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Okara, a renewable biomass resource, is a promising fermentative raw material for the bio-production of value-added chemicals due to its abundance and low-costs. we developed a process for the enzymatic hydrolysis of okara, and then engineered Bacillus subtilis to utilize mixed sugars to produce acetoin in okara hydrolysis without the addition of a supplemental nitrogen source. Okara was initially hydrolyzed with cellulase, β-glucosidase, and pectinase to obtain okara hydrolysate containing mixed sugars (32.78 ± 0.23 g/L glucose, 1.43 ± 0.064 g/L arabinose, 7.74 ± 0.11 g/L galactose) and amino acids. In this study, Bacillus subtilis 168 was used as the acetoin-producing strain, and the key genes bdhA and acoA of the acetoin catabolism pathway were knocked out to improve the fermentation yield of acetoin. In order to utilize the galactose in the hydrolysate, the recombinant strain BS03 (Bacillus subtilis168∆bdhA∆acoA) was used to overexpress the arabinose transporter-encoding gene (araE) drive heterologous expression of the Leloir pathway gene (galKTE). The corn dry powder concentration was optimized to 29 g/L in the reducing sugar okara hydrolysate. The results show that the recombinant bacterium BS03 could still synthesize 11.79 g/L acetoin without using corn dry powder as a nitrogen source. Finally, using okara enzymatic hydrolysate as the carbon and nitrogen source, 11.11 g/L and 29.7 g/L acetoin were obtained by batch fermentation and fed-batch fermentation, respectively, which was further converted to 5.33 g/L and 13.37 g/L tetramethylpyrazine (TTMP) by reaction with an ammonium salt. [ABSTRACT FROM AUTHOR]

Methyl farnesoate (MF), a crucial sesquiterpenoid hormone, plays a pivotal role in the reproduction of female crustaceans, particularly in the vitellogenesis process. Despite extensive research on its functions, the molecular mechanisms that regulate MF levels during the vitellogenic phase remain largely elusive. This study investigates the roles of microRNAs (miRNAs), significant post-transcriptional regulators of gene expression, in controlling MF levels in the swimming crab Portunus trituberculatus. Through bioinformatic analysis, four miRNAs were identified as potential regulators targeting two genes encoding Carboxylesterases (CXEs), which are key enzymes in MF degradation. Dual luciferase reporter assays revealed that let-7b and miR-141 suppress CXE1 and CXE2 expression by directly binding to their 3′ UTRs. In vivo overexpression of let-7b and miR-141 significantly diminished CXE1 and CXE2 levels, consequently elevating hemolymph MF and enhancing vitellogenin expression. Spatiotemporal expression profile analysis showed that these two miRNAs and their targets exhibited generally opposite patterns during ovarian development. These findings demonstrate that let-7b and miR-141 collaboratively modulate MF levels by targeting CXEs, thus influencing vitellogenesis in P. trituberculatus. Additionally, we found that the expression of let-7b and miR-141 were suppressed by MF, constituting a regulatory loop for the regulation of MF levels. The findings contribute novel insights into miRNA-mediated ovarian development regulation in crustaceans and offer valuable information for developing innovative reproduction manipulation techniques for P. trituberculatus. [ABSTRACT FROM AUTHOR]

Na/K-ATPases are membrane-associated enzymes responsible for the active transport of Na and K ions across cell membranes, generating chemical and electrical gradients. These enzymes' α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na/K-ATPase α-subunit cDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end methods. Analysis of the nucleotide sequence revealed that the cDNA had a full-length of 3 833 base pairs (bp), with an open reading frame of 3 120 bp, 5′ untranslated region (UTR) of 317 bp, and 3′ UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na/K-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of amino acid sequences showed that the P. trituberculatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit's transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab's Na/K-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress. [ABSTRACT FROM AUTHOR]

Astragaloside IV (AsIV) is the major effective component extracted from the Chinese herb Astragalus membranaceus, which has been widely used to treat cardiovascular disease. Recent studies have shown that AsIV can potentially protect the arteries from atherosclerosis; however the mechanisms underneath are unknown. The aim of this study was to investigate the effects of AsIV on blood lipids, CD40-CD40L signal system, and SDF-1/CXCR4 biological axis in high-fat diet apoE−/− mice and reveal the molecular mechanisms of AsIV against atherosclerosis. Here, we showed that AsIV alleviated the extent of atherosclerosis in aorta of apoE−/− mice. And AsIV can significantly downregulate PAC-1, CD40L, and CXCR4 expression on platelet surface in blood of high-fat diet apoE−/− mice. AsIV also can significantly downregulate mRNA and protein level of SDF-1 and CXCR4 in thoracic aorta. Consistent with aorta CXCR4 expression, CXCR4 in bone marrow-derived endothelial progenitor cell (EPC) was also reduced. Meanwhile biochemical analysis showed that AsIV could downregulate TG, TC, and LDL-C levels and upregulate HDL-C level in blood of high-fat diet apoE−/− mice. We concluded that the protective effects of AsIV in atherosclerosis injury may be related to regulating blood lipids, CD40-CD40L system, and SDF-1/CXCR4 biological axis. SDF-1/CXCR4 biological axis is probably one of the main targets of intervening atherosclerosis. [ABSTRACT FROM AUTHOR]

The trace element zinc is often used in the diet of weaned piglets, as high doses have resulted in positive effects on intestinal health. However, the majority of previous studies evaluated zinc supplementations for a short period only and focused on the small intestine. The hypothesis of the present study was that low, medium and high levels of dietary zinc (57, 164 and 2,425 mg Zn/kg from zinc oxide) would affect colonic morphology and innate host defense mechanisms across 4 weeks post-weaning. Histological examinations were conducted regarding the colonic morphology and neutral, acidic, sialylated and sulphated mucins. The mRNA expression levels of mucin (MUC) 1, 2, 13, 20, toll-like receptor (TLR) 2, 4, interleukin (IL)-1β, 8, 10, interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β) were also measured. The colonic crypt area increased in an age-depending manner, and the greatest area was found with medium concentration of dietary zinc. With the high concentration of dietary zinc, the number of goblet cells containing mixed neutral-acidic mucins and total mucins increased. Sialomucin containing goblet cells increased age-dependently. The expression of MUC2 increased with age and reached the highest level at 47 days of age. The expression levels of TLR2 and 4 decreased with age. The mRNA expression of TLR4 and the pro-inflammatory cytokine IL-8 were down-regulated with high dietary zinc treatment, while piglets fed with medium dietary zinc had the highest expression. It is concluded that dietary zinc level had a clear impact on colonic morphology, mucin profiles and immunological traits in piglets after weaning. Those changes might support local defense mechanisms and affect colonic physiology and contribute to the reported reduction of post-weaning diarrhea. [ABSTRACT FROM AUTHOR]

Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy ( TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC-SIGN, a C-type lectin, in membrane microdomains. DC-SIGN or its mutants were labeled with primary monoclonal antibodies ( mAbs) in either dendritic cells ( DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC-SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1 µm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3 T3 cells contains only 4-8 molecules of DC-SIGN, consistent with our preliminary super-resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (∼50 nm) pathogen, dengue virus, leading to infection of host cells. [ABSTRACT FROM AUTHOR]

In this study, a single tail vein injection of streptozotocin (STZ)-induced rat model was employed to study the effects of 1,25(OH)D supplementation, the active form of vitamin D, on diabetes-induced aortic injury. Aortas from different groups were assessed for histopathology, toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MyD88), and nuclear factor-kappa B (NF-κB) p65 expression by hematoxylin and eosin staining, immunohistochemistry staining, reverse transcription polymerase chain reaction, and Western blot analysis. High-dose 1,25(OH)D (0.3 μg/kg/day) significantly prevented diabetes-induced aortic pathological changes and collagen deposition and decreased the expression of TLR4, MyD88, and NF-κB at both mRNA and protein levels in the aorta of STZ-induced diabetic rats ( P < 0.01). In vitro studies in A7r5 cells (a rat embryonic thoracic aortic smooth muscle cell line) showed that high-dose glucose (25 mmol/L) enhanced TLR4 expression at both mRNA and protein levels by fourfold and twofold, respectively, at 24 h, which were significantly diminished by 1,25(OH)D (1 × 10 mol/L) by 50 and 36 %, respectively. Similar effects of high-dose 1,25(OH)D on the expression of MyD88 were observed. Our results indicate that vitamin D has protective effects on diabetes-induced aortic injury and attenuates the expressions of TLR4, MyD88, and NF-κB in diabetic rats. [ABSTRACT FROM AUTHOR]

Dominant-negative (DN) Ikaros isoforms, having important roles in pathogenesis of leukemia, are mainly studied in pediatric patients, but little is known about Chinese adult patients. We examined 339 Chinese adult patients with leukemia and demonstrated the different findings between our results and those in several previous studies showing that DN isoforms overexpressed in Philadelphia chromosome positive acute lymphoblastic leukemia (PhALL) and lymphoid/mixed blast crisis of chronic myelogenous leukemia. We confirmed that deletion of IKZF1 gene exons 4-7 is responsible for the generation of Ikaros 6 (Ik6). Moreover, we observed that expression of DN isoforms was dynamically consistent with BCR-ABL1 transcript levels, associated with higher incidence of relapse within 3 months or poor response to induction chemotherapy in PhALL, correlated with high white blood cell, blast cells, CD34 positive cells, and delayed achieving complete hematological remission in ALL patients. In conclusion, this study provides a rationale for the integration of aberrant Ikaros isoforms, notably Ik6 and Ik10, in the evaluation of adult ALL, particularly in PhALL patients. [ABSTRACT FROM AUTHOR]

