Construction of novel multi-epitope-based diagnostic biomarker HP16118P and its application in the differential diagnosis of Mycobacterium tuberculosis latent infection.

Tuberculosis (TB) is an infectious disease that significantly threatens human health. However, the differential diagnosis of latent tuberculosis infection (LTBI) and active tuberculosis (ATB) remains a challenge for clinicians in early detection and preventive intervention. In this study, we developed a novel biomarker named HP16118P, utilizing 16 helper T lymphocyte (HTL) epitopes, 11 cytotoxic T lymphocyte (CTL) epitopes, and 8 B cell epitopes identified from 15 antigens associated with LTBI-RD using the IEDB database. We analyzed the physicochemical properties, spatial structure, and immunological characteristics of HP16118P using various tools, which indicated that it is a hydrophilic and relatively stable alkaline protein. Furthermore, HP16118P exhibited good antigenicity and immunogenicity, while being non-toxic and non-allergenic, with the potential to induce immune responses. We observed that HP16118P can stimulate the production of high levels of IFN-γ+ T lymphocytes in individuals with ATB, LTBI, and health controls. IL-5 induced by HP16118P demonstrated potential in distinguishing LTBI individuals and ATB patients (p=0.0372, AUC=0.8214, 95% CI [0.5843 to 1.000]) with a sensitivity of 100% and specificity of 71.43%. Furthermore, we incorporated the GM-CSF, IL-23, IL-5, and MCP-3 induced by HP16118P into 15 machine learning algorithms to construct a model. It was found that the Quadratic discriminant analysis model exhibited the best diagnostic performance for discriminating between LTBI and ATB, with a sensitivity of 1.00, specificity of 0.86, and accuracy of 0.93. In summary, HP16118P has demonstrated strong antigenicity and immunogenicity, with the induction of GM-CSF, IL-23, IL-5, and MCP-3, suggesting their potential for the differential diagnosis of LTBI and ATB.

Investigating the Role of Extracellular Matrix Stiffness in Modulating the Ferroptosis Process in Hepatocellular Carcinoma Cells via Scanning Electrochemical Microscopy.

Extracellular matrix (ECM) stiffness modulates a variety of cellular processes, including ferroptosis, a process with significant potential implications for hepatocellular carcinoma (HCC) fibrosis and cirrhosis. However, the exact relationship between ECM stiffness and HCC ferroptosis is yet unclarified, partially due to the lack of in situ information on key parameters of the ferroptosis process of living HCC cells. This study pioneers the use of in vitro mechanical microenvironment models of HCC and the scanning electrochemical microscopy (SECM) technique for understanding this interplay. We first cultured HuH7 cells on 4.0, 18.0, and 44.0 kPa polyacrylamide (PA) gels to simulate early, intermediate, and advanced HCC ECM stiffness, respectively. Then, we used SECM to in situ monitor changes in cell membrane permeability, respiratory activity, and reactive oxygen species (ROS) levels of erastin-induced HuH7 cells on PA gels, finding that increasing ECM stiffness potentiates ferroptosis, including increased membrane permeabilization and H2O2 release as well as reduced respiratory activity. Through further transcriptome sequencing and molecular biology measurements, we identified a critical role for focal adhesion kinase (FAK)-mediated yes-associated protein (YAP) in regulating the ferroptosis process dependent on ECM stiffness, which provides novel insights into the mechanical regulation of ferroptosis in HCC cells and may pave the way for innovative therapeutic strategies.

Mycobacterium tuberculosis: immune response, biomarkers, and therapeutic intervention.

