TrkB-mediated neuroprotection in female hippocampal neurons is autonomous, estrogen receptor alpha-dependent, and eliminated by testosterone: a proposed model for sex differences in neonatal hippocampal neuronal injury.

BACKGROUND: Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of learning disabilities and memory deficits in children. In both human and animal studies, female neonate brains are less susceptible to HI than male brains. Phosphorylation of the nerve growth factor receptor TrkB has been shown to provide sex-specific neuroprotection following in vivo HI in female mice in an estrogen receptor alpha (ERα)-dependent manner. However, the molecular and cellular mechanisms conferring sex-specific neonatal neuroprotection remain incompletely understood. Here, we test whether female neonatal hippocampal neurons express autonomous neuroprotective properties and assess the ability of testosterone (T) to alter this phenotype. METHODS: We cultured sexed hippocampal neurons from ERα+/+ and ERα-/- mice and subjected them to 4 h oxygen glucose deprivation and 24 h reoxygenation (4-OGD/24-REOX). Sexed hippocampal neurons were treated either with vehicle control (VC) or the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following in vitro ischemia. End points at 24 h REOX were TrkB phosphorylation (p-TrkB) and neuronal survival assessed by immunohistochemistry. In addition, in vitro ischemia-mediated ERα gene expression in hippocampal neurons were investigated following testosterone (T) pre-treatment and TrkB antagonist therapy via q-RTPCR. Multifactorial analysis of variance was conducted to test for significant differences between experimental conditions. RESULTS: Under normoxic conditions, administration of 3 µM 7,8-DHF resulted an ERα-dependent increase in p-TrkB immunoexpression that was higher in female, as compared to male neurons. Following 4-OGD/24-REOX, p-TrkB expression increased 20% in both male and female ERα+/+ neurons. However, with 3 µM 7,8-DHF treatment p-TrkB expression increased further in female neurons by 2.81 ± 0.79-fold and was ERα dependent. 4-OGD/24-REOX resulted in a 56% increase in cell death, but only female cells were rescued with 3 µM 7,8-DHF, again in an ERα dependent manner. Following 4-OGD/3-REOX, ERα mRNA increased ~ 3 fold in female neurons. This increase was blocked with either the TrkB antagonist ANA-12 or pre-treatment with T. Pre-treatment with T also blocked the 7,8-DHF- dependent sex-specific neuronal survival in female neurons following 4-OGD/24-REOX. CONCLUSIONS: OGD/REOX results in sex-dependent TrkB phosphorylation in female neurons that increases further with 7,8-DHF treatment. TrkB phosphorylation by 7,8-DHF increased ERα mRNA expression and promoted cell survival preferentially in female hippocampal neurons. The sex-dependent neuroprotective actions of 7,8-DHF were blocked by either ANA-12 or by T pre-treatment. These results are consistent with a model for a female-specific neuroprotective pathway in hippocampal neurons in response to hypoxia. The pathway is activated by 7,8-DHF, mediated by TrkB phosphorylation, dependent on ERα and blocked by pre-exposure to T.

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

Co-delivery of Cas9 mRNA and guide RNAs for editing of LGMN gene represses breast cancer cell metastasis.

Legumain (or asparagine endopeptidase/AEP) is a lysosomal cysteine endopeptidase associated with increased invasive and migratory behavior in a variety of cancers. In this study, co-delivery of Cas9 mRNA and guide RNA (gRNA) by lipid nanoparticles (LNP) for editing of LGMN gene was performed. For in-vitro transcription (IVT) of gRNA, two templates were designed: linearized pUC57-T7-gRNA and T7-gRNA oligos, and the effectiveness of gRNA was verified in multiple ways. Cas9 plasmid was modified and optimized for IVT of Cas9 mRNA. The effects of LGMN gene editing on lysosomal/autophagic function and cancer cell metastasis were investigated. Co-delivery of Cas9 mRNA and gRNA resulted in impaired lysosomal/autophagic degradation, clone formation, migration, and invasion capacity of cancer cells in-vitro. Experimental lung metastasis experiment indicates co-delivery of Cas9 mRNA and gRNA by LNP reduced the migration and invasion capacity of cancer cells in-vivo. These results indicate that co-delivery of Cas9 mRNA and gRNA can enhance the efficiency of CRISPR/Cas9-mediated gene editing in-vitro and in-vivo, and suggest that Cas9 mRNA and gRNA gene editing of LGMN may be a potential treatment for breast tumor metastasis.

