Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification.

Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage and isothermal amplification is the primary obstacle to quantitative detection. Though several works have demonstrated enhanced detection sensitivity by reducing the inhibition of CRISPR on amplification in one pot, few paid attention to the amplification process and even dynamic reaction processes between the two. Herein, we find that DNA quantification can be realized by regulating either recombinase polymerase amplification (RPA) efficiency or CRISPR/Cas12a cleaving efficiency (namely, tuning the dynamic reaction balance) in one pot. The sensitive quantification is realized by utilizing dual PAM-free crRNAs for CRISPR/Cas12a recognition. The varied RPA primer concentration with stabilized CRISPR systems significantly affects the amplification efficiency and quantitative performances. Alternatively, quantitative detection can also be achieved by stabilizing the amplification process while regulating the CRISPR/Cas12a concentration. The quantitative capability is proved by detecting DNA targets from Lactobacillus acetotolerans and SARS-CoV-2. The quantitative performance toward real samples is comparable to quantitative real-time PCR for detecting L. acetotolerans spiked in fermented food samples and SARS-CoV-2 clinical samples. We expect that the presented method will be a powerful tool for quantifying other nucleic acid targets.

A novel multiscale framework for delineating cancer evolution from subclonal compositions.

OBJECTIVE: Owing to the heterogeneity in the evolution of cancer, distinguishing between diverse growth patterns and predicting long-term outcomes based on short-term measurements poses a great challenge. METHODS: A novel multiscale framework is proposed to unravel the connections between the population dynamics of cancer growth (i.e., aggressive, bounded, and indolent) and the cellular-subclonal dynamics of cancer evolution. This framework employs the non-negative lasso (NN-LASSO) algorithm to forge a link between an ordinary differential equation (ODE)-based population model and a cellular evolution model. RESULTS: The findings of our current work not only affirm the impact of subclonal composition on growth dynamics but also identify two significant subclones within heterogeneous growth patterns. Moreover, the subclonal compositions at the initial time are able to accurately discriminate diverse growth patterns through a machine learning algorithm. CONCLUSION: The proposed multiscale framework successfully delineates the intricate landscape of cancer evolution, bridging the gap between long-term growth dynamics and short-term measurements, both in simulated and real-world data. This methodology provides a novel avenue for thorough exploration into the realm of cancer evolution.

Wnt10b regulates osteogenesis of adipose-derived stem cells through Wnt/ß-catenin signalling pathway in osteoporosis.

Our previous finding revealed that the Wnt10b RNA expression of osteoporotic adipose-derived stem cells (OP-ASCs) with impaired osteogenic capacity was significantly reduced than that of ASCs. There are no ideas that the relationship between the OP-ASCs' impaired osteogenic potential and Wnt10b expression. This study aimed to indicate the potential molecular mechanisms and functional role of Wnt10b in OP-ASCs, as well as to investigate a potential application to reverse the OP-ASCs' impaired osteogenic differentiation potential. The OP-ASCs and ASCs were harvested from the inguinal fat of osteoporosis (OP) mice with bilateral ovariectomy (OVX) and normal mice. qPCR and WB were used to detect the different levels of the expression of the Wnt10b RNA in both OP-ASCs and ASCs. Lentiviral-mediated regulation of Wnt10b expression was employed for OP-ASCs, and the detection of the expression levels of key molecules in the Wnt signalling pathway and key osteogenic factors was performed through qPCR and WB in vitro experiments. The capacity of OP-ASCs to osteogenesis was determined using alizarin red staining. Lastly, the repair effect of the BCP scaffolds incorporating modified OP-ASCs on the critical-sized calvarial defects (CSCDs) in OP mice was scanned and detected by micro-computed tomography, haematoxylin and eosin staining, Masson's trichrome staining and immunohistochemistry. First, we discovered that both the RNA and protein expression levels of Wnt10b were significantly lower in OP-ASCs than that in ASCs. In vitro experiments, upregulation of Wnt10b could activate the Wnt signalling pathway, and increase expression of ß-catenin, Lef1, Runx2 and osteopontin (Opn), thereby enhancing the osteogenic ability of OP-ASCs. In addition, the OP-ASCs with Wnt10b-overexpressing could promote the repair of CSCD in osteoporotic mice with increasing new bone volume, bone mineral density, and increased expression of Opn in new bone in vivo. Taken together, overexpression of Wnt10b could partially facilitate the differentiation of OP-ASCs towards osteogenesis and accelerated the healing of bone defects by activating the Wnt/ß-catenin signalling pathway in vitro and in vivo experiments. This study confirmed the important role of Wnt10b in regulating the osteogenic differentiation capability of OP-ASCs and indicated Wnt10b could be a potential therapeutic target for reversing the impaired osteogenic capabilities of OP-ASCs to therapy bone defects of OP patients.

