Data-driven selection of analysis decisions in single-cell RNA-seq trajectory inference.

Single-cell RNA sequencing (scRNA-seq) experiments have become instrumental in developmental and differentiation studies, enabling the profiling of cells at a single or multiple time-points to uncover subtle variations in expression profiles reflecting underlying biological processes. Benchmarking studies have compared many of the computational methods used to reconstruct cellular dynamics; however, researchers still encounter challenges in their analysis due to uncertainty with respect to selecting the most appropriate methods and parameters. Even among universal data processing steps used by trajectory inference methods such as feature selection and dimension reduction, trajectory methods' performances are highly dataset-specific. To address these challenges, we developed Escort, a novel framework for evaluating a dataset's suitability for trajectory inference and quantifying trajectory properties influenced by analysis decisions. Escort evaluates the suitability of trajectory analysis and the combined effects of processing choices using trajectory-specific metrics. Escort navigates single-cell trajectory analysis through these data-driven assessments, reducing uncertainty and much of the decision burden inherent to trajectory inference analyses. Escort is implemented in an accessible R package and R/Shiny application, providing researchers with the necessary tools to make informed decisions during trajectory analysis and enabling new insights into dynamic biological processes at single-cell resolution.

Comprehensive analysis of transcriptomics and metabolomics to understand chronic ethanol induced murine cardiotoxicity.

Alcohol abuse has attracted public attention and long-term alcohol exposure can lead to alcohol-featured non-ischemic dilated cardiomyopathy. However, the precise underlying mechanisms of alcoholic cardiomyopathy remain to be elucidated. This study aimed to comprehensively characterize alcohol abuse-mediated effects on downstream metabolites and genes transcription using a multi-omics strategy. We established chronic ethanol intoxication model in adult male C57BL/6 mice through 8 weeks of 95% alcohol vapor administration and performed metabolomics analysis, mRNA-seq and microRNA-seq analysis with myocardial tissues. Firstly, ethanol markedly induced ejection fraction reductions, cardiomyocyte hypertrophy, and myocardial fibrosis in mice with myocardial oxidative injury. In addition, the omics analysis identified a total of 166 differentially expressed metabolites (DEMs), 241 differentially expressed genes (DEGs) and 19 differentially expressed microRNAs (DEmiRNAs), respectively. The results highlighted that alcohol abuse mainly interfered with endogenous lipids, amino acids and nucleotides production and the relevant genes transcription in mice hearts. Based on KEGG database, the affected signaling pathways are primarily mapped to the antigen processing and presentation, regulation of actin cytoskeleton, AMPK signaling pathway, tyrosine metabolism and PPAR signaling pathway, etc. Furthermore, 9 hub genes related to oxidative stress from DEGs were selected based on function annotation, and potential alcoholic cardiotoxic oxidative stress biomarkers were determined through establishing PPI network and DEmiRNAs-DEGs cross-talk. Altogether, our data strongly supported the conclusion that ethanol abuse characteristically affected amino acid and energy metabolism, nucleotide metabolism and especially lipids metabolism in mice hearts, and underlined the values of lipids signaling and oxidative stress in the treatment strategies.

Inhibition of Ceramide Synthesis Attenuates Chronic Ethanol Induced Cardiotoxicity by Restoring Lysosomal Function and Reducing Necroptosis.