[Inhibition of connexin 43-mediated hemichannel activity promotes odontoblast differentiation of human dental pulp cells induced by lipopolysaccharide].

PURPOSE: To investigate the role and mechanism of connexin 43（Cx43）in odontoblast differentiation of human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). METHODS: The maxillary first molar injury model of SD rats was established. The expression pattern of Cx43 in dental pulp repair after injury was detected by immunofluorescence(IF) staining. hDPCs was respectively stimulated with 0, 1, 10, 100 and 1 000 ng/mL LPS for 6 h to screen the optimal concentration, and then the expression of Cx43 was inhibited and overexpressed in hDPCs. Quantitative real-time PCR(qRT-PCR) and Western blot(WB) were used to detect the expression of Cx43 and dentin sialophosphoprotein (DSPP), dental matrix protein-1 (DMP-1), osterix (Osx) and extracellular signal-regulated kinase (ERK) activity. Furthermore, hDPCs were treated with specific Cx43 channel inhibitors to investigate the effect of Cx43-mediated channel activity in odontoblast differentiation of hDPCs, and to explore the role and mechanism of Cx43 in regulating odontoblast differentiation of hDPCs induced by LPS. Statistical analysis was performed with SPSS 26.0 software package. RESULTS: IF results showed that Cx43 was mainly expressed in the odontoblast layer in healthy dental pulp tissues. At 3-24 h after tooth injury, the expression of Cx43 decreased and then gradually increased to the normal level; from 3 days to 2 weeks after injury, the expression of Cx43 tended to be down-regulated which was in the odontoblast layer and pulp proper. The expression of DSPP mRNA was significantly up-regulated in the hDPCs stimulated with 10 ng/mL LPS for 6 h(P＜0.01). Inhibition of Cx43 significantly up-regulated the expression of DSPP, DMP-1 and Osx mRNA induced by LPS in hDPCs(P＜0.05), while overexpression of Cx43 obviously inhibited the expression of factors related to LPS-induced odontoblast differentiation(P＜0.01) and the fluorescence intensity of DSPP. 10 ng/mL LPS activated ERK signal in hDPCs, and overexpression of Cx43 significantly attenuated the activity of ERK signal induced by LPS(P＜0.01). Inhibition of Cx43-mediated hemichannel (HC) promoted mRNA expression of factors related to odontoblast differentiation in hDPCs and the activity of ERK signal induced by LPS(P＜0.05), while blocking Cx43-mediated gap junction channel (GJC) inhibited odontoblast differentiation. CONCLUSIONS: Cx43 participates in the regulation of dental pulp repair after injury, and its expression shows a downward trend as a whole. Inhibition of Cx43 or blocking of HC promotes LPS-induced ERK signal activity and odontoblast differentiation of hDPCs.

[Effects of M2-type macrophages and GKT137831 on oxidative stress in hepatic stellate cells].

