Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and Memory.

PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.

Stress/cell death pathways, neuroinflammation, and neuropathic pain.

Neuropathic pain is a common and debilitating modality of chronic pain induced by a lesion or disease of the somatosensory nervous system. Albeit the elucidation of numerous pathophysiological mechanisms and the development of potential treatment compounds, safe and reliable therapies of neuropathic pain remain poor. Multiple stress/cell death pathways have been shown to be implicated in neuroinflammation during neuropathic pain. Here, we summarize the current knowledge of stress/cell death pathways and present an overview of the roles and molecular mechanisms of stress/cell death pathways in neuroinflammation during neuropathic pain, covering intrinsic and extrinsic apoptosis, autophagy, mitophagy, ferroptosis, pyroptosis, necroptosis, and phagoptosis. Small molecule compounds that modulate stress/cell death pathways in alleviating neuropathic pain are discussed mainly based on preclinical neuropathic pain models. These findings will contribute to in-depth understanding of the pathological processes during neuropathic pain as well as bridge the gap between basic and translational research to uncover new neuroprotective interventions.

An ultrafast and facile nondestructive strategy to convert various inefficient commercial nanocarbons to highly active Fenton-like catalysts.

The Fenton-like process catalyzed by metal-free materials presents one of the most promising strategies to deal with the ever-growing environmental pollution. However, to develop improved catalysts with adequate activity, complicated preparation/modification processes and harsh conditions are always needed. Herein, we proposed an ultrafast and facile strategy to convert various inefficient commercial nanocarbons into highly active catalysts by noncovalent functionalization with polyethylenimine (PEI). The modified catalysts could be in situ fabricated by direct addition of PEI aqueous solution into the nanocarbon suspensions within 30 s and without any tedious treatment. The unexpectedly high catalytic activity is even superior to that of the single-atom catalyst and could reach as high as 400 times higher than the pristine carbon material. Theoretical and experimental results reveal that PEI creates net negative charge via intermolecular charge transfer, rendering the catalyst higher persulfate activation efficiency.

Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4-NOTCH-CLDN5 pathway and is vulnerable to neonatal hyperoxia.

The mechanisms governing brain vascularization during development remain poorly understood. A key regulator of developmental vascularization is delta like 4 (DLL4), a Notch ligand prominently expressed in endothelial cells (EC). Exposure to hyperoxia in premature infants can disrupt the development and functions of cerebral blood vessels and lead to long-term cognitive impairment. However, its role in cerebral vascular development and the impact of postnatal hyperoxia on DLL4 expression in mouse brain EC have not been explored. We determined the DLL4 expression pattern and its downstream signalling gene expression in brain EC using Dll4+/+ and Dll4+/LacZ mice. We also performed in vitro studies using human brain microvascular endothelial cells. Finally, we determined Dll4 and Cldn5 expression in mouse brain EC exposed to postnatal hyperoxia. DLL4 is expressed in various cell types, with EC being the predominant one in immature brains. Moreover, DLL4 deficiency leads to persistent abnormalities in brain microvasculature and increased vascular permeability both in vivo and in vitro. We have identified that DLL4 insufficiency compromises endothelial integrity through the NOTCH-NICD-RBPJ-CLDN5 pathway, resulting in the downregulation of the tight junction protein claudin 5 (CLDN5). Finally, exposure to neonatal hyperoxia reduces DLL4 and CLDN5 expression in developing mouse brain EC. We reveal that DLL4 is indispensable for brain vascular development and maintaining the blood-brain barrier's function and is repressed by neonatal hyperoxia. We speculate that reduced DLL4 signalling in brain EC may contribute to the impaired brain development observed in neonates exposed to hyperoxia. KEY POINTS: The role of delta like 4 (DLL4), a Notch ligand in vascular endothelial cells, in brain vascular development and functions remains unknown. We demonstrate that DLL4 is expressed at a high level during postnatal brain development in immature brains and DLL4 insufficiency leads to abnormal cerebral vasculature and increases vascular permeability both in vivo and in vitro. We identify that DLL4  regulates endothelial integrity through NOTCH-NICD-RBPJ-CLDN5 signalling. Dll4 and Cldn5 expression are decreased in mouse brain endothelial cells exposed to postnatal hyperoxia.