Objective: C-Jun plays a critical role in ultraviolet A (UVA) irradiation-induced photoaging. The exact mechanisms by which UVA irradiation up-regulates c-Jun expression in human dermal fibroblasts (HDFs) are still not completely understood. We undertook this study to investigate whether microRNA-155 (miR-155) directly regulates the expression of c-Jun in HDFs in vitro. Methods: Expression of c-Jun mRNA and protein and miR-155 in UVA-irradiated HDFs were detected using quantitative real-time RT-PCR and Western blotting. Luciferase reporter assays were performed to examine whether a miR-155 binding site in the 3′-untranslated region (3′-UTR) of the c-Jun gene is responsible for miR-155-mediated c-Jun regulation in HEK293A cells, and expression of c-Jun mRNA and protein in UVA non-exposed and exposed HDFs trasfected with a miR-155 mimic or a miR-155 inhibitor was detected by quantitative real-time RT-PCR and Western blotting. Results: Expression of miR-155 was markedly reduced and that of c-Jun mRNA and protein was significantly up-regulated in UVA-irradiated HDFs. Luciferase reporter assays indicated that c-Jun is a direct target of miR-155 in HEK293A cells. In both UVA non-exposed and exposed HDFs, miR-155 mimic decreased c-Jun protein levels, while miR-155 inhibitor increased c-Jun protein levels, but both had no effect on c-Jun mRNA expression, which suggest that miR-155-induced c-Jun inhibition occurs at the post-transcriptional level. Conclusions: Our results demonstrate that miR-155 directly controls c-Jun expression in HDFs at the post-transcriptional level and might function as a protective miRNA in HDFs. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

MicroRNA 520c and 373 (miR-520c and miR-373) have been characterized as oncogenes and play critical roles in cancer cell metastasis. However, the relationship between these two microRNAs and matrix metalloproteinases (MMPs), which are important in cancer cell metastasis, remains unknown. Here, we report new evidence in which miR-520c and miR-373 effects in human fibrosarcoma HT1080 cells are associated with MMP9 activity, and this upregulation of MMP9 is not only at the activity and protein levels, but also at that of its mRNA. Our experimental data demonstrate that these effects occur not by direct binding to the MMP9 promoter, but by miR-520c and miR-373 directly targeting the 3′-untranslational region (UTR) of mRNAs of mTOR and SIRT1 (negative regulators of expression of MMP9 via inactivating the Ras/Raf/MEK/Erk signaling pathway and transcription factor NF-κB activity); and thus suppressing translation levels of SIRT1 and mTOR. Moreover, inhibition of key kinases of the Ras/Raf/MEK/Erk signaling pathway and Western blots for selected proteins further identified miR-520c and miR-373 as activating this signaling pathway and NF-κB. In conclusion, miR-520c and miR-373 increased the expression of MMP9 by directly targeting the 3′-UTRs of mRNAs of mTOR and SIRT1 and suppressing their translation; resulting in activation of the Ras/Raf/MEK/Erk signaling pathway and NF-κB; and, finally, increasing the mRNA, protein, and activity of MMP9 and enhancing cell migration and cell growth in 3D type I collagen gels. J. Cell. Physiol. 227: 867-876, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the changes in Tenon''s capsule fibroblast (HTF) proliferation and their expressions of transforming growth factor-ββ (TGF-ββ) and matrix metalloproteinases (MMPs) in patients with long-term use of topical antiglaucoma medications, and to evaluate the potential effect of these medications on the outcome of glaucoma filtering surgery for primary open-angle glaucoma (POAG). Methods: HTFs isolated from the conjunctiva of ten POAG patients and ten control patients with cataract or retinal detachment were tested for their in vitro proliferation capacity. RT-PCR (reverse-transcription polymerase chain reaction) was performed to detect the expressions of TGF-ββ and MMP mRNAs in the HTFs, and the expression of TGF-ββ2 protein was detected immunohistochemically. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of TGF-ββ1 in the supernatant of the HTFs treated with the topical antiglaucoma agents. Results: The HTFs from the POAG patients showed significantly higher proliferative activity than those from the control patients ( P == 0.0013). The HTFs in the POAG group showed significantly higher expressions of TGF-ββ2 and TGF-ββ3 mRNA and lower MMP-9 mRNA than the control HTFs. MMP-3 mRNA was not detected in the HTFs in POAG groups, but lowly expressed in the control HTFs. The HTFs in the POAG group had a significantly higher expression of TGF-ββ2 protein; TGF-ββ1 protein was not detected in the control HTFs, but was highly expressed in the POAG group. Treatment of the HTFs with latanoprost resulted in a 4-fold increase in TGF-ββ1 expression in both groups, which was comparable for the expression after the exposure. Conclusions: Long-term use of some topical antiglaucoma medications might be associated with enhanced HTF proliferation and abnormal expressions of TGF-ββ and MMPs, which might have promoted scarring of the filtering blebs following glaucoma filtering surgery. [ABSTRACT FROM AUTHOR]

Abstract: Fenvalerate is a potential endocrine disruptor. Several studies have demonstrated that fenvalerate disrupts testosterone (T) synthesis in testes. T and estradiol (E2) are de novo synthesized in the developing brain. Thus, the aim of the present study was to investigate the effects of pubertal fenvalerate exposure on the synthesis of T and E2 and the expression of androgen receptor (AR) and estrogen receptors (ERs) in cerebral cortex. CD-1 mice were orally administered daily with either vehicle or fenvalerate (7.5 or 30mg/kg) from postnatal day (PND) 28 to PND56. The level of T and E2 in cerebral cortex was significantly decreased in males exposed to fenvalerate. In agreement with the decrease in T and E2 syntheses, the expression of 17β-HSD, a key enzyme for T synthesis, was significantly reduced in cerebral cortex of fenvalerate-exposed males. Conversely, in females, the expression of 17β-HSD in cerebral cortex was mildly up-regulated by fenvalerate and the level of T and E2 was mildly increased. Pubertal fenvalerate exposure had no effect on the expression of StAR, P45017α and P450scc, the key enzymes for T synthesis, and P450 aromatase, the key enzyme for E2 synthesis, in cerebral cortex of males and females. Interestingly, the expression of AR in cerebral cortex was up-regulated in male and female mice exposed to fenvalerate, whereas pubertal fenvalerate exposure did not affect the level of ERα and ERβ in cerebral cortex. Taken together, these results suggest that pubertal fenvalerate exposure disrupts T and E2 synthesis and the expression of AR in cerebral cortex. These changes of steroid status in the developing brain might be deleterious for neurobehavioral development. [Copyright &y& Elsevier]

Abstract: para-Nitrophenol (PNP) degradation cluster was cloned from a newly isolated Arthrobacter sp. strain NyZ415 by genome walking extending from a conserved region of gene encoding the oxygenase component of PNP monooxygenase. Sequence analysis indicated it contained 12 open reading frames including genes npdA1 and npdA2 which encoded the reductase and oxygenase components of PNP monooxygenase, respectively. Both NpdA1 and NpdA2 were expressed and purified to homogeneity. Hydroquinone and hydroxyquinol were captured when PNP was transformed by purified NpdA1A2 and para-benzoquinone was also captured for the first time as one of the products of PNP transformation by monooxygenase involved in the hydroxyquinol pathway. Sequence analysis showed four conserved residues (Arg100, Gln158, Arg161 and Thr193) related to the binding between the oxygenase component and substrate. Substitutions of any of above four residues to Ala have resulted in the complete loss of its activity on PNP transformation, indicating these four residues play important roles in the catalytic activity of PNP monooxygenase. [Copyright &y& Elsevier]

Summary: Human interferon alpha2b (IFN-α2b) is a pleiotropic cytokine used for the treatment of various cancers. Antibacterial peptide CM4 is a small peptide that can strongly inhibit many kinds of bacteria, fungi, and tumor cells, but it does no harm to normal cells. Here, we describe a protein expression system for the production of IFN-α2b/CM4 fusion protein in insoluble form in Escherichia coli, coupled to an efficient dialysis refolding and histidine-tag purification protocol. The expressed IFN-α2b/CM4 fusion protein not only displays significantly improved antitumor activity, but also retains the antibacterial–antifungal activity of CM4. [Copyright &y& Elsevier]

