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Sample records for cells suggests molecular

  1. Limitations of Time-Resolved Fluorescence Suggested by Molecular Simulations: Assessing the Dynamics of T cell Receptor Binding Loops

    PubMed Central

    Scott, Daniel R.; Vardeman, Charles F.; Corcelli, Steven A.; Baker, Brian M.

    2012-01-01

    Time-resolved fluorescence anisotropy (TRFA) has a rich history in evaluating protein dynamics. Yet as often employed, TRFA assumes that the motional properties of a covalently tethered fluorescent probe accurately portray the motional properties of the protein backbone at the probe attachment site. In an extensive survey using TRFA to study the dynamics of the binding loops of a αβ T cell receptor, we observed multiple discrepancies between the TRFA data and previously published results that led us to question this assumption. We thus simulated several of the experimentally probed systems using a protocol that permitted accurate determination of probe and protein time correlation functions. We found excellent agreement in the decays of the experimental and simulated correlation functions. However, the motional properties of the probe were poorly correlated with those of the backbone of both the labeled and unlabeled protein. Our results warrant caution in the interpretation of TRFA data and suggest further studies to ascertain the extent to which probe dynamics reflect those of the protein backbone. Meanwhile, the agreement between experiment and computation validates the use of molecular dynamics simulations as an accurate tool for exploring the molecular motion of T cell receptors and their binding loops. PMID:23260055

  2. Molecular and biochemical analysis of rainbow trout LCK suggests a conserved mechanism for T-cell signaling in gnathostomes

    USGS Publications Warehouse

    Laing, K.J.; Dutton, S.; Hansen, J.D.

    2007-01-01

    Two genes were identified in rainbow trout that display high sequence identity to vertebrate Lck. Both of the trout Lck transcripts are associated with lymphoid tissues and were found to be highly expressed in IgM-negative lymphocytes. In vitro analysis of trout lymphocytes indicates that trout Lck mRNA is up-regulated by T-cell mitogens, supporting an evolutionarily conserved function for Lck in the signaling pathways of T-lymphocytes. Here, we describe the generation and characterization of a specific monoclonal antibody raised against the N-terminal domains of recombinant trout Lck that can recognize Lck protein(s) from trout thymocyte lysates that are similar in size (???57 kDa) to mammalian Lck. This antibody also reacted with permeabilized lymphocytes during FACS analysis, indicating its potential usage for cellular analyses of trout lymphocytes, thus representing an important tool for investigations of salmonid T-cell function.

  3. Molecular profiling of blastic plasmacytoid dendritic cell neoplasm reveals a unique pattern and suggests selective sensitivity to NF-kB pathway inhibition

    PubMed Central

    Sapienza, MR; Fuligni, F; Agostinelli, C; Tripodo, C; Righi, S; Laginestra, MA; Pileri, A; Mancini, M; Rossi, M; Ricci, F; Gazzola, A; Melle, F; Mannu, C; Ulbar, F; Arpinati, M; Paulli, M; Maeda, T; Gibellini, D; Pagano, L; Pimpinelli, N; Santucci, M; Cerroni, L; Croce, CM; Facchetti, F; Piccaluga, PP; Pileri, SA

    2015-01-01

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease of controversial origin recently recognized as a neoplasm deriving from plasmacytoid dendritic cells (pDCs). Nevertheless, it remains an orphan tumor with obscure biology and dismal prognosis. To better understand the pathobiology of BPDCN and discover new targets for effective therapies, the gene expression profile (GEP) of 25 BPDCN samples was analyzed and compared with that of pDCs, their postulated normal counterpart. Validation was performed by immunohistochemistry (IHC), whereas functional experiments were carried out ex vivo. For the first time at the molecular level, we definitely recognized the cellular derivation of BPDCN that proved to originate from the myeloid lineage and in particular, from resting pDCs. Furthermore, thanks to an integrated bioinformatic approach we discovered aberrant activation of the NF-kB pathway and suggested it as a novel therapeutic target. We tested the efficacy of anti-NF-kB-treatment on the BPDCN cell line CAL-1, and successfully demonstrated by GEP and IHC the molecular shutoff of the NF-kB pathway. In conclusion, we identified a molecular signature representative of the transcriptional abnormalities of BPDCN and developed a cellular model proposing a novel therapeutic approach in the setting of this otherwise incurable disease. PMID:24504027

  4. A Suggested Molecular Pathology Curriculum for Residents: A Report of the Association for Molecular Pathology.

    PubMed

    Aisner, Dara L; Berry, Anna; Dawson, D Brian; Hayden, Randall T; Joseph, Loren; Hill, Charles E

    2016-03-01

    Molecular pathology is an essential element of pathology training. As more molecular tests have become available, there is an increasing need for pathology trainees to receive a strong foundation in molecular pathology. Appointed by the Training and Education Committee of the Association for Molecular Pathology, the Molecular Curriculum Task Force has developed a suggested curriculum in molecular pathology for residents. The foundations of molecular pathology are presented as a series of goals and objectives that residency programs can use to develop their educational programs. As pathologists continue to expand their roles to include regular clinical consultations in the realm of molecular testing, a strong foundation in molecular pathology and genomic medicine has become essential to the practice of pathology. PMID:26857063

  5. Molecular spectroscopic study for suggested mechanism of chrome tanned leather

    NASA Astrophysics Data System (ADS)

    Nashy, Elshahat H. A.; Osman, Osama; Mahmoud, Abdel Aziz; Ibrahim, Medhat

    2012-03-01

    Collagen represents the structural protein of the extracellular matrix, which gives strength of hides and/or skin under tanning process. Chrome tan is the most important tanning agent all over the world. The methods for production of leather evolved over several centuries as art and engineering with little understanding of the underlying science. The present work is devoted to suggest the most probable mechanistic action of chrome tan on hide proteins. First the affect of Cr upon hide protein is indicated by the studied mechanical properties. Then the spectroscopic characterization of the hide protein as well as chrome tanned leather was carried out with Horizontal Attenuated Total Reflection (HATR) FT-IR. The obtained results indicate how the chromium can attached with the active sites of collagen. Molecular modeling confirms that chromium can react with amino as well as carboxylate groups. Four schemes were obtained to describe the possible interactions of chrome tan with hide proteins.

  6. Molecular spectroscopic study for suggested mechanism of chrome tanned leather.

    PubMed

    Nashy, Elshahat H A; Osman, Osama; Mahmoud, Abdel Aziz; Ibrahim, Medhat

    2012-03-01

    Collagen represents the structural protein of the extracellular matrix, which gives strength of hides and/or skin under tanning process. Chrome tan is the most important tanning agent all over the world. The methods for production of leather evolved over several centuries as art and engineering with little understanding of the underlying science. The present work is devoted to suggest the most probable mechanistic action of chrome tan on hide proteins. First the affect of Cr upon hide protein is indicated by the studied mechanical properties. Then the spectroscopic characterization of the hide protein as well as chrome tanned leather was carried out with Horizontal Attenuated Total Reflection (HATR) FT-IR. The obtained results indicate how the chromium can attached with the active sites of collagen. Molecular modeling confirms that chromium can react with amino as well as carboxylate groups. Four schemes were obtained to describe the possible interactions of chrome tan with hide proteins. PMID:22225606

  7. Transcriptome Variability in Keratocystic Odontogenic Tumor Suggests Distinct Molecular Subtypes

    PubMed Central

    Hu, Shijia; Divaris, Kimon; Parker, Joel; Padilla, Ricardo; Murrah, Valerie; Wright, John Timothy

    2016-01-01

    Keratocystic Odontogenic Tumor (KCOT) is a locally aggressive developmental cystic neoplasm thought to arise from the odontogenic epithelium. A high recurrence rate of up to 30% has been found following conservative treatment. Aggressive tumor resection can lead to the need for extensive reconstructive surgery, resulting in significant morbidity and impacting quality of life. Most research has focused on candidate-genes with a handful of studies employing whole transcriptome approaches. There is also the question of which reference tissue is most biologically-relevant. This study characterizes the transcriptome of KCOT using whole genome microarray and compare it with gene expression of different odontogenic tissues (“dentome”). Laser capture microdissection was used to isolate the neoplastic epithelial tissue in 20 cases. KCOT gene expression was compared with the “dentome” and relevant pathways were examined. Cluster analysis revealed 2 distinct molecular subtypes of KCOT. Several inflammatory pathways were activated in both subtypes. The AKT pathway was activated in one subtype while MAP kinase pathway was activated in the other. Additionally, PTCH1 expression was downregulated in both clusters suggesting involvement in KCOT tumorigenesis. In conclusion, this study provides new insights into the transcriptome of KCOT and highlights pathways that could be of diagnostic and prognostic value. PMID:27066764

  8. PSF/SFPQ is a very common gene fusion partner in TFE3 rearrangement-associated perivascular epithelioid cell tumors (PEComas) and melanotic Xp11 translocation renal cancers: clinicopathologic, immunohistochemical, and molecular characteristics suggesting classification as a distinct entity.

    PubMed

    Rao, Qiu; Shen, Qin; Xia, Qiu-yuan; Wang, Zi-yu; Liu, Biao; Shi, Shan-shan; Shi, Qun-li; Yin, Hong-lin; Wu, Bo; Ye, Sheng-bing; Li, Li; Chen, Jie-Yu; Pan, Min-hong; Li, Qing; Li, Rui; Wang, Xuan; Zhang, Ru-song; Yu, Bo; Ma, Heng-hui; Lu, Zhen-feng; Zhou, Xiao-jun

    2015-09-01

    An increasing number of TFE3 rearrangement-associated tumors, such as TFE3 rearrangement-associated perivascular epithelioid cell tumors (PEComas), melanotic Xp11 translocation renal cancers, and melanotic Xp11 neoplasms, have recently been reported. We examined 12 such cases, including 5 TFE3 rearrangement-associated PEComas located in the pancreas, cervix, or pelvis and 7 melanotic Xp11 translocation renal cancers, using clinicopathologic, immunohistochemical, and molecular analyses. All the tumors shared a similar morphology, including a purely nested or sheet-like architecture separated by a delicate vascular network, purely epithelioid cells displaying a clear or granular eosinophilic cytoplasm, a lack of papillary structures and spindle cell or fat components, uniform round or oval nuclei containing small visible nucleoli, and, in most cases (11/12), melanin pigmentation. The levels of mitotic activity and necrosis varied. All 12 cases displayed moderately (2+) or strongly (3+) positive immunoreactivity for TFE3 and cathepsin K. One case labeled focally for HMB45 and Melan-A, whereas the others typically labeled moderately (2+) or strongly (3+) for 1 of these markers. None of the cases were immunoreactive for smooth muscle actin, desmin, CKpan, S100, or PAX8. PSF-TFE3 fusion genes were confirmed by reverse transcription polymerase chain reaction in cases (7/7) in which a novel PSF-TFE3 fusion point was identified. All of the cases displayed TFE3 rearrangement associated with Xp11 translocation. Furthermore, we developed a PSF-TFE3 fusion fluorescence in situ hybridization assay for the detection of the PSF-TFE3 fusion gene and detected it in all 12 cases. Clinical follow-up data were available for 7 patients. Three patients died, and 2 patients (cases 1 and 3) remained alive with no evidence of disease after initial resection. Case 2 experienced recurrence and remained alive with disease. Case 5, a recent case, remained alive with extensive abdominal cavity

  9. Global gene expression profiling of oral cavity cancers suggests molecular heterogeneity within anatomic subsites

    PubMed Central

    Severino, Patricia; Alvares, Adriana M; Michaluart, Pedro; Okamoto, Oswaldo K; Nunes, Fabio D; Moreira-Filho, Carlos A; Tajara, Eloiza H

    2008-01-01

    Background Oral squamous cell carcinoma (OSCC) is a frequent neoplasm, which is usually aggressive and has unpredictable biological behavior and unfavorable prognosis. The comprehension of the molecular basis of this variability should lead to the development of targeted therapies as well as to improvements in specificity and sensitivity of diagnosis. Results Samples of primary OSCCs and their corresponding surgical margins were obtained from male patients during surgery and their gene expression profiles were screened using whole-genome microarray technology. Hierarchical clustering and Principal Components Analysis were used for data visualization and One-way Analysis of Variance was used to identify differentially expressed genes. Samples clustered mostly according to disease subsite, suggesting molecular heterogeneity within tumor stages. In order to corroborate our results, two publicly available datasets of microarray experiments were assessed. We found significant molecular differences between OSCC anatomic subsites concerning groups of genes presently or potentially important for drug development, including mRNA processing, cytoskeleton organization and biogenesis, metabolic process, cell cycle and apoptosis. Conclusion Our results corroborate literature data on molecular heterogeneity of OSCCs. Differences between disease subsites and among samples belonging to the same TNM class highlight the importance of gene expression-based classification and challenge the development of targeted therapies. PMID:19014556

  10. Evidences Suggesting Involvement of Viruses in Oral Squamous Cell Carcinoma

    PubMed Central

    Gupta, Kanupriya; Metgud, Rashmi

    2013-01-01

    Oral cancer is one of the most common cancers and it constitutes a major health problem particularly in developing countries. Oral squamous cell carcinoma (OSCC) represents the most frequent of all oral neoplasms. Several risk factors have been well characterized to be associated with OSCC with substantial evidences. The etiology of OSCC is complex and involves many factors. The most clearly defined potential factors are smoking and alcohol, which substantially increase the risk of OSCC. However, despite this clear association, a substantial proportion of patients develop OSCC without exposure to them, emphasizing the role of other risk factors such as genetic susceptibility and oncogenic viruses. Some viruses are strongly associated with OSCC while the association of others is less frequent and may depend on cofactors for their carcinogenic effects. Therefore, the exact role of viruses must be evaluated with care in order to improve the diagnosis and treatment of OSCC. Although a viral association within a subset of OSCC has been shown, the molecular and histopathological characteristics of these tumors have yet to be clearly defined. PMID:24455418

  11. DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding

    PubMed Central

    Szambowska, Anna; Tessmer, Ingrid; Kursula, Petri; Usskilat, Christian; Prus, Piotr; Pospiech, Helmut; Grosse, Frank

    2014-01-01

    The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3′ protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3′–5′ polarity and, thereby acts as a molecular ‘wedge’ to initiate DNA strand displacement. PMID:24293646

  12. Improved detection suggests all Merkel cell carcinomas harbor Merkel polyomavirus.

    PubMed

    Rodig, Scott J; Cheng, Jingwei; Wardzala, Jacek; DoRosario, Andrew; Scanlon, Jessica J; Laga, Alvaro C; Martinez-Fernandez, Alejandro; Barletta, Justine A; Bellizzi, Andrew M; Sadasivam, Subhashini; Holloway, Dustin T; Cooper, Dylan J; Kupper, Thomas S; Wang, Linda C; DeCaprio, James A

    2012-12-01

    A human polyomavirus was recently discovered in Merkel cell carcinoma (MCC) specimens. The Merkel cell polyomavirus (MCPyV) genome undergoes clonal integration into the host cell chromosomes of MCC tumors and expresses small T antigen and truncated large T antigen. Previous studies have consistently reported that MCPyV can be detected in approximately 80% of all MCC tumors. We sought to increase the sensitivity of detection of MCPyV in MCC by developing antibodies capable of detecting large T antigen by immunohistochemistry. In addition, we expanded the repertoire of quantitative PCR primers specific for MCPyV to improve the detection of viral DNA in MCC. Here we report that a novel monoclonal antibody detected MCPyV large T antigen expression in 56 of 58 (97%) unique MCC tumors. PCR analysis specifically detected viral DNA in all 60 unique MCC tumors tested. We also detected inactivating point substitution mutations of TP53 in the two MCC specimens that lacked large T antigen expression and in only 1 of 56 tumors positive for large T antigen. These results indicate that MCPyV is present in MCC tumors more frequently than previously reported and that mutations in TP53 tend to occur in MCC tumors that fail to express MCPyV large T antigen. PMID:23114601

  13. Improved detection suggests all Merkel cell carcinomas harbor Merkel polyomavirus

    PubMed Central

    Rodig, Scott J.; Cheng, Jingwei; Wardzala, Jacek; DoRosario, Andrew; Scanlon, Jessica J.; Laga, Alvaro C.; Martinez-Fernandez, Alejandro; Barletta, Justine A.; Bellizzi, Andrew M.; Sadasivam, Subhashini; Holloway, Dustin T.; Cooper, Dylan J.; Kupper, Thomas S.; Wang, Linda C.; DeCaprio, James A.

    2012-01-01

    A human polyomavirus was recently discovered in Merkel cell carcinoma (MCC) specimens. The Merkel cell polyomavirus (MCPyV) genome undergoes clonal integration into the host cell chromosomes of MCC tumors and expresses small T antigen and truncated large T antigen. Previous studies have consistently reported that MCPyV can be detected in approximately 80% of all MCC tumors. We sought to increase the sensitivity of detection of MCPyV in MCC by developing antibodies capable of detecting large T antigen by immunohistochemistry. In addition, we expanded the repertoire of quantitative PCR primers specific for MCPyV to improve the detection of viral DNA in MCC. Here we report that a novel monoclonal antibody detected MCPyV large T antigen expression in 56 of 58 (97%) unique MCC tumors. PCR analysis specifically detected viral DNA in all 60 unique MCC tumors tested. We also detected inactivating point substitution mutations of TP53 in the two MCC specimens that lacked large T antigen expression and in only 1 of 56 tumors positive for large T antigen. These results indicate that MCPyV is present in MCC tumors more frequently than previously reported and that mutations in TP53 tend to occur in MCC tumors that fail to express MCPyV large T antigen. PMID:23114601

  14. Molecular pathogenesis of mantle cell lymphoma

    PubMed Central

    Jares, Pedro; Colomer, Dolors; Campo, Elias

    2012-01-01

    Mantle cell lymphoma is a B cell malignancy in which constitutive dysregulation of cyclin D1 and the cell cycle, disruption of DNA damage response pathways, and activation of cell survival mechanisms contribute to oncogenesis. A small number of tumors lack cyclin D1 overexpression, suggesting that its dysregulation is always not required for tumor initiation. Some cases have hypermutated IGHV and stable karyotypes, a predominant nonnodal disease, and an indolent clinical evolution, which suggests that they may correspond to distinct subtypes of the disease. In this review, we discuss the molecular pathways that contribute to pathogenesis, and how improved understanding of these molecular mechanisms offers new perspectives for the treatment of patients. PMID:23023712

  15. Use of multivariate analysis to suggest a new molecular classification of colorectal cancer

    PubMed Central

    Domingo, Enric; Ramamoorthy, Rajarajan; Oukrif, Dahmane; Rosmarin, Daniel; Presz, Michal; Wang, Haitao; Pulker, Hannah; Lockstone, Helen; Hveem, Tarjei; Cranston, Treena; Danielsen, Havard; Novelli, Marco; Davidson, Brian; Xu, Zheng-Zhou; Molloy, Peter; Johnstone, Elaine; Holmes, Christopher; Midgley, Rachel; Kerr, David; Sieber, Oliver; Tomlinson, Ian

    2013-01-01

    Abstract Molecular classification of colorectal cancer (CRC) is currently based on microsatellite instability (MSI), KRAS or BRAF mutation and, occasionally, chromosomal instability (CIN). Whilst useful, these categories may not fully represent the underlying molecular subgroups. We screened 906 stage II/III CRCs from the VICTOR clinical trial for somatic mutations. Multivariate analyses (logistic regression, clustering, Bayesian networks) identified the primary molecular associations. Positive associations occurred between: CIN and TP53 mutation; MSI and BRAF mutation; and KRAS and PIK3CA mutations. Negative associations occurred between: MSI and CIN; MSI and NRAS mutation; and KRAS mutation, and each of NRAS, TP53 and BRAF mutations. Some complex relationships were elucidated: KRAS and TP53 mutations had both a direct negative association and a weaker, confounding, positive association via TP53–CIN–MSI–BRAF–KRAS. Our results suggested a new molecular classification of CRCs: (1) MSI+ and/or BRAF-mutant; (2) CIN+ and/or TP53– mutant, with wild-type KRAS and PIK3CA; (3) KRAS- and/or PIK3CA-mutant, CIN+, TP53-wild-type; (4) KRAS– and/or PIK3CA-mutant, CIN–, TP53-wild-type; (5) NRAS-mutant; (6) no mutations; (7) others. As expected, group 1 cancers were mostly proximal and poorly differentiated, usually occurring in women. Unexpectedly, two different types of CIN+ CRC were found: group 2 cancers were usually distal and occurred in men, whereas group 3 showed neither of these associations but were of higher stage. CIN+ cancers have conventionally been associated with all three of these variables, because they have been tested en masse. Our classification also showed potentially improved prognostic capabilities, with group 3, and possibly group 1, independently predicting disease-free survival. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:23165447

  16. Characterization of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

    PubMed Central

    Stam, Remco; Howden, Andrew J. M.; Delgado-Cerezo, Magdalena; M. M. Amaro, Tiago M.; Motion, Graham B.; Pham, Jasmine; Huitema, Edgar

    2013-01-01

    Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centers on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signaling. Here, we characterized three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localization of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organization, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility. PMID:24155749

  17. Nanotubule and Tour Molecule Based Molecular Electronics: Suggestion for a Hybrid Approach

    NASA Technical Reports Server (NTRS)

    Srivastava, Deepak; Saini, Subhash (Technical Monitor)

    1998-01-01

    Recent experimental and theoretical attempts and results indicate two distinct broad pathways towards future molecular electronic devices and architectures. The first is the approach via Tour type ladder molecules and their junctions which can be fabricated with solution phase chemical approaches. Second are fullerenes or nanotubules and their junctions which may have better conductance, switching and amplifying characteristics but can not be made through well controlled and defined chemical means. A hybrid approach combining the two pathways to take advantage of the characteristics of both is suggested. Dimension and scale of such devices would be somewhere in between isolated molecule and nanotubule based devices but it maybe possible to use self-assembly towards larger functional and logicalunits.

  18. Molecular phenotypes in triple negative breast cancer from African American patients suggest targets for therapy.

    PubMed

    Lindner, Robert; Sullivan, Catherine; Offor, Onyinye; Lezon-Geyda, Kimberly; Halligan, Kyle; Fischbach, Neal; Shah, Mansi; Bossuyt, Veerle; Schulz, Vincent; Tuck, David P; Harris, Lyndsay N

    2013-01-01

    Triple negative breast cancer (TNBC) is characterized by high proliferation, poor differentiation and a poor prognosis due to high rates of recurrence. Despite lower overall incidence African American (AA) patients suffer from higher breast cancer mortality in part due to the higher proportion of TNBC cases among AA patients compared to European Americans (EA). It was recently shown that the clinical heterogeneity of TNBC is reflected by distinct transcriptional programs with distinct drug response profiles in preclinical models. In this study, gene expression profiling and immunohistochemistry were used to elucidate potential differences between TNBC tumors of EA and AA patients on a molecular level. In a retrospective cohort of 136 TNBC patients, a major transcriptional signature of proliferation was found to be significantly upregulated in samples of AA ethnicity. Furthermore, transcriptional profiles of AA tumors showed differential activation of insulin-like growth factor 1 (IGF1) and a signature of BRCA1 deficiency in this cohort. Using signatures derived from the meta-analysis of TNBC gene expression carried out by Lehmann et al., tumors from AA patients were more likely of basal-like subtypes whereas transcriptional features of many EA samples corresponded to mesenchymal-like or luminal androgen receptor driven subtypes. These results were validated in The Cancer Genome Atlas mRNA and protein expression data, again showing enrichment of a basal-like phenotype in AA tumors and mesenchymal subtypes in EA tumors. In addition, increased expression of VEGF-activated genes together with elevated microvessel area determined by the AQUA method suggest that AA patients exhibit higher tumor vascularization. This study confirms the existence of distinct transcriptional programs in triple negative breast cancer in two separate cohorts and that these programs differ by racial group. Differences in TNBC subtypes and levels of tumor angiogenesis in AA versus EA patients

  19. Atomistic Molecular Simulations Suggest a Kinetic Model for Membrane Translocation by Arginine-Rich Peptides.

    PubMed

    Sun, Delin; Forsman, Jan; Woodward, Clifford E

    2015-11-12

    Arginine-rich cell penetrating peptides (ARCPPs) are known to quickly permeate cell membranes through a non-endocytotic pathway. Potential clinical applications of this facility have prompted enormous effort, both experimental and theoretical, to better understand how ARCPPs manage to overcome the prodigious thermodynamic cost of lipid bilayer permeation by these highly charged peptides. In this work we report the results of all-atom simulations, which suggest that a kinetic (rather than thermodynamic) mechanism may explain how ARCPPs are able to achieve this. Our simulations reveal that octaarginine significantly hinders the closing of membrane pores, either individually or via aggregation in the membrane pore, while octalysine (not an ARCPP) lacks this ability. Our proposed mechanism is an alternative to current attempts to explain pore-mediated translocation of ARCPPs. It asserts that ARCPPs need not lower the equilibrium thermodynamic cost of pore formation. Instead, they can achieve rapid bilayer translocation by instead slowing down the kinetics of naturally occurring thermal pores. Linking the pore lifetime to the characteristic time for peptide diffusion out of the pore, ARCPPs are able to cooperatively permeate the membrane pore. PMID:26485313

  20. Mixed Signals? Morphological and Molecular Evidence Suggest a Color Polymorphism in Some Neotropical Polythore Damselflies

    PubMed Central

    Harding, Kathleen M.; Ankrom, Nikole; Sherratt, Thomas N.; Hoffmann, Joachim; Van Gossum, Hans; Ware, Jessica L.; Cordero-Rivera, Adolfo

    2015-01-01

    The study of color polymorphisms (CP) has provided profound insights into the maintenance of genetic variation in natural populations. We here offer the first evidence for an elaborate wing polymorphism in the Neotropical damselfly genus Polythore, which consists of 21 described species, distributed along the eastern slopes of the Andes in South America. These damselflies display highly complex wing colors and patterning, incorporating black, white, yellow, and orange in multiple wing bands. Wing colors, along with some components of the male genitalia, have been the primary characters used in species description; few other morphological traits vary within the group, and so there are few useful diagnostic characters. Previous research has indicated the possibility of a cryptic species existing in P. procera in Colombia, despite there being no significant differences in wing color and pattern between the populations of the two putative species. Here we analyze the complexity and diversity of wing color patterns of individuals from five described Polythore species in the Central Amazon Basin of Peru using a novel suite of morphological analyses to quantify wing color and pattern: geometric morphometrics, chromaticity analysis, and Gabor wavelet transformation. We then test whether these color patterns are good predictors of species by recovering the phylogenetic relationships among the 5 species using the barcode gene (COI). Our results suggest that, while highly distinct and discrete wing patterns exist in Polythore, these “wingforms” do not represent monophyletic clades in the recovered topology. The wingforms identified as P. victoria and P. ornata are both involved in a polymorphism with P. neopicta; also, cryptic speciation may have taking place among individuals with the P. victoria wingform. Only P. aurora and P. spateri represent monophyletic species with a single wingform in our molecular phylogeny. We discuss the implications of this polymorphism, and

  1. Cytogenetic and molecular evidence suggest multiple origins and geographical parthenogenesis in Nothoscordum gracile (Alliaceae)

    PubMed Central

    Souza, Luiz Gustavo Rodrigues; Crosa, Orfeo; Speranza, Pablo; Guerra, Marcelo

    2012-01-01

    Background and Aims Nothoscordum gracile is an apomitic tetraploid widely distributed throughout the Americas and naturalized in many temperate regions of other continents. It has been suggested to form a species complex with sexual and apomictic N. nudicaule and N. macrostemon. Tetraploids of these species also share a structurally heterozygous chromosome complement 2n = 19 (13M + 6A). In this work, the origin of N. gracile and its relationships with its related species was investigated based on cytological and molecular data. Methods Cytogenetic analyses were based on meiotic behaviour, CMA bands, localization of 5S and 45S rDNA sites, and genomic in situ hybridization (GISH). Nuclear ITS and plastidial trnL-trnF sequences were also obtained for most individuals. Key Results Proximal CMA bands were observed in the long arms of all acrocentrics of 2x and 4x N. macrostemon but not in diploid and some tetraploid cytotypes of N. nudicaule. Samples of N. gracile showed a variable number of CMA bands in the long arms of acrocentrics. Analysis of ITS sequences, dot-blot, GISH, and 5S and 45S rDNA sites, revealed no differentiation among the three species. The trnL-trnF cpDNA fragment showed variation with a trend to geographical structuring irrespective of morphospecies and fully congruent with karyotype variation. Conclusions The 2n = 19 karyotype was probably formed by a centric fusion event occurring in N. nudicaule and later transmitted to tetraploid cytotypes of N. macrostemon. Diploids of N. nudicaule and N. macrostemon appeared as consistent recently diverged species, whereas tetraploid apomicts seem to constitute an assemblage of polyploid hybrids originating from multiple independent hybridization events between them, part of which are morphologically recognizable as N. gracile. PMID:22362660

  2. Mixed signals? Morphological and molecular evidence suggest a color polymorphism in some neotropical polythore damselflies.

    PubMed

    Sánchez Herrera, Melissa; Kuhn, William R; Lorenzo-Carballa, Maria Olalla; Harding, Kathleen M; Ankrom, Nikole; Sherratt, Thomas N; Hoffmann, Joachim; Van Gossum, Hans; Ware, Jessica L; Cordero-Rivera, Adolfo; Beatty, Christopher D

    2015-01-01

    The study of color polymorphisms (CP) has provided profound insights into the maintenance of genetic variation in natural populations. We here offer the first evidence for an elaborate wing polymorphism in the Neotropical damselfly genus Polythore, which consists of 21 described species, distributed along the eastern slopes of the Andes in South America. These damselflies display highly complex wing colors and patterning, incorporating black, white, yellow, and orange in multiple wing bands. Wing colors, along with some components of the male genitalia, have been the primary characters used in species description; few other morphological traits vary within the group, and so there are few useful diagnostic characters. Previous research has indicated the possibility of a cryptic species existing in P. procera in Colombia, despite there being no significant differences in wing color and pattern between the populations of the two putative species. Here we analyze the complexity and diversity of wing color patterns of individuals from five described Polythore species in the Central Amazon Basin of Peru using a novel suite of morphological analyses to quantify wing color and pattern: geometric morphometrics, chromaticity analysis, and Gabor wavelet transformation. We then test whether these color patterns are good predictors of species by recovering the phylogenetic relationships among the 5 species using the barcode gene (COI). Our results suggest that, while highly distinct and discrete wing patterns exist in Polythore, these "wingforms" do not represent monophyletic clades in the recovered topology. The wingforms identified as P. victoria and P. ornata are both involved in a polymorphism with P. neopicta; also, cryptic speciation may have taking place among individuals with the P. victoria wingform. Only P. aurora and P. spateri represent monophyletic species with a single wingform in our molecular phylogeny. We discuss the implications of this polymorphism, and the

  3. Heterogeneous estrogen receptor expression in circulating tumor cells suggests diverse mechanisms of fulvestrant resistance.

    PubMed

    Paoletti, Costanza; Larios, Jose M; Muñiz, Maria C; Aung, Kimberly; Cannell, Emily M; Darga, Elizabeth P; Kidwell, Kelley M; Thomas, Dafydd G; Tokudome, Nahomi; Brown, Martha E; Connelly, Mark C; Chianese, David A; Schott, Anne F; Henry, N Lynn; Rae, James M; Hayes, Daniel F

    2016-08-01

    Fulvestrant is a dose dependent selective estrogen receptor (ER) down-regulator (SERD) used in ER-positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance. Preclinical studies were performed with cultured breast cancer cells spiked into human blood and analyzed on the CellSearch(®) system. Clinical data are limited to a subset of patients with ER-positive MBC from a previously reported pilot trial whose disease was progressing on fulvestrant (N = 7) or aromatase inhibitors (AIs) (N = 10). CTCs were enumerated and phenotyped for ER and B-cell lymphoma (BCL2) using the CellSearch(®) CXC kit. In preclinical modeling, tamoxifen and AIs resulted in stabilized ER expression, whereas fulvestrant eliminated it. Five of seven patients progressing on fulvestrant had ≥5CTC/7.5 ml WB. Two of these five, treated with 500 mg/month fulvestrant, had no detectable CTC-expression of ER and BCL2 (an ER regulated gene). Three patients had heterogeneous CTC-ER and BCL2 expression indicating incomplete degradation of the ER target by fulvestrant. Two of these patients received 250 mg/month whereas the third patient received 500 mg/month fulvestrant. Her cancer harbored a mutation (Y537S) in the estrogen receptor alpha gene (ESR1). All seven ER positive patients progressing on AIs had heterogeneous CTC-ER expression. These results suggest heterogeneous mechanisms of resistance to fulvestrant, including insufficient dosage, ESR1 mutation, or conversion to dependence on non-ER pathways. CTC enumeration, phenotyping, and genotyping might identify patients who would benefit from fulvestrant dose escalation versus switching to alternative therapies. PMID:27178224

  4. Coumarins as Potential Antioxidant Agents Complemented with Suggested Mechanisms and Approved by Molecular Modeling Studies.

    PubMed

    Al-Majedy, Yasameen K; Al-Duhaidahawi, Dunya L; Al-Azawi, Khalida F; Al-Amiery, Ahmed A; Kadhum, Abdul Amir H; Mohamad, Abu Bakar

    2016-01-01

    Syntheses of coumarins, which are a structurally interesting antioxidant activity, was done in this article. The modification of 7-hydroxycoumarin by different reaction steps was done to yield target compounds. Molecular structures were characterized by different spectroscopical techniques (Fourier transformation infrared and nuclear magnetic resonance). Antioxidant activities were performed by using various in vitro spectrophometric assays against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and hydrogen peroxide (H2O2). All compounds exhibited high efficiency as antioxidants compared to ascorbic acid. The highest efficiency scavenging activity was found for compound 3 (91.0 ± 5.0), followed by compounds 2 and 4 (88.0 ± 2.00; and 87.0 ± 3.00). Ascorbic acid C was used as a standard drug with a percentage inhibition of 91.00 ± 1.5. The mechanism of the synthesized compounds as antioxidants was also studied. Hartree-Fock-based quantum chemical studies have been carried out with the basis set to 3-21G, in order to obtain information about the three-dimensional (3D) geometries, electronic structure, molecular modeling, and electronic levels, namely HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital), to understand the antioxidant activity for the synthesized compounds. PMID:26805811

  5. Deceptive Desmas: Molecular Phylogenetics Suggests a New Classification and Uncovers Convergent Evolution of Lithistid Demosponges

    PubMed Central

    Schuster, Astrid; Erpenbeck, Dirk; Pisera, Andrzej; Hooper, John; Bryce, Monika; Fromont, Jane; Wörheide, Gert

    2015-01-01

    Reconciling the fossil record with molecular phylogenies to enhance the understanding of animal evolution is a challenging task, especially for taxa with a mostly poor fossil record, such as sponges (Porifera). ‘Lithistida’, a polyphyletic group of recent and fossil sponges, are an exception as they provide the richest fossil record among demosponges. Lithistids, currently encompassing 13 families, 41 genera and >300 recent species, are defined by the common possession of peculiar siliceous spicules (desmas) that characteristically form rigid articulated skeletons. Their phylogenetic relationships are to a large extent unresolved and there has been no (taxonomically) comprehensive analysis to formally reallocate lithistid taxa to their closest relatives. This study, based on the most comprehensive molecular and morphological investigation of ‘lithistid’ demosponges to date, corroborates some previous weakly-supported hypotheses, and provides novel insights into the evolutionary relationships of the previous ‘order Lithistida’. Based on molecular data (partial mtDNA CO1 and 28S rDNA sequences), we show that 8 out of 13 ‘Lithistida’ families belong to the order Astrophorida, whereas Scleritodermidae and Siphonidiidae form a separate monophyletic clade within Tetractinellida. Most lithistid astrophorids are dispersed between different clades of the Astrophorida and we propose to formally reallocate them, respectively. Corallistidae, Theonellidae and Phymatellidae are monophyletic, whereas the families Pleromidae and Scleritodermidae are polyphyletic. Family Desmanthidae is polyphyletic and groups within Halichondriidae – we formally propose a reallocation. The sister group relationship of the family Vetulinidae to Spongillida is confirmed and we propose here for the first time to include Vetulina into a new Order Sphaerocladina. Megascleres and microscleres possibly evolved and/or were lost several times independently in different

  6. Molecular phylogeny of beetle associated diplogastrid nematodes suggests host switching rather than nematode-beetle coevolution

    PubMed Central

    2009-01-01

    Background Nematodes are putatively the most species-rich animal phylum. They have various life styles and occur in a variety of habitats, ranging from free-living nematodes in aquatic or terrestrial environments to parasites of animals and plants. The rhabditid nematode Caenorhabditis elegans is one of the most important model organisms in modern biology. Pristionchus pacificus of the family of the Diplogastridae has been developed as a satellite model for comparison to C. elegans. The Diplogastridae, a monophyletic clade within the rhabditid nematodes, are frequently associated with beetles. How this beetle-association evolved and whether beetle-nematode coevolution occurred is still elusive. As a prerequisite to answering this question a robust phylogeny of beetle-associated Diplogastridae is needed. Results Sequences for the nuclear small subunit ribosomal RNA and for 12 ribosomal protein encoding nucleotide sequences were collected for 14 diplogastrid taxa yielding a dataset of 5996 bp of concatenated aligned sequences. A molecular phylogeny of beetle-associated diplogastrid nematodes was established by various algorithms. Robust subclades could be demonstrated embedded in a phylogenetic tree topology with short internal branches, indicating rapid ancestral divergences. Comparison of the diplogastrid phylogeny to a comprehensive beetle phylogeny revealed no major congruence and thus no evidence for a long-term coevolution. Conclusion Reconstruction of the phylogenetic history of beetle-associated Diplogastridae yields four distinct subclades, whose deep phylogenetic divergence, as indicated by short internal branch lengths, shows evidence for evolution by successions of ancient rapid radiation events. The stem species of the Diplogastridae existed at the same time period when the major radiations of the beetles occurred. Comparison of nematode and beetle phylogenies provides, however, no evidence for long-term coevolution of diplogastrid nematodes and their

  7. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

    PubMed Central

    Liberato, Marcelo V.; Silveira, Rodrigo L.; Prates, Érica T.; de Araujo, Evandro A.; Pellegrini, Vanessa O. A.; Camilo, Cesar M.; Kadowaki, Marco A.; Neto, Mario de O.; Popov, Alexander; Skaf, Munir S.; Polikarpov, Igor

    2016-01-01

    Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme. PMID:27032335

  8. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

    NASA Astrophysics Data System (ADS)

    Liberato, Marcelo V.; Silveira, Rodrigo L.; Prates, Érica T.; de Araujo, Evandro A.; Pellegrini, Vanessa O. A.; Camilo, Cesar M.; Kadowaki, Marco A.; Neto, Mario De O.; Popov, Alexander; Skaf, Munir S.; Polikarpov, Igor

    2016-04-01

    Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

  9. Molecular marker suggests rapid changes of sex-determining mechanisms in Australian dragon lizards.

    PubMed

    Ezaz, Tariq; Quinn, Alexander E; Sarre, Stephen D; O'Meally, Denis; Georges, Arthur; Graves, Jennifer A Marshall

    2009-01-01

    Distribution of sex-determining mechanisms across Australian agamids shows no clear phylogenetic segregation, suggesting multiple transitions between temperature-dependent (TSD) and genotypic sex determination (GSD). These taxa thus present an excellent opportunity for studying the evolution of sex chromosomes, and evolutionary transitions between TSD and GSD. Here we report the hybridization of a 3 kb genomic sequence (PvZW3) that marks the Z and W microchromosomes of the Australian central bearded dragon (Pogona vitticeps) to chromosomes of 12 species of Australian agamids from eight genera using fluorescence in-situ hybridization (FISH). The probe hybridized to a single microchromosome pair in 11 of these species, but to the tip of the long arm of chromosome pair 2 in the twelfth (Physignathus lesueurii), indicating a micro-macro chromosome rearrangement. Three TSD species shared the marked microchromosome, implying that it is a conserved autosome in related species that determine sex by temperature. C-banding identified the marked microchromosome as the heterochromatic W chromosome in two of the three GSD species. However, in Ctenophorus fordi, the probe hybridized to a different microchromosome from that shown by C-banding to be the heterochromatic W, suggesting an independent origin for the ZW chromosome pair in that species. Given the haphazard distribution of GSD and TSD in this group and the existence of at least two sets of sex microchromosomes in GSD species, we conclude that sex-determining mechanisms in this family have evolved independently, multiple times in a short evolutionary period. PMID:19172405

  10. Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation.