Genome-wide characterization of the CPA gene family in potato and a preliminary functional analysis of its role in NaCl tolerance.

BACKGROUND: The cation/proton antiporter (CPA) superfamily plays a crucial role in regulating ion homeostasis and pH in plant cells, contributing to stress resistance. However, in potato (Solanum tuberosum L.), systematic identification and analysis of CPA genes are lacking. RESULTS: A total of 33 StCPA members were identified and classified into StNHX (n = 7), StKEA (n = 6), and StCHX (n = 20) subfamilies. StCHX owned the highest number of conserved motifs, followed by StKEA and StNHX. The StNHX and StKEA subfamilies owned more exons than StCHX. NaCl stress induced the differentially expression of 19 genes in roots or leaves, among which StCHX14 and StCHX16 were specifically induced in leaves, while StCHX2 and StCHX19 were specifically expressed in the roots. A total of 11 strongly responded genes were further verified by qPCR. Six CPA family members, StNHX1, StNHX2, StNHX3, StNHX5, StNHX6 and StCHX19, were proved to transport Na+ through yeast complementation experiments. CONCLUSIONS: This study provides comprehensive insights into StCPAs and their response to NaCl stress, facilitating further functional characterization.

A Bioinspired Copper-Pair Catalyst in Metal-Organic Frameworks for Molecular Dioxygen Activation and Aerobic Oxidative C-N Coupling.

Open Cu sites were loaded to the UiO-67 metal-organic framework (MOF) skeleton by introduction of flexible Cu-binding pyridylmethylamine (pyma) side chains to the biphenyldicarboxylate linkers. Distance between Cu centers in the MOF pores was tuned by controlling the density of metal-binding side chains. "Interacted" Cu-pair or "isolated" monomeric Cu sites were achieved with high and low (pyma)Cu side chain loading, respectively. Spectroscopic and theoretical studies indicate that "interacted" Cu pairs can effectively bind and activate molecular dioxygen to form Cu2O2 clusters, which showed high catalytic activity for aerobic oxidative C-N coupling. On the contrary, MOF catalyst bearing isolated monomeric Cu sites only showed modest catalytic activity. Enhancement in catalytic performance for the Cu-pair catalyst is attributed to the remote synergistic effect of the paired Cu site, which binds molecular dioxygen and cleaves the O═O bond in a collaborative manner. This work demonstrates that noncovalently interacted metal-pair sites can effectively activate inert small molecules and promote heterogeneous catalytic processes.

Metabolic obesity phenotypes and all-cause mortality among the Chinese oldest-old population: a prospective cohort study.

BACKGROUND: The obesity paradox has been reported among older adults. However, whether the favorable effect of obesity is dependent on metabolic status remains largely unknown. We aimed to explore the association of metabolic obesity phenotypes and their changes with all-cause mortality among the Chinese oldest-old population. METHODS: This prospective cohort study included 1207 Chinese oldest old (mean age: 91.8 years). Metabolic obesity phenotypes were determined by central obesity and metabolic status, and participants were classified into metabolically healthy obesity (MHO), metabolically unhealthy obesity (MUO), metabolically healthy non-obesity (MHN), and metabolically unhealthy non-obesity (MUN). The hazard ratios (HRs) and 95% confidence intervals (95% CIs) were estimated by Cox regression models. RESULTS: During 5.3 years of follow-up, 640 deaths were documented. Compared with non-obesity, obesity was associated with a decreased mortality risk among participants with metabolically healthy (HR, 0.75; 95% CI, 0.63-0.91) while this association was insignificant among metabolically unhealthy. Compared to MHO, MHN (HR, 1.27; 95% CI, 1.06-1.53) and MUN (HR, 1.49; 95% CI, 1.10-2.02) were significantly associated with an increased mortality risk. Compared to those with stable MHO, those transited from MHO to MUO demonstrated a higher mortality risk (HR, 1.81; 95% CI, 1.06-3.11). CONCLUSIONS: MHO predicts better survival among the Chinese oldest-old population. These findings suggest that ensuring optimal management of metabolic health is beneficial and taking caution in weight loss based on the individual body weight for the metabolically healthy oldest-old adults.

The interactive effects of posture and biological sex on the control of muscle sympathetic nerve activity during rhythmic handgrip exercise.

