Frontiers and future perspectives of neuroimmunology.

Neuroimmunology is an interdisciplinary branch of biomedical science that emerges from the intersection of studies on the nervous system and the immune system. The complex interplay between the two systems has long been recognized. Research efforts directed at the underlying functional interface and associated pathophysiology, however, have garnered attention only in recent decades. In this narrative review, we highlight significant advances in research on neuroimmune interplay and modulation. A particular focus is on early- and middle-career neuroimmunologists in China and their achievements in frontier areas of "neuroimmune interface", "neuro-endocrine-immune network and modulation", "neuroimmune interactions in diseases", "meningeal lymphatic and glymphatic systems in health and disease", and "tools and methodologies in neuroimmunology research". Key scientific questions and future directions for potential breakthroughs in neuroimmunology research are proposed.

Research on Explosion Pressure Characteristics of Long Flame Coal Dust and the Inhibition Effect of Different Explosion Suppressants.

To discuss the inhibition of long flame coal dust explosion pressure, NaHCO3, KHCO3, and NH4H2PO4 are selected as explosion suppression dust for explosion pressure tests under different conditions. The results show that when 25-38 and 38-45 µm coal dust are mixed in 1:1 ratio, the maximum explosion pressure is the largest, the maximum pressure is 0.79 MPa, and the maximum pressure rise rate is 74.89 MPa·s-1. The suppression dusts have good inhibition effect on explosion, the order of inhibition is NaHCO3, KHCO3, and NH4H2PO4 from the smallest to the largest. With the reduction of particle size of NH4H2PO4, its inhibition effect on explosion pressure is increasing, because more NH4H2PO4 particles move around coal dust particles, blocking the heat transfer and kinetic energy exchange. The above three suppression dust and their suppression methods can provide important data for dust prevention and control and have certain reference significance for carrying out explosion suppression work.

Erratum: [Corrigendum] MicroRNA­23a inhibits endometrial cancer cell development by targeting SIX1.

[This corrects the article DOI: 10.3892/ol.2019.10694.].

Acetate controls endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT), a process initiated by activation of endothelial TGF-ß signaling, underlies numerous chronic vascular diseases and fibrotic states. Once induced, EndMT leads to a further increase in TGF-ß signaling, thus establishing a positive-feedback loop with EndMT leading to more EndMT. Although EndMT is understood at the cellular level, the molecular basis of TGF-ß-driven EndMT induction and persistence remains largely unknown. Here, we show that metabolic modulation of the endothelium, triggered by atypical production of acetate from glucose, underlies TGF-ß-driven EndMT. Induction of EndMT suppresses the expression of the enzyme PDK4, which leads to an increase in ACSS2-dependent Ac-CoA synthesis from pyruvate-derived acetate. This increased Ac-CoA production results in acetylation of the TGF-ß receptor ALK5 and SMADs 2 and 4 leading to activation and long-term stabilization of TGF-ß signaling. Our results establish the metabolic basis of EndMT persistence and unveil novel targets, such as ACSS2, for the potential treatment of chronic vascular diseases.

Experimental Study of Thermal and Fire Reaction Properties of Glass Fiber/Bismaleimide Composites for Aeronautic Application.

Thermal behavior and fire reaction properties of aerial glass fiber (GF)/bismaleimide (BMI) composites were tested using thermogravimetric analysis (TGA), thermogravimetric coupled with Fourier transform infrared spectroscopy (TG-FTIR), cone calorimeter, limiting oxygen index, and smoke density chamber. The results showed that the pyrolysis process was one stage in a nitrogen atmosphere with the prominent volatile components of CO2, H2O, CH4, NOx, and SO2. The release of heat and smoke increased with the increase in heat flux, while the time required to reach hazardous conditions decreased. The limiting oxygen index decreased monotonically from 47.8% to 39.0% with increasing experimental temperature. The maximum specific optical density within 20 min in the non-flaming mode was greater than that in the flaming mode. According to the four kinds of fire hazard assessment indicators, the greater the heat flux, the higher the fire hazard, for the contribution of more decomposed components. The calculations of two indices confirmed that the smoke release in the early stage of fire was more negative under flaming mode. This work can provide a comprehensive understanding of the thermal and fire characteristics of GF/BMI composites used for aircraft.