    PubMed

    Petrou, Vasileios I; Herrera, Carmen M; Schultz, Kathryn M; Clarke, Oliver B; Vendome, Jérémie; Tomasek, David; Banerjee, Surajit; Rajashankar, Kanagalaghatta R; Belcher Dufrisne, Meagan; Kloss, Brian; Kloppmann, Edda; Rost, Burkhard; Klug, Candice S; Trent, M Stephen; Shapiro, Lawrence; Mancia, Filippo

    2016-02-01

    Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes. PMID:26912703

  11. Molecular evolution of bovine Toll-like receptor 2 suggests substitutions of functional relevance

    PubMed Central

    2008-01-01

    Background There is accumulating evidence that polymorphism in Toll-like receptor (TLR) genes might be associated with disease resistance or susceptibility traits in livestock. Polymorphic sites affecting TLR function should exhibit signatures of positive selection, identified as a high ratio of non-synonymous to synonymous nucleotide substitutions (ω). Phylogeny based models of codon substitution based on estimates of ω for each amino acid position can therefore offer a valuable tool to predict sites of functional relevance. We have used this approach to identify such polymorphic sites within the bovine TLR2 genes from ten Bos indicus and Bos taurus cattle breeds. By analysing TLR2 gene phylogeny in a set of mammalian species and a subset of ruminant species we have estimated the selective pressure on individual sites and domains and identified polymorphisms at sites of putative functional importance. Results The ω were highest in the mammalian TLR2 domains thought to be responsible for ligand binding and lowest in regions responsible for heterodimerisation with other TLR-related molecules. Several positively-selected sites were detected in or around ligand-binding domains. However a comparison of the ruminant subset of TLR2 sequences with the whole mammalian set of sequences revealed that there has been less selective pressure among ruminants than in mammals as a whole. This suggests that there have been functional changes during ruminant evolution. Twenty newly-discovered non-synonymous polymorphic sites were identified in cattle. Three of them were localised at positions shaped by positive selection in the ruminant dataset (Leu227Phe, His305Pro, His326Gln) and in domains involved in the recognition of ligands. His326Gln is of particular interest as it consists of an exchange of differentially-charged amino acids at a position which has previously been shown to be crucial for ligand binding in human TLR2. Conclusion Within bovine TLR2, polymorphisms at amino

  12. Gene expression profiles from discordant monozygotic twins suggest that molecular pathways are shared among multiple systemic autoimmune diseases

    PubMed Central

    2011-01-01

    Introduction The objective of this study is to determine if multiple systemic autoimmune diseases (SAID) share gene expression pathways that could provide insights into pathogenic mechanisms common to these disorders. Methods RNA microarray analyses (Agilent Human 1A(V2) 20K oligo arrays) were used to quantify gene expression in peripheral blood cells from 20 monozygotic (MZ) twin pairs discordant for SAID. Six affected probands with systemic lupus erythematosus (SLE), six with rheumatoid arthritis (RA), eight with idiopathic inflammatory myopathies (IIM), and their same-gendered unaffected twins, were enrolled. Comparisons were made between discordant twin pairs and these were also each compared to 40 unrelated control subjects (matched 2:1 to each twin by age, gender and ethnicity) using statistical and molecular pathway analyses. Relative quantitative PCR was used to verify independently measures of differential gene expression assessed by microarray analysis. Results Probands and unrelated, matched controls differed significantly in gene expression for 104 probes corresponding to 92 identifiable genes (multiple-comparison adjusted P values < 0.1). Differentially expressed genes involved several overlapping pathways including immune responses (16%), signaling pathways (24%), transcription/translation regulators (26%), and metabolic functions (15%). Interferon (IFN)-response genes (IFI27, OASF, PLSCR1, EIF2AK2, TNFAIP6, and TNFSF10) were up-regulated in probands compared to unrelated controls. Many of the abnormally expressed genes played regulatory roles in multiple cellular pathways. We did not detect any probes expressed differentially in comparisons among the three SAID phenotypes. Similarly, we found no significant differences in gene expression when comparing probands to unaffected twins or unaffected twins to unrelated controls. Gene expression levels for unaffected twins appeared intermediate between that of probands and unrelated controls for 6535 probes

  13. A case of an alpha-fetoprotein-producing intrahepatic cholangiocarcinoma suggests probable cancer stem cell origin.

    PubMed

    Ishikawa, Kenji; Sasaki, Atsushi; Haraguchi, Naotsugu; Yoshikawa, Yasuji; Mori, Masaki

    2007-03-01

    Recent evidence suggests that some cancers may originate from cancer stem cells, which may derive from carcinogenesis of normal stem cells. A hepatic progenitor cell population, which gives rise to hepatocytes and cholangiocytes, has been suggested in humans, though whether these cells can give rise to malignant tumors has not been confirmed. We report here a case of an alpha-fetoprotein (AFP)-producing intrahepatic cholangiocarcinoma (ICC) in an 81-year-old woman with chronic hepatitis C viral infection, suggesting malignant transformation of hepatic stem cells as a mechanism for hepatic neoplasia. Abdominal computed tomography revealed a low-density mass with surrounding enhancement measuring 5 cm x 5 cm in segments IV and VIII of the liver. The preoperative serum levels of tumor markers were 1.7 ng/ml of carcinoembryonic antigen, 22 mAU/ml of protein induced by vitamin K absence or antagonist II, 43.4 U/ml of carbohydrate antigen 19-9, and 1,560 ng/ml of AFP. Following central bisegmentectomy of the liver, serum AFP levels decreased dramatically. Histologically, the tumor cells showed indistinct glandular structures with abundant fibrous stroma. Immunohistochemical analysis demonstrated that the neoplastic cells reacted strongly to antibodies against AFP and cytokeratin (CK) 7. In addition, cancer cells showed partially positive reaction to anti-CK14, a liver stem cell marker, and to anticluster designation (CD) 133, a hematopoietic stem cell marker, and negative reaction to antihepatocyte paraffin (HepPar) 1. These data may indicate that the tumor was derived from a normal liver stem cell that underwent oncogenic transformation. PMID:17405896

  14. PDGF beta targeting in cervical cancer cells suggest a fine-tuning of compensatory signalling pathways to sustain tumourigenic stimulation

    PubMed Central

    Tudoran, Oana Mihaela; Soritau, Olga; Balacescu, Loredana; Pop, Laura; Meurice, Guillaume; Visan, Simona; Lindberg, Staffan; Eniu, Alexandru; Langel, Ulo; Balacescu, Ovidiu; Berindan-Neagoe, Ioana

    2015-01-01

    The platelet-derived growth factor (PDGF) signalling pathway has been reported to play an important role in human cancers by modulating autocrine and paracrine processes such as tumour growth, metastasis and angiogenesis. Several clinical trials document the benefits of targeting this pathway; however, in cervical cancer the role of PDGF signalling in still unclear. In this study, we used siRNA against PDGF beta (PDGFBB) to investigate the cellular and molecular mechanisms of PDGFBB signalling in Ca Ski and HeLa cervical cancer cells. Our results show that PDGFBB inhibition in Ca Ski cells led to rapid alterations of the transcriptional pattern of 579 genes, genes that are known to have antagonistic roles in regulating tumour progression. Concomitantly, with the lack of significant effects on cervical cancer cells proliferation, apoptosis, migration or invasion, these findings suggests that cervical cancer cells shift between compensatory signalling pathways to maintain their behaviour. The observed autocrine effects were limited to cervical cancer cells ability to adhere to an endothelial cell (EC) monolayer. However, by inhibiting PDGFBB on cervical cells, we achieved reduced proliferation of ECs in co-culture settings and cellular aggregation in conditioned media. Because of lack of PDGF receptor expression on ECs, we believe that these effects are a result of indirect PDGFBB paracrine signalling mechanisms. Our results shed some light into the understanding of PDGFBB signalling mechanism in cervical cancer cells, which could be further exploited for the development of synergistic anti-tumour and anti-angiogenic therapeutic strategies. PMID:25311137

  15. Molecular Hallmarks of Adult T Cell Leukemia

    PubMed Central

    Yamagishi, Makoto; Watanabe, Toshiki

    2012-01-01

    The molecular hallmarks of adult T cell leukemia (ATL) comprise outstanding deregulations of signaling pathways that control the cell cycle, resistance to apoptosis, and proliferation of leukemic cells, all of which have been identified by early excellent studies. Nevertheless, we are now confronted the therapeutic difficulties of ATL that is a most aggressive T cell leukemia/lymphoma. Using next-generation strategies, emerging molecular characteristics such as specific surface markers and an additional catalog of signals affecting the fate of leukemic cells have been added to the molecular hallmarks that constitute an organizing principle for rationalizing the complexities of ATL. Although human T cell leukemia virus type 1 is undoubtedly involved in ATL leukemogenesis, most leukemic cells do not express the viral protein Tax. Instead, cellular gene expression changes dominate homeostasis disorders of infected cells and characteristics of ATL. In this review, we summarize the state of the art of ATL molecular pathology, which supports the biological properties of leukemic cells. In addition, we discuss the recent discovery of two molecular hallmarks of potential generality; an abnormal microRNA pattern and epigenetic reprogramming, which strongly involve the imbalance of the molecular network of lymphocytes. Global analyses of ATL have revealed the functional impact of crosstalk between multifunctional pathways. Clinical and biological studies on signaling inhibitory agents have also revealed novel oncogenic drivers that can be targeted in future. ATL cells, by deregulation of such pathways and their interconnections, may become masters of their own destinies. Recognizing and understanding of the widespread molecular applicability of these concepts will increasingly affect the development of novel strategies for treating ATL. PMID:23060864

  16. Detection of Molecular Charges at Cell Membrane

    NASA Astrophysics Data System (ADS)

    Sakata, Toshiya; Miyahara, Yuji

    2008-01-01

    Molecular charges at the cell membrane have been successfully detected using cell-based field-effect devices. Mouse fibroblast cells were adhered to the Si3N4 gate surface of the field-effect devices. The negative charges of sialic acid at the surface of the cell membrane could be detected as a shift of the flatband voltage of the field-effect devices. Quantitative analysis of molecular charges at the cell membrane could be demonstrated in relation to the number of adhered cells on the Si3N4 gate surface. The platform based on the field-effect devices is suitable for a simple, accurate and non-invasive system for cell functional analysis.

  17. Molecular multiproxy analysis of ancient root systems suggests strong alteration of deep subsoil organic matter by rhizomicrobial activity

    NASA Astrophysics Data System (ADS)

    Gocke, Martina; Huguet, Arnaud; Derenne, Sylvie; Kolb, Steffen; Wiesenberg, Guido L. B.

    2013-04-01

    Roots have a high potential capacity to store large amounts of CO2 in the subsoil. However, associated with rooting, microorganisms enter the subsoil and might contribute to priming effects of carbon mineralisation in the microbial hotspot rhizosphere. Although these processes are well known for recent surface soils, it remains questionable, if and how microorganisms contribute to priming effects in the subsoil and if these effects can be traced after the roots' lifetime. The current study implies several state-of-the-art techniques like DNA and lipid molecular proxies to trace remains of microbial biomass in ancient root systems. These can provide valuable information if parts of the root and rhizomicrobial biomass are preserved, e.g. by encrustation with secondary carbonate during the root's lifespan or shortly thereafter. At the Late Pleistocene loess-paleosol sequence near Nussloch (SW Germany), rhizoliths (calcified roots) occur highly abundant in the deep subsoil from 1 to 9 m depth and below. They were formed by Holocene woody vegetation. Their size can account for up to several cm in diameter and up to > 1 m length. Rhizoliths and surrounding sediment with increasing distances of up to 10 cm, as well as reference loess without visible root remains were collected at several depth intervals. Samples were analysed for n-fatty acids (FAs) and glycerol dialkyl glycerol tetraethers (GDGTs; membrane lipids from Archaea and some Bacteria), as well as structural diversity based on the RNA gene of the prokaryotic ribosome subunit 16S (16S rRNA). GDGT represent organic remains from microbial biomass, whereas FA comprise both microbial remains and degradation products. 16S rRNA indicates the presence of both living cells and/or cell fragments. Despite the general low RNA contents in the sample set, results pointed to a much higher abundance of bacterial compared to archaeal RNA. The latter occured in notable amounts only in some rhizoliths. This was in part enforced by

  18. The Molecular Control of Blood Cell Development

    NASA Astrophysics Data System (ADS)

    Sachs, Leo

    1987-12-01

    The establishment of a cell culture system for the clonal development of blood cells has made it possible to identify the proteins that regulate the growth and differentiation of different blood cell lineages and to discover the molecular basis of normal and abnormal cell development in blood forming tissues. A model system with myeloid blood cells has shown that (i) normal blood cells require different proteins to induce cell multiplication (growth inducers) and cell differentiation (differentiation inducers), (ii) there is a hierarchy of growth inducers as cells become more restricted in their developmental program, and (iii) a cascade of interactions between proteins determines the correct balance between immature and mature cells in normal blood cell development. Gene cloning has shown that there is a family of different genes for these proteins. Normal protein regulators of blood cell development can control the abnormal growth of certain types of leukemic cells and suppress malignancy by incuding differentiation to mature nondividing cells. Chromosome abnormalities that give rise to malignancy in these leukemic cells can be bypassed and their effects nullified by inducing differentiation, which stops cells from multiplying. These blood cell regulatory proteins are active in culture and in the body, and they can be used clinically to correct defects in blood cell development.

  19. Molecular Mechanisms of HTLV-1 Cell-to-Cell Transmission

    PubMed Central

    Gross, Christine; Thoma-Kress, Andrea K.

    2016-01-01

    The tumorvirus human T-cell lymphotropic virus type 1 (HTLV-1), a member of the delta-retrovirus family, is transmitted via cell-containing body fluids such as blood products, semen, and breast milk. In vivo, HTLV-1 preferentially infects CD4+ T-cells, and to a lesser extent, CD8+ T-cells, dendritic cells, and monocytes. Efficient infection of CD4+ T-cells requires cell-cell contacts while cell-free virus transmission is inefficient. Two types of cell-cell contacts have been described to be critical for HTLV-1 transmission, tight junctions and cellular conduits. Further, two non-exclusive mechanisms of virus transmission at cell-cell contacts have been proposed: (1) polarized budding of HTLV-1 into synaptic clefts; and (2) cell surface transfer of viral biofilms at virological synapses. In contrast to CD4+ T-cells, dendritic cells can be infected cell-free and, to a greater extent, via viral biofilms in vitro. Cell-to-cell transmission of HTLV-1 requires a coordinated action of steps in the virus infectious cycle with events in the cell-cell adhesion process; therefore, virus propagation from cell-to-cell depends on specific interactions between cellular and viral proteins. Here, we review the molecular mechanisms of HTLV-1 transmission with a focus on the HTLV-1-encoded proteins Tax and p8, their impact on host cell factors mediating cell-cell contacts, cytoskeletal remodeling, and thus, virus propagation. PMID:27005656

  20. Zika Virus Targets Different Primary Human Placental Cells, Suggesting Two Routes for Vertical Transmission.

    PubMed

    Tabata, Takako; Petitt, Matthew; Puerta-Guardo, Henry; Michlmayr, Daniela; Wang, Chunling; Fang-Hoover, June; Harris, Eva; Pereira, Lenore

    2016-08-10

    Zika virus (ZIKV) infection during pregnancy is linked to severe birth defects, but mother-to-fetus transmission routes are unknown. We infected different primary cell types from mid- and late-gestation placentas and explants from first-trimester chorionic villi with the prototype Ugandan and a recently isolated Nicaraguan ZIKV strain. ZIKV infects primary human placental cells and explants-cytotrophoblasts, endothelial cells, fibroblasts, and Hofbauer cells in chorionic villi and amniotic epithelial cells and trophoblast progenitors in amniochorionic membranes-that express Axl, Tyro3, and/or TIM1 viral entry cofactors. ZIKV produced NS3 and E proteins and generated higher viral titers in amniotic epithelial cells from mid-gestation compared to late-gestation placentas. Duramycin, a peptide that binds phosphatidylethanolamine in enveloped virions and precludes TIM1 binding, reduced ZIKV infection in placental cells and explants. Our results suggest that ZIKV spreads from basal and parietal decidua to chorionic villi and amniochorionic membranes and that targeting TIM1 could suppress infection at the uterine-placental interface. PMID:27443522

  1. Dictyostelium discoideum: Molecular approaches to cell biology

    SciTech Connect

    Spudich, J.A.

    1987-01-01

    The central point of this book is to present Dictyostelium as a valuable eukaryotic organism for those interested in molecular studies that require a combined biochemical, structural, and genetic approach. The book is not meant to be a comprehensive compilation of all methods involving Dictyostelium, but instead is a selective set of chapters that demonstrates the utility of the organism for molecular approaches to interesting cell biological problems.

  2. Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis*

    PubMed Central

    Alvarez-Navarro, Carlos; Cragnolini, Juan J.; Dos Santos, Helena G.; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A.

    2013-01-01

    Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na+-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27+ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA. PMID:23867464

  3. Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes

    PubMed Central

    Herce, Henry D.; Garcia, Angel E.

    2007-01-01

    The recombinant HIV-1 Tat protein contains a small region corresponding to residues 47YGRKKRRQRR57R, which is capable of translocating cargoes of different molecular sizes, such as proteins, DNA, RNA, or drugs, across the cell membrane in an apparently energy-independent manner. The pathway that these peptides follow for entry into the cell has been the subject of strong controversy for the last decade. This peptide is highly basic and hydrophilic. Therefore, a central question that any candidate mechanism has to answer is how this highly hydrophilic peptide is able to cross the hydrophobic barrier imposed by the cell membrane. We propose a mechanism for the spontaneous translocation of the Tat peptides across a lipid membrane. This mechanism involves strong interactions between the Tat peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of the Tat peptides by diffusing on the pore surface. This mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifically the arginine amino acids, contribute to the uptake. The proposed mechanism also illustrates the importance of membrane fluctuations. Indeed, mechanisms that involve large fluctuations of the membrane structure, such as transient pores and the insertion of charged amino acid side chains, may be common and perhaps central to the functions of many membrane protein functions. PMID:18093956

  4. Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes.

    PubMed

    Herce, Henry D; Garcia, Angel E

    2007-12-26

    The recombinant HIV-1 Tat protein contains a small region corresponding to residues (47)YGRKKRRQRR(57)R, which is capable of translocating cargoes of different molecular sizes, such as proteins, DNA, RNA, or drugs, across the cell membrane in an apparently energy-independent manner. The pathway that these peptides follow for entry into the cell has been the subject of strong controversy for the last decade. This peptide is highly basic and hydrophilic. Therefore, a central question that any candidate mechanism has to answer is how this highly hydrophilic peptide is able to cross the hydrophobic barrier imposed by the cell membrane. We propose a mechanism for the spontaneous translocation of the Tat peptides across a lipid membrane. This mechanism involves strong interactions between the Tat peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of the Tat peptides by diffusing on the pore surface. This mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifically the arginine amino acids, contribute to the uptake. The proposed mechanism also illustrates the importance of membrane fluctuations. Indeed, mechanisms that involve large fluctuations of the membrane structure, such as transient pores and the insertion of charged amino acid side chains, may be common and perhaps central to the functions of many membrane protein functions. PMID:18093956

  5. Molecular profiling of single circulating tumor cells with diagnostic intention

    PubMed Central

    Polzer, Bernhard; Medoro, Gianni; Pasch, Sophie; Fontana, Francesca; Zorzino, Laura; Pestka, Aurelia; Andergassen, Ulrich; Meier-Stiegen, Franziska; Czyz, Zbigniew T; Alberter, Barbara; Treitschke, Steffi; Schamberger, Thomas; Sergio, Maximilian; Bregola, Giulia; Doffini, Anna; Gianni, Stefano; Calanca, Alex; Signorini, Giulio; Bolognesi, Chiara; Hartmann, Arndt; Fasching, Peter A; Sandri, Maria T; Rack, Brigitte; Fehm, Tanja; Giorgini, Giuseppe; Manaresi, Nicolò; Klein, Christoph A

    2014-01-01

    Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance. PMID:25358515

  6. Molecular mechanisms of angioimmunoblastic T-cell lymphoma development.

    PubMed

    Sakata-Yanagimoto, Mamiko; Chiba, Shigeru

    2016-08-01

    The molecular pathogenesis of peripheral T-cell lymphoma (PTCL) has gradually been clarified in terms of genomic abnormalities. Insights into these genomic abnormalities have provided clues to understanding the pathogenesis of PTCL. Furthermore, the origins of lymphoma cells have been clarified by investigating the distribution of genomic abnormalities in tumor cells and non-tumor blood cells. Multistep tumorigenesis has been suggested to be a fundamental mechanism underlying the development of angioimmunoblastic T-cell lymphoma (AITL), a distinct subtype of PTCL: premalignant cells evolve from hematopoietic progenitors via mutations in epigenetic regulators. These cells then further differentiate into tumor cells via the addition of tumor-specific G17V RHOA mutations. Meanwhile, AITL are composed of various infiltrating cells as well as tumor cells. Most notably, AITL tissues are characterized by massive infiltration of B cells partially infected by Epstein-Barr virus, follicular dendritic cells, and high endothelial venules. Infiltration of these cell types has been thought to be a reactive process, promoted by cytokines and chemokines released from tumor cells. Considering the multistep mechanisms of AITL allows us to analyze whether these infiltrating cells are also derived from premalignant cells. Indeed, the mechanisms underlying massive infiltration of bystander cells might be more complicated than previously imagined. PMID:27599421

  7. Molecular phylogenetic analysis of nuclear genes suggests a Cenozoic over-water dispersal origin for the Cuban solenodon.

    PubMed

    Sato, Jun J; Ohdachi, Satoshi D; Echenique-Diaz, Lazaro M; Borroto-Páez, Rafael; Begué-Quiala, Gerardo; Delgado-Labañino, Jorge L; Gámez-Díez, Jorgelino; Alvarez-Lemus, José; Nguyen, Son Truong; Yamaguchi, Nobuyuki; Kita, Masaki

    2016-01-01

    The Cuban solenodon (Solenodon cubanus) is one of the most enigmatic mammals and is an extremely rare species with a distribution limited to a small part of the island of Cuba. Despite its rarity, in 2012 seven individuals of S. cubanus were captured and sampled successfully for DNA analysis, providing new insights into the evolutionary origin of this species and into the origins of the Caribbean fauna, which remain controversial. We conducted molecular phylogenetic analyses of five nuclear genes (Apob, Atp7a, Bdnf, Brca1 and Rag1; total, 4,602 bp) from 35 species of the mammalian order Eulipotyphla. Based on Bayesian relaxed molecular clock analyses, the family Solenodontidae diverged from other eulipotyphlan in the Paleocene, after the bolide impact on the Yucatan Peninsula, and S. cubanus diverged from the Hispaniolan solenodon (S. paradoxus) in the Early Pliocene. The strikingly recent divergence time estimates suggest that S. cubanus and its ancestral lineage originated via over-water dispersal rather than vicariance events, as had previously been hypothesised. PMID:27498968

  8. Molecular phylogenetic analysis of nuclear genes suggests a Cenozoic over-water dispersal origin for the Cuban solenodon

    PubMed Central

    Sato, Jun J.; Ohdachi, Satoshi D.; Echenique-Diaz, Lazaro M.; Borroto-Páez, Rafael; Begué-Quiala, Gerardo; Delgado-Labañino, Jorge L.; Gámez-Díez, Jorgelino; Alvarez-Lemus, José; Nguyen, Son Truong; Yamaguchi, Nobuyuki; Kita, Masaki

    2016-01-01

    The Cuban solenodon (Solenodon cubanus) is one of the most enigmatic mammals and is an extremely rare species with a distribution limited to a small part of the island of Cuba. Despite its rarity, in 2012 seven individuals of S. cubanus were captured and sampled successfully for DNA analysis, providing new insights into the evolutionary origin of this species and into the origins of the Caribbean fauna, which remain controversial. We conducted molecular phylogenetic analyses of five nuclear genes (Apob, Atp7a, Bdnf, Brca1 and Rag1; total, 4,602 bp) from 35 species of the mammalian order Eulipotyphla. Based on Bayesian relaxed molecular clock analyses, the family Solenodontidae diverged from other eulipotyphlan in the Paleocene, after the bolide impact on the Yucatan Peninsula, and S. cubanus diverged from the Hispaniolan solenodon (S. paradoxus) in the Early Pliocene. The strikingly recent divergence time estimates suggest that S. cubanus and its ancestral lineage originated via over-water dispersal rather than vicariance events, as had previously been hypothesised. PMID:27498968

  9. Differential DNA methylome profiling of nonfunctioning pituitary adenomas suggesting tumour invasion is correlated with cell adhesion.

    PubMed

    Gu, Ye; Zhou, Xinyao; Hu, Fan; Yu, Yong; Xie, Tao; Huang, Yuying; Zhao, Xinzhi; Zhang, Xiaobiao

    2016-08-01

    Global and gene-specific changes to the epigenome are hallmarks of most tumours including those of pituitary origin, and this fact might offer important clues about diagnostic and therapeutic applications. We performed global DNA methylation screening with 6 invasive and 6 noninvasive nonfunctioning pituitary adenomas (PA) to investigate whether DNA methylation was associated with the invasion of nonfunctioning pituitary adenomas. An additional seven PAs were included as an independent cohort to validate the initial results. Five thousand nine hundred thirty-one CpGs were selected (△β ≥0.15 and p value ≤0.01) as differentially methylated sites (DMSs). The hypomethylated DMSs in the invasive PAs were significantly more than the hypermethylated sites. Cluster analysis of 339 CpGs (△β ≥0.25 and p value ≤0.001) demonstrated a complete distinction between the invasive and noninvasive nonfunctioning groups. GO analysis of the three hundred seven corresponding genes shown they were involved in homophilic cell adhesion, cell-cell adhesion, cell adhesion and biological adhesion. The mRNA expression of GALNT9 which contain a validated DMS was significantly downregulated in invasive group. Our findings indicate that the differential DNA methylome profiling of invasive and noninvasive nonfunctioning PAs suggesting tumour invasion is correlated with cell adhesion. PMID:27168190

  10. Molecular biology of retinal ganglion cells.

    PubMed Central

    Xiang, M; Zhou, H; Nathans, J

    1996-01-01

    Retinal ganglion cells are the output neurons that encode and transmit information from the eye to the brain. Their diverse physiologic and anatomic properties have been intensively studied and appear to account well for a number of psychophysical phenomena such as lateral inhibition and chromatic opponency. In this paper, we summarize our current view of retinal ganglion cell properties and pose a number of questions regarding underlying molecular mechanisms. As an example of one approach to understanding molecular mechanisms, we describe recent work on several POU domain transcription factors that are expressed in subsets of retinal ganglion cells and that appear to be involved in ganglion cell development. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8570601

  11. Genomewide scans of red cell indices suggest linkage on chromosome 6q23

    PubMed Central

    Iliadou, A; Evans, D M; Zhu, G; Duffy, D L; Frazer, I H; Montgomery, G W; Martin, N G

    2007-01-01

    Background The red cell indices quantify the size, number and oxygen‐carrying ability of erythrocytes. Although the genetic basis of many monogenic forms of anaemia is well understood, comparatively little is known about the genes responsible for variation in the red cell indices among healthy participants. Objective To identify quantitative trait loci (QTLs) responsible for normal variation in the red cell indices of 391 pairs of dizygotic twins who were measured longitudinally at 12, 14 and 16 years of age. Results Evidence suggesting linkage of red cell indices to haemoglobin concentration (LOD  = 3.03) and haematocrit (LOD  = 2.95) on chromosome 6q23, a region previously identified as possibly harbouring a QTL for haematocrit, was found. Evidence for linkage to several other regions of the genome, including chromosome 4q32 for red cell count and 7q for mean cell volume, was also found. In contrast, there was little evidence of linkage to the chromosomal regions containing the genes for erythropoietin (7q21) and its receptor (19p13.2), nor to the regions containing the genes for the haemoglobin α (16p13.3) and β chains (11p15.5). Conclusion Findings provide additional evidence for a QTL affecting haemoglobin and haematocrit on chromosome 6q23. In contrast, polymorphisms in the genes coding for erythropoietin, its receptor and the haemoglobin α and β chains do not appear to contribute substantially to variation in the red cell indices between healthy persons. PMID:16950815

  12. Expression profiling and biochemical analysis suggest stress response as a potential mechanism inhibiting proliferation of polyamine-depleted cells.

    PubMed

    Landau, Guy; Ran, Avichai; Bercovich, Zippi; Feldmesser, Ester; Horn-Saban, Shirley; Korkotian, Eduard; Jacob-Hirsh, Jasmine; Rechavi, Gideon; Ron, David; Kahana, Chaim

    2012-10-19

    Polyamines are small organic polycations that are absolutely required for cell growth and proliferation; yet the basis for this requirement is mostly unknown. Here, we combined a genome-wide expression profiling with biochemical analysis to reveal the molecular basis for inhibited proliferation of polyamine-depleted cells. Transcriptional responses accompanying growth arrest establishment in polyamine-depleted cells or growth resumption following polyamine replenishment were monitored and compared. Changes in the expression of genes related to various fundamental cellular processes were established. Analysis of mirror-symmetric expression patterns around the G(1)-arrest point identified a set of genes representing a stress-response signature. Indeed, complementary biochemical analysis demonstrated activation of the PKR-like endoplasmic reticulum kinase arm of the unfolded protein response and of the stress-induced p38 MAPK. These changes were accompanied by induction of key growth-inhibitory factors such as p21 and Gadd45a and reduced expression of various cyclins, most profoundly cyclin D1, setting the basis for the halted proliferation. However, although the induced stress response could arrest growth, polyamine depletion also inhibited proliferation of PKR-like endoplasmic reticulum kinase and p38α-deficient cells and of cells harboring a nonphosphorylatable mutant eIF2α (S51A), suggesting that additional yet unidentified mechanisms might inhibit proliferation of polyamine-depleted cells. Despite lengthy persistence of the stress and activation of apoptotic signaling, polyamine-depleted cells remained viable, apparently due to induced expression of protective genes and development of autophagy. PMID:22942278

  13. Mutational spectrum of Barrett's stem cells suggests paths to initiation of a precancerous lesion

    PubMed Central

    Yamamoto, Yusuke; Wang, Xia; Bertrand, Denis; Kern, Florian; Zhang, Ting; Duleba, Marcin; Srivastava, Supriya; Khor, Chiea Chuen; Hu, Yuanyu; Wilson, Lane H.; Blaszyk, Hagen; Rolshud, Daniil; Teh, Ming; Liu, Jianjun; Howitt, Brooke E.; Vincent, Matthew; Crum, Christopher P.; Nagarajan, Niranjan; Ho, Khek Yu; McKeon, Frank; Xian, Wa

    2016-01-01

    The precancerous lesion known as Barrett's oesophagus can evolve to oesophageal adenocarcinoma in decades-long processes of regenerative growth. Here we report the isolation and propagation of distinct, patient-matched stem cells of Barrett's, gastric and oesophageal epithelia that yield divergent tumour types following in vitro transformation and xenografting. Genomic analyses reveal a broad mutational spectrum unique to Barrett's stem cells that likely reflects their risk for oncogenesis. Remarkably, 25% of cases show no cancer-related genomic changes, suggesting that Barrett's initiates without driver mutations. Most cases, however, sustain patterns of deletions almost identical to adenocarcinoma though tumour-associated gene amplifications were absent. Notably, those suspected of low-grade dysplasia have p53 mutations or undergo amplifications of proto-oncogenes and receptor tyrosine kinases, implicating these events in lethal transitions. Our findings suggest paths for the initiation and progression of Barrett's and define a discrete stem cell underlying its regenerative growth whose eradication could prevent oesophageal adenocarcinoma. PMID:26783136

  14. How a Small Change in Retinal Leads to G-Protein Activation: Initial Events Suggested by Molecular Dynamics Calculations

    PubMed Central

    Crozier, Paul S.; Stevens, Mark J.; Woolf, Thomas B.

    2010-01-01

    Rhodopsin is the prototypical G-protein coupled receptor, coupling light activation with high efficiency to signaling molecules. The dark-state X-ray structures of the protein provide a starting point for consideration of the relaxation from initial light activation to conformational changes that may lead to signaling. In this study we create an energetically unstable retinal in the light activated state and then use molecular dynamics simulations to examine the types of compensation, relaxation, and conformational changes that occur following the cis–trans light activation. The results suggest that changes occur throughout the protein, with changes in the orientation of Helices 5 and 6, a closer interaction between Ala 169 on Helix 4 and retinal, and a shift in the Schiff base counterion that also reflects changes in sidechain interactions with the retinal. Taken together, the simulation is suggestive of the types of changes that lead from local conformational change to light-activated signaling in this prototypical system. PMID:17109408

  15. Shared Genetic Factors Involved in Celiac Disease, Type 2 Diabetes and Anorexia Nervosa Suggest Common Molecular Pathways for Chronic Diseases

    PubMed Central

    Mostowy, Joanna; Montén, Caroline; Gudjonsdottir, Audur H.; Arnell, Henrik; Browaldh, Lars; Nilsson, Staffan; Agardh, Daniel

    2016-01-01

    Background and Objectives Genome-wide association studies (GWAS) have identified several genetic regions involved in immune-regulatory mechanisms to be associated with celiac disease. Previous GWAS also revealed an over-representation of genes involved in type 2 diabetes and anorexia nervosa associated with celiac disease, suggesting involvement of common metabolic pathways for development of these chronic diseases. The aim of this study was to extend these previous analyses to study the gene expression in the gut from children with active celiac disease. Material and Methods Thirty six target genes involved in type 2 diabetes and four genes associated with anorexia nervosa were investigated for gene expression in small intestinal biopsies from 144 children with celiac disease at median (range) age of 7.4 years (1.6–17.8) and from 154 disease controls at a median (range) age 11.4.years (1.4–18.3). Results A total of eleven of genes were differently expressed in celiac patients compared with disease controls of which CD36, CD38, FOXP1, SELL, PPARA, PPARG, AGT previously associated with type 2 diabetes and AKAP6, NTNG1 with anorexia nervosa remained significant after correction for multiple testing. Conclusion Shared genetic factors involved in celiac disease, type 2 diabetes and anorexia nervosa suggest common underlying molecular pathways for these diseases. PMID:27483138

  16. A Common Molecular Motif Characterizes Extracellular Allosteric Enhancers of GPCR Aminergic Receptors and Suggests Enhancer Mechanism of Action

    PubMed Central

    Bernstein, Robert Root; Dillon, Patrick F

    2014-01-01

    Several classes of compounds that have no intrinsic activity on aminergic systems nonetheless enhance the potency of aminergic receptor ligands three-fold or more while significantly increasing their duration of activity, preventing tachyphylaxis and reversing fade. Enhancer compounds include ascorbic acid, ethylenediaminetetraacetic acid, cortico-steroids, opioid peptides, opiates and opiate antagonists. This paper provides the first review of aminergic enhancement, demonstrating that all enhancers have a common, inobvious molecular motif and work through a common mechanism that is manifested by three common characteristics. First, aminergic enhancers bind directly to the amines they enhance, suggesting that the common structural motif is reflected in common binding targets. Second, one common target is the first extracellular loop of aminergic receptors. Third, at least some enhancers are antiphosphodiesterases. These observations suggest that aminergic enhancers act on the extracellular surface of aminergic receptors to keep the receptor in its high affinity state, trapping the ligand inside the receptor. Enhancer binding produces allosteric modifications of the receptor structure that interfere with phosphorylation of the receptor, thereby inhibiting down-regulation of the receptor. The mechanism explains how enhancers potentiate aminergic activity and increase duration of activity and makes testable predictions about additional compounds that should act as aminergic enhancers. PMID:25174918

  17. Free Energy of Translocating an Arginine-Rich Cell-Penetrating Peptide across a Lipid Bilayer Suggests Pore Formation

    PubMed Central

    Huang, Kun; García, Angel E.

    2013-01-01

    The molecular mechanism and energetics of the translocation of arginine-rich, cell-penetrating peptides through membranes are still under debate. One possible mechanism involves the formation of a water pore in the membrane such that the hydrophilic residues of the peptide are solvated throughout the translocating process. In this work, employing two different order parameters, we calculate the free energies of translocating a cyclic Arg9 peptide into a lipid bilayer along one path that involves a water-pore formation and another path that does not form a separate pore. The free-energy barrier of translocating the peptide along a pore path is 80 kJ/mol lower than along a pore-free path. This suggests that the peptide translocation is more likely associated with a water-pore formation. PMID:23442863

  18. The active stem cell specific expression of sponge Musashi homolog EflMsiA suggests its involvement in maintaining the stem cell state.

    PubMed

    Okamoto, Kazuko; Nakatsukasa, Mikiko; Alié, Alexandre; Masuda, Yoshiki; Agata, Kiyokazu; Funayama, Noriko

    2012-01-01

    A hallmark of stem cells is the ability to sustainably generate stem cells themselves (self-renew) as well as differentiated cells. Although a full understanding of this ability will require clarifying underlying the primordial molecular and cellular mechanisms, how stem cells maintain their stem state and their population in the evolutionarily oldest extant multicellular organisms, sponges, is poorly understood. Here, we report the identification of the first stem cell-specific gene in demosponges, a homolog of Musashi (an evolutionarily conserved RNA binding protein that regulates the stem cell state in various organisms). EflMsiA, a Musashi paralog, is specifically expressed in stem cells (archeocytes) in the freshwater sponge Ephydatia fluviatilis. EflMsiA protein is localized predominantly in the nucleus, with a small fraction in the cytoplasm, in archeocytes. When archeocytes enter M-phase, EflMsiA protein diffuses into the cytoplasm, probably because of the breakdown of the nuclear membrane. In the present study, the existence of two types of M-phase archeocytes [(M)-archeocytes] was revealed by a precise analysis of the expression levels of EflMsiA mRNA and protein. In Type I (M)-archeocytes, presumably archeocytes undergoing self-renewal, the expression levels of EflMsiA mRNA and protein were high. In Type II (M)-archeocytes, presumably archeocytes committed to differentiate (committed archeocytes), the expression levels of EflMsiA mRNA and protein were about 60% and 30% lower than those in Type I (M)-archeocytes. From these results, archeocytes can be molecularly defined for the first time as EflMsiA-mRNA-expressing cells. Furthermore, these findings shed light on the mode of cell division of archeocytes and suggest that archeocytes divide symmetrically for both self-renewal and differentiation. PMID:22464976

  19. Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues

    PubMed Central

    Ravenscroft, Stephanie M.; Pointon, Amy; Williams, Awel W.; Cross, Michael J.; Sidaway, James E.

    2016-01-01

    The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca2+ transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca2+ handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity. PMID:27125969

  20. Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues.

    PubMed

    Ravenscroft, Stephanie M; Pointon, Amy; Williams, Awel W; Cross, Michael J; Sidaway, James E

    2016-07-01

    The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca(2+ )transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca(2+ )handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity. PMID:27125969

  1. Exome Sequencing of Bilateral Testicular Germ Cell Tumors Suggests Independent Development Lineages12

    PubMed Central

    Brabrand, Sigmund; Johannessen, Bjarne; Axcrona, Ulrika; Kraggerud, Sigrid M.; Berg, Kaja G.; Bakken, Anne C.; Bruun, Jarle; Fosså, Sophie D.; Lothe, Ragnhild A.; Lehne, Gustav; Skotheim, Rolf I.