Body posture and biological sex exhibit independent effects on the sympathetic neural responses to dynamic exercise. However, the neural mechanisms (e.g., baroreflex) by which posture impacts sympathetic outflow during rhythmic muscular contractions, and whether biological sex affects posture-mediated changes in efferent sympathetic nerve traffic during exercise, remain unknown. Thus, we tested the hypotheses that increases in muscle sympathetic nerve activity (MSNA) would be greater during upright compared with supine rhythmic handgrip (RHG) exercise, and that females would demonstrate smaller increases in MSNA during upright RHG exercise than males. Twenty young (30 [6] yr; means [SD]) individuals (9 males, 11 females) underwent 6 min of supine and upright (head-up tilt 45°) RHG exercise at 40% maximal voluntary contraction with continuous measurements of MSNA (microneurography), blood pressure (photoplethysmography), and heart rate (electrocardiogram). In the pooled group, absolute MSNA burst frequency (P < 0.001), amplitude (P = 0.009), and total MSNA (P < 0.001) were higher during upright compared with supine RHG exercise. However, body posture did not impact the peak change in MSNA during RHG exercise (range: P = 0.063-0.495). Spontaneous sympathetic baroreflex gain decreased from rest to RHG exercise (P = 0.006) and was not impacted by posture (P = 0.347). During upright RHG exercise, males demonstrated larger increases in MSNA burst amplitude (P = 0.002) and total MSNA (P = 0.001) compared with females, which coincided with greater reductions in sympathetic baroreflex gain among males (P = 0.004). Collectively, these data indicate that acute attenuation of baroreflex-mediated sympathoinhibition permits increases in MSNA during RHG exercise and that males exhibit a greater reserve for efferent sympathetic neural recruitment during orthostasis than females.NEW & NOTEWORTHY The impact of posture and sex on cardiovascular control during rhythmic handgrip (RHG) exercise is unknown. We show that increases in muscle sympathetic nerve activity (MSNA) during RHG are partly mediated by a reduction in sympathetic baroreflex gain. In addition, males demonstrate larger increases in total MSNA during upright RHG than females. These data indicate that the baroreflex partly mediates increases in MSNA during RHG and that males have a greater sympathetic vasoconstrictor reserve than females.

Impact of maternal obesity on resting muscle sympathetic nerve activity during uncomplicated pregnancy: a longitudinal assessment.

Maternal obesity increases the risk of adverse pregnancy outcomes. The mechanisms that contribute to this elevated risk are unclear but may be related to greater activity of the sympathetic nervous system, which is associated with hypertensive disorders of pregnancy. We hypothesized that resting muscle sympathetic nerve activity (MSNA) would be greater in women with obesity during pregnancy when compared with normal-weight women. Blood pressure, heart rate, and MSNA were recorded during 5 min of supine rest in 14 normal-weight women [body mass index (BMI) 22.1 ± 2.1 (SD) kg/m2] and 14 women with obesity (BMI 33.9 ± 3.5 kg/m2) during (early and late) pregnancy and postpartum. All women had uncomplicated pregnancies. Resting MSNA burst frequency was not different between groups during early (normal weight 17 ± 10 vs. obesity 22 ± 15 bursts/min, P = 0.35) but was significantly greater in the obesity group during late pregnancy (23 ± 13 vs. 35 ± 15 bursts/min, P = 0.031) and not different postpartum (10 ± 6 vs. 9 ± 7 bursts/min, P = 0.74). These findings were also apparent when comparing burst incidence and total activity. Although still within the normotensive range, systolic blood pressure was greater in the obesity group across all time points (P = 0.002). Diastolic blood pressure was lower during pregnancy compared with postpartum (P < 0.001) and not different between groups (P = 0.488). Heart rate increased throughout pregnancy in both groups (P < 0.001). Our findings suggest that maternal obesity is associated with greater increases in sympathetic activity even during uncomplicated pregnancy. Future research is needed to determine if this is linked with an increased risk of adverse outcomes or is required to maintain homeostasis in pregnancy.NEW & NOTEWORTHY The impact of maternal obesity on resting muscle sympathetic nerve activity was examined during (early and late) and after uncomplicated pregnancy. Resting muscle sympathetic nerve activity is not different during early pregnancy or postpartum but is significantly elevated in women with obesity during late pregnancy when compared with normal-weight women. Future research is needed to determine if this is linked with an increased risk of adverse outcomes or is required to maintain homeostasis in pregnancy.