AIMS: Chronic alcohol misuse could cause alcoholic cardiomyopathy (ACM), and the specific mechanisms remained largely unknown. In this study, we aimed to explore the effects of endogenous ceramides on chronic ethanol-induced myocardial injury or cell loss (e.g. necroptosis). METHODS: We established chronic alcohol intoxication models in vivo (male C57BL/6 mice) and in vitro (H9c2 cardiomyoblasts). The ceramide profiles were analyzed in mice myocardium and cultured cardiomyocytes. Further research on the role of ceramides and underlying signaling pathways was carried out in H9c2 cells. RESULTS AND CONCLUSIONS: The ceramide profiles analysis revealed increased long and very long-chain ceramides in alcoholic myocardium and ethanol-treated cardiomyocytes. Next, we proved that endogenous ceramide inhibition could reduce necroptosis and alleviate cardiomyocytes injury as suggested by decreased levels of p-RIPK1, p-RIPK3 and p-MLKL proteins and cardiac injury factors expression. Furthermore, we found that lysosomal dysfunction also contributed to alcohol-induced cardiac damage and inhibiting ceramide biosynthesis could repaired this to some extent. Cells studies with exogenous C6 ceramide confirmed the pleotropic roles of ceramide in myocardial damage by causing both necroptosis and lysosomal dysfunction. Finally, our data suggested that lysosomal dysfunction could sensitize cardiomyocytes to induction of necroptosis due to the restriction on degradation of RIPK1/RIPK3 proteins. In conclusion, chronic ethanol treatment boosted myocardial ceramide synthesis in animal hearts and cultured cardiomyocytes. Moreover, ceramides exerted crucial roles in the intrinsic signaling pathways of alcohol-induced cardiotoxicity. Targeting ceramide biosynthesis to simultaneously attenuate necroptosis and lysosomal dysfunction might be a novel strategy for preventing alcoholic cardiotoxicity.

Chemical Enhancement and Quenching in Single-Molecule Tip-Enhanced Raman Spectroscopy.

Despite intensive research in surface enhanced Raman spectroscopy (SERS), the influence mechanism of chemical effects on Raman signals remains elusive. Here, we investigate such chemical effects through tip-enhanced Raman spectroscopy (TERS) of a single planar ZnPc molecule with varying but controlled contact environments. TERS signals are found dramatically enhanced upon making a tip-molecule point contact. A combined physico-chemical mechanism is proposed to explain such an enhancement via the generation of a ground-state charge-transfer induced vertical Raman polarizability that is further enhanced by the strong vertical plasmonic field in the nanocavity. In contrast, TERS signals from ZnPc chemisorbed flatly on substrates are found strongly quenched, which is rationalized by the Raman polarizability screening effect induced by interfacial dynamic charge transfer. Our results provide deep insights into the understanding of the chemical effects in TERS/SERS enhancement and quenching.

Induced antibodies directed to the angiotensin receptor type 1 provoke skin and lung inflammation, dermal fibrosis and act species overarching.

OBJECTIVE: To determine contributions and functions of autoantibodies (Abs) directed to the angiotensin receptor type 1 (AT1R), which are suggested to be involved in the pathogenesis of AT1R Abs-related diseases such as systemic sclerosis (SSc). METHODS: C57BL/6J mice were immunised with membrane-embedded human AT1R or empty membrane as control. Mice deficient for CD4+ or CD8+ T cells and B cells were immunised with membrane-embedded AT1R or an AT1R peptide proposed to be a dominant T cell epitope. A monoclonal (m)AT1R Ab was generated by hybridoma technique and transferred into C57BL/6J and AT1Ra/b knockout mice. The induced phenotype was examined by histology, immunohistochemistry, immunofluorescence, apoptosis assay and ELISA. In vitro, Abs responses towards AT1R were measured in cells of different origins and species. RESULTS: AT1R-immunised mice developed perivascular skin and lung inflammation, lymphocytic alveolitis, weak lung endothelial apoptosis and skin fibrosis accompanied by Smad2/3 signalling, not present in controls or mice deficient for CD4+ T and B cells. The AT1R peptide 149-172 provoked lung inflammation. Application of the mAT1R Ab induced skin and lung inflammation, not observed in AT1Ra/b knockout mice. In vitro, AT1R Abs activated rat cardiomyocytes and human monocytes, enhanced angiotensin II-mediated AT1R activation in AT1R-transfected HEK293 cells via AT1R binding and mAT1R Ab-activated monocytes mediated the induction of profibrotic markers in dermal fibroblasts. CONCLUSION: Our immunisation strategy successfully induced AT1R Abs, contributing to inflammation and, possibly, to fibrosis via activation of AT1R. Therefore, AT1R Abs are valuable targets for future therapies of SSc and other AT1R Ab-related diseases.

Bio-Inspired Antibacterial Hydrogel Adhesives with High Adhesion Strength.