    2015-01-01

    Intratubular germ cell neoplasia, the precursor of testicular germ cell tumors (TGCTs), is hypothesized to arise during embryogenesis from developmentally arrested primordial germ cells (PGCs) or gonocytes. In early embryonal life, the PGCs migrate from the yolk sac to the dorsal body wall where the cell population separates before colonizing the genital ridges. However, whether the malignant transformation takes place before or after this separation is controversial. We have explored the somatic exome-wide mutational spectra of bilateral TGCT to provide novel insight into the in utero critical time frame of malignant transformation and TGCT pathogenesis. Exome sequencing was performed in five patients with bilateral TGCT (eight tumors), of these three patients in whom both tumors were available (six tumors) and two patients each with only one available tumor (two tumors). Selected loci were explored by Sanger sequencing in 71 patients with bilateral TGCT. From the exome-wide mutational spectra, no identical mutations in any of the three bilateral tumor pairs were identified. Exome sequencing of all eight tumors revealed 87 somatic non-synonymous mutations (median 10 per tumor; range 5-21), some in already known cancer genes such as CIITA, NEB, platelet-derived growth factor receptor α (PDGFRA), and WHSC1. SUPT6H was found recurrently mutated in two tumors. We suggest independent development lineages of bilateral TGCT. Thus, malignant transformation into intratubular germ cell neoplasia is likely to occur after the migration of PGCs. We reveal possible drivers of TGCT pathogenesis, such as mutated PDGFRA, potentially with therapeutic implications for TGCT patients. PMID:25748235

  2. Exome sequencing of bilateral testicular germ cell tumors suggests independent development lineages.

    PubMed

    Brabrand, Sigmund; Johannessen, Bjarne; Axcrona, Ulrika; Kraggerud, Sigrid M; Berg, Kaja G; Bakken, Anne C; Bruun, Jarle; Fosså, Sophie D; Lothe, Ragnhild A; Lehne, Gustav; Skotheim, Rolf I

    2015-02-01

    Intratubular germ cell neoplasia, the precursor of testicular germ cell tumors (TGCTs), is hypothesized to arise during embryogenesis from developmentally arrested primordial germ cells (PGCs) or gonocytes. In early embryonal life, the PGCs migrate from the yolk sac to the dorsal body wall where the cell population separates before colonizing the genital ridges. However, whether the malignant transformation takes place before or after this separation is controversial. We have explored the somatic exome-wide mutational spectra of bilateral TGCT to provide novel insight into the in utero critical time frame of malignant transformation and TGCT pathogenesis. Exome sequencing was performed in five patients with bilateral TGCT (eight tumors), of these three patients in whom both tumors were available (six tumors) and two patients each with only one available tumor (two tumors). Selected loci were explored by Sanger sequencing in 71 patients with bilateral TGCT. From the exome-wide mutational spectra, no identical mutations in any of the three bilateral tumor pairs were identified. Exome sequencing of all eight tumors revealed 87 somatic non-synonymous mutations (median 10 per tumor; range 5-21), some in already known cancer genes such as CIITA, NEB, platelet-derived growth factor receptor α (PDGFRA), and WHSC1. SUPT6H was found recurrently mutated in two tumors. We suggest independent development lineages of bilateral TGCT. Thus, malignant transformation into intratubular germ cell neoplasia is likely to occur after the migration of PGCs. We reveal possible drivers of TGCT pathogenesis, such as mutated PDGFRA, potentially with therapeutic implications for TGCT patients. PMID:25748235

  3. Molecular Mechanisms of HTLV-1 Cell-to-Cell Transmission.

    PubMed

    Gross, Christine; Thoma-Kress, Andrea K

    2016-01-01

    The tumorvirus human T-cell lymphotropic virus type 1 (HTLV-1), a member of the delta-retrovirus family, is transmitted via cell-containing body fluids such as blood products, semen, and breast milk. In vivo, HTLV-1 preferentially infects CD4⁺ T-cells, and to a lesser extent, CD8⁺ T-cells, dendritic cells, and monocytes. Efficient infection of CD4⁺ T-cells requires cell-cell contacts while cell-free virus transmission is inefficient. Two types of cell-cell contacts have been described to be critical for HTLV-1 transmission, tight junctions and cellular conduits. Further, two non-exclusive mechanisms of virus transmission at cell-cell contacts have been proposed: (1) polarized budding of HTLV-1 into synaptic clefts; and (2) cell surface transfer of viral biofilms at virological synapses. In contrast to CD4⁺ T-cells, dendritic cells can be infected cell-free and, to a greater extent, via viral biofilms in vitro. Cell-to-cell transmission of HTLV-1 requires a coordinated action of steps in the virus infectious cycle with events in the cell-cell adhesion process; therefore, virus propagation from cell-to-cell depends on specific interactions between cellular and viral proteins. Here, we review the molecular mechanisms of HTLV-1 transmission with a focus on the HTLV-1-encoded proteins Tax and p8, their impact on host cell factors mediating cell-cell contacts, cytoskeletal remodeling, and thus, virus propagation. PMID:27005656

  4. Immunohistochemical Expression and Clinical Significance of Suggested Stem Cell Markers in Hepatocellular Carcinoma

    PubMed Central

    Sung, Jong Jin; Noh, Sang Jae; Bae, Jun Sang; Park, Ho Sung; Jang, Kyu Yun; Chung, Myoung Ja; Moon, Woo Sung

    2016-01-01

    Background: Increasing evidence has shown that tumor initiation and growth are nourished by a small subpopulation of cancer stem cells (CSCs) within the tumor mass. CSCs are posited to be responsible for tumor maintenance, growth, distant metastasis, and relapse after curative operation. We examined the expression of CSC markers in paraffin-embedded tissue sections of hepatocellular carcinoma (HCC) and correlated the results with clinicopathologic characteristics. Methods: Immunohistochemical staining for the markers believed to be expressed in the CSCs, including epithelial cell adhesion molecule (EpCAM), keratin 19 (K19), CD133, and CD56, was performed in 82 HCC specimens. Results: EpCAM expression was observed in 56% of the HCCs (46/82) and K19 in 6% (5/82). EpCAM expression in HCC significantly correlated with elevated α-fetoprotein level, microvessel invasion of tumor cells, and high histologic grade. In addition, EpCAM expression significantly correlated with K19 expression. The overall survival and relapsefree survival rates in patients with EpCAM-expressing HCC were relatively lower than those in patients with EpCAM-negative HCC. All but two of the 82 HCCs were negative for CD133 and CD56, respectively. Conclusions: Our results suggest that HCCs expressing EpCAM are associated with unfavorable prognostic factors and have a more aggressive clinical course than those not expressing EpCAM. Further, the expression of either CD133 or CD56 in paraffin-embedded HCC tissues appears to be rare. PMID:26581206

  5. T Cell Allorecognition via Molecular Mimicry

    SciTech Connect

    Macdonald, Whitney A.; Chen, Zhenjun; Gras, Stephanie; Archbold, Julia K.; Tynan, Fleur E.; Clements, Craig S.; Bharadwaj, Mandvi; Kjer-Nielsen, Lars; Saunders, Philippa M.; Wilce, Matthew C.J.; Crawford, Fran; Stadinsky, Brian; Jackson, David; Brooks, Andrew G.; Purcell, Anthony W.; Kappler, John W.; Burrows, Scott R.; Rossjohn, Jamie; McCluskey, James

    2010-08-16

    T cells often alloreact with foreign human leukocyte antigens (HLA). Here we showed the LC13 T cell receptor (TCR), selected for recognition on self-HLA-B*0801 bound to a viral peptide, alloreacts with B44 allotypes (HLA-B*4402 and HLA-B*4405) bound to two different allopeptides. Despite extensive polymorphism between HLA-B*0801, HLA-B*4402, and HLA-B*4405 and the disparate sequences of the viral and allopeptides, the LC13 TCR engaged these peptide-HLA (pHLA) complexes identically, accommodating mimicry of the viral peptide by the allopeptide. The viral and allopeptides adopted similar conformations only after TCR ligation, revealing an induced-fit mechanism of molecular mimicry. The LC13 T cells did not alloreact against HLA-B*4403, and the single residue polymorphism between HLA-B*4402 and HLA-B*4403 affected the plasticity of the allopeptide, revealing that molecular mimicry was associated with TCR specificity. Accordingly, molecular mimicry that is HLA and peptide dependent is a mechanism for human T cell alloreactivity between disparate cognate and allogeneic pHLA complexes.

  6. Consumption of the epidermis: a suggested precursor of ulceration associated with increased proliferation of melanoma cells.

    PubMed

    Bønnelykke-Behrndtz, Louise M; Schmidt, Henrik; Damsgaard, Tine E; Christensen, Ib Jarle; Bastholt, Lars; Møller, Holger J; Nørgaard, Peter; Steiniche, Torben

    2015-11-01

    It has recently been demonstrated that the extent of ulceration and the presence of epidermal involvement that theoretically precede ulceration (consumption of epidermis, COE) or seen subsequent to inflammation (reactive epidermal hyperplasia or re-epithelialization) allowed better prognostic stratification of ulcerated melanoma. Understanding why these histopathologic markers have prognostic potential is important, not least because accurate consensual assessment of ulceration lies at the root of proper staging and clinical management. The authors therefore performed immunohistochemical analyses of tumor cell proliferation (Melan-A/Ki67) and infiltration of inflammatory cells (CD66b neutrophils and CD163 macrophages) to better understand the biology of the epidermal changes described. Tumors with a COE configuration showed 37% (95% CI: 4-54, P = 0.0046) increased tumor cell proliferation compared with tumors of normal epidermal configuration. COE is therefore suggested a precursor of ulceration associated with increased proliferation of melanoma cells. There was no observed correlation between COE and an increased inflammatory response (CD163 macrophages or CD66b neutrophils), which supports that the proliferation drive is noninflammatory. In contrast, the presence of re-epithelialization and/or reactive epidermal hyperplasia demonstrated an 18% (95% CI: 6-53, P = 0.0021) increased density of neutrophils compared with tumor with no evidence of these possibly prolonged late-stage or resolved ulcerations. These results further support the relevance of including these epidermal changes into the definition of ulceration and to define ulceration of a primary melanoma as loss of epidermis with evidence of a host response (infiltration of neutrophils or fibrin deposition) and thinning, effacement, or reactive hyperplasia of the surrounding epidermis. PMID:26485240

  7. Molecular genetic analysis of cucumber mosaic virus populations infecting pepper suggests unique patterns of evolution in Korea.

    PubMed

    Kim, Mi-Kyeong; Seo, Jang-Kyun; Kwak, Hae-Ryun; Kim, Jeong-Soo; Kim, Kook-Hyung; Cha, Byeong-Jin; Choi, Hong-Soo

    2014-09-01

    Studying genetic structure and diversity of viruses is important to understand the evolutionary mechanisms that generate and maintain variations in viral populations. Cucumber mosaic virus (CMV) is endemic in most pepper fields in Korea. Currently, no effective methods for control of CMV are available due to many environmental and biological factors such as the extensive evolutionary capacity of CMV. Thus, analyzing the genetic structure of CMV populations may facilitate the development of strategies for the control of CMV. In this study, 252 pepper (Capsicum annuum) samples showing virus symptoms were collected by field surveys performed throughout Korea in 2007. Reverse-transcription polymerase chain reaction analyses revealed that, in total, 165 collected samples were infected with CMV. Forty-five CMV isolates were randomly selected within each regional subpopulation and analyzed by full-genome sequencing. Analyses of genetic diversity showed that the 2b gene of CMV is under weaker purifying selection than the other genes. Based on the phylogenetic analysis of RNA1, the CMV isolates from pepper were divided into three clusters in subgroup I. Our full-genome sequence-based molecular analyses of the CMV Korean population suggest that the subpopulations of CMV have been geographically localized in pepper fields in Korea. PMID:25116642

  8. Recent Advances in the Molecular Characterization of Circulating Tumor Cells

    PubMed Central

    Lowes, Lori E.; Allan, Alison L.

    2014-01-01

    Although circulating tumor cells (CTCs) were first observed over a century ago, lack of sensitive methodology precluded detailed study of these cells until recently. However, technological advances have now facilitated the identification, enumeration, and characterization of CTCs using a variety of methods. The majority of evidence supporting the use of CTCs in clinical decision-making has been related to enumeration using the CellSearch® system and correlation with prognosis. Growing evidence also suggests that CTC monitoring can provide an early indication of patient treatment response based on comparison of CTC levels before and after therapy. However, perhaps the greatest potential that CTCs hold for oncology lies at the level of molecular characterization. Clinical treatment decisions may be more effective if they are based on molecular characteristics of metastatic cells rather than on those of the primary tumor alone. Molecular characterization of CTCs (which can be repeatedly isolated in a minimally invasive fashion) provides the opportunity for a “real-time liquid biopsy” that allows assessment of genetic drift, investigation of molecular disease evolution, and identification of actionable genomic characteristics. This review focuses on recent advances in this area, including approaches involving immunophenotyping, fluorescence in situ hybridization (FISH), multiplex RT-PCR, microarray, and genomic sequencing. PMID:24633084

  9. Recent advances in the molecular characterization of circulating tumor cells.

    PubMed

    Lowes, Lori E; Allan, Alison L

    2014-01-01

    Although circulating tumor cells (CTCs) were first observed over a century ago, lack of sensitive methodology precluded detailed study of these cells until recently. However, technological advances have now facilitated the identification, enumeration, and characterization of CTCs using a variety of methods. The majority of evidence supporting the use of CTCs in clinical decision-making has been related to enumeration using the CellSearch® system and correlation with prognosis. Growing evidence also suggests that CTC monitoring can provide an early indication of patient treatment response based on comparison of CTC levels before and after therapy. However, perhaps the greatest potential that CTCs hold for oncology lies at the level of molecular characterization. Clinical treatment decisions may be more effective if they are based on molecular characteristics of metastatic cells rather than on those of the primary tumor alone. Molecular characterization of CTCs (which can be repeatedly isolated in a minimally invasive fashion) provides the opportunity for a "real-time liquid biopsy" that allows assessment of genetic drift, investigation of molecular disease evolution, and identification of actionable genomic characteristics. This review focuses on recent advances in this area, including approaches involving immunophenotyping, fluorescence in situ hybridization (FISH), multiplex RT-PCR, microarray, and genomic sequencing. PMID:24633084

  10. Drug screen in patient cells suggests quinacrine to be repositioned for treatment of acute myeloid leukemia

    PubMed Central

    Eriksson, A; Österroos, A; Hassan, S; Gullbo, J; Rickardson, L; Jarvius, M; Nygren, P; Fryknäs, M; Höglund, M; Larsson, R

    2015-01-01

    To find drugs suitable for repositioning for use against leukemia, samples from patients with chronic lymphocytic, acute myeloid and lymphocytic leukemias as well as peripheral blood mononuclear cells (PBMC) were tested in response to 1266 compounds from the LOPAC1280 library (Sigma). Twenty-five compounds were defined as hits with activity in all leukemia subgroups (<50% cell survival compared with control) at 10 μM drug concentration. Only one of these compounds, quinacrine, showed low activity in normal PBMCs and was therefore selected for further preclinical evaluation. Mining the NCI-60 and the NextBio databases demonstrated leukemia sensitivity and the ability of quinacrine to reverse myeloid leukemia gene expression. Mechanistic exploration was performed using the NextBio bioinformatic software using gene expression analysis of drug exposed acute myeloid leukemia cultures (HL-60) in the database. Analysis of gene enrichment and drug correlations revealed strong connections to ribosomal biogenesis nucleoli and translation initiation. The highest drug–drug correlation was to ellipticine, a known RNA polymerase I inhibitor. These results were validated by additional gene expression analysis performed in-house. Quinacrine induced early inhibition of protein synthesis supporting these predictions. The results suggest that quinacrine have repositioning potential for treatment of acute myeloid leukemia by targeting of ribosomal biogenesis. PMID:25885427

  11. Quiescent Innate Response to Infective Filariae by Human Langerhans Cells Suggests a Strategy of Immune Evasion

    PubMed Central

    Boyd, Alexis; Bennuru, Sasisekhar; Wang, Yuanyuan; Sanprasert, Vivornpun; Law, Melissa; Chaussabel, Damien; Nutman, Thomas B.

    2013-01-01

    Filarial infection is initiated by mosquito-derived third-stage larvae (L3) deposited on the skin that transit through the epidermis, which contains Langerhans cells (LC) and keratinocytes (KC), among other cells. This earliest interaction between L3 and the LC likely conditions the priming of the immune system to the parasite. To determine the nature of this interaction, human LC (langerin+ E-cadherin+ CD1a+) were generated in vitro and exposed to live L3. LC exposed to live L3 for 48 h showed no alterations in the cell surface markers CD14, CD86, CD83, CD207, E-cadherin, CD80, CD40, and HLA-DR or in mRNA expression of inflammation-associated genes, such as those for interleukin 18 (IL-18), IL-18BP, and caspase 1. In contrast to L3, live tachyzoites of Toxoplasma gondii, an intracellular parasite, induced production of CXCL9, IP-10, and IL-6 in LC. Furthermore, preexposure of LC to L3 did not alter Toll-like receptor 3 (TLR3)- or TLR4-mediated expression of the proinflammatory cytokines IL-1β, gamma interferon (IFN-γ), IL-6, or IL-10. Interestingly, cocultures of KC and LC produced significantly more IL-18, IL-1α, and IL-8 than did cultures of LC alone, although exposure of the cocultures to live L3 did not result in altered cytokine production. Microarray examination of ex vivo LC from skin blisters that were exposed to live L3 also showed few significant changes in gene expression compared with unexposed blisters, further underscoring the relatively muted response of LC to L3. Our data suggest that failure by LC to initiate an inflammatory response to the invasive stage of filarial parasites may be a strategy for immune evasion by the filarial parasite. PMID:23429540

  12. Quiescent innate response to infective filariae by human Langerhans cells suggests a strategy of immune evasion.

    PubMed

    Boyd, Alexis; Bennuru, Sasisekhar; Wang, Yuanyuan; Sanprasert, Vivornpun; Law, Melissa; Chaussabel, Damien; Nutman, Thomas B; Semnani, Roshanak Tolouei

    2013-05-01

    Filarial infection is initiated by mosquito-derived third-stage larvae (L3) deposited on the skin that transit through the epidermis, which contains Langerhans cells (LC) and keratinocytes (KC), among other cells. This earliest interaction between L3 and the LC likely conditions the priming of the immune system to the parasite. To determine the nature of this interaction, human LC (langerin(+) E-cadherin(+) CD1a(+)) were generated in vitro and exposed to live L3. LC exposed to live L3 for 48 h showed no alterations in the cell surface markers CD14, CD86, CD83, CD207, E-cadherin, CD80, CD40, and HLA-DR or in mRNA expression of inflammation-associated genes, such as those for interleukin 18 (IL-18), IL-18BP, and caspase 1. In contrast to L3, live tachyzoites of Toxoplasma gondii, an intracellular parasite, induced production of CXCL9, IP-10, and IL-6 in LC. Furthermore, preexposure of LC to L3 did not alter Toll-like receptor 3 (TLR3)- or TLR4-mediated expression of the proinflammatory cytokines IL-1β, gamma interferon (IFN-γ), IL-6, or IL-10. Interestingly, cocultures of KC and LC produced significantly more IL-18, IL-1α, and IL-8 than did cultures of LC alone, although exposure of the cocultures to live L3 did not result in altered cytokine production. Microarray examination of ex vivo LC from skin blisters that were exposed to live L3 also showed few significant changes in gene expression compared with unexposed blisters, further underscoring the relatively muted response of LC to L3. Our data suggest that failure by LC to initiate an inflammatory response to the invasive stage of filarial parasites may be a strategy for immune evasion by the filarial parasite. PMID:23429540

  13. Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation.

    PubMed

    Goulet, Brigitte; Sansregret, Laurent; Leduy, Lam; Bogyo, Matthew; Weber, Ekkehard; Chauhan, Shyam S; Nepveu, Alain

    2007-09-01

    It is generally accepted that the role of cathepsin L in cancer involves its activities outside the cells once it has been secreted. However, cathepsin L isoforms that are devoid of a signal peptide were recently shown to be present in the nucleus where they proteolytically process the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor. A role for nuclear cathepsin L in cell proliferation could be inferred from the observation that the CDP/Cux processed isoform can accelerate entry into S phase. Here, we report that in many transformed cells the proteolytic processing of CDP/Cux is augmented and correlates with increased cysteine protease expression and activity in the nucleus. Taking advantage of an antibody that recognizes the prodomain of human cathepsin L, we showed that human cells express short cathepsin L species that do not contain a signal peptide, do not transit through the endoplasmic reticulum, are not glycosylated, and localize to the nucleus. We also showed that transformation by the ras oncogene causes rapid increases both in the production of short nuclear cathepsin L isoforms and in the processing of CDP/Cux. Using a cell-based assay, we showed that a cell-permeable inhibitor of cysteine proteases is able to delay the progression into S phase and the proliferation in soft agar of ras-transformed cells, whereas the non-cell-permeable inhibitor had no effect. Taken together, these results suggest that the role of cathepsin L in cancer might not be limited to its extracellular activities but may also involve its processing function in the nucleus. PMID:17855659

  14. Early Events following Experimental Infection with Peste-Des-Petits Ruminants Virus Suggest Immune Cell Targeting

    PubMed Central

    Pope, Robert A.; Parida, Satya; Bailey, Dalan; Brownlie, Joe; Barrett, Thomas; Banyard, Ashley C.

    2013-01-01

    Peste-des-petits ruminants virus (PPRV) is a viral pathogen that causes a devastating plague of small ruminants. PPRV is an economically significant disease that continues to be a major obstacle to the development of sustainable agriculture across the developing world. The current understanding of PPRV pathogenesis has been heavily assumed from the closely related rinderpest virus (RPV) and other morbillivirus infections alongside data derived from field outbreaks. There have been few studies reported that have focused on the pathogenesis of PPRV and very little is known about the processes underlying the early stages of infection. In the present study, 15 goats were challenged by the intranasal route with a virulent PPRV isolate, Côte d’Ivoire ’89 (CI/89) and sacrificed at strategically defined time-points post infection to enable pre- and post-mortem sampling. This approach enabled precise monitoring of the progress and distribution of virus throughout the infection from the time of challenge, through peak viraemia and into a period of convalescence. Observations were then related to findings of previous field studies and experimental models of PPRV to develop a clinical scoring system for PPRV. Importantly, histopathological investigations demonstrated that the initial site for virus replication is not within the epithelial cells of the respiratory mucosa, as has been previously reported, but is within the tonsillar tissue and lymph nodes draining the site of inoculation. We propose that virus is taken up by immune cells within the respiratory mucosa which then transport virus to lymphoid tissues where primary virus replication occurs, and from where virus enters circulation. Based on these findings we propose a novel clinical scoring methodology for PPRV pathogenesis and suggest a fundamental shift away from the conventional model of PPRV pathogenesis. PMID:23418464

  15. Response dynamics of phosphorelays suggest their potential utility in cell signalling

    PubMed Central

    Csikász-Nagy, Attila; Cardelli, Luca; Soyer, Orkun S.

    2011-01-01

    Phosphorelays are extended two-component signalling systems found in diverse bacteria, lower eukaryotes and plants. Only few of these systems are characterized, and we still lack a full understanding of their signalling abilities. Here, we aim to achieve a global understanding of phosphorelay signalling and its dynamical properties. We develop a generic model, allowing us to systematically analyse response dynamics under different assumptions. Using this model, we find that the steady-state concentration of phosphorylated protein at the final layer of a phosphorelay is a linearly increasing, but eventually saturating function of the input. In contrast, the intermediate layers can display ultrasensitivity. We find that such ultrasensitivity is a direct result of the phosphorelay biochemistry; shuttling of a single phosphate group from the first to the last layer. The response dynamics of the phosphorelay results in tolerance of cross-talk, especially when it occurs as cross-deactivation. Further, it leads to a high signal-to-noise ratio for the final layer. We find that a relay length of four, which is most commonly observed, acts as a saturating point for these dynamic properties. These findings suggest that phosphorelays could act as a mechanism to reduce noise and effects of cross-talk on the final layer of the relay and enforce its input–response relation to be linear. In addition, our analysis suggests that middle layers of phosphorelays could embed thresholds. We discuss the consequence of these findings in relation to why cells might use phosphorelays along with enzymatic kinase cascades. PMID:20702449

  16. Cell list algorithms for nonequilibrium molecular dynamics

    NASA Astrophysics Data System (ADS)

    Dobson, Matthew; Fox, Ian; Saracino, Alexandra

    2016-06-01

    We present two modifications of the standard cell list algorithm that handle molecular dynamics simulations with deforming periodic geometry. Such geometry naturally arises in the simulation of homogeneous, linear nonequilibrium flow modeled with periodic boundary conditions, and recent progress has been made developing boundary conditions suitable for general 3D flows of this type. Previous works focused on the planar flows handled by Lees-Edwards or Kraynik-Reinelt boundary conditions, while the new versions of the cell list algorithm presented here are formulated to handle the general 3D deforming simulation geometry. As in the case of equilibrium, for short-ranged pairwise interactions, the cell list algorithm reduces the computational complexity of the force computation from O(N2) to O(N), where N is the total number of particles in the simulation box. We include a comparison of the complexity and efficiency of the two proposed modifications of the standard algorithm.

  17. Molecular regulation of plant cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1998-01-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized.

  18. Neoplastic cell transformation by high-LET radiation: Molecular mechanisms

    NASA Astrophysics Data System (ADS)

    Chui-Hsu Yang, Tracy; Craise, Laurie M.; Mei, Man-Tong; Tobias, Cornelius A.

    Experimental data on molecular mechanisms are essential for understanding the bioeffects of radiation and for developing biophysical models, which can help in determining the shape of dose-response curves at very low doses, e.g., doses less than 1 cGy. Although it has been shown that ionizing radiation can cause neoplastic cell transformation directly, that high-LET heavy ions in general can be more effective than photons in transforming cells, and that the radiogenic cell transformation is a multi-step processes, we know very little about the molecular nature of lesions important for cell transformation, the relationship between lethal and transformational damages, and the evolution of initial damages into final chromosomal aberrations which alter the growth control of cells. Using cultured mouse embryo cells (C3H10T1/2) as a model system, we have collected quantitative data on dose-response curves for heavy ions with various charges and energies. An analysis of these quantitative data suggested that two DNA breaks formed within 80 Å may cause cell transformation and that two DNA breaks formed within 20 Å may be lethal. Through studies with restriction enzymes which produce DNA damages at specific sites, we have found that DNA double strand breaks, including both blunt- and cohesive-ended breaks, can cause cell transformation in vitro. These results indicate that DNA double strand breaks can be important primary lesions for radiogenic cell transformation and that blunt-ended double strand breaks can form lethal as well as transformational damages due to misrepair or incomplete repair in the cell. The RBE-LET relationship is similar for HGPRT gene mutation, chromosomal deletion, and cell transformation, suggesting common lesions may be involved in these radiation effects. The high RBE of high-LET radiation for cell killing and neoplastic cell transformation is most likely related to its effectiveness in producing DNA double strand breaks in mammalian cells. At

  19. Molecular cytogenetic characterization of cancer cell alterations.

    PubMed

    Popescu, N C; Zimonjic, D B

    1997-01-01

    Chromosomal abnormalities are the hallmark of cancer cells. Recurring and highly consistent structural and numerical alterations have been identified in a large number of leukemias, lymphomas, and solid tumors. The identification of recurrent genetic alterations and the isolation of molecular markers have clinical applications in the diagnosis and prognosis of neoplasia and in the detection of minimal residual disease that are essential for designing the most effective therapeutic approach. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are powerful techniques for detection of genomic alterations. The battery of FISH methods and DNA probes that are available can resolve virtually any chromosomal alterations regardless of their complexity. Combined chromosome banding, multifluor or spectral karyotype, and comparative genomic hybridization (CGH) allow identification of structural and numerical alterations on a global basis, mapping of the DNA copy number on the entire tumor genome, complete derivation of complex rearrangements, and localization of the breakpoints of translocations and deletions. Regions of recurrent alterations can be microdisected, amplified, microclone libraries constructed and probes localized on extended chromosomes or chromatin fibers for construction of high resolution physical maps that are critical for positional cloning and gene identification. In this review we attempted to cover the current trends in cancer molecular cytogenetics, and to outline the importance of molecular chromosome analysis in the understanding of oncogenesis and its clinical applications. PMID:9062575

  20. Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle

    PubMed Central

    Atwell, Kathryn; Qin, Zhao; Gavaghan, David; Kugler, Hillel; Hubbard, E. Jane Albert; Osborne, James M.

    2015-01-01

    The Caenorhabditis elegans germ line is an outstanding model system in which to study the control of cell division and differentiation. Although many of the molecules that regulate germ cell proliferation and fate decisions have been identified, how these signals interact with cellular dynamics and physical forces within the gonad remains poorly understood. We therefore developed a dynamic, 3D in silico model of the C. elegans germ line, incorporating both the mechanical interactions between cells and the decision-making processes within cells. Our model successfully reproduces key features of the germ line during development and adulthood, including a reasonable ovulation rate, correct sperm count, and appropriate organization of the germ line into stably maintained zones. The model highlights a previously overlooked way in which germ cell pressure may influence gonadogenesis, and also predicts that adult germ cells might be subject to mechanical feedback on the cell cycle akin to contact inhibition. We provide experimental data consistent with the latter hypothesis. Finally, we present cell trajectories and ancestry recorded over the course of a simulation. The novel approaches and software described here link mechanics and cellular decision-making, and are applicable to modeling other developmental and stem cell systems. PMID:26428008

  1. Molecular Culprits Generating Brain Tumor Stem Cells

    PubMed Central

    Oh, Se-Yeong

    2013-01-01

    Despite current advances in multimodality therapies, such as surgery, radiotherapy, and chemotherapy, the outcome for patients with high-grade glioma remains fatal. Understanding how glioma cells resist various therapies may provide opportunities for developing new therapies. Accumulating evidence suggests that the main obstacle for successfully treating high-grade glioma is the existence of brain tumor stem cells (BTSCs), which share a number of cellular properties with adult stem cells, such as self-renewal and multipotent differentiation capabilities. Owing to their resistance to standard therapy coupled with their infiltrative nature, BTSCs are a primary cause of tumor recurrence post-therapy. Therefore, BTSCs are thought to be the main glioma cells representing a novel therapeutic target and should be eliminated to obtain successful treatment outcomes. PMID:24904883

  2. Paired octamer rings of retinoschisin suggest a junctional model for cell-cell adhesion in the retina.

    PubMed

    Tolun, Gökhan; Vijayasarathy, Camasamudram; Huang, Rick; Zeng, Yong; Li, Yan; Steven, Alasdair C; Sieving, Paul A; Heymann, J Bernard

    2016-05-10

    Retinoschisin (RS1) is involved in cell-cell junctions in the retina, but is unique among known cell-adhesion proteins in that it is a soluble secreted protein. Loss-of-function mutations in RS1 lead to early vision impairment in young males, called X-linked retinoschisis. The disease is characterized by separation of inner retinal layers and disruption of synaptic signaling. Using cryo-electron microscopy, we report the structure at 4.1 Å, revealing double octamer rings not observed before. Each subunit is composed of a discoidin domain and a small N-terminal (RS1) domain. The RS1 domains occupy the centers of the rings, but are not required for ring formation and are less clearly defined, suggesting mobility. We determined the structure of the discoidin rings, consistent with known intramolecular and intermolecular disulfides. The interfaces internal to and between rings feature residues implicated in X-linked retinoschisis, indicating the importance of correct assembly. Based on this structure, we propose that RS1 couples neighboring membranes together through octamer-octamer contacts, perhaps modulated by interactions with other membrane components. PMID:27114531

  3. Analysis of 18S rRNA gene sequences suggests significant molecular differences between Macrodasyida and Chaetonotida (Gastrotricha).

    PubMed

    Manylov, Oleg G; Vladychenskaya, Natalia S; Milyutina, Irina A; Kedrova, Olga S; Korokhov, Nikolai P; Dvoryanchikov, Gennady A; Aleshin, Vladimir V; Petrov, Nikolai B

    2004-03-01

    Partial 18S rRNA gene sequences of four macrodasyid and one chaetonotid gastrotrichs were obtained and compared with the available sequences of other gastrotrich species and representatives of various metazoan phyla. Contrary to the earlier molecular data, the gastrotrich sequences did not comprise a monophyletic group but formed two distinct clades, corresponding to the Macrodasyida and Chaetonotida, with the basal position occupied by the sequences of Tetranchyroderma sp. and Xenotrichula sp., respectively. Depending on the taxon sampling and methods of analysis, the two clades were separated by various combinations of clades Rotifera, Gnathostomulida, and Platyhelminthes, and never formed a clade with Nematoda. Thus, monophyly of the Gastrotricha is not confirmed by analysis of the presently available molecular data. PMID:15012964

  4. Viscoelastic analysis of high molecular weight, alkali-denatured DNA from mouse 3T3 cells.

    PubMed Central

    Uhlenhopp, E L

    1975-01-01

    Alkaline lysates of mouse 3T3 cells showed viscoelastic properties characteristic of very large molecules of single-stranded DNA. The viscoelastic retardation time and the sensitivity to low doses of nitrogen mustard and of X-irradiation suggest a molecular weight in excess of 10-10 daltons. Contact-inhibited cells yielded larger single strands than actively growing cells. PMID:235335

  5. Molecular mechanisms underlying progesterone-enhanced breast cancer cell migration.

    PubMed

    Wang, Hui-Chen; Lee, Wen-Sen

    2016-01-01

    Progesterone (P4) was demonstrated to inhibit migration in vascular smooth muscle cells (VSMCs), but to enhance migration in T47D breast cancer cells. To investigate the mechanism responsible for this switch in P4 action, we examined the signaling pathway responsible for the P4-induced migration enhancement in breast cancer cell lines, T47D and MCF-7. Here, we demonstrated that P4 activated the cSrc/AKT signaling pathway, subsequently inducing RSK1 activation, which in turn increased phosphorylation of p27 at T198 and formation of the p27pT198-RhoA complex in the cytosol, thereby preventing RhoA degradation, and eventually enhanced migration in T47D cells. These findings were confirmed in the P4-treated MCF-7. Comparing the P4-induced molecular events in between breast cancer cells and VSMCs, we found that P4 increased p27 phosphorylation at T198 in breast cancer cells through RSK1 activation, while P4 increased p27 phosphorlation at Ser10 in VSMCs through KIS activation. P27pT198 formed the complex with RhoA and prevented RhoA degradation in T47D cells, whereas p-p27Ser10 formed the complex with RhoA and caused RhoA degradation in VSMCs. The results of this study highlight the molecular mechanism underlying P4-enhanced breast cancer cell migration, and suggest that RSK1 activation is responsible for the P4-induced migration enhancement in breast cancer cells. PMID:27510838

  6. Molecular mechanisms underlying progesterone-enhanced breast cancer cell migration

    PubMed Central

    Wang, Hui-Chen; Lee, Wen-Sen

    2016-01-01

    Progesterone (P4) was demonstrated to inhibit migration in vascular smooth muscle cells (VSMCs), but to enhance migration in T47D breast cancer cells. To investigate the mechanism responsible for this switch in P4 action, we examined the signaling pathway responsible for the P4-induced migration enhancement in breast cancer cell lines, T47D and MCF-7. Here, we demonstrated that P4 activated the cSrc/AKT signaling pathway, subsequently inducing RSK1 activation, which in turn increased phosphorylation of p27 at T198 and formation of the p27pT198-RhoA complex in the cytosol, thereby preventing RhoA degradation, and eventually enhanced migration in T47D cells. These findings were confirmed in the P4-treated MCF-7. Comparing the P4-induced molecular events in between breast cancer cells and VSMCs, we found that P4 increased p27 phosphorylation at T198 in breast cancer cells through RSK1 activation, while P4 increased p27 phosphorlation at Ser10 in VSMCs through KIS activation. P27pT198 formed the complex with RhoA and prevented RhoA degradation in T47D cells, whereas p-p27Ser10 formed the complex with RhoA and caused RhoA degradation in VSMCs. The results of this study highlight the molecular mechanism underlying P4-enhanced breast cancer cell migration, and suggest that RSK1 activation is responsible for the P4-induced migration enhancement in breast cancer cells. PMID:27510838

  7. Molecular signature of Epstein Barr virus-positive Burkitt lymphoma and post-transplant lymphoproliferative disorder suggest different roles for Epstein Barr virus

    PubMed Central

    Navari, Mohsen; Fuligni, Fabio; Laginestra, Maria A.; Etebari, Maryam; Ambrosio, Maria R.; Sapienza, Maria R.; Rossi, Maura; De Falco, Giulia; Gibellini, Davide; Tripodo, Claudio; Pileri, Stefano A.; Leoncini, Lorenzo; Piccaluga, Pier P.

    2014-01-01

    Epstein Barr virus (EBV) infection is commonly associated with human cancer and, in particular, with lymphoid malignancies. Although the precise role of the virus in the pathogenesis of different lymphomas is largely unknown, it is well recognized that the expression of viral latent proteins and miRNA can contribute to its pathogenetic role. In this study, we compared the gene and miRNA expression profile of two EBV-associated aggressive B non-Hodgkin lymphomas known to be characterized by differential expression of the viral latent proteins aiming to dissect the possible different contribution of such proteins and EBV-encoded miRNAs. By applying extensive bioinformatic inferring and an experimental model, we found that EBV+ Burkitt lymphoma presented with significant over-expression of EBV-encoded miRNAs that were likely to contribute to its global molecular profile. On the other hand, EBV+ post-transplant diffuse large B-cell lymphomas presented a significant enrichment in genes regulated by the viral latent proteins. Based on these different viral and cellular gene expression patterns, a clear distinction between EBV+ Burkitt lymphoma and post-transplant diffuse large B-cell lymphomas was made. In this regard, the different viral and cellular expression patterns seemed to depend on each other, at least partially, and the latency type most probably played a significant role in their regulation. In conclusion, our data indicate that EBV influence over B-cell malignant clones may act through different mechanisms of transcriptional regulation and suggest that potentially different pathogenetic mechanisms may depend upon the conditions of the interaction between EBV and the host that finally determine the latency pattern. PMID:25566237

  8. Mouse Genetics Suggests Cell-Context Dependency for Myc-Regulated Metabolic Enzymes during Tumorigenesis

    PubMed Central

    Nilsson, Lisa M.; Kreutzer, Christiane; Pretsch, Walter; Bornkamm, Georg W.; Nilsson, Jonas A.

