental symmetric and asymmetric double resonant tunneling barriers are realized by molecular beam epitaxy and characterized by temperature dependent current-voltage measurements. The negative differential resistance signal is enhanced for asymmetric heterostructures, and remains unchanged between low- and room-temperatures. Within Tsu-Esaki description of the tunnel current, this observation can be explained by the voltage dependence of the tunnel transmission amplitude, which presents a resonance under finite bias for asymmetric structures. This effect is notably discussed with respect to series resistance. Different parameters related to the electronic transmission of the structure and the influence of these parameters on the current voltage characteristic are investigated, bringing insights on critical processes to optimize in double resonant tunneling barriers applied to hot carrier solar cells.

Identification of CD34+ and CD34− leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia

PubMed Central

Aoki, Yuki; Watanabe, Takashi; Saito, Yoriko; Kuroki, Yoko; Hijikata, Atsushi; Takagi, Masatoshi; Tomizawa, Daisuke; Eguchi, Mariko; Eguchi-Ishimae, Minenori; Kaneko, Akiko; Ono, Rintaro; Sato, Kaori; Suzuki, Nahoko; Fujiki, Saera; Koh, Katsuyoshi; Ishii, Eiichi; Shultz, Leonard D.; Ohara, Osamu; Mizutani, Shuki

2015-01-01

Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34+CD38+CD19+ and CD34−CD19+ cells initiated leukemia, and in MLL-AF9 patients, CD34−CD19+ cells were LICs. In MLL-ENL patients, either CD34+ or CD34− cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34+CD38−CD19−CD33− cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34+CD38−CD19−CD33− cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4+ single positive (SP), CD8+ SP, and CD4+CD8+ double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34+CD38+ and CD34− LICs but not in CD34+CD38−CD19−CD33− HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia. PMID:25538041

Development of an in silico stochastic 4D model of tumor growth with angiogenesis.

PubMed

Forster, Jake C; Douglass, Michael J J; Harriss-Phillips, Wendy M; Bezak, Eva

2017-04-01

A stochastic computer model of tumour growth with spatial and temporal components that includes tumour angiogenesis was developed. In the current work it was used to simulate head and neck tumour growth. The model also provides the foundation for a 4D cellular radiotherapy simulation tool. The model, developed in Matlab, contains cell positions randomised in 3D space without overlap. Blood vessels are represented by strings of blood vessel units which branch outwards to achieve the desired tumour relative vascular volume. Hypoxic cells have an increased cell cycle time and become quiescent at oxygen tensions less than 1 mmHg. Necrotic cells are resorbed. A hierarchy of stem cells, transit cells and differentiated cells is considered along with differentiated cell loss. Model parameters include the relative vascular volume (2-10%), blood oxygenation (20-100 mmHg), distance from vessels to the onset of necrosis (80-300 μm) and probability for stem cells to undergo symmetric division (2%). Simulations were performed to observe the effects of hypoxia on tumour growth rate for head and neck cancers. Simulations were run on a supercomputer with eligible parts running in parallel on 12 cores. Using biologically plausible model parameters for head and neck cancers, the tumour volume doubling time varied from 45 ± 5 days (n = 3) for well oxygenated tumours to 87 ± 5 days (n = 3) for severely hypoxic tumours. The main achievements of the current model were randomised cell positions and the connected vasculature structure between the cells. These developments will also be beneficial when irradiating the simulated tumours using Monte Carlo track structure methods. © 2017 American Association of Physicists in Medicine.

Characterisation of rapid progressors to type 1 diabetes among children with HLA-conferred disease susceptibility.

PubMed

Pöllänen, Petra M; Lempainen, Johanna; Laine, Antti-Pekka; Toppari, Jorma; Veijola, Riitta; Vähäsalo, Paula; Ilonen, Jorma; Siljander, Heli; Knip, Mikael

2017-07-01

In this study, we aimed to characterise rapid progressors to type 1 diabetes among children recruited from the general population, on the basis of HLA-conferred disease susceptibility. We monitored 7410 HLA-predisposed children participating in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study for the development of beta cell autoimmunity and type 1 diabetes from birth over a median follow-up time of 16.2 years (range 0.9-21.1 years). Islet cell antibodies (ICA) and autoantibodies to insulin (IAA), GAD (GADA) and islet antigen 2 (IA-2A) were assessed as markers of beta cell autoimmunity. Rapid progression was defined as progression to clinical type 1 diabetes within 1.5 years of autoantibody seroconversion. We analysed the association between rapid progression and demographic and autoantibody characteristics as well as genetic markers, including 25 non-HLA SNPs predisposing to type 1 diabetes. Altogether, 1550 children (21%) tested positive for at least one diabetes-associated autoantibody in at least two samples, and 248 (16%) of seroconverters progressed to type 1 diabetes by the end of 2015. The median time from seroconversion to diagnosis was 0.51 years in rapid progressors (n = 42, 17%) and 5.4 years in slower progressors. Rapid progression was observed both among young (<5 years) and early pubertal children (>7 years), resulting in a double-peak distribution of seroconversion age. Compared with slower progressors, rapid progressors had a higher frequency of positivity for multiple (≥2) autoantibodies and had higher titres of ICA, IAA and IA-2A at seroconversion, and there was a higher prevalence of the secretor genotype in the FUT2 gene among those carrying the high-risk HLA genotype. Compared with autoantibody-positive non-progressors, rapid progressors were younger, were more likely to carry the high-risk HLA genotype and a predisposing SNP in the PTPN22 gene, had higher frequency of ICA, IAA, GADA and IA-2A positivity and multipositivity, and had higher titres of all four autoantibodies at seroconversion. At seroconversion, individuals with rapid progression to type 1 diabetes were characterised by a younger age, higher autoantibody titres, positivity for multiple autoantibodies and higher prevalence of a FUT2 SNP. The double-peak profile for seroconversion age among the rapid progressors demonstrates for the first time that rapid progression may take place not only in young children but also in children in early puberty. Rapid progressors might benefit from careful clinical follow-up and early preventive measures.

Sol-Gel Deposited Double Layer TiO₂ and Al₂O₃ Anti-Reflection Coating for Silicon Solar Cell.

PubMed

Jung, Jinsu; Jannat, Azmira; Akhtar, M Shaheer; Yang, O-Bong

2018-02-01

In this work, the deposition of double layer ARC on p-type Si solar cells was carried out by simple spin coating using sol-gel derived Al2O3 and TiO2 precursors for the fabrication of crystalline Si solar cells. The first ARC layer was created by freshly prepared sol-gel derived Al2O3 precursor using spin coating technique and then second ARC layer of TiO2 was deposited with sol-gel derived TiO2 precursor, which was finally annealed at 400 °C. The double layer Al2O3/TiO2 ARC on Si wafer exhibited the low average reflectance of 4.74% in the wavelength range of 400 and 1000 nm. The fabricated solar cells based on double TiO2/Al2O3 ARC attained the conversion efficiency of ~13.95% with short circuit current (JSC) of 35.27 mA/cm2, open circuit voltage (VOC) of 593.35 mV and fill factor (FF) of 66.67%. Moreover, the fabricated solar cells presented relatively low series resistance (Rs) as compared to single layer ARCs, resulting in the high VOC and FF.

Online Ozonolysis Combined with Ion Mobility-Mass Spectrometry Provides a New Platform for Lipid Isomer Analyses

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

Poad, Berwyck L. J.; Zheng, Xueyun; Mitchell, Todd W.