Traditional adhesives such as cyanoacrylate glue are mostly solvent-based. They are facing the problem of insufficient adhesion to some substrates, and also from the drawback of volatilization and release of small organic molecules in the process of usage. Therefore, a novel adhesive with non-irritating, high adhesive strength, and antibacterial properties is highly required. In this study, a full physically crosslinked zwitterionic poly(betaine sulfonate methacrylate) (PSBMA) hydrogel is proposed. The physical crosslinking interactions endow the hydrogel with good self-healing properties. Furthermore, the pure physical crosslinking hydrogel can form PSBMA powder adhesive after lyophilization and return to the hydrogel state after hydration. The mechanical properties of PSBMA adhesive can be modulated via adjusting the solid content and initiator dosage. Following the cure process similar to that of snail mucus or insect exoskeletons in nature, the adhesion of the PSBMA adhesive is improved at least 100 times than its wet state. In addition, the PSBMA adhesive is easy to be removed due to the dissociation of cross-linked structures in saltwater environments. Moreover, PSBMA adhesive with antifouling properties can effectively prevent the adhesion of proteins and bacteria, which shows potential applications in the assembly of medical devices.

Effects of co-exposure to lead and manganese on learning and memory deficits.

Lead (Pb) and manganese (Mn) are common neurotoxins. However, individuals are subject to co-exposures in real life, and it is therefore important to study these metals in combination. Weaning Sprague-Dawley rats were given ad libitum access to drinking water solutions containing Pb (100 mg/L), Mn (2.5 mg/mL) or a mixture, and each treatment had its own minocycline (50 mg/(kg•day)) supplement group. The results showed a significant difference in spatial memory and induction levels of hippocampal long-term potentiation (LTP) in all exposure groups when compared with controls. The combined-exposure group exhibited the most pronounced effect when compared with each of the single-metal exposure groups. Microglia displayed activation at day 3 after exposure alone or in combination, while astrocytes showed activation at day 5, accompanied by decreased expression levels of GLAST, GLT-1, and GS. Furthermore, the levels of glutamate in the synaptic cleft increased significantly. When microglial activation was inhibited by minocycline, the activation of astrocytes and the expression of GLAST, GLT-1, and GS were both reversed. In addition, upon minocycline treatment, hippocampal LTP impairment and cognitive injury were significantly alleviated in each of the exposure groups. These results suggest that combined exposure to Pb and Mn can cause greater effects on cognition and synaptic plasticity when compared to single-metal exposure groups. The reason may involve abnormal activation of microglia leading to excessive regulation of astrocytes, resulting in glutamate reuptake dysfunction in astrocytes and leading to perturbed cognition and synaptic plasticity.

Pharmacological activation of CB2 receptor protects against ethanol-induced myocardial injury related to RIP1/RIP3/MLKL-mediated necroptosis.

Chronic ethanol abuse can lead to harmful consequences for the heart, resulting in systolic dysfunction, variability in the heart rate, arrhythmia, and cardiac remodelling. However, the precise molecular mechanism responsible for ethanol-induced cardiomyopathy is poorly understood. In this regard, the present study aimed to describe the RIP1/RIP3/MLKL-mediated necroptotic cell death that may be involved in ethanol-induced cardiomyopathy and characterize CBR-mediated effects on the signalling pathway and myocardial injury. We performed an ethanol vapour administration experiment to analyse the effects of ethanol on cardiac structure and function in male C57BL/6J mice. Ethanol induced a significant decline in the cardiac structure and function, as evidenced by a decline in ejection fraction and fractional shortening, and an increase in serum Creatine Kinase levels, myocardial collagen content, and inflammatory reaction. Furthermore, ethanol also upregulated the expression levels of necroptosis-related markers such as p-RIP1, p-RIP3, and p-MLKL in the myocardium. Nec-1 treatment exerted significant cardioprotective effects by salvaging the heart tissue, improving the cardiac function, and mitigating inflammation and necroptosis. In addition, ethanol abuse caused an imbalance in the endocannabinoid system and regulated two cannabinoid receptors (CB1R and CB2R) in the myocardium. Treatment with selective CB2R agonists, JWH-133 or AM1241, markedly improved the cardiac dysfunction and reduced the ethanol-induced necroptosis in the myocardium. Altogether, our data provide evidence that ethanol abuse-induced cardiotoxicity can possibly be attributed to the RIP1/RIP3/MLKL-mediated necroptosis. Moreover, pharmacological activation of CB2R may represent a new cardioprotective strategy against ethanol-induced cardiotoxicity.

