Faculty Bibliography
Searched for:
Department/Unit:Cell Biology
Total Results:
14243
Immunostaining of Macrophages, Endothelial Cells, and Smooth Muscle Cells in the Atherosclerotic Mouse Aorta
The atherosclerotic mouse aorta consists of a heterogeneous population of cells, including macrophages, endothelial cells, and smooth muscle cells that play critical roles in cardiovascular disease. Identification of these vascular cells in the vessel wall is important to understanding their function in pathological conditions. Immunohistochemistry is an invaluable technique used to detect the presence of cells in different tissues. Here, we describe immunohistochemical techniques commonly used for the detection of the vascular cells in the atherosclerotic mouse aorta using cell type-specific markers.
PMCID:4736500
PMID: 26445786
ISSN: 1940-6029
CID: 1793202
Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle
The C. elegans germ line is an outstanding model system to study the control of cell division and differentiation. While 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 may 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.
PMCID:4712881
PMID: 26428008
ISSN: 1477-9129
CID: 1789982
A single-cell assay for telomere DNA content shows increasing telomere length heterogeneity, as well as increasing mean telomere length in human spermatozoa with advancing age
PURPOSE: The effect of age on telomere length heterogeneity in men has not been studied previously. Our aims were to determine the relationship between variation in sperm telomere length (STL), men's age, and semen parameters in spermatozoa from men undergoing in vitro fertilization (IVF) treatment. METHODS: To perform this prospective cross-sectional pilot study, telomere length was estimated in 200 individual spermatozoa from men undergoing IVF treatment at the NYU Fertility Center. A novel single-cell telomere content assay (SCT-pqPCR) measured telomere length in individual spermatozoa. RESULTS: Telomere length among individual spermatozoa within an ejaculate varies markedly and increases with age. Older men not only have longer STL but also have more variable STL compared to younger men. STL from samples with normal semen parameters was significantly longer than that from samples with abnormal parameters, but STL did not differ between spermatozoa with normal versus abnormal morphology. CONCLUSION: The marked increase in STL heterogeneity as men age is consistent with a role for ALT during spermatogenesis. No data have yet reported the effect of age on STL heterogeneity. Based on these results, future studies should expand this modest sample size to search for molecular evidence of ALT in human testes during spermatogenesis.
PMCID:4651947
PMID: 26411311
ISSN: 1573-7330
CID: 1789682
Green-to-Red Photoconversion of GCaMP
Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs-including GCaMP3, GCaMP5 and GCaMP6-can be converted from green to red following exposure to blue-green light (450-500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.
PMCID:4575167
PMID: 26382605
ISSN: 1932-6203
CID: 1789412
A novel germ cell determinant reveals parallel pathways for germ line development in Caenorhabditis elegans
Despite the central importance of germ cells for transmission of genetic material, our understanding of the molecular programs that control primordial germ cell (PGC) specification and differentiation are limited. Here, we present findings that X nondisjunction factor-1 (xnd-1), known for its role in regulating meiotic crossover formation, is an early determinant of germ cell fates in Caenorhabditis elegans. xnd-1 mutant embryos display a novel "one PGC" phenotype due to G2 cell cycle arrest of the P4 blastomere. Larvae and adults display smaller germ lines and reduced brood sized consistent with a role for XND-1 in germ cell proliferation. Maternal XND-1 proteins are found in P4 and exclusively localized to the nucleus in the PGCs, Z2 and Z3. Zygotic XND-1 turns on shortly thereafter, at the approximately 300-cell stage, making XND-1 the earliest zygotically-expressed gene in the worm PGCs. Strikingly, a subset of xnd-1 mutants lack germ cells, a phenotype shared with nos-2, a member of the conserved Nanos family of germline determinants. We generated a nos-2 null allele and show that nos-2; xnd-1 double mutants display synthetic sterility. Further removal of nos-1 leads to almost complete sterility, with the vast majority of animals without germ cells. The sterility in xnd-1 mutants is correlated with an increase in the transcriptional activation-associated histone modification and aberrant expression of somatic transgenes. Together, these data strongly suggest that xnd-1 defines a new branch for PGC development that functions redundantly with nos-2 and nos-1 to promote germline fates by maintaining transcriptional quiescence and regulating germ cell proliferation.
