Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

Staged surgically created type B aortic dissection model with endovascular reintervention for different morphological features.

OBJECTIVES: Understanding morphology and how this relates to treatment strategy is critical for achieving remodelling in aortic dissection. A controllable and reproducible large animal model is required for investigating new therapeutic devices and interventions. METHODS: Our experimental protocol involved the development of surgically created type B aortic dissection (TBAD) and endovascular reintervention-induced TBAD porcine models. The sample was randomly divided into 2 groups: 1 underwent a secondary tear creation (STC) procedure and the other underwent a false lumen extension (FLE) procedure. Anatomical features were observed at 1 and 3 months, and 2 animals in each group were euthanized at 3 months after the procedures. The aorta and main branches were harvested en bloc, cross-sectioned and prepared for histological examination. RESULTS: All surgically created TBAD models were successfully generated, and no unintended complications occurred. The endovascular reintervention-induced TBAD model was successfully created in 11 of 12 animals, with 6 in the STC group and 5 in the FLE group. In the STC group, the intraoperative mean diameter of the new secondary tear was 7.23 mm, and a slight increase was observed at first 30 days (P = 0.0026). In the FLE group, the intraoperative new propagation length was (235.80 ± 84.94) mm. The FL propagation length at the 1-month follow-up was significantly longer than that measured intraoperatively (P = 0.0362). Histological evaluation demonstrated that the elastic fibres in the media layer of the aortic wall were disrupted and appeared to be significantly stretched on the adventitial side of the false lumen. CONCLUSIONS: Our endovascular reintervention is a reliable, minimally invasive approach for producing specific TBAD models with different morphologies.

Finite-element simulation of in-plane tear propagation in the dissected aorta: Implications for the propagation mechanism.

Computer modeling and numerical simulation are essential for understanding the progression of aortic dissection. However, tear propagation has not been properly modeled and simulated. The in-plane dissection propagation between concentrically distributed elastic lamellae is modeled using the cohesive zone method with a bilinear traction-separation law. The parameters of cohesive elements are calibrated for the three modes of interfacial damage in the media, enabling quantitative predictions of in-plane tear propagation. An idealized cylindrical tube-shaped bilayer thick-wall model of the aorta is employed to elucidate the influence of geometrical parameters, loading conditions and residual stress on the tear propagation. While the model is capable of replicating that deeper, larger tears are associated with lower critical pressure, new findings include: (i) Larger axial stretch leads to lower critical pressure; (ii) Increased magnitude of residual stress is associated with higher critical pressure; (iii) Pressure difference between true and false lumen alters the critical pressure; (iv) The interfacial damage is mostly opening mode in the axial direction, but shear-dominated in the circumferential direction; (v) Damage due to the opening mode is associated with smaller fracture energy, which makes it easier to propagate than the shear and the mixed modes. (vi) The deformed shape of the tear, which is related to its geometrical features and loading conditions, modulates the critical pressure via two pathways: (a) deformed shapes are associated with specific modes of damage, which influences the critical pressure, and (b) deformed shapes modulate the critical pressure directly via geometrical constraints.

Neutralizing IL-8 potentiates immune checkpoint blockade efficacy for glioma.

Malignant gliomas are largely refractory to immune checkpoint blockade (ICB) therapy. To explore the underlying immune regulators, we examine the microenvironment in glioma and find that tumor-infiltrating T cells are mainly confined to the perivascular cuffs and express high levels of CCR5, CXCR3, and programmed cell death protein 1 (PD-1). Combined analysis of T cell clustering with T cell receptor (TCR) clone expansion shows that potential tumor-killing T cells are mainly categorized into pre-exhausted/exhausted and effector CD8+ T subsets, as well as cytotoxic CD4+ T subsets. Notably, a distinct subpopulation of CD4+ T cells exhibits innate-like features with preferential interleukin-8 (IL-8) expression. With IL-8-humanized mouse strain, we demonstrate that IL-8-producing CD4+ T, myeloid, and tumor cells orchestrate myeloid-derived suppressor cell infiltration and angiogenesis, which results in enhanced tumor growth but reduced ICB efficacy. Antibody-mediated IL-8 blockade or the inhibition of its receptor, CXCR1/2, unleashes anti-PD-1-mediated antitumor immunity. Our findings thus highlight IL-8 as a combinational immunotherapy target for glioma.