Cannabinoid 1 Receptor Antagonists Play a Neuroprotective Role in Chronic Alcoholic Hippocampal Injury Related to Pyroptosis Pathway.

BACKGROUND: Alcohol use disorders affect millions of people worldwide, and there is growing evidence that excessive alcohol intake causes severe damage to the brain of both humans and animals. Numerous studies on chronic alcohol exposure in animal models have identified that many functional impairments are associated with the hippocampus, which is a structure exhibiting substantial vulnerability to alcohol exposure. However, the precise mechanisms that lead to structural and functional impairments of the hippocampus are poorly understood. Herein, we report a novel cell death type, namely pyroptosis, which accounts for alcohol neurotoxicity in mice. METHODS: For this study, we used an in vivo model to induce alcohol-related neurotoxicity in the hippocampus. Adult male C57BL/6 mice were treated with 95% alcohol vapor either alone or in combination with selective cannabinoid receptor antagonists or agonists, and VX765 (Belnacasan), which is a selective caspase-1 inhibitor. RESULTS: Alcohol-induced in vivo pyroptosis occurs because of an increase in the levels of pyroptotic proteins such as nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), caspase-1, gasdermin D (GSDMD), and amplified inflammatory response. Our results indicated that VX765 suppressed the expression of caspase-1 and inhibited the maturation of the proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-18. Additionally, chronic alcohol intake created an imbalance in the endocannabinoid system and regulated 2 cannabinoid receptors (CB1R and CB2R) in the hippocampus. Specific antagonists of CB1R (AM251 and AM281) significantly ameliorated alcohol-induced pyroptosis signaling and inactivated the inflammatory response. CONCLUSIONS: Alcohol induces hippocampal pyroptosis, which leads to neurotoxicity, thereby indicating that pyroptosis may be an essential pathway involved in chronic alcohol-induced hippocampal neurotoxicity. Furthermore, cannabinoid receptors are regulated during this process, which suggests promising therapeutic strategies against alcohol-induced neurotoxicity through pharmacologic inhibition of CB1R.

Ionically Conductive Hydrogel with Fast Self-Recovery and Low Residual Strain as Strain and Pressure Sensors.

Hydrogel-based sensors have attracted enormous interest due to their broad applications in wearable devices. However, existing hydrogel-based sensors cannot integrate satisfying mechanical performances with excellent conductivity to meet the requirements for practical application. Herein, an ionically conductive hydrogel with high strength, fast self-recovery, and low residual strain is constructed through a facile soaking strategy. The proposed ionically conductive double network hydrogel is achieved by combining chemically crosslinked polyacrylamide and physically crosslinked gelatin network followed by sodium citrate solution immersing. The obtained hydrogel has a tensile strength of 1.66 MPa and an elongation of 849%. The ionically conductive hydrogels can be utilized as both strain and pressure sensors with high sensitivity. Moreover, they can be used as ionic skin to monitor various human movements precisely, demonstrating their promising potential in wearable devices and flexible electronics.

Proteomic Analysis of Brain Regions Reveals Brain Regional Differences and the Involvement of Multiple Keratins in Chronic Alcohol Neurotoxicity.

