Significant challenges, including low expression and extracellular secretion of soluble protein, are encountered in expressing and purifying Bacillus acidopullulyticus pullulanase (BaPul) in Escherichia coli.

MicroRNAs (miRNAs) are being developed to enhance tissue regeneration. Here we show that a hyperbranched polymer with high miRNA-binding affinity and negligible cytotoxicity can self-assemble into nano-sized polyplexes with a 'double-shell' miRNA distribution and high transfection efficiency. These polyplexes are encapsulated in biodegradable microspheres to enable controllable two-stage (polyplexes and miRNA) delivery. The microspheres are attached to cell-free nanofibrous polymer scaffolds that spatially control the release of miR-26a. This technology is used to regenerate critical-sized bone defects in osteoporotic mice by targeting Gsk-3β to activate the osteoblastic activity of endogenous stem cells, thus addressing a critical challenge in regenerative medicine of achieving cell-free scaffold-based miRNA therapy for tissue engineering.

Differentiated cells retain the genetic information of the donor but the extent to which phenotypic differences between donors or batches of differentiated cells are explained by variation introduced during the differentiation process is not fully understood. In this study, we evaluated four separate batches of commercially available neurons originating from the same iPSCs to investigate whether the differentiation process used in manufacturing iPSCs to neurons affected genome-wide gene expression and modified cytosines, or neuronal sensitivity to drugs. No significant changes in gene expression, as measured by RNA-Seq, or cytosine modification levels, as measured by the Illumina 450K arrays, were observed between batches relative to changes over time. As expected, neurotoxic chemotherapeutics affected neuronal outgrowth, but no inter-batch differences were observed in sensitivity to paclitaxel, vincristine and cisplatin. As a testament to the utility of the model for studies of neuropathy, we observed that genes involved in neuropathy had relatively higher expression levels in these samples across different time points. Our results suggest that the process used to differentiate iPSCs into neurons is consistent, resulting in minimal intra-individual variability across batches. Therefore, this model is reasonable for studies of human neuropathy, druggable targets to prevent neuropathy, and other neurological diseases.

The apetalous genotype is a morphological ideotype for increasing seed yield and should be of considerable agricultural use; however, only a few studies have focused on the genetic control of this trait in Brassica napus. In the present study, a recombinant inbred line, the AH population, containing 189 individuals was derived from a cross between an apetalous line 'APL01' and a normally petalled variety 'Holly'. The Brassica 60 K Infinium BeadChip Array harboring 52,157 single nucleotide polymorphism (SNP) markers was used to genotype the AH individuals. A high-density genetic linkage map was constructed based on 2,755 bins involving 11,458 SNPs and 57 simple sequence repeats, and was used to identify loci associated with petalous degree (PDgr). The linkage map covered 2,027.53 cM, with an average marker interval of 0.72 cM. The AH map had good collinearity with the B. napus reference genome, indicating its high quality and accuracy. After phenotypic analyses across five different experiments, a total of 19 identified quantitative trait loci (QTLs) distributed across chromosomes A3, A5, A6, A9 and C8 were obtained, and these QTLs were further integrated into nine consensus QTLs by a meta-analysis. Interestingly, the major QTL qPD.C8-2 was consistently detected in all five experiments, and qPD.A9-2 and qPD.C8-3 were stably expressed in four experiments. Comparative mapping between the AH map and the B. napus reference genome suggested that there were 328 genes underlying the confidence intervals of the three steady QTLs. Based on the Gene Ontology assignments of 52 genes to the regulation of floral development in published studies, 146 genes were considered as potential candidate genes for PDgr. The current study carried out a QTL analysis for PDgr using a high-density SNP map in B. napus, providing novel targets for improving seed yield. These results advanced our understanding of the genetic control of PDgr regulation in B. napus.

