Protein tyrosine phosphatase non-receptor type 14 (PTPN14) is frequently mutated in a variety of human cancers. However, the cell signaling pathways regulated by PTPN14 largely remain to be elucidated. Here, we identify a list of potential substrates of PTPN14 using a phospho-proteomic approach. We show that p130Cas is a direct substrate of PTPN14 and that PTPN14 specifically regulates p130Cas phosphorylation at tyrosine residue 128 (Y128) in colorectal cancer (CRC) cells. We engineered CRC cells homozygous for a p130Cas Y128F knock-in mutant and found that these cells exhibit significantly reduced migration and colony formation, impaired anchorage-independent growth, slower xenograft tumor growth in nude mice, and have decreased phosphorylation of AKT. Furthermore, we demonstrate that SRC phosphorylates p130Cas Y128 and that CRC cell lines harboring high levels of pY128 Cas are more sensitive to SRC family kinase inhibitor Dasatinib. These findings suggest that p130Cas Y128 phosphorylation may be exploited as a predictive marker for Dasatinib response in cancer patients. In aggregate, our studies reveal a novel signaling pathway that plays an important role in colorectal tumorigenesis.
PTPN14; p130Cas; tumorigenesis; colorectal cancer
Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. In this study, we show that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped–Yorkie transcriptional complex and that the Hippo (Hpo) signaling pathway directly restricted ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promoted ISC proliferation. Our findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation.
Most tissues can generate new cells to repair damage or replace worn-out cells. The new cells are often generated from stem cells—cells that can either reproduce themselves or mature into other types of cells. In the fruit-fly Drosophila, for example, intestinal stem cells in the midgut are capable of producing more stem cells or they can differentiate to produce immature cells called enteroblasts that go on to become either enterocytes (the cells that line the gut) or enteroendocrine cells (which secrete hormones).
Researchers have identified a number of signalling pathways that are involved in the proliferation and differentiation of intestinal stem cells in the midgut of fruit flies. These include the Hippo pathway, which is important for regulating both cell proliferation and programmed cell death (apoptosis). Activation of the Hippo protein triggers a cascade of signals that culminate in the regulation of many of the genes involved in cell proliferation, division and apoptosis.
Another process that is important for controlling the proliferation and differentiation of cells is chromatin remodelling. Chromatin is the ‘packaging’ that keeps DNA tightly wound within the cell nucleus, and remodelling refers to the structural changes that allow proteins called transcription factors to reach the genes and transcribe them into messenger RNA (which then leaves the nucleus to generate the protein).
Now, Jin et al. have explored how the Hippo pathway and chromatin remodelling work together to regulate of stem cells. Using a technique called RNA interference to block the expression of various genes in intestinal stem cells and enteroblasts, Jin et al. found that a protein called Brahma—which is an essential part of a chromatin-remodelling complex—must be present for the stem cells to multiply normally.
Jin et al. also showed how the Hippo signalling pathway interacts with chromatin remodelling. Activation of the Hippo pathway inhibits gene expression by preventing two other proteins, Yorkie and Scalloped, from forming a complex in the nucleus. The new work shows that Brahma interacts physically with the Yorkie and Scalloped proteins to regulate the proliferation of the intestinal stem cells. It also shows that the Hippo protein regulates the activity of the Brahma protein by inducing a process called caspase-dependent cleavage. Because many of the proteins involved in these pathways are evolutionarily conserved and expressed in a variety of tissues, these findings may have implications for stem cell function and tissue repair in many species.
Hippo signaling; brahma; midgut; D. melanogaster
Anesthetics are variable in patients with obstructive jaundice. The minimum alveolar concentration awake of desflurane is reduced in patients with obstructive jaundice, while it has no effect on pharmacodynamics and pharmacokinetics of propofol. In this study, we investigated the influence of obstructive jaundice on the pharmacodynamics and blood concentration of rocuronium.
Included in this study were 26 control patients and 27 patients with obstructive jaundice. Neuromuscular block of rocuronium was monitored by acceleromyography. Onset time, spontaneous recovery of the height of twitch first (T1) to 25% of the final T1 value (Duration 25%, Dur 25%), recovery index (RI), and spontaneous recovery of train-of-four (TOF) ratios to 70% were measured. The plasma rocuronium concentrations were determined by high performance liquid chromatography using berberine as an internal standard.
