NY-ESO-1-specific T cell receptor-engineered T cells and Tranilast, a TRPV2 antagonist bivalent treatment enhances the killing of esophageal cancer: a dual-targeted cancer therapeutic route.

BACKGROUND: Esophageal cancer (EC) is a global canker notorious for causing high mortality due to its relentless incidence rate, convoluted with unyielding recurrence and metastasis. However, these intricacies of EC are associated with an immoderate expression of NY-ESO-1 antigen, presenting a lifeline for adoptive T cell therapy. We hypothesized that naturally isolated higher-affinity T cell receptors (TCRs) that bind to NY-ESO-1 would allow T lymphocytes to target EC with a pronounced antitumor response efficacy. Also, targeting TRPV2, which is associated with tumorigenesis in EC, creates an avenue for dual-targeted therapy. We exploited the dual-targeting antitumor efficacy against EC. METHODS: We isolated antigen-specific TCRs (asTCRs) from a naive library constructed with TCRs obtained from enriched cytotoxic T lymphocytes. The robustness of our asTCRs and their TCR-T cell derivatives, Tranilast (TRPV2 inhibitor), and their bivalent treatment were evaluated with prospective cross-reactive human-peptide variants and tumor cells. RESULTS: Our study demonstrated that our naive unenhanced asTCRs and their TCR-Ts perpetuated their cognate HLA-A*02:01/NY-ESO-1(157-165) specificity, killing varying EC cells with higher cytotoxicity compared to the known affinity-enhanced TCR (TCRe) and its wild-type (TCR0) which targets the same NY-ESO-1 antigen. Furthermore, the TCR-Ts and Tranilast bivalent treatment showed superior EC killing compared to any of their monovalent treatments of either TCR-T or Tranilast. CONCLUSION: Our findings suggest that dual-targeted immunotherapy may have a superior antitumor effect. Our study presents a technique to evolve novel, robust, timely therapeutic strategies and interventions for EC and other malignancies.

A Rationally Designed CRISPR/Cas12a Assay Using a Multimodal Reporter for Various Readouts.

The CRISPR/Cas systems offer a programmable platform for nucleic acid detection, and CRISPR/Cas-based diagnostics (CRISPR-Dx) have demonstrated the ability to target nucleic acids with greater accuracy and flexibility. However, due to the configuration of the reporter and the underlying labeling mechanism, almost all reported CRISPR-Dx rely on a single-option readout, resulting in limitations in end-point result readouts. This is also associated with high reagent consumption and delays in diagnostic reports due to protocol differences. Herein, we report for the first time a rationally designed Cas12a-based multimodal universal reporter (CAMURE) with improved sensitivity that harnesses a dual-mode reporting system, facilitating options in end-point readouts. Through systematic configurations and optimizations, our novel universal reporter achieved a 10-fold sensitivity enhancement compared to the DETECTR reporter. Our unique and versatile reporter could be paired with various readouts, conveying the same diagnostic results. We applied our novel reporter for the detection of staphylococcal enterotoxin A due to its high implication in staphylococcal food poisoning. Integrated with loop-mediated isothermal amplification, our multimodal reporter achieved 10 CFU/mL sensitivity and excellent specificity using a real-time fluorimeter, in-tube fluorescence, and lateral flow strip readouts. We also propose, using artificially contaminated milk samples, a fast (2-5 min) Triton X-100 DNA extraction approach with a comparable yield to the commercial extraction kit. Our CAMURE could be leveraged to detect all gene-encoding SEs by simply reprogramming the guide RNA and could also be applied to the detection of other infections and disease biomarkers.

Thermal stress involved in TRPV2 promotes tumorigenesis through the pathways of HSP70/27 and PI3K/Akt/mTOR in esophageal squamous cell carcinoma.

