This study was to investigate the effect of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on cardiomyocyte differentiation of bone marrow mesenchymal stem cells (MSCs) in vitro. Rat MSCs were isolated and induced to differentiate into cardiomyocyte with SAHA or 5-azacytidine (5-aza, a DNA methylation inhibitor) or their combination. Following 7 days of SAHA treatment, the transcriptional expression of the cardiomyocyte-specific genes GATA4, NKx2.5, and Mef2c was dose-dependently increased in the cells with up to 15-fold increase in their mRNA levels over baseline. However, the mRNA levels of these genes were only increased by 2-4 fold in 5-aza-treated cells. After 4 weeks of induction with SAHA, cTnT protein content was substantially increased dose-dependently by up to 8-fold in the cells over the baseline. In contrast, only minimal cTnT protein was found in 5-aza-treated cells. When MSCs were treated with both SAHA and 5-aza, the mRNA levels of GATA4, NKx2.5, and Mef2c and cTnT protein content were the same as those in the cells treated with SAHA alone. These results indicate that SAHA effectively promotes cardiomyocyte differentiation of rat MSCs in vitro. SAHA was a much more potent inducer for cardiac differentiation of MSCs then 5-aza. Our data also indicate that no synergistic or antagonistic effect between SAHA and 5-aza on cardiomyocyte differentiation of MSCs is present, and histone acetylation, not DNA demethylation, may be the dominant mechanism that determines the cardiac differentiation of rat MSCs.

This study was to investigate the effect of high glucose (HG) on vascular endothelial growth factor (VEGF) expression in bone marrow stem cells and JAK2/STAT-3 signalling. Adult rat bone marrow multipotent progenitor cells (rMAPCs) were cultured to evaluate VEGF expression (both mRNA and protein) with or without exposure to HG for up to 48 hrs using RT-PCR and ELISA. JAK2 and STAT3 phosphorylation in rMAPCs was analysed by Western blotting. With cells in normal media, VEGF mRNA level after 24 hrs of culture was significantly increased by 15 times over baseline (day 0) with detectable level of VEGF protein intracellularly using immunofluorescence staining. Although there was no measurable VEGF in the media after 24 hrs of culture, a significant amount of VEGF was detected in the media after 48 hrs of incubation. VEGF expression was associated with constitutive activation of JAK2 and STAT3 in rMAPCs. However, VEGF mRNA level was significantly reduced without detectable VEGF in the media when rMAPCs exposed to HG for 48 hrs. Tyrosine-phosphorylation of JAK2 and STAT3 and nuclear translocation of phosphorylated STAT3 were significantly decreased in the cells exposed to HG for 48 hrs. When JAK2 and STAT3 phosphorylation was blocked by the selective inhibitor AG490, VEGF mRNA level was significantly decreased in rMAPCs in normal media by 80% with no detectable VEGF in the media. VEGF expression was significantly suppressed in rMAPCs cultured in HG media that was further reduced by AG490. VEGF expression in rMAPCs is impaired by HG possibly through inhibition of JAK2/STAT3 signalling.

WRKY transcription factors play an important role in cold defense of plants. However, little information is available about the cold-responsive WRKYs in tomato (Solanum lycopersicum). In the present study, a complete characterization of this gene family was described. Eighty WRKY genes in the tomato genome were identified. Almost all WRKY genes contain putative stress-responsive cis-elements in their promoter regions. Segmental duplications contributed significantly to the expansion of the SlWRKY gene family. Transcriptional analysis revealed notable differential expression in tomato tissues and expression patterns under cold stress, which indicated wide functional divergence in this family. Ten WRKYs in tomato were strongly induced more than 2-fold during cold stress. These genes represented candidate genes for future functional analysis of WRKYs involved in the cold-related signal pathways. Our data provide valuable information about tomato WRKY proteins and form a foundation for future studies of these proteins, especially for those that play an important role in response to cold stress.