Elevated Expression of ADAM10 Induced by HPV E6 Influences the Prognosis of Cervical Cancer.

Objective: To explore the abnormal expression of ADAM10, its cause, and its clinical value in the prognosis of cervical lesions. Methods: The abnormal expression of ADAM10 was explored using the Gene Expression Profiling Interactive Analysis database, and the abnormal expression in cervical lesions was verified using immunohistochemistry (IHC). The transfection effect of shRNA was evaluated using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression of ADAM10 in cells was analyzed using western blotting. Results: ADAM10 was highly expressed in multiple cancers. As the disease progressed, the expression of ADAM10 gradually increased (p < 0.05). Patients with higher expression of ADAM10 had poorer survival outcomes than those with lower expression levels (p < 0.05). The expression levels of ADAM10 decreased after expression levels of E6 was inhibited. Conclusion: ADAM10 is highly expressed in cervical cancer; the higher the expression levels, the worse the survival outcome. HPV E6 is the critical driver of the elevated expression of ADAM10 in cervical cancer.

High-resolution visualization of pial surface vessels by flattened whole mount staining.

Understanding development of the cerebral vasculature is essential for the central nervous system (CNS) research and therapeutic developments. Here, we developed a simple, convenient, and fast method-the flattened cortex whole mount (FCWM) technique-for imaging of pial cerebral vessels. FCWM involves dissection of the whole cerebral cortex followed by flattening, sectioning and application of CLARITY technology. Compared to conventional methods, FCWM offers several advantages including (1) high-resolution visualization of the whole cortex pial surface vessel structures and distributions; (2) precise localization of a particular blood vessel, allowing observations of a desired blood vessel during normal development or in disease settings; (3) compatibility with confocal imaging. Application of FCWM for examination of cerebral vasculature during postnatal development or in stroke settings allowed us to demonstrate that cerebral blood vessels manifest type-specific maturation and remodeling which are linked to the rate of endothelial proliferation.

Betulinaldehyde exhibits effective anti-tumor effects in A549 cells by regulating intracellular autophagy.

It is of great significance to find new effective drugs for an adjuvant therapy targeting lung cancer to improve the survival rate and prognosis of patients with the disease. Previous studies have confirmed that certain Chinese herbal extracts have clear anti-tumor effects, and in our preliminary study, betulinaldehyde was screened for its potential anti-tumor effects. The current study thus aimed to confirm the anti-tumor effect of betulinaldehyde, using in vitro experiments to explore its underlying molecular mechanism. It was found that betulinaldehyde treatment significantly inhibited the viability, proliferation, and migration of A549 cells in a dose-dependent manner. In addition, betulinaldehyde inhibited the activation of Akt, MAPK, and STAT3 signaling pathways in A549 cells in a time-dependent manner. More importantly, betulinaldehyde also decreased the expression level of SQSTM1 protein, increased the expression level of LC3 II, and increased the autophagy flux in A549 cells. The pretreatment of A549 cells with the autophagy inhibitor, 3-methyladenine, could partially negate the anti-tumor effects of betulinaldehyde. These findings suggest that betulinaldehyde could significantly inhibit the oncological activity of A549 cells by regulating the intracellular autophagy level, making it a potentially effective option for the adjuvant therapy used to treat lung cancer in the future.

The larval, pupal and mitogenomic characteristics of Agrilusadelphinus Kerremans, 1895 (Coleoptera, Buprestidae) from China.