BACKGROUND: The transient receptor potential vanilloid receptor 2 (TRPV2) has been found to participate in the pathogenesis of various types of cancers, however, its role(s) in the tumorigenesis of ESCC remain poorly understood. METHODS: Western blotting and immunohistochemistry were performed to determine the expression profiles of TRPV2 in the ESCC patient tissues. A series of in vitro and in vivo experiments were conducted to reveal the role of TRPV2 in the tumorigenesis of ESCC. RESULTS: Our study first uncovered that the activation of TRPV2 by recurrent acute thermal stress (54 °C) or O1821 (20 µM) promoted cancerous behaviours in ESCC cells. The pro-angiogenic capacity of the ESCC cells was found to be enhanced profoundly and both tumour formation and metastasis that originated from the cells were substantially promoted in nude mouse models upon the activation of TRPV2. These effects were inhibited significantly by tranilast (120 µM) and abolished by TRPV2 knockout. Conversely, overexpression of TRPV2 could switch the cells to tumorigenesis upon activation of TRPV2. Mechanistically, the driving role of TRPV2 in the progression of ESCC is mainly regulated by the HSP70/27 and PI3K/Akt/mTOR signalling pathways. CONCLUSIONS: We revealed that TRPV2-PI3K/Akt/mTOR is a novel and promising target for the prevention and treatment of ESCC.

Transient Receptor Potential Ankyrin 1 Contributes to Lysophosphatidylcholine-Induced Intracellular Calcium Regulation and THP-1-Derived Macrophage Activation.

Lysophosphatidylcholine (LPC) is a major atherogenic lipid that stimulates an increase in mitochondrial reactive oxygen species (mtROS) and the release of cytokines under inflammasome activation. However, the potential receptors of LPC in macrophages are poorly understood. Members of the transient receptor potential (TRP) channel superfamily, which is crucially involved in transducing environmental irritant stimuli into nociceptor activity, are potential receptors of LPC. In this study, we investigated whether LPC can induce the activation of transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily. The functional expression of TRPA1 was first detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and calcium imaging in human acute monocytic leukemia cell line (THP-1)-derived macrophages. The mechanism by which LPC induces the activation of macrophages through TRPA1 was verified by cytoplasmic and mitochondrial calcium imaging, mtROS detection, a JC-1 assay, enzyme-linked immunosorbent assay, the CCK-8 assay and the lactate dehydrogenase (LDH) cytotoxic assay. LPC induced the activation of THP-1-derived macrophages via calcium influx, and this activation was suppressed by potent and selective inhibitors of TRPA1. These results indicated that TRPA1 can mediate mtROS generation, mitochondrial membrane depolarization, the secretion of IL-1ß and cytotoxicity through cellular and mitochondrial Ca2+ influx in LPC-treated THP-1-derived macrophages. Therefore, the inhibition of TRPA1 may protect THP-1-derived macrophages against LPC-induced injury.

Transient receptor potential ankyrin 1 contributes to the ATP-elicited oxidative stress and inflammation in THP-1-derived macrophage.

P2X7 receptor (P2X7R) is an ATP-gated non-selective cation channel which mediates ATP-induced inflammation in macrophages. Transient receptor potential (TRP) receptors are nociceptors in cellular membrane which can perceive the stimuli of environmental irritant. The interaction between TRP channels and P2X7R has been found while the details about inflammation are still unclear. In this study, we suggested that transient receptor potential ankyrin 1 (TRPA1), a member of TRP superfamily, participates in ATP-induced oxidative stress and inflammation in human acute monocytic leukemia cell line (THP-1)-derived macrophage. The co-localization between TRPA1 and P2X7R was detected using immunofluorescence in THP-1-derived macrophage and transfected human embryonic kidney cell line (HEK293T). The mechanism by which ATP or 3'-O-(4-Benzoylbenzoyl)-ATP (BzATP) induces the activation of macrophages was verified by calcium imaging, mitochondrial reactive oxygen species (mtROS) detection, mitochondrial membrane potential (∆Ψm) measurement, flow cytometry, enzyme-linked immunosorbent assay (ELISA), western blotting, CCK-8 assay, and the lactate dehydrogenase (LDH) release cytotoxic assay. The BzATP and ATP induced calcium overload, mitochondria injury, interleukin-1ß (IL-1ß) secretion, and cytotoxicity can be inhibited by TRPA1 antagonists. These results indicated that TRPA1 can co-localize with P2X7R and mediate ATP-induced oxidative stress and inflammation. Therefore, the inhibition of TRPA1 may provide a potential therapy for ATP-elicited inflammatory diseases, including atherosclerosis.