There was no significant difference in onset time between the two groups. The Dur 25%, the recovery index and the time of recovery of the TOF ratios to 70% were all prolonged in the obstructive jaundice group compared with the control group. The plasma concentration of rocuronium at 60, 90 and 120 min after bolus administration was significantly higher in the obstructive jaundice group.
The neuromuscular blockade by rocuronium is prolonged in obstructive jaundice patients, and therefore precautions should be taken in case of postoperative residual neuromuscular block. The possible reason is impedance of rocuronium excretion due to biliary obstruction and increased plasma unbound rocuronium because of free bilirubin competing with it for albumin binding.
AIM: To investigate the effect of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) upregulation in gene expression profile and oncogenicity of human esophageal squamous cell carcinoma (ESCC) cell line TE13.
METHODS: TE13 cells were transfected with pcDNA3.1(+)/RIZ1 and pcDNA3.1(+). Changes in gene expression profile were screened and the microarray results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Nude mice were inoculated with TE13 cells to establish ESCC xenografts. After two weeks, the inoculated mice were randomly divided into three groups. Tumors were injected with normal saline, transfection reagent pcDNA3.1(+) and transfection reagent pcDNA3.1(+)/RIZ1, respectively. Tumor development was quantified, and changes in gene expression of RIZ1 transfected tumors were detected by RT-PCR and Western blotting.
RESULTS: DNA microarray data showed that RIZ1 transfection induced widespread changes in gene expression profile of cell line TE13, with 960 genes upregulated and 1163 downregulated. Treatment of tumor xenografts with RIZ1 recombinant plasmid significantly inhibited tumor growth, decreased tumor size, and increased expression of RIZ1 mRNA compared to control groups. The changes in gene expression profile were also observed in vivo after RIZ1 transfection. Most of the differentially expressed genes were associated with cell development, supervision of viral replication, lymphocyte costimulatory and immune system development in esophageal cells. RIZ1 gene may be involved in multiple cancer pathways, such as cytokine receptor interaction and transforming growth factor beta signaling.
CONCLUSION: The development and progression of esophageal cancer are related to the inactivation of RIZ1. Virus infection may also be an important factor.
Retinoblastoma protein-interacting zinc finger gene 1; Microarray; Nude mice; Esophageal squamous cell carcinoma cells
The recent dramatic cost reduction of next-generation sequencing technology enables investigators to assess most variants in the human genome to identify risk variants for complex diseases. However, sequencing large samples remains very expensive. For a study sample with existing genotype data, such as array data from genome-wide association studies, a cost-effective approach is to sequence a subset of the study sample and then to impute the rest of the study sample, using the sequenced subset as a reference panel. The use of such an internal reference panel identifies population-specific variants and avoids the problem of a substantial mismatch in ancestry background between the study population and the reference population. To efficiently select an internal panel, we introduce an idea of phylogenetic diversity from mathematical phylogenetics and comparative genomics. We propose the “most diverse reference panel”, defined as the subset with the maximal “phylogenetic diversity”, thereby incorporating individuals that span a diverse range of genotypes within the sample. Using data both from simulations and from the 1000 Genomes Project, we show that the most diverse reference panel can substantially improve the imputation accuracy compared to randomly selected reference panels, especially for the imputation of rare variants. The improvement in imputation accuracy holds across different marker densities, reference panel sizes, and lengths for the imputed segments. We thus propose a novel strategy for planning sequencing studies on samples with existing genotype data.
coalescent; imputation; phylogenetic diversity; sequencing; study design
Linear repair of left ventricular aneurysm has been performed with mixed clinical results. By using finite element analysis, this study evaluated the effect of this procedure on end-systolic stress.
Nine sheep underwent myocardial infarction and aneurysm repair with a linear repair (13.4 ± 2.3 weeks postmyocardial infarction). Satisfactory magnetic resonance imaging examinations were obtained in 6 sheep (6.6 ± 0.5 weeks postrepair). Finite element models were constructed from in vivo magnetic resonance imaging-based cardiac geometry and postmortem measurement of myofiber helix angles using diffusion tensor magnetic resonance imaging. Material properties were iteratively determined by comparing the finite element model output with systolic tagged magnetic resonance imaging strain measurements.
