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Adipose-derived cells (ADCs) from white adipose tissue are promising stem cell candidates because of their large regenerative reserves and the potential for cardiac regeneration. However, given the heterogeneity of ADC and its unsolved mechanisms of cardiac acquisition, ADC-cardiac transition efficiency remains low. In this study, we explored the heterogeneity of ADCs and the cellular kinetics of 39,432 single-cell transcriptomes along the leukemia inhibitory factor (LIF)-induced ADC-cardiac transition. We identified distinct ADC subpopulations that reacted differentially to LIF when entering the cardiomyogenic program, further demonstrating that ADC-myogenesis is time-dependent and initiates from transient changes in nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. At later stages, pseudotime analysis of ADCs navigated a trajectory with 2 branches corresponding to activated myofibroblast or cardiomyocyte-like cells. Our findings offer a high-resolution dissection of ADC heterogeneity and cell fate during ADC-cardiac transition, thus providing new insights into potential cardiac stem cells.

To determine cell-specific gene expression profiles that contribute to development of abdominal aortic aneurysms (AAAs).AAAs represent the most common pathological aortic dilation leading to the fatal consequence of aortic rupture. Both immune and structural cells contribute to aortic degeneration, however, gene specific alterations in these cellular subsets are poorly understood.We performed single-cell RNA sequencing (scRNA-seq) analysis of AAAs and control tissues. AAA-related changes were examined by comparing gene expression profiles as well as detailed receptor-ligand interactions. An integrative analysis of scRNA-seq data with large genome-wide association study data was conducted to identify genes critical for AAA development.Using scRNA-seq we provide the first comprehensive characterization of the cellular landscape in human AAA tissues. Unbiased clustering analysis of transcriptional profiles identified seventeen clusters representing 8 cell lineages. For immune cells, clustering analysis identified 4 T-cell and 5 monocyte/macrophage subpopulations, with distinct transcriptional profiles in AAAs compared to controls. Gene enrichment analysis on immune subsets identified multiple pathways only expressed in AAA tissue, including those involved in mitochondrial dysfunction, proliferation, and cytokine secretion. Moreover, receptor-ligand analysis defined robust interactions between vascular smooth muscle cells and myeloid populations in AAA tissues. Lastly, integrated analysis of scRNA-seq data with genome-wide association study studies determined that vascular smooth muscle cell expression of SORT1 is critical for maintaining normal aortic wall function.Here we provide the first comprehensive evaluation of single-cell composition of the abdominal aortic wall and reveal how the gene expression landscape is altered in human AAAs.

Cutaneous lupus erythematosus (CLE) is a disfiguring and poorly understood condition frequently associated with systemic lupus. Previous studies suggest that nonlesional keratinocytes play a role in disease predisposition, but this has not been investigated in a comprehensive manner or in the context of other cell populations. To investigate CLE immunopathogenesis, normal-appearing skin, lesional skin, and circulating immune cells from lupus patients were analyzed via integrated single-cell RNA sequencing and spatial RNA sequencing. We demonstrate that normal-appearing skin of patients with lupus represents a type I interferon–rich, prelesional environment that skews gene transcription in all major skin cell types and markedly distorts predicted cell-cell communication networks. We also show that lupus-enriched CD16 + dendritic cells undergo robust interferon education in the skin, thereby gaining proinflammatory phenotypes. Together, our data provide a comprehensive characterization of lesional and nonlesional skin in lupus and suggest a role for skin education of CD16 + dendritic cells in CLE pathogenesis.

RASpathway mutations, which are present in 30% of patients with chronic myelomonocytic leukemia (CMML) at diagnosis, confer a high risk of resistance to and progression after hypomethylating agent (HMA) therapy, the current standard of care for the disease. Using single-cell, multi-omics technologies, we sought to dissect the biological mechanisms underlying the initiation and progression ofRASpathway–mutated CMML. We found thatRASpathway mutations induced the transcriptional reprogramming of hematopoietic stem and progenitor cells (HSPCs), which underwent proliferation and monocytic differentiation in response to cell-intrinsic and -extrinsic inflammatory signaling that also impaired immune cells’ functions. HSPCs expanded at disease progression and relied on the NF-KB pathway effector MCL1 to maintain their survival, which explains why patients withRASpathway– mutated CMML do not benefit from BCL2 inhibitors such as venetoclax. Our study has implications for developing therapies to improve the survival of patients withRASpathway– mutated CMML.

