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Background: Alzheimer's disease (AD) is the most common type of dementia, and patients with advanced AD frequently lose the ability to identify family members. The fusiform gyrus (FUS) of the brain is critical in facial recognition. However, AD etiology in the FUS of AD patients is poorly understood. New analytical strategies are needed to reveal the genetic and epigenetic basis of AD in FUS., Results: A complex of new analytical paradigms that integrates an array of transcriptomes and methylomes of normal controls, AD patients, and "AD-in-dish" models were used to identify genetic and epigenetic signatures of AD in FUS. Here we identified changes in gene expression that are specific to the FUS in brains of AD patients. These changes are closely linked to key genes in the AD network. Profiling of the methylome (5mC/5hmC/5fC/5caC) at base resolution identified 5 signature genes (COL2A1, CAPN3, COL14A1, STAT5A, SPOCK3) that exhibit perturbed expression, specifically in the FUS and display altered DNA methylome profiles that are common across AD-associated brain regions. Moreover, we demonstrate proof-of-principle that AD-associated methylome changes in these genes effectively predict the disease prognosis with enhanced sensitivity compared to presently used clinical criteria., Conclusions: This study identified a set of previously unexplored FUS-specific AD genes and their epigenetic characteristics, which may provide new insights into the molecular pathology of AD, attributing the genetic and epigenetic basis of FUS to AD development.

Background: Δ6-desaturase belonging to membrane-bound enzyme is a key enzyme involved in the synthesis of polyunsaturated fatty acids (PUFAs). This study aimed to clone and characterise Δ6-desaturase gene and its upstream regulatory region of Mortierella sp. AGED., Results: Glucose and soybean meal are best for lipid and arachidonic acid accumulation of Mortierella sp. AGED. A 1375-bp Δ6-desaturase gene AGfad6 which contains a 1275-bp open reading frame encoding 424 amino acids without signal peptide was cloned. The putative protein contained three conserved histidine-rich motifs and a conserved cytochrome b5 HPGG (H: Histine, P: Proline, G: Glycine, G: Glycine) motif, with a mass of 48.3 kDa and an isoelectric point of 5.96. AGfad6 was successfully expressed in Pichia pastoris GS115, which exerted the effect on converting linoleic acid to γ-linolenic acid. The 1712-bp upstream region contained basic transcriptional elements including TATA, GC and GATA box, putative target-binding sites for transcription factors such as TATA binding protein, transcription activator, CCAAT-enhancer-binding protein, activator protein 1, alcohol dehydrogenase gene regulator 1 and metabolic regulators p40x in fungi, stress-related elements including GT-1 (light-responsive, salicylic acid-inducible), stress response element, heat stress-responsive element, which might participate in regulation of PUFAs synthesis., Conclusion: The present finding could enable us to understand the evolution and regulatory mechanism of Δ6-desaturase gene., (© 2014 Society of Chemical Industry.)

The retinal pigment epithelium (RPE) performs numerous functions that are indispensable for photoreceptor health and vision. This monolayer of cells is also a major site of insult in inherited and age-related macular degenerations. In vitro models of primary RPE such as human fetal and adult RPE cultures have been invaluable for dissecting disease pathways at the cellular and molecular level. However, numerous studies show that it takes over four weeks for human RPE cell monolayers to become fully polarized after plating on semipermeable membrane supports. Poor persistence of transgene expression over this time period critically limits the applicability of human RPE cultures for live imaging studies required to follow dynamic processes like intracellular trafficking and organelle transport that occur over timescales of milliseconds. Here, we provide a detailed three-step protocol for live imaging of polarized primary RPE using high-speed spinning disk confocal microscopy. Step 1: establish porcine RPE monolayers that undergo differentiation within one week after plating on semipermeable membrane supports; step 2: transfect or transduce RPE using either of two different protocols that result in prolonged transgene expression; and step 3: perform multicolor high-speed live imaging of organelle transport in polarized RPE monolayers. Porcine RPE cells and photoreceptor outer segments were isolated from freshly harvested eyes and plated on collagen-coated Transwell® filters to generate polarized monolayers. After seven days, RPE monolayers were highly pigmented, had TER values ≥ 200 Ω.cm2 and cleared outer segments within 5 hours after phagocytosis. These cells expressed RPE65, localized ZO-1 to the tight junction, Na+,K+-ATPase to the apical membrane and acetylated tubulin to the primary cilium. There was an inverse relationship between initial plating density and the time to differentiation. We used nucleofection to express fluorescently tagged genes in RPE cells prior to plating on filters or baculovirus fusion constructs to transfect polarized monolayers. Both these methods resulted in transfection efficiencies over 40% and transgene expression lasted up to 8 days after plating. These filters were imaged by high-speed spinning disk microscopy to follow tubulovesicular trafficking of lysosomes and actin dynamics in the RPE. Four-dimensional image analysis performed using commercially available software was used to analyze live imaging data. In conclusion, this 3-step protocol describes a powerful method to investigate organelle trafficking and function in real time in the RPE that can be used for answering fundamental questions of RPE cell biology and pathobiology.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is resistant to almost all antibiotics. Eravacycline, a newer treatment option, has the potential to treat CRAB infections, however, the mechanism by which CRAB isolates develop resistance to eravacycline has yet to be clarified. This study sought to investigate the features and mechanisms of eravacycline heteroresistance among CRAB clinical isolates. A total of 287 isolates were collected in China from 2020 to 2022. The minimum inhibitory concentration (MIC) of eravacycline and other clinically available agents against A. baumannii were determined using broth microdilution. The frequency of eravacycline heteroresistance was determined by population analysis profiling (PAP). Mutations and expression levels of resistance genes in heteroresistant isolates were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR), respectively. Antisense RNA silencing was used to validate the function of eravacycline heteroresistant candidate genes. Twenty- five eravacycline heteroresistant isolates (17.36%) were detected among 144 CRAB isolates with eravacycline MIC values ≤4 mg/L while no eravacycline heteroresistant strains were detected in carbapenem-susceptible A. baumannii (CSAB) isolates. All eravacycline heteroresistant strains contained OXA-23 carbapenemase and the predominant multilocus sequence typing (MLST) was ST208 (72%). Cross-resistance was observed between eravacycline, tigecycline, and levofloxacin in the resistant subpopulations. The addition of efflux pump inhibitors significantly reduced the eravacycline MIC in resistant subpopulations and weakened the formation of eravacycline heteroresistance in CRAB isolates. The expression levels of adeABC and adeRS were significantly higher in resistant subpopulations than in eravacycline heteroresistant parental strains (P < 0.05). An ISAba1 insertion in the adeS gene was identified in 40% (10/25) of the resistant subpopulations. Decreasing the expression of adeABC or adeRS by antisense RNA silencing significantly inhibited eravacycline heteroresistance. In conclusion, this study identified the emergence of eravacycline heteroresistance in CRAB isolates in China, which is associated with high expression of AdeABC and AdeRS. [ABSTRACT FROM AUTHOR]

