Cholera toxin B subunit (CTB) has been extensively used in the past for monosynaptic mapping. For decades, it was thought to lack the ability of transneuronal tracing. In order to investigate whether biotin conjugates of CTB (b-CTB) would pass through transneurons in the rat spinal cord, it was injected into the crushed left sciatic nerve. For experimental control, the first order afferent neuronal projections were defined by retrograde transport of fluorogold (FG, a non-transneuronal labeling marker as an experimental control) injected into the crushed right sciatic nerve in the same rat. Neurons containing b-CTB or FG were observed in the dorsal root ganglia (DRG) at the L4-L6 levels ipsilateral to the tracer injection. In the spinal cord, b-CTB labeled neurons were distributed in all laminae ipsilaterally between C7 and S1 segments, but labeling of neurons at the cervical segment was abolished when the T10 segment was transected completely. The interneurons, distributed in the intermediate gray matter and identified as gamma-aminobutyric acid-ergic (GABAergic), were labeled by b-CTB. In contrast, FG labeling was confined to the ventral horn neurons at L4-L6 spinal segments ipsilateral to the injection. b-CTB immunoreactivity remained to be restricted to the soma of neurons and often appeared as irregular patches detected by light and electron microscopy. Detection of monosialoganglioside (GM1) in b-CTB labeled neurons suggests that GM1 ganglioside may specifically enhance the uptake and transneuronal passage of b-CTB, thus supporting the notion that it may be used as a novel transneuronal tracer.

Disruption in circadian gene expression, whether due to genetic variation or environmental factors (e.g., light at night, shiftwork), is associated with increased incidence of breast, prostate, gastrointestinal and hematologic cancers and gliomas. Circadian genes are highly expressed in the ovaries where they regulate ovulation; circadian disruption is associated with several ovarian cancer risk factors (e.g., endometriosis). However, no studies have examined variation in germline circadian genes as predictors of ovarian cancer risk and invasiveness. The goal of the current study was to examine single nucleotide polymorphisms (SNPs) in circadian genes BMAL1, CRY2, CSNK1E, NPAS2, PER3, REV1 and TIMELESS and downstream transcription factors KLF10 and SENP3 as predictors of risk of epithelial ovarian cancer (EOC) and histopathologic subtypes. The study included a test set of 3,761 EOC cases and 2,722 controls and a validation set of 44,308 samples including 18,174 (10,316 serous) cases and 26,134 controls from 43 studies participating in the Ovarian Cancer Association Consortium (OCAC). Analysis of genotype data from 36 genotyped SNPs and 4600 imputed SNPs indicated that the most significant association was rs117104877 in BMAL1 (OR = 0.79, 95% CI = 0.68-0.90, p = 5.59 × 10-4]. Functional analysis revealed a significant down regulation of BMAL1 expression following cMYC overexpression and increasing transformation in ovarian surface epithelial (OSE) cells as well as alternative splicing of BMAL1 exons in ovarian and granulosa cells. These results suggest that variation in circadian genes, and specifically BMAL1, may be associated with risk of ovarian cancer, likely through disruption of hormonal pathways.

We evaluated miR-371-5p expression in gastric cancer (GC) tissues and its influence on the expression of downstream genes, especially SOX2. MiR-371-5p expression (measured using qRT-PCR) was upregulated in GC tissues and correlated positively with TNM staging and lymph node (LN) metastasis. MiR-371-5p expression was higher in human GC cell lines (AGS, MKN-28, BGC-823, MGC-803, SGC-7901 and MKN-45) than in human normal gastric epithelial (GES-1) cells (all P < 0.05). MGC-803 tumor cell growth (measured with an MTT assay), migration, and invasion (measured with Transwell chamber assays) were severely inhibited in cells transfected with a miR-371-5p inhibitor, whereas they were stimulated in cells transfected with SOX2 siRNA or miR-371-5p inhibitor + SOX2 siRNA. Expression of SOX2 mRNA and protein (assessed with qRT-PCR and Western blot) were greatly enhanced in the miR-371-5p inhibitor group. These results indicate that miR-371-5p expression is strongly upregulated in GC tissues and negatively correlated with SOX2 expression, while miR-371-5p expression is inversely related to proliferation, TNM stage, and LN metastasis of GC cells. Suppression of miR-371-5p may inhibit the growth and invasion of MGC-803 GC cells by upregulating SOX2 expression.

HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability.

Clinical genetic testing is commercially available for rs61764370, an inherited variant residing in a KRAS 3' UTR microRNA binding site, based on suggested associations with increased ovarian and breast cancer risk as well as with survival time. However, prior studies, emphasizing particular subgroups, were relatively small. Therefore, we comprehensively evaluated ovarian and breast cancer risks as well as clinical outcome associated with rs61764370.

Metal nanoparticle probes were used as molecular imaging agents to detect the expression levels and spatial distributions of the CCR5 receptors on the cell surfaces. Alexa Fluor 647-labeled anti-CCR5 monoclonal antibodies (mAbs) were covalently bound to 20 nm silver nanoparticles to synthesize the mAb-metal complexes. We measured the single nanoparticle emission of the mAb-metal complexes, showing that the complexes displayed enhanced intensities and reduced lifetimes in comparison with the metal-free mAbs. Six HeLa cell lines with various CCR5 expressions were incubated with the mAb-metal complexes for the target-specific binding to the cell surfaces. Fluorescence cell images were recorded on a time-resolved confocal microscope. The collected images expressed clear CCR5 expression-dependent optical properties. Two regression curves were obtained on the basis of the emission intensity and lifetime over the entire cell images against the number of the CCR5 expression on the cells. The emission from the single mAb-metal complexes could be distinctly identified from the cellular autofluorescence on the cell images. The CCR5 spatial distributions on the cells were analyzed on the cell images and showed that the low-expression cells have the CCR5 receptors as individuals or small clusters but the high expression cells have them as the dense and discrete clusters on the cell surfaces.

The upregulation of Wnt/β-catenin signaling occurs in virtually all types of kidney disease and is associated with podocyte injury. However, the precise mechanisms involved in the development of kidney disease remain to be elucidated. MicroRNAs (miRNAs or miRs) are a class of short non-coding RNAs and they have been shown to be regulators of gene expression, mainly by binding to the untranslated region (UTR) of mRNAs. The aim of the present study was to determine the role of the 2 members of the miR-135 family (miR‑135a and miR‑135b) in podocyte injury and to elucidate the mechanisms responsible for the damage to podocytes. The results revealed that miR-135a and miR-135b were upregulated in models of podocyte injury and in glomeruli isolated from patients with focal segmental glomerulosclerosis (FSGS). The ectopic expression of miR-135a and miR‑135b led to severe podocyte injury and the disorder of the podocyte cytoskeleton. Our findings demonstrated that miR-135a and miR‑135b activated Wnt/β‑catenin signaling and induced the nuclear translocation of β-catenin. Using luciferase reporter assays, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, glycogen synthase kinase 3β (GSK3β) was identified as a target gene of miR-135a and miR‑135b. To the best of our knowledge, this is the first study to demonstrate that members of the miR-135 family (specifically miR-135a and miR‑135b) regulate the expression of GSK3β, thus playing a role in the development of podocyte injury and the disorder of the podocyte cytoskeleton. This is an important finding as it may contribute to the development of novel therapeutics for podocyte injury-associated glomerulopathies.