Although tuberculosis (TB) is an infectious disease, the progression of the disease following Mycobacterium tuberculosis (MTB) infection is closely associated with the host's immune response. In this review, a comprehensive analysis of TB prevention, diagnosis, and treatment was conducted from an immunological perspective. First, we delved into the host's immune response mechanisms against MTB infection as well as the immune evasion mechanisms of the bacteria. Addressing the challenges currently faced in TB diagnosis and treatment, we also emphasized the importance of protein, genetic, and immunological biomarkers, aiming to provide new insights for early and personalized diagnosis and treatment of TB. Building upon this foundation, we further discussed intervention strategies involving chemical and immunological treatments for the increasingly critical issue of drug-resistant TB and other forms of TB. Finally, we summarized TB prevention, diagnosis, and treatment challenges and put forward future perspectives. Overall, these findings provide valuable insights into the immunological aspects of TB and offer new directions toward achieving the WHO's goal of eradicating TB by 2035.

From immunology to artificial intelligence: revolutionizing latent tuberculosis infection diagnosis with machine learning.

Latent tuberculosis infection (LTBI) has become a major source of active tuberculosis (ATB). Although the tuberculin skin test and interferon-gamma release assay can be used to diagnose LTBI, these methods can only differentiate infected individuals from healthy ones but cannot discriminate between LTBI and ATB. Thus, the diagnosis of LTBI faces many challenges, such as the lack of effective biomarkers from Mycobacterium tuberculosis (MTB) for distinguishing LTBI, the low diagnostic efficacy of biomarkers derived from the human host, and the absence of a gold standard to differentiate between LTBI and ATB. Sputum culture, as the gold standard for diagnosing tuberculosis, is time-consuming and cannot distinguish between ATB and LTBI. In this article, we review the pathogenesis of MTB and the immune mechanisms of the host in LTBI, including the innate and adaptive immune responses, multiple immune evasion mechanisms of MTB, and epigenetic regulation. Based on this knowledge, we summarize the current status and challenges in diagnosing LTBI and present the application of machine learning (ML) in LTBI diagnosis, as well as the advantages and limitations of ML in this context. Finally, we discuss the future development directions of ML applied to LTBI diagnosis.

Immunotherapy and biomarkers in patients with lung cancer with tuberculosis: Recent advances and future Directions.

Lung cancer (LC) and tuberculosis (TB) are two major global public health problems, and the incidence of LC-TB is currently on the rise. Therefore effective clinical interventions are crucial for LC-TB. The aim of this review is to provide up-to-date information on the immunological profile and therapeutic biomarkers in patients with LC-TB. We discuss the immune mechanisms involved, including the immune checkpoints that play an important role in the treatment of patients with LC-TB. In addition, we explore the susceptibility of patients with LC to TB and summarise the latest research on LC-TB. Finally, we discuss future prospects in this field, including the identification of potential targets for immune intervention. In conclusion, this review provides important insights into the complex relationship between LC and TB and highlights new advances in the detection and treatment of both diseases.

Next-Generation TB Vaccines: Progress, Challenges, and Prospects.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a prevalent global infectious disease and a leading cause of mortality worldwide. Currently, the only available vaccine for TB prevention is Bacillus Calmette-Guérin (BCG). However, BCG demonstrates limited efficacy, particularly in adults. Efforts to develop effective TB vaccines have been ongoing for nearly a century. In this review, we have examined the current obstacles in TB vaccine research and emphasized the significance of understanding the interaction mechanism between MTB and hosts in order to provide new avenues for research and establish a solid foundation for the development of novel vaccines. We have also assessed various TB vaccine candidates, including inactivated vaccines, attenuated live vaccines, subunit vaccines, viral vector vaccines, DNA vaccines, and the emerging mRNA vaccines as well as virus-like particle (VLP)-based vaccines, which are currently in preclinical stages or clinical trials. Furthermore, we have discussed the challenges and opportunities associated with developing different types of TB vaccines and outlined future directions for TB vaccine research, aiming to expedite the development of effective vaccines. This comprehensive review offers a summary of the progress made in the field of novel TB vaccines.

Effect of Extracellular Matrix Stiffness on Candesartan Efficacy in Anti-Fibrosis and Antioxidation.