Background: Lymph node metastasis can independently predict oral squamous cell carcinoma patients' survival. This study would investigate the genetic and cellular differences between oral squamous cell carcinoma with positive and negative lymph node metastases. Methods: We gathered single‐cell RNA sequencing and bulk gene expression data from the Cancer Genome Atlas and Gene Expression Omnibus databases. Sixty lymph node‐metastasis‐related genes were discovered with refined single‐cell RNA sequencing data analysis, and consensus clustering provided three molecular subtypes of oral squamous cell carcinoma. Least absolute shrinkage and selection operator analyses were then utilized to establish a five‐gene risk model. CIBERSORT analysis revealed the immune infiltration profile of different risk subgroups. Results: Oral squamous cell carcinoma patients were classified into three subtypes based on the 60 lymph node‐metastasis‐related key genes identified by single‐cell RNA sequencing data. Patients in Subtype 3 showed a tendency for lymph node metastasis and poorer prognosis. Moreover, five biomarkers were selected from the 60 genes to construct a five‐gene risk model evaluating the risk of lymph node metastasis. A lower probability of lymph node metastasis and a better prognosis was observed in the low‐risk group. The immune infiltration of three different risk groups was explored with CIBERSORT. Besides, further analysis implied different sensitivities of anticancer drugs, including immunotherapy drugs and targeted compounds, in the three risk groups. Conclusion: In view of intratumoral heterogeneity, we found 60 genes associated with lymph node metastasis of oral squamous cell carcinoma. Subsequently, we constructed a five‐gene signature that could improve the prediction of lymph node metastasis, clinical outcome, and promote individualized treatment strategies for oral squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

Background: Mori Fructus is an economical and readily available traditional Chinese medicine and food. Polysaccharides in Mori Fructus have clear antioxidant activity and have been found to alleviate oxidative stress (OS)-induced liver damage in experimental studies. The mechanism of regulation of cellular antioxidant activity by mulberry polysaccharides has been suggested to be Nrf2, but it is not clear whether the Nrf2 pathway is mediated by activation of other targets, and the exact process of effects in hepatocytes has yet to be elucidated. Methods: In this study, the basic characterization of total polysaccharides extracted from mulberry fruits (Morus nigra Linn.) was analyzed. A model of oxidative damage induced by H2O2 in HepG2 cells was established. The levels of cellular oxidation-related markers, including ROS, SOD and Gpx, were then examined. Furthermore, Q-PCR and Western-blot were used to detect the expression of genes and proteins related to the PI3K/Akt-mediated Nrf2 signaling pathway. Results: The results showed that a total mulberry polysaccharides (TMP) has a molecular weight of 57.5 kDa with a pyranose ring mainly composed of glucose (48.81%), galactose (22.79%) and mannose (18.2%). TMP reduced the accumulation of ROS in HepG2 cells after H2O2 treatment and modulated the activity of SOD and Gpx. Q-PCR and Western-blot showed that TMP could up-regulate the expression of p-PI3K, p-AKT, Nrf2, NQO1 and HO-1. Conclusions: This study demonstrates that TMP can reduce ROS accumulation in H2O2-treated HepG2 cells and restore cell viability by activating the PI3K/AKT-mediated Nrf2 pathway. TMP may be a potent antioxidant agent that could slow down oxidative damage to the liver. [ABSTRACT FROM AUTHOR]