    2012-01-01

    c-Myc (hereafter called Myc) belongs to a family of transcription factors that regulates cell growth, cell proliferation, and differentiation. Myc initiates the transcription of a large cast of genes involved in cell growth by stimulating metabolism and protein synthesis. Some of these, like those involved in glycolysis, may be part of the Warburg effect, which is defined as increased glucose uptake and lactate production in the presence of adequate oxygen supply. In this study, we have taken a mouse-genetics approach to challenge the role of select Myc-regulated metabolic enzymes in tumorigenesis in vivo. By breeding λ-Myc transgenic mice, ApcMin mice, and p53 knockout mice with mouse models carrying inactivating alleles of Lactate dehydrogenase A (Ldha), 3-Phosphoglycerate dehydrogenase (Phgdh) and Serine hydroxymethyltransferase 1 (Shmt1), we obtained offspring that were monitored for tumor development. Very surprisingly, we found that these genes are dispensable for tumorigenesis in these genetic settings. However, experiments in fibroblasts and colon carcinoma cells expressing oncogenic Ras show that these cells are sensitive to Ldha knockdown. Our genetic models reveal cell context dependency and a remarkable ability of tumor cells to adapt to alterations in critical metabolic pathways. Thus, to achieve clinical success, it will be of importance to correctly stratify patients and to find synthetic lethal combinations of inhibitors targeting metabolic enzymes. PMID:22438825

  9. Molecular Profiling of Clear Cell Ovarian Cancers

    PubMed Central

    Friedlander, Michael L.; Russell, Kenneth; Millis, Sherri; Gatalica, Zoran; Bender, Ryan; Voss, Andreas

    2016-01-01

    Background Advanced stage/recurrent clear cell ovarian cancers (CCOCs) are characterized by a low response to chemotherapy and a poor prognosis. There is growing interest in investigating novel/molecular targeted therapies in patients with CCOC in histotype-specific trials. However, CCOCs are not a uniform entity and comprise a number of molecular subtypes and it is unlikely that a single approach to treatment will be appropriate for all patients. The aim of this study was to analyze the results of a multiplatform profiling panel in CCOCs to identify potential therapeutic targets. Patients and Methods Tumor profiling was performed on 521 CCOCs. They were grouped into pure (n = 422) and mixed (n = 99) CCOC for analysis. Testing included a combination of DNA sequencing (including next-generation sequencing) using a 46-gene panel, immunohistochemistry, fluorescent or chromogenic in situ hybridization, and RNA fragment analysis. Results The most common findings were in the PIK3CA/Akt/mTOR pathway, with 61% of all CCOCs showing a molecular alteration in one of these pathway components. Next-generation sequencing revealed PIK3CA mutations in 50% of pure CCOCs. Significant differences were observed between pure and mixed CCOCs with respect to hormone receptor expression (9% vs 34.7% for ER, 13.45 vs 26.4% for PR), cMET (24.1% vs 11.6%), PD-1 tumor infiltrating lymphocytes (48.1% vs 100%), expression of PD-L1 (7.4% vs 25%), and TOPO1 (41% vs 27.1%) on immunohistochemistry, whereas next-generation sequencing revealed significant differences in mutation frequency in PIK3CA (50% vs 18.5%), TP53 (18.1% vs 57.7%), KRAS (12.4% vs 3.7%), and cMET (1.9% vs 11.1%). Conclusions This large study confirms that the PIK3CA/Akt/mTOR pathway is commonly altered in CCOCs, and highlights the significant differences between pure and mixed CCOCs. Clear cell ovarian cancers are molecularly heterogeneous and there are a number of potential therapeutic targets which could be tested in clinical

  10. Protein conservation and variation suggest mechanisms of cell type-specific modulation of signaling pathways.

    PubMed

    Schaefer, Martin H; Yang, Jae-Seong; Serrano, Luis; Kiel, Christina

    2014-06-01

    Many proteins and signaling pathways are present in most cell types and tissues and yet perform specialized functions. To elucidate mechanisms by which these ubiquitous pathways are modulated, we overlaid information about cross-cell line protein abundance and variability, and evolutionary conservation onto functional pathway components and topological layers in the pathway hierarchy. We found that the input (receptors) and the output (transcription factors) layers evolve more rapidly than proteins in the intermediary transmission layer. In contrast, protein expression variability decreases from the input to the output layer. We observed that the differences in protein variability between the input and transmission layer can be attributed to both the network position and the tendency of variable proteins to physically interact with constitutively expressed proteins. Differences in protein expression variability and conservation are also accompanied by the tendency of conserved and constitutively expressed proteins to acquire somatic mutations, while germline mutations tend to occur in cell type-specific proteins. Thus, conserved core proteins in the transmission layer could perform a fundamental role in most cell types and are therefore less tolerant to germline mutations. In summary, we propose that the core signal transmission machinery is largely modulated by a variable input layer through physical protein interactions. We hypothesize that the bow-tie organization of cellular signaling on the level of protein abundance variability contributes to the specificity of the signal response in different cell types. PMID:24922536

  11. Molecular biology of testicular germ cell tumors.

    PubMed

    Gonzalez-Exposito, R; Merino, M; Aguayo, C

    2016-06-01

    Testicular germ cell tumors (TGCTs) are the most common solid tumors in young adult men. They constitute a unique pathology because of their embryonic and germ origin and their special behavior. Genetic predisposition, environmental factors involved in their development and genetic aberrations have been under study in many works throughout the last years trying to explain the susceptibility and the transformation mechanism of TGCTs. Despite the high rate of cure in this type of tumors because its particular sensitivity to cisplatin, there are tumors resistant to chemotherapy for which it is needed to find new therapies. In the present work, it has been carried out a literature review on the most important molecular aspects involved in the onset and development of such tumors, as well as a review of the major developments regarding prognostic factors, new prognostic biomarkers and the possibility of new targeted therapies. PMID:26482724

  12. Altered gene expression in dry age-related macular degeneration suggests early loss of choroidal endothelial cells

    PubMed Central

    Whitmore, S. Scott; Braun, Terry A.; Skeie, Jessica M.; Haas, Christine M.; Sohn, Elliott H.; Stone, Edwin M.; Scheetz, Todd E.

    2013-01-01

    Purpose Age-related macular degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE) and choroid from early AMD and control maculas with exon-based arrays. Methods Gene expression levels in nine human donor eyes with early AMD and nine control human donor eyes were assessed using Affymetrix Human Exon ST 1.0 arrays. Two controls did not pass quality control and were removed. Differentially expressed genes were annotated using the Database for Annotation, Visualization and Integrated Discovery (DAVID), and gene set enrichment analysis (GSEA) was performed on RPE-specific and endothelium-associated gene sets. The complement factor H (CFH) genotype was also assessed, and differential expression was analyzed regarding high AMD risk (YH/HH) and low AMD risk (YY) genotypes. Results Seventy-five genes were identified as differentially expressed (raw p value <0.01; ≥50% fold change, mean log2 expression level in AMD or control ≥ median of all average gene expression values); however, no genes were significant (adj. p value <0.01) after correction for multiple hypothesis testing. Of 52 genes with decreased expression in AMD (fold change <0.5; raw p value <0.01), 18 genes were identified by DAVID analysis as associated with vision or neurologic processes. The GSEA of the RPE-associated and endothelium-associated genes revealed a significant decrease in genes typically expressed by endothelial cells in the early AMD group compared to controls, consistent with previous histologic and proteomic studies. Analysis of the CFH genotype indicated decreased expression of ADAMTS9 in eyes with high-risk genotypes (fold change = –2.61; raw p value=0.0008). Conclusions GSEA results suggest that RPE transcripts are preserved or elevated in early AMD, concomitant with loss of endothelial cell marker

  13. Microsecond Molecular Dynamics Simulations of Influenza Neuraminidase Suggest a Mechanism for the Increased Virulence of Stalk-Deletion Mutants

    PubMed Central

    2016-01-01

    Deletions in the stalk of the influenza neuraminidase (NA) surface protein are associated with increased virulence, but the mechanisms responsible for this enhanced virulence are unclear. Here we use microsecond molecular dynamics simulations to explore the effect of stalk deletion on enzymatic activity, contrasting NA proteins from the A/swine/Shandong/N1/2009 strain both with and without a stalk deletion. By modeling and simulating neuraminidase apo glycoproteins embedded in complex-mixture lipid bilayers, we show that the geometry and dynamics of the neuraminidase enzymatic pocket may differ depending on stalk length, with possible repercussions on the binding of the endogenous sialylated-oligosaccharide receptors. We also use these simulations to predict previously unrecognized druggable “hotspots” on the neuraminidase surface that may prove useful for future efforts aimed at structure-based drug design. PMID:27141956

  14. Microsecond Molecular Dynamics Simulations of Influenza Neuraminidase Suggest a Mechanism for the Increased Virulence of Stalk-Deletion Mutants.

    PubMed

    Durrant, Jacob D; Bush, Robin M; Amaro, Rommie E

    2016-08-25

    Deletions in the stalk of the influenza neuraminidase (NA) surface protein are associated with increased virulence, but the mechanisms responsible for this enhanced virulence are unclear. Here we use microsecond molecular dynamics simulations to explore the effect of stalk deletion on enzymatic activity, contrasting NA proteins from the A/swine/Shandong/N1/2009 strain both with and without a stalk deletion. By modeling and simulating neuraminidase apo glycoproteins embedded in complex-mixture lipid bilayers, we show that the geometry and dynamics of the neuraminidase enzymatic pocket may differ depending on stalk length, with possible repercussions on the binding of the endogenous sialylated-oligosaccharide receptors. We also use these simulations to predict previously unrecognized druggable "hotspots" on the neuraminidase surface that may prove useful for future efforts aimed at structure-based drug design. PMID:27141956

  15. Molecular response of liver sinusoidal endothelial cells on hydrogels.

    PubMed

    Bartneck, Matthias; Topuz, Fuat; Tag, Carmen Gabriele; Sauer-Lehnen, Sibille; Warzecha, Klaudia Theresa; Trautwein, Christian; Weiskirchen, Ralf; Tacke, Frank

    2015-06-01

    There is a high demand for the isolation of primary endothelial cells for biomaterial endotheliazation studies, tissue engineering, and artificial organ development. Further, biomarkers for monitoring the response of endothelial cells in biomaterials science are required. We systematically compared two strategies for isolating liver sinusoidal endothelial cells (LSEC) from mouse liver. We demonstrate that fluorescence-activated cell sorting results in a considerably higher purity (~97%) compared to magnetic-assisted cell sorting (~80%), but is associated with a lower yield and recovery rate. Cell repellent polyethylene glycol (PEG) substrates affected the morphology of primary LSEC in culture and significantly downregulated the intracellular adhesion molecule (ICAM) and upregulated the vascular cell adhesion molecule (VCAM). This molecular response could partially be reverted by further modification with arginylglycylaspartic acid (RGD). Thus, usage of PEGylated materials may reduce, while applying RGD may support endotheliazation of materials, and we could relate LSEC attachment to their expression of ICAM and VCAM mRNA, suggesting their usage as biomarkers for endothelialization. PMID:25842109

  16. Immunoglobulin VH Gene Mutational Analysis Suggests that Primary Effusion Lymphomas Derive from Different Stages of B Cell Maturation

    PubMed Central

    Matolcsy, András; Nádor, Roland G.; Cesarman, Ethel; Knowles, Daniel M.

    1998-01-01

    Primary effusion lymphoma (PEL) is a recently described distinct subtype of non-Hodgkin’s lymphoma associated with infection by the Kaposi’s sarcoma-associated herpesvirus, also called human herpesvirus-8. Most cases of PEL are also associated with the Epstein-Barr virus (EBV). In order to better characterize the cellular origin of PEL, we investigated the immunoglobulin (Ig) heavy chain variable region (VH) genes expressed by tumor cells of the BC-1 and BC-3 cell lines derived from PELs and five original PEL specimens. In the six EBV-positive PELs examined, including the BC-1 cell line, the expressed VH gene sequences showed numerous point mutations relative to the putative germline VH gene sequences. In addition, the VH segment of one of these cases showed intraclonal sequence heterogeneity, indicating ongoing somatic mutation. In five cases, the distribution and type of mutations indicated that tumor cells had been selected by antigen. Because somatically mutated Ig genes are expressed by B cells that have reached a germinal center/post-germinal center stage of development, these findings suggest that the PEL cell of origin is a germinal center or post-germinal center B cell in most cases. In contrast, the VH gene segment expressed by tumor cells of the BC-3 cell line, which was originated from an EBV-negative PEL obtained from an HIV-negative patient, was unmutated, suggesting a pre-germinal center B cell origin for tumor cells of this particular PEL cell line. Taken together, these findings suggest that development of PELs may not be restricted to one stage of B cell differentiation and may represent transformation of B cells at different stages of ontogeny. PMID:9811353

  17. Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes.

    PubMed

    Senderskiy, Igor V; Timofeev, Sergey A; Seliverstova, Elena V; Pavlova, Olga A; Dolgikh, Viacheslav V

    2014-01-01

    Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs. PMID:24705470

  18. Secretion of Antonospora (Paranosema) locustae Proteins into Infected Cells Suggests an Active Role of Microsporidia in the Control of Host Programs and Metabolic Processes

    PubMed Central

    Senderskiy, Igor V.; Timofeev, Sergey A.; Seliverstova, Elena V.; Pavlova, Olga A.; Dolgikh, Viacheslav V.

    2014-01-01

    Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs. PMID:24705470

  19. Molecular genetics of testicular germ cell tumors

    PubMed Central

    Sheikine, Yuri; Genega, Elizabeth; Melamed, Jonathan; Lee, Peng; Reuter, Victor E.; Ye, Huihui

    2012-01-01

    Testicular germ cell tumors (TGCT) are the most common malignancy in young men. While most TGCT are potentially curable, approximately 5% of patients with TGCT may develop chemoresistance and die from the disease. This review article summarizes current knowledge in genetics underlying the development, progression and chemoresistance of TGCT. Most post-pubertal TGCT originate from intratubular germ cell neoplasia unclassified (IGCNU), which are transformed fetal gonocytes. Development of IGCNU may involve aberrantly activated KITLG/KIT pathway and overexpression of embryonic transcription factors such as NANOG and POU5F1, which leads to suppression of apoptosis, increased proliferation, and accumulation of mutations in gonocytes. Invasive TGCT consistently show gain of chromosome 12p, typically isochromosome 12p. Single gene mutations are uncommon in TGCT. KIT, TP53, KRAS/NRAS, and BRAF are genes most commonly mutated in TGCT and implicated in their pathogenesis. Different histologic subtypes of TGCT possess different gene expression profiles that reflect different directions of differentiation. Their distinct gene expression profiles are likely caused by epigenetic regulation, in particular DNA methylation, but not by gene copy number alterations. Resistance of TGCT to chemotherapy has been linked to karyotypic aberrations, single-gene mutations, and epigenetic regulation of gene expression in small-scale studies. The study of TGCT genetics could ultimately translate into development of new molecular diagnostic and therapeutic modalities for these tumors and improve the care of patients with these malignancies. PMID:22432056

  20. Giant cell tumour of the sacrum: a suggested algorithm for treatment

    PubMed Central

    Grimer, R. J.; Carter, S. R.; Stirling, A. J.; Spilsbury, J.; Spooner, D.

    2010-01-01

    To investigate the outcome of our management of patients with giant cell tumour of the sacrum and draw lessons from this. A retrospective review of medical records and scans for all patients treated at our unit over the past 20 years with a giant cell tumour of the sacrum. Of the 517 patients treated at our unit for giant cell tumour over the past 20 years, only 9 (1.7%) had a giant cell tumour in the sacrum. Six were female, three male with a mean age of 34 (range 15–52). All, but two tumours involved the entire sacrum and there was only one purely distal to S3. The mean size was 10 cm and the most common symptom was back or buttock pain. Five had abnormal neurology at diagnosis, but only one presented with cauda equina syndrome. The first four patients were treated by curettage alone, but two patients had intraoperative cardiac arrests and although both survived all subsequent curettages were preceded by embolisation of the feeding vessels. Of the seven patients who had curettage, three developed local recurrence, but all were controlled with a combination of further embolisation, surgery or radiotherapy. One patient elected for treatment with radiotherapy and another had excision of the tumour distal to S3. All the patients are alive and only two patients have worse neurology than at presentation, one being impotent and one with stress incontinence. Three patients required spinopelvic fusion for sacral collapse. All patients are mobile and active at a follow-up between 2 and 21 years. Giant cell tumour of the sacrum can be controlled with conservative surgery rather than subtotal sacrectomy. The excision of small distal tumours is the preferred option, but for larger and more extensive tumours conservative management may well avoid morbidity whilst still controlling the tumour. Embolisation and curettage are the preferred first option with radiotherapy as a possible adjunct. Spinopelvic fusion may be needed when the sacrum collapses. PMID:20076978

  1. Difficulty distinguishing benign notochordal cell tumor from chordoma further suggests a link between them

    PubMed Central

    2014-01-01

    Background Much discussion about benign notochordal cell tissue in vertebrae has centered on the nature of its relationship, if any, to chordoma. Often referred to as benign notochordal cell tumors (BNCTs), these lesions have unique morphological features, however, differentiating between notochordal cells in discs, BNCT, and chordoma can be difficult. They are described as radiologically distinct from chordoma, with lysis, contrast enhancement, and a soft tissue mass indicating chordoma. Methods All chordomas diagnosed at our institution, the Istituto Ortopedico Rizzoli (Bologna, Italy), prior to 2008 were reviewed, yielding 174 cases. Five were limited to bone; one was a recurrent chordoma without original data available. The remaining four were re-evaluated in detail. Results There were three women and one man, aged 33–57 years (mean, 48 years). Two were BNCTs and two were mixed lesions containing BNCT and chordoma. On computed tomography, all were radiopaque with areas of lysis. One BNCT was heterogeneous on magnetic resonance imaging, enhancing after contrast. Microscopically, one BNCT had a well-defined cystic area with a sclerotic border. The other had a minute atypical area; it recurred as chordoma. The mixed lesions had areas of definitive BNCT, definitive chordoma, and atypical areas that did not meet the criteria for either. The atypical areas in all three cases ‘blended’ with areas of chordoma or BNCT. Conclusion These cases illustrate the ongoing challenges in differentiating between BNCT and chordoma. All had unique imaging features; three had atypical microscopic areas blending with BNCT or chordoma, strengthening the argument for a relationship between the two entities and supporting the idea that some BNCTs may progress to chordoma. Our study dispels the notion that any single radiologic criterion used to distinguish between chordoma and BNCT is reliable, opening the discussion as to whether or how to monitor BNCTs. PMID:25609192

  2. Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome

    PubMed Central

    Beššeová, Ivana; Réblová, Kamila; Leontis, Neocles B.; Šponer, Jiří

    2010-01-01

    We present extensive explicit solvent molecular dynamics analysis of three RNA three-way junctions (3WJs) from the large ribosomal subunit: the 3WJ formed by Helices 90–92 (H90–H92) of 23S rRNA; the 3WJ formed by H42–H44 organizing the GTPase associated center (GAC) of 23S rRNA; and the 3WJ of 5S rRNA. H92 near the peptidyl transferase center binds the 3′-CCA end of amino-acylated tRNA. The GAC binds protein factors and stimulates GTP hydrolysis driving protein synthesis. The 5S rRNA binds the central protuberance and A-site finger (ASF) involved in bridges with the 30S subunit. The simulations reveal that all three 3WJs possess significant anisotropic hinge-like flexibility between their stacked stems and dynamics within the compact regions of their adjacent stems. The A-site 3WJ dynamics may facilitate accommodation of tRNA, while the 5S 3WJ flexibility appears to be essential for coordinated movements of ASF and 5S rRNA. The GAC 3WJ may support large-scale dynamics of the L7/L12-stalk region. The simulations reveal that H42–H44 rRNA segments are not fully relaxed and in the X-ray structures they are bent towards the large subunit. The bending may be related to L10 binding and is distributed between the 3WJ and the H42–H97 contact. PMID:20507916

  3. Comparative modeling and molecular dynamics suggest high carboxylase activity of the Cyanobium sp. CACIAM14 RbcL protein.

    PubMed

    Siqueira, Andrei Santos; Lima, Alex Ranieri Jerônimo; Dall'Agnol, Leonardo Teixeira; de Azevedo, Juliana Simão Nina; da Silva Gonçalves Vianez, João Lídio; Gonçalves, Evonnildo Costa

    2016-03-01

    Rubisco catalyzes the first step reaction in the carbon fixation pathway, bonding atmospheric CO2/O2 to ribulose 1,5-bisphosphate; it is therefore considered one of the most important enzymes in the biosphere. Genetic modifications to increase the carboxylase activity of rubisco are a subject of great interest to agronomy and biotechnology, since this could increase the productivity of biomass in plants, algae and cyanobacteria and give better yields in crops and biofuel production. Thus, the aim of this study was to characterize in silico the catalytic domain of the rubisco large subunit (rbcL gene) of Cyanobium sp. CACIAM14, and identify target sites to improve enzyme affinity for ribulose 1,5-bisphosphate. A three-dimensional model was built using MODELLER 9.14, molecular dynamics was used to generate a 100 ns trajectory by AMBER12, and the binding free energy was calculated using MM-PBSA, MM-GBSA and SIE methods with alanine scanning. The model obtained showed characteristics of form-I rubisco, with 15 beta sheets and 19 alpha helices, and maintained the highly conserved catalytic site encompassing residues Lys175, Lys177, Lys201, Asp203, and Glu204. The binding free energy of the enzyme-substrate complexation of Cyanobium sp. CACIAM14 showed values around -10 kcal mol(-1) using the SIE method. The most important residues for the interaction with ribulose 1,5-bisphosphate were Arg295 followed by Lys334. The generated model was successfully validated, remaining stable during the whole simulation, and demonstrated characteristics of enzymes with high carboxylase activity. The binding analysis revealed candidates for directed mutagenesis sites to improve rubisco's affinity. PMID:26936271

  4. Physiological and molecular responses to heavy metal stresses suggest different detoxification mechanism of Populus deltoides and P. x canadensis.

    PubMed

    Benyó, Dániel; Horváth, Edit; Németh, Edit; Leviczky, Tünde; Takács, Kinga; Lehotai, Nóra; Feigl, Gábor; Kolbert, Zsuzsanna; Ördög, Attila; Gallé, Róbert; Csiszár, Jolán; Szabados, László; Erdei, László; Gallé, Ágnes

    2016-08-20

    Plants have divergent defense mechanisms against the harmful effects of heavy metals present in excess in soils and groundwaters. Poplars (Populus spp.) are widely cultivated because of their rapid growth and high biomass production, and members of the genus are increasingly used as experimental model organisms of trees and for phytoremediation purposes. Our aim was to investigate the copper and zinc stress responses of three outstanding biomass producer bred poplar lines to identify such transcripts of genes involved in the detoxification mechanisms, which can play an important role in the protection against heavy metals. Poplar cuttings were grown hydroponically and subjected to short-term (one week) mild and sublethal copper and zinc stresses. We evaluated the effects of the applied heavy metals and the responses of plants by detecting the changes of multiple physiological and biochemical parameters. The most severe cellular oxidative damage was caused by 30μM copper treatment, while zinc was less harmful. Analysis of stress-related transcripts revealed genotype-specific differences that are likely related to alterations in heavy metal tolerance. P. deltoides clones B-229 and PE 19/66 clones were clearly more effective at inducing the expression of various genes implicated in the detoxification process, such as the glutathione transferases, metallothioneins, ABC transporters, (namely PtGSTU51, PxMT1, PdABCC2,3), while the P. canadensis line M-1 accumulated more metal, resulting in greater cellular oxidative damage. Our results show that all three poplar clones are efficient in stress acclimatization, but with different molecular bases. PMID:27448721

  5. Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Cid, V J; Durán, A; del Rey, F; Snyder, M P; Nombela, C; Sánchez, M

    1995-01-01

    In fungi and many other organisms, a thick outer cell wall is responsible for determining the shape of the cell and for maintaining its integrity. The budding yeast Saccharomyces cerevisiae has been a useful model organism for the study of cell wall synthesis, and over the past few decades, many aspects of the composition, structure, and enzymology of the cell wall have been elucidated. The cell wall of budding yeasts is a complex and dynamic structure; its arrangement alters as the cell grows, and its composition changes in response to different environmental conditions and at different times during the yeast life cycle. In the past few years, we have witnessed a profilic genetic and molecular characterization of some key aspects of cell wall polymer synthesis and hydrolysis in the budding yeast. Furthermore, this organism has been the target of numerous recent studies on the topic of morphogenesis, which have had an enormous impact on our understanding of the intracellular events that participate in directed cell wall synthesis. A number of components that direct polarized secretion, including those involved in assembly and organization of the actin cytoskeleton, secretory pathways, and a series of novel signal transduction systems and regulatory components have been identified. Analysis of these different components has suggested pathways by which polarized secretion is directed and controlled. Our aim is to offer an overall view of the current understanding of cell wall dynamics and of the complex network that controls polarized growth at particular stages of the budding yeast cell cycle and life cycle. PMID:7565410

  6. Molecular signatures in response to Isoliquiritigenin in lymphoblastoid cell lines

    SciTech Connect

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young; Hwang, Meeyul; Kim, Ji-Hyun; Han, Bok-Ghee; Jeon, Jae-Pil

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12 putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.

  7. Molecular properties of a venom allergen-like protein suggest a parasitic function in the pinewood nematode Bursaphelenchus xylophilus.

    PubMed

    Kang, Jae Soon; Koh, Young Ho; Moon, Yil Sung; Lee, Si Hyeock

    2012-01-01

    The pinewood nematode, Bursaphelenchus xylophilus, is a destructive pest in several countries including Japan, China and Korea. Of three genes encoding the venom allergen-like protein in B. xylophilus, Bxvap-1 showed the highest transcript levels at the pine-grown propagative stage. In addition, western blot and immunohistochemical analyses using anti-BxVap-1 polyclonal antibody verified a specific increase in BxVap-1 expression levels at the pine-grown propagative stage. Using immunohistochemistry, BxVap-1 was detected around the putative oesophageal glands and metacarpus, suggesting that BxVap-1 is secreted into the host pine tree and is involved in the parasitic mechanism. To explain the parasitic role of BxVap-1, we measured the migration rate inside pine seedlings of B. xylophilus either with or without Bxvap-1 knockdown by RNA interference. Bxvap-1 knockdown resulted in a significantly lower migration rate in the >6cm region compared with the control B. xylophilus. These results suggest that BxVap-1 is involved in B. xylophilus migration, perhaps by suppressing the pine tree defence mechanism. PMID:22142561

  8. BROADBAND TRANSMISSION SPECTROSCOPY OF THE SUPER-EARTH GJ 1214b SUGGESTS A LOW MEAN MOLECULAR WEIGHT ATMOSPHERE

    SciTech Connect

    Croll, Bryce; Jayawardhana, Ray; Albert, Loic; Kempton, Eliza Miller-Ricci; Fortney, Jonathan J.; Murray, Norman; Neilson, Hilding

    2011-08-01

    We use the Wide-field Infrared Camera (WIRCam) on the Canada-France-Hawaii Telescope to observe four transits of the super-Earth planet GJ 1214b in the near-infrared. For each transit, we observe GJ 1214 in two bands nearly simultaneously by rapidly switching the WIRCam filter wheel back and forth for the duration of the observations. By combining all our J-band ({approx}1.25 {mu}m) observations we find a transit depth, analogous to the planet-to-star radius ratio squared, in this band of (R{sub PJ} /R{sub *}){sup 2} = (1.338 {+-} 0.013)%-a value consistent with the optical transit depth reported by Charbonneau and collaborators. However, our best-fit combined K{sub s}-band ({approx}2.15 {mu}m) transit depth is deeper: (R{sub PKs} /R{sub *}){sup 2} = (1.438 {+-} 0.019)%. Formally, our K{sub s}-band transits are deeper than the J-band transits observed simultaneously by a factor of (R{sub PKs} /R{sub PJ}){sup 2} = 1.072 {+-} 0.018-a 4{sigma} discrepancy. The most straightforward explanation for our deeper K{sub s}-band transit depth is a spectral absorption feature from the limb of the atmosphere of the planet; for the spectral absorption feature to be this prominent, the atmosphere of GJ 1214b must have a large-scale height and a low mean molecular weight. That is, its atmosphere would have to be hydrogen/helium dominated and this planet would be better described as a mini-Neptune. However, recently published observations from 0.78 to 1.0 {mu}m, by Bean and collaborators, show a lack of spectral features and transit depths consistent with those obtained by Charbonneau and collaborators. The most likely atmospheric composition for GJ 1214b that arises from combining all these observations is less clear; if the atmosphere of GJ 1214b is hydrogen/helium dominated, then it must have either a haze layer that is obscuring transit-depth differences at shorter wavelengths or significantly different spectral features from what current models predict. Our observations

  9. A Diagnostic Assessment for Introductory Molecular and Cell Biology

    ERIC Educational Resources Information Center

    Shi, Jia; Wood, William B.; Martin, Jennifer M.; Guild, Nancy A.; Vicens, Quentin; Knight, Jennifer K.

    2010-01-01

    We have developed and validated a tool for assessing understanding of a selection of fundamental concepts and basic knowledge in undergraduate introductory molecular and cell biology, focusing on areas in which students often have misconceptions. This multiple-choice Introductory Molecular and Cell Biology Assessment (IMCA) instrument is designed…

  10. Ameloblastin expression and putative autoregulation in mesenchymal cells suggest a role in early bone formation and repair

    PubMed Central

    Tamburstuen, Margareth V.; Reseland, Janne E.; Spahr, Axel; Brookes, Steven J.; Kvalheim, Gunnar; Slaby, Ivan; Snead, Malcolm L.; Lyngstadaas, S. Petter

    2015-01-01

    Ameloblastin is mainly known as a dental enamel protein, synthesized and secreted into developing enamel matrix by the enamel-forming ameloblasts. The function of ameloblastin in tooth development remains unclear, but it has been suggested to be involved in processes varying from regulating crystal growth to activity as a growth factor or partaking in cell signaling. Recent studies suggest that some enamel matrix proteins also might have important functions outside enamel formation. In this context ameloblastin has recently been reported to induce dentin and bone repair, as well as being present in the early bone and cartilage extracellular matrices during embryogenesis. However, what cells express ameloblastin in these tissues still remain unclear. Thus, the expression of ameloblastin was examined in cultured primary mesenchymal cells and in vivo during healing of bone defects in a “proof of concept” animal study. The real time RT-PCR analysis revealed human ameloblastin (AMBN) mRNA expression in human mesenchymal stem cells and primary osteoblasts and chondrocytes. Expression of AMBN mRNA was also confirmed in human CD34 positive cells and osteoclasts. Western and dot blot analysis of cell lysates and medium confirmed the expression and secretion of ameloblastin from mesenchymal stem cells, primary human osteoblasts and chondrocytes. Expression of ameloblastin was also detected in newly formed bone in experimental bone defects in adult rats. Together these findings suggest a role of this protein in early bone formation and repair. PMID:20854943

  11. Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin

    PubMed Central

    Corrao, Simona; Anzalone, Rita; Lo Iacono, Melania; Corsello, Tiziana; Di Stefano, Antonino; D'Anna, Silvestro Ennio; Balbi, Bruno; Carone, Mauro; Sala, Anna; Corona, Davide; Timperio, Anna Maria; Zolla, Lello; Farina, Felicia; Conway de Macario, Everly; Macario, Alberto J. L.; Cappello, Francesco; La Rocca, Giampiero

    2014-01-01

    Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein. PMID:25355063

  12. Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin.

    PubMed

    Corrao, Simona; Anzalone, Rita; Lo Iacono, Melania; Corsello, Tiziana; Di Stefano, Antonino; D'Anna, Silvestro Ennio; Balbi, Bruno; Carone, Mauro; Sala, Anna; Corona, Davide; Timperio, Anna Maria; Zolla, Lello; Farina, Felicia; de Macario, Everly Conway; Macario, Alberto J L; Cappello, Francesco; La Rocca, Giampiero

    2014-10-01

    Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein. PMID:25355063

  13. HLA class I molecular variation and peptide-binding properties suggest a model of joint divergent asymmetric selection.

    PubMed

    Buhler, Stéphane; Nunes, José Manuel; Sanchez-Mazas, Alicia

    2016-07-01

    The main function of HLA class I molecules is to present pathogen-derived peptides to cytotoxic T lymphocytes. This function is assumed to drive the maintenance of an extraordinary amount of polymorphism at each HLA locus, providing an immune advantage to heterozygote individuals capable to present larger repertories of peptides than homozygotes. This seems contradictory, however, with a reduced diversity at individual HLA loci exhibited by some isolated populations. This study shows that the level of functional diversity predicted for the two HLA-A and HLA-B genes considered simultaneously is similar (almost invariant) between 46 human populations, even when a reduced diversity exists at each locus. We thus propose that HLA-A and HLA-B evolved through a model of joint divergent asymmetric selection conferring all populations an equivalent immune potential. The distinct pattern observed for HLA-C is explained by its functional evolution towards killer cell immunoglobulin-like receptor (KIR) activity regulation rather than peptide presentation. PMID:27233953

  14. Molecular modeling and docking of novel laccase from multiple serotype of Yersinia enterocolitica suggests differential and multiple substrate binding.

    PubMed

    Singh, Deepti; Sharma, Krishna Kant; Dhar, Mahesh Shanker; Virdi, Jugsharan Singh

    2014-06-20

    Multi-copper oxidases (MCOs) are widely distributed in bacteria, where they are responsible for metal homeostasis, acquisition and oxidation. Using specific primers, yacK coding for MCO was amplified from different serotypes of Yersinia enterocolitica biovar 1A. Homology modeling of the protein followed by docking with five well-known substrates for different MCO's (viz., 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid [ABTS], syringaldazine, L-tyrosine, ammonium ferrous sulfate and guaiacol), lignin monomers (Coniferyl alcohol, p-coumaryl alcohol and sinapyl alcohol) and two inhibitors i.e., kojic acid and N-hydroxyglycine was done. The docking gave maximum GoldScore i.e., 91.93 and 72.64 with ammonium ferrous sulfate and ABTS, respectively. Similarly, docking with ICM gave -82.10 and -83.61 docking score, confirming the protein to be true laccase with ferroxidase activity. Further, validation with ammonium ferrous sulfate as substrate gave laccase activity of 0.36Units/L/min. Guaiacol, L-tyrosine, and lignin monomers showed good binding affinity with protein models with GoldScores of 35.89, 41.82, 40.41, 41.12 and 43.10, respectively. The sequence study of all the cloned Yack genes showed serotype specific clade in dendrogram. There was distinct discrimination in the ligand binding affinity of Y. enterocolitica laccase, among strains of same clonal groups, suggesting it as a tool for phylogenetic studies. PMID:24832734

  15. Molecular Diversity of Anthracnose Pathogen Populations Associated with UK Strawberry Production Suggests Multiple Introductions of Three Different Colletotrichum Species

    PubMed Central

    Baroncelli, Riccardo; Zapparata, Antonio; Sarrocco, Sabrina; Sukno, Serenella A.; Lane, Charles R.; Thon, Michael R.; Vannacci, Giovanni; Holub, Eric; Sreenivasaprasad, Surapareddy

    2015-01-01

    Fragaria × ananassa (common name: strawberry) is a globally cultivated hybrid species belonging to Rosaceae family. Colletotrichum acutatum sensu lato (s.l.) is considered to be the second most economically important pathogen worldwide affecting strawberries. A collection of 148 Colletotrichum spp. isolates including 67 C. acutatum s.l. isolates associated with the phytosanitary history of UK strawberry production were used to characterize multi-locus genetic variation of this pathogen in the UK, relative to additional reference isolates that represent a worldwide sampling of the diversity of the fungus. The evidence indicates that three different species C. nymphaeae, C. godetiae and C. fioriniae are associated with strawberry production in the UK, which correspond to previously designated genetic groups A2, A4 and A3, respectively. Among these species, 12 distinct haplotypes were identified suggesting multiple introductions into the country. A subset of isolates was also used to compare aggressiveness in causing disease on strawberry plants and fruits. Isolates belonging to C. nymphaeae, C. godetiae and C. fioriniae representative of the UK anthracnose pathogen populations showed variation in their aggressiveness. Among the three species, C. nymphaeae and C. fioriniae appeared to be more aggressive compared to C. godetiae. This study highlights the genetic and pathogenic heterogeneity of the C. acutatum s.l. populations introduced into the UK linked to strawberry production. PMID:26086351

  16. Conformational Changes in the GM-CSF Receptor Suggest a Molecular Mechanism for Affinity Conversion and Receptor Signaling.

    PubMed

    Broughton, Sophie E; Hercus, Timothy R; Nero, Tracy L; Dottore, Mara; McClure, Barbara J; Dhagat, Urmi; Taing, Houng; Gorman, Michael A; King-Scott, Jack; Lopez, Angel F; Parker, Michael W

    2016-08-01

    The GM-CSF, IL-3, and IL-5 receptors constitute the βc family, playing important roles in inflammation, autoimmunity, and cancer. Typical of heterodimeric type I cytokine receptors, signaling requires recruitment of the shared subunit to the initial cytokine:α subunit binary complex through an affinity conversion mechanism. This critical process is poorly understood due to the paucity of crystal structures of both binary and ternary receptor complexes for the same cytokine. We have now solved the structure of the binary GM-CSF:GMRα complex at 2.8-Å resolution and compared it with the structure of the ternary complex, revealing distinct conformational changes. Guided by these differences we performed mutational and functional studies that, importantly, show GMRα interactions playing a major role in receptor signaling while βc interactions control high-affinity binding. These results support the notion that conformational changes underlie the mechanism of GM-CSF receptor activation and also suggest how related type I cytokine receptors signal. PMID:27396825

  17. Quantitative analysis of Plasmodium ookinete motion in three dimensions suggests a critical role for cell shape in the biomechanics of malaria parasite gliding motility.

    PubMed

    Kan, Andrey; Tan, Yan-Hong; Angrisano, Fiona; Hanssen, Eric; Rogers, Kelly L; Whitehead, Lachlan; Mollard, Vanessa P; Cozijnsen, Anton; Delves, Michael J; Crawford, Simon; Sinden, Robert E; McFadden, Geoffrey I; Leckie, Christopher; Bailey, James; Baum, Jake

    2014-05-01

    analysis suggests that the molecular basis of cell shape may, in addition to motor force, be a key adaptive strategy for malaria parasite dissemination and, as such, transmission. PMID:24612056

  18. Molecular Inferences Suggest Multiple Host Shifts of Rabies Viruses from Bats to Mesocarnivores in Arizona during 2001–2009

    PubMed Central

    Kuzmin, Ivan V.; Shi, Mang; Orciari, Lillian A.; Yager, Pamela A.; Velasco-Villa, Andres; Kuzmina, Natalia A.; Streicker, Daniel G.; Bergman, David L.; Rupprecht, Charles E.

    2012-01-01

    In nature, rabies virus (RABV; genus Lyssavirus, family Rhabdoviridae) represents an assemblage of phylogenetic lineages, associated with specific mammalian host species. Although it is generally accepted that RABV evolved originally in bats and further shifted to carnivores, mechanisms of such host shifts are poorly understood, and examples are rarely present in surveillance data. Outbreaks in carnivores caused by a RABV variant, associated with big brown bats, occurred repeatedly during 2001–2009 in the Flagstaff area of Arizona. After each outbreak, extensive control campaigns were undertaken, with no reports of further rabies cases in carnivores for the next several years. However, questions remained whether all outbreaks were caused by a single introduction and further perpetuation of bat RABV in carnivore populations, or each outbreak was caused by an independent introduction of a bat virus. Another question of concern was related to adaptive changes in the RABV genome associated with host shifts. To address these questions, we sequenced and analyzed 66 complete and 20 nearly complete RABV genomes, including those from the Flagstaff area and other similar outbreaks in carnivores, caused by bat RABVs, and representatives of the major RABV lineages circulating in North America and worldwide. Phylogenetic analysis demonstrated that each Flagstaff outbreak was caused by an independent introduction of bat RABV into populations of carnivores. Positive selection analysis confirmed the absence of post-shift changes in RABV genes. In contrast, convergent evolution analysis demonstrated several amino acids in the N, P, G and L proteins, which might be significant for pre-adaptation of bat viruses to cause effective infection in carnivores. The substitution S/T242 in the viral glycoprotein is of particular merit, as a similar substitution was suggested for pathogenicity of Nishigahara RABV strain. Roles of the amino acid changes, detected in our study, require

  19. Expression of essential B cell genes and immunoglobulin isotypes suggests active development and gene recombination during equine gestation.

    PubMed

    Tallmadge, Rebecca L; McLaughlin, Kristin; Secor, Erica; Ruano, Diana; Matychak, Mary Beth; Flaminio, M Julia B F

    2009-09-01

    Many features of the equine immune system develop during fetal life, yet the naïve or immature immune state of the neonate renders the foal uniquely susceptible to particular pathogens. RT-PCR and immunohistochemical experiments investigated the progressive expression of developmental B cell markers and immunoglobulins in lymphoid tissues from equine fetus, pre-suckle neonate, foal, and adult horses. Serum IgM, IgG isotype, and IgA concentrations were also quantified in pre-suckle foals and adult horses. The expression of essential B cell genes suggests active development and gene recombination during equine gestation, including immunoglobulin isotype switching. The corresponding production of IgM and IgG proteins is detectable in a limited scale at birth. Although the equine neonate humoral response seems competent, B cell activation factors derived from antigen presenting cells and T cells may control critical developmental regulation and immunoglobulin production during the initial months of life. PMID:19442687

  20. The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana

    PubMed Central

    Busse-Wicher, Marta; Gomes, Thiago C F; Tryfona, Theodora; Nikolovski, Nino; Stott, Katherine; Grantham, Nicholas J; Bolam, David N; Skaf, Munir S; Dupree, Paul

    2014-01-01

    The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan–cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy. PMID:24889696

  1. Molecular Pathogenesis of Sporadic Melanoma and Melanoma-Initiating Cells

    PubMed Central

    Kong, Yunyi; Kumar, Suresh M.; Xu, Xiaowei

    2014-01-01

    Recent advances in molecular genetics and cancer stem cell biology have shed some light on the molecular basis of melanomagenesis. In this review, we will focus on major genetic alterations in the melanoma, particularly pathways involved in cell proliferation, apoptosis, and tumor suppression. The potential role of melanoma-initiating cells during melanomagenesis and progression will also be discussed. Understanding pathogenesis of melanoma may uncover new diagnostic clues and therapeutic targets for this increasingly prevalent disease. PMID:21128770

  2. Quantum dot imaging platform for single-cell molecular profiling

    NASA Astrophysics Data System (ADS)

    Zrazhevskiy, Pavel; Gao, Xiaohu

    2013-03-01

    Study of normal cell physiology and disease pathogenesis heavily relies on untangling the complexity of intracellular molecular mechanisms and pathways. To achieve this goal, comprehensive molecular profiling of individual cells within the context of microenvironment is required. Here we report the development of a multicolour multicycle in situ imaging technology capable of creating detailed quantitative molecular profiles for individual cells at the resolution of optical imaging. A library of stoichiometric fluorescent probes is prepared by linking target-specific antibodies to a universal quantum dot-based platform via protein A in a quick and simple procedure. Surprisingly, despite the potential for multivalent binding between protein A and antibody and the intermediate affinity of this non-covalent bond, fully assembled probes do not aggregate or exchange antibodies, facilitating highly multiplexed parallel staining. This single-cell molecular profiling technology is expected to open new opportunities in systems biology, gene expression studies, signalling pathway analysis and molecular diagnostics.