One of the most significant challenges in contemporary lipidomics lies in the separation and identification of lipid isomers that differ only in site(s) of unsaturation or geometric configuration of the carbon-carbon double bonds. While analytical separation techniques including ion mobility spectrometry (IMS) and liquid chromatography (LC) can separate isomeric lipids under appropriate conditions, conventional tandem mass spectrometry cannot provide unequivocal identification. To address this challenge, we have implemented ozone-induced dissociation (OzID) in-line with LC, IMS and high resolution mass spectrometry. Modification of an IMS- capable quadrupole time-of-flight mass spectrometer was undertaken to allow the introduction of ozone into the high-pressuremore » trapping ion funnel region preceding the IMS cell. This enabled the novel LC-OzID-IMS-MS configuration where ozonolysis of ionized lipids occurred rapidly (10 ms) without prior mass-selection. LC-elution time alignment combined with accurate mass and arrival time extraction of ozonolysis products facilitated correlation of precursor and product ions without mass-selection (and associated reductions in duty cycle). Unsaturated lipids across 11 classes were examined using this workflow in both positive and negative ion modalities and in all cases the positions of carbon-carbon double bonds were unequivocally assigned based on predictable OzID transitions. Under these conditions geometric isomers exhibited different IMS arrival time distributions and distinct OzID product ion ratios providing a means for discrimination of cis/trans double bonds in complex lipids. The combination of OzID with multidimensional separations shows significant promise for facile profiling of unsaturation patterns within complex lipidomes.« less

Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

PubMed Central

Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G; Kristensen, Bjarne

2013-01-01

Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem cells. Targeting of lysosomes may be a promising novel therapeutic strategy against this highly malignant neoplasm. PMID:23826410

Cortical Double-Opponent Cells in Color Perception: Perceptual Scaling and Chromatic Visual Evoked Potentials.

PubMed

Nunez, Valerie; Shapley, Robert M; Gordon, James

2018-01-01

In the early visual cortex V1, there are currently only two known neural substrates for color perception: single-opponent and double-opponent cells. Our aim was to explore the relative contributions of these neurons to color perception. We measured the perceptual scaling of color saturation for equiluminant color checkerboard patterns (designed to stimulate double-opponent neurons preferentially) and uniformly colored squares (designed to stimulate only single-opponent neurons) at several cone contrasts. The spatially integrative responses of single-opponent neurons would produce the same response magnitude for checkerboards as for uniform squares of the same space-averaged cone contrast. However, perceived saturation of color checkerboards was higher than for the corresponding squares. The perceptual results therefore imply that double-opponent cells are involved in color perception of patterns. We also measured the chromatic visual evoked potential (cVEP) produced by the same stimuli; checkerboard cVEPs were much larger than those for corresponding squares, implying that double-opponent cells also contribute to the cVEP response. The total Fourier power of the cVEP grew sublinearly with cone contrast. However, the 6-Hz Fourier component's power grew linearly with contrast-like saturation perception. This may also indicate that cortical coding of color depends on response dynamics.

Cortical Double-Opponent Cells in Color Perception: Perceptual Scaling and Chromatic Visual Evoked Potentials

PubMed Central

Shapley, Robert M.; Gordon, James

2018-01-01

In the early visual cortex V1, there are currently only two known neural substrates for color perception: single-opponent and double-opponent cells. Our aim was to explore the relative contributions of these neurons to color perception. We measured the perceptual scaling of color saturation for equiluminant color checkerboard patterns (designed to stimulate double-opponent neurons preferentially) and uniformly colored squares (designed to stimulate only single-opponent neurons) at several cone contrasts. The spatially integrative responses of single-opponent neurons would produce the same response magnitude for checkerboards as for uniform squares of the same space-averaged cone contrast. However, perceived saturation of color checkerboards was higher than for the corresponding squares. The perceptual results therefore imply that double-opponent cells are involved in color perception of patterns. We also measured the chromatic visual evoked potential (cVEP) produced by the same stimuli; checkerboard cVEPs were much larger than those for corresponding squares, implying that double-opponent cells also contribute to the cVEP response. The total Fourier power of the cVEP grew sublinearly with cone contrast. However, the 6-Hz Fourier component’s power grew linearly with contrast-like saturation perception. This may also indicate that cortical coding of color depends on response dynamics. PMID:29375753

Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

PubMed

Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Distribution of protein kinase C isoforms in the cat retina.

PubMed

Fyk-Kolodziej, Bozena; Cai, Wenhui; Pourcho, Roberta G

2002-01-01

Immunocytochemical localization was carried out for five isoforms of protein kinase C (PKC) in the cat retina. In common with other mammalian species, PKCalpha was found in rod bipolar cells. Staining was also seen in a small population of cone bipolar cells with axon terminals ramifying near the middle of the inner plexiform layer (IPL). PKCbetaI was localized to rod bipolar cells, one class of cone bipolar cell, and numerous amacrine and displaced amacrine cells. Staining for PKCbetaI was seen in three types of cone bipolar cells as well as in amacrine and ganglion cells. Immunoreactivity for both PKCepsilon and PKCzeta was found in rod bipolar cells; PKCepsilon was also seen in a population of cone bipolar cells and a few amacrine and ganglion cells whereas PKCzeta was found in all ganglion cells. Double-label immunofluorescence studies showed that dendrites of the two PKCbetaII-positive OFF-cone bipolar cells exhibit immmunoreactivity for the kainate-selective glutamate receptor GluR5. The third PKCbetaII cone bipolar is an ON-type cell and did not stain for GluR5. The retinal distribution of these isoforms of PKC is consistent with a role in modulation of various aspects of neurotransmission including synaptic vesicle release and regulation of receptor molecules.

The presence and prognostic significance of human papillomavirus in squamous cell carcinoma of the larynx.

PubMed

Erkul, Evren; Yilmaz, Ismail; Narli, Gizem; Babayigit, Mustafa Alparslan; Gungor, Atila; Demirel, Dilaver

2017-07-01

The aim of the present study was to evaluate the role of HPV in laryngeal squamous cell carcinoma and correlate it with patients' clinicopathological data. In total, 78 laryngeal squamous cell carcinoma patients enrolled in this study. The presence of genotype-specific HPV DNA was evaluated using Genotyping Assay in formalin-fixed paraffin-embedded tissue which was diagnosed between 2005 and 2015. All samples were also evaluated for p16 immunohistochemical staining. HPV DNA and p16 status were assessed in terms of location, smoking, alcohol consumption, lymph node status, tumor stage, overall survival, disease-free survival, perineural invasion, and vascular invasion retrospectively. Five test samples were excluded from the study due to inadequate deoxyribonucleic acid purity. HPV DNA was detected in 19 of 73 (26.02%) in patients with laryngeal squamous cell carcinoma. Human papilloma virus genotyping revealed double human papilloma virus in one case (types 16 and 59) and HPV 16 in the remaining cases. Although HPV-positive cases showed slightly better 3 years survival than HPV-negative ones, this finding was not statistically significant (overall survival p = 0.417, HPV positive: 92.3%, HPV negative: 81.4%, and disease-free survival p = 0.526, HPV positive: 93.8%, HPV negative: 80.9%). The presence of HPV DNA was not significantly associated with any clinicopathological features (p > 0.05). Among 73 patients, only 4 had an immunohistochemical staining of p16 and these patients were also HPV DNA 16 positive. Although our study results revealed a slightly better survival in patients with HPV DNA positivity for HPV 16 compared to the negative ones, the difference was not statistically significant. However, an increasing rate in especially high-risk-type HPV-16 prevalence in laryngeal squamous cell carcinoma by RT-PCR method was observed compared to our previous study. Although the presence of HPV in laryngeal SCCs seems to be associated with slightly better prognosis, additional studies may be needed, since our results were not statistically significant. We believed that HPV is not an adequate biomarker for diagnostic and prognostic purposes in laryngeal squamous cell carcinoma.

