Ischemic Preconditioning Increases Endothelial Progenitor Cell Number to Attenuate Partial Nephrectomy-Induced Ischemia/Reperfusion Injury

Objectives

The objective of this study was to investigate the role of endothelial progenitor cells (EPCs) in the modulation of ischemia-reperfusion injury (IRI) in a partial nephrectomy (PN) rat model using early-phase ischemic preconditioning (IPC).

Materials and Methods

Ninety male Sprague-Dawley rats were randomly divided into three groups following right-side nephrectomy: Sham-operated rats (surgery without vascular clamping); PN rats (renal blood vessels were clamped for 40 min and PN was performed); and IPC rats (pretreated with 15 min ischemia and 10 min reperfusion). At 1, 3, 6, 12, 24 h, and 3 days after reperfusion, the pool of circulating EPCs and kidneys were harvested. The extent of renal injury was assessed, along with EPC number, cell proliferation, angiogenesis, and vascular growth factor expression.

Results

Pretreated rats exhibited significant improvements in renal function and morphology. EPC numbers in the kidneys were increased at 12 h following reperfusion in the IPC group as compared to the PN or Sham groups. Cell proliferation (including endothelial and tubular epithelial cells) and angiogenesis in peritubular capillaries were markedly increased in kidneys treated with IPC. In addition, vascular endothelial growth factor-A (VEGF-A) and stromal cell-derived factor-1α (SDF-1α) expression in the kidneys of pretreated rats was increased compared to rats subjected to PN.

Conclusions

Our investigation suggested that: (1) the early phase of IPC may attenuate renal IRI induced by PN; (2) EPCs play an important role in renal protection, involving promotion of cell proliferation and angiogenesis through release of several angiogenic factors.     

An Efficient Hierarchical Generalized Linear Mixed Model for Mapping QTL of Ordinal Traits in Crop Cultivars

Many important phenotypic traits in plants are ordinal. However, relatively little is known about the methodologies for ordinal trait association studies. In this study, we proposed a hierarchical generalized linear mixed model for mapping quantitative trait locus (QTL) of ordinal traits in crop cultivars. In this model, all the main-effect QTL and QTL-by-environment interaction were treated as random, while population mean, environmental effect and population structure were fixed. In the estimation of parameters, the pseudo data normal approximation of likelihood function and empirical Bayes approach were adopted. A series of Monte Carlo simulation experiments were performed to confirm the reliability of new method. The result showed that new method works well with satisfactory statistical power and precision. The new method was also adopted to dissect the genetic basis of soybean alkaline-salt tolerance in 257 soybean cultivars obtained, by stratified random sampling, from 6 geographic ecotypes in China. As a result, 6 main-effect QTL and 3 QTL-by-environment interactions were identified.     

Function and Expression of Cystic Fibrosis Transmembrane Conductance Regulator after Small Intestinal Transplantation in Mice

The secretion function of intestinal graft is one of the most important factors for successful intestinal transplantation. Cystic fibrosis transmembrane conductance regulator (CFTR) mediates HCO₃ ^- and Cl^- secretions in intestinal epithelial cells. In this study, we made investigation on the expression and function of CFTR in an experimental model of murine small intestinal transplantation. Heterotopic intestinal transplantations were performed in syngeneic mice. The mRNA and protein expressions of CFTR were analyzed by real time PCR and western blot. Murine intestinal mucosal HCO₃ ^- and Cl^- secretions were examined in vitro in Ussing chambers by the pH stat and short circuit current (I_sc) techniques. The results showed that forskolin, an activator of CFTR, stimulated jejunal mucosal epithelial HCO₃ ^- and Cl^- secretions in mice, but forskolin-stimulated HCO₃ ^- and Cl^- secretions in donor and recipient jejunal mucosae of mice after heterotopic jejunal transplantation were markedly decreased, compared with controls (P<0.001). The mRNA and protein expression levels of CFTR in donor and recipient jejunal mucosae of mice were also markedly lower than those in controls (P<0.001), and the mRNA and protein expression levels of tumor necrosis factor α (TNFα) were markedly increased in donor jejunal mucosae of mice (P<0.001), compared with controls. Further experiments showed that TNFα down-regulated the expression of CFTR mRNA in murine jejunal mucosa. In conclusion, after intestinal transplantation, the function of CFTR was impaired, and its mRNA and protein expressions were down-regulated, which may be induced by TNFα.     

Association between MMP1 -1607 1G>2G Polymorphism and Head and Neck Cancer Risk: A Meta-Analysis

Background

MMP1 is an important member of the MMP endopeptidase family that plays a critical role in the development of head and neck cancer (HNC). Several studies have investigated the association between the MMP1 -1607 1G>2G polymorphism and risk of HNC, but their results have been inconsistent. Here, we conducted a meta-analysis to further explore the role of the MMP1 -1607 1G>2G polymorphism in HNC development.