Promoting Osteogenesis of Human Umbilical Cord Mesenchymal Stem Cells Using 1α, 25-(OH)2D3 to Change Intracellular Calcium Concentration.

Vitamin D plays a major role in the regulation of calcium homeostasis and affects bone metabolism. There is currently limited detailed knowledge about the vitamin D endocrine system in human bone cells. Here, we investigated the direct effects of 1α, 25-dihydroxyvitamin D3 (1α, 25-(OH)2D3 or 'VD3') on osteogenesis of human umbilical cord mesenchymal stem cells (HUCMSCs). We also studied the impact of VD3 on intracellular Ca2+ concentrations in osteogenic cells. The results of alizarin red staining and alkaline phosphatase activity tests showed that VD3 could not induce osteogenic differentiation in HUCMSCs. However, addition of VD3 to the osteogenic differentiationinducing medium could promote HUCMSC to differentiate into osteoblasts. Calcium imaging showed that the addition of VD3 increased intracellular Ca2+ concentrations in osteogenic HUCMSCs. Thus, we concluded that adding VD3 increased intracellular Ca2+ concentrations in osteogenic HUCMSCs and promoted their osteogenesis.

Potential Target of CDK6 Signaling Pathway for Cancer Treatment.

BACKGROUND: Cancer involves uncontrolled cell growth due to genetic mutations. Tumors can form when CDK6, a gene essential for controlling cell growth, isn't working correctly. Researchers are investigating drugs that inhibit CDK6; some of them appear promising. Nevertheless, CDK6 is advantageous and harmful to cancer because it controls other cellular processes. By inhibiting CDK6 and CDK4, CDK4/6 inhibitors offer a novel therapeutic strategy that stops cell proliferation. The study investigates the function of CDK6 in cancer, the difficulties in targeting CDK6, and possible remedies. OBJECTIVE: Scientists have developed drugs designed to block CDK6 and prevent it from altering other proteins. These drugs, also known as CDK6 inhibitors, help treat cancer. Finding the best drugs for CDK6 is still tricky, though. The drugs' selectivity, potency, and cost are some difficulties. These factors depend on CDK6's structure and interactions with other proteins. The structure of CDK6 and how it influences its function and regulation are explained in this review. It also describes CDK6's function in cancer and its interaction with other molecules and proteins, which is crucial for cell division. Moreover, this review describes how CDK6 interacts with the drugs that block it and what the current and future treatments that target CDK6 are. CONCLUSION: This review presents the structure, current research, and overview of CDK6. It also reviews the role of CDK6 in cancer, function, and regulation. Additionally, it explores its role in cancer signaling networks and its interaction with CDK6 inhibitors. Lastly, it discusses the current status and prospects of therapies targeting CDK6.

Network Pharmacology-Based Virtual Screening of Chemical Constituents from Vitexnegundo Linn's Discovered Novel Molecular Targets for Breast Cancer Treatment.