The loss of retinal pericytes (RPCs) is a hallmark of early stage diabetic retinopathy (DR), but the mechanism underlying RPC death is unclear. Although it was postulated in previous studies using bovine RPCs that autoantibodies against RPCs might develop and induce RPC death, it is unknown whether autoantibodies against cell-surface antigens on human RPCs exist in DR patients, whether such autoantibodies contribute to RPC damage/loss, and if they do, through which mechanism. We screened serum samples from DR patients and controls using primary human RPCs and found that that levels of IgGs reactive to RPCs were significantly higher in the DR group than the control group. Serum samples with higher RPC-reactive IgG levels induced more severe complement-mediated RPC damage than those with lower RPC-reactive IgG levels. We also assessed levels of the complement-activation products C3a, C4a and C5a in these serum samples, and found that serum levels of C3a and C5a, but not C4a, were higher in the DR group than control group. These data provide evidence the first time showing that autoantibodies against RPCs can develop in DR patients, and that these autoantibodies could contribute to pericyte damage through complement activation.

Nogo-B receptor (NgBR), a type I single transmembrane domain receptor is the specific receptor for Nogo-B. Our previous work demonstrated that NgBR is highly expressed in breast cancer cells, where it promotes epithelial mesenchymal transition (EMT), an important step in metastasis. Here, we show that both in vitro and in vivo increased expression of NgBR contributes to the increased chemoresistance of Bel7402/5FU cells, a stable 5-FU (5-Fluorouracil) resistant cell line related Bel7402 cells. NgBR knockdown abrogates S-phase arrest in Bel7402/5FU cells, which correlates with a reduction in G1/S phase checkpoint proteins p53 and p21. In addition, NgBR suppresses p53 protein levels through activation of the PI3K/Akt/MDM2 pathway, which promotes p53 degradation via the ubiquitin proteasome pathway and thus increases the resistance of human hepatocellular cancer cells to 5-FU. Furthermore, we found that NgBR expression is associated with a poor prognosis of human hepatocellular carcinoma (HCC) patients. These results suggest that targeting NgBR in combination with chemotherapeutic drugs, such as 5-FU, could improve the efficacy of current anticancer treatments.

Although trastuzumab is an effective treatment in early stage HER2(+) breast cancer the majority of advanced HER2(+) breast cancers develop trastuzumab resistance, especially in the 40% of breast cancers with loss of PTEN. However, HER2(+) breast cancer patients continue to receive trastuzumab regardless PTEN status and the consequence of therapy in these patients is unknown. We demonstrate that continued use of trastuzumab in HER2(+) cells with loss of PTEN induces the epithelial-mesenchymal transition (EMT) and transform HER2(+) to a triple negative breast cancer. These transformed cells exhibited mesenchymal morphology and gene expression markers, while parent HER2(+) cells showed epithelial morphology and markers. The transformed cells exhibited loss of dependence on ERBB family signaling (such as HER2, HER3, HER4, BTC, HRG, EGF) and reduced estrogen and progesterone receptors. Continued use of trastuzumab in HER2(+) PTEN(-) cells increased the frequency of cancer stem cells (CSCs) and metastasis potential. Strikingly, parental HER2(+) cells and transformed resistant cells respond to treatment differently. Transformed resistant cells were sensitive to chemical probe (sulforaphane) through inhibition of IL-6/STAT3/NF-κB positive feedback loop whereas parental HER2(+) cells did not respond. This data suggests that trastuzumab resistance in HER2(+) PTEN- breast cancer induces EMT and subtype switching, which requires unique treatment options.