A randomized double-blind study of testosterone replacement therapy or placebo in testicular cancer survivors with mild Leydig cell insufficiency (Einstein-intervention).

PubMed

Bandak, Mikkel; Jørgensen, Niels; Juul, Anders; Lauritsen, Jakob; Kreiberg, Michael; Oturai, Peter Sandor; Helge, Jørn Wulff; Daugaard, Gedske

2017-07-03

Elevated serum levels of luteinizing hormone and slightly decreased serum levels of testosterone (mild Leydig cell insufficiency) is a common hormonal disturbance in testicular cancer (TC) survivors. A number of studies have shown that low serum levels of testosterone is associated with low grade inflammation and increased risk of metabolic syndrome. However, so far, no studies have evaluated whether testosterone substitution improves metabolic dysfunction in TC survivors with mild Leydig cell insufficiency. This is a single-center, randomized, double-blind, placebo-controlled study, designed to evaluate the effect of testosterone replacement therapy in TC survivors with mild Leydig cell insufficiency. Seventy subjects will be randomized to receive either testosterone replacement therapy or placebo. The subjects will be invited for an information meeting where informed consent will be obtained. Afterwards, a 52-weeks treatment period begins in which study participants will receive a daily dose of transdermal testosterone or placebo. Dose adjustment will be made three times during the initial 8 weeks of the study to a maximal daily dose of 40 mg of testosterone in the intervention arm. Evaluation of primary and secondary endpoints will be performed at baseline, 26 weeks post-randomization, at the end of treatment (52 weeks) and 3 months after completion of treatment (week 64). This study is the first to investigate the effect of testosterone substitution in testicular cancer survivors with mild Leydig cell insufficiency. If positive, it may change the clinical handling of testicular cancer survivors with borderline low levels of testosterone. ClinicalTrials.gov : NCT02991209 (November 25, 2016).

Low-dose ionizing irradiation triggers a 53BP1 response to DNA double strand breaks in mouse spermatogonial stem cells.

PubMed

Le, Wei; Qi, Lixin; Li, Jiaxuan; Wu, DengIong; Xu, Jun; Zhang, Jinfu

2016-01-01

The present study aims to examine the effect of low-dose ionizing irradiation on DNA double strand breaks (DSB) in mouse spermatogonial stem cells (SSCs) and reveal the underlying pathways for the DNA repair for DSB in SSCs. Eighteen one-month-old mice were divided into 6 groups and sacrificed separately at 45 minutes, 2 hours, 24 hours, 48 hours, and 72 hours after 0.1Gy X-ray irradiation (mice without receiving ionizing irradiation served as control). After perfusion fixation, testes were removed, sectioned, and followed by staining of γH2AX, 53BP1, Caspase 3, and promyelocytic leukemia zinc-finger (PLZF) for analysis among the different groups. The staining was observed by immunofluorescence visualized by confocal laser scanning. After low-dose irradiation, only 53BP1, but not Caspase3 or γH2AX was upregulated in PLZF positive SSCs within 45 minutes. The expression level of 53BP1 gradually decreased 24 hours after irradiation. Moreover, low-dose irradiation had no effect on the cell number and apoptotic status of SSCs. However other spermatogenic cells highly expressed γH2AX shortly after irradiation which was dramatically reduced following the events of DNA repair. It appears that low-dose ionizing irradiation may cause the DNA DSB of mouse spermatogenic cells. 53BP1, but not γH2AX, is involved in the DNA repair for DSB in SSCs. Our data indicates that 53BP1 plays an important role in the pathophysiological repair of DNA DSB in SSCs. This may open a new avenue to understanding the mechanisms of DNA repair of SSCs and male infertility.

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells.

PubMed

Ju, Young Seok; Tubio, Jose M C; Mifsud, William; Fu, Beiyuan; Davies, Helen R; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J; Tan, Benita K T; Aparicio, Samuel; Span, Paul N; Martens, John W M; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Myklebost, Ola; Flanagan, Adrienne M; Foster, Christopher; Neal, David E; Cooper, Colin; Eeles, Rosalind; Bova, Steven G; Lakhani, Sunil R; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L; Purdie, Colin A; Thompson, Alastair M; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J; Stratton, Michael R

2015-06-01

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells. © 2015 Ju et al.; Published by Cold Spring Harbor Laboratory Press.

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells

PubMed Central

Ju, Young Seok; Tubio, Jose M.C.; Mifsud, William; Fu, Beiyuan; Davies, Helen R.; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S.; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R.; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J.; Tan, Benita K.T.; Aparicio, Samuel; Span, Paul N.; Martens, John W.M.; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Flanagan, Adrienne M.; Foster, Christopher; Neal, David E.; Cooper, Colin; Eeles, Rosalind; Lakhani, Sunil R.; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L.; Purdie, Colin A.; Thompson, Alastair M.; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J.; Stratton, Michael R.

2015-01-01

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells. PMID:25963125

Reduce on the Cost of Photovoltaic Power Generation for Polycrystalline Silicon Solar Cells by Double Printing of Ag/Cu Front Contact Layer

NASA Astrophysics Data System (ADS)

Peng, Zhuoyin; Liu, Zhou; Chen, Jianlin; Liao, Lida; Chen, Jian; Li, Cong; Li, Wei

2018-06-01

With the development of photovoltaic industry, the cost of photovoltaic power generation has become the significant issue. And the metallization process has decided the cost of original materials and photovoltaic efficiency of the solar cells. Nowadays, double printing process has been introduced instead of one-step printing process for front contact of polycrystalline silicon solar cells, which can effectively improve the photovoltaic conversion efficiency of silicon solar cells. Here, the relative cheap Cu paste has replaced the expensive Ag paste to form Ag/Cu composite front contact of silicon solar cells. The photovoltaic performance and the cost of photovoltaic power generation have been investigated. With the optimization on structure and height of Cu finger layer for Ag/Cu composite double-printed front contact, the silicon solar cells have exhibited a photovoltaic conversion efficiency of 18.41%, which has reduced 3.42 cent per Watt for the cost of photovoltaic power generation.

Yield and proliferation rate of adipose-derived stromal cells as a function of age, body mass index and harvest site-increasing the yield by use of adherent and supernatant fractions?

PubMed

Buschmann, Johanna; Gao, Shuping; Härter, Luc; Hemmi, Sonja; Welti, Manfred; Werner, Clement M L; Calcagni, Maurizio; Cinelli, Paolo; Wanner, Guido A

2013-09-01

Adipose-derived stem cells are easily accessed and have a relatively high density compared with other mesenchymal stromal cells. Isolation protocols of adipose-derived stem cells (ASC) rely on the cell's ability to adhere to tissue culture plastic overnight. It was evaluated whether the floating ASC fractions are also of interest for cell-based therapies. In addition, the impact of age, body mass index (BMI) and harvest site was assessed. The surface protein profile with the use of flow cytometry, the cell yield and the doubling time of passages 4, 5 and 6 of ASC from 30 donors were determined. Adherent and supernatant fractions were compared. The impact of age, BMI and harvest site on cell yield and doubling times was determined. Both adherent and supernatant fractions showed high mean fluorescence intensities for CD13, CD29, CD44, CD73, CD90 and CD105 and comparatively low mean fluorescence intensities for CD11b, CD62L, intracellular adhesion molecule-1 and CD34. Doubling times of adherent and supernatant fractions did not differ significantly. Whereas the old age group had a significantly lower cell yield compared with the middle aged group, BMI and harvest site had no impact on cell yield. Finally, doubling times for passages 4, 5 and 6 were not influenced by the age and BMI of the donors, nor the tissue-harvesting site. The floating ASC fraction is an equivalent second cell source just like the adherent ASC fraction. Donor age, BMI and harvest site do not influence cell yield and proliferation rate. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Properties and Applications of Lossy Metamaterials