  3. Disc cell senescence in intervertebral disc degeneration: Causes and molecular pathways

    PubMed Central

    Feng, Chencheng; Liu, Huan; Yang, Minghui; Zhang, Yang; Huang, Bo; Zhou, Yue

    2016-01-01

    ABSTRACT The accumulation of senescent disc cells in degenerative intervertebral disc (IVD) suggests the detrimental roles of cell senescence in the pathogenesis of intervertebral disc degeneration (IDD). Disc cell senescence decreased the number of functional cells in IVD. Moreover, the senescent disc cells were supposed to accelerate the process of IDD via their aberrant paracrine effects by which senescent cells cause the senescence of neighboring cells and enhance the matrix catabolism and inflammation in IVD. Thus, anti-senescence has been proposed as a novel therapeutic target for IDD. However, the development of anti-senescence therapy is based on our understanding of the molecular mechanism of disc cell senescence. In this review, we focused on the molecular mechanism of disc cell senescence, including the causes and various molecular pathways. We found that, during the process of IDD, age-related damages together with degenerative external stimuli activated both p53-p21-Rb and p16-Rb pathways to induce disc cell senescence. Meanwhile, disc cell senescence was regulated by multiple signaling pathways, suggesting the complex regulating network of disc cell senescence. To understand the mechanism of disc cell senescence better contributes to developing the anti-senescence-based therapies for IDD. PMID:27192096

  4. Genomic and molecular characterization of esophageal squamous cell carcinoma

    PubMed Central

    Lin, De-Chen; Hao, Jia-Jie; Nagata, Yasunobu; Xu, Liang; Shang, Li; Meng, Xuan; Sato, Yusuke; Okuno, Yusuke; Varela, Ana Maria; Ding, Ling-Wen; Garg, Manoj; Liu, Li-Zhen; Yang, Henry; Yin, Dong; Shi, Zhi-Zhou; Jiang, Yan-Yi; Gu, Wen-Yue; Gong, Ting; Zhang, Yu; Xu, Xin; Kalid, Ori; Shacham, Sharon; Ogawa, Seishi; Wang, Ming-Rong; Koeffler, H. Phillip

    2014-01-01

    Esophageal squamous cell carcinoma (ESCC) is a world-wide prevalent cancer, which is particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified novel significantly mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to previously discovered ones (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Moreover, our approaches uncovered many novel druggable candidates, and XPO1 was further explored as a therapeutic target because of its mutation and protein overexpression. Together, our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets. PMID:24686850

  5. Genomic and molecular characterization of esophageal squamous cell carcinoma.

    PubMed

    Lin, De-Chen; Hao, Jia-Jie; Nagata, Yasunobu; Xu, Liang; Shang, Li; Meng, Xuan; Sato, Yusuke; Okuno, Yusuke; Varela, Ana Maria; Ding, Ling-Wen; Garg, Manoj; Liu, Li-Zhen; Yang, Henry; Yin, Dong; Shi, Zhi-Zhou; Jiang, Yan-Yi; Gu, Wen-Yue; Gong, Ting; Zhang, Yu; Xu, Xin; Kalid, Ori; Shacham, Sharon; Ogawa, Seishi; Wang, Ming-Rong; Koeffler, H Phillip

    2014-05-01

    Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide and particularly common in certain regions of Asia. Here we report the whole-exome or targeted deep sequencing of 139 paired ESCC cases, and analysis of somatic copy number variations (SCNV) of over 180 ESCCs. We identified previously uncharacterized mutated genes such as FAT1, FAT2, ZNF750 and KMT2D, in addition to those already known (TP53, PIK3CA and NOTCH1). Further SCNV evaluation, immunohistochemistry and biological analysis suggested their functional relevance in ESCC. Notably, RTK-MAPK-PI3K pathways, cell cycle and epigenetic regulation are frequently dysregulated by multiple molecular mechanisms in this cancer. Our approaches also uncovered many druggable candidates, and XPO1 was further explored as a therapeutic target because it showed both gene mutation and protein overexpression. Our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets. PMID:24686850

  6. A conundrum in molecular toxicology: molecular and biological changes during neoplastic transformation of human cells.

    PubMed

    Milo, G E; Shuler, C F; Lee, H; Casto, B C

    1995-12-01

    , the populations exhibit phenotypic diversity in that many of the transformed cells differentiate and fail to continue to divide in culture. Historically, we have assumed only a limited role for epigenetic modulation of molecular changes that occur during progression; however, our data suggest quite strongly that nonmalignant tumor populations can be converted to a more malignant phenotype without additional mutations taking place and, conversely, malignant populations can be downregulated to a nontumorigenic phenotype. Tumor cell plasticity is not only a fundamental characteristic of diverse types of human tumors, but also appears as an integral characteristic of carcinogen-transformed cells in vitro. PMID:8788209

  7. Posterior elongation in the annelid Platynereis dumerilii involves stem cells molecularly related to primordial germ cells.

    PubMed

    Gazave, Eve; Béhague, Julien; Laplane, Lucie; Guillou, Aurélien; Préau, Laetitia; Demilly, Adrien; Balavoine, Guillaume; Vervoort, Michel

    2013-10-01

    Like most bilaterian animals, the annelid Platynereis dumerilii generates the majority of its body axis in an anterior to posterior temporal progression with new segments added sequentially. This process relies on a posterior subterminal proliferative body region, known as the "segment addition zone" (SAZ). We explored some of the molecular and cellular aspects of posterior elongation in Platynereis, in particular to test the hypothesis that the SAZ contains a specific set of stem cells dedicated to posterior elongation. We cloned and characterized the developmental expression patterns of orthologs of 17 genes known to be involved in the formation, behavior, or maintenance of stem cells in other metazoan models. These genes encode RNA-binding proteins (e.g., tudor, musashi, pumilio) or transcription factors (e.g., myc, id, runx) widely conserved in eumetazoans. Most of these genes are expressed both in the migrating primordial germ cells and in overlapping ring-like patterns in the SAZ, similar to some previously analyzed genes (piwi, vasa). The SAZ patterns are coincident with the expression of proliferation markers cyclin B and PCNA. EdU pulse and chase experiments suggest that new segments are produced through many rounds of divisions from small populations of teloblast-like posterior stem cells. The shared molecular signature between primordial germ cells and posterior stem cells in Platynereis thus corresponds to an ancestral "stemness" program. PMID:23891818

  8. Gene Expression Profile of Adult Human Olfactory Bulb and Embryonic Neural Stem Cell Suggests Distinct Signaling Pathways and Epigenetic Control

    PubMed Central

    Marei, Hany E. S.; Ahmed, Abd-Elmaksoud; Michetti, Fabrizio; Pescatori, Mario; Pallini, Roberto; Casalbore, Patricia; Cenciarelli, Carlo; Elhadidy, Mohamed

    2012-01-01

    Global gene expression profiling was performed using RNA from human embryonic neural stem cells (hENSC), and adult human olfactory bulb-derived neural stem cells (OBNSCs), to define a gene expression pattern and signaling pathways that are specific for each cell lineage. We have demonstrated large differences in the gene expression profile of human embryonic NSC, and adult human OBNSCs, but less variability between parallel cultures. Transcripts of genes involved in neural tube development and patterning (ALDH1A2, FOXA2), progenitor marker genes (LMX1a, ALDH1A1, SOX10), proliferation of neural progenitors (WNT1 and WNT3a), neuroplastin (NPTN), POU3F1 (OCT6), neuroligin (NLGN4X), MEIS2, and NPAS1 were up-regulated in both cell populations. By Gene Ontology, 325 out of 3875 investigated gene sets were scientifically different. 41 out of the 307 investigated Cellular Component (CC) categories, 45 out of the 620 investigated Molecular Function (MF) categories, and 239 out of the 2948 investigated Biological Process (BP) categories were significant. KEGG Pathway Class Comparison had revealed that 75 out of 171 investigated gene sets passed the 0.005 significance threshold. Levels of gene expression were explored in three signaling pathways, Notch, Wnt, and mTOR that are known to be involved in NS cell fates determination. The transcriptional signature also deciphers the role of genes involved in epigenetic modifications. SWI/SNF DNA chromatin remodeling complex family, including SMARCC1 and SMARCE1, were found specifically up-regulated in our OBNSC but not in hENSC. Differences in gene expression profile of transcripts controlling epigenetic modifications, and signaling pathways might indicate differences in the therapeutic potential of our examined two cell populations in relation to in cell survival, proliferation, migration, and differentiation following engraftments in different CNS insults. PMID:22485144

  9. The molecular signature of CD8+ T cells undergoing deletional tolerance

    PubMed Central

    Rao, Sudha; Smyth, Gordon K.; Juelich, Torsten; Denyer, Gareth S.; Davey, Gayle M.; Strasser, Andreas

    2009-01-01

    Peripheral tolerance induction is critical for the maintenance of self-tolerance and can be mediated by immunoregulatory T cells or by direct induction of T-cell anergy or deletion. Although the molecular processes underlying anergy have been extensively studied, little is known about the molecular basis for peripheral T-cell deletion. Here, we determined the gene expression signature of peripheral CD8+ T cells undergoing deletional tolerance, relative to those undergoing immunogenic priming or lymphopenia-induced proliferation. From these data, we report the first detailed molecular signature of cells undergoing deletion. Consistent with defective cytolysis, these cells exhibited deficiencies in granzyme up-regulation. Furthermore, they showed antigen-driven Bcl-2 down-regulation and early up-regulation of the proapoptotic protein Bim, consistent with the requirement of this BH3-only protein for peripheral T-cell deletion. Bim up-regulation was paralleled by defective interleukin-7 receptor α (IL-7Rα) chain reexpression, suggesting that Bim-dependent death may be triggered by loss of IL-7/IL-7R signaling. Finally, we observed parallels in molecular signatures between deletion and anergy, suggesting that these tolerance pathways may not be as molecularly distinct as previously surmised. PMID:19204323

  10. Human BCAS3 Expression in Embryonic Stem Cells and Vascular Precursors Suggests a Role in Human Embryogenesis and Tumor Angiogenesis

    PubMed Central

    Siva, Kavitha; Venu, Parvathy; Mahadevan, Anita; S. K., Shankar; Inamdar, Maneesha S.

    2007-01-01

    Cancer is often associated with multiple and progressive genetic alterations in genes that are important for normal development. BCAS3 (Breast Cancer Amplified Sequence 3) is a gene of unknown function on human chromosome 17q23, a region associated with breakpoints of several neoplasms. The normal expression pattern of BCAS3 has not been studied, though it is implicated in breast cancer progression. Rudhira, a murine WD40 domain protein that is 98% identical to BCAS3 is expressed in embryonic stem (ES) cells, erythropoiesis and angiogenesis. This suggests that BCAS3 expression also may not be restricted to mammary tissue and may have important roles in other normal as well as malignant tissues. We show that BCAS3 is also expressed in human ES cells and during their differentiation into blood vascular precursors. We find that BCAS3 is aberrantly expressed in malignant human brain lesions. In glioblastoma, hemangiopericytoma and brain abscess we note high levels of BCAS3 expression in tumor cells and some blood vessels. BCAS3 may be associated with multiple cancerous and rapidly proliferating cells and hence the expression, function and regulation of this gene merits further investigation. We suggest that BCAS3 is mis-expressed in brain tumors and could serve as a human ES cell and tumor marker. PMID:18030336

  11. Recombinant T-cell Receptor Ligands: Immunomodulatory, Neuroprotective and Neuroregenerative Effects Suggest Application as Therapy for Multiple Sclerosis

    PubMed Central

    Offner, Halina; Sinha, Sushmita; Wang, Chunhe; Burrows, Gregory G.; Vandenbark, Arthur A.

    2009-01-01

    Recombinant TCR ligands (RTL) represent the minimal interactive surface with antigen-specific T cell receptors. These novel constructs fold similarly to native four-domain MHC/peptide complexes but deliver suboptimal and qualitatively different signals that cause a “cytokine switch” to anti-inflammatory factors in targeted encephalitogenic T cells. RTL treatment can reverse clinical and histological signs of EAE and most dramatically can promote myelin and axonal recovery in the CNS of mice with chronic disease. These properties of RTL suggest that this novel antigen-specific approach may hold unusual promise as a therapy for multiple sclerosis. PMID:19145988

  12. Nano-guided cell networks as conveyors of molecular communication

    PubMed Central

    Terrell, Jessica L.; Wu, Hsuan-Chen; Tsao, Chen-Yu; Barber, Nathan B.; Servinsky, Matthew D.; Payne, Gregory F.; Bentley, William E.

    2015-01-01

    Advances in nanotechnology have provided unprecedented physical means to sample molecular space. Living cells provide additional capability in that they identify molecules within complex environments and actuate function. We have merged cells with nanotechnology for an integrated molecular processing network. Here we show that an engineered cell consortium autonomously generates feedback to chemical cues. Moreover, abiotic components are readily assembled onto cells, enabling amplified and ‘binned' responses. Specifically, engineered cell populations are triggered by a quorum sensing (QS) signal molecule, autoinducer-2, to express surface-displayed fusions consisting of a fluorescent marker and an affinity peptide. The latter provides means for attaching magnetic nanoparticles to fluorescently activated subpopulations for coalescence into colour-indexed output. The resultant nano-guided cell network assesses QS activity and conveys molecular information as a ‘bio-litmus' in a manner read by simple optical means. PMID:26455828

  13. Nano-guided cell networks as conveyors of molecular communication.

    PubMed

    Terrell, Jessica L; Wu, Hsuan-Chen; Tsao, Chen-Yu; Barber, Nathan B; Servinsky, Matthew D; Payne, Gregory F; Bentley, William E

    2015-01-01

    Advances in nanotechnology have provided unprecedented physical means to sample molecular space. Living cells provide additional capability in that they identify molecules within complex environments and actuate function. We have merged cells with nanotechnology for an integrated molecular processing network. Here we show that an engineered cell consortium autonomously generates feedback to chemical cues. Moreover, abiotic components are readily assembled onto cells, enabling amplified and 'binned' responses. Specifically, engineered cell populations are triggered by a quorum sensing (QS) signal molecule, autoinducer-2, to express surface-displayed fusions consisting of a fluorescent marker and an affinity peptide. The latter provides means for attaching magnetic nanoparticles to fluorescently activated subpopulations for coalescence into colour-indexed output. The resultant nano-guided cell network assesses QS activity and conveys molecular information as a 'bio-litmus' in a manner read by simple optical means. PMID:26455828

  14. Autonomous molecular cascades for evaluation of cell surfaces.

    PubMed

    Rudchenko, Maria; Taylor, Steven; Pallavi, Payal; Dechkovskaia, Alesia; Khan, Safana; Butler, Vincent P; Rudchenko, Sergei; Stojanovic, Milan N

    2013-08-01

    Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs. Previously studied nucleic acid-based automata include game-playing molecular devices (MAYA automata) and finite-state automata for the analysis of nucleic acids, with the latter inspiring circuits for the analysis of RNA species inside cells. Here, we describe automata based on strand-displacement cascades directed by antibodies that can analyse cells by using their surface markers as inputs. The final output of a molecular automaton that successfully completes its analysis is the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells. PMID:23892986

  15. COMPREHENSIVE MOLECULAR CHARACTERIZATION OF CLEAR CELL RENAL CELL CARCINOMA

    PubMed Central

    2013-01-01

    Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (e.g. VHL) and the maintenance of chromatin states (e.g. PBRM1). We surveyed more than 400 tumors using different genomic platforms and identified 19 significantly mutated genes. The PI3K/Akt pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodeling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving down-regulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, up-regulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 and GRB10. Remodeling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumor stage and severity and offers new views on the opportunities for disease treatment. PMID:23792563

  16. Extending the molecular clutch beyond actin-based cell motility

    NASA Astrophysics Data System (ADS)

    Havrylenko, Svitlana; Mezanges, Xavier; Batchelder, Ellen; Plastino, Julie

    2014-10-01

    Many cell movements occur via polymerization of the actin cytoskeleton beneath the plasma membrane at the front of the cell, forming a protrusion called a lamellipodium, while myosin contraction squeezes forward the back of the cell. In what is known as the ‘molecular clutch’ description of cell motility, forward movement results from the engagement of the acto-myosin motor with cell-matrix adhesions, thus transmitting force to the substrate and producing movement. However during cell translocation, clutch engagement is not perfect, and as a result, the cytoskeleton slips with respect to the substrate, undergoing backward (retrograde) flow in the direction of the cell body. Retrograde flow is therefore inversely proportional to cell speed and depends on adhesion and acto-myosin dynamics. Here we asked whether the molecular clutch was a general mechanism by measuring motility and retrograde flow for the Caenorhabditis elegans sperm cell in different adhesive conditions. These cells move by adhering to the substrate and emitting a dynamic lamellipodium, but the sperm cell does not contain an acto-myosin cytoskeleton. Instead the lamellipodium is formed by the assembly of major sperm protein, which has no biochemical or structural similarity to actin. We find that these cells display the same molecular clutch characteristics as acto-myosin containing cells. We further show that retrograde flow is produced both by cytoskeletal assembly and contractility in these cells. Overall this study shows that the molecular clutch hypothesis of how polymerization is transduced into motility via adhesions is a general description of cell movement regardless of the composition of the cytoskeleton.

  17. Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency

    PubMed Central

    Frankowiack, Marcel; Kierczak, Marcin; Bergvall, Kerstin; Axelsson, Erik; Tintle, Linda; Marti, Eliane; Roosje, Petra; Leeb, Tosso; Hedhammar, Åke; Hammarström, Lennart; Lindblad-Toh, Kerstin

    2015-01-01

    Immunoglobulin A deficiency (IgAD) is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS) to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei) identified 35 genomic loci suggestively associated (p <0.0005) to IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9) were genome-wide significantly associated (p <0.0002) with IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005) to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development. PMID:26225558

  18. The cell as the smallest DNA-based molecular computer.

    PubMed

    Ji, S

    1999-10-01

    The pioneering work of Adleman (1994) demonstrated that DNA molecules in test tubes can be manipulated to perform a certain type of mathematical computation. This has stimulated a theoretical interest in the possibility of constructing DNA-based molecular computers. To gauge the practicality of realizing such microscopic computers, it was thought necessary to learn as much as possible from the biology of the living cell--presently the only known DNA-based molecular computer in existence. Here the recently developed theoretical model of the living cell (the Bhopalator) and its associated theories (e.g. cell language), principles, laws and concepts (e.g. conformons, IDS's) are briefly reviewed and summarized in the form of a set of five laws of 'molecular semiotics' (synonyms include 'microsemiotics', 'cellular semiotics', or 'cytosemiotics') the study of signs mediating measurement, computation, and communication on the cellular and molecular levels. Hopefully, these laws will find practical applications in designing DNA-based computing systems. PMID:10636037

  19. Molecular barriers to processes of genetic reprogramming and cell transformation.

    PubMed

    Chestkov, I V; Khomyakova, E A; Vasilieva, E A; Lagarkova, M A; Kiselev, S L

    2014-12-01

    Genetic reprogramming by ectopic expression of transcription factor genes induces the pluripotent state in somatic cells. This technology provides an opportunity to establish pluripotent stem cells for each person, as well as to get better understanding of epigenetic mechanisms controlling cell state. Interestingly, some of the molecular processes that accompany somatic cell reprogramming in vitro are also characteristic for tumor manifestation. Thus, similar "molecular barriers" that control the stability of epigenetic state exist for both processes of pluripotency induction and malignant transformation. The reprogramming of tumor cells is interesting in two aspects: first, it will determine the contribution of epigenetic changes in carcinogenesis; second, it gives an approach to evaluate tumor stem cells that are supposed to form the entire cell mass of the tumor. This review discusses the key stages of genetic reprogramming, the similarity and difference between the reprogramming process and malignant transformation. PMID:25716723

  20. Where's the glass? Biomarkers, molecular clocks, and microRNAs suggest a 200-Myr missing Precambrian fossil record of siliceous sponge spicules

    NASA Astrophysics Data System (ADS)

    Sperling, E. A.; Robinson, J.; Pisani, D.; Peterson, K.

    2010-12-01

    The earliest evidence for animal life comes from the fossil record of 24-isopropylcholestane, a sterane found in Cryogenian deposits, and whose precursors are found in modern demosponges, but not choanoflagellates, calcareans, hexactinellids, or eumetazoans. However, many modern demosponges are also characterized by the presence of siliceous spicules, and there are no convincing demosponge spicules in strata older than the Cambrian. This temporal disparity highlights a problem with our understanding of the Precambrian fossil record - either these supposed demosponge-specific biomarkers were derived from the sterols of some other organism and are simply retained in modern demosponges, or spicules do not primitively characterize crown-group demosponges. Resolving this issue requires resolving the phylogenetic placement of another group of sponges, the hexactinellids, which not only make a spicule thought to be homologous to the spicules of demosponges, but also make their first appearance near the Precambrian/Cambrian boundary. Using two independent analytical approaches and data sets - traditional molecular phylogenetic analyses and the presence or absence of specific microRNA genes - we show that demosponges are monophyletic, and that hexactinellids are their sister group (together forming the Silicea). Thus, spicules must have evolved before the last common ancestor of all living siliceans, suggesting the presence of a significant gap in the silicean spicule fossil record. Molecular divergence estimates date the origin of this last common ancestor well within the Cryogenian, consistent with the biomarker record, and strongly suggests that siliceous spicules were present during the Precambrian but were not preserved.

  1. Expression of the spexin gene in the rat adrenal gland and evidences suggesting that spexin inhibits adrenocortical cell proliferation.

    PubMed

    Rucinski, Marcin; Porzionato, Andrea; Ziolkowska, Agnieszka; Szyszka, Marta; Macchi, Veronica; De Caro, Raffaele; Malendowicz, Ludwik K

    2010-04-01

    Spexin (SPX, also called NPQ) is a recently identified, highly conserved peptide which is processed and secreted. We analysed the SPX gene and its protein product in the rat adrenal gland to ascertain whether SPX is involved in the regulation of corticosteroid secretion of and growth of adrenocortical cells. In adult rat adrenal glands the highest levels of SPX mRNA were present in the glomerulosa (ZG) and fasciculate/reticularis (ZF/R) zones. High SPX gene expression levels were found in freshly isolated adult rat ZG and ZF/R cells. In cultured adrenocortical cells the levels of SPX mRNA were lower than in freshly isolated cells. SPX mRNA expression levels were found to be 2-3 times higher during days 90-540 of postnatal development than found during days 2-45. Prolonged ACTH administration lowered and dexamethasone increased adrenal SPX mRNA levels in vivo. Adrenal enucleation produced a significant linear increase in SPX mRNA levels, with the highest value occurring at day 8 after surgery, with control values taken on day 30 after enucleation. Immunohistochemistry revealed SPX-like immunoreactivity in the entire cortex of the adult male rat and in enucleation-induced regenerating cortex. A concentration of 10-6M SPX peptide stimulated basal aldosterone secretion by freshly isolated ZG. In prolonged exposure of adrenocortical cell primary cultures to SPX (10-6M) resulted in a small increase in corticosterone secretion and a notable decrease in BrdU incorporation. The results suggest the direct involvement of SPX in the regulation of adrenocortical cell proliferation; however, the mechanism of action remains unknown. PMID:20045034

  2. Effect of morphine on PC12 cells with molecular radar

    NASA Astrophysics Data System (ADS)

    Shi, Chen; Yu, Xiaoli; Lu, Jiuyi; Zhang, Chunyang; Jin, Lei; Ma, Hui; Zhang, Dacheng; Chen, Die Yan

    2000-10-01

    Molecular Radar (MR) is a new method to detect biological processes in living cells at the level of molecular, it is also the newest means to get intracellular information. In this paper we study the effect of morphine on PC12 cells using MR. The results show that the effect of morphine on PC12 cells is time- and concentration-dependent. Morphine treating for short time induces the increase and fluctuation of intracellular (CA2+), while morphine treating for long time induces chromatin condensation, loss of mitochondria membrane potential apoptosis.

  3. Tunable Single-Cell Extraction for Molecular Analyses.

    PubMed

    Guillaume-Gentil, Orane; Grindberg, Rashel V; Kooger, Romain; Dorwling-Carter, Livie; Martinez, Vincent; Ossola, Dario; Pilhofer, Martin; Zambelli, Tomaso; Vorholt, Julia A

    2016-07-14

    Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level. PMID:27419874

  4. Photodynamic Efficiency: From Molecular Photochemistry to Cell Death

    PubMed Central

    Bacellar, Isabel O. L.; Tsubone, Tayana M.; Pavani, Christiane; Baptista, Mauricio S.

    2015-01-01

    Photodynamic therapy (PDT) is a clinical modality used to treat cancer and infectious diseases. The main agent is the photosensitizer (PS), which is excited by light and converted to a triplet excited state. This latter species leads to the formation of singlet oxygen and radicals that oxidize biomolecules. The main motivation for this review is to suggest alternatives for achieving high-efficiency PDT protocols, by taking advantage of knowledge on the chemical and biological processes taking place during and after photosensitization. We defend that in order to obtain specific mechanisms of cell death and maximize PDT efficiency, PSes should oxidize specific molecular targets. We consider the role of subcellular localization, how PS photochemistry and photophysics can change according to its nanoenvironment, and how can all these trigger specific cell death mechanisms. We propose that in order to develop PSes that will cause a breakthrough enhancement in the efficiency of PDT, researchers should first consider tissue and intracellular localization, instead of trying to maximize singlet oxygen quantum yields in in vitro tests. In addition to this, we also indicate many open questions and challenges remaining in this field, hoping to encourage future research. PMID:26334268

  5. The Molecular Basis of Communication between Cells.

    ERIC Educational Resources Information Center

    Snyder, Solomon H.

    1985-01-01

    Chemical messengers mediate long-range hormonal communication and short-range neural communication between cells. Background information on peptides, steroids, neuropeptides, and specialized enzymes is given. Investigations reveal that the two systems have many common intercellular messenger molecules. (DH)

  6. Cell and molecular biology of Chlamydomonas

    SciTech Connect

    Not Available

    1988-01-01

    This document contains only the abstracts of 92 presentations on the biology of Chlamydomonas. Topics include gene transformations, gene regulation, biosynthetic pathways, cell surfaces, circadian clocks, and the development and structure of the flagellar apparatus. (TEM)

  7. Characterization of V beta-bearing cells in athymic (nu/nu) mice suggests an extrathymic pathway for T cell differentiation.

    PubMed

    Rocha, B

    1990-04-01

    In the present article, the expression of the T cell receptor (TcR) beta chain and other T cell molecules was evaluated in surface immunoglobulin-negative spleen cell populations of young and old BALB/c and C57BL/6 nude mice, using a panel of monoclonal antibodies. The results obtained show that in young nude mice, most Thy-1high cells do not express other T cell markers. These mice have, however, a sizable population of Thy-1low cells with the same phenotype of alpha/beta+, CD4-CD8- thymocytes or MRL/lpr peripheral T cells, expressing predominantly genes of the V beta 8 family. The evolution of alpha/beta+ cells in aging nudes is strongly suggestive of an extrathymic pathway of differentiation of these cells since (a) the acquisition of high density TcR and CD3, as well as Thy-1 or CD4CD8 antigens at the cell surface of nude V beta+ T cells is not simultaneous; (b) alpha/beta+ cells in nude mice co-express other T cell markers at random and, even in old mice, they never completely resemble to the predominant high Thy-1+ CD3+ TcR alpha/beta+, CD4+CD8+ cells of euthymic controls; and (c) BALB/c nude T cells express V beta 11 genes, that are deleted in euthymic BALB/c mice. This latter finding may also indicate differences in the mechanisms of selection of T cells specificities in the thymus vs. the peripheral pools. PMID:1971795

  8. [Molecular pathology of plasma cell neoplasms].

    PubMed

    Fend, F

    2010-10-01

    Plasma cell myeloma (PCM) and related immunosecretory disorders are a group of B-cell proliferations with a wide clinical and prognostic spectrum, characterized by the production of monoclonal immunoglobulin by immortalized plasma cells. Recent years have seen an explosion in knowledge on the genetic basis and biology of these diseases, followed by improved clinical risk stratification and the introduction of novel therapeutic concepts, such as treatment with proteasome inhibitors or immunomodulatory substances. PCM is a common malignancy, accounting for approximately 10% of all hematological neoplasms. There is good evidence to support a multistep transformation process in plasma cell neoplasms, which corresponds to clinically discernible disease stages. Monoclonal gammopathy of unknown significance is a common asymptomatic precursor lesion for PCM which carries an approximately 1% annual risk for progression. Terminal disease stages are characterized by increasing genetic complexity and independence from bone marrow stromal cells and show a rapidly increasing tumour load with severe clinical symptoms. Modern diagnostics of plasma cell neoplasms require inclusion of clinical, morphological, immunophenotypical and cytogenetic features to allow for individual risk assessment and therapy planning. PMID:20852863

  9. Multispectral optical tweezers for molecular diagnostics of single biological cells

    NASA Astrophysics Data System (ADS)

    Butler, Corey; Fardad, Shima; Sincore, Alex; Vangheluwe, Marie; Baudelet, Matthieu; Richardson, Martin

    2012-03-01

    Optical trapping of single biological cells has become an established technique for controlling and studying fundamental behavior of single cells with their environment without having "many-body" interference. The development of such an instrument for optical diagnostics (including Raman and fluorescence for molecular diagnostics) via laser spectroscopy with either the "trapping" beam or secondary beams is still in progress. This paper shows the development of modular multi-spectral imaging optical tweezers combining Raman and Fluorescence diagnostics of biological cells.

  10. Transcriptomic Changes in Osteoblasts Following Endothelial Cell-Cocultivation Suggest a Role of Extracellular Matrix in Cellular Interaction.

    PubMed

    Lampert, Florian M; Simunovic, Filip; Finkenzeller, Günter; Pfeifer, Dietmar; Stark, G Björn; Winninger, Oscar; Steiner, Dominik

    2016-08-01

    Vascularization is important for bone development, fracture healing and engineering of artificial bone tissue. In the context of bone tissue engineering, it was shown that coimplantation of human primary umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) results in the formation of functional blood vessels and enhanced bone regeneration. Implanted endothelial cells do not only contribute to blood vessel formation, but also support proliferation, cell survival and osteogenic differentiation of coimplanted hOBs. These effects are partially mediated by direct heterotypic cell contacts. In a previous report we could show that cocultivated hOBs strongly increase the expression of genes involved in extracellular matrix (ECM) formation in HUVECs, suggesting that ECM may be involved in the intercellular communication between hOBs and HUVECs. The present study aimed at investigating whether comparable changes occur in hOBs. We therefore performed a microarray analysis of hOBs cultivated in direct contact with HUVECs, revealing 1,004 differentially expressed genes. The differentially expressed genes could be assigned to the functional clusters ECM, proliferation, apoptosis and osteogenic differentiation. The microarray data could be confirmed by performing quantitative real time RT-PCR on selected genes. Furthermore, we could show that the ECM produced by HUVECs increased the expression of the osteogenic differentiation marker alkaline phosphatase (ALP) in hOBs. In summary, our data demonstrate that HUVECs provoke complex changes in gene expression patterns in cocultivated hOBs and that ECM plays and important role in this interaction. J. Cell. Biochem. 117: 1869-1879, 2016. © 2016 Wiley Periodicals, Inc. PMID:26754918

  11. Molecular Recognition of Human Liver Cancer Cells Using DNA Aptamers Generated via Cell-SELEX

    PubMed Central

    Zhang, Liqin; Delgado, Stefanie; Champanhac, Carole; Cansiz, Sena; Wu, Cuichen; Shan, Hong; Tan, Weihong

    2015-01-01

    Most clinical cases of liver cancer cannot be diagnosed until they have evolved to an advanced stage, thus resulting in high mortality. It is well recognized that the implementation of early detection methods and the development of targeted therapies for liver cancer are essential to reducing the high mortality rates associated with this disease. To achieve these goals, molecular probes capable of recognizing liver cancer cell-specific targets are needed. Here we describe a panel of aptamers able to distinguish hepatocarcinoma from normal liver cells. The aptamers, which were selected by cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment), have Kd values in the range of 64-349 nM toward the target human hepatoma cell HepG2, and also recognize ovarian cancer cells and lung adenocarcinoma. The proteinase treatment experiment indicated that all aptamers could recognize target HepG2 cells through surface proteins. This outcome suggested that these aptamers could be used as potential probes for further research in cancer studies, such as developing early detection assays, targeted therapies, and imaging agents, as well as for the investigation of common membrane proteins in these distinguishable cancers. PMID:25938802

  12. Regulatory T Cells: Molecular Actions on Effector Cells in Immune Regulation

    PubMed Central

    Arce-Sillas, Asiel; Álvarez-Luquín, Diana Denisse; Tamaya-Domínguez, Beatriz; Gomez-Fuentes, Sandra; Trejo-García, Abel; Melo-Salas, Marlene; Cárdenas, Graciela; Rodríguez-Ramírez, Juan; Adalid-Peralta, Laura

    2016-01-01

    T regulatory cells play a key role in the control of the immune response, both in health and during illness. While the mechanisms through which T regulatory cells exert their function have been extensively described, their molecular effects on effector cells have received little attention. Thus, this revision is aimed at summarizing our current knowledge on those regulation mechanisms on the target cells from a molecular perspective. PMID:27298831

  13. Molecular mechanisms of cisplatin cytotoxicity in acute promyelocytic leukemia cells

    PubMed Central

    Kumar, Sanjay; Tchounwou, Paul B.

    2015-01-01

    Cis-diamminedichloroplatinum (II) (cisplatin) is a widely used anti-tumor drug for the treatment of a broad range of human malignancies with successful therapeutic outcomes for head and neck, ovarian, and testicular cancers. It has been found to inhibit cell cycle progression and to induce oxidative stress and apoptosis in acute promyelocytic leukemia (APL) cells. However, its molecular mechanisms of cytotoxic action are poorly understood. We hypothesized that cisplatin induces cytotoxicity through DNA adduct formation, oxidative stress, transcriptional factors (p53 and AP-1), cell cycle regulation, stress signaling and apoptosis in APL cells. We used the APL cell line as a model, and applied a variety of molecular tools to elucidate the cytototoxic mode of action of cisplatin. We found that cisplatin inhibited cell proliferation by a cytotoxicity, characterized by DNA damage and modulation of oxidative stress. Cisplatin also activated p53 and phosphorylated activator protein (AP-1) component, c-Jun at serine (63, 73) residue simultaneously leading to cell cycle arrest through stimulation of p21 and down regulation of cyclins and cyclin dependent kinases in APL cell lines. It strongly activated the intrinsic pathway of apoptosis through alteration of the mitochondrial membrane potential, release of cytochrome C, and up-regulation of caspase 3 activity. It also down regulated the p38MAPK pathway. Overall, this study highlights the molecular mechanisms that underline cisplatin toxicity to APL cells, and provides insights into selection of novel targets and/or design of therapeutic agents to treat APL. PMID:26486083

  14. Expression of PROKR1 and PROKR2 in Human Enteric Neural Precursor Cells and Identification of Sequence Variants Suggest a Role in HSCR

    PubMed Central

    Ruiz-Ferrer, Macarena; Torroglosa, Ana; Núñez-Torres, Rocío; de Agustín, Juan Carlos; Antiñolo, Guillermo; Borrego, Salud

    2011-01-01

    Background The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung's disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS. Principal Findings In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function. Conclusions Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR. PMID:21858136

  15. Cell and molecular biology for diagnostic and therapeutic technology

    NASA Astrophysics Data System (ADS)

    Tan, M. I.

    2016-03-01

    Our body contains 100 trillion cells. However, each cell has certain function and structure. For maintaining their integrity, cells will be collaborating with each other and with extracellular matrix surround them to form a tissue. These interactions effect internally on many networks or pathway such as signalling pathway, metabolic pathway and transport network in the cell. These networks interact with each other to maintain cell survival, cell structure and function and moreover the tissue as well as the organ which the cells built. Therefore, as part of a tissue, genetic and epigenetic abnormality of a cell can also alter these networks, and moreover disturb the function of the tissue itself. Hence, condition of genetic and epigenetic of the cell may affect other conditions in omics level such as transcriptomic, proteomic, metabolomics characteristics which can be differentiated by a particular unique molecular profile from each level, which can be used for diagnostic as well as for targeted therapy.

  16. Phylogenetic Analysis and Molecular Dating Suggest That Hemidactylus anamallensis Is Not a Member of the Hemidactylus Radiation and Has an Ancient Late Cretaceous Origin

    PubMed Central

    Bansal, Rohini; Karanth, K. Praveen

    2013-01-01

    Background of the Work The phylogenetic position and evolution of Hemidactylus anamallensis (family Gekkonidae) has been much debated in recent times. In the past it has been variously assigned to genus Hoplodactylus (Diplodactylidae) as well as a monotypic genus ‘Dravidogecko’ (Gekkonidae). Since 1995, this species has been assigned to Hemidactylus, but there is much disagreement between authors regarding its phylogenetic position within this genus. In a recent molecular study H. anamallensis was sister to Hemidactylus but appeared distinct from it in both mitochondrial and nuclear markers. However, this study did not include genera closely allied to Hemidactylus, thus a robust evaluation of this hypothesis was not undertaken. Methods The objective of this study was to investigate the phylogenetic position of H. anamallensis within the gekkonid radiation. To this end, several nuclear and mitochondrial markers were sequenced from H. anamallensis, selected members of the Hemidactylus radiation and genera closely allied to Hemidactylus. These sequences in conjunction with published sequences were subjected to multiple phylogenetic analyses. Furthermore the nuclear dataset was also subjected to molecular dating analysis to ascertain the divergence between H. anamallensis and related genera. Results and Conclusion Results showed that H. anamallensis lineage was indeed sister to Hemidactylus group but was separated from the rest of the Hemidactylus by a long branch. The divergence estimates supported a scenario wherein H. anamallensis dispersed across a marine barrier to the drifting peninsular Indian plate in the late Cretaceous whereas Hemidactylus arrived on the peninsular India after the Indian plate collided with the Eurasian plate. Based on these molecular evidence and biogeographical scenario we suggest that the genus Dravidogecko should be resurrected. PMID:23696785

  17. Equivocal p16 immunostaining in squamous cell carcinoma of the head and neck: staining patterns are suggestive of HPV status.

    PubMed

    Chen, Zhongchuan Will; Weinreb, Ilan; Kamel-Reid, Suzanne; Perez-Ordoñez, Bayardo

    2012-12-01

    p16 immunohistochemistry (IHC) is commonly used as a surrogate marker for human papillomavirus (HPV) detection in squamous cell carcinomas of the head and neck (SCCHN). However, the HPV status of tumors not staining strongly for p16 is difficult to interpret and may require the use of PCR, not available in all laboratories, as a final arbiter. We aim to determine if staining pattern in equivocal p16 staining and correlation to the percentage of positively stained tumor cells is predictive of HPV status. A retrospective review was performed on all SCCHN that underwent p16 IHC and PCR in our institution from 2007 to 2010. Descriptors of staining pattern in the original IHC report were retrieved. All available IHC slides were reviewed and reclassified using consensus staining pattern descriptors. Original and reclassified descriptors were compared to the final PCR HPV status for statistical significance using the χ(2) test. An estimate of the percentage of tumor cells that showed any form of staining was performed. Thirty-two SCCHN cases that underwent PCR HPV testing had equivocal p16 IHC results. Twenty-six cases available for review were reclassified into four staining patterns. Comparing age, sex, tumor site and diagnosis to HPV PCR status showed no statistically significant findings. However, comparing original descriptors to HPV status was statistically significant with isolated staining associated with negative HPV status (p = 0.0002). Analysis using reclassified descriptors showed strong association of membranous/cytoplasmic staining of isolated cells with negative HPV status and faint, diffuse nuclear and cytoplasmic staining with positive HPV status (p = 0.00006). HPV-negative cases with the former pattern had no more than 30 % positively-stained tumor cells and HPV-positive cases with the latter pattern had 50-90 % positively-stained cells. Our results suggest that pattern of staining in p16 IHC is associated with HPV status. For instance, a diffuse

  18. Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation

    PubMed Central

    Truong, David M.; Hewitt, F. Curtis; Hanson, Joseph H.; Cui, Xiaoxia; Lambowitz, Alan M.