BACKGROUND: The Chinese chaste tree Vitexnegundo (VN) is a popular herb in South and Southeast Asia that has several health benefits, including the ability to inhibit tumor growth and induce apoptosis in multiple tumors. Literature revealed scanty research on breast cancer, with little focus on the molecular mechanism of the disease and an emphasis on targets, biological networks, and active components. Exploring natural compounds as possible therapeutic options is an old but still promising approach for drug discovery and development. This study used a thorough computational and statistical method to screen potential drug candidates. METHODS: The active ingredients and targets of VN were identified using SwissADME, SwissTargetPrediction, STITCH, IMPPAT database, KNapSAcK database, and literature. The OMIM and GeneCards databases were searched for possible targets related to breast cancer. The PASS online server was used to check the probability of active metabolite (Pa) against breast cancer. To build protein-protein interactions (PPI) networking, the intersection of disease and drug targets was uploaded to the STITCH database. Cytoscape software was used to analyze the topology parameters of networking to identify hub targets. Gene Ontology (GO) was analyzed using Metascape and ShinyGO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using the David database and SR plot, and the site of expression and protein domain were studied using FunRich. We employed AutoDockvina, Discovery Studio, and UCSF ChimeraX software and auxiliary tools for molecular docking and analysis. Zincpharmer was used for pharmacophore mapping. ADMET analysis was conducted using ADMETsar, Swiss ADME, ADMETLab servers, and mypresto using GROMACS for molecular dynamics simulation (MDS). RESULTS: A total of 65 targets and 21 active ingredients were identified. Further investigation was conducted on 20 hub targets selected through PPI networking construction. The enrichment analysis results indicated that the key factors were P, amyloid-beta response, cellular response to amyloid- beta, Pos. reg. of G2/M transition of the mitotic cell cycle, and response to a toxic substance. The molecular docking, pharmacophore mapping, and MD simulation results indicated that apigenin, kaempferol, and luteolin positively interacted with CDK1 and CDK6 proteins. CONCLUSION: This study is the first to use network pharmacology, molecular docking, pharmacophore mapping, and MD simulation to identify the active ingredients, molecular targets, and critical biological pathways responsible for VN anti-breast cancer. The study provides a theoretical basis for further research in this area.

STING agonist diABZI enhances the cytotoxicity of T cell towards cancer cells.

Antigen-specific T cell receptor-engineered T cell (TCR-T) based immunotherapy has proven to be an effective method to combat cancer. In recent years, cross-talk between the innate and adaptive immune systems may be requisite to optimize sustained antigen-specific immunity, and the stimulator of interferon genes (STING) is a promising therapeutic target for cancer immunotherapy. The level of expression or presentation of antigen in tumor cells affects the recognition and killing of tumor cells by TCR-T. This study aimed at investigating the potential of innate immune stimulation of T cells and engineered T cells to enhance immunotherapy for low-expression antigen cancer cells. We systematically investigated the function and mechanism of cross-talk between STING agonist diABZI and adaptive immune systems. We established NY-ESO-1 full knockout Mel526 cells for this research and found that diABZI activated STING media and TCR signaling pathways. In addition, the results of flow cytometry showed that antigens presentation from cancer cells induced by STING agonist diABZI also improved the affinity of TCR-T cells function against tumor cells in vitro and in vivo. Our findings revealed that diABZI enhanced the immunotherapy efficacy of TCR-T by activating STING media and TCR signaling pathways, improving interferon-γ expression, and increasing antigens presentation of tumor cells. This indicates that STING agonist could be used as a strategy to promote TCR-T cancer immunotherapy.

SYVN1 ubiquitinates FoxO1 to induce ß-catenin nuclear translocation, PD-L1-mediated metastasis, and immune evasion of hepatocellular carcinoma.