A thermal receptor for nonvisual sunlight detection in myriapods.

Organisms from cyanobacteria to humans have evolved a wide array of photoreceptive strategies to detect light. Sunlight avoidance behavior is common in animals without vision or known photosensory genes. While indirect light perception via photothermal conversion is a possible scenario, there is no experimental evidence for this hypothesis. Here, we show a nonvisual and extraocular sunlight detection mechanism by identifying the broad-range thermal receptor 1 (BRTNaC1, temperature range = 33 to 48 °C) in centipede antennae. BRTNaC1, a heat-activated cation-permeable ion channel, is structurally related to members of the epithelial sodium channel family. At the molecular level, heat activation of BRTNaC1 exhibits strong pH dependence controlled by two protonatable sites. Physiologically, temperature-dependent activation of BRTNaC1 upon sunlight exposure comes from a striking photothermal effect on the antennae, where a slightly acidic environment (pH 6.1) of the body fluid leads to the protonation of BRTNaC1 and switches on its high thermal sensitivity. Furthermore, testosterone potently inhibits heat activation of BRTNaC1 and the sunlight avoidance behavior of centipedes. Taken together, our study suggests a sophisticated strategy for nonvisual sunlight detection in myriapods.

Challenges and perspectives of quantitative microbiome profiling in food fermentations.

Spontaneously fermented foods are consumed and appreciated for thousands of years although they are usually produced with fluctuated productivity and quality, potentially threatening both food safety and food security. To guarantee consistent fermentation productivity and quality, it is essential to control the complex microbiota, the most crucial factor in food fermentations. The prerequisite for the control is to comprehensively understand the structure and function of the microbiota. How to quantify the actual microbiota is of paramount importance. Among various microbial quantitative methods evolved, quantitative microbiome profiling, namely to quantify all microbial taxa by absolute abundance, is the best method to understand the complex microbiota, although it is still at its pioneering stage for food fermentations. Here, we provide an overview of microbial quantitative methods, including the development from conventional methods to the advanced quantitative microbiome profiling, and the application examples of these methods. Moreover, we address potential challenges and perspectives of quantitative microbiome profiling methods, as well as future research needs for the ultimate goal of rational and optimal control of microbiota in spontaneous food fermentations. Our review can serve as reference for the traditional food fermentation sector for stable fermentation productivity, quality and safety.

Identification of tumor microenvironment-related signature for predicting prognosis and immunotherapy response in patients with bladder cancer.

The tumor microenvironment (TME) not only provides fertile soil for tumor growth and development but also widely involves immune evasion as well as the resistance towards therapeutic response. Accumulating interest has been attracted from the biological function of TME to its effects on patient outcomes and treatment efficacy. However, the relationship between the TME-related gene expression profiles and the prognosis of bladder cancer (BLCA) remains unclear. The TME-related genes expression data of BLCA were collected from The Cancer Genome Atlas (TCGA) database. NFM algorithm was used to identify the distinct molecular pattern based on the significantly different TME-related genes. LASSO regression and Cox regression analyses were conducted to identify TME-related gene markers related to the prognosis of BLCA and to establish a prognostic model. The predictive efficacy of the risk model was verified through integrated bioinformatics analyses. Herein, 10 TME-related genes (PFKFB4, P4HB, OR2B6, OCIAD2, OAS1, KCNJ15, AHNAK, RAC3, EMP1, and PRKY) were identified to construct the prognostic model. The established risk scores were able to predict outcomes at 1, 3, and 5 years with greater accuracy than previously known models. Moreover, the risk score was closely associated with immune cell infiltration and the immunoregulatory genes including T cell exhaustion markers. Notably, the predictive power of the model in immunotherapy sensitivity was verified when it was applied to patients with metastatic urothelial carcinoma (mUC) undergoing immunotherapy. In conclusion, TME risk score can function as an independent prognostic biomarker and a predictor for evaluating immunotherapy response in BLCA patients, which provides recommendations for improving patients' response to immunotherapy and promoting personalized tumor immunotherapy in the future.