The extravasation of tumor cells is a key event in tumor metastasis. However, the mechanism underlying tumor cell extravasation remains unknown, mainly hindered by obstacles from the lack of complexity of biological tissues in conventional cell culture, and the costliness and ethical issues of in vivo experiments. Thus, a cheap, time and labor saving, and most of all, vascular microenvironment-mimicking research model is desirable. Herein, we report a microfluidic chip-based tumor extravasation research model which is capable of simultaneously simulating both mechanical and biochemical microenvironments of human vascular systems and analyzing their synergistic effects on the tumor extravasation. Under different mechanical conditions of the vascular system, the tumor cells (HeLa cells) had the highest viability and adhesion activity in the microenvironment of the capillary. The integrity of endothelial cells (ECs) monolayer was destroyed by tumor necrosis factor-α (TNF-α) in a hemodynamic background, which facilitated the tumor cell adhesion, this situation was recovered by the administration of platinum nanoparticles (Pt-NPs). This model bridges the gap between cell culture and animal experiments and is a promising platform for studying tumor behaviors in the vascular system.

Body size, one of the most important quantitative traits under evolutionary scrutiny, varies considerably among species and among populations within species. Revealing the genetic basis underlying this variation is very important, particularly in humans where there is a close relationship with diseases and in domestic animals as the selective patterns are associated with improvements in production traits. The Debao pony is a horse breed with small body size that is unique to China; however, it is unknown whether the size-related candidate genes identified in Western breeds also account for the small body size of the Debao pony. Here, we compared individual horses from the Debao population with other two Chinese horse populations using single nucleotide polymorphisms (SNPs) identified with the Equine SNP 65 Bead Chip. The previously reported size-related candidate gene HMGA2 showed a significant signature for selection, consistent with its role observed in human populations. More interestingly, we found a candidate gene TBX3, which had not been observed in previous studies on horse body size that displayed the highest differentiation and most significant association, and thus likely is the dominating factor for the small stature of the Debao pony. Further comparison between the Debao pony and other breeds of horses from around the world demonstrated that TBX3 was selected independently in the Debao pony, suggesting that there were multiple origins of small stature in the horse.

The von Willebrand factor (vWF) is a plasma glycoprotein that plays an essential role in hemostasis by supporting platelet adhesion and thrombus formation in response to vascular injury. Plasma levels of vWF are an independent risk factor for patients with acute myocardial infarction (AMI); however, clinical data have demonstrated a marked variation of vWF levels in patients with AMI, the reason for which has not yet been identified. In the present study, a rat model of ST-segment elevation AMI was established, and cardiac and peripheral blood was collected for a time-course examination of the plasma levels of vWF and tumor necrosis factor-α (TNF-α). The level of vWF in the blood plasma increased, peaked at 1 h and decreased to normal levels by day 7 following AMI, while the level of TNF-α peaked at 24 h and remained elevated until day 7. The effects of TNF-α on vWF secretion and expression were examined in cultured human umbilical vascular endothelial cells (HUVECs). TNF-α treatment increased vWF secretion from the HUVECs but inhibited the mRNA and protein expression of vWF in the HUVECs. These results indicate that vWF secretion from endothelial cells is transiently elevated following AMI, and then decreases as the expression of vWF is inhibited by TNF-α. The present study increases the understanding of the pathophysiology of vWF and indicates that the determination of vWF levels may be useful in the clinical evaluation of AMI.

Clinical observations suggest that incidence of cough in Chinese taking angiotensin converting enzyme inhibitors is much higher than other racial groups. Cough is the most common adverse reaction of enalapril. We investigate whether SLCO1B1 genetic polymorphisms, previously reported to be important determinants of inter-individual variability in enalapril pharmacokinetics, are associated with the enalapril-induced cough. A cohort of 450 patients with essential hypertension taking 10 mg enalapril maleate were genotyped for the functional SLCO1B1 variants, 388A > G (Asn130Asp, rs2306283) and 521T > C (Val174Ala, rs4149056). The primary endpoint was cough, which was recorded when participants were bothered by cough and respiratory symptoms during enalapril treatment without an identifiable cause. SLCO1B1 521C allele conferred a 2-fold relative risk of enalapril-induced cough (95% confidence interval [CI] = 1.34-3.04, P = 6.2 × 10(-4)), and haplotype analysis suggested the relative risk of cough was 6.94-fold (95% CI = 1.30-37.07, P = 0.020) in SLCO1B1*15/*15 carriers. Furthermore, there was strong evidence for a gene-dose effect (percent with cough in those with 0, 1, or 2 copy of the 521C allele: 28.2%, 42.5%, and 71.4%, trend P = 6.6 × 10(-4)). Our study highlights, for the first time, SLCO1B1 variants are strongly associated with an increased risk of enalapril-induced cough. The findings will be useful to provide pharmacogenetic markers for enalapril treatment.