Effects of preoperative oral enzyme-hydrolyzed rice flour solution on gastric emptying and insulin resistance in patients undergoing laparoscopic cholecystectomy: a prospective randomized controlled trial.

BACKGROUND: The effect of preoperative oral carbohydrates (POC) on insulin resistance (IR) of laparoscopic cholecystectomy (LC) remains debatable. Enzyme-hydrolyzed rice flour (EHR) is a kind of water-soluble micromolecular carbohydrates. This study aimed to investigate the impact of preoperative oral EHR solution on gastric emptying and IR in patients undergoing LC. METHODS: Patients (n = 100) undergoing LC were divided into oral-water group (group C) or oral-EHR solution (group E) randomly (n = 50 each), and the patients drank 300 ml water or EHR solution 2-3 h before surgery respectively. Gastric emptying which was quantized by gastric volume (GV) from antrum ultrasonography, IR indicators, subjective comfort indicators, handgrip strength, postoperative recovery indexes, and complications were recorded. RESULTS: There were no differences in GV between the two groups before oral administration (V0), immediately after oral administration (V1) and before anesthesia induction(V2). The GV at V2 (GV2) reduced to the level of V0 (GV0) in the two groups. Fasting glucose (FG), fasting insulin (FINS) and Homa-IR in the two groups increased at postoperative day 1 (Pos 1d) compared with those at preoperative day 1(Pre 1d). Homa-IS and Homa-ß in the two groups decreased at Pos 1d compared with those at Pre 1d. FG, FINS and Homa-IR in group E were lower than those in group C at Pos 1d, and Homa-IS and Homa-ß were higher in group E than those in group C at Pos 1d. Subjective comfort indictors (hunger, fatigue and anxiety) in group E were lower than those in group C at preoperative 15 min (Pre 15 min) and postoperative 1 h (Pos 1 h). Handgrip strength in group E was raised compared with that in group C at Pre 15 min, Pos 1 h and Pos 1d. There was a lower incidence of nausea and earlier exhaust time in group E. CONCLUSION: Oral 300 ml EHR solution 2-3 h before LC surgery did not increase the occurrence of reflux and aspiration during anesthesia induction with a normal gastric emptying, ameliorated postoperative IR, improved subjective comfort, and promoted postoperative gastrointestinal function recovery. TRIAL REGISTRATION: Prospectively registered at the China Clinical Trial Registry, registration number: ChiCTR2000039939, date of registration:14/11/2020.

Targeting ATAD3A-PINK1-mitophagy axis overcomes chemoimmunotherapy resistance by redirecting PD-L1 to mitochondria.

Only a small proportion of patients with triple-negative breast cancer benefit from immune checkpoint inhibitor (ICI) targeting PD-1/PD-L1 signaling in combination with chemotherapy. Here, we discovered that therapeutic response to ICI plus paclitaxel was associated with subcellular redistribution of PD-L1. In our immunotherapy cohort of ICI in combination with nab-paclitaxel, tumor samples from responders showed significant distribution of PD-L1 at mitochondria, while non-responders showed increased accumulation of PD-L1 on tumor cell membrane instead of mitochondria. Our results also revealed that the distribution pattern of PD-L1 was regulated by an ATAD3A-PINK1 axis. Mechanistically, PINK1 recruited PD-L1 to mitochondria for degradation via a mitophagy pathway. Importantly, paclitaxel increased ATAD3A expression to disrupt proteostasis of PD-L1 by restraining PINK1-dependent mitophagy. Clinically, patients with tumors exhibiting high expression of ATAD3A detected before the treatment with ICI in combination with paclitaxel had markedly shorter progression-free survival compared with those with ATAD3A-low tumors. Preclinical results further demonstrated that targeting ATAD3A reset a favorable antitumor immune microenvironment and increased the efficacy of combination therapy of ICI plus paclitaxel. In summary, our results indicate that ATAD3A serves not only as a resistant factor for the combination therapy of ICI plus paclitaxel through preventing PD-L1 mitochondrial distribution, but also as a promising target for increasing the therapeutic responses to chemoimmunotherapy.

Measurement of Layer-Specific Mechanical Properties of Intact Blood Vessels Based on Intravascular Optical Coherence Tomography.