The fungal microbiota is an important component of the human gut microbiome and may be linked to gastrointestinal disease. In this study, the fungal microbiota of biopsy samples from adenomas and adjacent tissues was characterized by deep sequencing. Ascomycota, Glomeromycota and Basidiomycota were identified as the dominant phyla in both adenomas and adjacent tissues from all subjects. Among the 60 genera identified, the opportunist pathogens Phoma and Candida represented an average of 45% of the fungal microbiota. When analyzed at the operational taxonomic unit (OTU) level, however, a decreased diversity in adenomas was observed, and three OTUs differed significantly from the adjacent tissues. Principal Component Analysis (PCA) revealed that the core OTUs formed separate clusters for advanced and non-advanced adenomas for which the abundance of four OTUs differed significantly. Moreover, the size of adenomas and the disease stage were closely related to changes in the fungal microbiota in subjects with adenomas. This study characterized the fungal microbiota profile of subjects with adenomas and identified potential diagnostic biomarkers closely related to different stages of adenomas.

The present study was to investigate the effect of hydrogen peroxide (H2O2) on bone marrow stem cells and their endothelial differentiation and the underlying mechanisms in vitro.

A challenge in gene therapy is the efficient delivery of DNA/siRNA to the diseased cells. The physicochemical characteristics of siRNA, such as high molecular weight, negative charges and hydrophilic nature-prevent passive diffusion across the plasma membrane for most cells. A therapeutically feasible carrier for intra-cellular delivery of gene materials should accomplish a series of tasks such as: condensing nucleic acid, protecting nucleic acid from leaking in vivo, facilitating endosome escape and releasing DNA/siRNA to the target site. To meet these requirements, an efficient gene vector based on polycation synthesis for siRNA delivery both in vitro and in vivo was developed.

Since the availability of recombinant human growth hormone (rhGH) enabled the application of human growth hormone both in clinical and research use in the 1980s, millions of patients were prescribed a daily injection of rhGH, but noncompliance rates were high. To address the problem of noncompliance, numerous studies have been carried out, involving: sustained-release preparations, prolonged half-life derivatives, new injectors that cause less pain, and other noninvasive delivery methods such as intranasal, pulmonary and transdermal deliveries. Some accomplishments have been made and launched already, such as the Nutropin Depot microsphere and injectors (Zomajet, Serojet, and NordiFlex). Here, we provide a review of the different technologies and illustrate the key points of these studies to achieve an improved rhGH product.

Transcriptome sequencing has become the main methodology for analyzing the relationship between genes and characteristics of interests, particularly those associated with diseases and economic traits. Because of its role of functional food for humans, commercial royal jelly (RJ) and its production are major research focuses in the field of apiculture. Multiple lines of evidence have demonstrated that many factors affect RJ output by activating or inhibiting various target genes and signaling pathways. Available coding sequences from the Honey Bee Genome Sequencing Consortium have permitted a pathway-based approach for investigating the development of the hypopharyngeal glands (HGs). In the present study, 3573941, 3562730, 3551541, 3524453, and 3615558 clean reads were obtained from the HGs of five full-sister honey bee samples using Solexa RNA sequencing technology. These reads were then assembled into 18378, 17785, 17065, 17105, and 17995 unigenes, respectively, and aligned to the DFCI Honey Bee Gene Index database. The differentially expressed genes (DEGs) data were also correlated with detailed morphological data for HGs acini.

Low-density lipoprotein (LDL) is non-atherogenic, while oxidized LDL (ox-LDL) is critical to atherosclerosis. N-acetylcysteine (NAC) has anti-atherosclerotic effect with largely unknown mechanisms. The present study aimed to determine if NAC could attenuate in vivo LDL oxidation and inhibit atherosclerosis. A single dose of human native LDL was injected intravenously into male C57BL/6 mice with and without NAC treatment. Serum human ox-LDL was detected 30 min after injection, reached the peak in 3 hours, and became undetectable in 12 hours. NAC treatment significantly reduced serum ox-LDL level without detectable serum ox-LDL 6 hours after LDL injection. No difference in ox-LDL clearance was observed in NAC-treated animals. NAC treatment also significantly decreased serum ox-LDL level in patients with coronary artery diseases and hyperlipidemia without effect on LDL level. Intracellular and extracellular reactive oxidative species (ROS) production was significantly increased in the animals treated with native LDL, or ox-LDL and in hyperlipidemic LDL receptor knockout (LDLR(-/-)) mice that was effectively prevented with NAC treatment. NAC also significantly reduced atherosclerotic plaque formation in hyperlipidemic LDLR(-/-) mice. NAC attenuated in vivo oxidation of native LDL and ROS formation from ox-LDL associated with decreased atherosclerotic plaque formation in hyperlipidemia.