At the mid-wall, fiber stress in the border zone decreased by 39% (sham = 32.5 ± 2.5 kPa, repair = 19.7 ± 3.6 kPa, P = .001) to the level of remote regions after repair. In the septum, however, border zone fiber stress remained high (sham = 31.3 ± 5.4 kPa, repair = 23.8 ± 5.8 kPa, P = .29). Cross-fiber stress at the mid-wall decreased by 41% (sham = 13.0 ± 1.5 kPa, repair = 7.7 ± 2.1 kPa, P = .01), but cross-fiber stress in the un-excluded septal infarct was 75% higher in the border zone than remote regions (remote = 5.9 ± 1.9 kPa, border zone = 10.3 ± 3.6 kPa, P < .01). However, end-diastolic fiber and cross-fiber stress were not reduced in the remote myocardium after plication.
With the exception of the retained septal infarct, end-systolic stress is reduced in all areas of the left ventricle after infarct plication. Consequently, we expect the primary positive effect of infarct plication to be in the infarct border zone. However, the amount of stress reduction necessary to halt or reverse nonischemic infarct extension in the infarct border zone and eccentric hypertrophy in the remote myocardium is unknown.
Aptamers are small molecular ligands composed of short oligonucleotides that bind targets with high affinity. In contrast to antibodies, as synthetic oligonucleotides, aptamers have lower production costs and elicit no antigenic reactions. Therefore, aptamers are potential agents for disease diagnosis and treatment. In this study, we validate a fluorescently labeled RNA aptamer, which has been reported to bind specifically to mouse CD30 proteins in solution, for human CD30 protein recognition on intact cells. The aptamer probe was tested with cultured anaplastic large cell lymphoma and Hodgkin′s lymphoma cells that express high levels of CD30. Flow cytometry and fluorescence microscopy showed specific and sensitive binding of the aptamer probe to CD30-expressing lymphoma cells at low concentrations (0.3 nM). Studies performed on multiple cell lines and nuclear cells from healthy donors confirmed that the CD30 aptamer and anti-CD30 antibody, the standard clinical probe, recognized the same set of cells. The potential application of multicolor flow cytometry analysis using the CD30 aptamer probe and antibodies was also shown. In conclusion, the developed CD30 aptamer probe could act as a replacement and/or a supplement for antibodies in the diagnosis of the CD30-expressing lymphomas.
aptamer probe; CD30 expression; cell binding; flow cytometry analysis; lymphoma diagnosis; RNA oligonucleotide
Transcription factor E2F1 exerts effects on many types of cancers. As an upstream regulator of a host of genes, E2F1 can trigger diverse aberrant transcription processes that may dominate malignancy. Clear cell renal cell carcinoma (ccRCC) is the most common subtype in renal cell carcinoma which displays high malignancy and has a shortage of biomarkers in clinics. Our study aimed to explore the function of E2F1 in ccRCC and its correlation with clinicopathological parameters.
Transcription factor E2F1 was mainly distributed in cancer cell nucleus and mRNA expression signiﬁcantly increased in 72 cases of clear cell renal cell carcinoma (ccRCC) tissues compared with adjacent non-cancerous kidney tissues (p<0.001). The protein expression was consistent with mRNA expression. Further analysis in 92 cases indicated that E2F1 mRNA level expression was associated with the tumor pathologic parameters embracing diameter, Fuhrman tumor grade, pT stage, TNM stage grouping and macrovascular infiltration (MAVI). These surgical specimens had high grade tumors accompanied with an elevated E2F1 expression. Moreover, E2F1 transfection was found to contribute significantly to cancer cell proliferation, migration and invasion in vitro.
Overexpression of E2F1 may be a key event in the local and vascular infiltration of ccRCC indicated by the activation of matrix metalloproteinase (MMP) 2 and MMP9. These findings highlighted the implication of E2F1’s function in the metastatic process. Furthermore, the clinical relevance of E2F1 in ccRCC pointed to a potential new therapeutic target.