    2015-01-01

    Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed “retrohoming”. Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the

  19. S(mu) mutation patterns suggest different progression pathways in follicular lymphoma: early direct or late from FL progenitor cells.

    PubMed

    Ruminy, Philippe; Jardin, Fabrice; Picquenot, Jean-Michel; Parmentier, Françoise; Contentin, Nathalie; Buchonnet, Gérard; Tison, Sandrine; Rainville, Vinciane; Tilly, Hervé; Bastard, Christian

    2008-09-01

    Follicular lymphoma (FL) is a B-cell malignancy characterized by the t(14;18) translocation. Although sensitive to treatment, the disease remains incurable and the reason why tumor cells invariably evade treatment, leading to clinical relapse, is still unknown. Here, we tracked the clonal history of tumor cells by studying mutations introduced by activation-induced cytidine deaminase on the switch mu region of the der(14)t(14;18) during the early phase of the class-switch recombination (CSR) process. We observed frequent intraclonal variations, suggesting that CSR often remains active after the acquisition of the fully transformed phenotype. However, mutations only rarely accumulated over time, but instead showed complex evolutionary scenarios and 2 different progression pathways. The first pathway was a direct and rapid evolution from the dominant clone. The second was indirect, arising from earlier subclones usually after years of remission. A better understanding of these mechanisms might influence the future choice of treatment strategies. PMID:18515657

  20. Cell Engineering and Molecular Pharming for Biopharmaceuticals

    PubMed Central

    Abdullah, M.A; Rahmah, Anisa ur; Sinskey, A.J; Rha, C.K

    2008-01-01

    Biopharmaceuticals are often produced by recombinant E. coli or mammalian cell lines. This is usually achieved by the introduction of a gene or cDNA coding for the protein of interest into a well-characterized strain of producer cells. Naturally, each recombinant production system has its own unique advantages and disadvantages. This paper examines the current practices, developments, and future trends in the production of biopharmaceuticals. Platform technologies for rapid screening and analyses of biosystems are reviewed. Strategies to improve productivity via metabolic and integrated engineering are also highlighted. PMID:19662143

  1. Molecular Mechanisms by Which a Fucus vesiculosus Extract Mediates Cell Cycle Inhibition and Cell Death in Pancreatic Cancer Cells.

    PubMed

    Geisen, Ulf; Zenthoefer, Marion; Peipp, Matthias; Kerber, Jannik; Plenge, Johannes; Managò, Antonella; Fuhrmann, Markus; Geyer, Roland; Hennig, Steffen; Adam, Dieter; Piker, Levent; Rimbach, Gerald; Kalthoff, Holger

    2015-07-01

    Pancreatic cancer is one of the most aggressive cancer entities, with an extremely poor 5-year survival rate. Therefore, novel therapeutic agents with specific modes of action are urgently needed. Marine organisms represent a promising source to identify new pharmacologically active substances. Secondary metabolites derived from marine algae are of particular interest. The present work describes cellular and molecular mechanisms induced by an HPLC-fractionated, hydrophilic extract derived from the Baltic brown seaweed Fucus vesiculosus (Fv1). Treatment with Fv1 resulted in a strong inhibition of viability in various pancreatic cancer cell lines. This extract inhibited the cell cycle of proliferating cells due to the up-regulation of cell cycle inhibitors, shown on the mRNA (microarray data) and protein level. As a result, cells were dying in a caspase-independent manner. Experiments with non-dividing cells showed that proliferation is a prerequisite for the effectiveness of Fv1. Importantly, Fv1 showed low cytotoxic activity against non-malignant resting T cells and terminally differentiated cells like erythrocytes. Interestingly, accelerated killing effects were observed in combination with inhibitors of autophagy. Our in vitro data suggest that Fv1 may represent a promising new agent that deserves further development towards clinical application. PMID:26204945

  2. Molecular Mechanisms by Which a Fucus vesiculosus Extract Mediates Cell Cycle Inhibition and Cell Death in Pancreatic Cancer Cells

    PubMed Central

    Geisen, Ulf; Zenthoefer, Marion; Peipp, Matthias; Kerber, Jannik; Plenge, Johannes; Managò, Antonella; Fuhrmann, Markus; Geyer, Roland; Hennig, Steffen; Adam, Dieter; Piker, Levent; Rimbach, Gerald; Kalthoff, Holger

    2015-01-01

    Pancreatic cancer is one of the most aggressive cancer entities, with an extremely poor 5-year survival rate. Therefore, novel therapeutic agents with specific modes of action are urgently needed. Marine organisms represent a promising source to identify new pharmacologically active substances. Secondary metabolites derived from marine algae are of particular interest. The present work describes cellular and molecular mechanisms induced by an HPLC-fractionated, hydrophilic extract derived from the Baltic brown seaweed Fucus vesiculosus (Fv1). Treatment with Fv1 resulted in a strong inhibition of viability in various pancreatic cancer cell lines. This extract inhibited the cell cycle of proliferating cells due to the up-regulation of cell cycle inhibitors, shown on the mRNA (microarray data) and protein level. As a result, cells were dying in a caspase-independent manner. Experiments with non-dividing cells showed that proliferation is a prerequisite for the effectiveness of Fv1. Importantly, Fv1 showed low cytotoxic activity against non-malignant resting T cells and terminally differentiated cells like erythrocytes. Interestingly, accelerated killing effects were observed in combination with inhibitors of autophagy. Our in vitro data suggest that Fv1 may represent a promising new agent that deserves further development towards clinical application. PMID:26204945

  3. Molecular Background of Estrogen Receptor Gene Expression in Endometriotic Cells.

    PubMed

    Izawa, Masao; Taniguchi, Fuminori; Harada, Tasuku

    2016-07-01

    The molecular background of estrogen receptor (ER) expression is important to understand the pathophysiology of the high estrogen environment in endometriosis. However, the molecular details have not been fully understood. The objective of this study is to evaluate the molecular background of ERα and ERβ messenger RNA (mRNA) expression in endometriotic cells. The following summarizes our observations: (1) ERα mRNA expression in endometriotic cells was estimated to be approximately one-tenth of that in endometrial cells. (2) Three mRNAs, which include 3 different 5'-untranslated exons tagged to an open reading frame of wild-type ERα, were detected. (3) Expression of ERβ mRNA depends mostly on 0N promoter and includes 2 open reading frames: one for a wild-type ERβ1 and another for a splice variant ERβ2. (4) Expression of ERβ1 mRNA was approximately 40-fold higher than that in endometrial cells. (5) Expression of ERβ2 mRNA was almost at a comparable level of the ERβ1. 9 (6) ERα and ERβ mRNAs are equivalently expressed in endometriotic cells. These observations show the molecular background of ER mRNA expression in endometriotic cells and provide a clue to further understanding the estrogen-dependent pathophysiology leading to clinical application in endometriosis. PMID:26704524

  4. Update on the protective molecular pathways improving pancreatic beta-cell dysfunction.

    PubMed

    Puddu, Alessandra; Sanguineti, Roberta; Mach, François; Dallegri, Franco; Viviani, Giorgio Luciano; Montecucco, Fabrizio

    2013-01-01

    The primary function of pancreatic beta-cells is to produce and release insulin in response to increment in extracellular glucose concentrations, thus maintaining glucose homeostasis. Deficient beta-cell function can have profound metabolic consequences, leading to the development of hyperglycemia and, ultimately, diabetes mellitus. Therefore, strategies targeting the maintenance of the normal function and protecting pancreatic beta-cells from injury or death might be crucial in the treatment of diabetes. This narrative review will update evidence from the recently identified molecular regulators preserving beta-cell mass and function recovery in order to suggest potential therapeutic targets against diabetes. This review will also highlight the relevance for novel molecular pathways potentially improving beta-cell dysfunction. PMID:23737653

  5. Cell and molecular biology of septins.

    PubMed

    Fung, Karen Y Y; Dai, Lu; Trimble, William S

    2014-01-01

    Septins are a family of GTP-binding proteins that assemble into cytoskeletal filaments. Unlike other cytoskeletal components, septins form ordered arrays of defined stoichiometry that can polymerize into long filaments and bundle laterally. Septins associate directly with membranes and have been implicated in providing membrane stability and serving as diffusion barriers for membrane proteins. In addition, septins bind other proteins and have been shown to function as multimolecular scaffolds by recruiting components of signaling pathways. Remarkably, septins participate in a spectrum of cellular processes including cytokinesis, ciliogenesis, cell migration, polarity, and cell-pathogen interactions. Given their breadth of functions, it is not surprising that septin abnormalities have also been linked to human diseases. In this review, we discuss the current knowledge of septin structure, assembly and function, and discuss these in the context of human disease. PMID:24725429

  6. A decade of molecular cell biology: achievements and challenges

    PubMed Central

    Akhtar, Asifa; Fuchs, Elaine; Mitchison, Tim; Shaw, Reuben J.; St Johnston, Daniel; Strasser, Andreas; Taylor, Susan; Walczak, Claire; Zerial, Marino

    2012-01-01

    Nature Reviews Molecular Cell Biology celebrated its 10-year anniversary during this past year with a series of specially commissioned articles. To complement this, here we have asked researchers from across the field for their insights into how molecular cell biology research has evolved during this past decade, the key concepts that have emerged and the most promising interfaces that have developed. Their comments highlight the broad impact that particular advances have had, some of the basic understanding that we still require, and the collaborative approaches that will be essential for driving the field forward. PMID:21941276

  7. Isolation, characterization, and molecular regulation of muscle stem cells

    PubMed Central

    Fukada, So-ichiro; Ma, Yuran; Ohtani, Takuji; Watanabe, Yoko; Murakami, Satoshi; Yamaguchi, Masahiko

    2013-01-01

    Skeletal muscle has great regenerative capacity which is dependent on muscle stem cells, also known as satellite cells. A loss of satellite cells and/or their function impairs skeletal muscle regeneration and leads to a loss of skeletal muscle power; therefore, the molecular mechanisms for maintaining satellite cells in a quiescent and undifferentiated state are of great interest in skeletal muscle biology. Many studies have demonstrated proteins expressed by satellite cells, including Pax7, M-cadherin, Cxcr4, syndecan3/4, and c-met. To further characterize satellite cells, we established a method to directly isolate satellite cells using a monoclonal antibody, SM/C-2.6. Using SM/C-2.6 and microarrays, we measured the genes expressed in quiescent satellite cells and demonstrated that Hesr3 may complement Hesr1 in generating quiescent satellite cells. Although Hesr1- or Hesr3-single knockout mice show a normal skeletal muscle phenotype, including satellite cells, Hesr1/Hesr3-double knockout mice show a gradual decrease in the number of satellite cells and increase in regenerative defects dependent on satellite cell numbers. We also observed that a mouse's genetic background affects the regenerative capacity of its skeletal muscle and have established a line of DBA/2-background mdx mice that has a much more severe phenotype than the frequently used C57BL/10-mdx mice. The phenotype of DBA/2-mdx mice also seems to depend on the function of satellite cells. In this review, we summarize the methodology of direct isolation, characterization, and molecular regulation of satellite cells based on our results. The relationship between the regenerative capacity of satellite cells and progression of muscular disorders is also summarized. In the last part, we discuss application of the accumulating scientific information on satellite cells to treatment of patients with muscular disorders. PMID:24273513

  8. Approaches to isolation and molecular characterization of disseminated tumor cells

    PubMed Central

    Magbanua, Mark Jesus M.; Das, Rishi; Polavarapu, Prithi; Park, John W.

    2015-01-01

    Micrometastatic cells in the bone marrow, now usually referred to as “disseminated tumor cells (DTCs)”, can be detected in early stage cancer patients. It has been hypothesized that DTCs represent key intermediates in the metastatic process as possible precursors of bone and visceral metastases, and are indicators of metastatic potential. Indeed, multiple clinical studies have unequivocally demonstrated the prognostic value of these cells in breast and other cancers, as DTCs have been associated with adverse outcomes, including inferior overall and disease-free survival. Despite this established clinical significance, the molecular nature of DTCs remains elusive. The complexity of the bone marrow poses a unique challenge in the isolation and direct characterization of these rare cells. However, recent advances in rare-cell technology along with technical improvements in analyzing limited cell inputs have enabled the molecular profiling of DTCs. In this review, we discuss research featuring the isolation and genomic analysis of DTCs. Emerging work on the molecular characterization of DTCs is now providing new insights into the biology of these cells. PMID:26378808

  9. Paired octamer rings of retinoschisin suggest a junctional model for cell–cell adhesion in the retina

    PubMed Central

    Tolun, Gökhan; Vijayasarathy, Camasamudram; Huang, Rick; Zeng, Yong; Li, Yan; Steven, Alasdair C.; Sieving, Paul A.

    2016-01-01

    Retinoschisin (RS1) is involved in cell–cell junctions in the retina, but is unique among known cell-adhesion proteins in that it is a soluble secreted protein. Loss-of-function mutations in RS1 lead to early vision impairment in young males, called X-linked retinoschisis. The disease is characterized by separation of inner retinal layers and disruption of synaptic signaling. Using cryo-electron microscopy, we report the structure at 4.1 Å, revealing double octamer rings not observed before. Each subunit is composed of a discoidin domain and a small N-terminal (RS1) domain. The RS1 domains occupy the centers of the rings, but are not required for ring formation and are less clearly defined, suggesting mobility. We determined the structure of the discoidin rings, consistent with known intramolecular and intermolecular disulfides. The interfaces internal to and between rings feature residues implicated in X-linked retinoschisis, indicating the importance of correct assembly. Based on this structure, we propose that RS1 couples neighboring membranes together through octamer–octamer contacts, perhaps modulated by interactions with other membrane components. PMID:27114531

  10. Largescale Transcriptomics Analysis Suggests Over-Expression of BGH3, MMP9 and PDIA3 in Oral Squamous Cell Carcinoma

    PubMed Central

    He, Yuan; Shao, Fangyang; Pi, Weidong; Shi, Cong; Chen, Yujia; Gong, Diping; Wang, Bingjie; Cao, Zhiwei; Tang, Kailin

    2016-01-01

    Oral squamous cell carcinoma (OSCC) has been reported as the most prevalent cancer of the head and neck region, while early diagnosis remains challenging. Here we took a comprehensive bioinformatics study on microarray data of 326 OSCC clinical samples with control of 165 normal tissues. The cell interaction pathways of ECM-receptor interaction and focal adhesion were found to be significantly regulated in OSCC samples. Further analysis of the topological properties and expression consistency identified that three hub genes in the gene interaction network, MMP9, PDIA3 and BGH3, were consistently up-expressed in OSCC samples. When being validated on additional microarray datasets of 41 OSCC samples, the validation rate of over-expressed BGH3, MMP9, and PDIA3 reached 90%, 90% and 84% respectively. At last, immuno-histochemical assays were done to test the protein expression of the three genes on newly collected clinical samples of 35 OSCC, 20 samples of pre-OSCC stage, and 12 normal oral mucosa specimens. Their protein expression levels were also found to progressively increase from normal mucosa to pre-OSCC stage and further to OSCC (ANOVA p = 0.000), suggesting their key roles in OSCC pathogenesis. Based on above solid validation, we propose BGH3, MMP9 and PDIA3 might be further explored as potential biomarkers to aid OSCC diagnosis. PMID:26745629

  11. The TUNEL assay suggests mandibular regression by programmed cell death during presoldier differentiation in the nasute termite Nasutitermes takasagoensis

    NASA Astrophysics Data System (ADS)

    Toga, Kouhei; Yoda, Shinichi; Maekawa, Kiyoto

    2011-09-01

    Termite soldiers are the most specialized caste of social insects in terms of their morphology and function. Soldier development requires increased juvenile hormone (JH) titer and the two molts via a presoldier stage. These molts are accompanied by dramatic morphological changes, including the exaggeration and regression of certain organs. Soldiers of the most apical termitid subfamily Nasutitermitinae possess not only a horn-like frontal tube, called the nasus, for the projection of defensive chemicals from the frontal gland reservoir but also regressed mandibles. Although candidate genes regulating soldier mandibular growth were reported in a relatively basal termite species, the regulatory mechanisms of mandibular regression remain unknown. To clarify these mechanisms, we performed morphological and histological examinations of the mandibles during soldier differentiation in Nasutitermes takasagoensis. Mandibular size reduced dramatically during soldier differentiation, and mandibular regression occurred just prior to the presoldier molt. Spotted TUNEL signals were observed in regressing mandibles of presoldiers, suggesting that the regression involved programmed cell death. Because soldiers of N. takasagoensis possess exaggerated organs (nasus and frontal gland), the present results suggest that JH-dependent regressive mechanisms exist in the mandibles without interfering with the formation of the exaggerated organs.

  12. Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance.

    PubMed

    Chaorattanakawee, Suwanna; Saunders, David L; Sea, Darapiseth; Chanarat, Nitima; Yingyuen, Kritsanai; Sundrakes, Siratchana; Saingam, Piyaporn; Buathong, Nillawan; Sriwichai, Sabaithip; Chann, Soklyda; Se, Youry; Yom, You; Heng, Thay Kheng; Kong, Nareth; Kuntawunginn, Worachet; Tangthongchaiwiriya, Kuntida; Jacob, Christopher; Takala-Harrison, Shannon; Plowe, Christopher; Lin, Jessica T; Chuor, Char Meng; Prom, Satharath; Tyner, Stuart D; Gosi, Panita; Teja-Isavadharm, Paktiya; Lon, Chanthap; Lanteri, Charlotte A

    2015-08-01

    Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure. PMID:26014942

  13. Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

    PubMed Central

    Chaorattanakawee, Suwanna; Saunders, David L.; Sea, Darapiseth; Chanarat, Nitima; Yingyuen, Kritsanai; Sundrakes, Siratchana; Saingam, Piyaporn; Buathong, Nillawan; Sriwichai, Sabaithip; Chann, Soklyda; Se, Youry; Yom, You; Heng, Thay Kheng; Kong, Nareth; Kuntawunginn, Worachet; Tangthongchaiwiriya, Kuntida; Jacob, Christopher; Takala-Harrison, Shannon; Plowe, Christopher; Lin, Jessica T.; Chuor, Char Meng; Prom, Satharath; Tyner, Stuart D.; Gosi, Panita; Teja-Isavadharm, Paktiya; Lon, Chanthap

    2015-01-01

    Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure. PMID:26014942

  14. Molecular pathways in myocardial development: a stem cell perspective.

    PubMed

    Solloway, Mark J; Harvey, Richard P

    2003-05-01

    The heart has long been considered to adapt to increased work or pathology through the cellular growth process of hypertrophy. However, recent evidence for the existence of endogenous stem cells and regenerative capacity in the adult heart has given new impetus to the quest for cell therapies for heart failure, which remains the number one killer in Western cultures. The molecular cues driving cardiac development are now being explored in detail and will come into sharp focus as regimes for stem cell differentiation and efforts to augment endogenous regeneration are trialed. This review briefly outlines the current state of knowledge on the molecular basis of the four modalities of myogenesis that have been identified in the developing vertebrate heart. Stem cell-mediated myogenic repair in the heart represents a fifth modality, and an exciting frontier with basic and practical implications that remain to be explored. PMID:12757862

  15. THE TRANSCRIPTIONAL SIGNATURES OF CELLS FROM THE HUMAN PEYRONIE'S DISEASE PLAQUE AND THE ABILITY OF THESE CELLS TO GENERATE A PLAQUE IN A RAT MODEL SUGGEST POTENTIAL THERAPEUTIC TARGETS

    PubMed Central

    Gelfand, R; Vernet, D; Kovanecz, I; Rajfer, J; Gonzalez-Cadavid, NF

    2015-01-01

    Introduction The success of medical therapies for Peyronie's disease (PD) has not been optimal, possibly because many of them went directly to clinical application without sufficient preclinical scientific research. Previous studies revealed cellular and molecular pathways involved in the formation of the PD plaque, and in particular the role of the myofibroblast. Aims The current work aimed to determine under normal and fibrotic conditions what differentiates PD cells from tunica albuginea (TA) and corpora cavernosa (CC) cells, by defining their global transcriptional signatures and testing in vivo whether PD cells can generate a PD like plaque Main Outcomes Measures Fibroproliferative features of PD cells and identification of related key genes as novel targets to reduce plaque size Methods Human TA, PD, and CC cells were grown with TGFβ1 (TA+, PD+, CC+) or without it (TA−, PD−, CC−) and assayed by: a) immunofluorescence, western blot and RT/PCR for myofibroblast, smooth muscle cell and stem cell markers; b) collagen content; and c) DNA microarray analysis. The ability of PD+ cells to induce a PD like plaque in an immuno-suppressed rat model was assessed by Masson trichrome and Picrosirius Red. Results Upon TGFβ1stimulation, collagen levels were increased by myofibroblasts in the PD+ but not in the CC+ cells. The transcriptional signature of the PD− cells identified fibroproliferative, myogenic (myofibroblasts), inflammatory, and collagen turnover genes, that differentiate them from TA− or CC− cells, and respond to TGFβ1 with a PD+ fibrotic phenotype, by upregulation of IGF1, ACTG2, MYF5, ACTC1, PSTN, COL III, MMP3, and others. The PD+ cells injected into the TA of the rat induce a PD like plaque. Conclusions This suggests a novel combination therapy to eliminate a PD plaque, by targeting the identified genes to: a) improve collagenase action by stimulating endogenous MMPs specific to key collagen types, and b) counteract fibromatosis by inhibiting

  16. Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death

    PubMed Central

    Garg, Abhishek D.; Galluzzi, Lorenzo; Apetoh, Lionel; Baert, Thais; Birge, Raymond B.; Bravo-San Pedro, José Manuel; Breckpot, Karine; Brough, David; Chaurio, Ricardo; Cirone, Mara; Coosemans, An; Coulie, Pierre G.; De Ruysscher, Dirk; Dini, Luciana; de Witte, Peter; Dudek-Peric, Aleksandra M.; Faggioni, Alberto; Fucikova, Jitka; Gaipl, Udo S.; Golab, Jakub; Gougeon, Marie-Lise; Hamblin, Michael R.; Hemminki, Akseli; Herrmann, Martin; Hodge, James W.; Kepp, Oliver; Kroemer, Guido; Krysko, Dmitri V.; Land, Walter G.; Madeo, Frank; Manfredi, Angelo A.; Mattarollo, Stephen R.; Maueroder, Christian; Merendino, Nicolò; Multhoff, Gabriele; Pabst, Thomas; Ricci, Jean-Ehrland; Riganti, Chiara; Romano, Erminia; Rufo, Nicole; Smyth, Mark J.; Sonnemann, Jürgen; Spisek, Radek; Stagg, John; Vacchelli, Erika; Vandenabeele, Peter; Vandenberk, Lien; Van den Eynde, Benoit J.; Van Gool, Stefaan; Velotti, Francesca; Zitvogel, Laurence; Agostinis, Patrizia

    2015-01-01

    The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called “damage-associated molecular patterns” (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation. PMID:26635802

  17. Molecular Programming of Immunological Memory in Natural Killer Cells.

    PubMed

    Beaulieu, Aimee M; Madera, Sharline; Sun, Joseph C

    2015-01-01

    Immunological memory is a hallmark of the adaptive immune system. Although natural killer (NK) cells have traditionally been classified as a component of the innate immune system, they have recently been shown in mice and humans to exhibit certain features of immunological memory, including an ability to undergo a clonal-like expansion during virus infection, generate long-lived progeny (i.e. memory cells), and mediate recall responses against previously encountered pathogens--all characteristics previously ascribed only to adaptive immune responses by B and T cells in mammals. To date, the molecular events that govern the generation of NK cell memory are not completely understood. Using a mouse model of cytomegalovirus infection, we demonstrate that individual pro-inflammatory IL-12, IL-18, and type I-IFN signaling pathways are indispensible and play non-redundant roles in the generation of virus-specific NK cell memory. Furthermore, we discovered that antigen-specific proliferation and protection by NK cells is mediated by the transcription factor Zbtb32, which is induced by pro-inflammatory cytokines and promotes a cell cycle program in activated NK cells. A greater understanding of the molecular mechanisms controlling NK cell responses will provide novel strategies for tailoring vaccines to target infectious disease. PMID:26324348

  18. Limited T-cell receptor beta-chain heterogeneity among interleukin 2 receptor-positive synovial T cells suggests a role for superantigen in rheumatoid arthritis.

    PubMed Central

    Howell, M D; Diveley, J P; Lundeen, K A; Esty, A; Winters, S T; Carlo, D J; Brostoff, S W

    1991-01-01

    Rheumatoid arthritis (RA) is a disease affecting the synovial membranes of articulating joints that is thought to result from T-cell-mediated autoimmune phenomena. T cells responsible for the pathogenesis of RA are likely present in that fraction of synovial T cells that expresses the interleukin 2 receptor (IL-2R), one marker of T-cell activation. We report herein an analysis of T-cell receptor (TCR) beta-chain gene expression by IL-2R-positive synovial T cells. These T cells were isolated from uncultured synovial tissue specimens by using IL-2R-specific monoclonal antibodies and magnetic beads, and TCR beta-chain transcription was analyzed by PCR-catalyzed amplification using a panel of primers specific for the human TCR beta-chain variable region (V beta). Multiple V beta gene families were found to be transcribed in these patients samples; however, three gene families, V beta 3, V beta 14, and V beta 17, were found in a majority of the five synovial samples analyzed, suggesting that T cells bearing these V beta s had been selectively retained in the synovial microenvironment. In many instances, the V beta 3, V beta 14, or V beta 17 repertoires amplified from an individual patient were dominated by a single rearrangement, indicative of clonal expansion in the synovium and supportive of a role for these T cells in RA. Of note is a high sequence similarity between V beta 3, V beta 14, and V beta 17 polypeptides, particularly in the fourth complementarity-determining region (CDR). Given that binding sites for superantigens have been mapped to the CDR4s of TCR beta chains, the synovial localization of T cells bearing V beta s with significant CDR4 homology indicates that V beta-specific T-cell activation by superantigen may play a role in RA. PMID:1660155

  19. Quantum Dot Platform for Single-Cell Molecular Profiling

    NASA Astrophysics Data System (ADS)

    Zrazhevskiy, Pavel S.

    In-depth understanding of the nature of cell physiology and ability to diagnose and control the progression of pathological processes heavily rely on untangling the complexity of intracellular molecular mechanisms and pathways. Therefore, comprehensive molecular profiling of individual cells within the context of their natural tissue or cell culture microenvironment is essential. In principle, this goal can be achieved by tagging each molecular target with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution, primarily via microscopy-based imaging. Yet, neither widely used conventional methods nor more advanced nanoparticle-based techniques have been able to address this task up to date. High multiplexing potential of fluorescent probes is heavily restrained by the inability to uniquely match probes with corresponding molecular targets. This issue is especially relevant for quantum dot probes---while simultaneous spectral imaging of up to 10 different probes is possible, only few can be used concurrently for staining with existing methods. To fully utilize multiplexing potential of quantum dots, it is necessary to design a new staining platform featuring unique assignment of each target to a corresponding quantum dot probe. This dissertation presents two complementary versatile approaches towards achieving comprehensive single-cell molecular profiling and describes engineering of quantum dot probes specifically tailored for each staining method. Analysis of expanded molecular profiles is achieved through augmenting parallel multiplexing capacity with performing several staining cycles on the same specimen in sequential manner. In contrast to other methods utilizing quantum dots or other nanoparticles, which often involve sophisticated probe synthesis, the platform technology presented here takes advantage of simple covalent bioconjugation and non-covalent self-assembly mechanisms for straightforward probe

  20. Membrane curvature in cell biology: An integration of molecular mechanisms.

    PubMed

    Jarsch, Iris K; Daste, Frederic; Gallop, Jennifer L

    2016-08-15

    Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists. PMID:27528656

  1. Roles of cell volume in molecular and cellular biology.

    PubMed

    Dubois, Jean-Marc; Rouzaire-Dubois, Béatrice

    2012-04-01

    Extracellular tonicity and volume regulation control a great number of molecular and cellular functions including: cell proliferation, apoptosis, migration, hormone and neuromediator release, gene expression, ion channel and transporter activity and metabolism. The aim of this review is to describe these effects and to determine if they are direct or are secondarily the result of the activity of second messengers. PMID:22192789

  2. Combination of Mechanical and Molecular Filtration for Enhanced Enrichment of Circulating Tumor Cells.

    PubMed

    Meunier, Anne; Hernández-Castro, Javier Alejandro; Turner, Kate; Li, Kebin; Veres, Teodor; Juncker, David

    2016-09-01

    Circulating tumor cells (CTCs) have been linked to cancer progression but are difficult to isolate, as they are very rare and heterogeneous, covering a range of sizes and expressing different molecular receptors. Filtration has emerged as a simple and powerful method to enrich CTCs but only captures cells above a certain size regardless of molecular characteristics. Here, we introduce antibody-functionalized microfilters to isolate CTCs based on both size and surface receptor expression. We present a 3D printed filtration cartridge with microfabricated polymer filters with 8, 10, 12, 15, or 20 μm-diameter pores. Pristine filters were used to optimize sample dilution, rinsing protocol, flow rate, and pore size, leading to >80% for the recovery of spiked cancer cells with very low white blood cell contamination (<1000). Then, filters were functionalized with antibodies against either epithelial cell adhesion molecule (EpCAM) or epidermal growth factor receptor (EGFR) and the cartridges were used to enrich breast (MDA-MB-231, MCF-7) and renal (786-O, A-498) cancer cells expressing various levels of EpCAM and EGFR. Cancer cells were spiked into human blood, and when using filters with antibodies specific to a molecular receptor expressed on a cell, efficiency was increased to >96%. These results suggest that filtration can be optimized to target specific CTC characteristics such as size and receptor expression and that a diverse range of CTCs may be captured using particular combinations of pore size, filtration parameters, and antibody functionalization. PMID:27442305

  3. Molecular evolution of the vertebrate mechanosensory cell and ear

    PubMed Central

    Fritzsch, Bernd; Beisel, Kirk W.; Pauley, Sarah; Soukup, Garrett

    2014-01-01

    The molecular basis of mechanosensation, mechanosensory cell development and mechanosensory organ development is reviewed with an emphasis on its evolution. In contrast to eye evolution and development, which apparently modified a genetic program through intercalation of genes between the master control genes on the top (Pax6, Eya1, Six1) of the hierarchy and the structural genes (rhodopsin) at the bottom, the as yet molecularly unknown mechanosensory channel precludes such a firm conclusion for mechanosensors. However, recent years have seen the identification of several structural genes which are involved in mechanosensory tethering and several transcription factors controlling mechanosensory cell and organ development; these warrant the interpretation of available data in very much the same fashion as for eye evolution: molecular homology combined with potential morphological parallelism. This assertion of molecular homology is strongly supported by recent findings of a highly conserved set of microRNAs that appear to be associated with mechanosensory cell development across phyla. The conservation of transcription factors and their regulators fits very well to the known or presumed mechanosensory specializations which can be mostly grouped as variations of a common cellular theme. Given the widespread distribution of the molecular ability to form mechanosensory cells, it comes as no surprise that structurally different mechanosensory organs evolved in different phyla, presenting a variation of a common theme specified by a conserved set of transcription factors in their cellular development. Within vertebrates and arthropods, some mechanosensory organs evolved into auditory organs, greatly increasing sensitivity to sound through modifications of accessory structures to direct sound to the specific sensory epithelia. However, while great attention has been paid to the evolution of these accessory structures in vertebrate fossils, comparatively less attention has

  4. CD30-positive peripheral T-cell lymphomas share molecular and phenotypic features

    PubMed Central

    Bisig, Bettina; de Reyniès, Aurélien; Bonnet, Christophe; Sujobert, Pierre; Rickman, David S.; Marafioti, Teresa; Delsol, Georges; Lamant, Laurence; Gaulard, Philippe; de Leval, Laurence

    2013-01-01

    Peripheral T-cell lymphoma, not otherwise specified is a heterogeneous group of aggressive neoplasms with indistinct borders. By gene expression profiling we previously reported unsupervised clusters of peripheral T-cell lymphomas, not otherwise specified correlating with CD30 expression. In this work we extended the analysis of peripheral T-cell lymphoma molecular profiles to prototypical CD30+ peripheral T-cell lymphomas (anaplastic large cell lymphomas), and validated mRNA expression profiles at the protein level. Existing transcriptomic datasets from peripheral T-cell lymphomas, not otherwise specified and anaplastic large cell lymphomas were reanalyzed. Twenty-one markers were selected for immunohistochemical validation on 80 peripheral T-cell lymphoma samples (not otherwise specified, CD30+ and CD30−; anaplastic large cell lymphomas, ALK+ and ALK−), and differences between subgroups were assessed. Clinical follow-up was recorded. Compared to CD30− tumors, CD30+ peripheral T-cell lymphomas, not otherwise specified were significantly enriched in ALK− anaplastic large cell lymphoma-related genes. By immunohistochemistry, CD30+ peripheral T-cell lymphomas, not otherwise specified differed significantly from CD30− samples [down-regulated expression of T-cell receptor-associated proximal tyrosine kinases (Lck, Fyn, Itk) and of proteins involved in T-cell differentiation/activation (CD69, ICOS, CD52, NFATc2); upregulation of JunB and MUM1], while overlapping with anaplastic large cell lymphomas. CD30− peripheral T-cell lymphomas, not otherwise specified tended to have an inferior clinical outcome compared to the CD30+ subgroups. In conclusion, we show molecular and phenotypic features common to CD30+ peripheral T-cell lymphomas, and significant differences between CD30− and CD30+ peripheral T-cell lymphomas, not otherwise specified, suggesting that CD30 expression might delineate two biologically distinct subgroups. PMID:23716562

  5. Bitter melon juice targets molecular mechanisms underlying gemcitabine resistance in pancreatic cancer cells.

    PubMed

    Somasagara, Ranganatha R; Deep, Gagan; Shrotriya, Sangeeta; Patel, Manisha; Agarwal, Chapla; Agarwal, Rajesh

    2015-04-01

    Pancreatic cancer (PanC) is one of the most lethal malignancies, and resistance towards gemcitabine, the front-line chemotherapy, is the main cause for dismal rate of survival in PanC patients; overcoming this resistance remains a major challenge to treat this deadly malignancy. Whereas several molecular mechanisms are known for gemcitabine resistance in PanC cells, altered metabolism and bioenergetics are not yet studied. Here, we compared metabolic and bioenergetic functions between gemcitabine-resistant (GR) and gemcitabine-sensitive (GS) PanC cells and underlying molecular mechanisms, together with efficacy of a natural agent bitter melon juice (BMJ). GR PanC cells showed distinct morphological features including spindle-shaped morphology and a decrease in E-cadherin expression. GR cells also showed higher ATP production with an increase in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Molecular studies showed higher expression of glucose transporters (GLUT1 and 4) suggesting an increase in glucose uptake by GR cells. Importantly, GR cells showed a significant increase in Akt and ERK1/2 phosphorylation and their inhibition decreased cell viability, suggesting their role in survival and drug resistance of these cells. Recently, we reported strong efficacy of BMJ against a panel of GS cells in culture and nude mice, which we expanded here and found that BMJ was also effective in decreasing both Akt and ERK1/2 phosphorylation and viability of GR PanC cells. Overall, we have identified novel mechanisms of gemcitabine resistance in PanC cells which are targeted by BMJ. Considering the short survival in PanC patients, our findings could have high translational potential in controlling this deadly malignancy. PMID:25672620

  6. Bitter melon juice targets molecular mechanisms underlying gemcitabine resistance in pancreatic cancer cells

    PubMed Central

    SOMASAGARA, RANGANATHA R.; DEEP, GAGAN; SHROTRIYA, SANGEETA; PATEL, MANISHA; AGARWAL, CHAPLA; AGARWAL, RAJESH

    2015-01-01

    Pancreatic cancer (PanC) is one of the most lethal malignancies, and resistance towards gemcitabine, the front-line chemotherapy, is the main cause for dismal rate of survival in PanC patients; overcoming this resistance remains a major challenge to treat this deadly malignancy. Whereas several molecular mechanisms are known for gemcitabine resistance in PanC cells, altered metabolism and bioenergetics are not yet studied. Here, we compared metabolic and bioenergetic functions between gemcitabine-resistant (GR) and gemcitabine-sensitive (GS) PanC cells and underlying molecular mechanisms, together with efficacy of a natural agent bitter melon juice (BMJ). GR PanC cells showed distinct morphological features including spindle-shaped morphology and a decrease in E-cadherin expression. GR cells also showed higher ATP production with an increase in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Molecular studies showed higher expression of glucose transporters (GLUT1 and 4) suggesting an increase in glucose uptake by GR cells. Importantly, GR cells showed a significant increase in Akt and ERK1/2 phosphorylation and their inhibition decreased cell viability, suggesting their role in survival and drug resistance of these cells. Recently, we reported strong efficacy of BMJ against a panel of GS cells in culture and nude mice, which we expanded here and found that BMJ was also effective in decreasing both Akt and ERK1/2 phosphorylation and viability of GR PanC cells. Overall, we have identified novel mechanisms of gemcitabine resistance in PanC cells which are targeted by BMJ. Considering the short survival in PanC patients, our findings could have high translational potential in controlling this deadly malignancy. PMID:25672620

  7. Identification of a molecular target of psychosine and its role in globoid cell formation.

    PubMed

    Im, D S; Heise, C E; Nguyen, T; O'Dowd, B F; Lynch, K R

    2001-04-16

    Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine. PMID:11309421

  8. Molecular Mechanisms of Treg-Mediated T Cell Suppression

    PubMed Central

    Schmidt, Angelika; Oberle, Nina; Krammer, Peter H.