AIMS: Alcohol abuse has attracted public attention and chronic alcohol exposure can result in irreversible structural changes in the brain. The molecular mechanisms underlying alcohol neurotoxicity are complex, mandating comprehensive mining of spatial protein expression profile. METHODS: In this study, mice models of chronic alcohol intoxication were established after 95% alcohol vapor administration for 30 consecutive days. On Day 30, striatum (the dorsal and ventral striatum) and hippocampus, the two major brain regions responsible for learning and memorizing while being sensitive to alcohol toxicity, were collected. After that, isobaric tags for relative and absolute quantitation -based quantitative proteomic analysis were carried out for further exploration of the novel mechanisms underlying alcohol neurotoxicity. RESULTS: Proteomic results showed that in the striatum, 29 proteins were significantly up-regulated and 17 proteins were significantly down-regulated. In the hippocampus, 72 proteins were significantly up-regulated, while 2 proteins were significantly down-regulated. Analysis of the overlay proteins revealed that a total of 102 proteins were consistently altered (P < 0.05) in both hippocampus and striatum regions, including multiple keratins such as Krt6a, Krt17 and Krt5. Ingenuity pathway analysis revealed that previously reported diseases/biofunctions such as dermatological diseases and developmental disorders were enriched in those proteins. Interestingly, the glucocorticoid receptor (GR) signaling was among the top enriched pathways in both brain regions, while multiple keratins from the GR signaling such as Krt1 and Krt17 exhibited significantly opposite expression patterns in the two brain nuclei. Moreover, there are several other involved pathways significantly differed between the hippocampus and striatum. CONCLUSIONS: Our data revealed brain regional differences upon alcohol consumption and indicated the critical involvement of keratins from GR signaling in alcohol neurotoxicity. The differences in proteomic results between the striatum and hippocampus suggested a necessity of taking into consideration brain regional differences and intertwined signaling pathways rather than merely focusing on single nuclei or molecule during the study of drug-induced neurotoxicity in the future.

Surrogate analyte-based quantification of main endocannabinoids in whole blood using liquid chromatography-tandem mass spectrometry.

Endocannabinoids (eCBs) are endogenous ligands of the endocannabinoid system that are known to regulate several physiological and behavioral processes. Previous studies have developed methods for the detection of main eCBs including arachidonylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), mostly in serum or plasma. Whole blood is a superior biomaterial for eCBs analysis owing to the nature of the shortened isolation procedure and decreased risk of 2-AG isomerization during preparation. In this study, a surrogate analyte-based liquid chromatography-tandem mass spectrometry assay was developed for the measurement of AEA, 2-AG and its isomer 1-arachidonoylglycerol (1-AG) using a maximum of 100 µL whole blood. Chromatographic separation was achieved using a reverse-phase column and a gradient elution. Detection was performed in selected reaction monitoring mode with an electrospray ionization source. The limits of detection of three eCBs were 0.05-0.1 ng/mL. Good linearity was observed over the concentration range. Intra- and inter-assay accuracy and precision were ≤10.9 and ≤8.7% at four quality control levels. The response factor and parallelism experiment illustrated that the surrogate analytes were suitable for accurate quantification of the main eCBs in whole blood. This surrogate analyte approach was successfully applied to authentic blood samples obtained from alcohol negative drivers and those under the influence of alcohol.

Spaceflight impacts xyloglucan oligosaccharide abundance in Arabidopsis thaliana root cell walls.

Over the course of more than a decade, space biology investigations have consistently indicated that cell wall remodeling occurs in a variety of spaceflight-grown plants. Here, we describe a mass spectrometric method to study the fundamental composition of xyloglucan, the most abundant hemicellulose in dicot cell walls, in space-grown plants. Four representative Arabidopsis root samples, from a previously conducted spaceflight experiment - Advanced Plant EXperiment - 04 (APEX-04), were used to investigate changes in xyloglucan oligosaccharides abundances in spaceflight-grown plants compared to ground controls. In situ localized enzymatic digestions and surface sampling mass spectrometry analysis provided spatial resolution of the changes in xyloglucan oligosaccharides abundances. Overall, the results showed that oligosaccharide XXLG/XLXG and XXFG branching patterns were more abundant in the lateral roots of spaceflight-grown plants, while XXXG, XLFG, and XLFG/XLFG were more abundant in the lateral roots of ground control plants. In the primary roots, XXFG had a higher abundance in ground controls than in spaceflight plants. This methodology of analyzing the basic components of the cell wall in this paper highlights two important findings. First, that are differences in the composition of xyloglucan oligosaccharides in spaceflight root cell walls compared to ground controls and, second, most of these differences are observed in the lateral roots. Thus, the methodology described in this paper provides insights into spaceflight cell wall modifications for future investigations.