Calcineurin (CaN) is a heterodimer composed of a catalytic subunit A (CaNA) and a regulatory subunit B (CaNB). We report here an active truncated mutation of the rat CaNAδ that contains only the catalytic domain (residues 1-347, also known as a/CaNA). The p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were higher than that of CaNA. Both p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were unaffected by CaM and the B-subunit; the B-subunit and CaM have relatively little effect on p-nitrophenyl phosphatase activity and a crucial effect on protein phosphatase activity of CaNA. Mn&sup2+; and Ni&sup2+; ions effeciently activated CaNA. The K[subm] of a/CaNA was about 16 mM, and the k[subcat] of a/CaNA was 10.03 s[sup-1] using pNPP as substrate. With RII peptide as a substrate, the K[subm] of a/CaNA was about 21 µM and the k[subcat] of a/CaNA was 0.51 s[sup-1]. The optimum reaction temperature was about 45°C, and the optimum reaction pH was about 7.2. Our results indicate that a/CaNA is the catalytic core of CaNA, and CaN and the B-sub-unit binding domain itself might play roles in the negative regulation of the phosphatase activity of CaN. The results provide the basis for future studies on the catalytic domain of CaN. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the possible relationship between changes in neuronal and endothelial nitric oxide synthase (nNOS and eNOS) and arginase expression in the vestibular nucleus complex and the hippocampus (CA1, CA2/3 and the dentate gyrus (DG) at 10 h or 2 weeks following a unilateral vestibular deafferentation (UVD) in rats. There were no significant differences in nNOS or arginase II expression in the ipsilateral or contralateral VNC at either 10 h or 2 weeks post-UVD. For eNOS, there was only a significant decrease in expression in the ipsilateral VNC at 2 weeks post-UVD (P<0.01). In the hippocampus, the only significant difference in nNOS expression was a decrease in the ipsilateral DG at 2 weeks post-UVD (P<0.05). There was a significant decrease in eNOS expression in the contralateral CA2/3 region at 10 h post-UVD (P<0.01). The only other significant change in eNOS was an increase in the contralateral DG at 10 h post-UVD (P<0.01). Although arginase II was expressed in all regions of the hippocampus, there were no significant differences in arginase II expression at any time point following UVD. These results suggest that the changes in NOS expression that occur in the VNC and hippocampus following UVD are not correlated with one another or with changes in arginase II. [Copyright &y& Elsevier]

Background: ETS transcription factors are known to act as either positive or negative regulators of the expression of genes involved in various biological processes. It was reported that ETS variant transcription factor 5 (ETV5), a key member of the ETS family, mainly plays a role as an potential oncogene in various malignant tumors. However, the role and mechanism of ETV5 in high-grade serous ovarian cancer (HGSOC) have not been elucidated. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to detect ETV5 messenger ribonucleic acid (mRNA) expression in 87 HGSOC tissues and 35 normal fallopian tube tissues. Western blotting and qRT-PCR were used to detect the protein and mRNA expression of ETV5 in six ovarian cancer (OC) and human embryonic cell lines. Knockdown or overexpression of ETV5 in HGSOC cell lines, Cell Counting Kit-8, colony formation, and transwell assays were used to detect HGSOC cell proliferation, invasion, and migration capabilities. The chi-square test was used to analyze the clinicopathological characteristics of HGSOC patients. Survival analysis was performed using the Kaplan-Meier method, and the log-rank test was used to analyze the correlation between ETV5 expression and HGSOC patient prognosis. Univariate and multivariate analyses using the Cox regression model were conducted to determine the independent significance of relevant clinical covariates. Results: Bioinformatic analysis demonstrated that ETV5 expression was significantly upregulated in OC (p < 0.05). qRT-PCR showed that ETV5 was significantly overexpressed in HGSOC tissues than in fallopian tube tissues (p < 0.05). qRT-PCR and western blotting assays demonstrated that ETV5 was relatively highly expressed in OC cell lines. ETV5 overexpression was positively associated with poor survival in HGSOC patients, therefore making it a high-risk factor for HGSOC progression. Furthermore, ETV5 promoted the proliferation, migration, and invasion capabilities of HGSOC cells. Conclusion: ETV5 has a carcinogenic effect in HGSOC and can be used as a clinically effective biomarker to determine the prognosis of HGSOC patients. [ABSTRACT FROM AUTHOR]

Background: Clinical variables may correlate with lack of response to treatment (primary resistance) or clinical benefit in patients with clear cell renal cell carcinoma (ccRCC) treated with anti-programmed death 1/ligand one antibodies.Methods: In this multi-institutional collaboration, clinical characteristics of patients with primary resistance (defined as progression on initial computed tomography scan) were compared to patients with clinical benefit using Two sample t-test and Chi-square test (or Fisher's Exact test). The Kaplan-Meier method was used to estimate the distribution of progression-free survival (PFS) and overall survival (OS) in all patients and the subsets of patients with clinical benefit or primary resistance. Cox's regression model was used to evaluate the correlation between survival endpoints and variables of interest. To explore clinical factors in a larger, independent patient sample, The Cancer Genome Atlas (TCGA) was analyzed. RNAseq gene expression data as well as demographic and clinical information were downloaded for primary tumors of 517 patients included within TCGA-ccRCC.Results: Of 90 patients, 38 (42.2%) had primary resistance and 52 (57.8%) had clinical benefit. Compared with the cohort of patients with initial benefit, primary resistance was more likely to occur in patients with worse ECOG performance status (p = 0.03), earlier stage at diagnosis (p = 0.04), had no prior nephrectomy (p = 0.04) and no immune-related adverse events (irAE) (p = 0.02). In patients with primary resistance, improved OS was significantly correlated with lower International Metastatic RCC Database Consortium risk score (p = 0.02) and lower neutrophil:lymphocyte ratio (p = 0.04). In patients with clinical benefit, improved PFS was significantly associated with increased BMI (p = 0.007) and irAE occurrence (p = 0.02) while improved OS was significantly correlated with overweight BMI (BMI 25-30; p = 0.03) and no brain metastasis (p = 0.005). The cohort TCGA-ccRCC was examined for the correlations between gene expression patterns, clinical factors, and survival outcomes observing associations of T-cell inflammation and angiogenesis signatures with histologic grade, pathologic stage and OS.Conclusions: Clinical characteristics including performance status, BMI and occurrence of an irAE associate with outcomes in patients with ccRCC treated with immunotherapy. The inverse association of angiogenesis gene signature with ccRCC histologic grade highlight opportunities for adjuvant combination VEGFR2 tyrosine kinase inhibitor and immune-checkpoint inhibition. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to investigate the molecular characteristics of cervical squamous cell carcinoma (CESC) by analyzing ferroptosis-related gene (FRG) expression data to predict prognosis. Methods: Gene expression and clinicopathological data of patients with CESC were collected from the Cancer Genome Atlas and the Genotype-Tissue Expression databases. Using Cox regression analysis, we identified 21 FRGs associated with prognosis. Cluster analysis categorized patients into subgroups based on these genes and compared their clinicopathological, biological, and immune infiltration features. FRG methylation levels were examined, and a risk model based on such FRG methylation levels was constructed using LASSO and Cox regression analyses. The model's predictive capacity was validated, and the relationships between the risk score and immune infiltration, tumor microenvironment, and drug sensitivity were explored. FRG methylation in CESC tissues was validated by immunohistochemistry. Results: We identified 21 FRGs associated with CESC prognosis. Patients were stratified into two subtypes based on these genes, they showed differences in prognosis, immune cell types, and immune checkpoint expression. A three-gene risk score (including AQP3, MGST1, and TFRC) was generated, and the low-risk group showed better overall survival. The high-risk and low-risk groups differed in terms of immune infiltration, gene mutations, and drug sensitivity. Experimental validation confirmed the upregulation of AQP3 and TFRC, whereas MGST1 expression was not significantly altered in CESC tissues compared with that in normal cervical tissues. Conclusion: This study highlights the potential role of FRG methylation in predicting CESC prognosis and provides a personalized assessment of immune responses in patients with CESC. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the expression of the excision repair cross-complementation group 1 (ERCC1) in oral squamous cell carcinoma (OSCC) and the possible association of ERCC1 polymorphisms with susceptibility and response to chemotherapy of OSCC in a Chinese Han population. The expression of ERCC1 was determined by real-time PCR in eight patients. Four single-nucleotide polymorphisms (SNPs) rs11615, rs3212948, rs3212961, and rs735482 of ERCC1 were genotyped in 113 OSCC patients and 184 healthy controls using a PCR restriction matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) assay. We found that a higher gene expression of ERCC1 was observed in tumor tissue as compared to pericarcinomatous tissue in OSCC patients. All genotypic and allelic frequencies of the tested ERCC1 polymorphisms were in Hardy-Weinberg equilibrium. The genotypic and allelic frequencies of rs11615, rs3212948, rs3212961, and rs735482 of ERCC1 were not different between OSCC patients and controls. No correlation was observed between ERCC1 polymorphisms and the response to chemotherapy. Our results show that ERCC1 is increased in the tumor tissue of OSCC patients. The investigated ERCC1 gene polymorphisms (rs11615, rs3212948, rs3212961, and rs735482) are not associated with the susceptibility and response to chemotherapy of OSCC in our investigated Chinese Han population. [ABSTRACT FROM AUTHOR]

Objective: Temozolomide is extensively applied in chemotherapy for glioblastoma with unclear exact action mechanisms. This article seeks to address the potential molecular mechanisms in temozolomide therapy for glioblastoma involving LINC00470. Methods: Bioinformatics analysis was conducted to predict the potential mechanism of LINC00470 in glioblastoma, which was validated by dual‐luciferase reporter, RIP, ChIP, and RNA pull‐down assays. LINC00470 expression and the predicted downstream transcription factor early growth response 2 (EGR2) were detected in the collected brain tissues from glioblastoma patients. Following temozolomide treatment and/or gain‐ and loss‐of‐function approaches in glioblastoma cells, cell viability, invasion, migration, cycle distribution, angiogenesis, autophagy, and apoptosis were measured. In addition, the expression of mesenchymal surface marker proteins was assessed by western blot. Tumor xenograft in nude mice was conducted for in vivo validation. Results: Mechanistic analysis and bioinformatics analysis revealed that LINC00470 transcriptionally activated SRY‐related high‐mobility‐group box 4 (SOX4) through the transcription factor EGR2. LINC00470 and EGR2 were highly expressed in brain tissues of glioblastoma patients. LINC00470 and EGR2 mRNA expression gradually decreased with increasing concentrations of temozolomide in glioblastoma cells, and SOX4 expression was reduced in cells by temozolomide and LINC00470 knockdown. Temozolomide treatment induced cell cycle arrest, diminished cell viability, migration, invasion, and angiogenesis, and increased apoptosis and autophagy in glioblastoma, which was counteracted by overexpressing LINC00470 or SOX4 but was further promoted by LINC00470 knockdown. Temozolomide restrained glioblastoma growth and angiogenesis in vivo, while LINC00470 or SOX4 overexpression nullified but LINC00470 knockdown further facilitated these trends. Conclusion: Conclusively, temozolomide repressed glioblastoma progression by repressing the LINC00470/EGR2/SOX4 axis. [ABSTRACT FROM AUTHOR]