DTIC Science & Technology

2011-12-01

Ring ENG Epsilon-Negative MNG Mu-Negative SNG Single-Negative NRI Negative Refractive Index FIT Finite Integration Technique xiv THIS PAGE...r r r rB ε µ ε µ′ ′′ ′′ ′= + parameters. A classification of double positive (DPS), double negative (DNG) and single negative ( SNG ) materials with...negative, and when 0B > the phase constant and the refractive index are both positive. The parameter A is negative in SNG materials but can be positive or

Comparison of DSB effects of the beta particles of iodine-131 and 6 MV X-ray at a dose of 2 Gy in the presence of 2-Methoxyestradiol, IUdR, and TPT in glioblastoma spheroids

NASA Astrophysics Data System (ADS)

Neshasteh-Riz, Ali; Eyvazzadeh, Nazila; Koosha, Fereshteh; Cheraghi, Susan

2017-02-01

Glioblastoma is one of the lethal brain tumors and one of the resistant tumors against radiotherapy. Multiple treatment methods and different types of radiation and Radiosensitizers drugs have been combined to optimize the efficacy of radiotherapy. Radiosensitizers are employed to reinforce tumor cell killing and have much fewer effects on the normal tissue. Inducing DNA double strand break in tumoral cells is a major goal of radiation sensitivity. In this study, the level of DNA double strand break in glioblastoma spheroids irradiated by 2 Gy beta particles of iodine-131 and 6 MV X-rays in the presence of 2-Methoxyestradiol (2ME2), iodo-deoxy-uridine (IUdR) and Topotecan (TPT) was measured using the PicoGreen method. Spheroids of the U87MG cell line were cultured to reach a 300 μm diameter. In the phase one of the study, the spheroids were treated in four groups individually, including 2 Gy of iodine-131, TPT+iodine-131, IUdR+iodine-131, IUdR+2ME2+iodine-131. In the next phase, the cells were treated with 2 Gy of 6 MV X-ray, TPT+6 MV X-ray, IUdR+6 MV X-ray, TPT+IUdR+6 MV X-ray. DSB lesions were measured by the Pico Green assay. The amount of DSB lesions in groups irradiated with iodine-131 individually was greater than the group irradiated with 6 MV X-ray (p<0.05). DNA double strand breaks became more significant in combination with TPT. However, the amount of DSBs in the two independent groups of TPT+IUdR+2ME2+iodine-131 and TPT+IUdR+2ME2+6 MV X-ray was approximately in the same range (P>0.05). The level of DNA double strand breaks in cells irradiated with Iodine-131 was higher than cells irradiated with 6 MV X-ray at the same dose and Topotecan had a positive effect on inducing the damage. The role of 2ME2+IUdR in increasing the damage caused by beta particles of iodine-131 was not significant. Iodine-131 could lead to major DSB damage than 6 MV X-ray at the same dose due to its cross fire effect and spatial distribution of energy in different angels. This study showed that a combination of chemotherapy and iodine-131 had better efficacy than radiotherapy with 6 MV X-ray in the treatment of glioblastoma.

Breast Cancer Detection

NASA Technical Reports Server (NTRS)

1976-01-01

NASA's Jet Propulsion Laboratory has come up with a technique to decrease exposure to harmful x-rays in mammographies or breast radiography. Usually, physicians make more than one exposure to arrive at an x-ray film of acceptable density. Now the same solar cells used to convert sunlight into electricity on space satellites can make a single exposure sufficient. When solar cell sensor is positioned directly beneath x-ray film, it can determine exactly when film has received sufficient radiation and has been exposed to optimum density. At that point associated electronic equipment sends signal to cut off x-ray source. Reduction of mammography to single exposures not only reduced x-ray hazard significantly, but doubled the number of patient examinations handled by one machine. The NASA laboratory used this control system at the Huntington Memorial Hospital with overwhelming success.

Apoplastic ROS production upon pollination by RbohH and RbohJ in Arabidopsis

PubMed Central

Kaya, Hidetaka; Iwano, Megumi; Takeda, Seiji; Kanaoka, Masahiro M; Kimura, Sachie; Abe, Mitsutomo; Kuchitsu, Kazuyuki

2015-01-01

Reactive oxygen species (ROS) accumulate at the tip of growing pollen tubes. In Arabidopsis, NADPH oxidases RbohH and RbohJ are localized at the plasma membrane of pollen tube tip and produce ROS in a Ca2+-dependent manner. The ROS produced by Rbohs and Ca2+ presumably play a critical role in the positive feedback regulation that maintains the tip growth. Ultrastructural cytochemical analysis revealed ROS accumulation in the apoplast/cell wall of the pollen grains on the stigmatic papillae in the wild type, but not in the rbohH rbohJ double mutant, suggesting that apoplastic ROS derived from RbohH and RbohJ are involved in pollen tube elongation into the stigmatic papillae by affecting the cell wall metabolism. PMID:25751652

Double Knockdown of Prolyyl Hydroxylase and Factor Inhibiting HIF with Non-Viral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction

PubMed Central

Huang, Mei; Nguyen, Patricia; Jia, Fangjun; Hu, Shijun; Gong, Yongquan; de Almeida, Patricia E.; Wang, Li; Nag, Divya; Kay, Mark A.; Giaccia, Amato J; Robbins, Robert C.; Wu, Joseph C.

2011-01-01

Background Under normoxic conditions, hypoxia inducible factor-1 alpha (HIF-1α) is rapidly degraded by two hydroxylases, prolyl hydroxylase (PHD) and factor inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its up-regulation may be an effective therapeutic option for ischemic heart failure. Methods and Results PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin sequences for inhibiting PHD isoenzyme 2 (shPHD2) and FIH (shFIH) were inserted into novel non-viral minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit+ cardiac progenitor cells (CPCs) demonstrated higher expression of angiogenesis factors in the double knockdown group compared to the single knockdown and shScramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially following LAD ligation in adult FVB mice (n=60). Functional studies using magnetic resonance imaging (MRI), echocardiography, and pressure-volume (PV) loops showed greater improvement in cardiac function in the double knockdown group. To assess mechanism(s) of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double knockdown group. Fluorescence activated cell sorting (FACS) showed significantly higher activation of endogenous c-kit+ cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser capture microdissection (LCM) analysis confirmed up-regulation of HIF-1α protein and angiogenesis genes, respectively. Conclusions We demonstrated that HIF-1α up-regulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function. PMID:21911818

GEM-loaded magnetic albumin nanospheres modified with cetuximab for simultaneous targeting, magnetic resonance imaging, and double-targeted thermochemotherapy of pancreatic cancer cells.