Myocardial fibrosis progression and imbalanced redox state are closely associated with increased extracellular matrix (ECM) stiffness. Candesartan (CAN), an angiotensin II (Ang II) receptor inhibitor, has shown promising anti-fibrosis and antioxidant efficacy in previous cardiovascular disease studies. However, the effect of ECM stiffness on CAN efficacy remains elusive. In this study, we constructed rat models with three different degrees of myocardial fibrosis and treated them with CAN, and then characterized the stiffness, cardiac function, and NADPH oxidase-2 (NOX2) expression of the myocardial tissues. Based on the obtained stiffness of myocardial tissues, we used polyacrylamide (PA) gels with three different stiffness to mimic the ECM stiffness of cardiac fibroblasts (CFs) at the early, middle, and late stages of myocardial fibrosis as the cell culture substrates and then constructed CFs mechanical microenvironment models. We studied the effects of PA gel stiffness on the migration, proliferation, and activation of CFs without and with CAN treatment, and characterized the reactive oxygen species (ROS) and glutathione (GSH) levels of CFs using fluorometry and scanning electrochemical microscopy (SECM). We found that CAN has the best amelioration efficacy in the cardiac function and NOX2 levels in rats with medium-stiffness myocardial tissue, and the most obvious anti-fibrosis and antioxidant efficacy in CFs on the medium-stiffness PA gels. Our work proves the effect of ECM stiffness on CAN efficacy in myocardial anti-fibrosis and antioxidants for the first time, and the results demonstrate that the effect of ECM stiffness on drug efficacy should also be considered in the treatment of cardiovascular diseases.

Effect of Exogenous Electric Stimulation on the Cardiac Tissue Function In Situ Monitored by Scanning Electrochemical Microscopy.

Cardiac tissue is sensitive to and can be easily damaged by exogenous electric stimulation. However, due to the thermal-electric coeffect and the limitation of in situ and quantitative information on the cardiac tissue function under electric stimulation, the detailed effect and the underlying mechanism of exogenous electric stimulation on the cardiac tissue remain elusive. To address this, in this work, we first constructed an in vitro cardiac tissue model and established a thermal-electric coupled theoretical model for simulating the electric field and temperature distributions around the cardiac tissue, from which we selected the electric field strengths (1.19, 2.37, and 3.39 kV cm-1) and electrical energies (0.001, 0.005, and 0.011 J) for electric stimulations without inducing a thermal effect. Then, we applied electric field stimulations on the cardiac tissue using these parameters and scanning electrochemical microscopy (SECM) to in situ and quantitatively monitor the dynamic changes in the key parameters of the cardiac tissue function, including respiratory activity, membrane permeability, and contraction frequency, after electric field stimulations. The SECM results showed that the oxygen consumption, cell membrane permeability coefficient, and contraction frequency of the cardiac tissue were strongly dependent on electrical energy, especially when the electrical energy was higher than 0.001 J. Our work, for the first time, achieves the in situ and quantitative monitoring of the cardiac tissue function under electric stimulation using SECM, which would provide important references for designing an electric stimulation regime for cardiac tissue engineering and clinical application of electrotherapy.

In Situ and Quantitatively Monitoring the Dynamic Process of Ferroptosis in Single Cancer Cells by Scanning Electrochemical Microscopy.

Ferroptosis, as a promising therapeutic strategy for cancers, has aroused great interest. Quantifying the quick dynamic changes in key parameters during the early course of ferroptosis can provide insights for understanding the underlying mechanisms of ferroptosis and help the development of therapies targeting ferroptosis. However, in situ and quantitatively monitoring the quick responses of living cancer cells to ferroptosis at the single-cell level remains technically challenging. In this work, we selected HuH7 cells (hepatocellular carcinoma (HCC) cells) as a cell model and Erastin as a typical ferroptosis inducer. We utilized scanning electrochemical microscopy (SECM) to quantitatively and in situ monitor the early course of ferroptosis in HuH7 cells by characterizing the three key parameters of cell ferroptosis (i.e., cell membrane permeability, respiratory activity, and the redox state). The SECM results show that the membrane permeability of ferroptotic HuH7 cells continuously increased from 0 to 8.1 × 10-5 m s-1, the cellular oxygen consumption was continuously reduced by half, and H2O2 released from the cells exhibited periodic bursts during the early course of ferroptosis, indicating the gradually destroyed cell membrane structure and intensified oxidative stress. Our work realizes, for the first time, the in situ and quantitative monitoring of the cell membrane permeability, respiratory activity, and H2O2 level of the early ferroptosis process of a single living cancer cell with SECM, which can contribute to the understanding of the physiological process and underlying mechanisms of ferroptosis.

miR-153-3p inhibits osteogenic differentiation of BMSCs by down-regulating the expression of RUNX2 in a high glucose environment.