Objective. We aimed to investigate whether apatinib has an inhibitory effect on the invasion and metastasis of liver cancer in vitro. Methods. The anti-invasion and antimetastasis effects of apatinib in HepG2, Hep3B,Huh7 and SMMC-7721 liver cancer cell lines were tested by the wound-healing and transwell invasion assays. Real-time PCR and Western blot were used to detect the influence of apatinib on the gene expression of MMPs, TIMPs, and constituents of the NF-κB signaling pathway in Hep3B and HepG2 liver cell lines. Results. Apatinib has a significant inhibitory effect on the metastasis and invasion of liver cancer cells. The expression levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, and MMP-16 were downregulated, while the expression levels of TIMP-3 and TIMP-4 were upregulated by apatinib treatment at both the mRNA and protein levels. The phosphorylation of IκBα and NF-κB p65 was significantly reduced compared with that in the control group. Conclusions. Apatinib inhibits the invasion and metastasis of human liver cancer cells by downregulating the expression of MMP-related genes. This may be achieved by inhibiting the activation of the NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Cutaneous squamous‐cell carcinoma (cSCC) is the second most common skin cancer, with an increasing incidence in recent years. To define the molecular basis that drive cSCC development and progression, this study aimed at identifying potential novel molecular targets for the diagnosis and therapy of patients with cSCC. Two data sets with the accession number GSE45164 and GSE66359 were downloaded from Gene Expression Omnibus (GEO) database. After the identification of differentially expressed genes (DEGs) from these two data sets, respectively, between cSCC samples and controls, a combination of DEGs from these two data sets were subjected to the following analyses, including functional annotation, protein–protein interaction (PPI) network and module construction, transcription factor (TF)‐target regulation prediction, and drug–gene interaction predictive analysis. A total of 204 upregulated genes and 213 downregulated genes were found in two data sets which were used for the follow‐up analysis. Upregulated and downregulated genes were mainly involved in the functions such as cell division, mitotic nuclear division, cell cycle, and p53 signaling pathway. Interferon induced protein family members and proteasome subunit members were involved in the TF‐target regulatory network, such as PSMB8, CXCL10, and IFIT3. Eight upregulated genes (TOP2A, CXCL8, RRM2, PSMB8, PSMB9, PBK, CXCL10, and ISG15) that were hub genes in the PPI network and significant modules were identified in the predicted drug–gene interaction. In conclusion, TOP2A, CXCL8, RRM2, PSMB8, PSMB9, PBK, CXCL10, and ISG15 may be potential targets for the diagnosis and therapy of patients with cSCC. [ABSTRACT FROM AUTHOR]

Elucidating the cold tolerance mechanism of Paeonia lactiflora, which is one of the most valuable ornamental and medicinal plants in Asia, fundamentally impacts its breeding and production. The glycerol-3-phosphate acyltransferase (GPAT) gene plays a pivotal role in cold resistance in a variety of plant species. Here, we cloned the P. lactiflora GPAT gene, determined its expression pattern, and tested its role in cold resistance. We obtained the full-length P. lactiflora GPAT gene using tissue-cultured seedlings and real-time polymerase chain reaction and rapid amplification of cDNA ends analyses. We named this gene PlGPAT in P. lactiflora. Phylogenetic analysis indicates that the PlGPAT gene is closely related with the GPAT genes in core eudicots. The phylogenetic tree containing 31 angiosperm species based on GPAT protein sequences is largely consistent with the known phylogeny in flowering plants. We conducted a time-course PlGPAT expression analysis and demonstrated that PlGPAT expression is correlated with low-temperature stress. Our results suggest that the PlGPAT gene plays an important role in regulating cold resistance in P. lactiflora. [ABSTRACT FROM AUTHOR]

Excessive free fatty acids (FFAs) accumulation is a leading risk factor for the pathogenesis of insulin resistance (IR) in metabolic tissues, including the liver. Ethanolamine-phosphate phospho-lyase (ETNPPL), a newly identified metabolic enzyme, catalyzes phosphoethanolamine (PEA) to ammonia, inorganic phosphate, and acetaldehyde and is highly expressed in hepatic tissue. Whether it plays a role in regulating FFA-induced IR in hepatocytes has yet to be understood. In this study, we established an in vitro palmitic acid (PA)-induced IR model in human HepG2 cells and mouse AML12 cells with chronic treatment of PA. Next, we overexpressed ETNPPL by using lentivirus-mediated ectopic to investigate the effects of ETNPPL per se on IR without PA stimulation. We show that ETNPPL expression is significantly elevated in PA-induced IR and that silencing ETNPPL ameliorates this IR in hepatocytes. Inversely, overexpressing ETNPPL under normal conditions without PA promotes IR, reactive oxygen species generation, and ARG2 activation in both HepG2 and AML12 cells. Moreover, ETNPPL depletion markedly down-regulates ARG2 expression in hepatocytes. Besides, silencing ARG2 prevents ETNPPL-induced ROS accumulation and inhibition of autophagic flux and IR in hepatocytes. Finally, we found that phytopharmaceutical disruption of ETNPPL by quercetin ameliorates PA-induced IR in hepatocytes. Our study discloses that ETNPPL inhibiting autophagic flux mediates insulin resistance triggered by PA in hepatocytes via ARG2/ROS signaling cascade. Our findings provide novel insights into elucidating the pathogenesis of obesity-associated hepatic IR, suggesting that targeting ETNPPL might represent a potential approach for T2DM therapy. [Display omitted] • ETNPPL is aberrantly upregulated in PA-induced insulin resistance in hepatocytes. • ETNPPL impairing hepatic autophagy via the ARG2-ROS signaling axis contributes to PA-induced hepatic insulin resistance. • Quercetin disrupting ETNPPL ameliorates PA-induced IR in hepatocytes. [ABSTRACT FROM AUTHOR]

Simple Summary: Exopalaemon carinicauda is a typical representative of crustaceans. However, the mechanism of ovary early development remains obscure. The purpose of this study is to identify and verify important genes related to ovary development by transcriptome sequencing and two-color fluorescent in situ hybridization, respectively. The result would provide clues for further study of ovary development in crustaceans. Eyestalk ablation is an effective method to promote ovarian development in crustaceans. Herein, we performed transcriptome sequencing of ovary and hepatopancreas tissues after eyestalk ablation in Exopalaemon carinicauda to identify genes related to ovarian development. Our analyses led to the identification of 97,383 unigenes and 190,757 transcripts, with an average N50 length of 1757 bp. In the ovary, four pathways related to oogenesis and three related to oocyte rapid growth were enriched. In the hepatopancreas, two vitellogenesis-associated transcripts were identified. Furthermore, short time-series expression miner (STEM) and gene ontology (GO) enrichment analyses revealed five terms related to gamete generation. In addition, two-color fluorescent in situ hybridization results suggested that dmrt1 might play a vital role in oogenesis during the early stage of ovarian development. Overall, our insights should support future studies focusing on investigating oogenesis and ovarian development in E. carinicauda. [ABSTRACT FROM AUTHOR]

Cortical and subcortical structural alteration has been extensively reported in schizophrenia, including the unusual expansion of gray matter volumes (GMVs) of basal ganglia (BG), especially putamen. Previous genome-wide association studies pinpointed kinectin 1 gene (KTN1) as the most significant gene regulating the GMV of putamen. In this study, the role of KTN1 variants in risk and pathogenesis of schizophrenia was explored. A dense set of SNPs (n = 849) covering entire KTN1 was analyzed in three independent European- or African-American samples (n = 6704) and one mixed European and Asian Psychiatric Genomics Consortium sample (n = 56,418 cases vs. 78,818 controls), to identify replicable SNP-schizophrenia associations. The regulatory effects of schizophrenia-associated variants on the KTN1 mRNA expression in 16 cortical or subcortical regions in two European cohorts (n = 138 and 210, respectively), the total intracranial volume (ICV) in 46 European cohorts (n = 18,713), the GMVs of seven subcortical structures in 50 European cohorts (n = 38,258), and the surface areas (SA) and thickness (TH) of whole cortex and 34 cortical regions in 50 European cohorts (n = 33,992) and eight non-European cohorts (n = 2944) were carefully explored. We found that across entire KTN1, only 26 SNPs within the same block (r2 > 0.85) were associated with schizophrenia across ≥ 2 independent samples (7.5 × 10–5 ≤ p ≤ 0.048). The schizophrenia-risk alleles, which increased significantly risk for schizophrenia in Europeans (q < 0.05), were all minor alleles (f < 0.5), consistently increased (1) the KTN1 mRNA expression in 12 brain regions significantly (5.9 × 10–12 ≤ p ≤ 0.050; q < 0.05), (2) the ICV significantly (6.1 × 10–4 ≤ p ≤ 0.008; q < 0.05), (3) the SA of whole (9.6 × 10–3 ≤ p ≤ 0.047) and two regional cortices potentially (2.5 × 10–3 ≤ p ≤ 0.042; q > 0.05), and (4) the TH of eight regional cortices potentially (0.006 ≤ p ≤ 0.050; q > 0.05), and consistently decreased (1) the BG GMVs significantly (1.8 × 10–19 ≤ p ≤ 0.050; q < 0.05), especially putamen GMV (1.8 × 10–19 ≤ p ≤ 1.0 × 10–4; q < 0.05, (2) the SA of four regional cortices potentially (0.010 ≤ p ≤ 0.048), and (3) the TH of four regional cortices potentially (0.015 ≤ p ≤ 0.049) in Europeans. We concluded that we identified a significant, functional, and robust risk variant block covering entire KTN1 that might play a critical role in the risk and pathogenesis of schizophrenia. [ABSTRACT FROM AUTHOR]