Objective: To investigate the effects of reduced nicotinamide adenine dinucleotide phosphooxidase 4 (NOX4) inhibitors GKT137831 and M2-type macrophages on oxidative stress markers NOX4, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the rat hepatic stellate cell line (HSC-T6). Methods: Rat bone marrow macrophages were extracted and induced using interleukin (IL)-4 to differentiate them into M2 phenotype macrophages. HSC-T6 activation was performed with 5 µg/L transforming growth factor ß1 (TGF-ß1). The proliferation condition of HSC-T6 cells stimulated by the NOX4 inhibitor GKT137831 at a concentration gradient of 5 to 80 µmol/L after 48 hours was detected using the Cell Counting Kit-8 (CCK-8) assay. The optimal drug concentration was chosen and divided into an HSC co-culture group (the control group) and five experimental groups: the TGF-ß1 stimulation group, the TGF-ß1 +GKT137831 stimulation group, the M2-type macrophage + HSC co-culture group, the M2-type macrophage +TGF-ß1 stimulation group, and the M2-type + TGF-ß1 + GKT137831 stimulation group. Reactive oxygen species (ROS) production level was detected in each cell using the DCFH-DA probe method. NOX4, α-smooth muscle actin (α-SMA), Nrf2, and HO-1 levels in each group of HSC cells were detected using the qRT-PCR method and the Western blot method. The t-test was used to compare the two groups. The one-way ANOVA method was used to compare multiple groups. Results: Intracellular ROS increased significantly following TGF-ß1 stimulation. ROS relative levels in each cell group were 1.03±0.11, 3.88±0.07, 2.90±0.08, 0.99±0.06, 3.30±0.05, 2.21±0.11, F = 686.1, P = 0.001, respectively. The mRNA and protein expressions of NOX4, α-SMA, Nrf2, and HO-1 were significantly increased (P < 0.05). After the addition of GKT137831, ROS, and NOX4, α-SMA mRNA and protein expression were comparatively decreased in the TGF-ß1 stimulation group (P < 0.05), while mRNA and protein expressions of Nrf2 and HO-1 were increased (P < 0.05). The expression of ROS and NOX4, as well as α-SMA mRNA and protein, produced by HSC were significantly decreased in the co-culture group compared to the single culture group after TGF-ß1 stimulation (P < 0.05). After the addition of GKT137831, ROS, NOX4, α-SMA mRNA, and protein expression were further reduced in the co-culture group compared with the single culture group (P < 0.05), while the mRNA and protein expression of Nrf2 and HO-1 were further increased (P < 0.05). Conclusion: NOX4 inhibitor GKT137831 can reduce RO, NOX4, and α-SMA levels while increasing Nrf2 and HO-1 levels in hepatic stellate cells. After M2-type macrophage co-culture, GKT137831 assists in lowering ROS, NOX4, and α-SMA levels while accelerating Nrf2 and HO-1 levels in hepatic stellate cells, which regulates the balance between oxidative stress and anti-oxidative stress systems, thereby antagonizing the fibrosis process.

Oral post-exercise garlic extract supplementation enhances glycogen replenishment but does not up-regulate mitochondria biogenesis mRNA expression in human-exercised skeletal muscle.

PURPOSE: Garlic extract (GA) is purported to enhance antioxidant and anti-inflammatory activity and glucose regulation in humans. The present study investigated the effects of post-exercise GA supplementation on GLUT4 expression, glycogen replenishment, and the transcript factors involved with mitochondrial biosynthesis in exercised human skeletal muscle. METHODS: The single-blinded crossover counterbalanced study was completed by 12 participants. Participants were randomly divided into either GA (2000 mg of GA) or placebo trials immediately after completing a single bout of cycling exercise at 75% Maximal oxygen uptake (VO2max) for 60 minutes. Participants consumed either GA (2000 mg) or placebo capsules with a high glycemic index carbohydrate meal (2 g carb/body weight) immediately after exercise. Muscle samples were collected at 0-h and 3-h post-exercise. Muscle samples were used to measure glycogen levels, GLUT4 protein expression, as well as transcription factors for glucose uptake, and mitochondria biogenesis. Plasma glucose, insulin, glycerol, non-esterified fatty acid (NEFA) concentrations, and respiratory exchange ratio (RER) were also analyzed during the post-exercise recovery periods. RESULTS: Skeletal muscle glycogen replenishment was significantly elevated during the 3-h recovery period for GA concurrent with no difference in GLUT4 protein expression between the garlic and placebo trials. PGC1-α gene expression was up-regulated for both GA and placebo after exercise (p < 0.05). Transcript factors corresponding to muscle mitochondrial biosynthesis were significantly enhanced under acute garlic supplementation as demonstrated by TFAM and FIS1. However, the gene expression of SIRT1, ERRα, NFR1, NFR2, MFN1, MFN2, OPA1, Beclin-1, DRP1 were not enhanced, nor were there any improvements in GLUT4 expression, following post-exercise garlic supplementation. CONCLUSION: Acute post-exercise garlic supplementation may improve the replenishment of muscle glycogen, but this appears to be unrelated to the gene expression for glucose uptake and mitochondrial biosynthesis in exercised human skeletal muscle.

