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.

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.

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.

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.

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.

The effect of gap junction-mediated transfer of miR-200b on osteogenesis and angiogenesis in a co-culture of MSCs and HUVECs.

For successful engineering of pre-vascularized bone tissue in vitro, understanding the interactions between vasculogenic cells and bone-forming cells is a prerequisite. Mounting evidence indicates that microRNAs can serve as intercellular signals that allow cell-cell communication. Here, the role of the transfer of the microRNA miR-200b between vasculogenic and osteogenic cells was explored in a co-culture system. Rat bone-marrow derived mesenchymal stem cells (BMSCs) formed functional gap junctions composed of connexin 43 (Cx43, also known as GJA1) with human umbilical vein endothelial cells (HUVECs), through which miR-200b could transfer from BMSCs to HUVECs to regulate osteogenesis and angiogenesis. As a negative regulator, the decrease in miR-200b level in BMSCs derepressed the expression of VEGF-A, leading to increased osteogenic differentiation. Once inside HUVECs, miR-200b reduced the angiogenic potential of HUVECs through downregulation of ZEB2, ETS1, KDR and GATA2 Additionally, TGF-ß was found to trigger the transfer of miR-200b to HUVECs. Upon adding the TGF-ß inhibitor SB431542 or TGF-ß-neutralizing antibody, the formation of capillary-like structures in co-culture could be partially rescued. These findings may be fundamental to the development of a cell-based bone regeneration strategy.

Effect of Substrate Stiffness on Redox State of Single Cardiomyocyte: A Scanning Electrochemical Microscopy Study.

Mechanical microenvironment plays a key role in the regulation of the phenotype and function of cardiac cells, which are strongly associated with the intracellular redox mechanism of cardiomyocytes. However, the relationship between the redox state of cardiomyocytes and their mechanical microenvironment remains elusive. In this work, we used polyacrylamide (PA) gels with varying stiffness (6.5-92.5 kPa) as the substrate to construct a mechanical microenvironment for cardiomyocytes. Then we employed scanning electrochemical microscopy (SECM) to in situ characterize the redox state of a single cardiomyocyte in terms of the apparent rate constant (kf) of the regeneration rate of ferrocenecarboxylic by glutathione (GSH) released from cardiomyocyte, which is the most abundant reactant of intracellular reductive-oxidative metabolic cycles in cells and can represent the redox level of cardiomyocytes. The obtained SECM results show that the cardiomyocytes cultured on the stiffer substrates present lower kf values than those on the softer ones, that is, the more oxidative state of cardiomyocytes on the stiffer substrates compared to those on the softer ones. It proves the relationship between mechanical factors and the redox state of cardiomyocytes. This work can contribute to understanding the intracellular chemical process of cardiomyocytes during physiopathologic conditions. Besides, it also provides a new SECM method to in situ investigate the redox mechanism of cardiomyocytes at a single-cell level.

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.

Resveratrol improves ex vivo expansion of CB-CD34+ cells via downregulating intracellular reactive oxygen species level.

Intracellular reactive oxygen species (ROS) play important roles in the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). In this study, the effects of resveratrol (RES), on the ex vivo expansion of HSPCs were investigated by analyzing CD34+ cells expansion and biological functions, with the objective to optimize ex vivo culture conditions for CD34 + cells. Among the five tested doses (0, 0.1, 1, 10, 20, and 50 µM), 10 µM RES was demonstrated to be the most favorable for ex vivo CD34 + cells expansion. In the primary cultures, 10 µM RES favored higher expansion folds of CD34 + cells, CD34 + CD38 - cells, and colony-forming units (CFUs) ( P < 0.05). It was found that the percentages of primitive HSPCs (CD34 + CD38 - CD45R - CD49f + CD90 + cells) in 10 µM RES cultures were higher than those without RES. Further, in the secondary cultures, expanded CD34 + cells derived from primary cultures with 10 µM RES exhibited significantly higher total cells and CD34 + cells expansion ( P < 0.05). In the semisolid cultures, the frequency of CFU-GM and total CFUs of 10 µM RES group were both higher than those of without RES group, demonstrating that CD34 + cells expanded with 10 µM RES possessed better biological function. Furthermore, the addition of 10 µM RES downregulated the intracellular ROS level via strengthening the scavenging capability of ROS, and meanwhile reducing the percentages of apoptotic cells in cultures. Collectively, RES could stimulate the ex vivo expansion of CD34 + cells, preserved more primitive HSPCs and maintain better biological function by alleviating intracellular ROS level and cell apoptosis in cultures.

Inhibiting expression of Cxcl9 promotes angiogenesis in MSCs-HUVECs co-culture.