In this study, the larva and pupa of Agrilusadelphinus are described and illustrated. DNA barcoding (COI gene) was used to associate the larval and pupal stages with adults based on the maximum-likelihood method. In the resulting phylogenetic tree, species from the same species-group were found to be clustered on a branch with high support value. To better understand A.adelphinus, the complete mitochondrial genome of this species was also sequenced and annotated. Comparing this genome to the known mitogenomes of Agrilus species, the newly sequenced genome is shorter, with 15,732 bp. However, its whole mitogenome composition and gene orientation were consistent with that of most species of Buprestidae. In the mitogenome of A.adelphinus, the ATGATAG sequence was observed between ATP8 and ATP6, which is ATGATAA in other insect mitogenomes. Leu2, Phe, Ile, Gly, and Ser2 were the five most frequently encoded amino acids. The results further prove that DNA barcoding can remove the limitation of traditional taxonomy which cannot identify to species all developmental stages. This study also provides valuable molecular and morphological data for species identification and phylogenetic analyses of the genus Agrilus.

Prophylactic exercise-derived circulating exosomal miR-125a-5p promotes endogenous revascularization after hindlimb ischemia by targeting endothelin converting enzyme 1.

Background: Prophylactic exercise improves clinical outcomes in patients experiencing severe ischemic diseases. Previous studies have shown that exercise could alter the amount or content of circulating exosomes. However, little is known about the role of precursory exercise-derived circulating exosomes (Exe-Exo) in ischemic diseases. We therefore aimed to explore the function and mechanism of Exe-Exo in endogenous revascularization and perfusion recovery in peripheral arterial disease. Methods and Results: We first determined that 4 weeks of precursory treadmill exercise improved perfusion recovery on days 7, 14 and 21 after unilateral femoral artery ligation (FAL) but had no effect immediately after ligation. Then, local muscle delivery of Exe-Exo promotes arteriogenesis, angiogenesis and perfusion recovery, which could be abolished by GW4869, a well-recognized pharmacological agent inhibiting exosome release. This suggests that Exe-Exo mediated exercise-induced revascularization. In vitro, Exe-Exo enhanced endothelial cell proliferation, migration and tube formation. In addition, we identified miR-125a-5p as a novel exerkine through exosomal miRNA sequencing and RT-qPCR validation. Inhibition of miR-125a-5p abrogated the beneficial effects of Exe-Exo both in vivo and in vitro. Mechanistically, these exercise-afforded benefits were attributed to the exosomal miR-125a-5p downregulation of ECE1 expression and the subsequent activation of the AKT/eNOS downstream signaling pathway. Specifically, skeletal muscle may be a major tissue source of exercise-induced exosomal miR-125a-5p via fluorescence in situ hybridization. Conclusions: Endogenous circulating exosomal miR-125a-5p promotes exercise-induced revascularization via targeting ECE1 and activating AKT/eNOS downstream signaling pathway. Identify exosomal miR-125a-5p as a novel exerkine, and highlight its potential therapeutic role in the prevention and treatment of peripheral arterial disease.

Prevalence and predictors of Helicobacter pylori infection in asymptomatic individuals: a hospital-based cross-sectional study in Shenzhen, China.

OBJECTIVE: Although a high Helicobacter pylori infection rate has been reported in dyspeptic patients, published data for the asymptomatic population are inadequate. We aimed to investigate the prevalence of H. pylori infection and the association between H. pylori infection diagnosed using the noninvasive 13C urea breath test (13C UBT) and risk factors in asymptomatic Chinese individuals. METHODS: Healthy subjects who underwent hospital health examinations in the hospital health management department from September 2020 to September 2021 were consecutively enrolled. Anthropometric and biochemical characteristics were measured, and all subjects underwent 13C UBT. Univariate and multivariate analyses were used to evaluate the factors associated with H. pylori infection. RESULTS: Of the 5007 participants included, the overall prevalence of H. pylori infection was 35.0% and was similar in men (35.5%) and women (34.2%). The prevalence of H. pylori increased by 84.8% in those aged ≥ 60 years compared with those aged < 30 years (P=0.006), and increased by 30.9% in the obese group compared with the normal weight groups (P = 0.034). Fasting blood glucose (FBG) and albumin levels showed a significant association with the prevalence of H. pylori. The prevalence of H. pylori increased by 6.9% following each 1-unit increase in FBG (P = 0.033) and decreased by 3.7% for each 1-unit increase in albumin (P = 0.012). Moreover, the results of the age stratification showed that albumin level was negatively associated with the prevalence of H. pylori in participants aged < 50 years (OR = 0.952, 95%CI: 0.921-0.985; P = 0.005). However, the prevalence of H. pylori in participants aged ≥ 50 years increased by 66.6% in the impaired glucose group compared to the normal glucose group (P = 0.003). CONCLUSION: These findings suggest that detailed and strict weight and glucose management is crucial to reduce the prevalence of H. pylori infection. Management strategies should be identified and implemented with regard to these identified factors to reduce the notable economic and healthcare burden in China.