Dopamine plays an important role in regulating neuronal functions in the central nervous system by activating the specific G-protein coupled receptors. Both D1 and D2 dopamine receptors are extensively distributed in the retinal neurons. In the present study, we investigated the effects of D1 receptor signaling on outward K(+) currents in acutely isolated rat retinal ganglion cells (RGCs) by patch-clamp techniques. Extracellular application of SKF81297 (10 μM), a specific D1 receptor agonist, significantly and reversibly suppressed outward K(+) currents of the cells, which was reversed by SCH23390 (10 μM), a selective D1 receptor antagonist. We further showed that SKF81297 mainly suppressed the glybenclamide (Gb)- and 4-aminopyridine (4-AP)-sensitive K(+) current components, but did not show effect on the tetraethylammonium (TEA)-sensitive one. Both protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways were likely involved in the SKF81297-induced suppression of the K(+) currents since either Rp-cAMP (10 μM), a cAMP/PKA signaling inhibitor, or KN-93 (10 μM), a specific CaMKII inhibitor, eliminated the SKF81297 effect. In contrast, neither protein kinase C (PKC) nor mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway seemed likely to be involved because both the PKC inhibitor bisindolylmaleimide IV (Bis IV) (10 μM) and the MAPK/ERK1/2 inhibitor U0126 (10 μM) did not block the SKF81297-induced suppression of the K(+) currents. These results suggest that activation of D1 receptors suppresses the Gb- and 4-AP-sensitive K(+) current components in rat RGCs through the intracellular PKA and CaMKII signaling pathways, thus modulating the RGC excitability.

A number of studies have showed that High mobility group box-1 (HMGB1), which played key role in inflammation activation by triggering the toll like receptor 4 (TLR4) signaling axis in hepatic fibrogenesis, may share similar inflammation stimulating mechanism with LPS. Herein, we introduced a recently established anti-LPS protein cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2) to investigate endogenous protection mechanism of HMGB1 induced inflammatory response. Our results shows that stromal cells and monocytes showed an evaluated pattern for CRISPLD2 expression after HMGB1 treatment, which was dependent on the integrity of TLR4 function. Pro-inflammatory Cytokines levels were significantly elevated after CRISPLD2 silencing despite the HMGB1 status. Soluble CRISPLD2 administration relieve the HMGB1 dependent pro-inflammatory cytokines release. Interestingly, we found that miRNA 155 play a key role in the process. Our data suggest that CRISPLD2 may have a unique anti-HMGB1 effect via miRNA and play an important role in immune balance.

5-HT1A autoreceptor stimulation can act to attenuate supraphysiological swings in extracellular dopamine levels following long-term levodopa treatment and may be useful in the treatment and prevention of the motor complications. The purpose of this study was to investigate cellular and behavioral effects of 5-HT1A receptor agonist 8-OH-DPAT in a rat model of levodopa-induced motor complications. Two sets of experiments were performed. First, animals were treated with levodopa (50 mg/kg with benserazide 12.5 mg/kg, twice daily), intraperitoneally (i.p.) for 22 days. On day 23, animals received either 8-OH-DPAT (1 mg/kg, i.p.) or 8-OH-DPAT plus WAY-100635 (0.1 mg/kg, i.p) or vehicle with each levodopa dose. In the second set, animals were treated either with levodopa (50 mg/kg, i.p.) plus 8-OH-DPAT (1 mg/kg, i.p.) or levodopa (50 mg/kg, i.p.) plus vehicle, administered twice daily for 22 consecutive days. Our study showed that 8-OH-DPAT plus levodopa both prolonged the duration of the motor response and reduced peak turning. 8-OH-DPAT plus levodopa also decreased the frequency of failures to levodopa. Co-administration of WAY-100635, a 5-HT1A receptor antagonist, with 8-OH-DPAT eliminated the effect of 8-OH-DPAT on motor complications indicating that the observed 8-OH-DPAT responses were probably mediated at the 5-HT1A autoreceptor. Moreover, 8-OH-DPAT plus levodopa significantly reduced hyperphosphorylation of GluR1 at serine 845, which was closely associated with levodopa-induced motor complications.