Characterization of the Dynamic Behavior of Multinanobubble System under Shock Wave Influence.

Shockwave-induced changes in nanobubbles cause cavitation erosion and membrane damage but can also be applied to biocarrier transport. Currently, research focuses on single nanobubbles; however, in reality, nanobubbles usually appear as a multibubble system. Therefore, this study proposes a method based on cutting and replicating to construct a multibubble model. This method can be widely applied to molecular dynamics (MD) models and enhance the customization capabilities of MD models. The dynamic behavior of a multinanobubble system with different numbers and arrangements of nanobubbles is investigated with the MD method under the influence of shock waves in a liquid argon system. The study also explores the range of influence between nanobubbles. The results show that in the case of two nanobubbles, when the distance between the bubbles is constant, the smaller the angle between the direction of the shock wave and the line connecting the bubbles, the greater is the influence between nanobubbles, and the moment of collapse of the nanobubbles farther away from the shock wave is slower. When three nanobubbles are arranged with a right offset, after the first bubble collapses, the effect on the other two bubbles is similar to the changes in bubbles when the angle of arrangement is 30° or 60°. Under a different arrangement, the change of shock wave velocity on the nanobubble size only affects its collapse time and contraction collapse rate. When the shock wave with a radian of about 2.87 or greater than 2.87 touches the bubbles, the collapse of the second nanobubble will not be affected.

Network analysis of posttraumatic stress and posttraumatic growth symptoms among women in subsequent pregnancies following pregnancy loss.

BACKGROUND: Pregnant women who have undergone pregnancy loss often display both posttraumatic stress (PTS) and posttraumatic growth (PTG). However, the precise relationship and structure of symptomatic levels of PTS and PTG have not been well understood. This study aimed to assess the associations between PTS and PTG symptoms in women during subsequent pregnancies following a previous pregnancy loss. METHODS: A total of 406 pregnant women with a history of pregnancy loss were included in this study. The Impact of Events Scale-6 (IES-6) and the Posttraumatic Growth Inventory Short Form (PTGI-SF) were used to assess symptoms of PTS and PTG, respectively. The Graphical Gaussian Model was employed to estimate the network model. Central symptoms and bridge symptoms were identified based on "expected influence" and "bridge expected influence" indices, respectively. The stability and accuracy of the network were examined using the case-dropping procedure and nonparametric bootstrapped procedure. RESULTS: The network analysis identified PTG3 ("Ability to do better things") as the most central symptom, followed by PTS3 ("Avoidance of thoughts") and PTG6 ("New path for life") in the sample. Additionally, PTS3 ("Avoidance of thoughts") and PTG9 ("Perception of greater personal strength") were bridge symptoms linking PTS and PTG clusters. The network structure was robust in stability and accuracy tests. CONCLUSIONS: Interventions targeting the central symptoms identified, along with key bridge symptoms, have the potential to alleviate the severity of PTS experienced by women with a history of pregnancy loss and promote their personal growth.

Biocontrol Efficacy and Induced Resistance of Paenibacillus polymyxa J2-4 Against Meloidogyne incognita Infection in Cucumber.

Meloidogyne incognita is one of the most destructive agricultural pathogens around the world, resulting in severe damage to yield and quality in agricultural production. Biological control promises to be a great potential alternative to chemical agents against M. incognita. Paenibacillus polymyxa J2-4, isolated from ginger plants injured by M. incognita, has shown excellent biocontrol efficacy against M. incognita in cucumber. In vitro experiments with the strain J2-4 resulted in a correct mortality rate of 88.79% (24 h) and 98.57% (48 h) for second-stage juveniles (J2s) of M. incognita. Strain J2-4 significantly suppressed nematode infection on potted plants, with a 65.94% reduction in galls and a 51.64% reduction in eggs compared with the control. The split-root assay demonstrated that strain J2-4 not only reduced J2s' invasion but also inhibited nematode development through the dependence on salicylic acid and jasmonic acid signaling of strain J2-4 induction of plant resistance in local and systemic roots of cucumbers. Genomic analysis of strain J2-4 indicated biosynthetic gene clusters encoding polymyxin, fusaricidin B, paenilan, and tridecaptin. In addition, genetic analysis showed that none of the genes encoding virulence factors were detected in the genome of J2-4 compared with the pathogenic Bacillus species. Taking all the data together, we conclude that P. polymyxa J2-4 has potential as a biological control agent against M. incognita on cucumbers and can be considered biologically safe when used in agriculture.