To study the effect of miR-153-3p on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a high glucose environment and its potential mechanism. The results showed that high glucose inhibited the osteogenic differentiation of BMSCs, and the expression of miR-153-3p increased during osteogenic differentiation. Further experiments found that in BMSCs induced by high glucose, overexpression of miR-153-3p inhibited the osteogenic differentiation of BMSCs, and the expressions of osteogenesis-related genes bone sialoprotein, Collagen I and alkaline phosphatase were down-regulated, while silencing of miR-153-3p alleviated the inhibition effect. The dual-luciferase reporter gene assay confirmed that the 3'-untranslated region (3'-UTR) of runt related transcription factor 2 (RUNX2) had a targeted binding site with miR-153-3p and a negative regulatory effect. Molecular studies further confirmed that miR-153-3p inhibited the osteogenic differentiation of BMSCs by targeting the 3'-UTR of RUNX2. In conclusion, our study found that as one key regulator of high glucose affecting the osteogenic differentiation of BMSCs, miR-153-3p may play a negative regulatory role by inhibiting the expression of RUNX2.

[Effect of fibroblasts on promoting the sprout and migration of endothelial cells in three-dimensional pre-vascularized microstructures].

Objective: To construct three-dimensional (3D) pre-vascularized microstructures and explore the promoting effect of human fibroblasts (HFs) on the sprout and migration of human umbilical vein endothelial cells (HUVECs) in 3D co-culture system. Methods: HUVECs and HFs were cultured and the 3rd to 5th generation cells were selected for subsequent experiments. In 2D co-culture system, HFs were stained with PKH26 and the cell density was fixed, which co-cultured with HUVECs in different ratios (1∶4, 1∶1, 4∶1) and inoculation methods (HUVECs inoculation at 48 hours after HFs, direct mixed inoculation). Then the formation of vascular like structures was observed under fluorescence microscope. In 3D co-culture system, HUVECs and HFs were labeled with green fluorescent protein and red fluorescent protein by lentivirus transfection, respectively. They were inoculated on porous micro-carriers followed by dynamically culturing in rotating bottles to prepare HF, HUVEC, HF-EC, or HF&EC microstructures. The cell growth in microstructures was testing by low permeability crystal violet staining. Subsequently, the microstructures were embedded in fibrin gel and the cell growth and adhesion in HF and HUVEC microstructures were observed by laser confocal microscopy. Laser confocal microscope were also used to observe the sprouts of 4 kinds of microstructures, as well as the cell composition, the number and length of sprouts from HF-EC and HF&EC microstructures. HFs conditioned medium was prepared to observe its effect on sprouts of HUVEC microstructures with DMEM as control group. Results: In 2D co-culture system, HFs pre-culturing was helpful to the formation and stability of vascular like structures, and the best effect was when the ratio of two kinds of cells was 1∶1. In 3D co-culture system, it was found that the cells grew well on micro-carriers and had the ability of pre-vascularization. HUVEC microstructures did not sprout, but HF, HF-EC, and HF&EC microstructures could which indicated a good vascularization ability. The HF-EC microstructures were superior to HF&EC microstructures in terms of sprouts length and number ( P<0.05). The tubes sprouting from co-cultured group were composed of HFs and HUVECs, and HF microstructures migration preceded HUVEC microstructures always, and their migration trajectories were the same. HUVEC microstructures could sprout when cultured in HFs conditioned media. Conclusion: HF-HUVEC pre-vascularized microstructures can be prepared by pre-culturing HFs before HUVECs and with the cell ratio at 1∶1 in a rotating bottle. In 3D co-culture system, HFs can promote and guide the sprout of HUVECs.