Simple Summary: Ammonia is the most common contaminant in aquaculture systems, including intensive hatchery systems for crustaceans. Although it has been demonstrated that ammonia can result in abortive moulting and massive mortality of juvenile crustaceans, the underlying mechanisms are still unknown. The present work is aimed at examining the effect of ammonia exposure on the moulting process as well as molt signaling in the swimming crab Portunus trituberculatus, an important aquaculture species in China. We examined the survival rate and moulting rate of the juvenile crabs (C2) and analyzed the expression pattern of the genes in key components of molt signaling during a complete moulting cycle under different concentrations of ammonia. Our results revealed that ammonia has dose-dependent biphasic effects on moulting in juvenile swimming crab. Specifically, low levels of ammonia (5 mg/L) stimulated moulting, while high levels of ammonia (20 mg/L) suppressed the moulting process and caused moulting death syndrome (MDS). The gene expression analysis indicated that low levels of ammonia can reduce the expression of MIH, which encodes the key negative regulator of moulting, and trigger ecdysteroid biosynthesis and ecdysteroid signaling in the juvenile crabs. In contrast, though the high level of ammonia increased MIH expression, it still resulted in excessive ecdysteroids and over-activation of ecdysteroid signaling, which may contribute to the depressed moulting and MDS in the juvenile crabs. The novel findings of this study improve the understanding of ammonia toxicity in brachyura and provide valuable information for hatchery management of P. trituberculatus. Ammonia is a significant concern during hatchery culture in brachyuran species, and its accumulation may lead to abortive moulting and large-scale deaths of the early juveniles. To date, the underlying mechanism for ammonia-induced alteration of the moulting process is still unknown. In this study, we aimed to investigate the effects of ammonia on the moulting as well as the potential mechanisms in early juveniles of the swimming crab Portunus trituberculatus, an important aquaculture species in China. We evaluated the survival rate and moulting rate of the juvenile crabs (C2) and analyzed the expression pattern of the genes in key components of molt signaling during a complete moulting cycle under different concentrations of ammonia nitrogen (the control group: <0.1 mg/L; the LA group: 5 mg/L; and the HA group: 20 mg/L). The results showed that: (1) the survival rate in the LA and HA groups was lower than that in the control group at the end of the experiment, and moulting death syndrome (MDS) was only observed in the HA group; (2) the moulting rate was higher in the LA group and lower in the HA group compared to the control group; (3) consistent with the results of the moulting experiment, MIH showed decreased expression, and genes related to ecdysteroid synthesis, ecdysteroid receptors, and responsive effectors exhibited increased expression in the LA group compared to the control group; and (4) although MIH expression was upregulated, increased expression of the genes associated with ecdysteroid synthesis, ecdysteroid receptors and downstream effectors still observed in the HA group. Our results indicated that low levels of ammonia can promote moulting in juvenile swimming crabs by inhibiting the expression of MIH and activating moult signaling, whereas high levels of ammonia inhibit moulting and lead to MDS through impairing moult signaling. [ABSTRACT FROM AUTHOR]

Background: Nonalcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is becoming a major health-related problem. The exploration of NASH-related prognostic biomarkers and therapeutic targets is necessary. Methods: Data were downloaded from the GEO database. The "glmnet" package was used to identify differentially expressed genes (DEGs). The prognostic model was constructed by the univariate Cox and LASSO regression analyses. Validation of the expression and prognosis by immunohistochemistry (IHC) in vitro. Drug sensitivity and immune cell infiltration were analyzed by CTR-DB and ImmuCellAI. Results: We constructed a prognostic model that identified the NASH-related gene set (DLAT, IDH3B, and MAP3K4), which was validated in a real-world cohort. Next, seven prognostic transcription factors (TFs) were identified. The prognostic ceRNA network included three mRNAs, four miRNAs, and seven lncRNAs. Finally, we found that the gene set was associated with drug response which was validated in six clinical trial cohorts. Moreover, the expression level of the gene set was inversely correlated with CD8 T cell infiltration in HCC. Conclusions: We established a NASH-related prognostic model. Upstream transcriptome analysis and the ceRNA network provided clues for mechanism exploration. The mutant profile, drug sensitivity, and immune infiltration analysis further guided precise diagnosis and treatment strategies. [ABSTRACT FROM AUTHOR]

With advances in next-generation sequencing(NGS) technologies, a large number of multiple types of high-throughput genomics data are available. A great challenge in exploring cancer progression is to identify the driver genes from the variant genes by analyzing and integrating multi-types genomics data. Breast cancer is known as a heterogeneous disease. The identification of subtype-specific driver genes is critical to guide the diagnosis, assessment of prognosis and treatment of breast cancer. We developed an integrated frame based on gene expression profiles and copy number variation (CNV) data to identify breast cancer subtype-specific driver genes. In this frame, we employed statistical machine-learning method to select gene subsets and utilized an module-network analysis method to identify potential candidate driver genes. The final subtype-specific driver genes were acquired by paired-wise comparison in subtypes. To validate specificity of the driver genes, the gene expression data of these genes were applied to classify the patient samples with 10-fold cross validation and the enrichment analysis were also conducted on the identified driver genes. The experimental results show that the proposed integrative method can identify the potential driver genes and the classifier with these genes acquired better performance than with genes identified by other methods. [ABSTRACT FROM AUTHOR]

Myocardial ischemia/reperfusion (I/R) injury seriously threats the health and life of patients with ischemia heart disease. Herein, we probed the potential influence of myosin 1b (myo1b) on hypoxia/reoxygenation- (H/R-) stimulated cardiomyocyte H9c2 cell apoptosis and autophagy. After H/R stimulation, the myo1b mRNA level in H9c2 cells was tested via qRT-PCR. Myo1b overexpression plasmid (OE-myo1b) and small interfering RNA (siRNA) targeting myo1b (si-myo1b) were transfected into H9c2 cells to alter myo1b expression in H9c2 cells. Following H/R stimulation and/or OE-myo1b (or si-myo1b) transfection, H9c2 cell apoptosis, proliferation, and autophagy were detected, respectively. We found that H/R stimulation reduced the mRNA level of myo1b in H9c2 cells and resulted in H9c2 cell apoptosis, proliferation inhibition, and autophagy. Overexpression of myo1b reversed the H/R-resulted H9c2 cell apoptosis, proliferation inhibition, and autophagy. Silence of myo1b had opposite effects, which promoted H9c2 cell apoptosis, reduced cell proliferation, and accelerated cell autophagy. Taken together, Myo1b took part in the modulation of H/R-stimulated cardiomyocyte apoptosis and autophagy, which might be serve as a potential endogenous target for prevention and therapy of I/R injury. [ABSTRACT FROM AUTHOR]

Background: Molting is an essential biological process throughout the life history of crustaceans, which is regulated by many neuropeptide hormones expressed in the eyestalk. To better understand the molting mechanism in Portunus trituberculatus, we used digital gene expression (DGE) to analyze single eyestalk samples during the molting cycle by high-throughput sequencing. Results: We obtained 14,387,942, 12,631,508 and 13,060,062 clean sequence reads from inter-molt (InM), pre-molt (PrM) and post-molt (PoM) cDNA libraries, respectively. A total of 1,394 molt-related differentially expressed genes (DEGs) were identified. GO and KEGG enrichment analysis identified some important processes and pathways with key roles in molting regulation, such as chitin metabolism, peptidase inhibitor activity, and the ribosome. We first observed a pattern associated with the neuromodulator-related pathways during the molting cycle, which were up-regulated in PrM and down-regulated in PoM. Four categories of important molting-related transcripts were clustered and most of them had similar expression patterns, which suggests that there is a connection between these genes throughout the molt cycle. Conclusion: Our work is the first molt-related investigation of P. trituberculatus focusing on the eyestalk at the whole transcriptome level. Together, our results, including DEGs, identification of molting-related biological processes and pathways, and observed expression patterns of important genes, provide a novel insight into the function of the eyestalk in molting regulation. [ABSTRACT FROM AUTHOR]