PURPOSE: The biomechanical analysis of stress and strain state of multilayered blood vessels has shown great importance in vascular pathology and physiology. However, there is a lack of method in measuring the mechanical property of each layer of a vascular sample without splitting up the wall. METHODS: Here we develop a vascular inflation test method based on intravascular optical coherence tomography (IVOCT) imaging and inverse parametric estimation. We propose a three-step inverse parametric estimation method to solve the six constitutive parameters of the GOH models for the intima-media and adventitia of the coronaries simultaneously. A bilayer silicone vascular phantom inflation test and a virtual deformation test using finite element simulated data are conducted to evaluate the accuracy of the proposed method. RESULTS: The virtual deformation test demonstrates that the errors of the constitutive constants are less than 2.56% determined by the proposed inverse parametric estimation method. The stress-strain curves of a bilayer silicone vascular phantom obtained based on the parameters determined by the proposed method match well with those obtained by the uniaxial test. CONCLUSION: The proposed layer-specific vascular mechanical property measurement method provides a new experimental method for mechanical properties characterization of blood vessels. It also has the potential to be used for patient-specific mechanical properties estimation with IVOCT imaging in vivo.

In Vivo Intravascular Optical Coherence Tomography (IVOCT) Structural and Blood Flow Imaging Based Mechanical Simulation Analysis of a Blood Vessel.

INTRODUCTION: Computer modelling of blood vessels based on biomedical imaging provides important hemodynamic and biomechanical information for vascular disease studies and diagnosis. However due to lacking well-defined physiological blood flow profiles, the accuracy of the simulation results is often not guaranteed. Flow velocity profiles of a specific cross section of a blood vessel were obtained by in vivo Doppler intravascular optical coherence tomography (IVOCT) lately. However due to the influence of the catheter, the velocity profile imaged by IVOCT can't be applied to simulation directly. METHODS: A simulation-experiment combined method to determine the inlet flow boundary based on in vivo porcine carotid Doppler IVOCT imaging is proposed. A single conduit carotid model was created from the 3D IVOCT structural images using an image processing-computer aided design combined method. RESULTS: With both high- resolution arterial model and near physiological blood flow profile, stress analysis by fluid-structure interaction and computational fluid dynamics were performed. The influence of the catheter to the wall shear stress, the hemodynamics and the stresses of the carotid wall under the measured inlet flow and various outlet pressure boundary conditions, are analyzed. CONCLUSION: This study provides a solution to the difficulty of getting inlet flow boundary for numerical simulation of arteries. It paves the way for developing IVOCT based vascular stress analysis and imaging methods for the studies and diagnosis of vascular diseases.

CD127 imprints functional heterogeneity to diversify monocyte responses in inflammatory diseases.

Inflammatory monocytes are key mediators of acute and chronic inflammation; yet, their functional diversity remains obscure. Single-cell transcriptome analyses of human inflammatory monocytes from COVID-19 and rheumatoid arthritis patients revealed a subset of cells positive for CD127, an IL-7 receptor subunit, and such positivity rendered otherwise inert monocytes responsive to IL-7. Active IL-7 signaling engaged epigenetically coupled, STAT5-coordinated transcriptional programs to restrain inflammatory gene expression, resulting in inverse correlation between CD127 expression and inflammatory phenotypes in a seemingly homogeneous monocyte population. In COVID-19 and rheumatoid arthritis, CD127 marked a subset of monocytes/macrophages that retained hypoinflammatory phenotypes within the highly inflammatory tissue environments. Furthermore, generation of an integrated expression atlas revealed unified features of human inflammatory monocytes across different diseases and different tissues, exemplified by those of the CD127high subset. Overall, we phenotypically and molecularly characterized CD127-imprinted functional heterogeneity of human inflammatory monocytes with direct relevance for inflammatory diseases.

A novel framework for quantifying the subject-specific three-dimensional residual stress field in the aortic wall.

BACKGROUND: Quantification of subject-specific residual stress field remains a challenge that prohibits accurate stress analysis and refined understanding of the biomechanical behavior of the aortic wall. METHOD: This study presents a framework combining experiments, constitutive modeling, and computer simulation to quantify the subject-specific three-dimensional residual stress field of the aortic wall. The material properties and residual deformations were acquired from the same porcine aortic sample, so that the subject-specific residual stress field was quantified analytically. Consequently, a novel stress-driven tissue growth model was developed and incorporated in a finite element aortic model to recover the subject-specific residual stress with the help of analytical solution. We then evaluated the framework's efficacy by simulating the residual stress distribution in the aortic dissection (AD). RESULT: Subject-specific residual stress field of the aortic sample was quantified analytically. No appreciable discrepancy was observed between the numerically simulated and analytically derived residual stress distributions, indicating the effectiveness of the tissue growth model. Errors arising from the numerically simulated circumferential opening angle and axial bending angle were within 5% relative to experimental results, highlighting that the framework was accurate in terms of subject-specific residual stress estimation. Finally, numerical simulations recovered the buckling behavior of the intimal flap of the dissected aorta and revealed the expansion of the false lumen and compression of the true lumen as the tear propagates circumferentially. CONCLUSION: The proposed framework is effective in quantifying the three-dimensional subject-specific residual stress field and it is potentially applicable in more sophisticated scenarios involving residual stress.