Endothelium-derived lactate is required for pericyte function and blood-brain barrier maintenance.

Endothelial cells differ from other cell types responsible for the formation of the vascular wall in their unusual reliance on glycolysis for most energy needs, which results in extensive production of lactate. We find that endothelium-derived lactate is taken up by pericytes, and contributes substantially to pericyte metabolism including energy generation and amino acid biosynthesis. Endothelial-pericyte proximity is required to facilitate the transport of endothelium-derived lactate into pericytes. Inhibition of lactate production in the endothelium by deletion of the glucose transporter-1 (GLUT1) in mice results in loss of pericyte coverage in the retina and brain vasculatures, leading to the blood-brain barrier breakdown and increased permeability. These abnormalities can be largely restored by oral lactate administration. Our studies demonstrate an unexpected link between endothelial and pericyte metabolisms and the role of endothelial lactate production in the maintenance of the blood-brain barrier integrity. In addition, our observations indicate that lactate supplementation could be a useful therapeutic approach for GLUT1 deficiency metabolic syndrome patients.

Pharmacokinetics of Flunarizine Hydrochloride After Single Oral Doses in Healthy Subjects: Bioequivalence Study and Food Effects.

We designed a study to compare the newly developed 5-mg flunarizine hydrochloride capsules (test) to that of its marketed counterpart (5-mg; reference) among healthy adult Chinese volunteers. We performed an open-label, single-center study that consisted of 2 randomized, crossover trials, including a fasting trial and a fed trial. In each part of the study, the subjects were randomly assigned to either receive the test or reference products (5-mg flunarizine) in a 1:1 ratio. Subjects then received the alternative products, following a 14-day washout period. Concentrations of plasma flunarizine were analyzed using liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters (noncompartmental model) were evaluated using the WinNonlin software. The analysis of variance and Food and Drug Administration bioequivalence statistical criterion of 90% confidence interval for 80% to 125% range (set at P ≤ .05) of geometric means ratios of test: reference product for peak plasma concentration, area under the plasma concentration-time curve (AUC) from time 0 to time t, and AUC from time 0 to infinity were determined. Tolerability was evaluated during the entire study period. Overall, 23 volunteers completed the fasting study, while 40 volunteers completed the fed study. The test formulation was found to be bioequivalent to the marketed formulation, as the 90% confidence interval for the ratio of geometric means of peak plasma concentration (fasting: 87.61%-101.67%; fed: 87.38%-104.06%), AUC from time 0 to time t (fasting: 89.44%-99.92%; fed: 92.65%-98.28%), and AUC from time 0 to infinity (fasting: 95.02%-104.33%; fed: 90.41%-96.96%) were within equivalence limits (80-125%) under both the fasting and fed conditions. When flunarizine was given alongside high-fat meals, time to maximum concentration was delayed ≈3.5 hours compared to fasting conditions. Meantime, high-fat meals increased its exposure by nearly 50%. Furthermore, there were no serious adverse events found among the subjects. This study confirmed that test and reference flunarizine hydrochloride capsules were bioequivalent under fasting and postprandial conditions.

MEKK3-TGFß crosstalk regulates inward arterial remodeling.

Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet, despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell-fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The gene's deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries, leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGFß-signaling both in vitro and in vivo. Endothelial-specific TGFßR1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGFßR1-Smad2/3 signaling and inward arterial remodeling in artery diseases.

Role of Venous Endothelial Cells in Developmental and Pathologic Angiogenesis.