Genome-wide association studies have reported 11 regions conferring risk of high-grade serous epithelial ovarian cancer (HGSOC). Expression quantitative trait locus (eQTL) analyses can identify candidate susceptibility genes at risk loci. Here we evaluate cis-eQTL associations at 47 regions associated with HGSOC risk (P≤10(-5)). For three cis-eQTL associations (P<1.4 × 10(-3), FDR<0.05) at 1p36 (CDC42), 1p34 (CDCA8) and 2q31 (HOXD9), we evaluate the functional role of each candidate by perturbing expression of each gene in HGSOC precursor cells. Overexpression of HOXD9 increases anchorage-independent growth, shortens population-doubling time and reduces contact inhibition. Chromosome conformation capture identifies an interaction between rs2857532 and the HOXD9 promoter, suggesting this SNP is a leading causal variant. Transcriptomic profiling after HOXD9 overexpression reveals enrichment of HGSOC risk variants within HOXD9 target genes (P=6 × 10(-10) for risk variants (P<10(-4)) within 10 kb of a HOXD9 target gene in ovarian cells), suggesting a broader role for this network in genetic susceptibility to HGSOC.

The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe human congenital and neurologic disorders including microcephaly in newborns and Guillain-Barré syndrome in adults. Studies showed that the epidemic ZIKV strains are phenotypically different from the historic strains, suggesting that the epidemic ZIKV has acquired mutations associated with the altered viral pathogenicity. However, what genetic changes are responsible for the changed viral pathogenicity remains largely unknown. One of our early studies suggested that the ZIKV structural proteins contribute in part to the observed virologic differences. The objectives of this study were to compare the historic African MR766 ZIKV strain with two epidemic Brazilian strains (BR15 and ICD) for their abilities to initiate viral infection and to confer neurocytopathic effects in the human brain's SNB-19 glial cells, and further to determine which part of the ZIKV structural proteins are responsible for the observed differences. Our results show that the historic African (MR766) and epidemic Brazilian (BR15 and ICD) ZIKV strains are different in viral attachment to host neuronal cells, viral permissiveness and replication, as well as in the induction of cytopathic effects. The analysis of chimeric viruses, generated between the MR766 and BR15 molecular clones, suggests that the ZIKV E protein correlates with the viral attachment, and the C-prM region contributes to the permissiveness and ZIKV-induced cytopathic effects. The expression of adenoviruses, expressing prM and its processed protein products, shows that the prM protein and its cleaved Pr product, but not the mature M protein, induces apoptotic cell death in the SNB-19 cells. We found that the Pr region, which resides on the N-terminal side of prM protein, is responsible for prM-induced apoptotic cell death. Mutational analysis further identified four amino-acid residues that have an impact on the ability of prM to induce apoptosis. Together, the results of this study show that the difference of ZIKV-mediated viral pathogenicity, between the historic and epidemic strains, contributed in part the functions of the structural prM-E proteins.

Noninvasive prenatal screening (NIPS) based on cell-free fetal DNA (cffDNA) has rapidly been applied into clinic. However, the reliability of this method largely depends on the concentration of cffDNA in the maternal plasma. The chance of test failure results or false negative results would increase when cffDNA fraction is low. In this study, we set out to develop a method to enrich the cffDNA for NIPS based on the size difference between cell-free DNA (cfDNA) of fetal origin and maternal origin, and to evaluate whether the new NIPS method can improve the test quality.

The canonical role of cohesin is to mediate sister chromatid cohesion. In addition, cohesin plays important roles in processes such as DNA repair and regulation of gene expression. Mounting evidence suggests that various post-translational modifications, including phosphorylation, acetylation and sumoylation regulate cohesin functions. Our mass spectrometry analysis of cohesin purified from Schizosaccharomyces pombe cells revealed that the cohesin subunit Psm1 is methylated on two evolutionarily conserved lysine residues, K536 and K1200. We found that mutations that prevent methylation of Psm1 K536 and K1200 render sensitivity to DNA-damaging agents and show positive genetic interactions with mutations in genes encoding the Mus81-Eme1 endonuclease. Yeast two-hybrid and co-immunoprecipitation assays showed that there were interactions between subunits of the cohesin and Mus81-Eme1 complexes. We conclude that cohesin is methylated and that mutations that prevent methylation of Psm1 K536 and K1200 show synthetic phenotypes with mutants defective in the homologous recombination DNA repair pathway.