Supplementary Data from Targeting the EIF2AK1 Signaling Pathway Rescues Red Blood Cell Production in SF3B1-Mutant Myelodysplastic Syndromes With Ringed Sideroblasts

SF3B1 mutations, which occur in 20% of patients with myelodysplastic syndromes (MDS), are the hallmarks of a specific MDS subtype, MDS with ringed sideroblasts (MDS-RS), which is characterized by the accumulation of erythroid precursors in the bone marrow and primarily affects the elderly population. Here, using single-cell technologies and functional validation studies of primary SF3B1-mutant MDS-RS samples, we show that SF3B1 mutations lead to the activation of the EIF2AK1 pathway in response to heme deficiency and that targeting this pathway rescues aberrant erythroid differentiation and enables the red blood cell maturation of MDS-RS erythroblasts. These data support the development of EIF2AK1 inhibitors to overcome transfusion dependency in patients with SF3B1-mutant MDS-RS with impaired red blood cell production.Significance:MDS-RS are characterized by significant anemia. Patients with MDS-RS die from a shortage of red blood cells and the side effects of iron overload due to their constant need for transfusions. Our study has implications for the development of therapies to achieve long-lasting hematologic responses.This article is highlighted in the In This Issue feature, p. 476

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell–matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor–endothelial cell interactions.Significance:A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor–endothelial cell interactions.

Effects of positive hits on endothelial barrier, cytoskeletal organization and tumor cell motility

Effect of drug hits on cortical actin and tumor cell migration and invasion

Phenotypes of the human pancreatic cell Ines and effects on endothelium

Methods and Reagents

Determination of niclosamide stability and approach for counting lung metastatic nodules

Analysis of scRNAseq findings

Work flow to assess effects of drug hits in vivo

Myelodysplastic syndromes (MDS) are heterogeneous neoplastic disorders of hematopoietic stem cells (HSCs). The current standard of care for patients with MDS is hypomethylating agent (HMA)-based therapy; however, almost 50% of MDS patients fail HMA therapy and progress to acute myeloid leukemia, facing a dismal prognosis due to lack of approved second-line treatment options. As cancer stem cells are the seeds of disease progression, we investigated the biological properties of the MDS HSCs that drive disease evolution, seeking to uncover vulnerabilities that could be therapeutically exploited. Through integrative molecular profiling of HSCs and progenitor cells in large patient cohorts, we found that MDS HSCs in two distinct differentiation states are maintained throughout the clinical course of the disease, and expand at progression, depending on recurrent activation of the anti-apoptotic regulator BCL-2 or nuclear factor-kappa B-mediated survival pathways. Pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden in experimental systems. Further, patients with MDS who progressed after failure to frontline HMA therapy and whose HSCs upregulated BCL-2 achieved improved clinical responses to venetoclax-based therapy in the clinical setting. Overall, our study uncovers that HSC architectures in MDS are potential predictive biomarkers to guide second-line treatments after HMA failure. These findings warrant further investigation of HSC-specific survival pathways to identify new therapeutic targets of clinical potential in MDS.

Tandem reactor module has superior catalytic capacity, operating flexibility and stability, and reusability for the 4-NP reduction. Here, Au NP-coated glass beads were chosen as packing materials, prepared by means of airpotato yam rhizome extract.

DNA damage resistance is a major barrier to effective DNA-damaging therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting ILF2, a DNA damage regulator that is overexpressed in 70% of MM patients whose disease has progressed after standard therapies have failed. Here, we show that MM cells undergo an adaptive metabolic rewiring and rely on oxidative phosphorylation to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identified the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells’ ability to overcome ILF2 ASO−induced DNA damage, as being essential to counteracting oxidative DNA damage and maintaining mitochondrial respiration. Our study revealed a novel vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation.STATEMENT OF SIGNIFICANCEMetabolic reprogramming is a mechanism through which cancer cells maintain survival and become resistant to DNA-damaging therapy. Here, we show that targeting DNA2 is synthetically lethal in myeloma cells that undergo metabolic adaptation and rely on oxidative phosphorylation to maintain survival after DNA damage activation.