The insufficient vascularization is a major challenge in bone tissue engineering, leading to partial necrosis of the implant. Pre-vascularization is a promising way via in vitro cells co-culture strategies using osteogenic cells and vasculogenic cells, and the cross-talk of cells is essential. In the present study, the effect of rat bone-marrow derived mesenchymal stem cells (BMSCs) on angiogenic capability of human umbilical vein endothelial cells (HUVECs) in growth medium (GM) and osteogenic induction medium (OIM) was investigated. It was demonstrated that cells co-cultured in OIM showed high efficiency in osteogenesis but failed to form capillary-like structure while the results of co-culture in GM were the opposite. By comparing the angiogenic capacity of co-cultures under GM and OIM, chemokine (C-X-C motif) ligand 9 (Cxcl9), secreted by BMSCs in OIM, was identified to be an angiostatic factor to counter-regulate vascular endothelial growth factor (VEGF) and prevent its binding to HUVECs, which abrogated angiogenesis of MSCs-ECs co-culture. Moreover, Cxcl9 was proved to suppress the osteogenic differentiation of BMSCs monoculture. The molecular mechanism of Cxcl9 activation in BMSCs involved mTOR/STAT1 signaling pathway. Therefore, blocking this signaling pathway via rapamycin addition resulted in the inhibition of Cxcl9 and improvement of osteogenic differentiation and angiogenic capacity of co-culture in OIM. These results reveal that Cxcl9 is a negative modulator of angiogenesis and osteogenesis, and its inhibition could promote pre-vascularization of bone tissue engineering.

Transforming growth factor-ß1 stimulates mesenchymal stem cell proliferation by altering cell cycle through FAK-Akt-mTOR pathway.

Background: Mesenchymal stem cells (MSCs) are promising for cell therapy and regenerative medicine. An increased need for expanding of MSCs under serum-free condition to achieve a sufficient quantity for therapeutic applications is inevitable. Transforming growth factor-ß1 (TGF-ß1) is widely used for expanding clinical-grade MSCs in vitro. This work focuses on the influence of TGF-ß1 on proliferation in rat bone marrow-derived MSCs (BMSCs) and the underlying mechanism. Materials and Methods: BMSCs were isolated and cultured with or without TGF-ß1 in a serum-free medium and Cell Counting Kit-8 assay was used to detect BMSCs proliferation. Cell cycle transition was also analyzed. Further, the expression levels of cyclin D1, phosphorylated focal adhesion kinase, and downstream effectors in Akt-mTOR-S6K1 signaling pathway were examined by western blotting. Results and Conclusion: TGF-ß1 triggered proliferation via accelerating G1/S cell cycle transition in BMSCs. The addition of TGF-ß1 can activate Akt-mTOR-S6K1 pathway. Additionally, FAK was found to be involved in the process. Upon adding the FAK inhibitor, both the activation of Akt-mTOR-S6K1 and TGF-ß1-induced cell proliferation were abrogated. Together, an insight understanding of how TGF-ß1 influences BMSCs proliferation is achieved. This study provides a possible strategy of supplementing TGF-ß1 in serum-free medium for in vitro expansion, which eventually would advance the production of clinical-grade MSCs for regenerative medicine.

Regulation of osteogenesis of human amniotic mesenchymal stem cells by sodium butyrate.

Human amniotic membrane-derived mesenchymal stem cells (hAMSCs) draw great interests for regenerative medicine due to convenient availability and low immunogenicity. However, suboptimal culture conditions limit their application. In recent years, small molecules have proven powerful in regulating stem cell fates and can be applied to stimulate their function. In the present study, the impacts of sodium butyrate (NaBu), a histone deacetylase inhibitor (HDACi), on hAMSCs were investigated. It was shown that NaBu at a low concentration inhibited cell proliferation by arresting cell cycle at G0/G1 rather than inducing apoptosis. When NaBu was supplemented at a concentration of <1.0 mM for 3 days during osteogenic induction, significantly more mineralized nodules were generated and the expression of osteogenesis-related genes (ALP, Runx2, Opn, and Ocn) and proteins (Col1a1, OPN, OCN, Runx2, and TAZ) were both significantly enhanced. However, a higher concentration (1.0 mM) and longer exposure time (14 days) of NaBu showed no such effects, which may be partially attributed to both the increased expression of histone deacetylase 8 (HDAC8) and reduced level of H3K9-Ace, thus leading to the transcriptional inhibition during osteogenesis. Further, it was indicated that ERK might be involved in the stimulatory effects of NaBu. These findings may be helpful to develop an efficient culture process for hAMSCs towards bone regeneration.