FGFBP1 as a potential biomarker predicting bacillus Calmette-Guérin response in bladder cancer.

Accurate prediction of Bacillus Calmette-Guérin (BCG) response is essential to identify bladder cancer (BCa) patients most likely to respond sustainably, but no molecular marker predicting BCG response is available in clinical routine. Therefore, we first identified that fibroblast growth factor binding protein 1 (FGFBP1) was upregulated in failures of BCG therapy, and the increased FGFBP1 had a poor outcome for BCa patients in the E-MTAB-4321 and GSE19423 datasets. These different expression genes associated with FGFBP1 expression are mainly involved in neutrophil activation, neutrophil-mediated immunity, and tumor necrosis factor-mediated signal pathways in biological processes. A significant positive correlation was observed between FGFBP1 expression and regulatory T-cell (Treg) infiltration by the Spearman correlation test in the BCG cohort (r = 0.177) and The Cancer Genome Atlas (TCGA) cohort (r = 0.176), suggesting that FGFBP1 may influence the response of BCa patients to BCG immunotherapy through immune escape. Though FGFBP1 expression was positively correlated with the expressions of PD-L1, CTLA4, and PDCD1 in TCGA cohort, a strong association between FGFBP1 and PD-L1 expression was only detected in the BCG cohort (r = 0.750). Furthermore, elevated FGFBP1 was observed in BCa cell lines and tissues in comparison to corresponding normal controls by RT-qPCR, Western blotting, and immunohistochemical staining. Increased FGFBP1 was further detected in the failures than in the responders by immunohistochemical staining. Notably, FGFBP1 is positively associated with PD-L1 expression in BCa patients with BCG treatment. To sum up, FGFBP1 in BCa tissue could be identified as a promising biomarker for the accurate prediction of BCG response in BCa.

Multifunctional Surface Construction for Long-Term Cycling Stability of Li-Rich Mn-Based Layered Oxide Cathode for Li-Ion Batteries.

Li-rich Mn-based layered oxides (LMLOs) are promising cathode material candidate for the next-generation Li-ion batteries (LIBs) of high energy density. However, the fast capacity fading and voltage decay as well as low Coulombic efficiency caused by irreversible oxygen release and phase transition during the electrochemical process hinder their practical application. To solve these problems, in the present study, a multifunctional surface construction involving a coating layer, spinel-layered heterostructure, and rich-in oxygen vacancies is successfully conducted by a facile thermal reduction of the LMLO particles with potassium borohydride (KBH4 ) as the reducing agent. The multifunctional surface structure plays synergistic effects on suppressing the interface side reaction, reducing the dissolution of transition metal, increasing electron conductivity and lithium diffusion rate. As a result, electrochemical performances of the LMLO cathode are effectively enhanced. With optimization of the addition of KBH4 , the electrode delivers a reversible capacity of 280 mAh g-1 at 0.1 C, which maintains after 100 cycles. The capacity retention with respect to the initial capacity is as high as 98% at 1 C after 400 cycles. The present work provides insights into designing a highly effective functional surface structure of LMLO cathode materials for high-performance LIBs.

Safety and immunogenicity of a quadrivalent, inactivated, split-virion influenza vaccine (IIV4-W) in healthy people aged 3-60 years: a phase III randomized clinical noninferiority trial.