An easy to use tool for the analysis of subcellular mRNA transcript colocalisation in smFISH data.

Single molecule fluorescence in situ hybridisation (smFISH) has become a valuable tool to investigate the mRNA expression of single cells. However, it requires a considerable amount of programming expertise to use currently available open-source analytical software packages to extract and analyse quantitative data about transcript expression. Here, we present FISHtoFigure, a new software tool developed specifically for the analysis of mRNA abundance and co-expression in QuPath-quantified, multi-labelled smFISH data. FISHtoFigure facilitates the automated spatial analysis of transcripts of interest, allowing users to analyse populations of cells positive for specific combinations of mRNA targets without the need for computational image analysis expertise. As a proof of concept and to demonstrate the capabilities of this new research tool, we have validated FISHtoFigure in multiple biological systems. We used FISHtoFigure to identify an upregulation in the expression of Cd4 by T-cells in the spleens of mice infected with influenza A virus, before analysing more complex data showing crosstalk between microglia and regulatory B-cells in the brains of mice infected with Trypanosoma brucei brucei. These analyses demonstrate the ease of analysing cell expression profiles using FISHtoFigure and the value of this new tool in the field of smFISH data analysis.

Comparative miRNome and transcriptome analyses reveal the expression of novel miRNAs in the panicle of rice implicated in sustained agronomic performance under terminal drought stress.

MAIN CONCLUSION: Differential expression of 128 known and 111 novel miRNAs in the panicle of Nagina 22 under terminal drought stress targeting transcription factors, stress-associated genes, etc., enhances drought tolerance and helps sustain agronomic performance under terminal drought stress. Drought tolerance is a complex multigenic trait, wherein the genes are fine-tuned by coding and non-coding components in mitigating deleterious effects. MicroRNA (miRNA) controls gene expression at post-transcriptional level either by cleaving mRNA (transcript) or by suppressing its translation. miRNAs are known to control developmental processes and abiotic stress tolerance in plants. To identify terminal drought-responsive novel miRNA in contrasting rice cultivars, we constructed small RNA (sRNA) libraries from immature panicles of drought-tolerant rice [Nagina 22 (N 22)] and drought-sensitive (IR 64) cultivars grown under control and terminal drought stress. Our analysis of sRNA-seq data resulted in the identification of 169 known and 148 novel miRNAs in the rice cultivars. Among the novel miRNAs, 68 were up-regulated while 43 were down-regulated in the panicle of N 22 under stress. Interestingly, 31 novel miRNAs up-regulated in N 22 were down-regulated in IR 64, whereas 4 miRNAs down-regulated in N 22 were up-regulated in IR 64 under stress. To detect the effects of miRNA on mRNA expression level, transcriptome analysis was performed, while differential expression of miRNAs and their target genes was validated by RT-qPCR. Targets of the differentially expressed miRNAs include transcription factors and stress-associated genes involved in cellular/metabolic/developmental processes, response to abiotic stress, programmed cell death, photosynthesis, panicle/seed development, and grain yield. Differential expression of the miRNAs could be validated in an independent set of the samples. The findings might be useful in genetic improvement of drought-tolerant rice.