Basic region/leucine zipper (bZIP) transcription factors perform as crucial regulators in ABA-mediated stress response in plants. Nevertheless, the functions for most bZIP family members in tomato remain to be deciphered.

Elevated retinol-binding protein 4 (RBP4) levels may contribute to the development of metabolic abnormalities, but prospective studies evaluating the association between childhood RBP4 levels and metabolic syndrome (MS) in adulthood are lacking. We investigated whether RBP4 levels during childhood predict cardiometabolic risk at 10-year follow-up.

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations.

Programmed cell death 5 (PDCD5) was first identified as a gene upregulated in cells undergoing apoptosis. We recently demonstrated the inhibitory effect of PDCD5 on experimentally induced autoimmune encephalomyelitis. In this study, we investigated the anti-inflammatory effects of recombinant human PDCD5 (rhPDCD5) in a rat collagen-induced arthritis (CIA) model. We find that vaccination of collagen II (CII) induced CIA rats with rhPDCD5 significantly delayed the occurrence and reduced the severity of CIA rats. rhPDCD5 also restored the loss of Foxp3(+) regulatory T (Treg) cells and decreased the population of Th1 and Th17 in CIA rats. Simultaneously, rhPDCD5 treatment suppressed the production of pro-inflammatory cytokines (interleukin (IL)-6, IL-17A, tumor necrosis factor-α (TNF-α), and interferon gamma (IFN-γ)) and increased the secretion of anti-inflammatory cytokines (transforming growth factor beta 1 (TGF-β1) and IL-10) in CIA rats. In addition, rhPDCD5 inhibited the ability of CII to induce proliferation of splenocytes and lymph node cells (LNCs) and promoted the CII-activated CD4(+) cell apoptosis. These results of rhPDCD5-treated CIA rats were similar with those of recombinant human TNF-α receptor IgG Fc (rhTNFR:Fc). Thus, to our knowledge, we provide the first evidence that rhPDCD5 may be an efficient approach to diminishing exacerbated immune responses in CIA, indicating its therapeutic potential in the treatment of rheumatoid arthritis and other autoimmune diseases.

The purpose of this study was to compare the binding affinity and selective targeting of aptamer- and antibody-coated hollow gold nanospheres (HAuNS) targeted to epidermal growth factor receptors (EGFR). EGFR-targeting aptamers were conjugated to HAuNS (apt-HAuNS) by attaching a thiol-terminated single-stranded DNA to the HAuNS and then adding the complementary RNA targeted to EGFR. Apt-HAuNS was characterized in terms of size, surface charge, absorption, and number of aptamers per particle. The in vivo pharmacokinetics, in vivo biodistribution, and micro-SPECT/CT imaging of (111)In-labeled apt-HAuNS and anti-EGFR antibody (C225)-conjugated HAuNS were evaluated in nude mice bearing highly malignant human OSC-19 oral tumors. (111)In-labeled PEG-HAuNS was used as a control (n = 5/group). Apt-HAuNS did not have an altered absorbance profile or size (λmax = 800 nm; diameter = 55 nm) compared to C225-HAuNS or PEG-HAuNS. The surface charge became more negative upon conjugation of the aptamer (-51.4 vs -19.0 for PEG-HAuNS and -25.0 for C225-HAuNS). The number of aptamers/particle was ∼250. In vitro cell binding and in vivo biodistribution showed selective binding of the apt-HAuNS to EGFR. μSPECT/CT imaging confirmed that there was more tumor uptake of apt-HAuNS than C225-HAuNS. Aptamer is a promising ligand for image-guided delivery of nanoparticles for treatment of tumor cells overexpressing EGFR.