CNOT3 is the central component of the CCR4-NOT protein complex, which is a global regulator of RNA polymerase II transcription. Loss of function mutations in CNOT3 lead to intellectual developmental disorder with speech delay, autism, and dysmorphic facies (IDDSADF), which is very rare. Herein, we reported two novel heterozygous frameshift mutations (c.1058_1059insT and c.724delT) and one novel splice site variant (c.387 + 2 T > C) in CNOT3 (NM_014516.3) gene in three Chinese patients with dysmorphic features, developmental delay, and behavior anomalies. The functional study showed that the CNOT3 mRNA levels were significantly decreased in the peripheral blood of two patients with c.1058_1059insT and c.387 + 2 T > C variants, respectively, and minigene assay demonstrated that the splice variant (c.387 + 2 T > C) resulted in exon skipping. We also found that CNOT3 deficiency was linked to alterations of expression levels of other CCR4-NOT complex subunits in mRNA level in the peripheral blood. By analyzing the clinical manifestations of all these patients with CNOT3 variants, including our three cases and 22 patients previously reported, we did not observe a correlation between genotypes and phenotypes. In summary, this is the first time to report cases with IDDSADF in the Chinese population, and three novel CNOT3 variants in these patients expand its mutational spectrum. [ABSTRACT FROM AUTHOR]

Background: Neuroinflammatory factors, especially chemokines, have been widely reported to be involved in the pathogenesis of Alzheimer's disease (AD). It is unclear how chemokines are altered in AD, and whether dysregulation of chemokines is the cause, or the consequence, of the disease. Methods: We initially screened the transcriptomic profiles of chemokines from publicly available datasets of brain tissues of AD patients and mouse models. Expression alteration of chemokines in the blood from AD patients was also measured to explore whether any chemokine might be used as a potential biomarker for AD. We further analyzed the association between the coding variants of chemokine genes and genetic susceptibility of AD by targeted sequencing of a Han Chinese case–control cohort. Mendelian randomization (MR) was performed to infer the causal association of chemokine dysregulation with AD development. Results: Three chemokine genes (CCL5, CXCL1, and CXCL16) were consistently upregulated in brain tissues from AD patients and the mouse models and were positively correlated with Aβ and tau pathology in AD mice. Peripheral blood mRNA expression of CXCL16 was upregulated in mild cognitive impairment (MCI) and AD patients, indicating the potential of CXCL16 as a biomarker for AD development. None of the coding variants within any chemokine gene conferred a genetic risk to AD. MR analysis confirmed a causal role of CCL5 dysregulation in AD mediated by trans-regulatory variants. Conclusions: In summary, we have provided transcriptomic and genomic evidence supporting an active role of dysregulated CXCL16 and CCL5 during AD development. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) have seen an elevated use in clinical works like regenerative medicine. Its potential therapeutic properties increases when used in tandem with complementary agents like bio-based materials. Therefore, the present study is the first to investigate the cytotoxicity of a highly valued medicinal plant, Moringa oleifera, on human Wharton's Jelly mesenchymal stem cells (hWJMSCs) and its effects on the cells' gene expression when used as a pre-treatment agent in vitro. M. oleifera leaves (MOL) were dried and subjected to UHPLC-QTOF/MS analysis, revealing several major compounds like apigenin, kaempferol, and quercetin in the MOL, with various biological activities like antioxidant and anti-cancer properties. We then treated the hWJMSCs with MOL and noticed a dose-dependant inhibition on the cells' proliferation. RNA-sequencing was performed to explain the possible mechanism of action and revealed genes like PPP1R1C, SULT2B1, CDKN1A, mir-154 and CCNB1, whose expression patterns were closely associated with the negative cell cycle regulation and cell cycle arrest process. This is also evident from gene set enrichment analysis where the GO and KEGG terms for down-regulated pathways were closely related to the cell cycle regulation. The Ingenuity pathway analysis (IPA) software further predicted the significant activation of (p < 0.05, z-score > 2) of the G2/M DNA damage checkpoint regulation pathway. The present study suggests that MOL exhibits an antiproliferative effect on hWJMSCs via cell cycle arrest and apoptotic pathways. We believe that this study provides an important baseline reference for future works involving MOL's potential to accompany MSCs for clinical works. Future works can take advantage of the cell's strong anti-cancer gene expression found in this study, and evaluate our MOL treatment on various cancer cell lines. [ABSTRACT FROM AUTHOR]

HAS2 antisense RNA 1 (HAS2-AS1) is a long noncoding RNA that has increased expression in mature granulosa cells (GCs) and contributes to cumulus expansion by regulating HAS2 expression. However, the roles of HAS2-AS1 during the pathological process of polycystic ovary syndrome (PCOS) are still unclear. This study investigated the roles of HAS2-AS1 in patients with PCOS. Here, a significant upregulation of HAS2-AS1 was found in the primary GCs from patients with PCOS, which was positively correlated with the level of the protein HAS2. The knockdown of HAS2 restored the upregulation of HAS2-AS1 in promoting migration but could not restore the effects of HAS2-AS1 overexpression in promoting proliferation and repressing apoptosis. Transforming growth factor β (TGF-β) upregulated HAS2-AS1 levels, while HAS2-AS1 functioned as a feedback inhibition factor repressing TGF-β signaling by inhibiting TGF-β receptor type 2 (TGFBR2) expression. HAS2-AS1 bonded with EZH2 and guided the polycomb complex 2 to the TGFBR2 promoter region. HAS2-AS1 overexpression induced H3K27 hypermethylation in the TGFBR2 promoter region and then repressed TGFBR2 transcription in KGN cells and primary GCs. In conclusion, we identified for the first time that HAS2-AS1 is upregulated in patients with PCOS and represses TGF-β signaling via inducing TGFBR2 promoter region hypermethylation, which allowed us to explore the pathological processes of PCOS. [ABSTRACT FROM AUTHOR]