Cardioprotective Potential of Cymbopogon citratus Essential Oil against Isoproterenol-induced Cardiomyocyte Hypertrophy: Possible Involvement of NLRP3 Inflammasome and Oxidative Phosphorylation Complex Subunits.

OBJECTIVE: Cymbopogon citratus (DC.) Stapf is a medicinal and edible herb that is widely used for the treatment of gastric, nervous and hypertensive disorders. In this study, we investigated the cardioprotective effects and mechanisms of the essential oil, the main active ingredient of Cymbopogon citratus, on isoproterenol (ISO)-induced cardiomyocyte hypertrophy. METHODS: The compositions of Cymbopogon citratus essential oil (CCEO) were determined by gas chromatography-mass spectrometry. Cardiomyocytes were pretreated with 16.9 µg/L CCEO for 1 h followed by 10 µmol/L ISO for 24 h. Cardiac hypertrophy-related indicators and NLRP3 inflammasome expression were evaluated. Subsequently, transcriptome sequencing (RNA-seq) and target verification were used to further explore the underlying mechanism. RESULTS: Our results showed that the CCEO mainly included citronellal (45.66%), geraniol (23.32%), and citronellol (10.37%). CCEO inhibited ISO-induced increases in cell surface area and protein content, as well as the upregulation of fetal gene expression. Moreover, CCEO inhibited ISO-induced NLRP3 inflammasome expression, as evidenced by decreased lactate dehydrogenase content and downregulated mRNA levels of NLRP3, ASC, CASP1, GSDMD, and IL-1ß, as well as reduced protein levels of NLRP3, ASC, pro-caspase-1, caspase-1 (p20), GSDMD-FL, GSDMD-N, and pro-IL-1ß. The RNA-seq results showed that CCEO inhibited the increase in the mRNA levels of 26 oxidative phosphorylation complex subunits in ISO-treated cardiomyocytes. Our further experiments confirmed that CCEO suppressed ISO-induced upregulation of mt-Nd1, Sdhd, mt-Cytb, Uqcrq, and mt-Atp6 but had no obvious effects on mt-Col expression. CONCLUSION: CCEO inhibits ISO-induced cardiomyocyte hypertrophy through the suppression of NLRP3 inflammasome expression and the regulation of several oxidative phosphorylation complex subunits.

Comprehensive analysis of circRNA expression profiles in postmenopausal women differing in bone mineral density.

Postmenopausal osteoporosis (PMOP) seriously endangers the bone health of older women. Although there are currently indicators to diagnose PMOP, early diagnostic biomarkers are lacking. Circular ribonucleic acid (circRNA) has a stable structure, regulates gene expression, participates in the pathological process of disease, and has the potential to become a biomarker. The purpose of this study was to investigate circRNAs that could be used to predict patients with early PMOP. Ribonucleic acid (RNA) sequencing was performed on peripheral blood leukocytes from 15 female patients to identify differential circRNAs between different groups. Using bioinformatics analysis, enrichment analysis was performed to discover relevant functions and pathways. CircRNA-micro ribonucleic acid (miRNA) interaction analysis and messenger ribonucleic acid (mRNA) prediction and network construction help us to understand the relationship between circRNA, miRNA, and mRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the gene expression of candidate circRNAs. We screened out 2 co-expressed differential circRNAs, namely hsa_circ_0060849 and hsa_circ_0001394. By analyzing the regulatory network, a total of 54 miRNAs and 57 osteoporosis-related mRNAs were identified, which, as potential downstream target genes of hsa_circ_0060849 and hsa_circ_0001394, may play a key role in the occurrence and development of PMOP. The occurrence and development of PMOP is regulated by circRNAs, and hsa_circ_0060849 and hsa_circ_0001394 can be used as new diagnostic markers and therapeutic targets for early PMOP.