Stretch-induced relaxation has not been clearly identified in gastrointestinal tract. The present study is to explore the role of large conductance calcium-activated potassium channels (BKCa) in stretch-induced relaxation of colon. The expression and currents of BKCa were detected and the basal muscle tone and contraction amplitude of colonic smooth muscle strips were measured. The expression of BKCa in colon is higher than other GI segments (P < 0.05). The density of BKCa currents was very high in colonic smooth muscle cells (SMCs). BKCa in rat colonic SMCs were sensitive to stretch. The relaxation response of colonic SM strips to stretch was attenuated by charybdotoxin (ChTX), a nonspecific BKCa blocker (P < 0.05). After blocking enteric nervous activities by tetrodotoxin (TTX), the stretch-induced relaxation did not change (P > 0.05). Still, ChTX and iberiotoxin (IbTX, a specific BKCa blocker) attenuated the relaxation of the colonic muscle strips enduring stretch (P < 0.05). These results suggest stretch-activation of BKCa in SMCs was involved in the stretch-induced relaxation of colon. Our study highlights the role of mechanosensitive ion channels in SMCs in colon motility regulation and their physiological and pathophysiological significance is worth further study. [ABSTRACT FROM AUTHOR]

Objective: To identify differentially expressed genes (DEGs) in endometriosis and further analyze molecular mechanisms implicated in disease pathogenesis.Materials and Methods: Gene expression data (ID: GSE7846) of human endometrial endothelial cells (HEECs) collected from eutopic endometria tissue of patients with and without endometriosis were downloaded from Gene Expression Omnibus. DEGs were screened using Limma package, followed by enrichment analysis using clusterProfiler package in R. Thereafter, protein-protein interactions (PPIs) were analyzed using STRING (Search Tool for the Retrieval of Interacting Genes) database and visualized by Cytoscape software. Meanwhile, transcription factors were screened from the DEGs based on TRANSFA database, followed by construction of regulatory network using Cytoscape.Results: A total of 2255 up- and 408 down-regulated genes were identified in endometriosis patients as compared with control patients. Those DEGs were predominantly enriched in focal adhesion (e.g., FN1, EGF, FYN, EGFR, RAC1, CCND1 and JUN), regulation of actin cytoskeleton (e.g., FN1, EGF, EGFR, RAC1 and JUN) and MAPK signaling pathway (e.g., EGF, EGFR, RAC1, JUN, TGFB1 and MYC). Importantly, EGF, EGFR, JUN, FN1, RAC1, TGFB1, CCND1 and FYN were hub nodes in the PPI network. Additionally, TGFB1, SMAD1 and SMAD4 showed up-regulation in TGFB signaling pathway. Transcription factor MYC had a regulatory effect on the most DEGs, including TGFB1, RAC1 and CCND1.Conclusions: Focal adhesion, regulation of actin cytoskeleton, MAPK and TGFB/SMAD signaling pathway may be important molecular mechanism underlying the pathogenesis of endometriosis. [ABSTRACT FROM AUTHOR]

Growing evidence indicates that transient receptor potential canonical (TRPC) channels play important roles in various Ca-mediated physiological and pathophysiological processes, including development. Many types of TRPC proteins are expressed in the heart. However, limited data are available comparing the expression and localization among TRPC proteins in the ventricular myocyte at various developmental stages. Our purpose is to investigate the expression and localization profile of TRPC proteins in ventricular myocytes of fetal (18.5 days), neonatal (< 24 h after birth) and adult (8 week old) rats. Western blotting, immunofluorescence and confocal laser scanning microscopy were employed. TRPC1/3-6 proteins were expressed in the rat ventricle throughout the three developmental stages. The expression profile of TRPC1/3/4 in the ventricle followed an upward trend from the fetus to the adult. By contrast, TRPC6 in the ventricle was expressed at the highest level in the fetal group and was sharply down-regulated immediately after birth. TRPC5 expression in the ventricle did not change significantly during the three stages. TRPC1/3/5/6 proteins were localized to the T-tubule and TRPC1/3/4/6 to intercalated disks in adult myocytes. The wide spatiotemporal overlap and dynamic regulation of TRPC expression in ventricular myocytes indicates potential complex combinations and redundancy of native TRPC proteins in the heart and gives important clues for further investigations into the exact subunit compositions and functional properties of native TRPC channels in the heart. [ABSTRACT FROM AUTHOR]

Cervical cancer (CC), as a common female malignant tumor in the world, is an important risk factor endangering women's health worldwide. The purpose of this study was to investigate the role of RBM15 in CC. The TCGA database was used to screen differentially expressed m6A genes in normal and tumor tissues. QRT-PCR was used to quantify HEIH, miR-802, EGFR, cell stemness, and epithelial-mesenchymal transition (EMT)-related genes. The interaction between HEIH and miR-802 was verified by dual-luciferase reporter assay and RIP assay. The occurrence of tumor cells after different treatments was detected by CCK-8, transwell and EdU staining. BALB/c nude mice were used to examine the effects of different treatments on tumor growth and cell stemness in vivo. RBM15 was upregulated in tumor tissues and cells. M6A was highly enriched in HEIH and enhances its RNA stability. HEIH acts as an oncogenic lncRNA to promote CC cell proliferation, migration and tumor growth. Mechanistically, HEIH regulates tumor cell stemness and promotes the proliferation and migration of CC cells by competitively adsorbing miR-802 and up-regulating the expression of EGFR. In short, our data shown that the m6A methyltransferase RBM15 could affect tumor cell proliferation, metastasis and cell stemness by stabilizing HEIH expression. [Display omitted] • We used TCGA dataset to screen the differentially expressed RNA methylation molecule RBM15 and verify its role in cervical cancer. • This study reports for the first time the regulatory role of RBM15 in the stemness of cervical cancer tumor cells. • RBM15 regulates the proliferation, metastasis and cell stemness of cervical cancer cells through the lncRNA HEIH/miR-802/EGFR pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: Circulating miRNAs are promising biomarkers for detection of various cancers. As a "developmental" disorder, cancer showed great similarities with embryos. OBJECTIVE: A comprehensive analysis of circulating miRNAs in umbilical cord blood (UCB) and pan-cancers was conducted to identify circulating miRNAs with potential for cancer detection. METHODS: A total of 3831 cancer samples (2050 serum samples from 15 types of cancers and 1781 plasma samples from 13 types of cancers) and 248 UCB samples (120 serum and 128 plasma samples) with corresponding NCs from Chinese populations were analyzed via consistent experiment workflow with Exiqon panel followed by multiple-stage validation with qRT-PCR. RESULTS: Thirty-four serum and 32 plasma miRNAs were dysregulated in at least one type of cancer. Eighteen serum and 16 plasma miRNAs were related with embryos. Among them, 9 serum and 8 plasma miRNAs with consistent expression patterns between pan-cancers and UCB were identified as circulating oncofetal miRNAs. Retrospective analysis confirmed the diagnostic ability of circulating oncofetal miRNAs for specific cancers. And the oncofetal miRNAs were mainly up-regulated in tissues of pan-cancers. CONCLUSIONS: Our study might serve as bases for the potential application of the non-invasive biomarkers in the future clinical. [ABSTRACT FROM AUTHOR]

Background: This study aimed to investigate the effects of STOML2 and the relationship between STOML2 and PAI-1 in the development of multiple myeloma (MM). Methods: Cell proliferation was tested using CCK-8 assay and cell colony formation assay. Glucose consumption, lactate production and ATP level were measured using commercial kits. The mRNA and protein expression were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Results: Both mRNA and protein expression of STOML2 were upregulated in MM patients compared to healthy volunteers. CCK-8 and colony formation assays demonstrated that STOML2 silencing inhibited cell proliferation in MM cells. Knockdown of STOML2 reduced glucose consumption, lactate production and ATP/ADP ratios. STOML2 silencing by shSTOML2 led to reduced PAI-1 expression. Overexpression of PAI-1 reversed the inhibitory effects of shSTOML2 on MM cell growth. Conclusion: Results from this study demonstrated that STOML2 silencing inhibits cell proliferation and glycolysis through downregulation of PAI-1 expression, suggesting a new therapeutic target for MM. [ABSTRACT FROM AUTHOR]

Abstract: Withangulatin A (WA), a bioactive withanolide isolated from Physalis angulata L., has a strong immunosuppressive activity. We have demonstrated that WA significantly suppressed the over-proliferation of ConA-induced T lymphocytes in a dose- and time-dependent manner, which modulated Th1/Th2-type balance dramatically. Moreover, WA-induced heme oxygenase 1 (HO-1) expression in mice T lymphocytes through the ERK/MAPK pathway and inhibited ConA-induced nuclear factor κB (NF-κB p65) nuclear translocation, directly or indirectly mediated by HO-1. Notably, WA affected the expression of COX-2 and PPAR-γ in ConA-induced T lymphocytes. The synergistic manner in which WA enhanced HO-1 expression through the ERK/MAPK pathway and NF-κB nuclear translocation and other regulatory proteins, thereby restricting T lymphocytes from over-expression and modulating Th1/Th2-type balance, could be the mechanism for the immunosuppressive properties of WA, the immunosuppressive effect of which may protect against autoimmune disorder diseases. [Copyright &y& Elsevier]

Abstract: Upregulation of the inducible nitric oxide synthase (iNOS) gene is associated with many pathological conditions such as endoplasmic reticulum (ER) stress, and X-box binding protein 1 (XBP1) is critical in mediating ER-stress responsive genes, including iNOS. Nonetheless, the mechanism by which XBP1 regulates iNOS during ER stress remains unexplored. Here we show that the active/spliced form of XBP1 protein, XBP1S, directly binds to the AABS (A-activator-binding site) in the iNOS promoter in vitro and in living cells. XBP1S exhibits dose-dependent activation of iNOS-specific reporter gene activity and endogenous iNOS expression. XBP1S is elevated whereas the unspliced form of XBP1, XBP1U, reduced in ER stress in HepG2 cells. In addition, XBP1U binds to XBP1S and this complex is associated with the iNOS promoter in response to ER stress. Furthermore, XBP1U acts as a negative mediator and suppresses XBP1S-mediated induction of iNOS. Collectively, we present the first evidence demonstrating the regulation of iNOS gene induction by the interaction between the spliced and unspliced forms of XBP1 in response to ER stress. [Copyright &y& Elsevier]