Restored UBE2C expression in islets promotes ß-cell regeneration in mice by ubiquitinating PER1.

Insulin deficiency may be due to the reduced proliferation capacity of islet ß-cell, contributing to the onset of diabetes. It is therefore imperative to investigate the mechanism of the ß-cell regeneration in the islets. NKX6.1, one of the critical ß-cell transcription factors, is a pivotal element in ß-cell proliferation. The ubiquitin-binding enzyme 2C (UBE2C) was previously reported as one of the downstream molecules of NKX6.1 though the exact function and mechanism of UBE2C in ß-cell remain to be elucidated. Here, we determined a subpopulation of islet ß-cells highly expressing UBE2C, which proliferate actively. We also discovered that ß-cell compensatory proliferation was induced by UBE2C via the cell cycle renewal pathway in weaning and high-fat diet (HFD)-fed mice. Moreover, the reduction of ß-cell proliferation led to insulin deficiency in ßUbe2cKO mice and, therefore, developed type 2 diabetes. UBE2C was found to regulate PER1 degradation through the ubiquitin-proteasome pathway via its association with a ubiquitin ligase, CUL1. PER1 inhibition rescues UBE2C knockout-induced ß-cell growth inhibition both in vivo and in vitro. Notably, overexpression of UBE2C via lentiviral transduction in pancreatic islets was able to relaunch ß-cell proliferation in STZ-induced diabetic mice and therefore partially alleviated hyperglycaemia and glucose intolerance. This study indicates that UBE2C positively regulates ß-cell proliferation by promoting ubiquitination and degradation of the biological clock suppressor PER1. The beneficial effect of UBE2C on islet ß-cell regeneration suggests a promising application in treating diabetic patients with ß-cell deficiency.

Construction of Prognostic Model and miRNA-mRNA Regulatory Network for Lung Squamous Cell Carcinoma.

The purpose of this paper was to construct a prognostic model, miRNA-mRNA regulatory network and protein-protein interaction (PPI) network for lung squamous cell carcinoma (LUSC) used data from the cancer genome atlas (TCGA) database. In this study, we first downloaded and sorted out the expression matrix containing 19962 mRNA transcripts (including 502 LUSC and 51 normal control (NC) samples) and the expression matrix containing 2205 miRNA transcripts (including 478 LUSC and 45 NC samples) from the TCGA database. We obtained 389 differentially expressed miRNAs (DE-miRNAs), of which 305 were upregulated and 84 down-regulated DE-miRNAs. Next, a total of 7 prognosis-related DE-miRNAs (PDE-miRNAs) were identified by Cox regression analysis, and the prognosis model consisting of three PDE-miRNAs (hsa-miR-4746-5p, hsa-miR-556-3p and hsa-miR-489-3p) was optimized. Then, we drew the survival curves and found that the survival rates of the three PDE-miRNA high and low expression groups and the survival rates of the high-risk and low-risk patients in the prognosis model had significant statistical differences. In addition, the receiver operating characteristics (ROC) curve analysis and independent prognostic analysis confirmed that the prognostic model we built has a relatively accurate ability to predict the grouping and prognosis of LUSC patients. Finally, Cox regression analysis were used to construct the miRNA-mRNA regulatory network, which showed the regulatory relationship between PDE-miRNAs and targeted mRNAs. Moreover, we constructed the PPI network composed of 145 targeted mRNAs and the subnetwork composed of 10 hub-targeted mRNAs (FCGR3A, IL13, CCR2, PPARGC1A, FCGR3B, ACSL1, PLXNA4, LPL, KAT2B and AOC3), which showed the interaction between targeted mRNAs. The above results indicated that the prognosis model we built can predict LUSC patients relatively accurately. The miRNA-mRNA regulatory network and the PPI network of targeted mRNAs illustrated the regulatory mechanisms and interactions between RNAs, which were of certain reference significance for us to further understand the molecular pathogenesis of LUSC and for clinical early diagnosis and treatment.