PubMed

Wang, Ling; An, Yanli; Yuan, Chenyan; Zhang, Hao; Liang, Chen; Ding, Fengan; Gao, Qi; Zhang, Dongsheng

2015-01-01

Targeted delivery is a promising strategy to improve the diagnostic imaging and therapeutic effect of cancers. In this paper, novel cetuximab (C225)-conjugated, gemcitabine (GEM)-containing magnetic albumin nanospheres (C225-GEM/MANs) were fabricated and applied as a theranostic nanocarrier to conduct simultaneous targeting, magnetic resonance imaging (MRI), and double-targeted thermochemotherapy against pancreatic cancer cells. Fe3O4 nanoparticles (NPs) and GEM co-loaded albumin nanospheres (GEM/MANs) were prepared, and then C225 was further conjugated to synthesize C225-GEM/MANs. Their morphology, mean particle size, GEM encapsulation ratio, specific cell-binding ability, and thermal dynamic profiles were characterized. The effects of discriminating different EGFR-expressing pancreatic cancer cells (AsPC-1 and MIA PaCa-2) and monitoring cellular targeting effects were assessed by targeted MRI. Lastly, the antitumor efficiency of double/C225/magnetic-targeted and nontargeted thermochemotherapy was compared with chemotherapy alone using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and flow cytometry (FCM) assay. When treated with targeted nanospheres, AsPC-1 cells showed a significantly less intense MRI T2 signal than MIA PaCa-2 cells, while both cells had similar signal strength when incubated with nontargeted nanospheres. T2 signal intensity was significantly lower when magnetic and C225 targeting were combined, rather than used alone. The inhibitory and apoptotic rates of each thermochemotherapy group were significantly higher than those of the chemotherapy-alone groups. Additionally, both MTT and FCM analysis verified that double-targeted thermochemotherapy had the highest targeted killing efficiency among all groups. The C225-GEM/MANs can distinguish various EGFR-expressing live pancreatic cancer cells, monitor diverse cellular targeting effects using targeted MRI imaging, and efficiently mediate double-targeted thermochemotherapy against pancreatic cancer cells.

Anti-tumor effect of in vivo IL-2 and GM-CSF electrogene therapy in murine hepatoma model.

PubMed

Chi, Chau-Hwa; Wang, Yu-Shan; Lai, Yen-Shuae; Chi, Kwan-Hwa

2003-01-01

We evaluated the effect of in vivo electrogene therapy (EGT), a newly-developed gene transfer method using electroporation on the induction of anti-cancer immunity. The in vivo EGT was carried out by direct injection of plasmid DNAs encoding mouse interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in a subcutaneous murine hepatoma model of 1MEA.7R.1 cells. Six electric pulses were generated in situ from a square-wave electroporator fitted with a circular, six-needle electrode array. 1MEA.7R1 cells in vitro were modified to secret IL-2 (1MEA.7R.1/IL-2 cells). The 1MEA.7R.1/IL-2 cells had a similar cell doubling-time as their parent cells but showed a much slower growth rate on Balb/C mice. One, or 3 rounds of single gene EGT with IL-2 gene showed a dose-responsive effect of growth retardation. Co-administration of 3 rounds of IL-2/GM-CSF double genes EGT had a stronger growth inhibition effect than 3 rounds of IL-2 single gene EGT. Three rounds of IL-2/GM-CSF EGT rendered the tumor to a growth rate of stably transfected 1MEA.7R.1/IL-2 cells. Seven rounds of IL-2/GM-CSF EGT markedly inhibited the tumor growth. Reverse transciptase-polymerase chain reaction confirmed the expression of IL-2, GM-CSF and interferon-gamma within treated tumors. Systemic inhibitory effects can be demonstrated from tumor-re-challenged experiments on mice which received 3 rounds of double-gene EGT. The T cell proliferation assay revealed an increased T cell proliferation in double-gene EGT-treated mice. This experiment showed that partial systemic immunity can be provoked by IL-2/GM-CSF double-gene EGT. These findings suggest that our immuno-gene therapy protocol has the potential for future clinical applications.

Heme exporter FLVCR is required for T cell development and peripheral survival.

PubMed

Philip, Mary; Funkhouser, Scott A; Chiu, Edison Y; Phelps, Susan R; Delrow, Jeffrey J; Cox, James; Fink, Pamela J; Abkowitz, Janis L

2015-02-15

All aerobic cells and organisms must synthesize heme from the amino acid glycine and the tricarboxylic acid cycle intermediate succinyl CoA for incorporation into hemoproteins, such as the cytochromes needed for oxidative phosphorylation. Most studies on heme regulation have been done in erythroid cells or hepatocytes; however, much less is known about heme metabolism in other cell types. The feline leukemia virus subgroup C receptor (FLVCR) is a 12-transmembrane domain surface protein that exports heme from cells, and it was shown to be required for erythroid development. In this article, we show that deletion of Flvcr in murine hematopoietic precursors caused a complete block in αβ T cell development at the CD4(+)CD8(+) double-positive stage, although other lymphoid lineages were not affected. Moreover, FLVCR was required for the proliferation and survival of peripheral CD4(+) and CD8(+) T cells. These studies identify a novel and unexpected role for FLVCR, a major facilitator superfamily metabolite transporter, in T cell development and suggest that heme metabolism is particularly important in the T lineage. Copyright © 2015 by The American Association of Immunologists, Inc.

Red blood cell membrane viscoelasticity, agglutination and zeta potential measurements with double optical tweezers

NASA Astrophysics Data System (ADS)

Fontes, Adriana; Fernandes, Heloise P.; Barjas-Castro, Maria L.; de Thomaz, André A.; de Ysasa Pozzo, Liliana; Barbosa, Luiz C.; Cesar, Carlos L.

2006-02-01

The red blood cell (RBC) viscoelastic membrane contains proteins and glycolproteins embedded in, or attached, to a fluid lipid bilayer and are negatively charged, which creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. There are techniques, however, to decrease the zeta potential to allow cell agglutination which are the basis of most of the tests of antigen-antibody interactions in blood banks. This report shows the use of a double optical tweezers to measure RBC membrane viscosity, agglutination and zeta potential. In our technique one of the optical tweezers trap a silica bead that binds strongly to a RBC at the end of a RBCs rouleaux and, at the same time, acts as a pico-Newton force transducer, after calibration through its displacement from the equilibrium position. The other optical tweezers trap the RBC at the other end. To measure the membrane viscosity the optical force is measured as a function of the velocity between the RBCs. To measure the adhesion the tweezers are slowly displaced apart until the RBCs disagglutination happens. The RBC zeta potential is measured in two complimentary ways, by the force on the silica bead attached to a single RBC in response to an applied electric field, and the conventional way, by the measurement of terminal velocity of the RBC after released from the optical trap. These two measurements provide information about the RBC charges and, also, electrolytic solution properties. We believe this can improve the methods of diagnosis in blood banks.

Observations, Analysis, and Orbital Calculation of the Visual Double Star STTA 123 AB

NASA Astrophysics Data System (ADS)

Brashear, Nicholas; Camama, Angel; Drake, Miles; Smith, Miranda; Johnson, Jolyon; Arnold, Dave; Chamberlain, Rebecca

2012-04-01

As part of a research workshop at Pine Mountain Observatory, four students from Evergreen State College met with an instructor and an experienced double star observer to learn the methods used to measure double stars and to contribute observations to the Washington Double Star (WDS) Catalog. The students then observed and analyzed the visual double star STTA 123 AB with few past observations in the WDS Catalog to determine if it is optical or binary in nature. The separation of this double star was found to be 69.9" and its position angle to be 148.0°. Using the spectral types, stellar parallaxes, and proper motion vectors of these two stars, the students determined that this double star is likely physically bound by gravity in a binary system. Johnson calculated a preliminary circular orbit for the system using Newton's version of Kepler's third law. The masses of the two stars were estimated based on their spectral types (F0) to be 1.4 Msun. Their separation was estimated to be 316 AU based on their distance from Earth (about 216.5 light years) and their orbital period was estimated to be 3357 years. Arnold compared the observations made by the students to what would be predicted by the orbit calculation. A discrepancy of 14° was found in the position angle. The authors suggest that the orbit is both eccentric and inclined to our line of sight, making the observed position angle change less than predicted.