STAT3 function in hematopoietic stem and progenitor cells (HSPCs) has been difficult to discern asStat3deficiency in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. To address this, we established mixed bone marrow (BM) chimeric mice with CreER-mediatedStat3deletion in 20% of the hematopoietic compartment.Stat3-deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM in contrast toStat3-sufficient (CreER) controls. Single-cell RNA sequencing of Lin−ckit+Sca1+BM cells revealed altered transcriptional responses inStat3-deficient hematopoietic stem cells (HSCs) and multipotent progenitors, including intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation,Stat3-deficient Lin−ckit+Sca1+cells accumulated γH2AX over time. Following secondary BM transplantation,Stat3-deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs.Key PointsSTAT3 is critical for hematopoietic activity and hematopoietic stem cell maintenance in non-inflammatory conditionsSTAT3 has a cell-intrinsic role in the suppression of interferon signaling and myeloid-skewed transcription in hematopoietic stem cells

The induction of proinflammatory T cells by dendritic cell (DC) subtypes is critical for antitumor responses and effective immune checkpoint blockade (ICB) therapy. Here, we show that human CD1c + CD5 + DCs are reduced in melanoma-affected lymph nodes, with CD5 expression on DCs correlating with patient survival. Activating CD5 on DCs enhanced T cell priming and improved survival after ICB therapy. CD5 + DC numbers increased during ICB therapy, and low interleukin-6 (IL-6) concentrations promoted their de novo differentiation. Mechanistically, CD5 expression by DCs was required to generate optimally protective CD5 hi T helper and CD8 + T cells; further, deletion of CD5 from T cells dampened tumor elimination in response to ICB therapy in vivo. Thus, CD5 + DCs are an essential component of optimal ICB therapy.

Palmoplantar skin is structurally and functionally unique, but the transcriptional programs driving this specialization are unclear. Here, we use bulk and single-cell RNA sequencing of human palm, sole, and hip skin to describe the distinguishing characteristics of palmoplantar and non-palmoplantar skin while also uncovering differences between palmar and plantar sites. Our approach reveals an altered immune environment in palmoplantar skin, with downregulation of diverse immunological processes and decreased immune cell populations. Further, we identify specific fibroblast populations that appear to orchestrate key differences in cell-cell communication in palm, sole, and hip. Dedicated keratinocyte analysis highlights major differences in basal cell fraction among the three sites and demonstrates the existence of two spinous keratinocyte populations constituting parallel, site-selective epidermal differentiation trajectories. In summary, this deep characterization of highly adapted palmoplantar skin contributes key insights into the fundamental biology of human skin and provides a valuable data resource for further investigation.

Many inflammatory skin diseases are characterized by altered epidermal differentiation. Whether this altered differentiation promotes inflammatory responses has been unknown. Here, we show that IRAK2, a member of the signaling complex downstream of IL-1 and IL-36, correlates positively with disease severity in both atopic dermatitis and psoriasis. Inhibition of epidermal IRAK2 normalizes differentiation and inflammation in two mouse models of psoriasis- and atopic dermatitis-like inflammation. Specifically, we demonstrate that IRAK2 ties together proinflammatory and differentiation-dependent responses and show that this function of IRAK2 is specific to keratinocytes and acts through the differentiation-associated transcription factor ZNF750. Taken together, our findings suggest that IRAK2 has a critical role in promoting feed-forward amplification of inflammatory responses in skin through modulation of differentiation pathways and inflammatory responses.

The ketogenic diet (KD) has demonstrated benefits in numerous clinical studies and animal models of disease in modulating the immune response and promoting a systemic anti-inflammatory state. Here we investigate the effects of a KD on systemic toxicity in mice following SARS-CoV-2 infection. Our data indicate that under KD, SARS-CoV-2 reduces weight loss with overall improved animal survival. Muted multi-organ transcriptional reprogramming and metabolism rewiring suggest that a KD initiates and mitigates systemic changes induced by the virus. We observed reduced metalloproteases and increased inflammatory homeostatic protein transcription in the heart, with decreased serum pro-inflammatory cytokines (i.e., TNF-α, IL-15, IL-22, G-CSF, M-CSF, MCP-1), metabolic markers of inflammation (i.e., kynurenine/tryptophane ratio), and inflammatory prostaglandins, indicative of reduced systemic inflammation in animals infected under a KD. Taken together, these data suggest that a KD can alter the transcriptional and metabolic response in animals following SARS-CoV-2 infection with improved mice health, reduced inflammation, and restored amino acid, nucleotide, lipid, and energy currency metabolism.