BACKGROUND: A quadrivalent split influenza vaccine IIV4-W against both influenza A and B viruses is urgently needed. METHODS: To evaluate the safety and immunogenicity of IIV4-W in people aged 3-60 years, 2400 participants recruited in a double-blind phase III trial and were randomly assigned to the IIV4-W, TIV1 and TIV2 groups. The immunogenicity indicators were measured at 28 days postvaccination and for 180 days for safety follow-up. RESULTS: Adverse events (AEs) occurred in 162 (20.28%), 116 (14.55%) and 123 (15.41%) participants in the IIV4-W, TIV1 and TIV2 groups, respectively. All these AEs were mild and self-limiting, and no serious AEs related to the vaccines were observed. IIV4-W elicited a non-inferior immune response for matched strains (the lower limit of 95% CI for GMT ratio >0.67, for SCR and SPR difference >-10%) and superior immune response for the additional B strains (the lower limit of 95% CI for GMT ratio >1.5, for SCR difference >10%) versus TIVs. The lower limit of the 95% confidence interval of the GMT increase fold, the seroconversion rate and the seroprotection rate exceeded 2.5, 40% and 70% for the four strains in IIV4-W respectively. CONCLUSIONS: IIV4-W was noninferior to the TIV-matched strains and was superior to the additional B strain. IIV4-W was safe in the participants and elicited high antibody titers.

Potent CaV3.2 channel inhibitors exert analgesic effects in acute and chronic pain models.

Pain is the most common presenting physical symptom and a primary reason for seeking medical care, which chronically affects people's mental health and social life. CaV3.2 channel plays an essential role in the peripheral processing maintenance of pain states. This study was designed to identify novel drug candidates targeting the CaV3.2 channel. Whole-cell patch-clamp, cellular thermal shift assay, FlexStation, in vivo and in vitro CaV3.2 knock-down, site-directed mutagenesis, and double-mutant cycle analysis were employed to explore the pain-related receptors and ligand-receptor direct interaction. We found that toddaculin efficiently inhibits the CaV3.2 channel and significantly reduced the excitability of dorsal root ganglion neurons and pain behaviors. The Carbonyl group of coumarins directly interacts with the pore domain of CaV3.2 via van der Waals (VDW) force. Docking with binding pockets further led us to identify glycycoumarin, which exhibited more potent inhibition on the CaV3.2 channel and better analgesic activity than the parent compound. Toddaculin and its analog showed beneficial therapeutic effects in pain models. Toddaculin binding pocket on CaV3.2 might be a promising docking site for the design of drugs.

The acquisition of cold sensitivity during TRPM8 ion channel evolution.

To cope with temperature fluctuations, molecular thermosensors in animals play a pivotal role in accurately sensing ambient temperature. Transient receptor potential melastatin 8 (TRPM8) is the most established cold sensor. In order to understand how the evolutionary forces bestowed TRPM8 with cold sensitivity, insights into both emergence of cold sensing during evolution and the thermodynamic basis of cold activation are needed. Here, we show that the trpm8 gene evolved by forming and regulating two domains (MHR1-3 and pore domains), thus determining distinct cold-sensitive properties among vertebrate TRPM8 orthologs. The young trpm8 gene without function can be observed in the closest living relatives of tetrapods (lobe-finned fishes), while the mature MHR1-3 domain with independent cold sensitivity has formed in TRPM8s of amphibians and reptiles to enable channel activation by cold. Furthermore, positive selection in the TRPM8 pore domain that tuned the efficacy of cold activation appeared late among more advanced terrestrial tetrapods. Interestingly, the mature MHR1-3 domain is necessary for the regulatory mechanism of the pore domain in TRPM8 cold activation. Our results reveal the domain-based evolution for TRPM8 functions and suggest that the acquisition of cold sensitivity in TRPM8 facilitated terrestrial adaptation during the water-to-land transition.

METTL3-m6 A methylase regulates the osteogenic potential of bone marrow mesenchymal stem cells in osteoporotic rats via the Wnt signalling pathway.