BACKGROUND: Angiogenesis is a dynamic process that involves expansion of a preexisting vascular network that can occur in a number of physiological and pathological settings. Despite its importance, the origin of the new angiogenic vasculature is poorly defined. In particular, the primary subtype of endothelial cells (capillary, venous, arterial) driving this process remains undefined. METHODS: Endothelial cells were fate-mapped with the use of genetic markers specific to arterial and capillary cells. In addition, we identified a novel venous endothelial marker gene (Gm5127) and used it to generate inducible venous endothelium-specific Cre and Dre driver mouse lines. Contributions of these various types of endothelial cells to angiogenesis were examined during normal postnatal development and in disease-specific setting. RESULTS: Using a comprehensive set of endothelial subtype-specific inducible reporter mice, including tip, arterial, and venous endothelial reporter lines, we showed that venous endothelial cells are the primary endothelial subtype responsible for the expansion of an angiogenic vascular network. During physiological angiogenesis, venous endothelial cells proliferate, migrating against the blood flow and differentiating into tip, capillary, and arterial endothelial cells of the new vasculature. Using intravital 2-photon imaging, we observed venous endothelial cells migrating against the blood flow to form new blood vessels. Venous endothelial cell migration also plays a key role in pathological angiogenesis. This was observed both in formation of arteriovenous malformations in mice with inducible endothelium-specific Smad4 deletion mice and in pathological vessel growth seen in oxygen-induced retinopathy. CONCLUSIONS: Our studies establish that venous endothelial cells are the primary endothelial subtype responsible for normal expansion of vascular networks, formation of arteriovenous malformations, and pathological angiogenesis. These observations highlight the central role of the venous endothelium in normal development and disease pathogenesis.

Method development and validation of sildenafil, N-desmethylsildenafil and N1,N4-desmethylsildenafil by LC-MS/MS and its application.

This study aimed to develop an LC-MS/MS method for determining sildenafil and its metabolites N-desmethylsildenafil and N1,N4-desmethylsildenafil in human plasma and applying it to a pharmacokinetic study of sildenafil in healthy volunteers. Sildenafil-d8 was used as the internal standard. Plasma samples were pretreated via protein precipitation with acetonitrile. The extractives were then separated on an ACQUITY UPLC BEH C18 (50-mm × 2.1-mm, 1.7-µm) column using gradient elution. The aqueous and organic mobile phases were ammonium formate 2 mM supplemented with 0.1% formic acid in water and acetonitrile, respectively, and the flow rate was 0.3 mL/min. An electrospray ionization source was applied, and multiple reaction monitoring was operated in the positive mode with selective channels at m/z 475.30 â†’ 100.10, 461.20 â†’ 283.30, 483.30 â†’ 108.10, and 449.00 â†’ 283.00 for sildenafil, sildenafil-d8, N-desmethylsildenafil, and N1,N4-desmethylsildenafil, respectively. The linear calibration curves of sildenafil and its metabolites spanned 1.0-1000 ng/mL. The lower limit of quantification was 1.0 ng/mL. The extractive recovery of analytes from the biological matrix was more than 90% and the matrix effect complied with relevant provisions. The intra- and inter-day precisions of sildenafil and its metabolite were <10%. The intra- and inter-day accuracy of sildenafil, N-desmethylsildenafil, and N1,N4-desmethylsildenafil was more than 99%. The method is highly sensitive and selective, and it was successfully applied to the bioequivalence studies of 100-mg sildenafil citrate tablets in 40 healthy Chinese volunteers.

B7-H6 as a Diagnostic Biomarker for Cervical Squamous Cell Carcinoma.