Puerarin (daidzein 8-C-glucoside) has potential on preventing osteoporosis. This study aims to investigate the effects of puerarin on osteogenesis and adipogenesis in vitro.
CCK-8 assay, alkaline phosphatase (ALP) activity and Alizarin Red S were used to measure the effects of puerarin on proliferation, osteoblastic differentiation, and mineralization in osteoblast-like MC3T3-E1 cells. The effects of puerarin on adipogenesis were measured by Oil Red O staining and intracellular triglyceride level in preadipocyte 3T3-L1 cells. The mRNA and protein levels of osteogenesis- and adiopogenesis-related factors were detected by qRT-PCR and western blot, respectively. Further, the secreted osteocalcin levels and nuclear translocation of β-catenin were detected by ELISA and immunofluorescence assay, respectively.
As to osteogenesis, puerarin could stimulate proliferation (1 μM, P = 0.012; 10 μM, P = 0.015; 20 μM, P = 0.050), ALP activity (20 μM, P = 0.008) and calcium nodule formation (20 μM, P = 0.011) in a dose-dependent manner. Puerarin (20 μM) promoted osteocalcin secretion (P = 0.004) and the protein expression of both osteopontin (P = 0.001) and osteoprotegerin (P = 0.003). As to adipogenesis, puerarin suppressed adipocytes formation and intracellular triglyceride level (P = 0.001). In addition, puerarin (20 μM) decreased the mRNA and protein levels of CCAAT/enhancer binding protein α (P = 0.001, P = 0.002), proliferator-activated receptor γ (P = 0.005, P = 0.003), and adipocyte lipid-binding protein 4 (P = 0.001, P = 0.001). Moreover, phosphorylation of AKT1-Ser437 (10 μM, P = 0.003; 20 μM, P = 0.007) and GSK-Ser9 (10 μM, P = 0.005; 20 μM, P = 0.003), and the nuclear translocation of β-catenin (10 μM, P = 0.006; 10 μM, P = 0.002) were increased in 3T3-L1 cells treated by puerarin.
Puerarin promoted osteogenesis and inhibited adipogenesis in vivo, and Akt/GSK-3β/β-catenin signaling pathway was involved in the suppression of adipogenesis.
Our quantitative proteomic study showed that selenium-binding protein 1 (SELENBP1) was progressively decreased in human bronchial epithelial carcinogenic process. However, there is little information on expression and function of SELENBP1 during human lung squamous cell cancer (LSCC) carcinogenesis.
iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed proteins in the human bronchial epithelial carcinogenic process. SELENBP1, member of selenoproteins family and progressively downregulated in this process, was selected to further study. Both Western blotting and immunohistochemistry were performed to detect SELENBP1 expression in independent sets of tissues of bronchial epithelial carcinogenesis, and ability of SELENBP1 for discriminating NBE (normal bronchial epithelium) from preneoplastic lesions from invasive LSCC was evaluated. The effects of SELENBP1 downregulation on the susceptibility of benzo(a)pyrene (B[a]P)-induced human bronchial epithelial cell transformation were determined.
102 differentially expressed proteins were identified by quantitative proteomics, and SELENBP1 was found and confirmed being progressively decreased in the human bronchial epithelial carcinogenic process. The sensitivity and specificity of SELENBP1 were 80% and 79% in discriminating NBE from preneoplastic lesions, 79% and 82% in discriminating NBE from invasive LSCC, and 77% and 71% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, knockdown of SELENBP1 in immortalized human bronchial epithelial cell line 16HBE cells significantly increased the efficiency of B[a]P-induced cell transformation.
The present data shows for the first time that decreased SELENBP1 is an early event in LSCC, increases B[a]P-induced human bronchial epithelial cell transformation, and might serve as a novel potential biomarker for early detection of LSCC.
This paper reports on a simple method to prepare a hydrophobic surface on black silicon, which is fabricated by metal-assisted wet etching. To increase the reaction rate, the reaction device was placed in a heat collection-constant temperature type magnetic stirrer and set at room temperature. It was demonstrated that the micro- and nanoscale spikes on the black silicon made the surface become hydrophobic. As the reaction rate increases, the surface hydrophobicity becomes more outstanding and presents self-cleaning until the very end. The reflectance of the black silicon is drastically suppressed over a broad spectral range due to the unique geometry, which is effective for the enhancement of absorption.