Effect of Different Local Antibiotic Regimens on Prevention of Postoperative Infection in Clean Surgical Wounds: A Systematic Review and Network Meta-analysis.

OBJECTIVE: To compare the efficacy of several local antibiotic regimens in preventing surgical site infection (SSI) in clean surgical wounds. DATA SOURCES: The authors searched CNKI (China National Knowledge Infrastructure), the VIP (VIP information resource integration service platform), Wanfang Data knowledge service platform (WANFANG), SinoMed, Cochrane Library, EMBASE, and PubMed. STUDY SELECTION: A total of 20 randomized controlled trials published between January 1, 2000 and April 1, 2021 were included in this meta-analysis. DATA EXTRACTION: Authors extracted the name of the first author, publication date, country, type of surgery, follow-up time, mean age of participants, sample size of each group, interventions, outcome indicators, and study type from each article. DATA SYNTHESIS: The overall effectiveness of eight local managements in reducing the incidence of the SSI effect were compared through the SUCRA (surface under the cumulative ranking curve) probabilities. The results of a network meta-analysis demonstrated that gentamicin ointment (odds ratio [OR], 0.16; 95% CI, 0.04-0.60), mupirocin ointment (OR, 0.44; 95% CI, 0.21-0.94), and gentamicin soaking of the graft (OR, 0.63; 95% CI, 0.44-0.91) significantly reduced the incidence of SSI compared with control. Further, vancomycin soaking of the graft (86.7%) ranked first, followed by gentamicin ointment (81.1%), gentamicin irrigation (79.9%), mupirocin ointment (56.8%), triple antibiotic ointment (47.8%), gentamicin soaking of the graft (42.3%), and vancomycin powder (22.1%); ampicillin powder (17.8%) was the least effective drug. CONCLUSIONS: The findings indicate that local antibiotics combined with conventional antibiotics in the wound before wound closure are effective in reducing the incidence of SSI in clean surgical wounds. Vancomycin inoculation of the graft exhibited the best effect.

Single-cell RNA sequencing combined with single-cell proteomics identifies the metabolic adaptation of islet cell subpopulations to high-fat diet in mice.

AIMS/HYPOTHESIS: Islets have complex heterogeneity and subpopulations. Cell surface markers representing alpha, beta and delta cell subpopulations are urgently needed for investigations to explore the compositional changes of each subpopulation in obesity progress and diabetes onset, and the adaptation mechanism of islet metabolism induced by a high-fat diet (HFD). METHODS: Single-cell RNA sequencing (scRNA-seq) was applied to identify alpha, beta and delta cell subpopulation markers in an HFD-induced mouse model of glucose intolerance. Flow cytometry and immunostaining were used to sort and assess the proportion of each subpopulation. Single-cell proteomics was performed on sorted cells, and the functional status of each alpha, beta and delta cell subpopulation in glucose intolerance was deeply elucidated based on protein expression. RESULTS: A total of 33,999 cells were analysed by scRNA-seq and clustered into eight populations, including alpha, beta and delta cells. For alpha cells, scRNA-seq revealed that the Ace2low subpopulation had downregulated expression of genes related to alpha cell function and upregulated expression of genes associated with beta cell characteristics in comparison with the Ace2high subpopulation. The impaired function and increased fragility of ACE2low alpha cells exposure to HFD was further suggested by single-cell proteomics. As for beta cells, the CD81high subpopulation may indicate an immature signature of beta cells compared with the CD81low subpopulation, which had robust function. We also found differential expression of Slc2a2 in delta cells and a potentially stronger cellular function and metabolism in GLUT2low delta cells than GLUT2high delta cells. Moreover, an increased proportion of ACE2low alpha cells and CD81low beta cells, with a constant proportion of GLUT2low delta cells, were observed in HFD-induced glucose intolerance. CONCLUSIONS/INTERPRETATION: We identified ACE2, CD81 and GLUT2 as surface markers to distinguish, respectively, alpha, beta and delta cell subpopulations with heterogeneous maturation and function. The changes in the proportion and functional status of islet endocrine subpopulations reflect the metabolic adaptation of islets to high-fat stress, which weakened the function of alpha cells and enhanced the function of beta and delta cells to bring about glycaemic homeostasis. Our findings provide a fundamental resource for exploring the mechanisms maintaining each islet endocrine subpopulation's fate and function in health and disease. DATA AVAILABILITY: The scRNA-seq analysis datasets from the current study are available in the Gene Expression Omnibus (GEO) repository under the accession number GSE203376.