The node and notochord (and their equivalents in other species) are essential signaling centers, positioned along the plane of bilateral symmetry in developing vertebrate embryos. However, genes and mechanisms regulating morphogenesis of these structures and their placement along the embryonic midline are not well understood. In this work, we provide the first evidence that the position of the node and the notochord along the bilateral plane of symmetry are under genetic control and are regulated by integrin α5β1 and fibronectin in mice. We found that the shape of the node is often inverted in integrin α5-null and fibronectin-null mutants, and that the positioning of node and the notochord is often skewed away from the perceived plane of embryonic bilateral of symmetry. Our studies also show that the shape and position of the notochord are dependent on the shape and embryonic placement of the node. Our studies suggest that fibronectin regulates the shape of the node by affecting apico-basal polarity of the nodal cells. Taken together, our data indicate that cell-extracellular matrix interactions mediated by integrin α5β1 and fibronectin regulate the geometry of the node as well as the placement of the node and notochord along the plane of bilateral symmetry in the mammalian embryo.

Because of the methodological complexity of network meta-analyses (NMAs), NMAs may be more vulnerable to methodological risks than conventional pair-wise meta-analysis. Our study aims to investigate epidemiology characteristics, conduction of literature search, methodological quality and reporting of statistical analysis process in the field of cancer based on PRISMA extension statement and modified AMSTAR checklist. We identified and included 102 NMAs in the field of cancer. 61 NMAs were conducted using a Bayesian framework. Of them, more than half of NMAs did not report assessment of convergence (60.66%). Inconsistency was assessed in 27.87% of NMAs. Assessment of heterogeneity in traditional meta-analyses was more common (42.62%) than in NMAs (6.56%). Most of NMAs did not report assessment of similarity (86.89%) and did not used GRADE tool to assess quality of evidence (95.08%). 43 NMAs were adjusted indirect comparisons, the methods used were described in 53.49% NMAs. Only 4.65% NMAs described the details of handling of multi group trials and 6.98% described the methods of similarity assessment. The median total AMSTAR-score was 8.00 (IQR: 6.00-8.25). Methodological quality and reporting of statistical analysis did not substantially differ by selected general characteristics. Overall, the quality of NMAs in the field of cancer was generally acceptable.

The human immunodeficiency virus type 1 (HIV-1) genome (~9 kb RNA) is flanked by two long terminal repeats (LTR) promoter regions with nine open reading frames, which encode Gag, Pol and Env polyproteins, four accessory proteins (Vpu, Vif, Vpr, Nef) and two regulatory proteins (Rev, Tat). In this study, we carried out a genome-wide and functional analysis of the HIV-1 genome in fission yeast (Schizosaccharomyces pombe).

Type 2 diabetes results from defects in both insulin sensitivity and insulin secretion. Elevated cholesterol content within pancreatic β-cells has been shown to reduce β-cell function and increase β-cell apoptosis. Hyperglycemia and dyslipidemia contribute to glucolipotoxicity that leads to type 2 diabetes. Here we examined the capacity of glucolipotoxicity to induce free cholesterol accumulation in human pancreatic islets and the INS-1 insulinoma cell line. Glucolipotoxicity treatment increased free cholesterol in β-cells, which was accompanied by increased reactive oxygen species (ROS) production and decreased insulin secretion. Addition of AAPH, a free radical generator, was able to increase filipin staining indicating a link between ROS production and increased cholesterol in β-cells. We also showed the ability of stigmasterol, a common food-derived phytosterol with anti-atherosclerotic potential, to prevent the increase in both free cholesterol and ROS levels induced by glucolipotoxicity in INS-1 cells. Stigmasterol addition also inhibited early apoptosis, increased total insulin, promoted actin reorganization, and improved insulin secretion in cells exposed to glucolipotoxicity. Overall, these data indicate cholesterol accumulation as an underlying mechanism for glucolipotoxicity-induced defects in insulin secretion and stigmasterol treatment as a potential strategy to protect β-cell function during diabetes progression.