BACKGROUND: A high incidence of hepatocellular carcinoma (HCC), the most frequently diagnosed form of liver cancer, is observed in Africa and Asia. SYVN1 is upregulated in HCC; however, the biological roles of SYVN1 in immune evasion remain unclear. METHODS: RT-qPCR and western blot were employed to detect the expression levels of SYVN1 and the key molecules in HCC cells and tissues. Flow cytometry was used to determine the proportion of T cells, and an ELISA assay was used to determine the amount of IFN-γ secreted. Cell viability was monitored by CCK-8 and colony formation assays. The metastatic properties of HCC cells were detected by Transwell assays. Bioinformatics analysis, ChIP, and luciferase assays were used to study the transcriptional regulation of PD-L1. Co-IP was used to detect direct interaction between SYVN1 and FoxO1, as well as the ubiquitination of FoxO1. The in vitro findings were validated in xenograft and lung metastasis models. RESULTS: In HCC cells and tissues, SYVN1 was upregulated while FoxO1 was downregulated. SYVN1 knockdown or FoxO1 overexpression reduced PD-L1 expression, and inhibited immune evasion, cell growth, and metastasis in HCC cells. Mechanistically, FoxO1 regulated PD-L1 transcription in a ß-catenin-independent or -dependent manner. Functional studies further showed that SYVN1 promoted immune evasion, cell proliferation, migration and invasion via facilitating ubiquitin-proteasome-dependent degradation of FoxO1. In vivo investigations showed that silencing of SYVN1 inhibited immune evasion and metastasis of HCC cells, possible via the FoxO1/PD-L1 axis. CONCLUSION: SYVN1 regulates FoxO1 ubiquitination to stimulate ß-catenin nuclear translocation and promotes PD-L1-mediated metastasis and immune evasion in HCC.

Large-scale expansion of human umbilical cord-derived mesenchymal stem cells using PLGA@PLL scaffold.

Mesenchymal stem cells (MSCs) are highly important in biomedicine and hold great potential in clinical treatment for various diseases. In recent years, the capabilities of MSCs have been under extensive investigation for practical application. Regarding therapy, the efficacy usually depends on the amount of MSCs. Nevertheless, the yield of MSCs is still limited due to the traditional cultural methods. Herein, we proposed a three-dimensional (3D) scaffold prepared using poly lactic-co-glycolic acid (PLGA) nanofiber with polylysine (PLL) grafting, to promote the growth and proliferation of MSCs derived from the human umbilical cord (hUC-MSCs). We found that the inoculated hUC-MSCs adhered efficiently to the PLGA scaffold with good affinity, fast growth rate, and good multipotency. The harvested cells were ideally distributed on the scaffold and we were able to gain a larger yield than the traditional culturing methods under the same condition. Thus, our cell seeding with a 3D scaffold could serve as a promising strategy for cell proliferation in the large-scale production of MSCs. Moreover, the simplicity and low preparation cost allow this 3D scaffold to extend its potential application beyond cell culture. Supplementary Information: The online version contains supplementary material available at 10.1186/s40643-023-00635-6.

Harnessing enhanced CRISPR/Cas12a trans-cleavage activity with extended reporters and reductants for early diagnosis of Helicobacter pylori, the causative agent of peptic ulcers and stomach cancer.

Developing rapid and non-invasive diagnostics for Helicobacter pylori (HP) is imperative to prevent associated diseases such as stomach gastritis, ulcers, and cancers. Owing to HP strain heterogeneity, not all HP-infected individuals incur side effects. Cytotoxin-associated gene A (CagA), and vacuolating cytotoxin A (VacA) genes predominantly drive HP pathogenicity. Therefore, diagnosing CagA and VacA genotypes could alert active infection and decide suitable therapeutics. We report an enhanced LbCas12a trans-cleavage activity with extended reporters and reductants (CEXTRAR) for early detection of HP. We demonstrate that extended ssDNA reporter acts as an excellent signal amplifier, making it a potential alternative substrate for LbCas12a collateral activity. Through a systematic investigation of various buffer components, we demonstrate that reductants improve LbCas12a trans-cleavage activity. Overall, our novel reporter and optimal buffer increased the trans-cleavage activity to an order of 16-fold, achieving picomolar sensitivity (171 pM) without target pre-amplification. Integrated with loop-mediated isothermal amplification (LAMP), CEXTRAR successfully attained attomolar sensitivity for HP detection using real-time fluorescence (43 and 96 aM), in-tube fluorescence readouts (430 and 960 aM), and lateral flow (4.3 and 9.6 aM) for CagA and VacA, respectively. We also demonstrate a rapid 2-min Triton X-100 lysis for clinical sample analysis, which could provide clinicians with actionable information for rapid diagnosis. CEXTRAR could potentially spot the 13C urea breath test false-negatives. For the first time, our study unveils an experimental outlook to manipulate reporters and reconsider precise cysteine substitution via protein engineering for Cas variants with enhanced catalytic activities for use in diagnostics and genetic engineering.