The interactive effects of age and sex on the neuro-cardiovascular responses during fatiguing rhythmic handgrip exercise.

The impact of age on exercise pressor responses is equivocal, likely because of sex-specific neuro-cardiovascular changes with age. However, assessments of the interactive effects of age and sex on muscle sympathetic nerve activity (MSNA) responses to exercise are lacking. We tested the hypothesis that older females would exhibit exaggerated increases in blood pressure (BP) and MSNA discharge patterns during handgrip exercise compared with similarly aged males and young adults. Twenty-five young (25 (2) years; mean (SD)) males (YM; n = 12) and females (YF; n = 13) and 23 older (71 (5) years) males (OM; n = 11) and females (OF; n = 12) underwent assessments of BP, total peripheral resistance (TPR; Modelflow) and MSNA action potential (AP) discharge patterns (microneurography) during incremental rhythmic handgrip exercise and post-exercise circulatory occlusion (PECO). OM demonstrated larger ∆BP and ∆TPR from baseline than YM (both P < 0.001) despite smaller increases in ∆APs/burst (OM: 0.4 (3) vs. YM: 5 (3) spikes/burst, P < 0.001) and ∆AP clusters/burst (OM: 0.1 (1) vs. YM: 1.8 (1) clusters/burst, P < 0.001) during exercise. Testosterone was lower in OM than YM (P < 0.001) and was inversely related to ∆BP but positively related to ∆AP clusters/burst in males (both P = 0.03). Conversely, YF and OF demonstrated similar ∆BP and ∆AP discharge during exercise (range: P = 0.75-0.96). Age and sex did not impact haemodynamics or AP discharge during PECO (range: P = 0.08-0.94). Altogether, age-related changes in neuro-cardiovascular reactivity exist in males but not females during fatiguing exercise and seem to be related to testosterone. This sex-specific impact of age underscores the importance of considering biological sex when assessing age-related changes in neuro-cardiovascular control during exercise. KEY POINTS: Older males have the largest increase in blood pressure despite having the smallest increases in sympathetic vasomotor outflow during rhythmic handgrip exercise. Young males demonstrate greater increases in sympathetic action potential (AP) discharge compared with young females during rhythmic handgrip exercise. Older adults (regardless of sex) demonstrate smaller increases in muscle sympathetic nerve activity (MSNA) burst amplitude and total AP clusters compared with young adults during exercise, as well as smaller increases in integrated MSNA burst frequency, incidence and total MSNA activity during post-exercise circulatory occlusion (i.e. independent effect of age). Males, but not females (regardless of age), reflexively modify AP conduction velocity during exercise. Our results indicate that age and sex independently and interactively impact the neural and cardiovascular homeostatic adjustments to fatiguing small muscle mass exercise.

CRISPR/Cas9-mediated knockin of IRES-tdTomato at Ins2 locus reveals no RFP-positive cells in mouse islets.

Using the CRISPR/Cas9 genomic editing technology, we constructed a transgenic mouse model to express specific fluorescent protein in pancreatic ß cells, which harbor tdTomato exogenous gene downstream of the Ins2 promoter in C57BL/6 J mice. The Ins2-specific single-guide RNA-targeted exon2 was designed for the CRISPR/Cas9 system and Donor vector was constructed at the same time. Then Cas9, sgRNA, and Donor vector were microinjected in vitro into the mouse zygotes that were implanted into pseudo-pregnant mice. We obtained homozygotes through mating heterozygotes, and verified the knockin effect through genotype identification, in vivo imaging, and frozen section. Six F0 mice and stable inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed that the knockin group had no change in the Ins2 exon compared with the control group, while only the base sequence of tdTomato was added and no base mutation occurred. However, in vivo imaging and frozen section did not observe the expression of red fluorescent protein (RFP), and the protein expression of knockin gene tdTomato was negative. As a result, the expressions of tdTomato protein and fluorescence intensity were low and the detection threshold was not reached. In the CRISP/Cas9 technique, the exogenous fragment of IRES connection would affect the transcription level of the preceding gene, which in turn would lead to low-level expression of the downstream gene and affect the effect of gene insertion.