Ectopic Expression of Nolz-1 in Neural Progenitors Promotes Cell Cycle Exit/Premature Neuronal Differentiation Accompanying with Abnormal Apoptosis in the Developing Mouse Telencephalon

PubMed Central

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU−, Ki67− and phospho-histone 3-positive cells in E11.5–12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon. PMID:24073229

Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon.

PubMed

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU-, Ki67- and phospho-histone 3-positive cells in E11.5-12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon.

Modeling of single event transients with dual double-exponential current sources: Implications for logic cell characterization

DOE PAGES

Black, Dolores Archuleta; Robinson, William H.; Wilcox, Ian Zachary; ...

2015-08-07

Single event effects (SEE) are a reliability concern for modern microelectronics. Bit corruptions can be caused by single event upsets (SEUs) in the storage cells or by sampling single event transients (SETs) from a logic path. Likewise, an accurate prediction of soft error susceptibility from SETs requires good models to convert collected charge into compact descriptions of the current injection process. This paper describes a simple, yet effective, method to model the current waveform resulting from a charge collection event for SET circuit simulations. The model uses two double-exponential current sources in parallel, and the results illustrate why a conventionalmore » model based on one double-exponential source can be incomplete. Furthermore, a small set of logic cells with varying input conditions, drive strength, and output loading are simulated to extract the parameters for the dual double-exponential current sources. As a result, the parameters are based upon both the node capacitance and the restoring current (i.e., drive strength) of the logic cell.« less

Mature Hepatocytes Exhibit Unexpected Plasticity by Direct Dedifferentiation into Liver Progenitor Cells in Culture

PubMed Central

Chen, Yixin; Wong, Philip P.; Sjeklocha, Lucas; Steer, Clifford J.; Sahin, M. Behnan

2011-01-01

Although there have been numerous reports describing the isolation of liver progenitor cells from adult liver, their exact origin has not been clearly defined; and the role played by mature hepatocytes as direct contributors to the hepatic progenitor cell pool has remained largely unknown. Here we report strong evidence that mature hepatocytes in culture have the capacity to dedifferentiate into a population of adult liver progenitors without genetic or epigenetic manipulations. By using highly-purified mature hepatocytes, which were obtained from untreated, healthy rat liver and labeled with fluorescent dye PKH2, we found that hepatocytes in culture gave rise to a population of PKH2-positive liver progenitor cells. These cells, Liver Derived Progenitor Cells or LDPCS, which share phenotypic similarities with oval cells, were previously reported to be capable of forming mature hepatocytes both in culture and in animals. Studies done at various time points during the course of dedifferentiation cultures revealed that hepatocytes rapidly transformed into liver progenitors within one week through a transient oval cell-like stage. This finding was supported by lineage-tracing studies involving double-transgenic AlbuminCreXRosa26 mice expressing β-galactosidase exclusively in hepatocytes. Cultures set up with hepatocytes obtained from these mice resulted in generation of β-galactosidase-positive liver progenitor cells demonstrating that they were a direct dedifferentiation product of mature hepatocytes. Additionally, these progenitors differentiated into hepatocytes in vivo when transplanted into rats that had undergone retrorsine pretreatment and partial hepatectomy. Conclusion Our studies provide strong evidence for the unexpected plasticity of mature hepatocytes to dedifferentiate into progenitor cells in culture; and this may potentially have a significant impact on the treatment of liver diseases requiring liver or hepatocyte transplantation. PMID:21953633

T-cell-specific deletion of Mof blocks their differentiation and results in genomic instability in mice

PubMed Central

Pandita, Tej K.

2013-01-01

Ataxia telangiectasia patients develop lymphoid malignancies of both B- and T-cell origin. Similarly, ataxia telangiectasia mutated (Atm)-deficient mice exhibit severe defects in T-cell maturation and eventually develop thymomas. The function of ATM is known to be influenced by the mammalian orthologue of the Drosophila MOF (males absent on the first) gene. Here, we report the effect of T-cell-specific ablation of the mouse Mof (Mof) gene on leucocyte trafficking and survival. Conditional Mof Flox/Flox (Mof F/F) mice expressing Cre recombinase under control of the T-cell-specific Lck proximal promoter (Mof F/F/Lck-Cre +) display a marked reduction in thymus size compared with Mof F/F/Lck-Cre – mice. In contrast, the spleen size of Mof F/F/Lck-Cre + mice was increased compared with control Mof F/F/Lck-Cre – mice. The thymus of Mof F/F/Lck-Cre + mice contained significantly reduced T cells, whereas thymic B cells were elevated. Within the T-cell population, CD4+CD8+ double-positive T-cell levels were reduced, whereas the immature CD4–CD8– double-negative (DN) population was elevated. Defective T-cell differentiation is also evident as an increased DN3 (CD44–CD25+) population, the cell stage during which T-cell receptor rearrangement takes place. The differentiation defect in T cells and reduced thymus size were not rescued in a p53-deficient background. Splenic B-cell distributions were similar between Mof F/F/Lck-Cre + and Mof F/F/Lck-Cre – mice except for an elevation of the κ light-chain population, suggestive of an abnormal clonal expansion. T cells from Mof F/F/Lck-Cre + mice did not respond to phytohaemagglutinin (PHA) stimulation, whereas LPS-stimulated B cells from Mof F/F/Lck-Cre + mice demonstrated spontaneous genomic instability. Mice with T-cell-specific loss of MOF had shorter lifespans and decreased survival following irradiation than did Mof F/F/Lck-Cre – mice. These observations suggest that Mof plays a critical role in T-cell differentiation and that depletion of Mof in T cells reduces T-cell numbers and, by an undefined mechanism, induces genomic instability in B cells through bystander mechanism. As a result, these mice have a shorter lifespan and reduced survival after irradiation. PMID:23386701

T-cell-specific deletion of Mof blocks their differentiation and results in genomic instability in mice.

PubMed

Gupta, Arun; Hunt, Clayton R; Pandita, Raj K; Pae, Juhee; Komal, K; Singh, Mayank; Shay, Jerry W; Kumar, Rakesh; Ariizumi, Kiyoshi; Horikoshi, Nobuo; Hittelman, Walter N; Guha, Chandan; Ludwig, Thomas; Pandita, Tej K

2013-05-01

Ataxia telangiectasia patients develop lymphoid malignancies of both B- and T-cell origin. Similarly, ataxia telangiectasia mutated (Atm)-deficient mice exhibit severe defects in T-cell maturation and eventually develop thymomas. The function of ATM is known to be influenced by the mammalian orthologue of the Drosophila MOF (males absent on the first) gene. Here, we report the effect of T-cell-specific ablation of the mouse Mof (Mof) gene on leucocyte trafficking and survival. Conditional Mof(Flox/Flox) (Mof (F/F)) mice expressing Cre recombinase under control of the T-cell-specific Lck proximal promoter (Mof(F/F)/Lck-Cre(+)) display a marked reduction in thymus size compared with Mof(F/F)/Lck-Cre(-) mice. In contrast, the spleen size of Mof(F/F)/Lck-Cre(+) mice was increased compared with control Mof(F/F)/Lck-Cre(-) mice. The thymus of Mof(F/F)/Lck-Cre(+) mice contained significantly reduced T cells, whereas thymic B cells were elevated. Within the T-cell population, CD4(+)CD8(+) double-positive T-cell levels were reduced, whereas the immature CD4(-)CD8(-) double-negative (DN) population was elevated. Defective T-cell differentiation is also evident as an increased DN3 (CD44(-)CD25(+)) population, the cell stage during which T-cell receptor rearrangement takes place. The differentiation defect in T cells and reduced thymus size were not rescued in a p53-deficient background. Splenic B-cell distributions were similar between Mof(F/F)/Lck-Cre(+) and Mof(F/F)/Lck-Cre(-) mice except for an elevation of the κ light-chain population, suggestive of an abnormal clonal expansion. T cells from Mof(F/F)/Lck-Cre(+) mice did not respond to phytohaemagglutinin (PHA) stimulation, whereas LPS-stimulated B cells from Mof(F/F)/Lck-Cre(+) mice demonstrated spontaneous genomic instability. Mice with T-cell-specific loss of MOF had shorter lifespans and decreased survival following irradiation than did Mof(F/F)/Lck-Cre(-) mice. These observations suggest that Mof plays a critical role in T-cell differentiation and that depletion of Mof in T cells reduces T-cell numbers and, by an undefined mechanism, induces genomic instability in B cells through bystander mechanism. As a result, these mice have a shorter lifespan and reduced survival after irradiation.