The immunological response occurring during periapical inflammation includes expression of nucleotide binding oligomerization domain containing 2 and hepcidin. Nucleotide binding oligomerization domain containing 2 deficiency increases infiltration of inflammatory cells close to alveolar bone. Hepcidin has an important role in iron metabolism affecting bone metabolism.We investigated the role of nucleotide binding oligomerization domain containing 2 and hepcidin in inflammatory periapical periodontitis. Periapical periodontitis was induced in rats and confirmed by micro-computed tomography. Nucleotide binding oligomerization domain 2 and hepcidin were evaluated through immunohistochemistry. Bioinformatics analysis was undertaken usingthe Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases. Micro-computer tomography revealed alveolar bone resorption in the periapical region and furcation area of mandibular molars in rats of the periapical periodontitis group. Immunohistochemistry showed increased expressionof nucleotide binding oligomerization domain containing 2 and hepcidin around root apices in rats of the periapical periodontitis group. Bioinformatics analysis of differentially expressed genes in inflamed and non-inflamed tissues revealed enrichment in the NOD-like receptor signaling pathway. Our data suggest that nucleotide binding oligomization domain contain2 and hepcidin have important roles in periapical periodontitis severity because they can reduce alveolar bone loss.They could elicit new perspectives for development of novel strategies for periapical periodontitis treatment. [ABSTRACT FROM AUTHOR]

To obtain novel insights into the tumor biology and therapeutic targets of LUAD, we performed a comprehensive analysis of the KCTD family genes. The expression patterns and clinical significance of the KCTD family were identified through multiple bioinformatics mining. Moreover, the molecular functions and potential mechanisms of differentially expressed KCTDs were evaluated using TIMER 2.0, cBioPortal, GeneMANIA, LinkedOmics and GSEA. The results indicated that the mRNA and protein expression levels of KCTD9, KCTD10, KCTD12, KCTD15 and KCTD16 were significantly decreased in LUAD, while those of KCTD5 were significantly increased. High KCTD5 expression was significantly associated with advanced tumor stage, lymph node metastasis, TP53 mutation and poor prognosis. In addition, KCTD5 was positively correlated with CD8 + T cell, neutrophil, macrophage and dendritic cell infiltration. Additionally, KCTDs demonstrate promising prospects in the diagnosis of LUAD. Importantly, high KCTD5 expression was enriched in signaling pathways associated with the malignant progression of tumors, including the inflammatory response, the IL6/JAK/STAT3 signaling pathway, EMT and hypoxia. Further association analysis showed that KCTD5 was positively correlated with hypoxia-related genes such as HIF1. Overall, KCTDs can be used as molecular targets for the treatment of LUAD, as well as effective molecular biomarkers for diagnosis and prognosis prediction. [ABSTRACT FROM AUTHOR]

The a ccessory g ene r egulator (agr) quorum-sensing system is an important global regulatory system of Staphylococcus aureus and contributes to its pathogenicity. The S. aureus agr system is divided into four agr groups based on the amino acid polymorphisms of AgrB, AgrD, and AgrC. The agr activation is group-specific, resulting in variations in agr activity and pathogenicity among the four agr groups. Strains with divergent agr system always have different phenotypes. In the present report, we, respectively, exchanged the agr system of a certain S. aureus with other three agr alleles and assessed the corresponding phenotypes of these congenic strains. Replacement of the agr system led to significant variations in hemolytic activity, protein expression, and virulence gene expression comparing with that of the parental strain. Interestingly, we found that the biological characteristics of these agr congenic strains in the same strain background were highly similar to each other, and the allele-dependent differences of the agr systems were weakened. These findings indicate that the allele-dependent agr predilections of S. aureus are determined by some factors in addition to the polymorphisms of AgrB, AgrD, and AgrC. Future studies may reveal the novel mechanism to improve our understanding of the agr network. [ABSTRACT FROM AUTHOR]

Objective: To investigate the attenuating effect of Hydroxysafflor yellow A (HSYA) on inflammatory injury in chronic obstructive pulmonary disease (COPD). Methods: Rats were randomly assigned to 7 groups according to body weight including normal control group, HSYA blank group (76.8 mg/kg), COPD group, COPD+HSYA (30, 48, 76.8 mg/kg) groups and COPD+dexamethasone (2 mg/kg), 10 in each group. Passive cigarette smoke and intratracheal instillation of lipopolysaccharides were used to establish a COPD model in rats. Hematoxylin and eosin staining of lung tissue sections was used, real-time polymerase chain reaction (PCR) was used to assay mRNA levels of some cytokines in lung tissues, the cytokines in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA), Western blot analysis was used to determine phosphorylated p38 mitogen-activated protein kinase (MAPK) levels in lung tissues, and nuclear factor-κB (NF-κB) p65 protein levels in lung tissues were detected by immunohistochemistry. Results: Lung alveolar septa destruction, alveolus fusion, inflammatory cell infiltration, and bronchiole exudation were observed. These pathological changes were alleviated in the COPD+HSYA group. The mRNA expression of inflammatory factors were significantly increased in lung tissues from COPD rats (all P<0.01) and were inhibited by HSYA. Levels of inflammatory cytokines in BALF of COPD rats were significantly increased (all P<0.01) which were inhibited by HSYA (all P<0.01, 48, 76.8 mg/kg). The levels of p38 MAPK phosphorylation and p65 in lung tissues of COPD rats were significantly increased (all P<0.01) and were suppressed by HSYA (all P<0.01, 48, 76.8 mg/kg). Conclusions: HSYA could alleviate inflammatory cell infiltration and other pathological changes in the lungs of COPD rats. HSYA inhibited inflammatory cytokine expression, and increase phosphorylation of p38 MAPK and NF-κB p65 in the lungs of COPD rats. The protective mechanism of HSYA to inhibit COPD inflammation might be by attenuating NF-κB and p38MAPK signal transduction. [ABSTRACT FROM AUTHOR]