Ameliorating Effect of Umbilical Cord Mesenchymal Stem Cells in a Human Induced Pluripotent Stem Cell Model of Dravet Syndrome.

Dravet syndrome (DS) is a form of severe childhood-onset refractory epilepsy typically caused by a heterozygous loss-of-function mutation. DS patient-derived induced pluripotent stem cells (iPSCs) are appropriate human cells for exploring disease mechanisms and testing new therapeutic strategies in vitro. Repeated spontaneous seizures can cause neuroinflammatory reactions and oxidative stress, resulting in neuronal toxicity, neuronal dysfunction, blood-brain barrier disruption, and hippocampal inflammation. Antiepileptic drug therapy does not delay the development of chronic epilepsy. The application of mesenchymal stem cells (MSCs) is one therapeutic strategy for thwarting epilepsy development. This study evaluated the effects of human umbilical cord mesenchymal stem cell-conditioned medium (HUMSC-CM) in a new in vitro model of neurons differentiated from DS patient-derived iPSCs. In the presence of HUMSC-CM, increases in superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), glutathione peroxidase (GPX), and glutathione (GSH) levels were found to contribute to a reduction in reactive oxygen species (ROS) levels. In parallel, inflammation was rescued in DS patient-derived neuronal cells via increased expression of anti-inflammatory cytokines (TGF-ß, IL-6, and IL-10) and significant downregulation of tumor necrosis factor-α and interleukin-1ß expression. The intracellular calcium concentration ([Ca2+]i) and malondialdehyde (MDA) and ROS levels were decreased in DS patient-derived cells. In addition, action potential (AP) firing ability was enhanced by HUMSC-CM. In conclusion, HUMSC-CM can effectively eliminate ROS, affect migration and neurogenesis, and promote neurons to enter a highly functional state. Therefore, HUMSC-CM is a promising therapeutic strategy for the clinical treatment of refractory epilepsy such as DS.

Mechanistic insights of CRISPR/Cas nucleases for programmable targeting and early-stage diagnosis: A review.

Conventional and routine diagnostics such as polymerase chain reaction (PCR) and serological tests are less sensitive, costly, and require sample pretreatment procedures. CRISPR/Cas systems that inherently assist bacteria and archaea in destroying invading phage genetic materials via an RNA-mediated interference strategy have been reconstituted in vitro and harnessed for nucleic and non-nucleic acid diagnostics. CRISPR/Cas-based diagnostics (CRISPR-Dx) are cost-effective, possess excellent sensitivity (attomolar) and specificity (single base distinction), exhibit fast turnaround response, and support nucleic acid extraction-free workflow. However, CRISPR-Dx still needs to address various challenges to translate the laboratory work into end-user tailored solutions. In this perspective, we review the relevant progress of CRISPR/Cas systems-based diagnostics, focusing on the comprehensive customization and applications of leading and trending CRISPR/Cas systems as platform technologies for fluorescence, colorimetric, and electrical signal detection. The impact of the CRISPR game-changing technology on the COVID-19 pandemic is highlighted. We also demonstrate the role of CRISPR/Cas systems for carryover contamination prevention. The advancements in signal amplification strategies using engineered crRNAs, novel reporters, nanoparticles, artificial genetic circuits, microfluidics, and smartphones are also covered. Furthermore, we critically discuss the translation of CRISPR-Dx's basic research into end-user diagnostics for commercialization success in the near future. Finally, we discuss the complex challenges and alternative solutions to harness the CRISPR/Cas potential in detail.