Abstract: A cytosolic manganese superoxide dismutase (cMnSOD) cDNA was cloned from the haemocytes of swimming crab Portunus trituberculatus using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the cMnSOD full-length cDNA consisted of 1106bp with an open reading frame of 861bp encoding a protein of 286 amino acids. Four conserved amino acids responsible for binding manganese (H110, H158, D243 and H247) and MnSOD signatures from 243 to 250 (DVWHHAYY) were observed. Sequence comparison showed that the cMnSOD deduced amino acid sequence of P. trituberculatus had similarity of 92%, 78%, 77% and 75% to that of blue crab Callinectes sapidus, white shrimp Litopenaeus vannamei, giant tiger prawn Penaeus monodon and freshwater prawn Macrobrachium rosenbergii, respectively. Quantitative real-time RT-PCR analysis revealed that P. trituberculatus cMnSOD transcript was strongly detected in haemocytes, hepatopancreas, heart, stomach, intestine, gill, ovary and muscle. RT-PCR analysis indicated that cMnSOD transcripts both in haemocytes and hepatopancreas increased at 3h and 6h after infection. These facts indicate that cMnSOD is potentially involved in the acute response against invading bacteria in P. trituberculatus. [ABSTRACT FROM AUTHOR]

Cholestatic Liver Fibrosis (CLF) is a hepatobiliary disease that typically arises as a late-stage complication of cholestasis, which can have multiple underlying causes. There are no satisfactory chemical or biological drugs for CLF. Total Astragalus saponins (TAS) are considered to be the main active constituents of the traditional Chinese herb Astragali Radix (AR), which has the obvious improvement effects for treating CLF. However, the mechanism of anti-CLF effects of TAS is still unclear. The present study was undertaken to investigate the therapeutic effects of TAS against bile duct ligation (BDL) and 3, 5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) -induced CLF models and to reveal the potential mechanism to support its clinic use with scientific evidence. In this study, BDL-induced CLF rats were treated with TAS (20 mg/kg, 40 mg/kg) and DDC-induced CLF mice were treated with 56 mg/kg TAS. The therapeutic effects of TAS on extrahepatic and intrahepatic CLF models were evaluated by serum biochemical analysis, liver histopathology and hydroxyproline (Hyp). Thirty-nine individual bile acids (BAs) in serum and liver were quantified by using UHPLC-Q-Exactive Orbitrap HRMS. qRT-PCR, Western blot and immunohistochemistry analysis were used to measure the expression of liver fibrosis and ductular reaction markers, inflammatory factors and BAs related metabolic transporters, along with nuclear receptor farnesoid X receptor (FXR). The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBiL), direct bilirubin (DBiL) and contents of liver Hyp were dose-dependently improved after treatment for TAS in BDL and DDC- induced CLF models. And the increased levels of ALT and AST were significantly improved by total extract from Astragali radix (ASE) in BDL model. The liver fibrosis and ductular reaction markers, α-smooth muscle actin (α-SMA) and cytokeratin 19 (CK19), were significantly ameliorated in TAS group. And the liver expression of inflammatory factors: interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) were significantly decreased after TAS treatment. In addition, TAS significantly ameliorated taurine-conjugated BAs (tau-BAs) levels, particularly α-TMCA, β-TMCA and TCA contents in serum and liver, which correlated with induced expressions of hepatic FXR and BAs secretion transporters. Furthermore, TAS significantly improved short heterodimer partner (SHP), cholesterol 7α-hydroxylase (Cyp7a1), Na+ taurocholate cotransport peptide (NTCP) and bile-salt export pump (BSEP) mRNA and protein expression. TAS exerted a hepatoprotective effect against CLF by ameliorating liver injury, inflammation and restoring the altered tau-BAs metabolism to produce a positive regulatory effect on FXR-related receptors and transporters. [Display omitted] • TAS improve cholestatic liver fibrosis in BDL and DDC models. • TAS regulate tau-BAs contents, especially α-TMCA, β-TMCA and TCA. • FXR signaling pathways are critical for the treatment of TAS in CLF. [ABSTRACT FROM AUTHOR]

Beclin1, also known as ATG6, has been shown to be closely related to coronavirus, however, the link between Beclin1 and nephropathogenic infectious bronchitis virus (NIBV) has been poorly investigated and there are no available antibodies specifically targeting the chicken Beclin1 protein. The study aimed to prepare and assay a polyclonal antibody to Beclin1, enabling a deeper understanding of the mechanism of action of Beclin1 in NIBV. In this study, we amplified the chicken Beclin1 target gene and constructed a recombinant plasmid using prokaryotic expression techniques, then obtained the recombinant target protein by induced expression. Finally, the serum is obtained by immunizing rabbits with the purified and concentrated protein. The results show that the antiserum potency of the ELISA assay was >1:204800. By western blotting and immunofluorescence, the antibodies we prepared specifically recognized the chicken Beclin1 protein, which is mainly found in the nucleus of trachea, lung, kidney, spleen and fabricant cells. NIBV infection significantly decreased the expression of Beclin1 in the trachea, but increased in others. We have successfully prepared specific rabbit anti-chicken Beclin1 polyclonal antibodies, and detected changes in tissues of diseased chickens infected with NIBV, laying the foundation for further studies on the role of Beclin1 in avian diseases. [ABSTRACT FROM AUTHOR]

• A comprehensive single cell miRNA activities map in BCG vaccine injected mice modeling tuberculous meningitis (TBM) brain infection; • Cell type enriched miRNAs were identified in TBM brain infection; • The functional miRNA-mRNA networks of macrophages and microglia, neurons, ependymal cells, and vascular smooth muscle cell was established. • Elevated miR-21a-3p activity is found in Microglia of TBM tissue. Our previous study revealed the transcriptome atlas of specific cell types in tuberculous meningitis (TBM) model mice injected with the BCG vaccine via scRNA sequencing. However, the activities of miRNAs in TBM at single-cell resolution remain to be explored. Cell type-specific miRNA activities were investigated by using motif enrichment analyses (miReact) on the transcriptome data of 15 cell types. The target mRNAs of miRNAs were predicted and subjected to enrichment analysis. Furthermore, miRNAs and their target mRNAs with opposite expression trends were chosen to construct functional networks. Besides, qRT-PCR and RNA scope were performed to verify the expression level of representative miRNA. The tSNE dimensionality reduction presented 15 cell types in TBM model mice, in which microglia and endothelial cells accounted for the majority. Target mRNAs of each cell type were predicted for verification or network construction. The immune and inflammation-related miRNA-mRNA networks of macrophages and microglia, oxidative phosphorylation-related miRNA-mRNA networks of neurons, ion and protein transport-related networks of epididymal cells, and angiogenesis-related miRNA-mRNA networks of VSMCs were constructed. The miRNA activity analysis revealed that miR-21a-3p activity was increased in microglia, macrophages, neurons and epididymal cells. The result of qRT-PCR and RNA scope indicate that miR-21a-3p was significantly higher-expressed in TBM brain tissue compared with normal brain tissue. In our study, an in-depth exploration of the mRNA expression and miRNA activity of macrophages, microglia, epididymal cells, neurons and vascular smooth muscle cells during TBM progression was conducted using scRNA-Seq, which provided novel insights into the immune cell engagement in TBM patients. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor α/δ (PPAR α/δ), regulating glucolipid metabolism and immune inflammation, has been identified as an effective therapeutic target in non-alcoholic steatohepatitis (NASH). Dual PPAR α/δ agonist, such as GFT505 (also known as elafibranor), demonstrated potential therapeutic effect for NASH in clinical trials. To profile the regulatory network of PPAR α/δ agonist in NASH, the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) induced NASH model was used to test the pharmacodynamics and transcriptome regulation of GFT505 in this study. The results showed that GFT505 ameliorated hepatic steatosis, inflammation and fibrosis in CDAHFD mice model. RNA-sequencing yielded 3995 up-regulated and 3576 down-regulated genes with GFT505 treatment. And the most significant differentialy expressed genes involved in glucolipid metabolism (Pparα, Acox1, Cpt1b, Fabp4, Ehhadh, Fabp3), inflammation (Ccl6, Ccl9, Cxcl14) and fibrosis (Timp1, Lamc3, Timp2, Col3a1, Col1a2, Col1a1, Hapln4, Timp3, Pik3r5, Pdgfα, Pdgfβ, Tgfβ1, Tgfβ2) were confirmed by RT-qPCR. The down-regulated genes were enriched in cytokine-cytokine receptor interaction pathway and ECM-receptor interaction pathway, while the up-regulated genes were enriched in PPAR signaling pathway and fatty acid degradation pathway. This study provides clues and basis for further understanding on the mechanism of PPAR α/δ agonist on NASH. [ABSTRACT FROM AUTHOR]