PMID: 26395476
ISSN: 1477-9129
CID: 1786812
Antitumor activity of melinjo (Gnetum gnemon L.) seed extract in human and murine tumor models in vitro and in a colon-26 tumor-bearing mouse model in vivo
Melinjo (Gnetum gnemon L.) seed extract (MSE) and its active ingredient gnetin C (GC), a resveratrol dimer, have been shown to possess a broad spectrum of pharmacological activities. In this study, we investigated the antitumor activity of MSE and GC using human and murine tumor cell culture models in vitro. The antitumor activity of GC was compared with trans-resveratrol (tRV), a stilbenoid polyphenol. Our results show that MSE and GC at clinically achievable concentrations significantly inhibited the proliferation of pancreatic, prostate, breast, and colon cancer cell types (P < 0.05), without affecting normal cells. Interestingly, GC exerts enhanced antitumor activity than that of tRV (P < 0.05). MSE and GC significantly induced apoptosis in all the cancer cells, indicating MSE and GC inhibit tumor cell growth by inducing apoptosis (P < 0.001). Our findings provide evidence that MSE might induce apoptosis in cancer cells via caspase-3/7-dependent and -independent mechanisms. However, GC might trigger both early and late stage apoptosis in cancer cells, at least in part by activating caspase 3/7-dependent mechanisms. Furthermore, the antitumor efficacy of MSE observed in vitro was also validated in a widely used colon-26 tumor-bearing mouse model. Oral administration of MSE at 50 and 100 mg/kg per day significantly inhibited tumor growth, intratumoral angiogenesis, and liver metastases in BALB/c mice bearing colon-26 tumors (P < 0.05). In conclusion, our findings provide evidence that MSE and GC have potent antitumor activity. Most importantly, we provide the first evidence that MSE inhibits tumor growth, intratumoral angiogenesis, and liver metastasis in a colon-26 tumor-bearing mice.
PMCID:4674003
PMID: 26408414
ISSN: 2045-7634
CID: 1787112
Suppression of T cell responses in the tumor microenvironment
The immune system recognizes protein antigens expressed in transformed cells evidenced by accumulation of antigen-specific T cells in tumor and tumor draining lymph nodes. However, despite demonstrable immune response, cancers grow progressively suggesting that priming of antitumor immunity is insufficiently vigorous or that antitumor immunity is suppressed, or both. Compared to virus infection, antitumor T cells are low abundance that likely contributes to tumor escape and enhancement of priming is a long-sought goal of experimental vaccination therapy. Furthermore, patient treatment with antigen-specific T cells can in some cases overcome deficient priming and cause tumor regression supporting the notion that low numbers of T cells permits tumor outgrowth. However, tumor-induced suppression of antitumor immune response is now recognized as a significant factor contributing to cancer growth and reversal of the inhibitory influences within the tumor microenvironment is a major research objective. Multiple cell types and factors can inhibit T cell functions in tumors and may be grouped in two general classes: T cell intrinsic and T cell extrinsic. T cell intrinsic factors are exemplified by T cell expression of cell surface inhibitory signaling receptors that, after contact with cells expressing a cognate ligand, inactivate proximal T Cell Receptor-mediated signal transduction therein rendering T cells dysfunctional. T cell extrinsic factors are more diverse in nature and are produced by tumors and various non-tumor cells in the tumor microenvironment including: regulatory T cells, B cells, myeloid-derived cells, cancer associated fibroblasts, pericytes, mast cells, or vascular endothelia. These include proteins secreted by tumor or stromal cells (some which may be retained in the extracellular matrix), highly reactive soluble oxygen and nitrogen species, cytokines, chemokines, gangliosides, and toxic metabolites. These factors may restrict T cell entrance into the tumor parenchyma, cause inactivation of effector phase T cell functions, or induce T cell apoptosis ultimately causing diminished cancer elimination. Here, we review the contributions of inhibitory factors to tumor T cell dysfunction leading to tumor escape.