Black silicon; Metal-assisted wet etching; Hydrophobic surface; Reflectance; Absorption
Nonalcoholic fatty liver disease is associated with obesity and insulin resistance. Factors that regulate the disposal of hepatic triglycerides contribute to the development of hepatic steatosis. G0/G1 switch gene 2 (G0S2) is a target of peroxisome proliferator-activated receptors and plays an important role in regulating lipolysis in adipocytes. Therefore, we investigated whether G0S2 plays a role in hepatic lipid metabolism. Adenovirus-mediated expression of G0S2 (Ad-G0S2) potently induced fatty liver in mice. The liver mass of Ad-G0S2-infected mice was markedly increased with excess triglyceride content compared to the control mice. G0S2 did not change cellular cholesterol levels in hepatocytes. G0S2 was found to be co-localized with adipose triglyceride lipase at the surface of lipid droplets. Hepatic G0S2 overexpression resulted in an increase in plasma Low-density lipoprotein (LDL)/Very-Low-density (VLDL) lipoprotein cholesterol level. Plasma High-density lipoprotein (HDL) cholesterol and ketone body levels were slightly decreased in Ad-G0S2 injected mice. G0S2 also increased the accumulation of neutral lipids in cultured HepG2 and L02 cells. However, G0S2 overexpression in the liver significantly improved glucose tolerance in mice. Livers expressing G0S2 exhibited increased 6-(N-(7-nitrobenz-2-oxa-1-3-diazol-4-yl) amino)-6-deoxyglucose uptake compared with livers transfected with control adenovirus. Taken together, our results provide evidence supporting an important role for G0S2 as a regulator of triglyceride content in the liver and suggest that G0S2 may be a molecular target for the treatment of insulin resistance and other obesity-related metabolic disorders.
Our pilot study using miRNA arrays found that miRNA-29c (miR-29c) is differentially expressed in the paired low-metastatic lung cancer cell line 95C compared to the high-metastatic lung cancer cell line 95D. Bioinformatics analysis shows that integrin β1 and matrix metalloproteinase 2 (MMP2) could be important target genes of miR-29c. Therefore, we hypothesized that miR-29c suppresses lung cancer cell adhesion to extracellular matrix (ECM) and metastasis by targeting integrin β1 and MMP2. The gain-of-function studies that raised miR-29c expression in 95D cells by using its mimics showed reductions in cell proliferation, adhesion to ECM, invasion and migration. In contrasts, loss-of-function studies that reduced miR-29c by using its inhibitor in 95C cells promoted proliferation, adhesion to ECM, invasion and migration. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29c inhibited the expression of the luciferase gene containing the 3′-UTRs of integrin β1 and MMP2 mRNA. Western blotting indicated that miR-29c downregulated the expression of integrin β1 and MMP2 at the protein level. Gelatin zymography analysis further confirmed that miR-29c decreased MMP2 enzyme activity. Nude mice with xenograft models of lung cancer cells confirmed that miR-29c inhibited lung cancer metastasis in vivo, including bone and liver metastasis. Taken together, our results demonstrate that miR-29c serves as a tumor metastasis suppressor, which suppresses lung cancer cell adhesion to ECM and metastasis by directly inhibiting integrin β1 and MMP2 expression and by further reducing MMP2 enzyme activity. The results show that miR-29c may be a novel therapeutic candidate target to slow lung cancer metastasis.
Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEOr was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase–PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (p<0.05). In addition, the growth performance of transgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows.
They demonstrate the successful production of a transgenic mouse expressing a lysine-rich gene in milk and suggest its potential application for drug development.