Analysis of Single-Cell RNA-seq Data.

As single-cell RNA sequencing experiments continue to advance scientific discoveries across biological disciplines, an increasing number of analysis tools and workflows for analyzing the data have been developed. In this chapter, we describe a standard workflow and elaborate on relevant data analysis tools for analyzing single-cell RNA sequencing data. We provide recommendations for the appropriate use of commonly used methods, with code examples and analysis interpretations.

PRR11 is a prognostic biomarker and correlates with immune infiltrates in bladder urothelial carcinoma.

Abnormal proline-rich protein 11 (PRR11) expression is associated with various tumors. However, there are few reports concerning PRR11 with prognostic risk, immune infiltration, or immunotherapy of bladder urothelial carcinoma (BLCA). This study is based on online databases, such as Oncomine, GEPIA, HPA, LinkedOmics, TIMER, ESTIMATE and TISIDB, and BLCA data downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus, we employed an array of bioinformatics methods to explore the potential oncogenic roles of PRR11, including analyzing the relationship between PRR11 and prognosis, tumor mutational burden (TMB), microsatellite instability, and immune cell infiltration in BLCA. The results depict that PRR11 is highly expressed in BLCA, and BLCA patients with higher PRR11 expression have worse outcomes. In addition, there was a significant correlation between PRR11 expression and TMB and tumor immune infiltration. These findings suggest that PRR11 can be used as a potential marker for BLCA patient assessment and risk stratification to improve clinical prognosis, and its potential regulatory mechanism in the BLCA tumor microenvironment and targeted therapy is worthy of further investigation.

Data-driven selection of analysis decisions in single-cell RNA-seq trajectory inference.

Single-cell RNA sequencing (scRNA-seq) experiments have become instrumental in developmental and differentiation studies, enabling the profiling of cells at a single or multiple time-points to uncover subtle variations in expression profiles reflecting underlying biological processes. Benchmarking studies have compared many of the computational methods used to reconstruct cellular dynamics, however researchers still encounter challenges in their analysis due to uncertainties in selecting the most appropriate methods and parameters. Even among universal data processing steps used by trajectory inference methods such as feature selection and dimension reduction, trajectory methods' performances are highly dataset-specific. To address these challenges, we developed Escort, a framework for evaluating a dataset's suitability for trajectory inference and quantifying trajectory properties influenced by analysis decisions. Escort navigates single-cell trajectory analysis through data-driven assessments, reducing uncertainty and much of the decision burden associated with trajectory inference. Escort is implemented in an accessible R package and R/Shiny application, providing researchers with the necessary tools to make informed decisions during trajectory analysis and enabling new insights into dynamic biological processes at single-cell resolution.

A Zwitterionic Hydrogel with Anti-Oxidative and Anti-Inflammatory Properties for the Prevention of Peritoneal Adhesion by Inhibiting Mesothelial-Mesenchymal Transition.

Postoperative peritoneal adhesion is a serious clinical complication. Various hydrogel barriers have been developed to prevent peritoneal adhesion. However, it remains a challenge to design a hydrogel with desirable physicochemical properties and bioactivities. In this study, a zwitterionic polysaccharide-based multifunctional hydrogel is developed using epigallocatechin-3-gallate (EGCG) to prevent postoperative abdominal adhesion. This hydrogel is simple to use and has desirable properties, such as excellent injectability, self-healing, and non-swelling properties. The hydrogel also has ultralow fouling capabilities, such as superior bactericidal performance, cell and protein adhesion, and low immunogenicity resistance. Moreover, the hydrogel exhibits good antioxidant activity, which is attributed to the integration of EGCG. Furthermore, the detailed mechanism from in vivo and in vitro experimental studies illustrates that hydrogel compositions can synergistically prevent adhesion formation through multiple pathways, including anti-inflammatory and antioxidant capabilities and inhibition effects on the mesothelial-mesenchymal transition (MMT) process induced by transforming growth factor (TGF-ß). In summary, this zwitterionic multifunctional hydrogel has great potential to prevent postoperative adhesion formation in the clinical setting.