Relation of follicular dendritic reticulum cells to Reed-Sternberg cells of Hodgkin's disease with emphasis on the expression of CD21 antigen.

PubMed Central

Delsol, G.; Meggetto, F.; Brousset, P.; Cohen-Knafo, E.; al Saati, T.; Rochaix, P.; Gorguet, B.; Rubin, B.; Voigt, J. J.; Chittal, S.

1993-01-01

Based on observations of 66 cases, in which tissues were specially processed to optimize the simultaneous preservation of cell membrane antigens and morphology, we provide evidence in favor of a relationship between follicular dendritic reticulum cells (FDRC) and Reed-Sternberg (RS) cells of Hodgkin's disease (HD) other than the lymphocyte predominance subtype. RS cells were intimately related to the FDRC network (75% of cases), and the expression of CD21 antigen was frequent (41% of cases). Exclusive expression of CD21 antigen was found in 11 cases of HD, while the expression of other B-cell-associated markers (CD19, CD20, CD22) was both variable and inconsistent. The expression of T-cell antigens (CD3, CD4, CD8) was rare. Null phenotype of RS cells was observed in 27 of 66 cases (41%). Epstein-Barr virus (EBV) nucleic acids were found in 34 of 66 (51.5%) cases. Double labeling techniques showed the presence of EBV-positive RS cells within the FDRC network. A non-B-cell origin of RS cells was supported by the differential expression of EBV latent antigens in HD (latent membrane protein+, EB nuclear antigen 2-), which is unusual in EBV-driven lymphoblastoid cell lines and EBV-positive B-cell lymphomas. FDRC and RS cells are known to share morphological traits (binucleated cells), and both cell types possess Fc receptor for IgG. The hypothesis is further backed by the findings of CD15 antigen expression by occasional RS-like dysplastic FDRC in Castleman's disease (five cases), which is characterized by hyperplasia of FDRC. Whether FDRC might be the only cells involved in the conversion to RS cells by the loss or gain of antigens remains to be determined. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7685151

Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions.

PubMed

Rimkute, Lina; Kraujalis, Tadas; Snipas, Mindaugas; Palacios-Prado, Nicolas; Jotautis, Vaidas; Skeberdis, Vytenis A; Bukauskas, Feliksas F

2018-01-01

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pH i ) and cytosolic magnesium ion concentration ([Mg 2+ ] i ), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pH i and [Mg 2+ ] i affect junctional conductance (g j ) in an interdependent manner; in other words, intracellular acidification cause increase or decay in g j depending on whether [Mg 2+ ] i is high or low, respectively, and intracellular alkalization cause reduction in g j independently of [Mg 2+ ] i . Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pH i and [Mg 2+ ] i . Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pH i and [Mg 2+ ] i . Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg 2+ ] i , while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36 * E8Q lost the initial increase of g j at low [Mg 2+ ] i and double mutation lost the sensitivity to high [Mg 2+ ] i . These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg 2+ and H + ions.

[The recent advances of HAM/TSP research].

PubMed

Osame, M

1999-12-01

The ninth international conference on HTLVs and related disorders was held on April 5-9, 1999 at Kagoshima, Japan under the conference chairperson, Dr. Mitsuhiro Osame. In this meeting, world-wide epidemiological data on HTLV-I carriers, ATL patients, and HAM/TSP patients were summarized as shown in the table. The total number of them was supposed to be more than 2.2 millions, 1,200, and 3,000, respectively. To elucidate the localization of HTLV-I proviral DNA directly, double staining using immunohistochemistry and PCR in situ hybridization in the spinal cords of HAM/TSP patients were performed. HTLV-I proviral DNA was localized only to OPD 4-positive cells (Matsuoka et al, 1998). The localization of HTLV-I messenger RNA was the same (Moritoyo et al, 1996). A novel technique to detect HTLV-I tax protein was also developed. In HAM/TSP patients, 0.04-1.16% of the CSF cells and 0.02-0.54% of PBMCs were positive for HTLV-I tax protein (Moritoyo et al, 1999). It was also hypothesized that HLA alleles control HTLV-I proviral load and thus influence susceptibility to HAM/TSP. Two hundred and thirty-two cases of HAM/TSP were compared with 201 randomly selected HTLV-I seropositive asymptomatic blood donors. It was shown that, after infection with HTLV-I, the class I allele HLA-A*02 halves the odds of HAM/TSP (p < 0.0001), preventing 28% of potential cases of HAM/TSP. Furthermore, HLA-A*02 positive healthy HTLV-I carriers have a proviral load one-third that (p = 0.0114) of HLA-A*02 negative HTLV-I carriers. An association of HLA-DRB1*0101 with disease susceptibility was also identified, which doubled the odds of HAM/TSP in the absence of the protective effect of HLA-A*02 (Jeffery and Usuku et al, 1999).

Influence of the charge double layer on solid oxide fuel cell stack behavior

NASA Astrophysics Data System (ADS)

Whiston, Michael M.; Bilec, Melissa M.; Schaefer, Laura A.

2015-10-01

While the charge double layer effect has traditionally been characterized as a millisecond phenomenon, longer timescales may be possible under certain operating conditions. This study simulates the dynamic response of a previously developed solid oxide fuel cell (SOFC) stack model that incorporates the charge double layer via an equivalent circuit. The model is simulated under step load changes. Baseline conditions are first defined, followed by consideration of minor and major deviations from the baseline case. This study also investigates the behavior of the SOFC stack with a relatively large double layer capacitance value, as well as operation of the SOFC stack under proportional-integral (PI) control. Results indicate that the presence of the charge double layer influences the SOFC stack's settling time significantly under the following conditions: (i) activation and concentration polarizations are significantly increased, or (ii) a large value of the double layer capacitance is assumed. Under normal (baseline) operation, on the other hand, the charge double layer effect diminishes within milliseconds, as expected. It seems reasonable, then, to neglect the charge double layer under normal operation. However, careful consideration should be given to potential variations in operation or material properties that may give rise to longer electrochemical settling times.

Genome editing with CompoZr custom zinc finger nucleases (ZFNs).

PubMed

Hansen, Keith; Coussens, Matthew J; Sago, Jack; Subramanian, Shilpi; Gjoka, Monika; Briner, Dave

2012-06-14

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes. Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA. Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency. While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site. The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator's cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

A medulloblastoma showing an unusually long doubling time: reflection of its singular nature.