The bacterial endotoxin lipopolysaccharide (LPS), a key pathogenic stimulator, can induce the activation of macrophages. Activin receptor-interacting protein 2 (ARIP2), an intracellular signaling protein, has a wide histological distribution, however, whether ARIP2 is involved in regulation of activation of macrophages was not well characterized. Here, by immunocytochemical staining, we found that ARIP2 protein existed in monocyte-macrophage cell line RAW264.7 cells and peritoneal macrophages of mouse, and ARIP2 expression in RAW264.7 cells was up-regulated by LPS. Furthermore, the results revealed that ARIP2 overexpression in the LPS-activated RAW264.7 cells inhibited the productions of IL-1β and TNFα, phagocytic activities and CD14 expression, whereas did not alter expressions of MyD88, TLR2 and TLR4. Additionally, in vivo ARIP2 overexpression also reduced the productions of IL-1β and TNFα from the LPS-stimulated peritoneal macrophages of mouse. These data suggest that ARIP2 may play an anti-inflammatory role in macrophages via inhibiting CD14 expression.

HIGHLIGHTS The aim of the research was to determine the functional effects and molecular mechanisms of GABAB receptor on ischemia reperfusion-induced gastric injury in rats.The lateral hypothalamus area GABAB receptor attenuated the ischemia reperfusion-induced gastric injury by up-regulating the production of GABA, GABABR, and down-regulating P-GABABR in the brain.This work would provide a new therapeutic strategy for acute gastric injury. Gastric ischemia-reperfusion (GI-R) injury progression is largely associated with excessive activation of the greater splanchnic nerve (GSN). This study aims to investigate the protective effects of GABAB receptor (GABABR) in the lateral hypothalamic area (LHA) on GI-R injury. A model of GI-R injury was established by clamping the celiac artery for 30 min and then reperfusion for 1 h. The coordinate of FN and LHA was identified in Stereotaxic Coordinates and then the L-Glu was microinjected into FN, GABAB receptor agonist baclofen, or GABAB receptor antagonist CGP35348 was microinjected into the LHA, finally the GI-R model was prepared. The expression of GABABR, P-GABABR, NOX2, NOX4, and SOD in the LHA was detected by western blot, PCR, and RT-PCR. The expression of IL-1β, NOX2, and NXO4 in gastric mucosa was detected by western blot. We found that microinjection of L-Glu into the FN or GABAB receptor agonist (baclofen) into the LHA attenuated GI-R injury. Pretreatment with GABAB receptor antagonist CGP35348 reversed the protective effects of FN stimulation or baclofen into the LHA. Microinjection of baclofen into the LHA obviously reduced the expression of inflammatory factor IL-1β, NOX2, and NOX4 in the gastric mucosa. Conclusion: The protective effects of microinjection of GABABR agonist into LHA on GI-R injury in rats could be mediated by up-regulating the production of GABA, GABABR, and down-regulating P-GABABR in the LHA.

Previously, we found several genes are involved in myocardial ischemia-reperfusion (M-I/R) injury. In this report, we first developed a mouse model of M-I/R injury and demonstrated microRNA-126a-5p was associated with the M-I/R injury by using high-throughput microRNA expression analysis. We further investigated the expression and function of microRNA-126a-5p during mouse M-I/R injury. We observed high expression of microRNA-126a-5p in the M-I/R mice and increased levels of LDH and CK-MB (damage markers) in the serum. H2O2 and hypoxia/reoxygenation (H/R) treatment significantly increased the expression of microRNA-126a-5p in H9C2 cells in concentration- and time-dependent manners. Moreover, microRNA-126a-5p overexpression in H9C2 cells inhibited cell viability but increased LDH release and caspase 3 activity. Cardiac function analysis based on the measurements of hemodynamic parameters showed that microRNA-126a-5p expression ablation in M-I/R injured mice led to the reversal of the symptoms caused by M-I/R injury. Transesophageal echocardiography also revealed that the values of LVIDd and LVIDs were decreased while the values of LVFS% and LVEF% were increased in M-I/R injured mice after treatment with microRNA-126a-5p inhibitor, compared with the M-I/R injured mice treated with the control. Bioinformatic analysis demonstrated that Hspb8, a protective protein in myocardium, was the target of microRNA-126a-5p. Thus, these findings indicated that microRNA-126a-5p was up-regulated in mouse M-I/R model and promoted M-I/R injury in vivo through suppressing the expression of Hspb8, which may shed light on the development of potential therapeutic target for M-I/R injury.