Methods

We identified all eligible studies in the electronic databases of PubMed, ISI Web of Knowledge, MEDLINE, Embase, and Google Scholar (from January 2000 to June 2012). A meta-analysis was performed to evaluate the association between the MMP1 -1607 1G>2G polymorphism and risk of HNC by calculating odds ratios (OR) and 95% confidence interval (CIs).

Results

Twelve studies were included in this meta-analysis. In overall comparison, significant associations were found using the recessive and allelic contrast models (OR, 1.38; 95% CI, 1.07–1.79 and OR, 1.27; 95% CI, 1.05–1.53, respectively), but no association was detected using the dominant model. In the stratified analyses by several variables, significant associations were observed using the recessive, dominant, and allelic contrast models in the Asian population (OR, 1.64; 95% CI, 1.29–2.08; OR, 1.39; 95% CI, 1.06–1.82; and OR, 1.41; 95% CI, 1.21–1.65, respectively), European population (OR, 0.58; 95% CI, 0.40–0.84; OR, 0.64; 95% CI, 0.44–0.92; and OR, 0.68; 95% CI, 0.54–0.85, respectively), and population-based subgroup (OR, 1.24; 95% CI,1.05–1.47; OR,1.48; 95% CI,1.04–2.12; and OR, 1.22; 95% CI, 1.07–1.38, respectively). Furthermore, significant associations were detected in oral cavity cancer and nasopharyngeal cancer under the recessive model.

Conclusion

Our results suggest that the MMP1 -1607 1G>2G polymorphism is associated with risk of HNC and that it plays different roles in Asian and European populations. Further studies with large sample size are needed to validate our findings.     

PKCδ as a Regulator for TGFβ1-Induced α-SMA Production in a Murine Nonalcoholic Steatohepatitis Model

The precise mechanism of TGFβ1 signaling in the progression of non-alcoholic steatohepatitis (NASH) has remained unclear. In particular, a potential regulatory mechanism by which PKCδ affects profibrogenic gene expression had never been explored. In this study, therefore, the role of PKCδ in TGFβ1 mediated α-SMA expression was investigated using NASH model mice. In preparation of the NASH model, male C57BL6/J mice were fed a methionine-choline-deficient (MCD) diet for 3 weeks, after which time they were intraperitoneally injected with lipopolysaccharide (LPS). In addition, Tlr4^Lps-d (CH3/HeJ) mice were used to demonstrate the TGFβ1 signaling’s dependency on TLR4 induction. Liver histology and hepatic hepatitis markers were investigated, and hepatic gene expression levels were determined by real-time PCR. Acute liver injury by LPS injection specifically elevated not only α-SMA expression but also phospho-PKCδ in this model. In contrast, Tlr4^Lps-d (CH3/HeJ) and blockade of TGFβ1 receptor by SB431542 resulted in a significant reduction of PKCδ activation and α-SMA expression. Moreover, the TGFβ1-induced α-SMA production was significantly reduced by a specific PKCδ inhibitor. These findings suggested that PKCδ plays a critical role in TGFβ1-induced α-SMA production in a NASH model. Thus, this was the first demonstration of the involvement of PKCδ in the regulation of α-SMA expression in NASH liver tissues, and the impaired induction of PKCδ phosphorylation by LPS in a steatohepatitis condition. Interestingly, treatment by PKCδ inhibitor caused dramatic reduction of myofibroblast activation, indicating that PKCδ represents a promising target for treating NASH.     

Influenza Nucleoprotein Delivered with Aluminium Salts Protects Mice from an Influenza A Virus That Expresses an Altered Nucleoprotein Sequence

Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes.     

An Integrated Approach to Uncover Driver Genes in Breast Cancer Methylation Genomes

Background

Cancer cells typically exhibit large-scale aberrant methylation of gene promoters. Some of the genes with promoter methylation alterations play “driver” roles in tumorigenesis, whereas others are only “passengers”.

Results

Based on the assumption that promoter methylation alteration of a driver gene may lead to expression alternation of a set of genes associated with cancer pathways, we developed a computational framework for integrating promoter methylation and gene expression data to identify driver methylation aberrations of cancer. Applying this approach to breast cancer data, we identified many novel cancer driver genes and found that some of the identified driver genes were subtype-specific for basal-like, luminal-A and HER2+ subtypes of breast cancer.

Conclusion

The proposed framework proved effective in identifying cancer driver genes from genome-wide gene methylation and expression data of cancer. These results may provide new molecular targets for potential targeted and selective epigenetic therapy.     