In sugar cataract formation in rats, aldose reductase (AR) actitvity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 hrs in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galctose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose / galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osomotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation.
diabetes; lens; cataracts; in vitro lens culture; aldose reductase inhibitors; sorbitol dehydrogenase inhibitors; iso-osmotic culture; growth factors; b-FGF; TGF-β; cell signaling; P-42/44 MAPK; P-SAPK/JNK; P-Akt
In this study, a five-generation Chinese family (family F013) with progressive autosomal dominant hearing loss was mapped to a critical region spanning 28.54 Mb on chromosome 9q31.3-q34.3 by linkage analysis, which was a novel DFNA locus, assigned as DFNA56. In this interval, there were 398 annotated genes. Then, whole exome sequencing was applied in three patients and one normal individual from this family. Six single nucleotide variants and two indels were found co-segregated with the phenotypes. Then using mass spectrum (Sequenom, Inc.) to rank the eight sites, we found only the TNC gene be co-segregated with hearing loss in 53 subjects of F013. And this missense mutation (c.5317G>A, p.V1773M ) of TNC located exactly in the critical linked interval. Further screening to the coding region of this gene in 587 subjects with nonsyndromic hearing loss (NSHL) found a second missense mutation, c.5368A>T (p. T1796S), co-segregating with phenotype in the other family. These two mutations located in the conserved region of TNC and were absent in the 387 normal hearing individuals of matched geographical ancestry. Functional effects of the two mutations were predicted using SIFT and both mutations were deleterious. All these results supported that TNC may be the causal gene for the hearing loss inherited in these families. TNC encodes tenascin-C, a member of the extracellular matrix (ECM), is present in the basilar membrane (BM), and the osseous spiral lamina of the cochlea. It plays an important role in cochlear development. The up-regulated expression of TNC gene in tissue repair and neural regeneration was seen in human and zebrafish, and in sensory receptor recovery in the vestibular organ after ototoxic injury in birds. Then the absence of normal tenascin-C was supposed to cause irreversible injuries in cochlea and caused hearing loss.
Rice gall dwarf virus (RGDV), a member of the family Reoviridae, causes repeated epidemics in rice fields in southern China. An RGDV isolate collected from Guangdong Province (southern China) is mainly transmitted by leafhopper vector Recilia dorsalis in a persistent-propagative manner. The infection by RGDV induces the formation of virus-containing tubules in the plant host and insect vector. In this study, we established continuous cell cultures of the leafhopper R. dorsalis to investigate the functional role of these tubules within the insect vector. Cytopathologic studies revealed that the tubules, which comprised viral non-structural protein Pns11 and contained viral particles, were able to protrude from the surface of cultured leafhopper cells. Tubule-like structures formed in non-host insect cells after the expression of Pns11 in a baculovirus system, suggesting that Pns11 was the minimal viral factor required for the formation of the tubules induced by RGDV infection. In cultured leafhopper cells, knockdown of Pns11 expression from RNA interference, induced by synthesized dsRNA from the Pns11 gene, abolished the formation of such tubules, preventing the direct cell-to-cell spread of RGDV without significant effects on viral multiplication. All these results show that RGDV exploits virus-containing tubules to facilitate viral spread among its insect vector cells.
rice gall dwarf virus; leafhopper vector Recilia dorsalis; tubules; viral spread; continuous cell cultures of leafhopper
Negative allosteric modulators (NAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) have shown promising results in preclinical models for anxiety and drug abuse. Here we describe a series of aryl-substituted alkynyl analogues of the prototypic mGluR5 NAM 2-methyl-6-(phenylethynyl)pyridine (MPEP, 1). Displacement of [3H]1 binding in rat brain membranes showed that several of these novel compounds displayed high affinity binding (Ki < 10 nM) for mGluR5, with up to a 24-fold increase in affinity over 1. Replacements of the 2-position Me on the pyridyl ring of 1 along with various 3’-CN, 5’-substitutions were generally well tolerated. All of the active analogues in this series had cLogP values in the 2–5 range and displayed inverse agonist characteristics in an ELISA-based assay of Gqα-mediated IP3 production. Compounds 7i and 7j produced in vivo effects in mouse models of anxiety-like behaviors more potently than 1 or 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP, 2), supporting their utility as in vivo tools.