Diabetic nephropathy (DN) is a major complication of type 1 diabetes mellitus, and hyperglycemia and hypertension are the main risk factors for the development of DN. N-Acetyl-Cysteine (NAC) has a variety of effects, interfering with the production and scavenging of free radicals and regulating the metabolic activity of tissue cells. However, the efficacy of NAC on DN treatment is unclear. Thus, this study investigated the protective mechanism of NAC combined with insulin on renal injury in dogs with DN. The forty dogs were selected and divided into control group, DM group, INS group, INS + NAC group and NAC group to establish the model for a trial period of 4 months. The results revealed that INS + NAC was effective in reducing and stabilizing blood glucose levels. Biochemical results showed that INS + NAC treatment significantly regulated the stability of UREA, CREA and fructosamine indicators. Meanwhile, histopathology staining showed significant glomerular wrinkling and fibrosis in the DM group, which could be reversed after INS + NAC treatment. In addition, INS + NAC could restore mitochondria homeostasis by upregulating the levels of mitochondrial fission (MFN1, MFN2 and OPA1) and inhibiting of mitochondrial fusion (DRP1, FIS1 and MFF) related indicators. Further studies revealed that INS + NAC regulated the expression levels of renal BNIP3, NIX and FUNDC1 in the DM group, thereby alleviating mitophagy. Collectively, these results suggested that NAC combined with insulin protects DN by regulating the mitochondrial dynamics and FUNDC1-mediated mitophagy.

Type 1 diabetes mellitus (T1DM) is a metabolic disease characterized by insulin deficiency and often accompanied by hypercholesterolemia. NAC is an effective antioxidative drug, but its application in the treatment of diabetes is still rare. A total of forty beagles were randomly divided into five groups: control group, DM group, INS group, INS with NAC group, and NAC group. The experiment lasted for 120 days. Results revealed that biochemical criterion increased in the DM group, while the indicators significantly decreased on the INS combined with NAC treatment group. Moreover, the insulin released test demonstrated that the model of T1DM was successfully constructed. The result of B ultrasound of gallbladder showed remarkable cholestasis in DM group. The cholesterol metabolism-related enzyme activity (HMGCR and SQLE) was evidently increased in DM group, but decreased in INS and NAC group. The content of TG, LDL-c, and HDL-c in liver was detected by the kit, and it was found that the content of TG, LDL-c, and HDL-c in DM group were reduced. Histopathological observation revealed that the cholestasis of liver cells and hepatic cords were disordered in DM group, the symptoms were alleviated under INS and NAC treatment. Additionally, the protein and mRNA expression of HMGCR and LDLR were obviously increased in DM group, but down regulated in INS and NAC treatment group. Overall, the liver function injury and secondary hypercholesterolemia can be found in T1DM canines, and NAC can relieve cholesterol metabolism disorder in the treatment of canine T1DM.

A large amount of copper (Cu) used in production activities can lead to the enrichment of Cu in the environment, which can cause toxicity to animals. However, the toxicity mechanism of Cu on the cerebrum is still uncertain. Hence, a total of 240 chickens were separated into four groups in this study to reveal the potential connection between mitophagy and endoplasmic reticulum (ER) stress-mediated apoptosis in the chicken cerebrum in the case of excess Cu exposure. The cu exposure situation was simulated by diets containing various levels of copper (11 mg/kg, control group; 110 mg/kg, group I; 220 mg/kg, group II and 330 mg/kg, group III) for 49 days. The results of histology showed that vacuolar degeneration was observed in the treated groups, and the mitochondria swell and autophagosomes formation were found under excess Cu treatment. Additionally, the expression of mitophagy (PINK1, Parkin, LC3I, LC3II and p62) and ER stress (GRP78, PERK, ATF6, IRE1α, XBP1, CHOP, and JNK) indexes were significantly upregulated under excess Cu exposure. Furthermore, the mRNA and protein expression of Bcl-2 were decreased, while Bak1, Bax, Caspase12, and Caspase3 were increased compared to the control group. In summary, this study demonstrated that an overdose of Cu could induce mitophagy and ER stress-mediated apoptosis in the chicken cerebrum. These findings revealed an important potential connection between Cu toxicity and cerebrum damage, which provided a new insight into Cu neurotoxicity.