More than 1000 candidate-gene association studies on genetic susceptibility to lung cancer have been published over the last two decades but with few consensuses for the likely culprits. We conducted a comprehensive review, meta-analysis and evidence strength evaluation of published candidate-gene association studies in lung cancer up to November 1, 2015. The epidemiological credibility of cumulative evidence was assessed using the Venice criteria. A total of 1018 publications with 2910 genetic variants in 754 different genes or chromosomal loci were eligible for inclusion. Main meta-analyses were performed on 246 variants in 138 different genes. Twenty-two variants from 21 genes (APEX1 rs1130409 and rs1760944, ATM rs664677, AXIN2 rs2240308, CHRNA3 rs6495309, CHRNA5 rs16969968, CLPTM1L rs402710, CXCR2 rs1126579, CYP1A1 rs4646903, CYP2E1 rs6413432, ERCC1 rs11615, ERCC2 rs13181, FGFR4 rs351855, HYKK rs931794, MIR146A rs2910164, MIR196A2 rs11614913, OGG1 rs1052133, PON1 rs662, REV3L rs462779, SOD2 rs4880, TERT rs2736098, and TP53 rs1042522) showed significant associations with lung cancer susceptibility with strong cumulative epidemiological evidence. No significant associations with lung cancer risk were found for other 150 variants in 98 genes; however, seven variants demonstrated strong cumulative evidence. Our findings provided the most updated summary of genetic risk effects on lung cancer and would help inform future research direction.

To develop a useful score for predicting the prognosis of severe corona virus disease 2019 (COVID-19) patients.We retrospectively analyzed patients with severe COVID-19 who were admitted from February 10, 2020 to April 5, 2020. First, all patients were randomly assigned to a training cohort or a validation cohort. By univariate analysis of the training cohort, we developed combination scores and screened the superior score for predicting the prognosis. Subsequently, we identified the independent factors influencing prognosis. Finally, we demonstrated the predictive efficiency of the score in validation cohort.A total of 145 patients were enrolled. In the training cohort, nonsurvivors had higher levels of lactic dehydrogenase than survivors. Among the 7 combination scores that were developed, lactic dehydrogenase-lymphocyte ratio (LLR) had the highest area under the curve (AUC) value for predicting prognosis, and it was associated with the incidence of liver injury, renal injury, and higher disseminated intravascular coagulation (DIC) score on admission. Univariate logistic regression analysis revealed that C-reactive protein, DIC score ≥2 and LLR >345 were the factors associated with prognosis. Multivariate analysis showed that only LLR >345 was an independent risk factor for prognosis (odds ratio [OR] = 9.176, 95% confidence interval [CI]: 2.674-31.487, P < .001). Lastly, we confirmed that LLR was also an independent risk factor for prognosis in severe COVID-19 patients in the validation cohort where the AUC was 0.857 (95% CI: 0.718-0.997).LLR is an accurate predictive score for poor prognosis of severe COVID-19 patients.