In Situ and Quantitative Monitoring of Cardiac Tissues Using Programmable Scanning Electrochemical Microscopy.

In vitro cardiac tissue model holds great potential as a powerful platform for drug screening. Respiratory activity, contraction frequency, and extracellular H2O2 levels are the three key parameters for determining the physiological functions of cardiac tissues, which are technically challenging to be monitored in an in situ and quantitative manner. Herein, we constructed an in vitro cardiac tissue model on polyacrylamide gels and applied a pulsatile electrical field to promote the maturation of the cardiac tissue. Then, we built a scanning electrochemical microscopy (SECM) platform with programmable pulse potentials to in situ characterize the dynamic changes in the respiratory activity, contraction frequency, and extracellular H2O2 level of cardiac tissues under both normal physiological and drug (isoproterenol and propranolol) treatment conditions using oxygen, ferrocenecarboxylic acid (FcCOOH), and H2O2 as the corresponding redox mediators. The SECM results showed that isoproterenol treatment induced enhanced oxygen consumption, accelerated contractile frequency, and increased released H2O2 level, while propranolol treatment induced dynamically decreased oxygen consumption and contractile frequency and no obvious change in H2O2 levels, suggesting the effects of activation and inhibition of ß-adrenoceptor on the metabolic and electrophysiological activities of cardiac tissues. Our work realizes the in situ and quantitative monitoring of respiratory activity, contraction frequency, and secreted H2O2 level of living cardiac tissues using SECM for the first time. The programmable SECM methodology can also be used to real-time and quantitatively monitor electrochemical and electrophysiological parameters of cardiac tissues for future drug screening studies.

[Identification of the glycosylation sites of Opsin3 and its glycosylation modification function].

Opsin3 (OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain. In recent years, it has been reported that OPN3 is also highly expressed in adipose tissue, and the protein is associated with the production of skin melanin. We found that the N82 site is the glycosylation site of OPN3. SNAP-tagTM has diverse functions and can be applied to a variety of different studies. By constructing a SNAP-tagged OPN3 recombinant protein, the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface® 549 and SNAP-Cell® OregonGreen®, which provides a new method for studying the function of OPN3. It also shows that SNAP-tag does not affect the function of OPN3. Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.

Enamel matrix derivative expedites osteogenic differentiation of BMSCs via Wnt/ß-catenin pathway in high glucose microenvironment.

INTRODUCTION: The influence of enamel matrix derivative (EMD) on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was explored in high glucose (HG) microenvironment with interaction of Wnt/ß-catenin pathway. MATERIALS AND METHODS: Extraction of BMSCs from Sprague-Dawley rats, culture, and identification were manifested. The cells were treated with different concentration of EMD in HG to figure out the most available concentration for proliferation and osteogenic differentiation. Then, observation of cell growth curve and cell cycle changes, and detection of Osterix, runt-related transcription factor 2 (Runx2), COL-I, early osteogenic indexes, Calcium salt deposition, and ß-catenin protein in Wnt/ß-catenin pathway were assured. After adding Wnt/ß-catenin pathway inhibitor (XAV-939) in the cells with osteogenesis induction, detection of binding of ß-catenin to Osterix was clarified. RESULTS: Via identification BMSCs cultured in vitro was qualified. Different concentrations of EMD could accelerate cell proliferation in HG and osteogenesis induction, and 75 µg/mL EMD had the best effect. The HG augmented BMSCs proliferation and the propidium iodide index of flow cytometry cycle was elevated in HG, which were strengthened via the EMD. After BMSCs' osteogenesis induction, Osterix, Runx2, CoL-1, early osteogenic indexes, and calcium salt deposition were reduced, but elevated via EMD. ß-Catenin was the lowest in the HG, but elevated after EMD. After addition of XAV-939, reduction of ß-catenin and the downstream (Osterix and Runx2) were manifested. Detection of binding protein bands was in ß-catenin and Osterix of the HG after EMD treatment. CONCLUSION: EMD may facilitate the osteogenic differentiation of BMSCs via activating the Wnt/ß-catenin pathway in HG.