The MMP-9 promoter genetic variant rs3918242, mRNA and protein expression in advanced carotid plaque tissue.

BACKGROUND: The MMP-9 is a known player in atherosclerosis, yet associations of the MMP-9 -1562 C/T variant (rs3918242) with various atherosclerotic phenotypes and tissue mRNA expression are still contradictory. This study aimed to investigate the MMP-9 -1562 C/T variant, its mRNA and protein expression in carotid plaque (CP) tissue, as a risk factor for CP presence and as a marker of different plaque phenotypes (hyperechoic and hypoechoic) in patients undergoing carotid endarterectomy. The MnSOD as an MMP-9 negative regulator was also studied in relation to CP phenotypes. METHODS AND RESULTS: Genotyping of 770 participants (285 controls/485 patients) was done by tetra-primer ARMS PCR. The MMP-9 mRNA expression in 88 human CP tissues was detected by TaqMan® technology. The protein levels of MMP-9 and MnSOD were assessed by Western blot analysis. The MMP-9 -1562 C/T variant was not recognized as a risk factor for plaque presence or in predisposing MMP-9 mRNA and protein levels in plaque tissue. Patients with hypoechoic plaques had significantly lower MMP-9 mRNA and protein levels than those with hyperechoic plaque (p = 0.008, p = 0.003, respectively). MnSOD protein level was significantly higher in hypoechoic plaque compared to hyperechoic (p = 0.039). MMP-9 protein expression in CP tissue was significantly affected by sex and plaque type interaction (p = 0.009). CONCLUSIONS: Considering the differences of MMP-9 mRNA and protein expression in CP tissue regarding different plaque phenotypes and the observed sex-specific effect, the role of MMP-9 in human atherosclerotic plaques should be further elucidated.

Discovery of YS-1 as a cell line of gastric inflammatory cancer-associated fibroblasts.

BACKGROUND: Inflammatory cancer-associated fibroblasts (iCAFs) was first identified by co-culture of pancreatic stellate cells and tumor organoids. The key feature of iCAFs is IL-6high/αSMAlow. We examine this phenomenon in gastric cancer using two cell lines of gastric fibroblasts (HGF and YS-1). METHODS AND RESULTS: HGF or YS-1 were co-cultured with MKN7 (a gastric adenocarcinoma cell line) in Matrigel. IL-6 protein levels in the culture supernatant were measured by ELISA. The increased production of IL-6 was not observed in any of the combinations. Instead, the supernatant of YS-1 exhibited the higher levels of IL-6. YS-1 showed IL-6high/αSMA (ACTA2)low in real-time PCR, mRNA-seq and immunohistochemistry. In mRNA-seq, iCAFs-associated genes and signaling pathways were up-regulated in YS-1. No transition to myofibroblastic phenotype was observed by monolayer culture, or the exposure to sonic hedgehog (SHH) or TGF-ß. YS-1 conditioned medium induced changes of morphology and stem-ness/differentiation in NUGC-3 (a human gastric adenocarcinoma cell line) and UBE6T-15 (a human bone marrow-derived mesenchymal stem cell line). CONCLUSIONS: YS-1 is a stable cell line of gastric iCAFs. This discovery will promote further research on iCAFs for many researchers.

[Dynamic observation on capillarization of liver sinusoidal endothelial cells induced by Echinococcus multilocularis infection].