Copper (Cu) is a common environmental pollutant which has been identified to cause toxic effects on animal bodies. MicroRNAs (miRNAs) are a type of non-coding RNAs involved in the regulation of various cellular activities including autophagy, but the potential regulatory mechanisms after excess Cu intake are still uncertain. Our previous study has prompted that Cu exposure reduced liver miR-455-3p levels. Herein, miR-455-3p was found to be an important molecule in the regulation of Cu-induced autophagy in vivo and in vitro. Histopathology observation of liver tissue indicated that Cu-induced severe hepatic damage including cellular swelling and vacuolization. Meanwhile, excessive Cu exposure not only heighten the mRNA and protein expression levels of Beclin1, Atg5, LC3Ⅰ and LC3Ⅱ, but also decreased miR-455-3p levels. In vitro experiment, Cu-induced autophagy can be attenuated by miR-455-3p overexpression. Additionally, oxidative stress-responsive 1 (OXSR1) was identified as a direct downstream target of miR-455-3p by dual luciferase reporter assays. Moreover, knockdown of OXSR1 can attenuate the autophagy induced by Cu treatment and the miR-455-3p inhibitor. Overall, the miR-455-3p-OXSR1 axis works as a regulator of autophagy under Cu stress, which provides a basis for further revealing the mechanism of chronic Cu poisoning.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1β. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response. Modlin and colleagues examined the skin lesions of human leprosy patients using single-cell RNA sequencing coupled to cellular spatial mapping. Their analysis maps the architecture of granulomas in leprosy lesions from patients with leprosy with localized disease (tuberculoid leprosy, reversal reaction) to those with progressive infection (lepromatous leprosy).

While most cases of vascular dementia represent complex interactions between host genetics and environmental factors, mendelian forms of vascular dementia also exist. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is a mendelian disease characterized by progressive vascular deterioration, cognitive deficits, and strokes. Mutations in the NOTCH3 receptor underlies the pathologies in CADASIL. NOTCH3 is primarily expressed in vascular smooth muscle cells (vSMCs) and its’ expression is critical for differentiation and functional integrity of arterial vSMCs, albeit through unclear mechanism(s). To elucidate the contribution of NOTCH3 in the maintenance of cerebral vascular architecture and function, we performed micro-computed tomography (micro-CT) on the brains of aged Notch3 -deficient animals. Micro-CT assessment of the cerebral vasculature architecture showed significant abnormalities including severe vessel dilation and tortuosity (dolicoectasia) of the middle cerebral artery and its branches in the Notch3 -/- compared to aged-match controls. To identify the molecular pathway from NOTCH3 dysregulation to the observed cerebral vascular dysfunction, we performed single-cell RNASeq on cerebral arteries isolated from young (4w) and old (104w) Notch3 -/- animals. Evaluation of the vSMC-specific transcriptomes indicated significant loss of proteins associated with muscle contraction and increased extracellular matrix production in animals that lack NOTCH3. Using a combination of immunofluorescence microscopy and in vitro functional assays, we confirmed that continued expression of Notch3 is a critical requirement for maintenance of vSMC contractile function. Impaired contractility also affected flow of cerebrospinal fluid in the parenchyma of Notch3 -/- . MRI and behavioral assessments were performed in the Notch3 -/- animals to elucidate the relationship between impaired vascular contractility to cognitive function. Taken together these findings link the molecular dysfunction of NOTCH3 through its regulation of vascular contractility and cerebral vessel architecture to altered neurological function and clarify the molecular pathways to cellular pathology of Notch3 driven dementias.

Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNA-seq and protein expression by LC-MS directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interations by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We identify novel flow depedent genes, as well as, genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information and physical forces.