    2012-01-01

    CD4+CD25highFoxp3+ regulatory T cells (Tregs) can suppress other immune cells and, thus, are critical mediators of peripheral self-tolerance. On the one hand, Tregs avert autoimmune disease and allergies. On the other hand, Tregs can prevent immune reactions against tumors and pathogens. Despite the importance of Tregs, the molecular mechanisms of suppression remain incompletely understood and controversial. Proliferation and cytokine production of CD4+CD25− conventional T cells (Tcons) can be inhibited directly by Tregs. In addition, Tregs can indirectly suppress Tcon activation via inhibition of the stimulatory capacity of antigen presenting cells. Direct suppression of Tcons by Tregs can involve immunosuppressive soluble factors or cell contact. Different mechanisms of suppression have been described, so far with no consensus on one universal mechanism. Controversies might be explained by the fact that different mechanisms may operate depending on the site of the immune reaction, on the type and activation state of the suppressed target cell as well as on the Treg activation status. Further, inhibition of T cell effector function can occur independently of suppression of proliferation. In this review, we summarize the described molecular mechanisms of suppression with a particular focus on suppression of Tcons and rapid suppression of T cell receptor-induced calcium (Ca2+), NFAT, and NF-κB signaling in Tcons by Tregs. PMID:22566933

  9. CAM and cell fate targeting: molecular and energetic insights into cell growth and differentiation.

    PubMed

    Ventura, Carlo

    2005-09-01

    Evidence-based medicine is switching from the analysis of single diseases at a time toward an integrated assessment of a diseased person. Complementary and alternative medicine (CAM) offers multiple holistic approaches, including osteopathy, homeopathy, chiropractic, acupuncture, herbal and energy medicine and meditation, all potentially impacting on major human diseases. It is now becoming evident that acupuncture can modify the expression of different endorphin genes and the expression of genes encoding for crucial transcription factors in cellular homeostasis. Extremely low frequency magnetic fields have been found to prime the commitment to a myocardial lineage in mouse embryonic stem cells, suggesting that magnetic energy may direct stem cell differentiation into specific cellular phenotypes without the aid of gene transfer technologies. This finding may pave the way to novel approaches in tissue engineering and regeneration. Different ginseng extracts have been shown to modulate growth and differentiation in pluripotent cells and to exert wound-healing and antitumor effects through opposing activities on the vascular system, prompting the hypothesis that ancient compounds may be the target for new logics in cell therapy. These observations and the subtle entanglement among different CAM systems suggest that CAM modalities may deeply affect both the signaling and transcriptional level of cellular homeostasis. Such a perception holds promises for a new era in CAM, prompting reproducible documentation of biological responses to CAM-related strategies and compounds. To this end, functional genomics and proteomics and the comprehension of the cell signaling networks may substantially contribute to the development of a molecular evidence-based CAM. PMID:16136206

  10. Molecular basis of red cell membrane disorders.

    PubMed

    Delaunay, Jean

    2002-01-01

    We will consider an array of genetic disorders of the red cell membrane. Some affect well-known genes. The mutations of most cases of hereditary spherocytosis (HS) are located in the following genes: ANK1, SPTB, SLC4A1, EPB42 and SPTA1, which encode ankyrin, spectrin beta-chain, the anion exchanger 1 (band 3), protein 4.2 and spectrin alpha-chain, respectively. A dominant form of distal renal tubular acidosis also stems from distinct mutations in the SLC4A1 gene. The mutations responsible for hereditary elliptocytosis (HE) and its aggravated form, poikilocytosis (HP), lie in the SPTA1 and SPTB gene, already mentioned, and in the EPB41 gene encoding protein 4.1. Whereas in HS, the SPTA1 and SPTB gene mutations tend to abolish the synthesis of the corresponding chains, in HE/HP, they hinder spectrin tetramerization. Allele alpha(LELY) is a common polymorphic allele which plays the role of an aggravating factor when it occurs in trans of an elliptocytogenic allele of the SPTA1 gene. Southeast Asian ovalocytosis results from a 27- nucleotide deletion in the SLC4A1 gene. Besides these conditions in which the mutations were reached from known alterations in the proteins, other conditions required a positional cloning approach. Such are the genetic disorders of membrane permeability to monovalent cations. Knowledge is the most advanced as regards dehydrated hereditary stomatocytois (DHS). DHS was shown to belong to a pleiotropic syndrome: DHS + fetal edema + pseudohyperkalemia, which maps to 16q23-24. Concerning DHS and another disease of the same class, overhydrated hereditary stomatocytosis, splenectomy almost certainly appears to elicit thromboembolic accidents. PMID:12432217

  11. Gene Mapping of a Mutant Mungbean (Vigna radiata L.) Using New Molecular Markers Suggests a Gene Encoding a YUC4-like Protein Regulates the Chasmogamous Flower Trait.

    PubMed

    Chen, Jingbin; Somta, Prakit; Chen, Xin; Cui, Xiaoyan; Yuan, Xingxing; Srinives, Peerasak

    2016-01-01

    Mungbean (Vigna radiata L.) is a cleistogamous plant in which flowers are pollinated before they open, which prevents yield improvements through heterosis. We previously generated a chasmogamous mutant (CM) mungbean in which open flowers are pollinated. In this study, we developed insertion/deletion (indel) markers based on the transcriptome differences between CM and Sulu-1 (i.e., normal flowering) plants. An F2 population derived from a cross between CM and Sulu-1 was used for gene mapping. Segregation analyses revealed that a single recessive gene regulates the production of chasmogamous flowers. Using newly developed indel and simple sequence repeat markers, the cha gene responsible for the chasmogamous flower trait was mapped to a 277.1-kb segment on chromosome 6. Twelve candidate genes were detected in this segment, including Vradi06g12650, which encodes a YUCCA family protein associated with floral development. A single base pair deletion producing a frame-shift mutation and a premature stop codon in Vradi06g12650 was detected only in CM plants. This suggested that Vradi06g12650 is a cha candidate gene. Our results provide important information for the molecular breeding of chasmogamous mungbean lines, which may serve as new genetic resources for hybrid cultivar development. PMID:27375671

  12. Gene Mapping of a Mutant Mungbean (Vigna radiata L.) Using New Molecular Markers Suggests a Gene Encoding a YUC4-like Protein Regulates the Chasmogamous Flower Trait

    PubMed Central

    Chen, Jingbin; Somta, Prakit; Chen, Xin; Cui, Xiaoyan; Yuan, Xingxing; Srinives, Peerasak

    2016-01-01

    Mungbean (Vigna radiata L.) is a cleistogamous plant in which flowers are pollinated before they open, which prevents yield improvements through heterosis. We previously generated a chasmogamous mutant (CM) mungbean in which open flowers are pollinated. In this study, we developed insertion/deletion (indel) markers based on the transcriptome differences between CM and Sulu-1 (i.e., normal flowering) plants. An F2 population derived from a cross between CM and Sulu-1 was used for gene mapping. Segregation analyses revealed that a single recessive gene regulates the production of chasmogamous flowers. Using newly developed indel and simple sequence repeat markers, the cha gene responsible for the chasmogamous flower trait was mapped to a 277.1-kb segment on chromosome 6. Twelve candidate genes were detected in this segment, including Vradi06g12650, which encodes a YUCCA family protein associated with floral development. A single base pair deletion producing a frame-shift mutation and a premature stop codon in Vradi06g12650 was detected only in CM plants. This suggested that Vradi06g12650 is a cha candidate gene. Our results provide important information for the molecular breeding of chasmogamous mungbean lines, which may serve as new genetic resources for hybrid cultivar development. PMID:27375671

  13. Checkpoint kinase 1 inhibitors as targeted molecular agents for clear cell carcinoma of the ovary

    PubMed Central

    KOBAYASHI, HIROSHI; SHIGETOMI, HIROSHI; YOSHIMOTO, CHIHARU

    2015-01-01

    In clear cell carcinoma of the ovary, chemoresistance frequently results in treatment failure. The present study aimed to review the potential association of transcription factor hepatocyte nuclear factor (HNF)-1β with cell cycle checkpoint machinery, as a mechanism for chemoresistance. The English-language literature on the subject was reviewed to identify genomic alterations and aberrant molecular pathways interacting with chemoresistance in clear cell carcinoma. Oxidative stress induced by repeated hemorrhage induces greater susceptibility of endometriotic cells to DNA damage, and subsequent malignant transformation results in endometriosis-associated ovarian cancer. Molecular changes, including those in HNF-1β and checkpoint kinase 1 (Chk1), may be a manifestation of essential alterations in cell cycle regulation, detoxification and chemoresistance in clear cell carcinoma. Chk1 is a critical signal transducer in the cell cycle checkpoint machinery. DNA damage, in turn, increases persistent phosphorylation of Chk1 and induction of G2/M phase cell cycle arrest in cells overexpressing HNF-1β. HNF-1β deletion induces apoptosis, suggesting that enhanced levels of HNF-1β may be associated with chemoresistance. Targeted therapy with Chk1 inhibitors may be explored as a potential treatment modality for patients with clear cell carcinoma. This provides a novel direction for combination therapy, including targeting of Chk1, which may overcome drug resistance and improve treatment efficacy. PMID:26622535

  14. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    PubMed

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  15. Comprehensive Molecular Characterization of Papillary Renal Cell Carcinoma

    PubMed Central

    Linehan, W. Marston; Spellman, Paul T.; Ricketts, Christopher J.; Creighton, Chad J.; Fei, Suzanne S.; Davis, Caleb; Wheeler, David A.; Murray, Bradley A.; Schmidt, Laura; Vocke, Cathy D.; Peto, Myron; Al Mamun, Abu Amar M.; Shinbrot, Eve; Sethi, Anurag; Brooks, Samira; Rathmell, W. Kimryn; Brooks, Angela N.; Hoadley, Katherine A.; Robertson, A. Gordon; Brooks, Denise; Bowlby, Reanne; Sadeghi, Sara; Shen, Hui; Weisenberger, Daniel J.; Bootwalla, Moiz; Baylin, Stephen B.; Laird, Peter W.; Cherniack, Andrew D.; Saksena, Gordon; Haake, Scott; Li, Jun; Liang, Han; Lu, Yiling; Mills, Gordon B.; Akbani, Rehan; Leiserson, Mark D.M.; Raphael, Benjamin J.; Anur, Pavana; Bottaro, Donald; Albiges, Laurence; Barnabas, Nandita; Choueiri, Toni K.; Czerniak, Bogdan; Godwin, Andrew K.; Hakimi, A. Ari; Ho, Thai; Hsieh, James; Ittmann, Michael; Kim, William Y.; Krishnan, Bhavani; Merino, Maria J.; Mills Shaw, Kenna R.; Reuter, Victor E.; Reznik, Ed; Shelley, Carl Simon; Shuch, Brian; Signoretti, Sabina; Srinivasan, Ramaprasad; Tamboli, Pheroze; Thomas, George; Tickoo, Satish; Burnett, Kenneth; Crain, Daniel; Gardner, Johanna; Lau, Kevin; Mallery, David; Morris, Scott; Paulauskis, Joseph D.; Penny, Robert J.; Shelton, Candace; Shelton, W. Troy; Sherman, Mark; Thompson, Eric; Yena, Peggy; Avedon, Melissa T.; Bowen, Jay; Gastier-Foster, Julie M.; Gerken, Mark; Leraas, Kristen M.; Lichtenberg, Tara M.; Ramirez, Nilsa C.; Santos, Tracie; Wise, Lisa; Zmuda, Erik; Demchok, John A.; Felau, Ina; Hutter, Carolyn M.; Sheth, Margi; Sofia, Heidi J.; Tarnuzzer, Roy; Wang, Zhining; Yang, Liming; Zenklusen, Jean C.; Zhang, Jiashan (Julia); Ayala, Brenda; Baboud, Julien; Chudamani, Sudha; Liu, Jia; Lolla, Laxmi; Naresh, Rashi; Pihl, Todd; Sun, Qiang; Wan, Yunhu; Wu, Ye; Ally, Adrian; Balasundaram, Miruna; Balu, Saianand; Beroukhim, Rameen; Bodenheimer, Tom; Buhay, Christian; Butterfield, Yaron S.N.; Carlsen, Rebecca; Carter, Scott L.; Chao, Hsu; Chuah, Eric; Clarke, Amanda; Covington, Kyle R.; Dahdouli, Mahmoud; Dewal, Ninad; Dhalla, Noreen; Doddapaneni, HarshaVardhan; Drummond, Jennifer; Gabriel, Stacey B.; Gibbs, Richard A.; Guin, Ranabir; Hale, Walker; Hawes, Alicia; Hayes, D. Neil; Holt, Robert A.; Hoyle, Alan P.; Jefferys, Stuart R.; Jones, Steven J.M.; Jones, Corbin D.; Kalra, Divya; Kovar, Christie; Lewis, Lora; Li, Jie; Ma, Yussanne; Marra, Marco A.; Mayo, Michael; Meng, Shaowu; Meyerson, Matthew; Mieczkowski, Piotr A.; Moore, Richard A.; Morton, Donna; Mose, Lisle E.; Mungall, Andrew J.; Muzny, Donna; Parker, Joel S.; Perou, Charles M.; Roach, Jeffrey; Schein, Jacqueline E.; Schumacher, Steven E.; Shi, Yan; Simons, Janae V.; Sipahimalani, Payal; Skelly, Tara; Soloway, Matthew G.; Sougnez, Carrie; Tam, Angela; Tan, Donghui; Thiessen, Nina; Veluvolu, Umadevi; Wang, Min; Wilkerson, Matthew D.; Wong, Tina; Wu, Junyuan; Xi, Liu; Zhou, Jane; Bedford, Jason; Chen, Fengju; Fu, Yao; Gerstein, Mark; Haussler, David; Kasaian, Katayoon; Lai, Phillip; Ling, Shiyun; Radenbaugh, Amie; Van Den Berg, David; Weinstein, John N.; Zhu, Jingchun; Albert, Monique; Alexopoulou, Iakovina; Andersen, Jeremiah J; Auman, J. Todd; Bartlett, John; Bastacky, Sheldon; Bergsten, Julie; Blute, Michael L.; Boice, Lori; Bollag, Roni J.; Boyd, Jeff; Castle, Erik; Chen, Ying-Bei; Cheville, John C.; Curley, Erin; Davies, Benjamin; DeVolk, April; Dhir, Rajiv; Dike, Laura; Eckman, John; Engel, Jay; Harr, Jodi; Hrebinko, Ronald; Huang, Mei; Huelsenbeck-Dill, Lori; Iacocca, Mary; Jacobs, Bruce; Lobis, Michael; Maranchie, Jodi K.; McMeekin, Scott; Myers, Jerome; Nelson, Joel; Parfitt, Jeremy; Parwani, Anil; Petrelli, Nicholas; Rabeno, Brenda; Roy, Somak; Salner, Andrew L.; Slaton, Joel; Stanton, Melissa; Thompson, R. Houston; Thorne, Leigh; Tucker, Kelinda; Weinberger, Paul M.; Winemiller, Cythnia; Zach, Leigh Anne; Zuna, Rosemary

    2016-01-01

    Background Papillary renal cell carcinoma, accounting for 15% of renal cell carcinoma, is a heterogeneous disease consisting of different types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal cell carcinoma; no effective forms of therapy for advanced disease exist. Methods We performed comprehensive molecular characterization utilizing whole-exome sequencing, copy number, mRNA, microRNA, methylation and proteomic analyses of 161 primary papillary renal cell carcinomas. Results Type 1 and Type 2 papillary renal cell carcinomas were found to be different types of renal cancer characterized by specific genetic alterations, with Type 2 further classified into three individual subgroups based on molecular differences that influenced patient survival. MET alterations were associated with Type 1 tumors, whereas Type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-ARE pathway. A CpG island methylator phenotype (CIMP) was found in a distinct subset of Type 2 papillary renal cell carcinoma characterized by poor survival and mutation of the fumarate hydratase (FH) gene. Conclusions Type 1 and Type 2 papillary renal cell carcinomas are clinically and biologically distinct. Alterations in the MET pathway are associated with Type 1 and activation of the NRF2-ARE pathway with Type 2; CDKN2A loss and CIMP in Type 2 convey a poor prognosis. Furthermore, Type 2 papillary renal cell carcinoma consists of at least 3 subtypes based upon molecular and phenotypic features. PMID:26536169

  16. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease

    PubMed Central

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-01-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the ‘gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  17. Optogenetic Control of Molecular Motors and Organelle Distributions in Cells

    PubMed Central

    Duan, Liting; Che, Daphne; Zhang, Kai; Ong, Qunxiang; Guo, Shunling; Cui, Bianxiao

    2015-01-01

    SUMMARY Intracellular transport and distribution of organelles play important roles in diverse cellular functions, including cell polarization, intracellular signaling, cell survival and apoptosis. Here we report an optogenetic strategy to control the transport and distribution of organelles by light. This is achieved by optically recruiting molecular motors onto organelles through the heterodimerization of Arabidopsis thaliana cryptochrome 2 (CRY2) and its interacting partner CIB1. CRY2 and CIB1 dimerize within subseconds upon blue light exposure, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven towards the cell periphery upon light-induced recruitment of kinesin, or towards the cell nucleus upon recruitment of dynein. Light-induced motor recruitment and organelle movements are repeatable, reversible and can be achieved at subcellular regions. This light-controlled organelle redistribution provides a new strategy for studying the causal roles of organelle transport and distribution in cellular functions in living cells. PMID:25963241

  18. [Physiological regulation of hematopoietic stem cell and its molecular basis].

    PubMed

    Dong, Fang; Hao, Sha; Cheng, Hui; Cheng, Tao

    2016-08-25

    As a classical type of tissue stem cells, hematopoietic stem cell (HSC) is the earliest discovered and has been widely applied in the clinic as a great successful example for stem cell therapy. Thus, HSC research represents a leading field in stem cell biology and regenerative medicine. Self-renewal, differentiation, quiescence, apoptosis and trafficking constitute major characteristics of functional HSCs. These characteristics also signify different dynamic states of HSC through physiological interactions with the microenvironment cues in vivo. This review covers our current knowledge on the physiological regulation of HSC and its underlying molecular mechanisms. It is our hope that this review will not only help our colleagues to understand how HSC is physiologically regulated but also serve as a good reference for the studies on stem cell and regenerative medicine in general. PMID:27546503

  19. Molecular Connections between Cancer Cell Metabolism and the Tumor Microenvironment

    PubMed Central

    Justus, Calvin R.; Sanderlin, Edward J.; Yang, Li V.

    2015-01-01

    Cancer cells preferentially utilize glycolysis, instead of oxidative phosphorylation, for metabolism even in the presence of oxygen. This phenomenon of aerobic glycolysis, referred to as the “Warburg effect”, commonly exists in a variety of tumors. Recent studies further demonstrate that both genetic factors such as oncogenes and tumor suppressors and microenvironmental factors such as spatial hypoxia and acidosis can regulate the glycolytic metabolism of cancer cells. Reciprocally, altered cancer cell metabolism can modulate the tumor microenvironment which plays important roles in cancer cell somatic evolution, metastasis, and therapeutic response. In this article, we review the progression of current understandings on the molecular interaction between cancer cell metabolism and the tumor microenvironment. In addition, we discuss the implications of these interactions in cancer therapy and chemoprevention. PMID:25988385

  20. Force probing cell shape changes to molecular resolution.

    PubMed

    Stewart, Martin P; Toyoda, Yusuke; Hyman, Anthony A; Muller, Daniel J

    2011-08-01

    Atomic force microscopy (AFM) is a force sensing nanoscopic tool that can be used to undertake a multiscale approach to understand the mechanisms that underlie cell shape change, ranging from the cellular to molecular scale. In this review paper, we discuss the use of AFM to characterize the dramatic shape changes of mitotic cells. AFM-based mechanical assays can be applied to measure the considerable rounding force and hydrostatic pressure generated by mitotic cells. A complementary AFM technique, single-molecule force spectroscopy, is able to quantify the interactions and mechanisms that functionally regulate individual proteins. Future developments of these nanomechanical methods, together with advances in light microscopy imaging and cell biological and genetic tools, should provide further insight into the biochemical, cellular and mechanical processes that govern mitosis and other cell shape change phenomena. PMID:21646023

  1. Biphasic components of sarcomatoid clear cell renal cell carcinomas are molecularly similar to each other, but distinct from, non-sarcomatoid renal carcinomas.

    PubMed

    Sircar, Kanishka; Yoo, Suk-Young; Majewski, Tadeusz; Wani, Khalida; Patel, Lalit R; Voicu, Horatiu; Torres-Garcia, Wandaliz; Verhaak, Roel G W; Tannir, Nizar; Karam, Jose A; Jonasch, Eric; Wood, Christopher G; Tamboli, Pheroze; Baggerly, Keith A; Aldape, Kenneth D; Czerniak, Bogdan

    2015-10-01

    Sarcomatoid transformation, wherein an epithelioid carcinomatous tumour component coexists with a sarcomatoid histology, is a predictor of poor prognosis in clear cell renal cell carcinoma. Our understanding of sarcomatoid change has been hindered by the lack of molecular examination. Thus, we sought to characterize molecularly the biphasic epithelioid and sarcomatoid components of sarcomatoid clear cell renal cell carcinoma and compare them to non-sarcomatoid clear cell renal cell carcinoma. We examined the transcriptome of the epithelioid and sarcomatoid components of advanced stage sarcomatoid clear cell renal cell carcinoma (n=43) and non-sarcomatoid clear cell renal cell carcinoma (n=37) from independent discovery and validation cohorts using the cDNA microarray and RNA-seq platforms. We analyzed DNA copy number profiles, generated using SNP arrays, from patients with sarcomatoid clear cell renal cell carcinoma (n=10) and advanced non-sarcomatoid clear cell renal cell carcinoma (n=155). The epithelioid and sarcomatoid components of sarcomatoid clear cell renal cell carcinoma had similar gene expression and DNA copy number signatures that were, however, distinct from those of high-grade, high-stage non-sarcomatoid clear cell renal cell carcinoma. Prognostic clear cell renal cell carcinoma gene expression profiles were shared by the biphasic components of sarcomatoid clear cell renal cell carcinoma and the sarcomatoid component showed a partial epithelial-to-mesenchymal transition signature. Our genome-scale microarray-based transcript data were validated in an independent set of sarcomatoid and non-sarcomatoid clear cell renal cell carcinomas using RNA-seq. Sarcomatoid clear cell renal cell carcinoma is molecularly distinct from non-sarcomatoid clear cell renal cell carcinoma, with its genetic programming largely shared by its biphasic morphological components. These data explain why a low percentage of sarcomatoid histology augurs a poor prognosis; suggest the

  2. Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy.

    PubMed

    Rosenthal, Adam Z; Zhang, Xinning; Lucey, Kaitlyn S; Ottesen, Elizabeth A; Trivedi, Vikas; Choi, Harry M T; Pierce, Niles A; Leadbetter, Jared R

    2013-10-01

    Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a "lower," wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based gene-species associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche. PMID:24043823

  3. Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy

    PubMed Central

    Rosenthal, Adam Z.; Zhang, Xinning; Lucey, Kaitlyn S.; Ottesen, Elizabeth A.; Trivedi, Vikas; Choi, Harry M. T.; Pierce, Niles A.; Leadbetter, Jared R.

    2013-01-01

    Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a “lower,” wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based gene–species associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche. PMID:24043823

  4. Reviewing and Updating the Major Molecular Markers for Stem Cells

    PubMed Central

    Calloni, Raquel; Cordero, Elvira Alicia Aparicio; Henriques, João Antonio Pêgas

    2013-01-01

    Stem cells (SC) are able to self-renew and to differentiate into many types of committed cells, making SCs interesting for cellular therapy. However, the pool of SCs in vivo and in vitro consists of a mix of cells at several stages of differentiation, making it difficult to obtain a homogeneous population of SCs for research. Therefore, it is important to isolate and characterize unambiguous molecular markers that can be applied to SCs. Here, we review classical and new candidate molecular markers that have been established to show a molecular profile for human embryonic stem cells (hESCs), mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs). The commonly cited markers for embryonic ESCs are Nanog, Oct-4, Sox-2, Rex-1, Dnmt3b, Lin-28, Tdgf1, FoxD3, Tert, Utf-1, Gal, Cx43, Gdf3, Gtcm1, Terf1, Terf2, Lefty A, and Lefty B. MSCs are primarily identified by the expression of CD13, CD29, CD44, CD49e, CD54, CD71, CD73, CD90, CD105, CD106, CD166, and HLA-ABC and lack CD14, CD31, CD34, CD45, CD62E, CD62L, CD62P, and HLA-DR expression. HSCs are mainly isolated based on the expression of CD34, but the combination of this marker with CD133 and CD90, together with a lack of CD38 and other lineage markers, provides the most homogeneous pool of SCs. Here, we present new and alternative markers for SCs, along with microRNA profiles, for these cells. PMID:23336433

  5. Molecular sequelae of proteasome inhibition in human multiple myeloma cells

    PubMed Central

    Mitsiades, Nicholas; Mitsiades, Constantine S.; Poulaki, Vassiliki; Chauhan, Dharminder; Fanourakis, Galinos; Gu, Xuesong; Bailey, Charles; Joseph, Marie; Libermann, Towia A.; Treon, Steven P.; Munshi, Nikhil C.; Richardson, Paul G.; Hideshima, Teru; Anderson, Kenneth C.

    2002-01-01

    The proteasome inhibitor PS-341 inhibits IκB degradation, prevents NF-κB activation, and induces apoptosis in several types of cancer cells, including chemoresistant multiple myeloma (MM) cells. PS-341 has marked clinical activity even in the setting of relapsed refractory MM. However, PS-341-induced apoptotic cascade(s) are not yet fully defined. By using gene expression profiling, we characterized the molecular sequelae of PS-341 treatment in MM cells and further focused on molecular pathways responsible for the anticancer actions of this promising agent. The transcriptional profile of PS-341-treated cells involved down-regulation of growth/survival signaling pathways, and up-regulation of molecules implicated in proapoptotic cascades (which are both consistent with the proapoptotic effect of proteasome inhibition), as well as up-regulation of heat-shock proteins and ubiquitin/proteasome pathway members (which can correspond to stress responses against proteasome inhibition). Further studies on these pathways showed that PS-341 decreases the levels of several antiapoptotic proteins and triggers a dual apoptotic pathway of mitochondrial cytochrome c release and caspase-9 activation, as well as activation of Jun kinase and a Fas/caspase-8-dependent apoptotic pathway [which is inhibited by a dominant negative (decoy) Fas construct]. Stimulation with IGF-1, as well as overexpression of Bcl-2 or constitutively active Akt in MM cells also modestly attenuates PS-341-induced cell death, whereas inhibitors of the BH3 domain of Bcl-2 family members or the heat-shock protein 90 enhance tumor cell sensitivity to proteasome inhibition. These data provide both insight into the molecular mechanisms of antitumor activity of PS-341 and the rationale for future clinical trials of PS-341, in combination with conventional and novel therapies, to improve patient outcome in MM. PMID:12391322

  6. Molecular anatomy and physiology of exocytosis in sensory hair cells.

    PubMed

    Rutherford, Mark A; Pangršič, Tina

    2012-01-01

    Hair cells mediate our senses of hearing and balance by synaptic release of glutamate from somatic active zones (AZs). They share conserved mechanisms of exocytosis with neurons and other secretory cells of diverse form and function. Concurrently, AZs of these neuro-epithelial hair cells employ several processes that differ remarkably from those of neuronal synaptic terminals of the brain. Their unique molecular anatomy enables them to better respond to small, graded changes in membrane potential and to produce unsurpassed rates of exocytosis. Here, we focus on the AZs of cochlear inner hair cells (IHCs). As in other hair cells, these AZs are occupied by a cytoplasmic extension of the presynaptic density, called the synaptic ribbon: a specialized protein complex required for normal physiological function. Some proteins found at IHC synapses are uniquely expressed or enriched there, where their disruption can beget deafness in humans and in animal models. Other proteins, essential for regulation of conventional neuronal Ca(2+)-triggered fusion, are apparently absent from IHCs. Certain common synaptic proteins appear to have extra significance at ribbon-type AZs because of their interactions with unique molecules, their unusual concentrations, or their atypical localization and regulation. We summarize the molecular-anatomical specializations that underlie the unique synaptic physiology of hair cells. PMID:22682011

  7. Androgen receptor- and PIAS1-regulated gene programs in molecular apocrine breast cancer cells.

    PubMed

    Malinen, Marjo; Toropainen, Sari; Jääskeläinen, Tiina; Sahu, Biswajyoti; Jänne, Olli A; Palvimo, Jorma J

    2015-10-15

    We have analyzed androgen receptor (AR) chromatin binding sites (ARBs) and androgen-regulated transcriptome in estrogen receptor negative molecular apocrine breast cancer cells. These analyses revealed that 42% of ARBs and 39% androgen-regulated transcripts in MDA-MB453 cells have counterparts in VCaP prostate cancer cells. Pathway analyses showed a similar enrichment of molecular and cellular functions among AR targets in both breast and prostate cancer cells, with cellular growth and proliferation being among the most enriched functions. Silencing of the coregulator SUMO ligase PIAS1 in MDA-MB453 cells influenced AR function in a target-selective fashion. An anti-apoptotic effect of the silencing suggests involvement of the PIAS1 in the regulation of cell death and survival pathways. In sum, apocrine breast cancer and prostate cancer cells share a core AR cistrome and target gene signature linked to cancer cell growth, and PIAS1 plays a similar coregulatory role for AR in both cancer cell types. PMID:26219822

  8. A Novel Small Molecular STAT3 Inhibitor, LY5, Inhibits Cell Viability, Cell Migration, and Angiogenesis in Medulloblastoma Cells*

    PubMed Central

    Xiao, Hui; Bid, Hemant Kumar; Jou, David; Wu, Xiaojuan; Yu, Wenying; Li, Chenglong; Houghton, Peter J.; Lin, Jiayuh

    2015-01-01

    Signal transducers and activators of transcription 3 (STAT3) signaling is persistently activated and could contribute to tumorigenesis of medulloblastoma. Numerous studies have demonstrated that inhibition of the persistent STAT3 signaling pathway results in decreased proliferation and increased apoptosis in human cancer cells, indicating that STAT3 is a viable molecular target for cancer therapy. In this study, we investigated a novel non-peptide, cell-permeable small molecule, named LY5, to target STAT3 in medulloblastoma cells. LY5 inhibited persistent STAT3 phosphorylation and induced apoptosis in human medulloblastoma cell lines expressing constitutive STAT3 phosphorylation. The inhibition of STAT3 signaling by LY5 was confirmed by down-regulating the expression of the downstream targets of STAT3, including cyclin D1, bcl-XL, survivin, and micro-RNA-21. LY5 also inhibited the induction of STAT3 phosphorylation by interleukin-6 (IL-6), insulin-like growth factor (IGF)-1, IGF-2, and leukemia inhibitory factor in medulloblastoma cells, but did not inhibit STAT1 and STAT5 phosphorylation stimulated by interferon-γ (IFN-γ) and EGF, respectively. In addition, LY5 blocked the STAT3 nuclear localization induced by IL-6, but did not block STAT1 and STAT5 nuclear translocation mediated by IFN-γ and EGF, respectively. A combination of LY5 with cisplatin or x-ray radiation also showed more potent effects than single treatment alone in the inhibition of cell viability in human medulloblastoma cells. Furthermore, LY5 demonstrated a potent inhibitory activity on cell migration and angiogenesis. Taken together, these findings indicate LY5 inhibits persistent and inducible STAT3 phosphorylation and suggest that LY5 is a promising therapeutic drug candidate for medulloblastoma by inhibiting persistent STAT3 signaling. PMID:25313399

  9. Differential Expression of Wnt Signaling Molecules Between Pre- and Postmenopausal Endometrial Epithelial Cells Suggests a Population of Putative Epithelial Stem/Progenitor Cells Reside in the Basalis Layer

    PubMed Central

    Nguyen, Hong P. T.; Sprung, Carl N.

    2012-01-01

    The human endometrium undergoes extensive monthly regeneration in response to fluctuating levels of circulating estrogen and progesterone in premenopausal (Pre-M) women. In contrast, postmenopausal (Post-M) endometrium is thin and quiescent with low mitotic activity, similar to the Pre-M endometrial basalis layer. Clonogenic epithelial stem/progenitor (ESP) cells, likely responsible for regenerating endometrial epithelium, have been identified in Pre-M and Post-M endometrium, but their location is unknown. We undertook transcriptional profiling of highly purified epithelial cells from full-thickness Pre-M and Post-M endometrium to identify differentially regulated genes that may indicate a putative ESP cell population resides in the basalis of Pre-M and basalis-like Post-M endometrium. Of 1077 differentially expressed genes identified, the Wnt signaling pathway, important in endometrial development and stem cell regulation, was one of the main gene families detected, including 22 Wnt-associated genes. Twelve genes were validated using quantitative RT-PCR, and all were concordant with microarray data. Immunostaining showed glandular epithelial location of Wnt-regulated genes, Axin-related protein 2 and β-catenin. Axin2 localized to the nucleus of basalis Pre-M and Post-M and cytoplasm of functionalis Pre-M endometrium, suggesting that it regulates β-catenin. Comparison of our Post-M gene profile with published gene microarray datasets revealed similarities to Pre-M basalis epithelial profiles. This differential expression of multiple Wnt-associated genes in human Pre-M and Post-M endometrial epithelial cells and the similar gene profile of Post-M and Pre-M basalis epithelium suggests that a population of putative endometrial ESP may reside in the basalis of Pre-M endometrium, which may be responsible for regenerating glandular epithelium each month. PMID:22474188

  10. ADVANCES IN MOLECULAR IMAGING OF PANCREATIC BETA CELLS

    PubMed Central

    Lin, Mai; Lubag, Angelo; McGuire, Michael J.; Seliounine, Serguei Y.; Tsyganov, Edward N.; Antich, Peter P.; Sherry, A. Dean; Brown, Kathlynn C.; Sun, Xiankai

    2009-01-01

    The development of non-invasive imaging methods for early diagnosis of the beta cell associated metabolic diseases, including type 1 and type 2 diabetes (T1D and T2D), has recently drawn considerable interest from the molecular imaging community as well as clinical investigators. Due to the challenges imposed by the location of the pancreas, the sparsely dispersed beta cell population within the pancreas, and the poor understanding of the pathogenesis of the diseases, clinical diagnosis of beta cell abnormalities is still limited. Current diagnostic methods are invasive, often inaccurate, and usually performed post-onset of the disease. Advances in imaging techniques for probing beta cell mass and function are needed to address this critical health care problem. A variety of currently available imaging techniques have been tested for the assessment of the pancreatic beta cell islets. Here we discuss the current advances in magnetic resonance imaging (MRI), bioluminescence imaging (BLI), and nuclear imaging for the study of beta cell diseases. Spurred by early successes in nuclear imaging techniques for beta cells, especially positron emission tomography (PET), the need for beta cell specific ligands has expanded. Progress in the field for obtaining such ligands is presented. Additionally, we report our preliminary efforts of developing such a peptidic ligand for PET imaging of the pancreatic beta cells. PMID:18508529

  11. Microbe Associated Molecular Pattern Signaling in Guard Cells

    PubMed Central

    Ye, Wenxiu; Murata, Yoshiyuki

    2016-01-01

    Stomata, formed by pairs of guard cells in the epidermis of terrestrial plants, regulate gas exchange, thus playing a critical role in plant growth and stress responses. As natural openings, stomata are exploited by microbes as an entry route. Recent studies reveal that plants close stomata upon guard cell perception of molecular signatures from microbes, microbe associated molecular patterns (MAMPs), to prevent microbe invasion. The perception of MAMPs induces signal transduction including recruitment of second messengers, such as Ca2+ and H2O2, phosphorylation events, and change of transporter activity, leading to stomatal movement. In the present review, we summarize recent findings in signaling underlying MAMP-induced stomatal movement by comparing with other signalings. PMID:27200056

  12. Microbe Associated Molecular Pattern Signaling in Guard Cells.

    PubMed

    Ye, Wenxiu; Murata, Yoshiyuki

    2016-01-01

    Stomata, formed by pairs of guard cells in the epidermis of terrestrial plants, regulate gas exchange, thus playing a critical role in plant growth and stress responses. As natural openings, stomata are exploited by microbes as an entry route. Recent studies reveal that plants close stomata upon guard cell perception of molecular signatures from microbes, microbe associated molecular patterns (MAMPs), to prevent microbe invasion. The perception of MAMPs induces signal transduction including recruitment of second messengers, such as Ca(2+) and H2O2, phosphorylation events, and change of transporter activity, leading to stomatal movement. In the present review, we summarize recent findings in signaling underlying MAMP-induced stomatal movement by comparing with other signalings. PMID:27200056

  13. Effect of molecular electrical doping on polyfuran based photovoltaic cells

    SciTech Connect

    Yu, Shuwen; Opitz, Andreas; Salzmann, Ingo; Frisch, Johannes; Cohen, Erez; Bendikov, Michael; Koch, Norbert

    2015-05-18

    The electronic, optical, and morphological properties of molecularly p-doped polyfuran (PF) films were investigated over a wide range of doping ratio in order to explore the impact of doping in photovoltaic applications. We find evidence for integer-charge transfer between PF and the prototypical molecular p-dopant tetrafluoro-tetracyanoquinodimethane (F4TCNQ) and employed the doped polymer in bilayer organic solar cells using fullerene as acceptor. The conductivity increase in the PF films at dopant loadings ≤2% significantly enhances the short-circuit current of photovoltaic devices. For higher doping ratios, however, F4TCNQ is found to precipitate at the heterojunction between the doped donor polymer and the fullerene acceptor. Ultraviolet photoelectron spectroscopy reveals that its presence acts beneficial to the energy-level alignment by doubling the open-circuit voltage of solar cells from 0.2 V to ca. 0.4 V, as compared to pristine PF.

  14. Molecular Characterization of Circulating Plasma Cells in Patients with Active Systemic Lupus Erythematosus

    PubMed Central

    Lugar, Patricia L.; Love, Cassandra; Grammer, Amrie C.; Dave, Sandeep S.; Lipsky, Peter E.

    2012-01-01

    Systemic lupus erythematosus (SLE) is a generalized autoimmune disease characterized by abnormal B cell activation and the occurrence of increased frequencies of circulating plasma cells (PC). The molecular characteristics and nature of circulating PC and B cells in SLE have not been completely characterized. Microarray analysis of gene expression was used to characterize circulating PC in subjects with active SLE. Flow cytometry was used to sort PC and comparator B cell populations from active SLE blood, normal blood and normal tonsil. The gene expression profiles of the sorted B cell populations were then compared. SLE PC exhibited a similar gene expression signature as tonsil PC. The differences in gene expression between SLE PC and normal tonsil PC and tonsil plasmablasts (PB) suggest a mature Ig secreting cell phenotype in the former population. Despite this, SLE PC differed in expression of about half the genes from previously published gene expression profiles of normal bone marrow PC, indicating that these cells had not achieved a fully mature status. Abnormal expression of several genes, including CXCR4 and S1P1, suggests a mechanism for the persistence of SLE PC in the circulation. All SLE B cell populations revealed an interferon (IFN) gene signature previously only reported in unseparated SLE peripheral blood mononuclear cells. These data indicate that SLE PC are a unique population of Ig secreting cells with a gene expression profile indicative of a mature, but not fully differentiated phenotype. PMID:23028528

  15. Molecular variants of human papillomavirus type 16 from four continents suggest ancient pandemic spread of the virus and its coevolution with humankind.

    PubMed Central

    Chan, S Y; Ho, L; Ong, C K; Chow, V; Drescher, B; Dürst, M; ter Meulen, J; Villa, L; Luande, J; Mgaya, H N

    1992-01-01

    We have amplified by the polymerase chain reaction, cloned, and sequenced genomic segments of 118 human papillomavirus type 16 (HPV-16) isolates from 76 cervical biopsy, 14 cervical smear, 3 vulval biopsy, 2 penile biopsy, 2 anal biopsy, and 1 vaginal biopsy sample and two cell lines. The specimens were taken from patients in four countries--Singapore, Brazil, Tanzania, and Germany. The sequence of a 364-bp fragment of the long control region of the virus revealed 38 variants, most of which differed by one or several point mutations. Phylogenetic trees were constructed by distance matrix methods and a transformation series approach. The trees based on the long control region were supported by another set based on the complete E5 protein-coding region. Both sets had two main branches. Nearly all of the variants from Tanzania were assigned to one (African) branch, and all of the German and most of the Singaporean variants were assigned to the other (Eurasian) branch. While some German and Singaporean variants were identical, each group also contained variants that formed unique branches. In contrast to the group-internal homogeneity of the Singaporean, German, and Tanzanian variants, the Brazilian variants were clearly divided between the two branches. Exceptions to this were the seven Singaporean isolates with mutational patterns typical of the Tanzanian isolates. The data suggest that HPV-16 evolved separately for a long period in Africa and Eurasia. Representatives of both branches may have been transferred to Brazil via past colonial immigration. The comparable efficiencies of transfer of the African and the Eurasian variants to the New World suggest pandemic spread of HPV-16 in past centuries. Representatives of the African branch were possibly transferred to the Far East along old Arab and Indonesian sailing routes. Our data also support the view that HPV-16 is a well-defined virus type, since the variants show only a maximal genomic divergence of about 5%. The

  16. Different sucrose-isomaltase response of Caco-2 cells to glucose and maltose suggests dietary maltose sensing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using the small intestine enterocyte Caco-2 cell model, sucrase-isomaltase (SI, the mucosal alpha-glucosidase complex) expression and modification were examined relative to exposure to different mono- and disaccharide glycemic carbohydrates. Caco-2/TC7 cells were grown on porous supports to post-con...