Transcription factor EB-mediated mesenchymal stem cell therapy induces autophagy and alleviates spinocerebellar ataxia type 3 defects in neuronal cells model.

Defects in ataxin-3 proteins and CAG repeat expansions in its coding gene ATXN3 cause Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph disease (MJD) polyglutamine neurodegenerative disease. The mutant proteins aggregate as inclusion bodies in cells and compete with wild-type ataxin-3, which leads to neuronal dysfunction or death and impairs Beclin1-mediated autophagy. It has been reported that Mesenchymal stem cells (MSCs) can reliably treat several neurodegenerative diseases. Herein, we used a Transcription Factor EB (TFEB) nuclear translocation-mediated MSCs co-culture approach to reconstitute autophagy and lysosomal biogenesis, and reduce SCA3-like behaviors in induced pluripotent stem cells (iPSCs)-derived neuron cells models. Our iPSCs model showed enhanced expression of autophagy proteins, attenuated the expression and toxic effects of mutant ataxin-3 on neurons, and alleviated the effects of ataxin-3 on autophagy. Therefore, MSCs are associated with autophagy-inducing therapy and compared to animal models, our MSCs co-culture could be used as a novel and potential therapeutic approach to study SCA3 disease and other neurodegenerative diseases.

S100A4 plays a key role in TRPV3 ion channel expression and its electrophysiological function.

Transient receptor potential vanilloid 3 (TRPV3), a non-selective cation ion channel, is regulated by small molecules such as Ca2+ and calmodulin (CaM). Together with S100A4 (S100 calcium-binding protein family), is critical in cell proliferation and progression. Although TRPV3 has been proved to play a role in Ca2+ regulation and participate in Ca2+-related cellular processes, its molecular mechanism remains unclear. In this study, we found that TRPV3 and S100A4 were co-expressed in the same region of the cell, and surprisingly, the protein expression level of TRPV3 significantly increased with the overexpression of S100A4. Moreover, co-immunoprecipitation results showed that these two proteins could bind with each other. Functionally, we found that when S100A4 was simultaneously expressed in cells, more Ca2+ would be transferred into the cells through the TRPV3 ion channel. Consistent with Ca2+ regulation results, electrophysiological recordings demonstrated that S100A4 improved the function of TRPV3 in ions' flux, suggesting that the S100A4 could bind with TRPV3 and simultaneously promoted its expression, thus affecting its functions on related ions' flux. Our findings identified the link between S100A4 and TRPV3 and provided a novel molecular mechanism for TRPV3 regulation.

Generation of UCiPSC-derived neurospheres for cell therapy and its application.

BACKGROUND: Neural stem cell (NSC) therapy remains one of the most potential approaches for the treatment of neurological disorders. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized the technique of cell therapy. Meanwhile, it is often required that NSCs are stored and transported to a long distance for research or treatment purposes. Although high survival rates could be maintained, conventional methods for cell transportation (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore, the establishment of a safe, affordable, and low-cost strategy to store and transport easily accessible hiPSCs and hiNSCs, with characteristics that match fetal hNSCs, is incredibly urgent. METHODS: We reprogrammed human urinary cells to iPSCs using a non-integrating, virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis, and in vitro as well as in vivo differentiation capabilities, etc.). RESULTS: Here, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique. Next, we demonstrated that the iNSCs differentiated into mature cerebral cortical neurons and neural networks. Interestingly, hiNSCs survived longer as neurospheres at ambient temperature (AT) than those cultured in a monolayer. Within 7 days approximately, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to AT that were placed under standard culture conditions (37 °C, 5% CO2) recovered their typical morphology, and retained their proliferation and differentiation abilities. CONCLUSIONS: In this study, we provided a simple method for the storage of NSCs as neurospheres at AT as an alternative method to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at AT and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.