Type 1 diabetes mellitus (T1DM) is a chronic and represented by insulin-causing pancreatic β-cell disruption and hyperglycemia. N-Acetyl-Cysteine (NAC) is regarded as facilitating endothelial cell function and angiogenesis and may have treatment effect in the case of diabetes. However, the impact of NAC on T1DM are unknown. Here we reported that inflammatory pathogenesis of canine type 1 diabetes liver disease and the therapeutic effect of NAC combined with insulin. For this purpose, the model was established by intravenous injection of streptozotocin (20 mg/kg). Forty adult dogs were used and divided into 5 groups: control group, DM group, insulin treatment group, NAC combined with insulin therapy, and NAC group, while study lasted for 16 weeks. Results showed that the level of liver function enzyme activity were apparently increased in DM group, while the NAC with insulin treatment remarkable decreased liver function enzyme levels. Histopathology revealed that obvious changes in liver structure of all DM group, as evidenced by hepatocyte disorder and cellular swelling. Liver structure was evaluated by Periodic Acid Schiff (PAS) and Masson staining, the tissues appeared glycogen deposition and collagen deposition, indicating that DM aggravated liver injury. Compared with control group, the protein and mRNA expression of NLRP3, Caspase-1, ASC, and GSDMD were significantly induced in the DM group, while INS and NAC combined with INS treatment reversed the above changes. The levels of NF-κB P65, p-NF-κB, and IFN γ were availably enhanced in the DM group, which decreased through insulin and NAC combined with insulin treatment. This study demonstrated that NAC combined with INS exerted protective effects against STZ-induced liver injury by inhibiting the NLRP3/NF-κB pathway. The findings indicated that NAC combined with INS may serve as a potential candidate therapy for the treatment of T1DM.

Purpose of review Single-cell RNA sequencing (scRNA-seq) can capture the transcriptional profile of thousands of individual cells concurrently from complex tissues and with remarkable resolution. Either with the goal of seeking information about distinct cell subtypes or responses to a stimulus, the approach has provided robust information and promoted impressive advances in cardiovascular research. The goal of this review is to highlight strategies and approaches to leverage this technology and bypass potential caveats related to evaluation of the vascular cells. Recent findings As the most recent technological development, details associated with experimental strategies, analysis, and interpretation of scRNA-seq data are still being discussed and scrutinized by investigators across the vascular field. Compilation of this information is valuable for those using the technology but particularly important to those about to start utilizing scRNA-seq to seek transcriptome information of vascular cells. Summary As our field progresses to catalog transcriptomes from distinct vascular beds, it is undeniable that scRNA-seq technology is here to stay. Sharing approaches to improve the quality of cell dissociation procedures, analysis, and a consensus of best practices is critical as information from this powerful experimental platform continues to emerge.

Copper (Cu) is an essential trace element, but it is also a ubiquitous environmental pollutant that threatens public health. Cuproptosis is a recently discovered cell death mode that unlike other programmed cell death, characterized by proteotoxic stress due to lipoylated protein aggregation and iron-sulfur cluster protein loss. Chickens as a high-trophic-level non-mammalian vertebrate that easily absorb and accumulate copper from the environment and food, but it is unclear whether the underlying molecular mechanisms that cause their hepatotoxicity under natural copper stress are related to cuproptosis. Therefore, we established animal models of chickens with different concentrations of copper exposure to dissect the role and mechanism of cuproptosis in chicken hepatotoxicity under natural copper stress. Our histopathological and biochemical results demonstrated that the liver structure with copper-treated exhibited dose-dependent damage. Meanwhile, copper treatment also dramatically increased serum and liver copper content and activated the expression of the membrane-associated copper transporter ATP7B. Furthermore, we found that Cu-exposure significantly increased the MDA content, and reduced the levels of T-AOC and SOD in serum and liver. Additionally, we found that the mRNA and protein levels of FDX1 were significantly upregulated in the 220 and 330 mg/kg Cu-treated groups. In our further studies, we found that copper did not alter protein levels of DLAT and DLST in chicken liver, but significantly increased Lipoylated-DLAT levels and oligomerization of Lipoylated-DLAT in the 330 mg/kg Cu-treatment group. Overall, we identified that FDX1-mediated protein lipoylation and proteotoxic stress indeed participate in copper-induced hepatotoxicity in chickens. Our results present novel insight into the pathogenesis of copper-induced hepatotoxicity in chickens and provide data to support filling in the role of cuproptosis in birds.