glutamate; negative allosteric modulator; inverse agonist; anxiety; light-dark box
Krüppel-like factor 4 (KLF4) is a transcription factor with diverse functions in various cancer types; however, the function of KLF4 in clear cell renal cell carcinoma (ccRCC) carcinogenesis remains unknown. In this study, we initially examined KLF4 expression by using a cohort of surgically removed ccRCC specimens and cell lines. Results indicated that the transcription and translation of KLF4 were lower in ccRCC tissues than in patient-matched normal tissues. Furthermore, the KLF4 expression was significantly downregulated in the five ccRCC cell lines at protein and mRNA levels compared with that in normal renal proximal tubular epithelial cell lines (HKC). KLF4 downregulation was significantly correlated with tumor stage and tumor diameter. Promoter hypermethylation may contribute to its low expression. In addition, in vitro studies indicated that the KLF4 overexpression significantly inhibited proliferation in human ccRCC cell lines 786-O and ACHN. Moreover, the KLF4 overexpression arrested the cell cycle progress at the G1/S phase transition by upregulating p21WAF1/CIP1 expression and downregulating cyclin D1 expression, KLF4 knockdown in HKC cells did the opposite. In vivo studies confirmed the anti-proliferative effect of KLF4. Our results suggested that KLF4 had an important function in suppressing the growth of ccRCC.
Handheld computers for data collection (HCDC) and management have become increasingly common in health research. However, current knowledge about the use of HCDC in health research in China is very limited. In this study, we administered a survey to a hard-to-reach population in China using HCDC and assessed the acceptability and adoption of HCDC in China.
Handheld computers operating Windows Mobile and Questionnaire Development Studio (QDS) software (Nova Research Company) were used for this survey. Questions on tobacco use and susceptibility were drawn from the Global Adult Tobacco Survey (GATS) and other validated instruments, and these were programmed in Chinese characters by local staff. We conducted a half-day training session for survey supervisors and a three-day training session for 20 interviewers and 9 supervisors. After the training, all trainees completed a self-assessment of their skill level using HCDC. The main study was implemented in fall 2010 in 10 sites, with data managed centrally in Beijing. Study interviewers completed a post-survey evaluation questionnaire on the acceptability and utility of HCDC in survey research.
Twenty-nine trainees completed post-training surveys, and 20 interviewers completed post-data collection questionnaires. After training, more than 90% felt confident about their ability to collect survey data using HCDC, to transfer study data from a handheld computer to a laptop, and to encrypt the survey data file. After data collection, 80% of the interviewers thought data collection and management were easy and 60% of staff felt confident they could solve problems they might encounter. Overall, after data collection, nearly 70% of interviewers reported that they would prefer to use handheld computers for future surveys. More than half (55%) felt the HCDC was a particularly useful data collection tool for studies conducted in China.
We successfully conducted a health-related survey using HCDC. Using handheld computers for data collection was a feasible, acceptable, and preferred method by Chinese interviewers. Despite minor technical issues that occurred during data collection, HCDC is a promising methodology to be used in survey-based research in China.
Surveys; Electronic data collection; Handheld computers; China
The aim of this study was to examine the contribution of side population (SP) cells from kidney and bone marrow for reconstitution of kidney SP pools after ischemia-reperfusion injury (IRI). The SP and non-SP cells in kidneys following IRI were isolated and serially assessed by fluorescence-activated cell sorting. The apoptosis, proliferation, phenotype, and paracrine actions of SP cells were evaluated in vitro and in vivo. Results indicated that the SP cells from ischemic kidney were acutely depleted within one day following renal IRI and were progressively restored to baseline within 7 days after IRI, through both proliferation of remaining kidney SP cells and homing of bone marrow-derived cells to ischemic kidney. Either hypoxia or serum deprivation alone increased apoptosis of SP cells, and a combination of both further aggravated it. Furthermore, hypoxia in vivo and in vitro induced the increase in the secretion of vascular endothelial growth factor, insulin-like growth factor 1, hepatocyte growth factor, and stromal cell-derived factor-1α in kidney SP but not non-SP cells. In summary, these results suggest that following renal IRI, kidney SP cells are acutely depleted and then progressively restored to baseline levels by both self-proliferation and extrarenal source, that is, bone marrow-derived cell homing.
Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals.
Wheat yellow mosaic virus (WYMV) is an important pathogen in China and other countries. It is the member of the genus Bymovirus and transmitted primarily by Polymyxa graminis. The incidence of wheat infections in endemic areas has risen in recent years. Prompt and dependable identification of WYMV is a critical component of response to suspect cases.