Background. Notch/Dll4 involvement in cornea neovascularization (CRNV) and lymphangiogenesis is unclear. This study aimed to explore the role of notch signaling in basic fibroblast growth factor- (bFGF-) induced corneal lymphangiogenesis and hemangiogenesis. Methods. Corneal stroma of C57BL/6 mice was implanted with bFGF- or phosphate-buffered saline- (PBS-) soaked pellets. Corneal lymphangiogenesis and neovascularization were evaluated by immunofluorescence. Vascular endothelial growth factor-A (VEGF-A), Delta-like ligand 4 (Dll4), and Notch1 mRNA and protein expression were examined on days 1, 3, 7, and 14 by real-time polymerase chain reaction and western blot. Corneal cells were treated with ranibizumab, dexamethasone, and γ-secretase inhibitor (GSI). Microspheres were used to evaluate corneal hemangiogenesis in vivo. Results. Corneal hemangiogenesis reached its peak on day 7 after bFGF implantation, and corneal lymphangiogenesis was significantly higher on day 7 and 14, compared with PBS. mRNA and protein expression of VEGF-A, Dll4, and Notch1 were higher in bFGF-induced animal models compared with controls. Corneal hemangiogenesis and lymphangiogenesis decreased after 7 days of ranibizumab or dexamethasone treatment. After adding GSI for 24 h in bFGF-induced cells, the expression of Notch1 and Dll4 were downregulated compared with that in the control group whereas the expression level of VEGF-A was upregulated. Fluorescent particle number was higher in the GSI group. Ranibizumab and dexamethasone decreased the fluorescence signal. Conclusion. The notch signaling pathway plays a role in regulating VEGF expression, affecting corneal lymphangiogenesis and hemangiogenesis in mice. The molecular imaging probe technique can visualize the changes in the VEGF-A expression level of corneal limbus hemangiogenesis. [ABSTRACT FROM AUTHOR]

Background: The five‐year survival rate of non‐small cell lung cancer (NSCLC) patients is very low. MiR‐873 is involved in the growth, metastasis, and differentiation of tumors. Herein, we determined the target gene and influence of miR‐873 in NSCLC. Methods: MiRanda and Targetscan websites were used to predict the target gene of miR‐873 in NSCLC. Luciferase activity was examined using a dual luciferase reporter gene assay kit. The viability, tube formation, and proliferation of cells were analyzed by cell counting kit‐8, angiogenic analysis, and flow cytometry, respectively. The levels of miR‐873 and GLI1 were evaluated using quantitative real‐time PCR and Western blot assays. Results: Low levels of GLI1 and high levels of miR‐873 were observed in an NSCLC cell line (PC9) highly sensitive to EGFR‐tyrosine kinase inhibitors. There was a negative correlation between miR‐873 and GLI1 expression in PC9 and PC9/GR cells. The inhibition of miR‐873 enhanced GLI1 levels. MiR‐873 expression was inhibited by gefitinib. Gefitinib markedly reduced the viability, tube formation, and cell number in PC9 cells. However, suppression of miR‐873 enhanced the resistance and knockdown of GLI1 enhanced the sensitivity of PC9 cells to gefitinib. Conclusions: GLI1 is a target gene of miR‐873 in NSCLC. The inhibition of miR‐873 increased gefitinib resistance of NSCLC cells via the upregulation of GLI1. These results indicate that miR‐873‐GLI1 signaling is involved in gefitinib resistance in NSCLC. [ABSTRACT FROM AUTHOR]

Background/Aims: Cisplatin-based treatment is first-line chemotherapy for several cancers including ovarian cancer. The development of cisplatin resistance results in treatment failure, but the underlying mechanisms are not fully understood. Histone deacetylases (HDACs) are a large family of enzymes that deacetylate lysine residues on histones and non-histone proteins. High expression of HDAC1 is associated with poor outcomes in ovarian cancer. Furthermore, resistance to chemotherapeutic agents is associated with HDAC1 overexpression in ovarian cancer cells. The goals of this study were to determine whether targeting HDAC1 can sensitize ovarian cancer cells to cisplatin and to explore the underlying mechanisms. Methods: Small interfering RNA (siRNA)-targeting HDAC1 was designed to silence HDAC1 in the cisplatin-resistant ovarian cancer cell line A2780CDDP and its cisplatin-sensitive cell line A2780. The effects of targeting HDAC1 on cell viability assay, colony formation, and apoptosis were detected. c-Myc re-expression or miR-34a inhibitors were used to examine the relationship among HDAC1, c-Myc, and miR-34a expression, which was assessed by western blot analysis and quantitative reverse transcription PCR. We established stable transfectants of A2780CDDP/HDAC1 short hairpin RNA (shRNA) and A2780/HDAC1 shRNA. The therapeutic effectiveness of cisplatin in murine xenograft models was assessed following shRNA-mediated HDAC1 silencing in A2780CDDP and A2780 cells. The mechanism of cell death was studied in tumor sections obtained from different mouse tumors. Results: In cisplatin-resistant A2780CDDP cells, HDAC1 knockdown by siRNA suppressed cell proliferation, and increased apoptosis and chemosensitivity by downregulating c-Myc and upregulating miR-34a. In cisplatin-sensitive A2780 cells, HDAC1 knockdown did not affect cell proliferation and apoptosis. Cisplatin treatment activated HDAC1 and c-Myc and inactivated miR-34a. Inhibition of HDAC1 with siRNA reduced c-Myc expression, increased miR-34a expression, and sensitized A2780 cells to cisplatin-induced apoptosis. c-Myc re-expression or miR-34a targeting by miR-34a inhibitors protected cells from apoptosis or reversed cisplatin resistance following HDAC1 knockdown or/and cisplatin exposure. Finally, in vivo studies showed that targeting HDAC1 inhibited A2780CDDP-induced xenograft tumor growth but not A2780-induced xenograft tumor growth. Targeting HDAC1 sensitized both A2780- and A2780CDDP-induced xenograft tumors to cisplatin treatment. Conclusions: Upregulation of HDAC1 is a crucial event in the development of drug resistance to current treatments in ovarian cancer. Thus, targeting HDAC1 by enhancing c-Myc-dependent miR-34a expression might be an effective strategy for increasing the efficacy of cisplatin treatment. [ABSTRACT FROM AUTHOR]

Apoptosis of human lens epithelial (HLE) cells is a process closely associated with cataract formation. The aim of the present study was to explore the effects of microRNA (miR)‑181a against hydrogen peroxide (H2O2)‑induced apoptosis in HLE cells in vitro. The recombinant lentiviral plasmid pLKO. 1‑puro‑miR‑181a was constructed and used to transfect human HLE‑B3 cells with the short hairpin (sh)RNA to silence the expression of miR‑181a. The apoptotic rate of both HLE‑B3 cells in which miR‑181a expression was stably silenced and in untransfected HLE‑B3 cells was assessed in the presence of H2O2 using flow cytometry. The mRNA expression levels of the apoptosis‑related genes caspase-3 (CASP3) and B‑cell lymphoma‑2‑associated X protein (BAX), and of the potential target genes for miR‑181a, c‑MET, cyclooxygenase 2 (COX‑2) and snail family transcriptional repressor 2 (SNAI2) were measured using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were assessed using ELISA. RT‑qPCR analysis revealed that miR‑181a expression was downregulated in HLE‑B3 cells following transfection with miR‑181a‑shRNA. Treatment with H2O2 significantly reduced the viability of HLE‑B3 cells, whereas miR‑181a knockdown was revealed to attenuate the effects on cell viability following H2O2 treatment. In addition, the downregulation of miR‑181a expression significantly decreased H2O2‑induced cell apoptosis, which was accompanied by a downregulation in CASP3 and BAX and COX‑2 expression. Furthermore, the levels of MDA were decreased, whereas the levels of SOD and CAT were increased following miR‑181a silencing. The present findings suggested that miR‑181a knockdown may protect HLE‑B3 cells against H2O2‑induced apoptosis in vitro. The molecular mechanisms involved in the protective effects of miR‑181a silencing may involve the suppression of CASP3, BAX and COX‑2 expression, and the inhibition of MDA generation. [ABSTRACT FROM AUTHOR]

Cataracts are the most common eye disease to cause blindness in patients. The abnormal deposition of laminins (LMs) in the lens capsule and the disruption of capsular epithelium contribute to cataract development, although the mechanism by which this occurs is currently unclear. The present study aimed to reproduce HLE B‑3 basement membranes (BMs) using HLE B‑3 cells and to analyze the similarities of LM expression between HLE B‑3 BMs and human anterior lens capsule (ALC). Immunohistochemistry (IHC), ELISA, western blot analysis and immunoprecipitation (IP)‑western blot analysis were used to detect total LMs, LM trimers and 11 LM subunits in HLE B‑3 cells, HLE B‑3 BMs and human ALCs. In IHC staining, HLE B‑3 cells and human ALCs were positive for LMs. In LM ELISA, all samples analyzed were positive for LMs. Western blot analysis detected all LM subunits except for LMγ3 in HLE B‑3 cell lysate, 4 subunits (LMα4, LMα2, LMα1 and LMγ1) in HLE B‑3 cell culture supernatant, 5 subunits (LMα4, LMα2, LMα1, LMβ3 and LMγ1) in HLE B‑3 BMs, and 3 subunits (LMα4, LMγ2 and LMγ1) in human ALCs. The results of IP‑western blot analysis revealed that the LM411 trimer was detected in HLE B‑3 cell culture supernatant. These results indicated that HLE B‑3 BMs were similar to human ALCs in terms of LM expression. Therefore, HLE B‑3 BMs could be used as an in vitro ALC model to determine the role of LMs in ALC in the pathogenesis of cataracts and to select potential anti‑cataract drugs. [ABSTRACT FROM AUTHOR]