Abstract Aims Clinical and epidemiological findings point to an association between type 2 diabetes (T2D) and osteoporosis. Genome-wide association studies (GWASs) have been fruitful in identifying some loci potentially associated with osteoporosis and T2D respectively. However, the total genetic variance for each of these two diseases and the shared genetic determination between them are largely unknown. The aim of this study was to identify novel genetic variants for osteoporosis and/or T2D. Methods First, using a pleiotropic conditional false discovery rate (cFDR) method, we analyzed two GWAS summary data of femoral neck bone mineral density (FN_BMD, n = 53,236) and T2D (n = 159,208) to identify novel shared genetic loci. FN_BMD is an important risk factor for osteoporosis. Next, to explore the potential functions of the identified potential pleiotropic genes, differential expression analysis was performed for them in monocytes and peripheral blood mononuclear cells (PBMCs) as these cells are relevant to the etiology of osteoporosis and/or T2D. Further, weighted gene co-expression analysis (WGCNA) was conducted to identify functional connections between novel pleiotropic genes and known osteoporosis/T2D susceptibility genes by using transcriptomic expression datasets in bone biopsies (E-MEXP-1618) and pancreatic islets (GSE50397). Finally, multi-trait fine mapping for the detected pleiotropic risk loci were conducted to identify the SNPs that have the highest probability of being causal for both FN_BMD and T2D. Results We identified 27 significant SNPs with cFDR<0.05 for FN_BMD and 61 SNPs for T2D respectively. Four loci, rs7068487 (PLEKHA1), rs10885421 (TCF7L2), rs944082 (GNG12-AS1 (WLS)) and rs2065929 (PIFO||PGCP1), were found to be potentially pleiotropic and shared between FN_BMD and T2D (ccFDR<0.05). PLEKHA1 was found differentially expressed in circulating monocytes between high and low BMD subjects, and PBMCs between diabetic and non-diabetic conditions. WGCNA showed that PLEKHA1 and TCF7L2 were interconnected with multiple osteoporosis and T2D associated genes in bone biopsy and pancreatic islets, such as JAG, EN1 and CPE. Fine mapping showed that rs11200594 was a potentially causal variant in the locus of PLEKHA1. rs11200594 is also an eQTL of PLEKHA1 in multiple tissue (e.g. peripheral blood cells, adipose and ovary) and is in strong LD with a number of functional variants. Conclusions Four potential pleiotropic loci were identified for shared genetic determination of osteoporosis and T2D. Our study highlights PLEKHA1 as an important potentially pleiotropic gene. The findings may help us gain a better understanding of the shared genetic determination between these two important disorders. Highlights • Identified potential novel variants for complex diseases by cFDR with GWAS statistics • Identified 27 potential novel variants for osteoporosis and 61 for type 2 diabetes • Identified 4 potential pleiotropic variants for osteoporosis and type 2 diabetes [ABSTRACT FROM AUTHOR]

The number of patients with type 2 diabetes mellitus (T2DM) is increasing rapidly worldwide. Glucose transporter 4 (GLUT4) is one of the main proteins that transport blood glucose into the cells and is a target in the treatment of T2DM. In this study, we investigated the mechanism of action of dandelion chloroform extract (DCE) on glucose uptake in L6 cells. The glucose consumption of L6 cell culture supernatant was measured by a glucose uptake assay kit, and the dynamic changes of intracellular GLUT4 and calcium (Ca2+) levels were monitored by laser scanning confocal microscopy in L6 cell lines stably expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM) was traced via myc-GLUT4-mOrange. GLUT4 expression and AMP-activated protein kinase (AMPK), protein kinase B (PKB/Akt), protein kinase C (PKC), and phosphorylation levels were determined by performing western blotting. GLUT4 mRNA expression was detected by real-time PCR. DCE up-regulated GLUT4 expression, promoted GLUT4 translocation and fusion to the membrane eventually leading to glucose uptake, and induced AMPK phosphorylation in L6 cells. The AMPK inhibitory compound C significantly inhibited DCE-induced GLUT4 expression and translocation while no inhibitory effect was observed by the phosphatidylinositol 3-kinase (PI3K) inhibitor Wortmannin and PKC inhibitor Gö6983. These data suggested that DCE promoted GLUT4 expression and transport to the membrane through the AMPK signaling pathway, thereby stimulating GLUT4 fusion with PM to enhance glucose uptake in L6 cells. DCE-induced GLUT4 translocation was also found to be Ca2+-independent. Together, these findings indicate that DCE could be a new hypoglycemic agent for the treatment of T2DM. [ABSTRACT FROM AUTHOR]