To examine if the hospitalisation trends of liver cirrhosis are changing with the changes of risk factors of the disease in China.

Substantial evidence has shown that most exogenous substances are metabolized by multiple cytochrome P450 (P450) enzymes instead of by merely one P450 isoform. Thus, multi-P450 inhibition leads to greater drug-drug interaction risk than specific P450 inhibition. Herein, we innovatively established an artificial neural network cascade (NNC) model composed of 23 cascaded networks in a ladder-like framework to identify potential multi-P450 inhibitors among natural compounds by integrating 12 molecular descriptors into a P450 inhibition score (PIS). Experimental data reporting in vitro inhibition of five P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) were obtained for 8,148 compounds from the Cytochrome P450 Inhibitors Database (CPID). The results indicate significant positive correlation between the PIS values and the number of inhibited P450 isoforms (Spearman's ρ = 0.684, p < 0.0001). Thus, a higher PIS indicates a greater possibility for a chemical to inhibit the enzyme activity of at least three P450 isoforms. Ten-fold cross-validation of the NNC model suggested an accuracy of 78.7% for identifying whether a compound is a multi-P450 inhibitor or not. Using our NNC model, 22.2% of the approximately 160,000 natural compounds in TCM Database@Taiwan were identified as potential multi-P450 inhibitors. Furthermore, chemical similarity calculations suggested that the prevailing parent structures of natural multi-P450 inhibitors were alkaloids. Our findings show that dissection of chemical structure contributes to confident identification of natural multi-P450 inhibitors and provides a feasible method for virtually evaluating multi-P450 inhibition risk for a known structure.

Apolipoprotein E ɛ4 allele is a common susceptibility gene for late-onset Alzheimer's disease. Brain vascular and metabolic deficits can occur in cognitively normal apolipoprotein E ɛ4 carriers decades before the onset of Alzheimer's disease. The goal of this study was to determine whether early intervention using rapamycin could restore neurovascular and neurometabolic functions, and thus impede pathological progression of Alzheimer's disease-like symptoms in pre-symptomatic Apolipoprotein E ɛ4 transgenic mice. Using in vivo, multimodal neuroimaging, we found that apolipoprotein E ɛ4 mice treated with rapamycin had restored cerebral blood flow, blood-brain barrier integrity and glucose metabolism, compared to age- and gender-matched wild-type controls. The preserved vasculature and metabolism were associated with amelioration of incipient learning deficits. We also found that rapamycin restored the levels of the proinflammatory cyclophilin A in vasculature, which may contribute to the preservation of cerebrovascular function in the apolipoprotein E ɛ4 transgenics. Our results show that rapamycin improves functional outcomes in this mouse model and may have potential as an effective intervention to block progression of vascular, metabolic and early cognitive deficits in human Apolipoprotein E ɛ4 carriers. As rapamycin is FDA-approved and neuroimaging is readily used in humans, the results of the present study may provide the basis for future Alzheimer's disease intervention studies in human subjects.

N-acetyltransferase 2 (NAT2) is involved in both carcinogen detoxification through hepatic N-acetylation and carcinogen activation through local O-acetylation. NAT2 slow acetylation status is significantly associated with increased bladder cancer risk among European populations, but its association in Asian populations is inconclusive.

Starting chest compressions immediately after a defibrillation shock might be harmful, if the victim already had a return of spontaneous circulation (ROSC) and yet was still being subjected to external compressions at the same time. The objective of this study was to study the influence of chest compressions on circulation during the peri-cardiac arrest period.