Danggui-Shaoyao-San improves cognitive impairment through inhibiting O-GlcNAc-modification of estrogen α receptor in female db/db mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine formula Danggui-Shaoyao-San (DSS) has been reported to show therapeutic effect on dementia. AIM OF THE STUDY: The present study aims to investigate whether DSS treatment could alleviate diabetes-induced cognitive dysfunction, and explores its neuroprotective mechanism on db/db mice. MATERIALS AND METHODS: The female db/db mice were randomly divided into model group, DSS low-dose group and DSS high-dose group. Homologous female db/m mice were used as the control group. DSS was intragastric administrated for 15 weeks. Glucose tolerance, insulin tolerance, blood glucose and blood lipid levels were measured. Morris water maze was used to measure spatial learning and memory ability in mice. Nissl staining and Tunel staining were used to measure the changes of brain neurons, and ELISA kits were used to measure levels of inflammatory mediators (PGE2, TXB2 and LTB4). The kits detected oxidative stress (MDA, SOD, CAT, GSH-PX), nitrosative stress (NO, iNOS, TNOS) and glucose metabolism (LDH, PK, HK) levels. Western blot and immunofluorescence detected neurotrophic factors (PSD95, BDNF, NGF and SYN), apoptosis (Bcl-2, Bax, Bcl-xl, Caspase-3) and changes of ERα, O-GlcNAc, OGT, OGA levels. RESULTS: Morris water maze results showed that DSS could improve the learning and memory abilities of female db/db mice. Nissl staining showed that DSS could relieve hippocampal neurons damage of db/db mice. In addition, the serological tests showed that DSS could improve the impaired glucose tolerance and insulin resistance, while reduce hyperlipemia in db/db mice. Besides, DSS treatment increased the activities of SOD, GSH-PX, and CAT, and reduced MDA, NO, iNOs, tNOS, PGE2, TXB2 and LTB4 levels. Western blot and immunofluorescence results of PSD95, BDNF, NGF, and SYN showed that DSS could improve the expressions of neurotrophic factors. Meanwhile, Tunel staning and Western blot (Bcl-2, Bax, Bcl-xl, Caspase-3) results indicated that DSS could reduce neuronal apoptosis. Finally, Western blot (ERα, O-GlcNAc, OGA, and OGT) and immunofluorescence (ERα and O-GlcNAc) results indicated that DSS could increase the levels of ERα and OGA, decrease the levels of O-GlcNAc and OGT. CONCLUSION: DSS alleviate DE might be related to improve the abnormal O-GlcNAc-modification of ERα.

COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.

CD127 imprints functional heterogeneity to diversify monocyte responses in human inflammatory diseases.

Studies on human monocytes historically focused on characterization of bulk responses, whereas functional heterogeneity is largely unknown. Here, we identified an inducible population of CD127-expressing human monocytes under inflammatory conditions and named the subset M127. M127 is nearly absent in healthy individuals yet abundantly present in patients with infectious and inflammatory conditions such as COVID-19 and rheumatoid arthritis. Multiple genomic and functional approaches revealed unique gene signatures of M127 and unified anti-inflammatory properties imposed by the CD127-STAT5 axis. M127 expansion correlated with mild COVID-19 disease outcomes. Thereby, we phenotypically and molecularly characterized a human monocyte subset marked by CD127 that retained anti-inflammatory properties within the pro-inflammatory environments, uncovering remarkable functional diversity among monocytes and signifying M127 as a potential therapeutic target for human inflammatory disorders.

SOSTDC1-producing follicular helper T cells promote regulatory follicular T cell differentiation.