OBJECTIVE: To investigate the capillarization of liver sinusoidal endothelial cells (LSECs) and its association with hepatic fibrosis during the development of alveolar echinococcosis, so as to provide the basis for unraveling the mechanisms underlying the role of LSEC in the development and prognosis of hepatic injuries and hepatic fibrosis caused by alveolar echinococcosis. METHODS: Forty C57BL/6 mice at ages of 6 to 8 weeks were randomly divided into a control group and 1-, 2- and 4-week infection groups, of 10 mice in each group. Each mouse in the infection groups was intraperitoneally injected with 2 000 Echinococcus multilocularis protoscoleces, while each mouse in the control group was given an equal volume of phosphate-buffered saline using the same method. All mice were sacrificed 1, 2 and 4 weeks post-infection and mouse livers were collected. The pathological changes of livers were observed using hematoxylin-eosin (HE) staining, and hepatic fibrosis was evaluated through semi-quantitative analysis of Masson's trichrome staining-positive areas. The activation of hepatic stellate cells (HSCs) and extracellular matrix (ECM) deposition were examined using immunohistochemical staining of α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1A1), and the fenestrations on the surface of LSECs were observed using scanning electron microscopy. Primary LSECs were isolated from mouse livers, and the mRNA expression of LSEC marker genes Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf was quantified using real-time fluorescence quantitative PCR (qPCR) assay. RESULTS: Destruction of local liver lobular structure was observed in mice 2 weeks post-infection with E. multilocularis protoscoleces, and hydatid cysts, which were surrounded by granulomatous tissues, were found in mouse livers 4 weeks post-infection. Semi-quantitative analysis of Masson's trichrome staining showed a significant difference in the proportion of collagen fiber contents in mouse livers among the four groups (F = 26.060, P < 0.001), and a higher proportion of collagen fiber contents was detected in mouse livers in the 4-week infection group [(11.29 ± 2.58)%] than in the control group (P < 0.001). Immunohistochemical staining revealed activation of a few HSCs and ECM deposition in mouse livers 1 and 2 weeks post-infection, and abundant brown-yellow stained α-SMA and COL1A1 were deposited in the lesion areas in mouse livers 4 weeks post-infection, which spread to surrounding tissues. Semi-quantitative analysis revealed significant differences in α-SMA (F = 7.667, P < 0.05) and COL1A1 expression (F = 6.530, P < 0.05) in mouse levers among the four groups, with higher α-SMA [(7.13 ± 3.68)%] and COL1A1 expression [(13.18 ± 7.20)%] quantified in mouse livers in the 4-week infection group than in the control group (both P values < 0.05). Scanning electron microscopy revealed significant differences in the fenestration frequency (F = 37.730, P < 0.001) and porosity (F = 16.010, P < 0.001) on the surface of mouse LSECs among the four groups, and reduced fenestration frequency and porosity were observed in the 1-[(1.22 ± 0.48)/µm2 and [(3.05 ± 0.91)%] and 2-week infection groups [(3.47 ± 0.10)/µm2 and (7.57 ± 0.23)%] groups than in the control group (all P values < 0.001). There was a significant difference in the average fenestration diameter on the surface of mouse LSECs among the four groups (F = 15.330, P < 0.001), and larger average fenestration diameters were measured in the 1-[(180.80 ± 16.42) nm] and 2-week infection groups [(161.70 ± 3.85) nm] than in the control group (both P values < 0.05). In addition, there were significant differences among the four groups in terms of Stabilin-1 (F = 153.100, P < 0.001), Stabilin-2 (F = 57.010, P < 0.001), Ehd3 (F = 31.700, P < 0.001), CD209b (F = 177.400, P < 0.001), GATA4 (F = 17.740, P < 0.001), and Maf mRNA expression (F = 72.710, P < 0.001), and reduced mRNA expression of Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf genes was quantified in three infection groups than in the control group (all P values < 0.001). CONCLUSIONS: E. multilocularis infections may induce capillarization of LSECs in mice, and result in a reduction in the expression of functional and phenotypic marker genes of LSECs, and capillarization of LSECs occurs earlier than activation of HSC and development of hepatic fibrosis.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Particulate polycyclic aromatic hydrocarbons and metals, DNA methylation and DNA methyltransferase among middle-school students in China.