In this study, a one step real-time RT-PCR, followed by standard curve analysis for the detection and identification of WYMV, was developed. Two reference genes, 18s RNA and β-actin were selected in order to adjust the veracity of the real-time RT-PCR assay.
We developed a one-step Taqman-based real-time quantitative RT-PCR (RT-qPCR) assay targeting the conserved region of the 879 bp long full-length WYMV coat protein gene. The accuracy of normalized data was analyzed along with appropriate internal control genes: β-actin and 18s rRNA which were included in detecting of WYMV-infected wheat leaf tissues. The detectable end point sensitivity in RT-qPCR assay was reaching the minimum limit of the quantitative assay and the measurable copy numbers were about 30 at106-fold dilution of total RNA. This value was close to 104-fold more sensitive than that of indirect enzyme-linked immunosorbent assay. More positive samples were detected by RT-qPCR assay than gel-based RT-PCR when detecting the suspected samples collected from 8 regions of China. Based on presented results, RT-qPCR will provide a valuable method for the quantitative detection of WYMV.
The Taqman-based RT-qPCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of WYMV than other currently used methods.
Wheat yellow mosaic virus (WYMV); RT-qPCR; Quantitation
Anxiety disorders are commonly comorbid with bipolar disorder (BP) and may worsen course of illness, but differential impact of specific anxiety disorders in men and women remains unknown.
We measured the impact of comorbid panic disorder (PD), social phobia, specific phobia, and obsessive-compulsive disorder (OCD) in 460 women and 276 men with Bipolar I Disorder (BPI) or schizoaffective disorder, bipolar type from the National Institute of Mental Health Bipolar Genetics Initiative. We compared clinical characteristics in BP with and without each anxiety disorder in men and women separately correcting for family relatedness.
Comorbid PD, OCD, and specific phobia were more common in women with BP than men. Comorbid social phobia correlated with increased risk of alcohol abuse in BP women, but not men. Women with comorbid PD attended fewer years of school. Comorbidity with OCD was associated with earlier age at the onset of BP for both genders. Comorbid PD, OCD, and specific phobia were associated with more antidepressant trials in BP, across both genders, compared to BP patients without these anxiety disorders.
In BP, comorbid anxiety disorders are associated with increased risk for functional impairment, and women had differently associated risks than men. Clinicians should be aware of an increased risk for comorbid PD, OCD, and specific phobia in women with BP, and an increased risk of alcohol abuse in women with BD and comorbid social phobia.
psychiatry; panic; obsessive-compulsive; social phobia; specific phobia; depression; mania; alcohol; female
Background and Aims
Preliminary field observations in 2001 and 2002 suggested that Kingdonia uniflora (Circaeasteraceae, Ranunculales) exhibits heterodichogamy, an unusual kind of reproductive heteromorphy, hitherto unreported in Ranunculales and known from only one other genus in basal eudicots.
During several subsequent years flowers were observed in the field. Flowers were fixed in FAA and studied with microtome sections series and with the scanning electron microscope.
The flowers proved to be heterodichogamous, with protandrous and protogynous morphs, which have a 1 : 1 ratio. Both morphs equally set fruit. Each year a single flower is formed at the tip of a rhizome or more rarely two flowers. The flowers are already open when they appear at the soil surface, before they are receptive and before pollen is dispersed. In both floral morphs the styles elongate early and the stigmas are positioned above the anthers before anthesis begins. In protogynous flowers the stigmas become receptive in this position; later the styles become reflexed and then the anthers dehisce. In contrast, in protandrous flowers the stamen filaments elongate during early anthesis such that the dehiscing anthers come to lie above the (still unreceptive) stigmas; after dehiscence of all anthers in a flower the styles begin to elongate and become receptive.
This is the first record of heterodichogamy in a representative of Ranunculales, in an herbaceous eudicot, and in a plant with uniflorous ramets. The occurrence of heterodichogamy in Kingdonia in which clonal reproduction appears to be dominant might be an adaptation to avoid mating between the ramets from a common mother individual (genet).
Kingdonia; Circaeasteraceae; Ranunculales; heterodichogamy; reproductive heteromorphy