Germinal center (GC) responses potentiate the generation of follicular regulatory T (TFR) cells. However, the molecular cues driving TFR cell formation remain unknown. Here, we show that sclerostin domain-containing protein 1 (SOSTDC1), secreted by a subpopulation of follicular helper T (TFH) cells and T-B cell border-enriched fibroblastic reticular cells, is developmentally required for TFR cell generation. Fate tracking and transcriptome assessment in reporter mice establishes SOSTDC1-expressing TFH cells as a distinct T cell population that develops after SOSTDC1- TFH cells and loses the ability to help B cells for antibody production. Notably, Sostdc1 ablation in TFH cells results in substantially reduced TFR cell numbers and consequently elevated GC responses. Mechanistically, SOSTDC1 blocks the WNT-ß-catenin axis and facilitates TFR cell differentiation.

Spermidine and spermine delay brain aging by inducing autophagy in SAMP8 mice.

The natural polyamine spermidine and spermine have been reported to ameliorate aging and aging-induced dementia. However, the mechanism is still confused. An aging model, the senescence accelerated mouse-8 (SAMP8), was used in this study. Novel object recognition and the open field test results showed that oral administration of spermidine, spermine and rapamycin increased discrimination index, modified number, inner squares distance and times. Spermidine and spermine increased the activity of SOD, and decreased the level of MDA in the aging brain. Spermidine and spermine phosphorylate AMPK and regulate autophagy proteins (LC3, Beclin 1 and p62). Spermidine and spermine balanced mitochondrial and maintain energy for neuron, with the regulation of MFN1, MFN2, DRP1, COX IV and ATP. In addition, western blot results (Bcl-2, Bax and Caspase-3, NLRP3, IL-18, IL-1ß) showed that spermidine and spermine prevented apoptosis and inflammation, and elevate the expression of neurotrophic factors, including NGF, PSD95and PSD93 and BDNF in neurons of SAMP8 mice. These results indicated that the effect of spermidine and spermine on anti-aging is related with improving autophagy and mitochondrial function.

Carnosine ameliorates age-related dementia via improving mitochondrial dysfunction in SAMP8 mice.

Dementia is a kind of age-related neurodegenerative disease. Carnosine, an endogenous dipeptide consisting of ß-alanine and l-histidine, has been shown to have neuroprotective effects. However, the exact mechanism is still obscure. In this study, senescence-accelerated mouse prone 8 (SAMP8) mice, an age-related animal model, were used. Carnosine (100 and 200 mg kg-1 day-1) was orally administered to the mice once daily for six weeks. Behavioral tests, western blotting, and detection kits were used to evaluate the potential effects of carnosine on SAMP8 mice. Open-field and new object recognition experiments have shown that carnosine improved cognitive deficits in SAMP8 mice. Carnosine decreased the levels of malondialdehyde (MDA) and reactive oxygen species (ROS), increased the activity of superoxide dismutase (SOD) and the level of adenosine triphosphate (ATP) in SAMP8 mice. Concomitantly, western blotting results proved that carnosine increased the protein expressions of Mitofusin-1, Mitofusin-2, and Bcl-2 and reduced the protein expressions of P-Drp1, Bax, cleaved Caspase-3 and NLRP3 inflammasomes in the hippocampus of SAMP8 mice. The present data provided evidence that carnosine might improve cognitive impairment in SAMP8 mice through modulating mitochondrial dysfunction.

Ligustilide improves aging-induced memory deficit by regulating mitochondrial related inflammation in SAMP8 mice.

Alzheimer's disease (AD) is an age-related neurodegenerative disease. The main active component in Angelica sinensis, ligustilide, has been reported to have the protective effect on AD. Whether ligustilide could protect against age-induced dementia is still unknown. In this study, we used an aging model, SAMP8 mice to investigate the neuroprotective effect of ligustilide. The behavioral tests (Morris water maze, object recognition task, open field test and elevated plus maze) results showed that ligustilide could improve the memory deficit in SAMP8 mice. For mechanism study, we found that the protein level of P-Drp1 (fission) was decreased and the levels of Mfn1 and Mfn2 (fusion) were increased after ligustilide treatment in animals and cells. Ligustilide increased P-AMPK and ATP levels. Malondialdehyde and superoxide dismutase activity results indicated that ligustilide exerts antioxidant effects by reducing the level of oxidative stress markers. In addition, ligustilide improved neural function and alieved apoptosis and neuroinflammation. These findings have shown that ligustilide treatment improves mitochondrial function in SAMP8 mice, and improves memory loss.