Antimicrobial resistance leads to failure of clinical antimicrobial therapy, and has raised urgent global public health concern. Humans can acquire antimicrobial resistance from drugs through the food chain or the environment (contaminated water, air, soil, or manure). While antimicrobials have been regular supplements in animal feed that maintain health and improve productivity of livestock, their over-use in feeding forage has led to a rise in antibacterial resistance. This review summarizes the current use of antimicrobials in livestock, the harmful effects of antimicrobial resistance, and the comprehensive combat measures.

Leukocytes and endothelial cells frequently cooperate to resolve inflammatory events. In most cases, these interactions are transient in nature and triggered by immunological insults. Here, we report that, in areas of disturbed blood flow, aortic endothelial cells permanently and intimately associate with a population of specialized macrophages. These macrophages are recruited at birth from the closing ductus arteriosus and share the luminal surface with the endothelium, becoming interwoven in the tunica intima. Anatomical changes that affect hemodynamics, such as in patent ductus arteriosus, alter macrophage seeding to coincide with regions of disturbed flow. Aortic resident macrophages expand in situ via direct cell renewal. Induced depletion of intimal macrophages leads to thrombin-mediated endothelial cell contraction, progressive fibrin accumulation and formation of microthrombi that, once dislodged, cause blockade of vessels in several organs. Together the findings reveal that intravascular resident macrophages are essential to regulate thrombin activity and clear fibrin deposits in regions of disturbed blood flow.

Viral infection outcomes are sex-biased, with males generally more susceptible than females. Paradoxically, the numbers of anti-viral natural killer (NK) cells are increased in males compared to females. Using samples from mice and humans, we demonstrate that while numbers of male NK cells are increased compared to females, they display impaired production of the anti-viral cytokine IFN-γ. These sex differences were not due solely to divergent levels of gonadal hormones, since these differences persisted in gonadectomized mice. Instead, these differences can be attributed to lower male expression of X-linkedKdm6a(UTX), an epigenetic regulator which escapes X inactivation in female NK cells. NK cell-specific UTX deletion in females phenocopied multiple features of male NK cells, which include increased numbers and reduced IFN-γ production. Integrative ATAC-seq and RNA-seq analysis revealed a critical role for UTX in the regulation of chromatin accessibility and gene expression at loci important in NK cell homeostasis and effector function. Consequently, NK cell-intrinsic UTX levels are critical for optimal anti-viral immunity, since mice with NK cell-intrinsic UTX deficiency show increased lethality to mouse cytomegalovirus (MCMV) challenge. Taken together, these data implicate UTX as a critical molecular determinant of NK cell sex differences and suggest enhancing UTX function as a new strategy to boost endogenous NK cell anti-viral responses.

Natural killer (NK) cells are innate lymphocytes that possess traits of adaptive immunity, such as memory formation. However, the molecular mechanisms by which NK cells persist to form memory cells are not well understood. Using single-cell RNA sequencing, we identified two distinct effector NK cell (NKeff) populations following mouse cytomegalovirus infection. Ly6C- memory precursor (MP) NK cells showed enhanced survival during the contraction phase in a Bcl2-dependent manner, and differentiated into Ly6C+ memory NK cells. MP NK cells exhibited distinct transcriptional and epigenetic signatures compared with Ly6C+ NKeff cells, with a core epigenetic signature shared with MP CD8+ T cells enriched in ETS1 and Fli1 DNA-binding motifs. Fli1 was induced by STAT5 signaling ex vivo, and increased levels of the pro-apoptotic factor Bim in early effector NK cells following viral infection. These results suggest that a NK cell-intrinsic checkpoint controlled by the transcription factor Fli1 limits MP NK formation by regulating early effector NK cell fitness during viral infection.