OBJECTIVES: Bone marrow mesenchymal stem cells (BMSCs) hold a high osteogenic differentiation potential, but the mechanisms that control the osteogenic ability of BMSCs from osteoporosis (OP-BMSCs) need further research. The purpose of this experiment is to discuss the osteogenic effect of Mettl3 on OP-BMSCs and explore new therapeutic target that can enhance the bone formation ability of OP-BMSCs. MATERIALS AND METHODS: The bilateral ovariectomy (OVX) method was used to establish the SD rat OP model. Dot blots were used to reveal the different methylation levels of BMSCs and OP-BMSCs. Lentiviral-mediated overexpression of Mettl3 was applied in OP-BMSCs. QPCR and WB detected the molecular changes of osteogenic-related factors and Wnt signalling pathway in vitro experiment. The staining of calcium nodules and alkaline phosphatase detected the osteogenic ability of OP-BMSCs. Micro-CT and histological examination evaluated the osteogenesis of Mettl3 in OP rats in vivo. RESULTS: The OP rat model was successfully established by OVX. Methylation levels and osteogenic potential of OP-BMSCs were decreased in OP-BMSCs. In vitro experiment, overexpression of Mettl3 could upregulate the osteogenic-related factors and activate the Wnt signalling pathway in OP-BMSCs. However, osteogenesis of OP-BMSCs was weakened by treatment with the canonical Wnt inhibitor Dickkopf-1. Micro-CT showed that the Mettl3(+) group had an increased amount of new bone formation at 8 weeks. Moreover, the results of histological staining were the same as the micro-CT results. CONCLUSIONS: Taken together, the methylation levels and osteogenic potential of OP-BMSCs were decreased in OP-BMSCs. In vitro and in vivo studies, overexpression of Mettl3 could partially rescue the decreased bone formation ability of OP-BMSCs by the canonical Wnt signalling pathway. Therefore, Mettl3 may be a key targeted gene for bone generation and therapy of bone defects in OP patients.

Carbon dots capped cerium oxide nanoparticles for highly efficient removal and sensitive detection of fluoride.

Since the excess exposure to F- may induce serious issues to human health, the effective adsorption and sensitive detection of F- is essential. Therefore, carbon dots (CDs) capped CeO2 (CeO2@CDs) was synthesized via hydrothermal treatment of tannic acid and CeCl3. Due to abundant phenolic hydroxyl are reserved and excellent hydrophilicity, CeO2@CDs possess high F- adsorption capacity. The partition coefficient parameters (PC) are determined to be 2.65 L/g, which is comparable with previous work. The kinetics results and adsorption isotherm are consistent with pseudo-second-order model and Freundlich model, respectively, indicating the chemisorption dominate the adsorption, mainly via the ion exchange between hydroxyl and F-. Since phenolic hydroxyl existed on the CeO2@CDs, synergetic effect of CDs and CeO2 contribute to superior ROS eliminating capacity, even at acidic conditions. Moreover, due to the ROS scavenging of CeO2 @CDs abilities can be potentiated by F-, colorimetric detection of F- can be realized via horseradish peroxidase as an indicator. The linear range is 0.3-2.1 mM with limit of detection is 0.13 mg/L. The current results imply that CeO2@CDs possess potential in both efficient removal and sensitive detection of F- related contamination issues and elucidation of development to address other anions related issues.

A Unique Nanoflake-Shape Bimetallic Ti-Nb Oxide of Superior Catalytic Effect for Hydrogen Storage of MgH2.

MgH2 is one of the most promising solid hydrogen storage materials due to its high capacity, excellent reversibility, and low cost. However, its operation temperature needs to be greatly reduced to realize its practical applications, especially in the highly desired fuel cell fields. This work synthesizes a 2D nanoflake-shape bimetallic Ti-Nb oxide of TiNb2 O7 , which has high surface area and shows superior catalytic effect for the hydrogen storage of MgH2 . Incorporated with the TiNb2 O7 nanoflakes as low as 3 wt%, MgH2 shows a low onset dehydrogenation temperature of 178 °C, which is lowered by 100 °C compared with the pristine one. A dehydrogenation capacity as high as 7.0 wt% H2 is achieved upon heating to 300 °C. The capacity retention is as high as 96% after 30 cycles. The mechanism of the improved hydrogen storage properties is analyzed by density functional theory (DFT) calculation and the microstructural evolution during dehydrogenation and hydrogenation. This work provides an MgH2 system with high available capacity and low operation temperature by a unique structural design of the catalyst. The high surface area feature of the TiNb2 O7 nanoflakes and the synthesis method hopefully can develop the application of TiNb2 O7 .