Objective: To observe the effect of hydroxysafflor yellow A (HSYA), an active ingredient of a traditional Chinese herbal medicine Carthamus tinctorius L., on lung inflflammation and pulmonary fibrosis induced by bleomycin (BLM) in rats. Methods: Animals were divided into 6 groups including normal group, model group, three HSYA groups and dexamethasone (DXM) group. Three doses of HSYA (35.6, 53.3, and 80.0 mg•kg•day) were intraperitoneally (i.p.) injected in rats for 3 weeks after BLM administration and DXM was used as the positive control ( n=8 or 12). Arterial blood gas was assayed and morphological changes were observed. Lung mRNA expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and some cytokines in lung tissue were detected by real-time polymerase chain reaction. Nuclear factor-κB p65 or α-smooth muscle actin (α-SMA) protein distribution in rat lung tissue was observed by immunohistochemistry. Results: On the 7th day after BLM administration, lung tissue showed serious inflammation. Treatment with HSYA or DXM ameliorated lung inflammation. After treatment with HSYA or DXM, oxygen partial pressure (PaO) increased (HSYA 80.0 mg•kg, P<0.01) and CO partial pressure (PaCO) decreased (HSYA 53.3, 80.0 mg•kg, P<0.05). Moreover, the mRNA expression of TNF-α, IL-1β, and IL-6; and the number of NF-κB p65 positive cells was lower in HSYA 53.3 and 80.0 mg•kg groups than those in the model group (all P<0.05). Twenty-one days after BLM administration, HSYA or DXM treatment ameliorated fibrosis, increased PaO2 (HSYA 53.3, 80.0 mg•kg-1, P<0.01), and decreased PaCO2 (53.3 and 80.0 mg•kg-1, P<0.05). Further, the mRNA expression of TGF-β1, α-SMA, and collagen I as well as the number of α-SMA positive cells increased in the model group and HSYA can attenuate these changes (53.3, 80.0 mg•kg, P<0.05). Hematoxylin and eosin and Masson's trichrome staining indicated that the fibrosis and collagen deposition were ameliorated in HSYA groups (53.3, 80.0 mg•kg, P<0.05). Conclusion: HSYA could alleviate acute lung inflflammation and chronic pulmonary fibrosis induced by BLM in rats. [ABSTRACT FROM AUTHOR]

Osteoporosis is characterized by increased bone loss and deterioration of bone microarchitecture, which will lead to reduced bone strength and increased risk of fragility fractures. Previous studies have identified many genetic loci associated with osteoporosis, but functional mechanisms underlying the associations have rarely been explored. In order to explore the potential molecular functional mechanisms underlying the associations for osteoporosis, we performed integrative analyses by using the publically available datasets and resources. We searched 128 identified osteoporosis associated SNPs (P<10−6), and 8 SNPs exert cis-regulation effects on 11 eQTL target genes. Among the 8 SNPs, 2 SNPs (RPL31 rs2278729 and LRP5 rs3736228) were confirmed to impact the expression of 3 genes (RPL31, CPT1A and MTL5) that were differentially expressed between human subjects of high BMD group and low BMD group. All of the functional evidence suggested the important functional mechanisms underlying the associations of the 2 SNPs (rs2278729 and rs3736228) and 3 genes (RPL31, CPT1A and MTL5) with osteoporosis. This study may provide novel insights into the functional mechanisms underlying the osteoporosis associated genetic variants, which will help us to comprehend the potential mechanisms underlying the genetic association for osteoporosis. [ABSTRACT FROM AUTHOR]

BACKGROUND Some oleaginous fungi can produce large amounts of polyunsaturated fatty acids ( PUFAs) which serve many physiological functions. Numerous desaturases are critical for the synthesis of PUFAs. This study aimed to investigate the regulation of lipid production and desaturase gene expression in Mortierella sp. AGED in response to different environmental factors, and the relationships between lipid production and desaturase gene expression. RESULTS The fatty acid composition and mRNA levels of desaturase genes were significantly changed under low temperatures. With the exception of Δ5-desaturase, the transcript levels of all desaturase genes increased at a temperature of 20 °C. Changes in content of lipid and PUFAs responding to low temperature were consistent with desaturase gene expression. Time course studies on gene expression showed that mRNA levels of four desaturase genes increased rapidly after transferring the cells to low temperature. Ethanol (1.5% v/v) increased the transcript levels of Δ9-, Δ6- and Δ5-desaturase genes significantly and of Δ12-desaturase gene slightly. Different metal ions such as Ca2+, Zn2+ and Fe3+ could stimulate PUFA synthesis and up-regulate desaturase gene transcription, while Cu2+ inhibited desaturase gene expression and lipid accumulation. CONCLUSION This study should enable us to understand the regulatory mechanism of desaturase gene expression and lipid synthesis. It is helpful to improve PUFA productivity in Mortierella sp. AGED. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα. [ABSTRACT FROM AUTHOR]

Integration of multiple profiling data and construction of functional gene networks may provide additional insights into the molecular mechanisms of complex diseases. Osteoporosis is a worldwide public health problem, but the complex gene-gene interactions, post-transcriptional modifications and regulation of functional networks are still unclear. To gain a comprehensive understanding of osteoporosis etiology, transcriptome gene expression microarray, epigenomic miRNA microarray and methylome sequencing were performed simultaneously in 5 high hip BMD (Bone Mineral Density) subjects and 5 low hip BMD subjects. SPIA (Signaling Pathway Impact Analysis) and PCST (Prize Collecting Steiner Tree) algorithm were used to perform pathway-enrichment analysis and construct the interaction networks. Through integrating the transcriptomic and epigenomic data, firstly we identified 3 genes (FAM50A, ZNF473 and TMEM55B) and one miRNA (hsa-mir-4291) which showed the consistent association evidence from both gene expression and methylation data; secondly in network analysis we identified an interaction network module with 12 genes and 11 miRNAs including AKT1, STAT3, STAT5A, FLT3, hsa-mir-141 and hsa-mir-34a which have been associated with BMD in previous studies. This module revealed the crosstalk among miRNAs, mRNAs and DNA methylation and showed four potential regulatory patterns of gene expression to influence the BMD status. In conclusion, the integration of multiple layers of omics can yield in-depth results than analysis of individual omics data respectively. Integrative analysis from transcriptomics and epigenomic data improves our ability to identify causal genetic factors, and more importantly uncover functional regulation pattern of multi-omics for osteoporosis etiology. [ABSTRACT FROM AUTHOR]