  17. Construction, molecular modeling, and simulation of Mycobacterium tuberculosis cell walls.

    PubMed

    Hong, Xuan; Hopfinger, A J

    2004-01-01

    The mycobacterial cell wall is extraordinarily thick and tight consisting mainly of (1). long chain fatty acids, the mycolic acids, and (2). a unique polysaccharide, arabinogalactan (AG). These two chemical constituents are covalently linked through ester bonds. Minnikin (The Biology of the Mycobacteria; Academic: London, 1982) proposed that the mycobacterial cell wall is composed of an asymmetric lipid bilayer. The inner leaflet of the cell wall contains mycolic acids covalently linked to AG. This inner leaflet is believed to have the lowest permeability to organic compounds of the overall cell wall. Conformational search and molecular dynamics simulation were used to explore the conformational profile of AG and the conformations and structural organization of the mycolic acid-AG complex, and overall, an inner leaflet molecular model of the cell wall was constructed. The terminal arabinose residues of AG that serve as linkers between AG and mycolic acids were found to exist in four major chemical configurations. The mycolate hydrocarbon chains were determined to be tightly packed and perpendicular to the "plane" formed by the oxygen atoms of the 5-hydroxyl groups of the terminal arabinose residues. For Mycobacterium tuberculosis, the average packing distance between mycolic acids is estimated to be approximately 7.3 A. Thus, Minnikin's model is supported by this computational study. Overall, this modeling and simulation approach provides a way to probe the mechanism of low permeability of the cell wall and the intrinsic drug resistance of M. tuberculosis. In addition, monolayer models were built for both dipalmitoylphosphatidylethanolamine and dimyristoylphosphatidylcholine, two common phospholipids in bacterial and animal membranes, respectively. Structural comparisons of these cell wall phospholipid membrane models were made to the M. tuberculosis cell wall model. PMID:15132700

  18. Single-cell approaches for molecular classification of endocrine tumors

    PubMed Central

    Koh, James; Allbritton, Nancy L.; Sosa, Julie A.

    2015-01-01

    Purpose of review In this review, we summarize recent developments in single-cell technologies that can be employed for the functional and molecular classification of endocrine cells in normal and neoplastic tissue. Recent findings The emergence of new platforms for the isolation, analysis, and dynamic assessment of individual cell identity and reactive behavior enables experimental deconstruction of intratumoral heterogeneity and other contexts, where variability in cell signaling and biochemical responsiveness inform biological function and clinical presentation. These tools are particularly appropriate for examining and classifying endocrine neoplasias, as the clinical sequelae of these tumors are often driven by disrupted hormonal responsiveness secondary to compromised cell signaling. Single-cell methods allow for multidimensional experimental designs incorporating both spatial and temporal parameters with the capacity to probe dynamic cell signaling behaviors and kinetic response patterns dependent upon sequential agonist challenge. Summary Intratumoral heterogeneity in the provenance, composition, and biological activity of different forms of endocrine neoplasia presents a significant challenge for prognostic assessment. Single-cell technologies provide an array of powerful new approaches uniquely well suited for dissecting complex endocrine tumors. Studies examining the relationship between clinical behavior and tumor compositional variations in cellular activity are now possible, providing new opportunities to deconstruct the underlying mechanisms of endocrine neoplasia. PMID:26632769

  19. Asymmetric Aneuploidy in Mesenchymal Stromal Cells Detected by In Situ Karyotyping and Fluorescence In Situ Hybridization: Suggestions for Reference Values for Stem Cells

    PubMed Central

    Kim, Seon Young; Im, Kyongok; Park, Si Nae; Kwon, Jiseok; Kim, Jung-Ah; Choi, Qute; Hwang, Sang Mee; Han, Sung-Hee; Kwon, Sunghoon; Oh, Il-Hoan

    2015-01-01

    Cytogenetic testing is important to ensure patient safety before therapeutic application of mesenchymal stromal cells (MSCs). However, the standardized methods and criteria for the screening of chromosomal abnormalities of MSCs have not yet been determined. We investigated the frequency of cytogenetic aberrations in MSCs using G-banding and fluorescence in situ hybridization (FISH) and suggest reference values for aneuploidy in MSCs. Cytogenetic analysis was performed on 103 consecutive cultures from 68 MSCs (25 adipose-origin, 20 bone marrow-origin, 18 cord blood-origin, and 5 neural stem cells; 8 from adipose tissue of patients with breast cancer and 60 from healthy donors). We compared the MSC aneuploidy patterns with those of hematological malignancies and benign hematological diseases. Interphase FISH showed variable aneuploid clone proportions (1%–20%) in 68 MSCs. The aneuploidy patterns were asymmetric, and aneuploidy of chromosomes 16, 17, 18, and X occurred most frequently. Clones with polysomy were significantly more abundant than those with monosomy. The cutoff value of maximum polysomy rates (upper 95th percentile value) was 13.0%. By G-banding, 5 of the 61 MSCs presented clonal chromosomal aberrations. Aneuploidy was asymmetric in the malignant hematological diseases, while it was symmetric in the benign hematological diseases. We suggest an aneuploidy cutoff value of 13%, and FISH for aneuploidy of chromosomes 16, 17, 18, and X would be informative to evaluate the genetic stability of MSCs. Although it is unclear whether the aneuploid clones might represent the senescent cell population or transformed cells, more attention should be focused on the safety of MSCs, and G-banding combined with FISH should be performed. PMID:25019198

  20. Proteomic analysis of the molecular response of Raji cells to maslinic acid treatment.

    PubMed

    Yap, W H; Khoo, K S; Lim, S H; Yeo, C C; Lim, Y M

    2012-01-15

    Maslinic acid, a natural pentacyclic triterpene has been shown to inhibit growth and induce apoptosis in some tumour cell lines. We studied the molecular response of Raji cells towards maslinic acid treatment. A proteomics approach was employed to identify the target proteins. Seventeen differentially expressed proteins including those involved in DNA replication, microtubule filament assembly, nucleo-cytoplasmic trafficking, cell signaling, energy metabolism and cytoskeletal organization were identified by MALDI TOF-TOF MS. The down-regulation of stathmin, Ran GTPase activating protein-1 (RanBP1), and microtubule associated protein RP/EB family member 1 (EB1) were confirmed by Western blotting. The study of the effect of maslinic acid on Raji cell cycle regulation showed that it induced a G1 cell cycle arrest. The differential proteomic changes in maslinic acid-treated Raji cells demonstrated that it also inhibited expression of dUTPase and stathmin which are known to induce early S and G2 cell cycle arrests. The mechanism of maslinic acid-induced cell cycle arrest may be mediated by inhibiting cyclin D1 expression and enhancing the levels of cell cycle-dependent kinase (CDK) inhibitor p21 protein. Maslinic acid suppressed nuclear factor-kappa B (NF-κB) activity which is known to stimulate expression of anti-apoptotic and cell cycle regulatory gene products. These results suggest that maslinic acid affects multiple signaling molecules and inhibits fundamental pathways regulating cell growth and survival in Raji cells. PMID:21893403

  1. Molecular mechanism of extrinsic factors affecting anti-aging of stem cells

    PubMed Central

    Wong, Tzyy Yue; Solis, Mairim Alexandra; Chen, Ying-Hui; Huang, Lynn Ling-Huei

    2015-01-01

    Scientific evidence suggests that stem cells possess the anti-aging ability to self-renew and maintain differentiation potentials, and quiescent state. The objective of this review is to discuss the micro-environment where stem cells reside in vivo, the secreted factors to which stem cells are exposed, the hypoxic environment, and intracellular factors including genome stability, mitochondria integrity, epigenetic regulators, calorie restrictions, nutrients, and vitamin D. Secreted tumor growth factor-β and fibroblast growth factor-2 are reported to play a role in stem cell quiescence. Extracellular matrices may interact with caveolin-1, the lipid raft on cell membrane to regulate quiescence. N-cadherin, the adhesive protein on niche cells provides support for stem cells. The hypoxic micro-environment turns on hypoxia-inducible factor-1 to prevent mesenchymal stem cells aging through p16 and p21 down-regulation. Mitochondria express glucosephosphate isomerase to undergo glycolysis and prevent cellular aging. Epigenetic regulators such as p300, protein inhibitors of activated Stats and H19 help maintain stem cell quiescence. In addition, calorie restriction may lead to secretion of paracrines cyclic ADP-ribose by intestinal niche cells, which help maintain intestinal stem cells. In conclusion, it is crucial to understand the anti-aging phenomena of stem cells at the molecular level so that the key to solving the aging mystery may be unlocked. PMID:25815136

  2. Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell

    PubMed Central

    Winer-Jones, Jessamine P.; Vahidi, Behrad; Arquilevich, Norma; Fang, Cong; Ferguson, Samuel; Harkins, Darren; Hill, Cory; Klem, Erich; Pagano, Paul C.; Peasley, Chrissy; Romero, Juan; Shartle, Robert; Vasko, Robert C.; Strauss, William M.; Dempsey, Paul W.

    2014-01-01

    Background Contemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available. Methodology/Principal Findings Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL. Conclusions/Significance The LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for

  3. High molecular weight insulating polymers can improve the performance of molecular solar cells

    NASA Astrophysics Data System (ADS)

    Huang, Ye; Wen, Wen; Kramer, Edward; Bazan, Guillermo

    2014-03-01

    Solution-processed molecular semiconductors for the fabrication of solar cells have emerged as a competitive alternative to their conjugated polymer counterparts, primarily because such materials systems exhibit no batch-to-batch variability, can be purified to a greater extent and offer precisely defined chemical structures. Highest power conversion efficiencies (PCEs) have been achieved through a combination of molecular design and the application of processing methods that optimize the bulk heterojunction (BHJ) morphology. However, one finds that the methods used for controlling structural order, for example the use of high boiling point solvent additives, have been inspired by examination of the conjugated polymer literature. It stands to reason that a different class of morphology modifiers should be sought that address challenges unique to molecular films, including difficulties in obtaining thicker films and avoiding the dewetting of active photovoltaic layers. Here we show that the addition of small quantities of high molecular weight polystyrene (PS) is a very simple to use and economically viable additive that improves PCE. Remarkably, the PS spontaneously accumulates away from the electrodes as separate domains that do not interfere with charge extraction and collection or with the arrangement of the donor and acceptor domains in the BHJ blend.

  4. Tracking neuronal marker expression inside living differentiating cells using molecular beacons

    PubMed Central

    Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole; Dufva, Martin

    2013-01-01

    Monitoring gene expression is an important tool for elucidating mechanisms of cellular function. In order to monitor gene expression during nerve cell development, molecular beacon (MB) probes targeting markers representing different stages of neuronal differentiation were designed and synthesized as 2'-O-methyl RNA backbone oligonucleotides. MBs were transfected into human mesencephalic cells (LUHMES) using streptolysin-O-based membrane permeabilization. Mathematical modeling, simulations and experiments indicated that MB concentration was equal to the MB in the transfection medium after 10 min transfection. The cells will then each contain about 60,000 MBs. Gene expression was detected at different time points using fluorescence microscopy. Nestin and NeuN mRNA were expressed in approximately 35% of the LUHMES cells grown in growth medium, and in 80–90% of cells after differentiation. MAP2 and tyrosine hydroxylase mRNAs were expressed 2 and 3 days post induction of differentiation, respectively. Oct 4 was not detected with MB in these cells and signal was not increased over time suggesting that MB are generally stable inside the cells. The gene expression changes measured using MBs were confirmed using qRT-PCR. These results suggest that MBs are simple to use sensors inside living cell, and particularly useful for studying dynamic gene expression in heterogeneous cell populations. PMID:24431988

  5. Tracking neuronal marker expression inside living differentiating cells using molecular beacons.

    PubMed

    Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole; Dufva, Martin

    2013-12-19

    Monitoring gene expression is an important tool for elucidating mechanisms of cellular function. In order to monitor gene expression during nerve cell development, molecular beacon (MB) probes targeting markers representing different stages of neuronal differentiation were designed and synthesized as 2'-O-methyl RNA backbone oligonucleotides. MBs were transfected into human mesencephalic cells (LUHMES) using streptolysin-O-based membrane permeabilization. Mathematical modeling, simulations and experiments indicated that MB concentration was equal to the MB in the transfection medium after 10 min transfection. The cells will then each contain about 60,000 MBs. Gene expression was detected at different time points using fluorescence microscopy. Nestin and NeuN mRNA were expressed in approximately 35% of the LUHMES cells grown in growth medium, and in 80-90% of cells after differentiation. MAP2 and tyrosine hydroxylase mRNAs were expressed 2 and 3 days post induction of differentiation, respectively. Oct 4 was not detected with MB in these cells and signal was not increased over time suggesting that MB are generally stable inside the cells. The gene expression changes measured using MBs were confirmed using qRT-PCR. These results suggest that MBs are simple to use sensors inside living cell, and particularly useful for studying dynamic gene expression in heterogeneous cell populations. PMID:24431988

  6. Molecular mechanisms of CD8(+) T cell trafficking and localization.

    PubMed

    Nolz, Jeffrey C

    2015-07-01

    Cytotoxic CD8(+) T cells are potent mediators of host protection against disease due to their ability to directly kill cells infected with intracellular pathogens and produce inflammatory cytokines at the site of infection. To fully achieve this objective, naïve CD8(+) T cells must be able to survey the entire body for the presence of foreign or "non-self" antigen that is delivered to draining lymph nodes following infection or tissue injury. Once activated, CD8(+) T cells undergo many rounds of cell division, acquire effector functions, and are no longer restricted to the circulation and lymphoid compartments like their naïve counterparts, but rather are drawn to inflamed tissues to combat infection. As CD8(+) T cells transition from naïve to effector to memory populations, this is accompanied by dynamic changes in the expression of adhesion molecules and chemokine receptors that ultimately dictate their localization in vivo. Thus, an understanding of the molecular mechanisms regulating CD8(+) T cell trafficking and localization is critical for vaccine design, control of infectious diseases, treatment of autoimmune disorders, and cancer immunotherapy. PMID:25577280

  7. Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness.

    PubMed

    Sellick, Christopher A; Croxford, Alexandra S; Maqsood, Arfa R; Stephens, Gill M; Westerhoff, Hans V; Goodacre, Royston; Dickson, Alan J

    2015-09-01

    Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC-MS) analytics to define the molecular loci by which two yield-enhancing feeds improve recombinant antibody yields from a model GS-CHO cell line. With data across core metabolic pathways, that report on metabolism within several cellular compartments, these data identify key metabolites and events associated with increased cell survival and specific productivity of cells. Of particular importance, increased process efficiency was linked to the functional activity of the mitochondria, with the amount and time course of use/production of intermediates of the citric acid cycle, for uses such as lipid biosynthesis, precursor generation and energy production, providing direct indicators of cellular status with respect to productivity. The data provide clear association between specific cellular metabolic indicators and cell process efficiency, extending from prior indications of the relevance of lactate metabolic balance to other redox sinks (glycerol, sorbitol and threitol). The information, and its interpretation, identifies targets for engineering cell culture efficiency, either from genetic or environmental perspectives, and greater understanding of the significance of specific medium components towards overall CHO cell bioprocessing. PMID:26198903

  8. Mechanistic modeling confronts the complexity of molecular cell biology.

    PubMed

    Phair, Robert D

    2014-11-01

    Mechanistic modeling has the potential to transform how cell biologists contend with the inescapable complexity of modern biology. I am a physiologist-electrical engineer-systems biologist who has been working at the level of cell biology for the past 24 years. This perspective aims 1) to convey why we build models, 2) to enumerate the major approaches to modeling and their philosophical differences, 3) to address some recurrent concerns raised by experimentalists, and then 4) to imagine a future in which teams of experimentalists and modelers build-and subject to exhaustive experimental tests-models covering the entire spectrum from molecular cell biology to human pathophysiology. There is, in my view, no technical obstacle to this future, but it will require some plasticity in the biological research mind-set. PMID:25368428

  9. Molecular features of a human rhabdomyosarcoma cell line with spontaneous metastatic progression

    PubMed Central

    Scholl, F A; Betts, D R; Niggli, F K; Schäfer, B W

    2000-01-01

    A novel human cell line was established from a primary botryoid rhabdomyosarcoma. Reverse transcription polymerase chain reaction investigations of this cell line, called RUCH-2, demonstrated expression of the regulatory factors PAX3, Myf3 and Myf5. After 3.5 months in culture, cells underwent a crisis after which Myf3 and Myf5 could no longer be detected, whereas PAX3 expression remained constant over the entire period. Karyotype analysis revealed breakpoints in regions similar to previously described alterations in primary rhabdomyosarcoma tumour samples. Interestingly, cells progressed to a metastatic phenotype, as observed by enhanced invasiveness in vitro and tumour growth in nude mice in vivo. On the molecular level, microarray analysis before and after progression identified extensive changes in the composition of the extracellular matrix. As expected, down-regulation of tissue inhibitors of metalloproteinases and up-regulation of matrix metalloproteinases were observed. Extensive down-regulation of several death receptors of the tumour necrosis factor family suggests that these cells might have an altered response to appropriate apoptotic stimuli. The RUCH-2 cell line represents a cellular model to study multistep tumorigenesis in human rhabdomyosarcoma, allowing molecular comparison of tumorigenic versus metastatic cancer cells. © 2000 Cancer Research Campaign PMID:10735512

  10. Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing

    PubMed Central

    Salvianti, Francesca; Rotunno, Giada; Galardi, Francesca; De Luca, Francesca; Pestrin, Marta; Vannucchi, Alessandro Maria; Di Leo, Angelo; Pazzagli, Mario; Pinzani, Pamela

    2015-01-01

    The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch® and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer. Upon whole genome amplification, samples were analysed by NGS on the Ion Torrent PGM™ system (Life Technologies) using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies), designed to investigate genomic “hot spot” regions of 50 oncogenes and tumor suppressor genes. We successfully sequenced five single cells, a pool of 5 cells and DNA from a cellular pellet of the same cell line with a mean depth of the sequencing reaction ranging from 1581 to 3479 reads. We found 27 sequence variants in 18 genes, 15 of which already reported in the COSMIC or dbSNP databases. We confirmed the presence of two somatic mutations, in the BRAF and TP53 gene, which had been already reported for this cells line, but also found new mutations and single nucleotide polymorphisms. Three variants were common to all the analysed samples, while 18 were present only in a single cell suggesting a high heterogeneity within the same cell line. This paper presents an optimized workflow for the molecular characterization of multiple genes in single cells by NGS. The described pipeline can be easily transferred to the study of single CTCs from oncologic patients. PMID:27077040

  11. Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing.

    PubMed

    Salvianti, Francesca; Rotunno, Giada; Galardi, Francesca; De Luca, Francesca; Pestrin, Marta; Vannucchi, Alessandro Maria; Di Leo, Angelo; Pazzagli, Mario; Pinzani, Pamela

    2015-09-01

    The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch(®) and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer. Upon whole genome amplification, samples were analysed by NGS on the Ion Torrent PGM™ system (Life Technologies) using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies), designed to investigate genomic "hot spot" regions of 50 oncogenes and tumor suppressor genes. We successfully sequenced five single cells, a pool of 5 cells and DNA from a cellular pellet of the same cell line with a mean depth of the sequencing reaction ranging from 1581 to 3479 reads. We found 27 sequence variants in 18 genes, 15 of which already reported in the COSMIC or dbSNP databases. We confirmed the presence of two somatic mutations, in the BRAF and TP53 gene, which had been already reported for this cells line, but also found new mutations and single nucleotide polymorphisms. Three variants were common to all the analysed samples, while 18 were present only in a single cell suggesting a high heterogeneity within the same cell line. This paper presents an optimized workflow for the molecular characterization of multiple genes in single cells by NGS. The described pipeline can be easily transferred to the study of single CTCs from oncologic patients. PMID:27077040

  12. Serological & molecular diagnostic surveys combined with examining hematological profiles suggest increased levels of infection & hematological response of cattle to babesiosis infections compared to native buffaloes in Egypt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Babesiosis threatens the development of the cattle and buffaloes industries in Egypt and improved control is needed. The main objectives of this study are surveying the presence of bovine babesiosis in distinct selected bovine and buffalo populations in Egypt using novel molecular and pr...

  13. Multi-locus molecular phylogeny and allelic variation in a transcription factor gene suggest the multiple independent origins of kabuli chickpea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To examine the patterns of molecular diversity in wild crop relatives and the cultivated gene pool of chickpea we genotyped a set of 98 wild annual and 224 cultivated accessions with a 768 feature assay that monitored SNPs in low-copy orthologous loci. Analyses of the resulting multi-locus genotypin...

  14. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

    PubMed Central

    Galluzzi, L; Vitale, I; Abrams, J M; Alnemri, E S; Baehrecke, E H; Blagosklonny, M V; Dawson, T M; Dawson, V L; El-Deiry, W S; Fulda, S; Gottlieb, E; Green, D R; Hengartner, M O; Kepp, O; Knight, R A; Kumar, S; Lipton, S A; Lu, X; Madeo, F; Malorni, W; Mehlen, P; Nuñez, G; Peter, M E; Piacentini, M; Rubinsztein, D C; Shi, Y; Simon, H-U; Vandenabeele, P; White, E; Yuan, J; Zhivotovsky, B; Melino, G; Kroemer, G

    2012-01-01

    In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including ‘apoptosis', ‘necrosis' and ‘mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features. PMID:21760595

  15. Nitric oxide and thermogenesis--challenge in molecular cell physiology.

    PubMed

    Otasevic, Vesna; Korac, Aleksandra; Buzadzic, Biljana; Stancic, Ana; Jankovic, Aleksandra; Korac, Bato

    2011-01-01

    Only recently we can link thermogenesis, mitochondria, nitric oxide, and redox regulation in biochemical terms. Currently, we are discussing these processes from the aspect of fundamental principles of molecular physiology. Thus, the present article highlights both cell physiology and the principles of the maintenance of energy homeostasis in organisms. Energy homeostasis means much more than simple combustion; adipose tissues at this point of evolution development are related to a broad spectrum of metabolic disturbances and all aspects of cellular remodeling (i.e. structural, metabolic and endocrine changes). Therefore, this paper addresses not only thermogenesis but also energy homeostasis, oxidative phosphorylation and ATP production, proliferation and differentiation of brown adipocytes, their life and death, mitochondriogenesis and angiogenesis. These processes will be united by molecular players of oxidation/reduction reactions, thus creating the principles based on the redox regulation. PMID:21622264

  16. Molecular differential diagnosis of renal cell carcinomas by microsatellite analysis.

    PubMed Central

    Bugert, P.; Kovacs, G.

    1996-01-01

    Recent application of molecular cytogenetic techniques has resulted in a new type of genetic classification of renal cell tumors. The key aspect of the novel diagnostic concept is reflected by biologically distinct entities, each characterized by a specific combination of genetic changes. To work out a diagnostic/prognostic approach, we have applied polymorphic microsatellite markers for a quick analysis, based on polymerase chain reaction, of 82 tumor specimens. We compared the results to previously evaluated cytogenetic and histological data. All nonpapillary and chromophobe renal cell carcinomas, which make up approximately 90% of all malignant renal cell tumors, and a subset of renal oncocytomas were correctly diagnosed by detection of loss of heterozygosity at chromosomal sites 1, 2, and 3p. Allelic losses at chromosomal regions 8p, 9p, and 14q are associated with an advanced pathological stage of nonpapillary renal cell carcinomas. A loss of heterozygosity at chromosomes 6, 10, 13, 17, and 21, in addition to those at chromosomes 1 and 2, confirm the diagnosis of chromophobe renal cell tumors. Using this approach, the differential diagnosis of renal cell tumors could be carried out within 1 or 2 days. Images Figure 2 PMID:8952540

  17. The molecular basis of the memory T cell response: differential gene expression and its epigenetic regulation

    PubMed Central

    Weng, Nan-ping; Araki, Yasuto; Subedi, Kalpana

    2015-01-01

    How the immune system remembers a previous encounter with a pathogen and responds more efficiently to a subsequent encounter has been one of the central enigmas for immunologists for over a century. The identification of pathogen-specific memory lymphocytes that arise after an infection provided a cellular basis for immunological memory. But the molecular mechanisms of immunological memory remain only partially understood. The emerging evidence suggests that epigenetic changes have a key role in controlling the distinct transcriptional profiles of memory lymphocytes and thus in shaping their function. In this Review, we summarize the recent progress that has been made in assessing the differential gene expression and chromatin modifications in memory CD4+ and CD8+ T cells, and we present our current understanding of the molecular basis of memory T cell function. PMID:22421787

  18. Neoplastic cell transformation by high-LET radiation - Molecular mechanisms

    NASA Technical Reports Server (NTRS)

    Yang, Tracy Chui-Hsu; Craise, Laurie M.; Tobias, Cornelius A.; Mei, Man-Tong

    1989-01-01

    Quantitative data were collected on dose-response curves of cultured mouse-embryo cells (C3H10T1/2) irradiated with heavy ions of various charges and energies. Results suggests that two breaks formed on DNA within 80 A may cause cell transformation and that two DNA breaks formed within 20 A may be lethal. From results of experiments with restriction enzymes which produce DNA damages at specific sites, it was found that DNA double strand breaks are important primary lesions for radiogenic cell transformation and that blunt-ended double-strand breaks can form lethal as well as transformational damages due to misrepair or incomplete repair in the cell. The RBE-LET relationship for high-LET radiation is similar to that for HGPRT locus mutation, chromosomal deletion, and cell transformation, indicating that common lesions may be involved in these radiation effects.

  19. Towards quantitative molecular mapping of cells by Raman microscopy: using AFM for decoupling molecular concentration and cell topography.

    PubMed

    Boitor, Radu; Sinjab, Faris; Strohbuecker, Stephanie; Sottile, Virginie; Notingher, Ioan

    2016-06-23

    Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in vitro. However, obtaining quantitative molecular information from Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least squares fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 25 ± 6 mg ml(-1), while proteins were distributed more uniformly and reached concentrations as high as ∼50 ± 12 mg ml(-1). The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman maps of fixed cells (n = 10), we found a linear relationship between the scores corresponding to the first component (PC1) and the cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n = 10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were used to obtain concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing

  20. Proteomic Shifts in Embryonic Stem Cells with Gene Dose Modifications Suggest the Presence of Balancer Proteins in Protein Regulatory Networks

    PubMed Central

    Mao, Lei; Zabel, Claus; Herrmann, Marion; Nolden, Tobias; Mertes, Florian; Magnol, Laetitia; Chabert, Caroline; Hartl, Daniela; Herault, Yann; Delabar, Jean Maurice; Manke, Thomas; Himmelbauer, Heinz; Klose, Joachim

    2007-01-01

    Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of “balancer” proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the “elasticity” of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions. PMID:18043732

  1. Pathway-based evaluation of 380 candidate genes and lung cancer susceptibility suggests the importance of the cell cycle pathway

    PubMed Central

    Hosgood, H.Dean; Menashe, Idan; Shen, Min; Yeager, Meredith; Yuenger, Jeff; Rajaraman, Preetha; He, Xingzhou; Chatterjee, Nilanjan; Caporaso, Neil E.; Zhu, Yong; Chanock, Stephen J.; Zheng, Tongzhang; Lan, Qing

    2008-01-01

    Common genetic variation may play an important role in altering lung cancer risk. We conducted a pathway-based candidate gene evaluation to identify genetic variations that may be associated with lung cancer in a population-based case–control study in Xuan Wei, China (122 cases and 111 controls). A total of 1260 single-nucleotide polymorphisms (SNPs) in 380 candidate genes for lung cancer were successfully genotyped and assigned to one of 10 pathways based on gene ontology. Logistic regression was used to assess the marginal effect of each SNP on lung cancer susceptibility. The minP test was used to identify statistically significant associations at the gene level. Important pathways were identified using a test of proportions and the rank truncated product methods. The cell cycle pathway was found as the most important pathway (P = 0.044) with four genes significantly associated with lung cancer (PLA2G6 minP = 0.001, CCNA2 minP = 0.006, GSK3β minP = 0.007 and EGF minP = 0.013), after adjusting for multiple comparisons. Interestingly, most cell cycle genes that were associated with lung cancer in this analysis were concentrated in the AKT signaling pathway, which is essential for regulation of cell cycle progression and cellular survival, and may be important in lung cancer etiology in Xuan Wei. These results should be viewed as exploratory until they are replicated in a larger study. PMID:18676680

  2. Tuning Open-Circuit Voltage in Organic Solar Cells with Molecular Orientation.

    PubMed

    Kitchen, Brent; Awartani, Omar; Kline, R Joseph; McAfee, Terry; Ade, Harald; O'Connor, Brendan T

    2015-06-24

    The role of molecular orientation of a polar conjugated polymer in polymer-fullerene organic photovoltaic (OPV) cells is investigated. A planar heterojunction (PHJ) OPV cell composed of poly(3-hexylthiophene) (P3HT) and [6,6]-phenyl C61-butyric acid methyl ester (PCBM) is used as a model system to isolate the effect of the interfacial orientation on the photovoltaic properties. The molecular orientation of the aggregate P3HT relative to the PCBM layer is varied from highly edge-on (conjugated ring plane perpendicular to the interface plane) to appreciably face-on (ring plane parallel to the interface). It is found that as the P3HT stacking becomes more face-on there is a positive correlation to the OPV open-circuit voltage (V(OC)), attributed to a shift in the highest occupied molecular orbital (HOMO) energy level of P3HT. In addition, the PHJ OPV cell with a broad P3HT stacking orientation distribution has a V(OC) comparable to an archetypal bulk heterojunction (BHJ) device. These results suggest that, in the BHJ OPV cell, the hole energy level in the charge transfer state is defined in part by the orientation distribution of the P3HT at the interface with PCBM. Finally, the photoresponses of the devices are also shown to have a dependence on P3HT stacking orientation. PMID:26027430

  3. Enhanced sampling molecular dynamics simulation captures experimentally suggested intermediate and unfolded states in the folding pathway of Trp-cage miniprotein

    NASA Astrophysics Data System (ADS)

    Shao, Qiang; Shi, Jiye; Zhu, Weiliang

    2012-09-01

    The ability of molecular dynamics simulation to capturing the transient states within the folding pathway of protein is important to the understanding of protein folding mechanism. In the present study, the integrated-tempering-sampling molecular dynamics (ITS-MD) simulation was performed to investigate the transient states including intermediate and unfolded ones in the folding pathway of a miniprotein, Trp-cage. Three force fields (FF03, FF99SB, and FF96) were tested, and both intermediate and unfolded states with their characteristics in good agreement with experiments were observed during the simulations, which supports the hypothesis that observable intermediates might present in the folding pathway of small polypeptides. In addition, it was demonstrated that FF03 force field as combined with ITS-MD is in overall a more proper force field than the others in reproducing experimentally recorded properties in UVRS, ECD, and NMR, Photo-CIDNP NMR, and IR T-jump experiments, and the folding/unfolding thermodynamics parameters, such as ΔGU, ΔCp, and ΔHU (Tm). In summary, the present study showed that using suitable force field and energy sampling method, molecular dynamics simulation could capture the transient states within the folding pathway of protein which are consistent with the experimental measurements, and thus provide information of protein folding mechanism and thermodynamics.

  4. Transcriptional analysis of Volvox photoreceptors suggests the existence of different cell-type specific light-signaling pathways.

    PubMed

    Kianianmomeni, Arash; Hallmann, Armin

    2015-02-01

    Photosynthetic organisms, e.g., plants including green algae, use a sophisticated light-sensing system, composed of primary photoreceptors and additional downstream signaling components, to monitor changes in the ambient light environment towards adjust their growth and development. Although a variety of cellular processes, e.g., initiation of cleavage division and final cellular differentiation, have been shown to be light-regulated in the green alga Volvox carteri, little is known about the underlying light perception and signaling pathways. This multicellular alga possesses at least 12 photoreceptors, i.e., one phototropin (VcPhot), four cryptochromes (VcCRYa, VcCRYp, VcCRYd1, and VcCRYd2), and seven members of rhodopsin-like photoreceptors (VR1, VChR1, VChR2, VcHKR1, VcHKR2, VcHKR3, and VcHKR4), which display distinct light-dependent chemical processes based on their protein architectures and associated chromophores. Gene expression analyses could show that the transcript levels of some of the photoreceptor genes (e.g., VChR1 and VcHKR1) accumulate during division cleavages, while others (e.g., VcCRYa, VcCRYp, and VcPhot) accumulate during final cellular differentiation. However, the pattern of transcript accumulation changes when the alga switches to the sexual development. Eight photoreceptor genes, e.g., VcPhot, VcCRYp, and VcHKR1, are highly expressed in the somatic cells, while only the animal-type rhodopsin VR1 was found to be highly expressed in the reproductive cells/embryos during both asexual and sexual life cycles. Moreover, accumulation of VChR1 and VcCRYa transcripts is more sensitive to light and changes in response to more than one light quality. Obviously, different regulatory mechanisms underlying gene expression control transcript accumulation of photoreceptors not only during development, but also in a cell-type specific way and in response to various external signals such as light quality. The transcriptional patterns described in this study

  5. Electron Transfer Dynamics in Efficient Molecular Solar Cells

    SciTech Connect

    Meyer, Gerald John

    2014-10-01

    This research provided new mechanistic insights into surface mediated photochemical processes relevant to solar energy conversion. In this past three years our research has focused on oxidation photo-redox chemistry and on the role surface electric fields play on basic spectroscopic properties of molecular-semiconductor interfaces. Although this research as purely fundamental science, the results and their interpretation have relevance to applications in dye sensitized and photogalvanic solar cells as well as in the storage of solar energy in the form of chemical bonds.

  6. How Cells Feel: Stochastic Model for a Molecular Mechanosensor

    PubMed Central

    Escudé, Matteo; Rigozzi, Michelle K.; Terentjev, Eugene M.

    2014-01-01

    Understanding mechanosensitivity (i.e., how cells sense the stiffness of their environment) is very important, yet there is a fundamental difficulty in understanding its mechanism: to measure an elastic modulus one requires two points of application of force—a measuring and a reference point. The cell in contact with substrate has only one (adhesion) point to work with, and thus a new method of measurement needs to be invented. The aim of this theoretical work is to develop a self-consistent physical model for mechanosensitivity, a process by which a cell detects the mechanical stiffness of its environment (e.g., a substrate it is attached to via adhesion points) and generates an appropriate chemical signaling to remodel itself in response to this environment. The model uses the molecular mechanosensing complex of latent TGF-β attached to the adhesion point as the biomarker. We show that the underlying Brownian motion in the substrate is the reference element in the measuring process. The model produces a closed expression for the rate of release of active TGF-β, which depends on the substrate stiffness and the pulling force coming from the cell in a subtle and nontrivial way. It is consistent with basic experimental data showing an increase in signal for stiffer substrates and higher pulling forces. In addition, we find that for each cell there is a range of stiffness where a homeostatic configuration of the cell can be achieved, outside of which the cell either relaxes its cytoskeletal forces and detaches from the very weak substrate, or generates an increasingly strong pulling force through stress fibers with a positive feedback loop on very stiff substrates. In this way, the theory offers the underlying mechanism for the myofibroblast conversion in wound healing and smooth muscle cell dysfunction in cardiac disease. PMID:24411244

  7. How cells feel: stochastic model for a molecular mechanosensor.

    PubMed

    Escudé, Matteo; Rigozzi, Michelle K; Terentjev, Eugene M

    2014-01-01

    Understanding mechanosensitivity (i.e., how cells sense the stiffness of their environment) is very important, yet there is a fundamental difficulty in understanding its mechanism: to measure an elastic modulus one requires two points of application of force-a measuring and a reference point. The cell in contact with substrate has only one (adhesion) point to work with, and thus a new method of measurement needs to be invented. The aim of this theoretical work is to develop a self-consistent physical model for mechanosensitivity, a process by which a cell detects the mechanical stiffness of its environment (e.g., a substrate it is attached to via adhesion points) and generates an appropriate chemical signaling to remodel itself in response to this environment. The model uses the molecular mechanosensing complex of latent TGF-β attached to the adhesion point as the biomarker. We show that the underlying Brownian motion in the substrate is the reference element in the measuring process. The model produces a closed expression for the rate of release of active TGF-β, which depends on the substrate stiffness and the pulling force coming from the cell in a subtle and nontrivial way. It is consistent with basic experimental data showing an increase in signal for stiffer substrates and higher pulling forces. In addition, we find that for each cell there is a range of stiffness where a homeostatic configuration of the cell can be achieved, outside of which the cell either relaxes its cytoskeletal forces and detaches from the very weak substrate, or generates an increasingly strong pulling force through stress fibers with a positive feedback loop on very stiff substrates. In this way, the theory offers the underlying mechanism for the myofibroblast conversion in wound healing and smooth muscle cell dysfunction in cardiac disease. PMID:24411244

  8. Molecular mechanisms of Ebola virus pathogenesis: focus on cell death.

    PubMed

    Falasca, L; Agrati, C; Petrosillo, N; Di Caro, A; Capobianchi, M R; Ippolito, G; Piacentini, M

    2015-08-01

    Ebola virus (EBOV) belongs to the Filoviridae family and is responsible for a severe disease characterized by the sudden onset of fever and malaise accompanied by other non-specific signs and symptoms; in 30-50% of cases hemorrhagic symptoms are present. Multiorgan dysfunction occurs in severe forms with a mortality up to 90%. The EBOV first attacks macrophages and dendritic immune cells. The innate immune reaction is characterized by a cytokine storm, with secretion of numerous pro-inflammatory cytokines, which induces a huge number of contradictory signals and hurts the immune cells, as well as other tissues. Other highly pathogenic viruses also trigger cytokine storms, but Filoviruses are thought to be particularly lethal because they affect a wide array of tissues. In addition to the immune system, EBOV attacks the spleen and kidneys, where it kills cells that help the body to regulate its fluid and chemical balance and that make proteins that help the blood to clot. In addition, EBOV causes liver, lungs and kidneys to shut down their functions and the blood vessels to leak fluid into surrounding tissues. In this review, we analyze the molecular mechanisms at the basis of Ebola pathogenesis with a particular focus on the cell death pathways induced by the virus. We also discuss how the treatment of the infection can benefit from the recent experience of blocking/modulating cell death in human degenerative diseases. PMID:26024394

  9. Molecular mechanisms of Ebola virus pathogenesis: focus on cell death

    PubMed Central

    Falasca, L; Agrati, C; Petrosillo, N; Di Caro, A; Capobianchi, M R; Ippolito, G; Piacentini, M

    2015-01-01

    Ebola virus (EBOV) belongs to the Filoviridae family and is responsible for a severe disease characterized by the sudden onset of fever and malaise accompanied by other non-specific signs and symptoms; in 30–50% of cases hemorrhagic symptoms are present. Multiorgan dysfunction occurs in severe forms with a mortality up to 90%. The EBOV first attacks macrophages and dendritic immune cells. The innate immune reaction is characterized by a cytokine storm, with secretion of numerous pro-inflammatory cytokines, which induces a huge number of contradictory signals and hurts the immune cells, as well as other tissues. Other highly pathogenic viruses also trigger cytokine storms, but Filoviruses are thought to be particularly lethal because they affect a wide array of tissues. In addition to the immune system, EBOV attacks the spleen and kidneys, where it kills cells that help the body to regulate its fluid and chemical balance and that make proteins that help the blood to clot. In addition, EBOV causes liver, lungs and kidneys to shut down their functions and the blood vessels to leak fluid into surrounding tissues. In this review, we analyze the molecular mechanisms at the basis of Ebola pathogenesis with a particular focus on the cell death pathways induced by the virus. We also discuss how the treatment of the infection can benefit from the recent experience of blocking/modulating cell death in human degenerative diseases. PMID:26024394

  10. A Drosophila XPD model links cell cycle coordination with neuro-development and suggests links to cancer

    PubMed Central

    Stettler, Karin; Li, Xiaoming; Sandrock, Björn; Braga-Lagache, Sophie; Heller, Manfred; Dümbgen, Lutz; Suter, Beat

    2015-01-01

    XPD functions in transcription, DNA repair and in cell cycle control. Mutations in human XPD (also known as ERCC2) mainly cause three clinical phenotypes: xeroderma pigmentosum (XP), Cockayne syndrome (XP/CS) and trichothiodystrophy (TTD), and only XP patients have a high predisposition to developing cancer. Hence, we developed a fly model to obtain novel insights into the defects caused by individual hypomorphic alleles identified in human XP-D patients. This model revealed that the mutations that displayed the greatest in vivo UV sensitivity in Drosophila did not correlate with those that led to tumor formation in humans. Immunoprecipitations followed by targeted quantitative MS/MS analysis showed how different xpd mutations affected the formation or stability of different transcription factor IIH (TFIIH) subcomplexes. The XP mutants most clearly linked to high cancer risk, Xpd R683W and R601L, showed a reduced interaction with the core TFIIH and also an abnormal interaction with the Cdk-activating kinase (CAK) complex. Interestingly, these two XP alleles additionally displayed high levels of chromatin loss and free centrosomes during the rapid nuclear division phase of the Drosophila embryo. Finally, the xpd mutations showing defects in the coordination of cell cycle timing during the Drosophila embryonic divisions correlated with those human mutations that cause the neurodevelopmental abnormalities and developmental growth defects observed in XP/CS and TTD patients. PMID:25431422