Enamel matrix derivative (EMD) enhances the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

To investigate the EMD's capacity in BMSCs osteogenic differentiation. In vivo and in vitro, BMSCs were treated with EMD, scanning electron microscopy, and Alizarin Red staining were used to detect the changes in the osteogenic ability of BMSCs, and the proliferation ability of BMSCs was evaluated by CCK8. In addition, by adding xav939, a typical inhibitor of Wnt/ß-catenin signaling pathway, the regulatory function of Wnt/ß-catenin signaling was clarified. The results showed that EMD promote cell proliferation and 25 µg/ml EMD had the most significant effect. Cells inducing osteogenesis for 2 and 3 even 4 weeks, the cell staining is deeper in EMD treated group than that of the control (P < 0.05) by alizarin Red staining, suggesting more mineralization of BMSCs. In vivo implanting the titanium plate wrapped with 25 µg/ml EMD treated-BMSC film into nude mice for 8 weeks, more nodules were formed on the surface of the titanium plate than that the control (P < 0.05). HE showed that there is a little blue-violet immature bone-like tissue block. Besides, the expression of RUNX Family Transcription Factor 2 (Runx2), Osterix, Osteocalcin (OCN), collagen I (COLI), alkaline phosphatase (ALP) and ß-catenin were inhibited in xav939 group (P < 0.05); Inversely, all were activated in EMD group (P < 0.05). In conclusion, EMD promoted the proliferation and osteogenic differentiation of BMSCs. EMD's function on BMSCs might be associated with the Wnt/ß-catenin signaling pathway.

Investigating the Effect of Substrate Stiffness on the Redox State of Cardiac Fibroblasts Using Scanning Electrochemical Microscopy.

Cardiac fibrosis, in which cardiac fibroblasts differentiate into myofibroblasts, leads to oversecretion of the extracellular matrix, results in increased stiffness, and facilitates disequilibrium of cellular redox state, further leading to oxidative stress and various degrees of cell death. However, the relationship between the matrix stiffness and the redox status of cardiac fibroblasts remains unclear. In this work, we constructed an in vitro cardiac fibrosis model by culturing cardiac fibroblasts on polyacrylamide gels with tunable stiffness and characterized the differentiation of cardiac fibroblasts to myofibroblasts by immunofluorescence staining of α-smooth muscle actin. We then applied scanning electrochemical microscopy (SECM) with a depth scan mode to in situ and quantitatively assess the redox status by monitoring the glutathione (GSH) efflux rate (k) through the redox reaction between GSH (a typical indicator of cellular redox level) released from cardiac fibroblasts and SECM probe-oxidized ferrocenecarboxylic acid ([FcCOOH]+). The SECM results demonstrate that the GSH efflux from the cardiac fibroblasts decreased with increasing substrate stiffness (i.e., mimicking the increased fibrosis degree), indicating that a more oxidizing microenvironment facilitates the cell differentiation and GSH may serve as a biomarker to predict the degree of cardiac fibrosis. This work provides an SECM approach to quantify the redox state of cardiac fibroblasts by recording the GSH efflux rate. In addition, the newly established relationship between the redox balance and the substrate stiffness would help to better understand the redox state of cardiac fibroblasts during cardiac fibrosis.

In situ ornamenting poly(ε-caprolactone) electrospun fibers with different fiber diameters using chondrocyte-derived extracellular matrix for chondrogenesis of mesenchymal stem cells.