Zwitterionic Polysaccharide-Based Hydrogel Dressing as a Stem Cell Carrier to Accelerate Burn Wound Healing.

Stem cell therapy integrated with hydrogels has shown promising potential in wound healing. However, the existing hydrogels usually cannot reach the desired therapeutic efficacy for burn wounds due to the inadaptability to wound shape and weak anti-infection ability. Moreover, it is difficult to improve the environment for the survival and function of stem cells under complicated wound microenvironments. In this study, an injectable and self-healing hydrogel (DSC), comprising sulfobetaine-derived dextran and carboxymethyl chitosan, is fabricated through a Schiff-base reaction. Meanwhile, the DSC hydrogel shows high nonfouling properties, including resistance to bacteria and nonspecific proteins; moreover, the prepared hydrogel can provide a biomimetic microenvironment for cell proliferation whilst maintaining the stemness of adipose-derived stem cells (ADSCs) regardless of complex microenvironments. In burnt murine animal models, the ADSCs-laden hydrogel can significantly accelerate wound healing rate and scarless skin tissue regeneration through multiple pathways. Specifically, the ADSCs-laden DSC hydrogel can avoid immune system recognition and activation and thus reduce the inflammatory response. Moreover, the ADSCs-laden DSC hydrogel can promote collagen deposition, angiogenesis, and enhance macrophage M2 polarization in the wound area. In summary, sulfobetaine-derived polysaccharide hydrogel can serve as a versatile platform for stem cell delivery to promote burn wound healing.

Responders to low-dose ATG induce CD4+ T cell exhaustion in type 1 diabetes.

BACKGROUNDLow-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of the response remain unclear. Here, we characterized the post hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production).METHODSWe assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n = 29), ATG plus granulocyte colony-stimulating factor (ATG/G-CSF, n = 28), or placebo (n = 31).RESULTSTreatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (P < 0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNF-α (P < 0.05 for all) 2 weeks after treatment and a durable CD4+ exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [P = 0.011] and PD1+CD4+ Temra MFI [P < 0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG nonresponders displayed higher proportions of senescent T cells (at baseline and after treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker).CONCLUSIONAltogether in these exploratory analyses, Th1 inflammation-associated serum and CD4+ exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.TRIAL REGISTRATIONClinicalTrials.gov NCT02215200.FUNDINGThe Leona M. and Harry B. Helmsley Charitable Trust (2019PG-T1D011), the NIH (R01 DK106191 Supplement, K08 DK128628), NIH TrialNet (U01 DK085461), and the NIH NIAID (P01 AI042288).

T follicular helper 17 (Tfh17) cells are superior for immunological memory maintenance.

A defining feature of successful vaccination is the ability to induce long-lived antigen-specific memory cells. T follicular helper (Tfh) cells specialize in providing help to B cells in mounting protective humoral immunity in infection and after vaccination. Memory Tfh cells that retain the CXCR5 expression can confer protection through enhancing humoral response upon antigen re-exposure but how they are maintained is poorly understood. CXCR5+ memory Tfh cells in human blood are divided into Tfh1, Tfh2, and Tfh17 cells by the expression of chemokine receptors CXCR3 and CCR6 associated with Th1 and Th17, respectively. Here, we developed a new method to induce Tfh1, Tfh2, and Tfh17-like (iTfh1, iTfh2, and iTfh17) mouse cells in vitro. Although all three iTfh subsets efficiently support antibody responses in recipient mice with immediate immunization, iTfh17 cells are superior to iTfh1 and iTfh2 cells in supporting antibody response to a later immunization after extended resting in vivo to mimic memory maintenance. Notably, the counterpart human Tfh17 cells are selectively enriched in CCR7+ central memory Tfh cells with survival and proliferative advantages. Furthermore, the analysis of multiple human cohorts that received different vaccines for HBV, influenza virus, tetanus toxin or measles revealed that vaccine-specific Tfh17 cells outcompete Tfh1 or Tfh2 cells for the persistence in memory phase. Therefore, the complementary mouse and human results showing the advantage of Tfh17 cells in maintenance and memory function supports the notion that Tfh17-induced immunization might be preferable in vaccine development to confer long-term protection.