Diabetic nephropathy (DN) is known as a major microvascular complication in type 1 diabetes. The effect of insulin treatment alone on controlling blood glucose is unsatisfactory. N-acetylcysteine (NAC), a chemical agent with thiol group, is found to confer a protective effect in renal injury. However, whether NAC combined with insulin treatment can further enhance the therapeutic effect in DN remains unclear. Here, we firstly used large mammal beagle as DN model to explore the effect of NAC combined with insulin treatment on DN during 120 d. Our results showed that NAC further alleviated mitochondrial oxidative damage and ferroptosis by enhancing activity of mitochondria GSH and maintaining mitochondrial redox homeostasis in DN. Additionally, the upregulated acetylation level of SOD2 was further abrogated by NAC treatment. In MDCK cells, NAC reduced high glucose (HG)-caused ferroptosis via activating Gpx4 expression. Of note, inhibition of Gpx4 by FIN56 abolished the protective effects of NAC on HG-induced ferroptosis. More importantly, 3-TYP reversed the effect of NAC on the mitochondria ROS under HG treatment, as well as eliminated its following beneficial effects for ferroptosis against HG-stimulated cells. These results reveal that NAC attenuated ferroptosis in DN via maintaining mitochondrial redox homeostasis through activating SIRT3-SOD2-Gpx4 signaling pathway.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Estimating cell type composition of blood and tissue samples is a biological challenge relevant in both laboratory studies and clinical care. In recent years, a number of computational tools have been developed to estimate cell type abundance using gene expression data. While these tools use a variety of approaches, they all leverage expression profiles from purified cell types to evaluate the cell type composition within samples. In this study, we compare ten deconvolution tools and evaluate their performance while using each of eleven separate reference profiles. Specifically, we have run deconvolution tools on over 4,000 samples with known cell type proportions, spanning both immune and stromal cell types. Twelve of these represent in vitro synthetic mixtures and 300 represent in silico synthetic mixtures prepared using single cell data. A final 3,728 clinical samples have been collected from the Framingham Cohort, for which cell populations have been quantified using electrical impedance cell counting. When tools are applied to the Framingham dataset, the tool EPIC produces the highest correlation while GEDIT produces the lowest error. The best tool for other datasets is varied, but CIBERSORT and GEDIT most consistently produce accurate results. In terms of reference choice, we find that the Human Primary Cell Atlas (HPCA) and references published by the EPIC authors produce accurate results for the largest number of tools and datasets. When applying deconvolution to blood samples, the leukocyte reference matrix LM22 is also a suitable choice, usually (but not always) outperforming HPCA and EPIC. Running time varies substantially across tools. For as many as 5052 samples, SaVanT and dtangle reliably finish in under one minute, while slower tools may require up to two hours. However, when using custom references, CIBERSORT can run very slowly, taking over 24 hours to complete for large datasets. We conclude that combining the best tools with optimal reference datasets can provide significant gains in accuracy when carrying out deconvolution tasks.

The molecular mechanisms that drive hematopoietic stem cell functional decline under conditions of telomere shortening are not completely understood. In light of recent advances in single-cell technologies, we sought to redefine the transcriptional and epigenetic landscape of mouse and human hematopoietic stem cells under telomere attrition, as induced by pathogenic germline variants in telomerase complex genes. Here, we show that telomere attrition maintains hematopoietic stem cells under persistent metabolic activation and differentiation towards the megakaryocytic lineage through the cell-intrinsic upregulation of the innate immune signaling response, which directly compromises hematopoietic stem cells’ self-renewal capabilities and eventually leads to their exhaustion. Mechanistically, we demonstrate that targeting members of the Ifi20x/IFI16 family of cytosolic DNA sensors using the oligodeoxynucleotide A151, which comprises four repeats of the TTAGGG motif of the telomeric DNA, overcomes interferon signaling activation in telomere-dysfunctional hematopoietic stem cells and these cells’ skewed differentiation towards the megakaryocytic lineage. This study challenges the historical hypothesis that telomere attrition limits the proliferative potential of hematopoietic stem cells by inducing apoptosis, autophagy, or senescence, and suggests that targeting IFI16 signaling axis might prevent hematopoietic stem cell functional decline in conditions affecting telomere maintenance., The molecular mechanisms that drive hematopoietic stem cell functional decline under conditions of telomere shortening are not completely understood. Here the authors demonstrate that hematopoietic stem cells with short telomeres induced by mutations affecting telomerase complex genes undergo differentiation towards megakaryopoiesis through the activation of the IFI16-mediated interferon response.