Biomimetic instructive tissue engineering scaffolds are critical for achieving successful tissue regeneration. In the present study, we developed a novel scaffold via ornamenting poly(ε-caprolactone) (PCL) electrospun fibers with a chondrocyte-derived extracellular matrix (ECM)-coating, which was applied for chondrogenesis of mesenchymal stem cells (MSCs). PCL fibrous films with different fiber diameters (1282±121 nm, 549±61 nm and 285±38 nm) were first prepared via electrospinning. Rabbit articular chondrocytes (rACs) were cultured on PCL fibrous scaffolds, followed by a decellularization treatment to generate decellularized ECM (dECM)-coated PCL scaffolds (dECM/PCL). Rabbit bone marrow-derived MSCs (rMSCs) were then seeded onto these scaffolds and adhesion, proliferation and chondrogenic differentiation were evaluated. dECM/PCL scaffolds displayed distinct surface microstructural features with varying fiber diameters and fibrous mesh-like ECM with more developed collagen fibers was observed on nanofibers. On dECM/PCL scaffolds, rMSCs tended to spread more at 24 h post-seeding and proliferated better within 7 d compared to those on uncoated PCL scaffolds. Based on analysis of gene expression, rMSCs underwent the best chondrogenic differentiation on dECM/PCL scaffolds of 549-nm fibers. Collectively, such dECM/PCL composite scaffolds are very promising for cartilage tissue regeneration.

ß-mercaptoethanol promotes osteogenesis of human mesenchymal stem cells via sirt1-ERK pathway.

Human umbilical cord-derived mesenchymal stem cells (hUMSCs) hold strong self-renewal capacity and low immunogenicity, which have attracted attention as potential candidates for bone repair and regeneration. However, insufficient osteogenic differentiation markedly hinders the clinical applications of hUMSCs. In the present study, the effect of ß-mercaptoethanol (BME), a small molecule antioxidant which has been identified to regulate cell proliferation and differentiation, on osteogenic differentiation of hUMSCs and underlying signaling mechanism were investigated. The results indicated that under osteogenic induction conditions, BME treatment increased the alkaline phosphatase (ALP) activity and promoted calcium mineralization in hUMSCs. The gene and protein expression of osteogenesis-related markers such as ALP, osteopontin (OPN), osteocalcin (OCN) and collagen type I (COLI) were also significantly up-regulated. Besides, BME promoted the protein expression of silent information regulator type 1 (sirt1) and stimulated the activation of extracellular signal-related kinase (ERK), contributing to increased Runx2 expression. Furthermore, blocking the expression of sirt1 attenuated BME-enhanced ERK phosphorylation and osteogenic differentiation of hUMSCs. These results indicated that BME accelerated osteogenic differentiation of hUMSCs by activating the sirt1-ERK signaling pathway, thereby providing insights into the development of MSCs-based bone regeneration strategies.

Methylmercury disrupts autophagic flux by inhibiting autophagosome-lysosome fusion in mouse germ cells.

Methylmercury (MeHg) is an extremely toxic environmental pollutant that can cause serious male reproductive developmental dysplasia in humans and animals. However, the molecular mechanisms underlying MeHg-induced male reproductive injury are not fully clear. The purpose of this study was to explore whether mitophagy and lysosome dysfunction contribute to MeHg-induced apoptosis of germ cell and to determine the potential mechanism. First, we confirmed the exposure of GC2-spd cells to mercury. In GC2-spd cells (a mouse spermatocyte cell line), we found that MeHg treatment led to an obvious increase of cell apoptosis accompanied by a marked rise of LC3-II expression and an elevated number of autophagosomes. These results were associated with the induction of oxidative stress and mitophagy. Interestingly, we found that MeHg did not promote but prevented autophagosome-lysosome fusion by impairing the lysosome function. Furthermore, as a lysosome inhibitor, chloroquine pre-treatment obviously enhanced LC3-II expression and mitophagy formation in MeHg-treated cells. This further proved that the induction of mitophagy and the injury of the lysosome played an important role in the GC2-spd cell apoptosis induced by MeHg. Our findings indicate that MeHg caused apoptosis in the GC2-spd cells, which were dependent on oxidative stress-mediated mitophagy and the lysosome damaging-mediated inhibition of autophagic flux induced by MeHg.