Formononetin (FMNT) is a major bioactive compound from Astragalus membranaceus (Fisch.) Bunge, and has been widely used to treat conditions related to vascular insufficiency. However, the molecular mechanism for the therapeutic effect has not been well defined. This study aimed to investigate the effect and mechanism of FMNT on endothelial function. The potential targets and signaling pathways of FMNT in the setting of ischemia were predicted using network pharmacology analysis. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro studies and C57BL/6 mice were used for in vivo experiments. The results of the network pharmacology analysis showed that multiple signaling molecules including MAPK and PI3K-Akt pathways could be involved in the pharmacological actions of FMNT against ischemic diseases. The experimental validation data showed that FMNT significantly promoted the growth, proliferation, migration and tube formation of HUVECs in association with activation of endothelial nitric oxide synthase (eNOS) and promotion of intracellular nitric oxide (NO) production. FMNT also markedly activated Erk1/2 and Akt signaling in HUVECs. The enhanced endothelial function by FMNT was abolished when the cells were pre-treated with eNOS inhibitor. FMNT-induced eNOS/NO activation, endothelial function and angiogenesis was also effectively attenuated when Erk1/2 or Akt signaling pathway was inhibited. In addition, FMNT significantly promoted wound healing in C57BL/6 mice associated with activation of Erk1/2 and Akt signaling. Enhanced wound healing by FMNT in mice was prevented when eNOS-, Erk1/2, or Akt-medicated signaling was inhibited. Moreover, when Akt signaling was inhibited in HUVECs, FMNT was still able to activate Erk1/2 signaling without promotion of endothelial function. Similarly, FMNT could activate Akt signaling with no change in endothelial function when Erk1/2 signaling was attenuated in HUVECs. Conclusively, the present study demonstrated that FMNT significantly enhanced endothelial function and promoted angiogenesis in vitro and in vivo through activating Erk1/2- and Akt-mediated eNOS/NO signaling pathway. The data also suggested that simultaneous activation of Erk1/2 and Akt signaling was required for FMNT-induced promotion of endothelial function. Results from the present study might provide support and evidence for the application of FMNT during the clinical treatment of conditions related to vascular insufficiency.

The polyandrous mating system of honeybees (Apis mellifera L.) has garnered widespread attention. Long-lived honeybee queens only mate early in maturation, and the sperm obtained from the aerial mating is stored in the spermatheca. The maintenance of sperm viability in the spermatheca is an intriguing and complex process. However, the key physiological and biochemical adaptations underlying the long-term storage of sperm remain unclear. Analysis of the metabolite profile could help better understand the biology of the spermatheca and offer insights into the breeding and conservation of honeybees and even pest control strategies. Here, the changes in metabolites in the spermatheca were quantified between virgin queens and new-laying queens (with stored sperm) via liquid chromatography-mass spectrometry. Compared with virgin queens, changes occurred in lipids and lipid-like molecules, including fatty acyls and glycerophospholipids (GPL), prenol lipids, and sterol lipids, during storage of sperm in new-laying honeybee queens. Furthermore, the metabolic pathways that were enriched with the differentially expressed metabolites were identified and included GPL metabolism, biosynthesis of amino acids, and the mTOR signaling pathway. The likely roles of the pathways in the maintenance and protection of sperm are discussed. The study identifies key metabolites and pathways in the complex interplay of substances that contribute to the long-term storage of sperm and ultimately reproductive success of honeybee queens.

UDP-glucuronosyltransferases (UGTs), being multifunctional detoxification enzymes, play a major role in the process of resistance to various pesticides in insects. However, the mechanism underlying the molecular regulation of pesticide resistance remains unclear, especially in Apis cerana cerana. In this study, all of the UGTs in Apis cerana cerana (AccUGT) have been identified through the multiple alignment and phylogenetic analysis. Expression of AccUGT genes under different pesticides, and antioxidant genes after silencing of AccUGT2B20-like, were detected by qRT-PCR. The resistance of overexpressed AccUGT2B20-like to oxidative stress was investigated by an Escherichia coli overexpression system. Also, antioxidant-related enzyme activity was detected after silencing of the AccUGT2B20-like gene. Expression pattern analysis showed that almost all UGT genes were upregulated under different pesticide treatments. This result indicated that AccUGTs participate in the detoxification process of pesticides. AccUGT2B20-like was the major gene because it was more highly induced than the others. Overexpression of AccUGT2B20-like in E. coli could effectively improve oxidative stress resistance. Specifically, silencing the AccUGT2B20-like gene increased oxidative stress by repressing the expression of oxidation-related genes, decreasing antioxidant-related enzyme activity, and increasing malondialdehyde concentration. Taken together, our results indicate that AccUGTs are involved in pesticide resistance, among which, AccUGT2B20-like contributes to the detoxification of pesticides by eliminating oxidative stress in Apis cerana cerana. This study explains the molecular basis for the resistance of bees to pesticides and provides an important safeguard for maintaining ecological balance.