Age- and sex-related differences in sympathetic vascular transduction and neurohemodynamic balance in humans.

Bursts of muscle sympathetic nerve activity (MSNA) and the ensuing vasoconstriction are pivotal determinants of beat-by-beat blood pressure regulation. Although age and sex impact blood pressure regulation, how these factors affect the central and peripheral arcs of the baroreflex remains unclear. In 27 young [25 (SD 3) yr] males (YM; n = 14) and females (YF; n = 13) and 23 older [71 (SD 5) yr] males (OM; n = 11) and females (OF; n = 12), femoral artery blood flow, blood pressure, and MSNA were recorded for 10 min of supine rest. Sympathetic baroreflex sensitivity (i.e., central arc) was quantified as the relationship between diastolic blood pressure and MSNA burst incidence. Signal averaging was used to determine sympathetic vascular transduction into leg vascular conductance (LVC) for 12 cardiac cycles following MSNA bursts (i.e., peripheral arc). Older adults demonstrated attenuated sympathetic transduction into LVC (both P < 0.001) following MSNA bursts, and smaller increases in sympathetic transduction as a function of MSNA burst size and firing pattern compared with young adults (range, P = 0.004-0.032). YM (r2 = 0.36; P = 0.032) and OM (r2 = 0.51; P = 0.014) exhibited an inverse relationship between the central and peripheral arcs of the baroreflex, whereas females did not (YF, r2 = 0.03, P = 0.621; OF, r2 = 0.06, P = 0.445). MSNA burst incidence was inversely related to sympathetic transduction in YM and OF (range, P = 0.03-0.046) but not in YF or OM (range, P = 0.360-0.603). These data indicate that age is associated with attenuated sympathetic vascular transduction, whereas age- and sex-specific changes are present in the relationship between the central and peripheral arcs of the baroreflex regulation of blood pressure.NEW & NOTEWORTHY Sympathetic vascular transduction is attenuated in older compared with young adults, regardless of biological sex. Males, but not females (regardless of age), demonstrate an inverse relationship between central (sympathetic baroreflex sensitivity) and peripheral (sympathetic vascular transduction) components of the baroreflex arc. Young males and older females exhibit an inverse relationship between resting sympathetic outflow and sympathetic vascular transduction. Our results indicate that age and sex exert independent and interactive effects on sympathetic vascular transduction and sympathetic neurohemodynamic balance in humans.

Greater resting muscle sympathetic nerve activity reduces cold pressor autonomic reactivity in older women, but not older men.

Previous work demonstrates augmented muscle sympathetic nerve activity (MSNA) responses to the cold pressor test (CPT) in older women. Given its interindividual variability, however, the influence of baseline MSNA on CPT reactivity in older adults remains unknown. Sixty volunteers (60-83y; 30 women) completed testing where MSNA (microneurography), blood pressure (BP), and heart rate (HR) were recorded during baseline and a 2-min CPT (~4°C). Participant data were terciled by baseline MSNA (n=10/group); comparisons were made between the high baseline men (HM) and women (HW), and low baseline men (LM) and women (LW). By design, HM and HW, vs. LM and LW, had greater baseline MSNA burst frequency (37±5 and 38±3 vs. 9±4 and 15±5 bursts/min) and burst incidence (59±14 and 60±8 vs. 16±10 and 23±7 bursts/100hbs; both P<0.001). However, baseline BP and HR were not different between the groups (all P>0.05). During the CPT, there were no differences in the increase in BP and HR (all P>0.05). Conversely, ΔMSNA burst frequency was lower in HW vs. LW (8±9 vs. 22±12 bursts/min; P=0.012) yet was similar in HM vs. LM (17±12 vs. 19±10 bursts/min, P=0.994). Further, ΔMSNA burst incidence was lower in HW vs. LW (9±13 vs. 28±16 bursts/100hbs; P=0.020), with no differences between HM vs. LM (21±17 vs. 31±17 bursts/100hbs; P=0.455). Our findings suggest that heightened baseline activity in older women attenuates the typical CPT-mediated increase in MSNA without changing cardiovascular reactivity. While the underlying mechanisms remain unknown, altered sympathetic recruitment or neurovascular transduction may contribute to these disparate responses.