Mesenchymal stem cells (MSCs) are bone marrow stem cells which play an important role in tissue repair. The treatment with MSCs will be likely to aggravate the degree of fibrosis. The Wnt/β-catenin signaling pathway is involved in developmental and physiological processes, such as fibrosis. Dickkopfs (DKKs) are considered as an antagonist to block Wnt/β-catenin signaling pathway by binding the receptor of receptor-related protein (LRP5/6). DKK1 was chosen in attempt to inhibit fibrosis of MSCs by lowering activity of Wnt/β-catenin signaling pathway.

Aberrant c-Met has been implicated in the development of many cancers. The objective of this study was to identify an unfavorable prognostic marker that might guide decisions regarding clinical treatment strategies for high-grade gliomas. C-Met expression was measured using immunohistochemistry in 783 gliomas, and we further analyzed c-Met mRNA levels using the Agilent Whole Genome mRNA Microarray in 286 frozen samples. In vitro, we performed cell migration and invasion assays. Cell sensitivity to temozolomide (TMZ) chemotherapy was determined using MTT assays. Both mRNA and protein levels of c-Met were significantly associated with tumor grade progression and inversely correlated with overall and progression-free survival in high-grade gliomas (all P < 0.0001). These findings were nearly consistent at the mRNA level across 3 independent cohorts. Multivariable analysis indicated that c-Met was an independent prognostic marker after adjusting for age, preoperative Karnofsky Performance Status (KPS) score, the extent of resection, radiotherapy, TMZ chemotherapy, and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status. Further analysis in vitro revealed that downregulating the expression of c-Met dramatically inhibited cell migration and invasion capacities, enhanced sensitivity to TMZ chemotherapy in H4 and U87 glioma cells. Our results suggest that c-Met may serve as a potential predictive maker for clinical decision making.

Although crizotinib has demonstrated promising efficacy and acceptable toxicity in patients with advanced non-small cell lung cancer (NSCLC), the available evidence in Chinese populations is currently limited. This study compared the progression-free survival (PFS) of Chinese patients with anaplastic lymphoma kinase (ALK)-positive, advanced lung adenocarcinoma who received first-line crizotinib therapy with that of patients who received first-line standard chemotherapy, and also the PFS benefit of first-line versus second-line crizotinib treatment. Data on 80 patients with ALK-positive, advanced lung adenocarcinoma who received crizotinib or standard chemotherapy as first-line treatments between June 2013 and December 2014 were retrospectively collected; 26 of the patients received crizotinib as second-line therapy after progressive disease (PD) occurred on first-line chemotherapy. Tumor responses were assessed using Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. The median PFS was 13.3 months (95% CI: 6.5-20.0 months) in patients who received first-line crizotinib as compared with 5.4 months (95% CI: 4.4-6.5 months) in patients who received first-line standard chemotherapy (adjusted hazard ratio for progression or death with crizotinib, 0.20; 95% CI: 0.11-0.36; P < 0.001). In patients who received second-line crizotinib therapy, the median PFS was 9.9 months (95% CI: 6.4-13.4 months). The difference between first-line and second-line crizotinib treatment was not statistically significant (adjusted hazard ratio for progression, 0.56; 95% CI: 0.29-1.11; P = 0.092). Thus, there was a significant PFS benefit of first-line crizotinib versus first-line standard chemotherapy in Chinese patients with ALK-positive lung adenocarcinoma. Additionally, crizotinib showed promising efficacy in patients who received it as second-line therapy after PD had occurred on first-line chemotherapy.

Trimetazidine, as an anti-ischemic and antioxidant agent, has been demonstrated to have many cardioprotective effects. However, whether early administration of trimetazidine has an effect on diabetic cardiomyopathy and the mechanisms underlying the effect have not yet been elucidated.