PMID: 26403368
ISSN: 1873-2518
CID: 1786942
L(59) TGF-beta LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice
BACKGROUND: Hepatic fibrosis, which is the excessive accumulation of extracellular matrices (ECMs) produced mainly from activated hepatic stellate cells (HSCs), develops to cirrhosis over several decades. There are no validated biomarkers that can non-invasively monitor excessive production of ECM (i.e., fibrogenesis). Transforming growth factor (TGF)-beta, a key driver of fibrogenesis, is produced as an inactive latent complex, in which active TGF-beta is enveloped by its pro-peptide, the latency-associated protein (LAP). Thus, active TGF-beta must be released from the complex for binding to its receptor and inducing ECM synthesis. We recently reported that during the pathogenesis of liver fibrosis, plasma kallikrein (PLK) activates TGF-beta by cleavage between R(58) and L(59) residues within LAP and that one of its by-products, the N-terminal side LAP degradation products ending at residue R(58) (R(58) LAP-DPs), can be detected mainly around activated HSCs by specific antibodies against R(58) cleavage edges and functions as a footprint of PLK-dependent TGF-beta activation. Here, we describe a sandwich enzyme-linked immunosorbent assay (ELISA) that detects the other by-products, the C-terminal side LAP-DPs starting from residue L(59) (L(59) LAP-DPs). We demonstrated that the L(59) LAP-DPs are a potentially novel blood biomarker reflecting hepatic fibrogenesis. RESULTS: We established a specific sandwich ELISA to quantify L(59) LAP-DPs as low as 2 pM and measured L(59) LAP-DP levels in the culture media of a human activated HSC line, TWNT-4 cells. L(59) LAP-DPs could be detected in their media, and after treatment of TWNT-4 cells with a TGF-beta receptor kinase inhibitor, SB431542, a simultaneous reduction was observed in both L(59) LAP-DP levels in the culture media and the mRNA expression levels of collagen type (I) alpha1. In carbon tetrachloride- and bile duct ligation-induced liver fibrosis models in mice, plasma L(59) LAP-DP levels increased prior to increase of hepatic hydroxyproline (HDP) contents and well correlated with alpha-smooth muscle actin (alphaSMA) expression in liver tissues. At this time, alphaSMA-positive cells as well as R(58) LAP-DPs were seen in their liver tissues. CONCLUSIONS: L(59) LAP-DPs reflect PLK-dependent TGF-beta activation and the increase in alphaSMA-positive activated HSCs in liver injury, thereby serving as a novel blood biomarker for liver fibrogenesis.
PMCID:4570586
PMID: 26379781
ISSN: 1755-1536
CID: 1779352
Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture
The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF
PMCID:4755714
PMID: 26367794
ISSN: 1476-4687
CID: 1779142
Rationale and design of the Investigation of Motivational Interviewing and Prevention Consults to Achieve Cardiovascular Targets (IMPACT) trial
BACKGROUND: Patients undergoing cardiovascular procedures remain at increased risk for myocardial infarction, stroke, and cardiovascular death. Risk factor control in this patient population remains suboptimal and would likely benefit from strategies targeting education, lifestyle, and healthy behaviors. DESIGN: The IMPACT trial is a 400-subject prospective randomized trial designed to compare different cardiovascular prevention strategies in subjects following a cardiovascular intervention. The trial began enrollment in the Spring of 2012 and is randomizing subjects in a 1:1:1 manner to usual care, a one-time cardiovascular prevention consult, or a one-time cardiovascular prevention consult plus behavioral intervention program (telephone-based motivational interviewing and tailored text messages) over a 6-month period. The primary end point is non-high-density lipoprotein cholesterol. Secondary end points include other plasma lipid values, metabolic risk, smoking cessation, physical activity, dietary intake, medication use and adherence, and quality of life. CONCLUSIONS: The IMPACT trial provides data on different management strategies for risk factor optimization in subjects following cardiovascular procedures. The results will provide a platform for the continued development of novel multidisciplinary interventions in this high-risk population.
PMID: 26385025
ISSN: 1097-6744
CID: 1779482