Highlights: [•] We present a detailed 3-step protocol for live imaging of polarized primary RPE. [•] Step 1: a protocol for primary porcine retinal pigment epithelial culture. [•] This method results in highly polarized monolayers within 7 days. [•] Step 2: two gene delivery protocols that yield efficient transgene expression. [•] Step 3: High speed live imaging to visualize intracellular traffic in the RPE. [ABSTRACT FROM AUTHOR]

Highlights: [•] H19 is expressed in Bcr-Abl transformants in an Abl kinase-dependent manner. [•] Silencing H19 expression promotes K562 leukemic cells to undergo apoptosis. [•] H19 deficiency inhibits the growth of Bcr-Abl-positive tumor in nude mice. [•] The expression of H19 is regulated by c-Myc in the K562 cells. [Copyright &y& Elsevier]

Atrazine is one of the most widely used herbicides for controlling weeds and grasses. Due to its intensive use, it has become a serious contaminant in soil and water. To evaluate impact of atrazine on graminaceous crops, experiments focusing on atrazine accumulation and toxic response in rice (Oryza sativa) were carried out. Treatment with atrazine at 0.05–0.8mgL−1 for 6d reduced elongation of shoot and root. Compared with a mock treatment, the elongation of shoot with atrazine was 67.1 percent of the control, whereas that of root was 79.5 percent, indicating that the shoot was more affected than the root. Atrazine was readily absorbed by rice from media. Although the quantitative absorption of atrazine was positively correlated with the external supply of the herbicide, translocation of atrazine from roots to the above-ground was reduced from 39.88±6.26 (at 0.05mgL−1) to 9.25±0.27 (0.8mgL−1). While accumulation of atrazine in rice plants led to toxic responses such as over-generation of hydrogen peroxide and superoxide anions, it triggered the plant defense system against the herbicide-induced oxidative stress. This was best presented by the enhanced activities of several antioxidant enzymes (e.g. superoxide dismutase, catalase and peroxidase) and expression of genes responsible for the tolerance to atrazine toxicity. [Copyright &y& Elsevier]

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR) pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs) or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKβ) and IKK-related (IKKε and TBK1) kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser910 and Ser935 as pharmacodynamic biomarkers. [ABSTRACT FROM AUTHOR]

To identify susceptibility loci for schizophrenia, we performed a two-stage genome-wide association study (GWAS) of schizophrenia in the Han Chinese population (GWAS: 746 individuals with schizophrenia and 1,599 healthy controls; validation: 4,027 individuals with schizophrenia and 5,603 healthy controls). We identified two susceptibility loci for schizophrenia at 6p21-p22.1 (rs1233710 in an intron of ZKSCAN4, Pcombined = 4.76 × 10?11, odds ratio (OR) = 0.79; rs1635 in an exon of NKAPL, Pcombined = 6.91 × 10?12, OR = 0.78; rs2142731 in an intron of PGBD1, Pcombined = 5.14 × 10?10, OR = 0.79) and 11p11.2 (rs11038167 near the 5? UTR of TSPAN18, Pcombined = 1.09 × 10?11, OR = 1.29; rs11038172, Pcombined = 7.21 × 10?10, OR = 1.25; rs835784, Pcombined = 2.73 × 10?11, OR = 1.27). These results add to previous evidence of susceptibility loci for schizophrenia at 6p21-p22.1 in the Han Chinese population. We found that NKAPL and ZKSCAN4 were expressed in postnatal day 0 (P0) mouse brain. These findings may lead to new insights into the pathogenesis of schizophrenia. [ABSTRACT FROM AUTHOR]

We have analysed the role of RBR (retinoblastoma related), the Arabidopsis homologue of the tumour suppressor Retinoblastoma protein (pRb), during meiosis. We characterise the rbr-2 mutation, which causes a loss of RBR in male meiocytes. The rbr-2 plants exhibit strongly reduced fertility, while vegetative growth is generally unaffected. The reduced fertility is due to a meiotic defect that results in reduced chiasma formation and subsequent errors in chromosome disjunction. Immunolocalisation studies in wild-type meiocytes reveal that RBR is recruited as foci to the chromosomes during early prophase I in a DNA double-strand-break-dependent manner. In the absence of RBR, expression of several meiotic genes is reduced. The localisation of the recombinases AtRAD51 and AtDMC1 is normal. However, localisation of the MutS homologue AtMSH4 is compromised. Additionally, polymerisation of the synaptonemal complex protein AtZYP1 is abnormal. Together, these data indicate that loss of RBR during meiosis results in a reduction of crossover formation and an associated failure in chromosome synapsis. Our results indicate that RBR has an important role in meiosis affecting different aspects of this complex process. [ABSTRACT FROM AUTHOR]