Tryptase Promotes Atherosclerotic Plaque Haemorrhage in ApoE-/- Mice

Tryptase, the most abundant mast cell (MC) granule protein, plays an important role in atherosclerosis plaque development. To test the hypothesis that tryptase participates directly in atherosclerosis plaque haemorrhage, the gene sequence and siRNA for tryptase were cloned into a lentivirus carrier and atherosclerosis plaque haemorrhage models in ApoE-/- mice were constructed. After a cuffing-cervical artery operation, the mice were randomly divided into 6 groups. Hematoxylin and eosin(HE) staining showed that the cervical artery plaque area was much larger in the tryptase overexpression group compared to the other groups, and there was greater artery stenosis. The artery stenosis from the cuff-side in all groups was more than 90%, except the siRNA group. Tryptase promotes plaque haemorrhage distinctively because 50% of the mice in the tryptase overexpression group had plaque haemorrhage, while only 10% in the siRNA group did. The immunohistochemistry of the cervical artery plaque showed that plasminogen activator inhibitor-1 (PAI-1) expression was the lowest while tissue plasminogen activator (tPA), CD31, CD34 and VEGF was the highest in the tryptase overexpression groups. This observation was completely contrary to what was observed in the siRNA group. Tryptase promoted bEnd.3 cell growth, migration and capillary-like tube formation, which suggests that tryptase can promote microvessel angiogenesis. PAI-1 expression was inhibited, while tPA expression was increased by tryptase in bEnd.3 cells. Our in vivo and in vitro studies suggest that trypase can promote atherosclerotic plaque haemorrhage by promoting angiogenesis and regulating the balance of PAI-1 and tPA. Thus, regulating tryptase expression in MCs may provide a potential target for atherosclerosis treatment.     

Digital Gene Expression Analysis of Corky Split Vein Caused by Boron Deficiency in ‘Newhall’ Navel Orange (Citrus sinensis Osbeck) for Selecting Differentially Expressed Genes Related to Vascular Hypertrophy

Corky split vein caused by boron (B) deficiency in ‘Newhall’ Navel Orange was studied in the present research. The boron-deficient citrus exhibited a symptom of corky split vein in mature leaves. Morphologic and anatomical surveys at four representative phases of corky split veins showed that the symptom was the result of vascular hypertrophy. Digital gene expression (DGE) analysis was performed based on the Illumina HiSeq™ 2000 platform, which was applied to analyze the gene expression profilings of corky split veins at four morphologic phases. Over 5.3 million clean reads per library were successfully mapped to the reference database and more than 22897 mapped genes per library were simultaneously obtained. Analysis of the differentially expressed genes (DEGs) revealed that the expressions of genes associated with cytokinin signal transduction, cell division, vascular development, lignin biosynthesis and photosynthesis in corky split veins were all affected. The expressions of WOL and ARR12 involved in the cytokinin signal transduction pathway were up-regulated at 1^st phase of corky split vein development. Furthermore, the expressions of some cell cycle genes, CYCs and CDKB, and vascular development genes, WOX4 and VND7, were up-regulated at the following 2^nd and 3^rd phases. These findings indicated that the cytokinin signal transduction pathway may play a role in initiating symptom observed in our study.     

The Ambiguous Base-Pairing and High Substrate Efficiency of T-705 (Favipiravir) Ribofuranosyl 5′-Triphosphate towards Influenza A Virus Polymerase

T-705 (Favipiravir) is a broad-spectrum antiviral molecule currently in late stage clinical development for the treatment of influenza virus infection. Although it is believed that T-705 potency is mediated by its ribofuranosyl triphosphate (T-705 RTP) metabolite that could be mutagenic, the exact molecular interaction with the polymerase of influenza A virus (IAVpol) has not been elucidated. Here, we developed a biochemical assay to measure the kinetics of nucleotide incorporation by IAVpol in the elongation mode. In this assay, T-705 RTP was recognized by IAVpol as an efficient substrate for incorporation to the RNA both as a guanosine and an adenosine analog. Compared to natural GTP and ATP, the discrimination of T-705 RTP was about 19- and 30-fold, respectively. Although the single incorporation of the ribonucleotide monophosphate form of T-705 did not efficiently block RNA synthesis, two consecutive incorporation events prevented further primer extension. In comparison, 3′-deoxy GTP caused immediate chain termination but was incorporated less efficiently by the enzyme, with a discrimination of 4,900-fold relative to natural GTP. Collectively, these results provide the first detailed biochemical characterization to evaluate the substrate efficiency and the inhibition potency of nucleotide analogs against influenza virus polymerase. The combination of ambiguous base-pairing with low discrimination of T-705 RTP provides a mechanistic basis for the in vitro mutagenic effect of T-705 towards influenza virus.