Elevated thyroid hormones caused by high concentrate diets participate in hepatic metabolic disorders in dairy cows.

OBJECTIVE: High concentrate diets are widely used to satisfy high-yielding dairy cows; however, long-term feeding of high concentrate diets can cause subacute ruminal acidosis (SARA). The endocrine disturbance is one of the important reasons for metabolic disorders caused by SARA. However, there is no current report about thyroid hormones involved in liver metabolic disorders induced by a high concentrate diet. METHODS: In this study, 12 mid-lactating dairy cows were randomly assigned to HC (high concentrate) group (60% concentrate of dry matter, n = 6) and LC (low concentrate) group (40% concentrate of dry matter, n = 6). All cows were slaughtered on the 21st day, and the samples of blood and liver were collected to analyze the blood biochemistry, histological changes, thyroid hormones, and the expression of genes and proteins. RESULTS: Compared with LC group, HC group showed decreased serum triglyceride, free fatty acid, total cholesterol, low-density lipoprotein cholesterol, increased hepatic glycogen, and glucose. For glucose metabolism, the gene and protein expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1 in the liver were significantly up-regulated in HC group. For lipid metabolism, the expression of sterol regulatory element-binding protein 1, long-chain acyl-CoA synthetase 1, and fatty acid synthase in the liver was decreased in HC group, whereas carnitine palmitoyltransferase 1α and peroxisome proliferator activated receptor α were increased. Serum triiodothyronine, thyroxin, free triiodothyronine (FT3), and hepatic FT3 increased in HC group, accompanied by increased expression of thyroid hormone receptor (THR) in the liver. CONCLUSION: Taken together, thyroid hormones may increase hepatic gluconeogenesis, ß-oxidation and reduce fatty acid synthesis through the THR pathway to participate in the metabolic disorders caused by a high concentrate diet.

Whole transcriptome analysis of RNA expression profiles reveals the potential regulating action of long noncoding RNA in lactating cows fed a high concentrate diet.

Subacute ruminal acidosis (SARA) is a common metabolic disease in the dairy farming industry which is usually caused by an excessive amount of high concentrate diet. SARA not only threatens animal welfare but also leads to economic losses in the farming industry. The liver plays an important role in the distribution of nutritional substances and metabolism; however, a high concentrate diet can cause hepatic metabolic disorders and liver injury. Recently, noncoding RNA has been considered as a critical regulator of hepatic disease, however, its role in the bovine liver is limited. In this study, 12 mid-lactating dairy cows were randomly assigned to a control (CON) group (40% concentrate of dry matter, n = 6) and a SARA group (60% concentrate of dry matter, n = 6). After 21 d of treatment, all cows were sacrificed, and liver tissue samples were collected. Three dairy cows were randomly selected from the CON and SARA groups respectively to perform whole transcriptome analysis. More than 20,000 messenger RNA (mRNA), 10,000 long noncoding RNA (lncRNA), 3,500 circular RNA (circRNA) and 1,000 micro RNA (miRNA) were identified. Furthermore, 43 mRNA, 121 lncRNA and 3 miRNA were differentially expressed, whereas no obvious differentially expressed circRNA were detected between the 2 groups. Gene Ontology (GO) annotation revealed that the differentially expressed genes were mainly enriched in oxidoreductase activity, stress, metabolism, the immune response, cell apoptosis, and cell proliferation. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the deferentially expressed genes were highly enriched in the phosphatidylinositol 3 kinase (PI3K)-serine/threonine kinase (AKT) signaling pathway (P < 0.05). According to KEGG pathway analysis, the differentially expressed lncRNA (DElncRNA) target genes were mainly related to proteasomes, peroxisomes, and the hypoxia-inducible factor-1 signaling pathway (P < 0.005). Further bioinformatics and integrative analyses revealed that the lncRNA were strongly correlated with mRNA; therefore, it is reasonable to speculate that lncRNA potentially play important roles in the liver dysfunction induced by SARA. Our study provides a valuable resource for future investigations on the mechanisms of SARA to facilitate an understanding of the importance of lncRNA, and offer functional RNA information.