Simple Summary: Programmed cell death ligand 1 (PD-L1) is an essential immune checkpoint molecule that helps tumor cells to escape the immune surveillance. The aim of the current study was to investigate the epigenetic mechanisms underlying the aberrant expression of PD-L1 in breast cancer cells. Here, we identified TET2 as a negative regulator of PD-L1 gene transcription in breast cancer cells. Mechanistically, TET2 recruits HDAC1/2 to the PD-L1 promoter and facilitates the deacetylation of H3K27ac, resulting to the suppression of PD-L1 gene transcription. Our work reveals an unanticipated role of TET2-HDAC1/2 complex in the regulation of PD-L1 gene expression, providing new insights into the epigenetic mechanisms that drive immune evasion during breast cancer pathogenesis. Activation of PD-1/PD-L1 checkpoint is a critical step for the immune evasion of malignant tumors including breast cancer. However, the epigenetic mechanism underlying the aberrant expression of PD-L1 in breast cancer cells remains poorly understood. To investigate the role of TET2 in the regulation of PD-L1 gene expression, quantitative reverse transcription PCR (RT-qPCR), Western blotting, chromatin immunoprecipitation (ChIP) assay and MeDIP/hMeDIP-qPCR were performed on MCF7 and MDA-MB-231 human breast cancer cells. Here, we reported that TET2 depletion upregulated PD-L1 gene expression in MCF7 cells. Conversely, ectopic expression of TET2 inhibited PD-L1 gene expression in MDA-MB-231 cells. Mechanistically, TET2 protein recruits histone deacetylases (HDACs) to PD-L1 gene promoter and orchestrates a repressive chromatin structure to suppress PD-L1 gene transcription, which is likely independent of DNA demethylation. Consistently, treatment with HDAC inhibitors upregulated PD-L1 gene expression in wild-type (WT) but not TET2 KO MCF7 cells. Furthermore, analysis of the CCLE and TCGA data showed a negative correlation between TET2 and PD-L1 expression in breast cancer. Taken together, our results identify a new epigenetic regulatory mechanism of PD-L1 gene transcription, linking the catalytic activity-independent role of TET2 to the anti-tumor immunity in breast cancer. [ABSTRACT FROM AUTHOR]

Abstract The programmed death-ligand 1 (PD-L1) expression by tumors results in potent antitumor immune suppression through binding to programmed death-1 (PD-1) on T cells and subsequent inhibition of T cells activity. Although recent pathological studies have shown that PD-L1 is actively expressed in certain ERα-negative breast cancer, little is known about whether ER signaling regulates PD-L1 gene expression. Here, we investigated the relationship between ERα and PD-L1 in breast cancer. Analysis of Comprehensive Cell Line Encyclopedia (CCLE) data showed that the average mRNA level of PD-L1 in ERα-positive breast cancer cell lines was significantly lower than that in ERα-negative breast cancer cell lines. E2 treatment inhibited PD-L1 mRNA expression in hormone-depleted ERα-positive MCF7 cells. Moreover, ectopic expression of ERα in triple-negative MDA-MB-231 cells reduced PD-L1 mRNA and protein expression. Consistently, analysis of The Cancer Genome Atlas (TCGA) data revealed an inverse correlation between ERα and PD-L1 expression in ERα-positive breast cancer. Taken together, our results identify ERα as a negative regulator of PD-L1 gene transcription in breast cancer cells, suggesting that ERα loss-of-function may facilitate the immune evasion of breast cancer cells via up-regulation of PD-L1. Highlights • PD-L1 mRNA level is much lower in ERα-positive breast cancer cell lines. • E2 treatment inhibits PD-L1 gene transcription in ERα-positive MCF7 cells. • Ectopic expression of ERα down-regulates PD-L1 gene expression in ERα-negative MDA-MB-231 cells. • ERα expression is negatively correlated with PD-L1 expression in ERα-positive breast cancer. [ABSTRACT FROM AUTHOR]

Highlights: [•] Histamine alleviates astrocyte damage induced by OGD through H1 and H2 receptors. [•] OGD for 6h suppresses GS expression in astrocytes. [•] Histamine reverses GS expression in astrocytes exposed to OGD through H1 receptor. [•] PKC pathway is involved in the regulatory action of histamine on GS expression. [Copyright &y& Elsevier]

Abstract: Peak bone mass (PBM) is an important determinant of osteoporosis. Circulating monocytes serve as early progenitors of osteoclasts and produce important molecules for bone metabolism. To search for genes functionally important for PBM variation, we performed a whole genome gene differential expression study of circulating monocytes in human premenopausal subjects with extremely low (N =12) vs. high (N =14) PBM. We used Affymetrix HG-U133 plus2.0 GeneChip® arrays. We identified 70 differential expression probe sets (p <0.01) corresponding to 49 unique genes. After false discovery rate adjustment, three genes [STAT1, signal transducer and activator of transcription 1; GBP1, guanylate binding protein 1; CXCL10, Chemokine (C-X-C motif) ligand 10] expressed significantly differentially (p <0.05). The RT-PCR results independently confirmed the significantly differential expression of GBP1 gene, and the differential expression trend of STAT1. Functional analyses suggested that the three genes are associated with the osteoclastogenic processes of proliferation, migration, differentiation, migration, chemotaxis, adhesion. Therefore, we may tentatively hypothesize that the three genes may potentially contribute to differential osteoclastogenesis, which may in the end lead to differential PBM. Our results indicate that the GBP1, STAT1 and CXCL10 may be novel risk genes for the differentiation of PBM at the monocyte stage. [Copyright &y& Elsevier]