Hair follicle-derived mesenchymal stem cells decrease alopecia areata mouse hair loss and reduce inflammation around the hair follicle.

BACKGROUND: Alopecia areata (AA) is a common autoimmune hair loss disease with increasing incidence. Corticosteroids are the most widely used for hair loss treatment; however, long-term usage of hormonal drugs is associated with various side effects. Mesenchymal stem cells (MSCs) therapy has been studied extensively to curb autoimmune diseases without affecting immunity against diseases. METHODS: Hair follicle-derived MSCs (HF-MSCs) were harvested from the waste material of hair transplants, isolated and expanded. The therapeutic effect of HF-MSCs for AA treatment was investigated in vitro AA-like hair follicle organ model and in vivo C3H/HeJ AA mice model. RESULTS: AA-like hair follicle organ in vitro model was successfully established by pre-treatment of mouse vibrissa follicles by interferon-γ (IFN-γ). The AA-like symptoms were relieved when IFN-γ induced AA in vitro model was co-cultured with HF-MSC for 2 days. In addition, when skin grafted C3H/HeJ AA mice models were injected with 106 HF-MSCs once a week for 3 weeks, the transcription profiling and immunofluorescence analysis depicted that HF-MSCs treatment significantly decreased mouse hair loss and reduced inflammation around HF both in vitro and in vivo. CONCLUSIONS: This study provides a new therapeutic approach for alopecia areata based on HF-MSCs toward its future clinical application.

circRNA_100859 functions as an oncogene in colon cancer by sponging the miR-217-HIF-1α pathway.

Circular RNAs (circRNAs) play an important role in cancer development and progression by regulating gene expression. The present study aimed to investigate the function of circRNA_100859 in colon cancer. circRNA expression profiles from a human circRNAs chip were analyzed. The effects of circRNA_100859 on cell proliferation and apoptosis were assessed in vitro and interactions between circRNA_100859 and its micro (mi)RNA and target genes were analyzed. The diagnostic and prognostic significance of circRNA_100859 was also investigated. It was identified that circRNA_100859 was overexpressed in colon cancer tissues and promoted cell proliferation and inhibited cell apoptosis. Additionally, bioinformatics and a dual-luciferase reporter assay confirmed that circRNA_100859 acted as a miR-217 sponge, and miR-217 directly targeted hypoxia-inducible factor (HIF)-1α. Rescue assays demonstrated that HIF-1α protein and mRNA expression levels and cell proliferation were regulated by the circRNA_100859/miR-217 axis (P<0.05). Furthermore, statistical analysis showed that the circRNA_100859-miR-217-HIF-1α axis was associated with Tumor-Node-Metastasis (TNM) stage, histological grade, and KRAS mutations, and also showed high diagnostic and prognostic value for patients with colon cancer (P<0.05). Therefore, it was concluded that circRNA_100859 functions as an oncogene in colon cancer by sponging the miR-217-HIF-1α pathway. In addition, the circRNA_100859-miR-217-HIF-1α axis may serve as a novel diagnostic and prognostic biomarker for patients with colon cancer.

A heterozygous SCN1A (c.A5768G/+) mutant human induced pluripotent stem cell line (USTCi002-A) generated using TALEN-mediated precise gene editing.

Severe mycological epilepsy of infancy is a catastrophic disease with preferential dysfunction of interneurons, frequentepisoderate, cognitive and sudden death. The disease is mainly caused by heterozygous loss-of-function mutation of SCN1A gene encoding α subunit of the sodium channel Nav1.1. To generate mutations in normal iPSC, Transcription activator-like effector nucleases was used to introduce the epilepsy-causing mutation A5768G into the endogenous locus of SCN1A gene. The gene editing induced pluripotent stem cell line and normal iPSC were obtained from the same donor to eliminate significantly the genetic background noise.