PubMed

Doron, Omer; Zauberman, Jacob; Feldman, Ze'ev

2016-06-01

In this paper, we present a case of a 4-year-old male diagnosed with a desmoplastic, SHH-type medulloblastoma. Retrospectively, we discovered that the patient underwent an MRI scan at 21 months for reasons unrelated, revealing a T1-enhanced lesion at the vermis, later recognized as the source of the tumor. This unique case provides us with a glimpse into the natural history of this tumor. Our ability to measure tumor volume at two defined time points, 31 months apart, enabled us to deduce the tumor's doubling time. This is defined as the time of one cell cycle divided by the amount of cycling cells, multiplied by cell loss factor. Potential doubling time (Tpot) and actual doubling time (Td), calculated using the Gompertzian model, are the most clinically relevant with regard to a tumor's response to radiotherapy. Here, we show an actual doubling-time (Td) of 78 days, and an extrapolated tumor diameter at the time of birth of 0.25 mm. These results both support the medulloblastoma's embryonic origin, and indicating a threefold longer actual doubling time when compared to previous studies. Taking into account the reported range of medulloblastoma potential doubling time, we deduced a cell loss factor of between 48.9 and 95.5 %. These percentages fall in line with other malignant tumors. Although limited due to the obvious reliance on only two points in time and using the Gompertzian model to complete the remainder, to the best of our knowledge, this is the longest follow-up period reported for medulloblastoma. We have described how a unique turn of events enabled us to get a glimpse into the in situ development of a medulloblastoma over a 31-month period. Regarded sometimes as an idiosyncratic tumor comprised of an array of molecular changes, the complexity of medulloblastoma is displayed here, by revealing for the first time an actual doubling time three- to fourfold the previously known length.

Polysulfide intercalated layered double hydroxides for metal capture applications

DOEpatents

Kanatzidis, Mercouri G.; Ma, Shulan

2017-04-04

Polysulfide intercalated layered double hydroxides and methods for their use in vapor and liquid-phase metal capture applications are provided. The layered double hydroxides comprise a plurality of positively charged host layers of mixed metal hydroxides separated by interlayer spaces. Polysulfide anions are intercalated in the interlayer spaces.

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.

PubMed

Bañuelos, C A; Banáth, J P; MacPhail, S H; Zhao, J; Eaves, C A; O'Connor, M D; Lansdorp, P M; Olive, P L

2008-09-01

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.

MYC protein expression is detected in plasma cell myeloma but not in monoclonal gammopathy of undetermined significance (MGUS).

PubMed

Xiao, Ruobing; Cerny, Jan; Devitt, Katherine; Dresser, Karen; Nath, Rajneesh; Ramanathan, Muthalagu; Rodig, Scott J; Chen, Benjamin J; Woda, Bruce A; Yu, Hongbo

2014-06-01

It has been recognized that monoclonal gammopathy of undetermined significance (MGUS) precedes a diagnosis of plasma cell myeloma in most patients. Recent gene expression array analysis has revealed that an MYC activation signature is detected in plasma cell myeloma but not in MGUS. In this study, we performed immunohistochemical studies using membrane CD138 and nuclear MYC double staining on bone marrow biopsies from patients who met the diagnostic criteria of plasma cell myeloma or MGUS. Our study demonstrated nuclear MYC expression in CD138-positive plasma cells in 22 of 26 (84%) plasma cell myeloma samples and in none of the 29 bone marrow samples from patients with MGUS. In addition, our data on the follow-up biopsies from plasma cell myeloma patients with high MYC expression demonstrated that evaluation of MYC expression in plasma cells can be useful in detecting residual disease. We also demonstrated that plasma cells gained MYC expression in 5 of 8 patients (62.5%) when progressing from MGUS to plasma cell myeloma. Analysis of additional lymphomas with plasmacytic differentiation, including lymphoplasmacytic lymphoma, marginal zone lymphoma, and plasmablastic lymphoma, reveals that MYC detection can be a useful tool in the diagnosis of plasma cell myeloma.

Three-dimensional particle simulation of back-sputtered carbon in electric propulsion test facility

NASA Astrophysics Data System (ADS)

Zheng, Hongru; Cai, Guobiao; Liu, Lihui; Shang, Shengfei; He, Bijiao

2017-03-01

The back-sputtering deposition on thruster surface caused by ion bombardment on chamber wall material affects the performance of thrusters during the ground based electric propulsion endurance tests. In order to decrease the back-sputtering deposition, most of vacuum chambers applied in electric propulsion experiments are equipped with anti-sputtering targets. In this paper, a three-dimensional model of plume experimental system (PES) including double layer anti-sputtering target is established. Simulation cases are made to simulate the plasma environment and sputtering effects when an ion thruster is working. The particle in cell (PIC) method and direct simulation Monte Carlo (DSMC) method is used to calculate the velocity and position of particles. Yamamura's model is used to simulate the sputtering process. The distribution of sputtered anti-sputtering target material is presented. The results show that the double layer anti-sputtering target can significantly reduce the deposition on thruster surface. The back-sputtering deposition rates on thruster exit surface for different cases are compared. The chevrons on the secondary target are rearranged to improve its performance. The position of secondary target has relation with the ion beam divergence angle, and the radius of the vacuum chamber. The back-sputtering deposition rate is lower when the secondary target covers the entire ion beam.

The N-terminus of RPA large subunit and its spatial position are important for the 5'->3' resection of DNA double-strand breaks.

PubMed

Tammaro, Margaret; Liao, Shuren; McCane, Jill; Yan, Hong

2015-10-15

The first step of homology-dependent repair of DNA double-strand breaks (DSBs) is the resection of the 5' strand to generate 3' ss-DNA. Of the two major nucleases responsible for resection, EXO1 has intrinsic 5'->3' directionality, but DNA2 does not. DNA2 acts with RecQ helicases such as the Werner syndrome protein (WRN) and the heterotrimeric eukaryotic ss-DNA binding protein RPA. We have found that the N-terminus of the RPA large subunit (RPA1N) interacts with both WRN and DNA2 and is essential for stimulating WRN's 3'->5' helicase activity and DNA2's 5'->3' ss-DNA exonuclease activity. A mutant RPA complex that lacks RPA1N is unable to support resection in Xenopus egg extracts and human cells. Furthermore, relocating RPA1N to the middle subunit but not to the small subunit causes severe defects in stimulating DNA2 and WRN and in supporting resection. Together, these findings suggest that RPA1N and its spatial position are critical for restricting the directionality of the WRN-DNA2 resection pathway. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The N-terminus of RPA large subunit and its spatial position are important for the 5′->3′ resection of DNA double-strand breaks

PubMed Central

Tammaro, Margaret; Liao, Shuren; McCane, Jill; Yan, Hong

2015-01-01

The first step of homology-dependent repair of DNA double-strand breaks (DSBs) is the resection of the 5′ strand to generate 3′ ss-DNA. Of the two major nucleases responsible for resection, EXO1 has intrinsic 5′->3′ directionality, but DNA2 does not. DNA2 acts with RecQ helicases such as the Werner syndrome protein (WRN) and the heterotrimeric eukaryotic ss-DNA binding protein RPA. We have found that the N-terminus of the RPA large subunit (RPA1N) interacts with both WRN and DNA2 and is essential for stimulating WRN's 3′->5′ helicase activity and DNA2's 5′->3′ ss-DNA exonuclease activity. A mutant RPA complex that lacks RPA1N is unable to support resection in Xenopus egg extracts and human cells. Furthermore, relocating RPA1N to the middle subunit but not to the small subunit causes severe defects in stimulating DNA2 and WRN and in supporting resection. Together, these findings suggest that RPA1N and its spatial position are critical for restricting the directionality of the WRN-DNA2 resection pathway. PMID:26227969