The main components of particulate matter (PM) had been reported to change DNA methylation levels. However, the mixed effect of PM and its constituents on DNA methylation and the underlying mechanism in children has not been well characterized. To investigate the association between single or mixture exposures and global DNA methylation or DNA methyltransferases (DNMTs), 273 children were recruited (110 in low-exposed area and 163 in high-exposed area) in China. Serum benzo[a]pyridin-7,8-dihydroglycol-9, 10-epoxide (BPDE)-albumin adduct and urinary metals were determined as exposure markers. The global DNA methylation (% 5mC) and the mRNA expression of DNMT1, and DNMT3A were measured. The linear regression, quantile-based g-computation (QGC), and mediation analyses were performed to investigate the effects of individual and mixture exposure. We found that significantly lower levels of % 5mC (P < 0.001) and the mRNA expression of DNMT3A in high-PM exposed group (P = 0.031). After adjustment for age, gender, BMI z-score, detecting status of urinary cotinine, serum folate, and white blood cells, urinary arsenic (As) was negatively correlated with the % 5mC. One IQR increase in urinary As (19.97 µmol/mol creatinine) was associated with a 11.06 % decrease in % 5mC (P = 0.026). Serum BPDE-albumin adduct and urinary cadmium (Cd) were negatively correlated with the levels of DNMT1 and DNMT3A (P < 0.05). Mixture exposure was negatively associated with expression of DNMT3A in QGC analysis (ß: -0.19, P < 0.001). Mixture exposure was significantly associated with decreased % 5mC in the children with non-detected cotinine or normal serum folate (P < 0.05), which the most contributors were PAHs and As. The mediated effect of hypomethylation through DNMT1 or DNMT3A pathway was not observed. Our findings indicated that individual and mixture exposure PAHs and metal components had negative associations with global DNA methylation and decreased DNMT3A expression significantly in school-age individuals.

Post-transcriptional splicing can occur in a slow-moving zone around the gene.

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

m6A methyltransferase AflIme4 orchestrates mycelial growth, development and aflatoxin B1 biosynthesis in Aspergillus flavus.

Aflatoxin B1 (AFB1), a highly toxic secondary metabolite produced by Aspergillus flavus, poses a severe threat to agricultural production, food safety and human health. The methylation of mRNA m6A has been identified as a regulator of both the growth and AFB1 production of A. flavus. However, its intracellular occurrence and function needs to be elucidated. Here, we identified and characterized a m6A methyltransferase, AflIme4, in A. flavus. The enzyme was localized in the cytoplasm, and knockout of AflIme4 significantly reduced the methylation modification level of mRNA. Compared with the control strains, ΔAflIme4 exhibited diminished growth, conidial formation, mycelial hydrophobicity, sclerotium yield, pathogenicity and increased sensitivity to CR, SDS, NaCl and H2O2. Notably, AFB1 production was markedly inhibited in the A. flavus ΔAflIme4 strain. RNA-Seq coupled with RT-qPCR validation showed that the transcriptional levels of genes involved in the AFB1 biosynthesis pathway including aflA, aflG, aflH, aflK, aflL, aflO, aflS, aflV and aflY were significantly upregulated. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) analysis demonstrated a significant increase in m6A methylation modification levels of these pathway-specific genes, concomitant with a decrease in mRNA stability. These results suggest that AflIme4 attenuates the mRNA stability of genes in AFB1 biosynthesis by enhancing their mRNA m6A methylation modification, leading to impaired AFB1 biosynthesis. Our study identifies a novel m6A methyltransferase AflIme4 and highlights it as a potential target to control A. flavus growth, development and aflatoxin pollution.

Effects of moxibustion on intestinal barrier function and TLR4/NF-κB p65 signaling pathway in obese rats. / è‰¾ç¸å¯¹è‚¥èƒ–å¤§é¼ è‚ é“å±éšœåŠŸèƒ½åŠTLR4/NF-κB p65信号通路的影响.

OBJECTIVES: To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. METHODS: Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) ℃. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) ℃. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. RESULTS: Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). CONCLUSIONS: Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.