Self-Assembling Peptide Nanofibrous Hydrogel on Immediate Hemostasis and Accelerative Osteosis.

The use of local agents to achieve hemostasis of bone that does not interfere with repair and recovery is a complex and emergency subject in surgery. In this study, the dual functional biodegradable self-assembling nanopeptide (SAP) RADA16-I was synthesized by solid phase synthesis and was shown to exhibit immediate hemostasis and accelerative osteosis. The RADA16-I showed good performance as a hemostatic agent, which was investigated by comparison with the effects of bone wax in the ilium bone defect model of New Zealand rabbits. The RADA16-I exhibited efficient function of bone regeneration in both radiographic analysis and histological examination, while the bone wax inhibited osteogenesis. Moreover, in in vivo experiment, the RADA16-I was shown to exhibit excellent biocompatibility, while the group with bone wax showed a severe inflammatory response at the interface with bone. Thus, the RADA16-I is proven to be an excellent biocompatible material with effective dual function of hemostasis and osteosis.

Structural genomics studies of human caries pathogen Streptococcus mutans.

Gram-positive bacterium Streptococcus mutans is the primary causative agent of human dental caries. To better understand this pathogen at the atomic structure level and to establish potential drug and vaccine targets, we have carried out structural genomics research since 2005. To achieve the goal, we have developed various in-house automation systems including novel high-throughput crystallization equipment and methods, based on which a large-scale, high-efficiency and low-cost platform has been establish in our laboratory. From a total of 1,963 annotated open reading frames, 1,391 non-membrane targets were selected prioritized by protein sequence similarities to unknown structures, and clustered by restriction sites to allow for cost-effective high-throughput conventional cloning. Selected proteins were over-expressed in different strains of Escherichia coli. Clones expressed soluble proteins were selected, expanded, and expressed proteins were purified and subjected to crystallization trials. Finally, protein crystals were subjected to X-ray analysis and structures were determined by crystallographic methods. Using the previously established procedures, we have so far obtained more than 200 kinds of protein crystals and 100 kinds of crystal structures involved in different biological pathways. In this paper we demonstrate and review a possibility of performing structural genomics studies at moderate laboratory scale. Furthermore, the techniques and methods developed in our study can be widely applied to conventional structural biology research practice.

In situ controlled release of rhBMP-2 in gelatin-coated 3D porous poly(ε-caprolactone) scaffolds for homogeneous bone tissue formation.

In tissue engineering, incorporation of bone morphogenetic protein-2 (BMP-2) into biomaterial scaffolds is an attractive strategy to stimulate bone repair. However, suboptimal release of BMP-2 remains a great concern, which may cause unfavorable bone formation as well as severe inflammation. In this study, genipin-cross-linked gelatin entrapped with recombinant human BMP-2 (rhBMP-2) was exploited to decorate the interior surface of three-dimensional porous poly(ε-caprolactone) (PCL) scaffolds. With gelatin-coating, PCL scaffolds demonstrated enhanced water uptake and improved compressive moduli. Intriguingly, a unique release profile of rhBMP-2 composed of a transient burst release followed by a sustained release was achieved in coated scaffolds. These coated scaffolds well supported growth and osteogenesis of human mesenchymal stem cells (hMSCs) in vitro, indicating the retaining of rhBMP-2 bioactivity. When hMSCs-seeded scaffolds were implanted subcutaneously in nude mice for 4 weeks, better bone formation was observed in gelatin/rhBMP-2-coated scaffolds. Specifically, the spatial distribution of newly formed bone was more uniform in gelatin-coated scaffolds than in uncoated scaffolds, which displayed preferential bone formation at the periphery. These results collectively demonstrated that gelatin-coating of porous PCL scaffolds is a promising approach for delivering rhBMP-2 to stimulate improved bone regeneration.

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.

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.

Modular multifunctional poly(ethylene glycol) hydrogels for stem cell differentiation.

Synthetic polymers are employed to create highly defined microenvironments with controlled biochemical and biophysical properties for cell culture and tissue engineering. Chemical modification is required to input biological or chemical ligands, which often changes the fundamental structural properties of the material. Here, we report on a simple modular biomaterial design strategy that employs functional cyclodextrin nanobeads threaded onto poly(ethylene glycol) polymer necklaces to form multifunctional hydrogels. Nanobeads with desired chemical or biological functionalities can be simply threaded onto the PEG chains to form hydrogels, creating an accessible platform for users. We describe the design and synthesis of these multifunctional hydrogels, elucidate structure-property relationships, and demonstrate applications ranging from stem cell culture and differentiation to tissue engineering.