Decreased autophagy impairs osteogenic differentiation of adipose-derived stem cells via Notch signaling in diabetic osteoporosis mice.

BACKGROUND: The osteogenic differentiation ability of adipose-derived stem cells (ASCs) is attenuated in type 2 diabetic osteoporosis (Dop) mice. Several studies suggest autophagy and Notch signaling pathway play vital roles in cell proliferation, differentiation, and osteogenesis. However, the mechanisms of autophagy and Notch signaling in the osteogenic differentiation of Dop ASCs were unclear. Thus, it is meaningful to reveal potential correlations between autophagy, Notch signaling, and osteogenesis, and explore involved molecular mechanisms in Dop ASCs. MATERIALS AND METHODS: The diabetic osteoporosis C57BL/6 mouse model, which was confirmed by micro-CT and HE & Masson staining, was established through high-sugar and high-fat diet and streptozotocin injection. ASCs were obtained from the inguinal subcutaneous fat of Dop mice. The multi-differentiation potential of ASCs was evaluated by staining with Alizarin Red (osteogenesis), Oil Red O (adipogenesis), and Alcian blue (chondrogenesis). Cell viability was assessed by Cell Counting Kit-8 assay. Torin1, an inhibitor of mTOR, was used to stimulate the autophagy signaling pathway. DAPT, a γ-secretase inhibitor, was used to suppress Notch signaling pathway activity. Gene and protein expression of autophagy, Notch signaling pathway, and osteogenic factors were detected by real-time quantitative PCR, western blot, and immunofluorescence microscopy. RESULTS: Our findings showed autophagy and osteogenic differentiation ability of Dop ASCs exhibited downward trends that were both rescued by Torin1. Notch signaling was suppressed in Dop ASCs, but upregulated when autophagy was activated. After activation of autophagy, DAPT treatment led to decreased Notch signaling pathway activation and attenuated osteogenic differentiation ability in Dop ASCs. CONCLUSIONS: Downregulated autophagy suppressed Notch signaling, leading to a reduced osteogenic differentiation capacity of Dop ASCs, and Torin1 can rescue this process by activating autophagy. Our findings contribute to understanding the mechanism underlying impairment of the osteogenic differentiation ability of Dop ASCs.

Role of Fzd6 in Regulating the Osteogenic Differentiation of Adipose-derived Stem Cells in Osteoporotic Mice.

OBJECTIVE: Although it has been demonstrated that adipose-derived stem cells (ASCs) from osteoporotic mice (OP-ASCs) exhibited impaired osteogenic differentiation potential, the molecular mechanism has not yet been elucidated. We found that Fzd6 was decreased in OP-ASCs compared with ASCs. This study investigates effects and underlying mechanisms of Fzd6 in the osteogenic potential of OP-ASCs, and explores methods to enhance osteogenic capacity of OP-ASCs. METHODS: Fzd6 overexpression and silencing lentiviruses were used to evaluate the role of Fzd6 in the osteogenic differentiation of OP-ASCs. Real-time PCR (qPCR) and western blotting (WB) was performed to detect the expression of Fzd6 and bone-related molecules, including runt-related transcription factor 2 (Runx2) and osteopontin (Opn). Alizarin red staining and Alkaline phosphatase (ALP) staining were performed following osteogenic induction. Microscopic CT (Micro-CT), hematoxylin and eosin staining (HE) staining, and Masson staining were used to assess the role of Fzd6 in osteogenic differentiation of osteoporosis (OP) mice in vivo. RESULTS: Expression of Fzd6 was decreased significantly in OP-ASCs. Fzd6 silencing down-regulated the osteogenic ability of OP-ASCs in vitro. Overexpression of Fzd6 rescued the impaired osteogenic capacity in OP-ASCs in vitro. We obtained similar results in vivo. CONCLUSIONS: Fzd6 plays an important role in regulating the osteogenic ability of OP-ASCs both in vivo and in vitro. Overexpression of Fzd6 promotes the osteogenic ability of OP-ASCs, which provides new insights for the prevention and treatment of OP mice.