Background: B7-H6, a newly discovered member of the immunoglobulin superfamily, exerts antitumor effects by binding to NKP30 receptor on natural killer cells; it has important clinical implications. Cell surface ectodomain shedding of B7-H6 generates soluble B7-H6 (sB7-H6), which is highly expressed and serves as a valuable biomarker in multiple tumors, but the clinical significance and diagnostic value of B7-H6 in cervical squamous cell carcinoma (CSCC) remains unclear. Objective: To assess the expression and diagnostic value of B7-H6 in CSCC. Methods: In this study, 69 cervical specimens were analyzed for B7-H6 expression: 25 paired CSCC tissues were examined using quantitative real-time polymerase chain reaction, and 24 paraffin-embedded CSCC tissues and 20 normal tissues were analyzed immunohistochemically. Furthermore, plasma samples from 30 CSCC patients and 24 healthy controls were examined using ELISA. Results: B7-H6 mRNA and protein levels were significantly higher in CSCC tissues than in adjacent normal cervical tissues (p < 0.05). Immunohistochemical analysis revealed that high B7-H6 expression correlated with stromal invasion (p = 0.043), lymphovascular space involvement (p = 0.005), lymph node metastasis (p = 0.019), and International Federation of Gynecology and Obstetrics (FIGO) stage (p = 0.002). Moreover, ELISA results demonstrated that the sB7-H6 concentration in peripheral blood was higher in CSCC patients than in healthy controls (p < 0.0001). Notably, at the optimal cutoff point of 0.076 ng/mL, sB7-H6 showed 93.3% sensitivity and 62.5% specificity in the discrimination of CSCC patients from healthy controls. Conclusions: B7-H6 mRNA and protein levels are markedly increased in CSCC tissues and peripheral blood samples, and the B7-H6 level can be used as a biomarker for predicting the severity of CSCC disease and discriminating CSCC patients from healthy controls.

B7 homolog 6 promotes the progression of cervical cancer.

B7 homolog 6 (B7-H6) was recently discovered to act as a co-stimulatory molecule. In particular, the expression of B7-H6 has been found to play an important biological role in several types of tumors. The aim of the present study was to determine the role of B7-H6 in cervical cancer. Immunohistochemistry was used to analyze the expression levels of B7-H6 in cervical precancerous and cancerous tissues. Furthermore, the expression of B7-H6 was knocked down in HeLa cells using short hairpin RNA and the effects of B7-H6 on HeLa cell proliferation, migration and invasion were determined using Cell Counting Kit-8, colony formation, wound healing and Transwell invasion assays, respectively. In addition, flow cytometry was used to analyze the levels of cell apoptosis and the cell cycle distribution. The results of the immunohistochemical staining revealed that the expression levels of B7-H6 were upregulated in cervical lesions. Furthermore, the expression levels of B7-H6 were positively associated with the clinical stage of the cervical lesions. B7-H6 knockdown suppressed the invasive, migratory and proliferative abilities of HeLa cells, and promoted G1 cell cycle arrest and apoptosis. In conclusion, the findings of the present study suggested that B7-H6 may serve as a novel oncogene and may hold promise as a potential therapeutic target for cervical cancer.

LINC01133 and LINC01243 are positively correlated with endometrial carcinoma pathogenesis.

PURPOSE: To characterize the role of two long non-coding RNAs (lncRNAs), LINC01133 and LINC01243, in endometrial carcinoma (EC) pathogenesis. LINC01133 is an lncRNA that has been implicated in many cancers, and LINC01243 is a newly identified lncRNA identified from the NCBI GEO database. METHODS: We studied the effect of LINC01133 and LINC01243 on EC malignancy using siRNA knockdown and real-time quantitative polymerase chain reaction (RT-qPCR), flow cytometry, Annexin V-FITC/propidium iodide double staining, Transwell, and scratch invasion assays in two EC cell lines (Ishikawa and HEC-1-A cells). RESULTS: We first confirmed the partial knockdown of both LINC01133 and LINC01243 expression in Ishikawa and HEC-1-A cells using RT-qPCR. Following confirmation of lncRNA knockdown, we assessed the effect of knockdown on EC malignancy. We observed reduced EC cell proliferation using the CCK-8 assay, as well as cell cycle arrest and increased apoptosis in both EC cell lines. Furthermore, Transwell and scratch invasion assays revealed decreased migration and invasion of the two EC cell lines, respectively. CONCLUSION: We demonstrated that LINC01133 and LINC01243 expression are associated with EC development and progression. Our findings suggest a potential role for these lncRNAs as novel EC biomarkers.