Safety of Dabigatran as an Anticoagulant: A Systematic Review and Meta-Analysis.

Background: Dabigatran is a univalent low-molecular-weight direct thrombin inhibitor which was developed as an alternative to vitamin K antagonists (VKAs). However, the safety of dabigatran remains controversial so far. In this study, we aimed to compare the risk of bleeding, fatal adverse events, and the all-cause mortality of dabigatran with those of the control group by a systematic review and meta-analysis of randomized controlled trials. Methods: We systematically searched PubMed, Web of Science, Cochrane Library, Medline, Embase, Wanfang database, Clinical trial, China National Knowledge Infrastructure Chinese Scientific Journal database (VIP), and Chinese Biological Medicine database (CBM), for clinical trials on conventional treatments compared with dabigatran, published between January 2014 and July 2020. The reported outcomes, including the endpoints of primary safety, were systematically investigated. Results: Seven RCTs (n = 10,743) were included in the present systematic review. Compared to the control groups, dabigatran was not associated with an increased risk of major bleeding (relative risk [RR] 0.86, 95% confidence interval [CI]: 0.61 to 1.21, p = 0.06), intracranial hemorrhage (RR 0.89, 95% CI: 0.58 to 1.36, p = 0.41), fatal adverse reactions (RR 0.87, 95% CI: 0.65 to 1.17, p = 0.66), all-cause mortality (RR 0.88, 95% CI: 0.70 to 1.11, p = 0.45, I2 = 0%), and significantly reduced risk of clinically relevant non-major bleeding (RR 0.96, 95% CI: 0.65 to 1.42, p = 0.0007). However, dabigatran is associated with an increased risk of gastrointestinal (GI) bleeding (RR 1.78, 95% CI: 1.02 to 3.13, p = 0.05). Conclusion: Dabigatran has a favorable safety profile in terms of major bleeding, intracranial hemorrhage, and life-threatening events, among other safety outcomes. The present study suggested that dabigatran may be a suitable alternative to VKAs as an oral anticoagulant. However, more data are necessary to clarify the incidence of other adverse events and serious adverse reactions.

Glucocorticoid receptors involved in melatonin inhibiting cell apoptosis and NLRP3 inflammasome activation caused by bacterial toxin pyocyanin in colon.

The immunoinhibitory effect of glucocorticoid and immunoenhancing attributes of melatonin (MEL) are well known, however, the involvement of glucocorticoid receptor (GR) in melatonin modulation of bacterial toxins caused-inflammation has not been studied in colon. Pyocyanin (PCN), a toxin released by Pseudomonas aeruginosa, can destroy cells through generating superoxide products and inflammatory response. Here we report that PCN treatment elevated the generation of reactive oxygen species (ROS), which further lead to mitochondrial swelling and caspase cascades activation both in vivo and in vitro. However, MEL treatment alleviated the oxidative stress caused by PCN on cells through scavenging ROS and restoring the expression of antioxidant enzyme so that to effectively alleviate the apoptosis. Large amounts of ROS can activate the NLRP3 signaling pathway, so MEL inhibited PCN induced NLRP3 inflammasome activation and inflammatory cytokines (IL-1ß, IL-8, and TNF-α) secretion. In order to further investigate the molecular mechanism, goblet cells were exposed to MEL and PCN in the presence of luzindole and RU486, inhibitors of MEL receptors and GR respectively. It was found that PCN significantly inhibited the expression level of GR, and MEL effectively alleviated the inhibition phenomenon. Moreover, we found that MEL mainly upregulated the expression of GR to achieve its anti-inflammatory and anti-apoptotic functions rather than through its own receptor (MT2) in colon goblet cells. Therefore, MEL can reverse the inhibitory effects of PCN on GR/p-GR expression to present its anti-oxidative and anti-apoptotic function.