IL-33 Induces Cellular and Exosomal miR-146a Expression as a Feedback Inhibitor of Mast Cell Function.

IL-33 is an inflammatory cytokine that promotes allergic disease by activating group 2 innate lymphoid cells, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. Homeostatic control of IL-33 signaling is poorly understood. Because the IL-33 receptor, ST2, acts via cascades used by the TLR family, similar feedback mechanisms may exist. MicroRNA (miR)-146a is induced by LPS-mediated TLR4 signaling and serves as a feedback inhibitor. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow-derived mast cells (BMMCs). BMMCs transfected with a miR-146a antagonist or derived from miR-146a knockout mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. In vivo, miR-146a expression in plasma exosomes was elevated after i.p. injection of IL-33 in wild-type but not mast cell-deficient KitW-sh/W-sh mice. Finally, KitW-sh/W-sh mice acutely reconstituted with miR-146a knockout BMMCs prior to IL-33 challenge had elevated plasma IL-6 levels compared with littermates receiving wild-type BMMCs. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

Extracellular RNA Sensing Mediates Inflammation and Organ Injury in a Murine Model of Polytrauma.

Severe traumatic injury leads to marked systemic inflammation and multiorgan injury. Endogenous drivers such as extracellular nucleic acid may play a role in mediating innate immune response and the downstream pathogenesis. Here, we explored the role of plasma extracellular RNA (exRNA) and its sensing mechanism in inflammation and organ injury in a murine model of polytrauma. We found that severe polytrauma-bone fracture, muscle crush injury, and bowel ischemia-induced a marked increase in plasma exRNA, systemic inflammation, and multiorgan injury in mice. Plasma RNA profiling with RNA sequencing in mice and humans revealed a dominant presence of miRNAs and marked differential expression of numerous miRNAs after severe trauma. Plasma exRNA isolated from trauma mice induced a dose-dependent cytokine production in macrophages, which was almost abolished in TLR7-deficient cells but unchanged in TLR3-deficient cells. Moreover, RNase or specific miRNA inhibitors against the selected proinflammatory miRNAs (i.e., miR-7a-5p, miR-142, let-7j, miR-802, and miR-146a-5p) abolished or attenuated trauma plasma exRNA-induced cytokine production, respectively. Bioinformatic analyses of a group of miRNAs based on cytokine readouts revealed that high uridine abundance (>40%) is a reliable predictor in miRNA mimic-induced cytokine and complement production. Finally, compared with the wild-type, TLR7-knockout mice had attenuated plasma cytokine storm and reduced lung and hepatic injury after polytrauma. These data suggest that endogenous plasma exRNA of severely injured mice and ex-miRNAs with high uridine abundance prove to be highly proinflammatory. TLR7 sensing of plasma exRNA and ex-miRNAs activates innate immune responses and plays a role in inflammation and organ injury after trauma.

Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations.

Hotspot driver mutations presented by human leukocyte antigens might be recognized by anti-tumor T cells. Based on their advantages of tumor-specificity and immunogenicity, neoantigens derived from hotspot mutations, such as PIK3CAH1047L, may serve as emerging targets for cancer immunotherapies. NetMHCpan V4.1 was utilized for predicting neoepitopes of PIK3CA hotspot mutation. Using in vitro stimulation, antigen-specific T cells targeting the HLA-A*11:01-restricted PIK3CA mutation were isolated from healthy donor-derived peripheral blood mononuclear cells. T cell receptors (TCRs) were cloned using single-cell PCR and sequencing. Their functionality was assessed through T cell activation markers, cytokine production and cytotoxic response to cancer cell lines pulsed with peptides or transduced genes of mutant PIK3CA. Immunogenic mutant antigens from PIK3CA and their corresponding CD8+ T cells were identified. These PIK3CA mutation-specific CD8+ T cells were subsequently enriched, and their TCRs were isolated. The TCR clones exhibited mutation-specific and HLA-restricted reactivity, demonstrating varying degrees of functional avidity. Identified TCR genes were transferred into CD8+ Jurkat cells and primary T cells deficient of endogenous TCRs. TCR-expressing cells demonstrated specific recognition and reactivity against the PIK3CAH1047L peptide presented by HLA-A*11:01-expressing K562 cells. Furthermore, mutation-specific TCR-T cells demonstrated an elevation in cytokine production and profound cytotoxic effects against HLA-A*11:01+ malignant cell lines harboring PIK3CAH1047L. Our data demonstrate the immunogenicity of an HLA-A*11:01-restricted PIK3CA hotspot mutation and its targeting therapeutic potential, together with promising candidates of TCR-T cell therapy.

M2 Macrophage Membrane-Camouflaged Fe3 O4 -Cy7 Nanoparticles with Reduced Immunogenicity for Targeted NIR/MR Imaging of Atherosclerosis.

Atherosclerosis （AS） is the primary reason behind cardiovascular diseases, leading to approximately one-third of global deaths. Developing a novel multi-model probe to detect AS is urgently required. Macrophages are the primary cells from which AS genesis occurs. Utilizing natural macrophage membranes coated on the surface of nanoparticles is an efficient delivery method to target plaque sites. Herein, Fe3 O4 -Cy7 nanoparticles (Fe3 O4 -Cy7 NPs), functionalized using an M2 macrophage membrane and a liposome extruder for Near-infrared fluorescence and Magnetic resonance imaging, are synthesized. These macrophage membrane-coated nanoparticles (Fe3 O4 @M2 NPs) enhance the recognition and uptake using active macrophages. Moreover, they inhibit uptake using inactive macrophages and human coronary artery endothelial cells. The macrophage membrane-coated nanoparticles (Fe3 O4 @M0 NPs, Fe3 O4 @M1 NPs, Fe3 O4 @M2 NPs) can target specific sites depending on the macrophage membrane type and are related to C-C chemofactor receptor type 2 protein content. Moreover, Fe3 O4 @M2 NPs demonstrate excellent biosafety in vivo after injection, showing a significantly higher Fe concentration in the blood than Fe3 O4 -Cy7 NPs. Therefore, Fe3 O4 @M2 NPs effectively retain the physicochemical properties of nanoparticles and depict reduced immunological response in blood circulation. These NPs mainly reveal enhanced targeting imaging capability for atherosclerotic plaque lesions.

Sepsis-Induced Coagulopathy: A Comprehensive Narrative Review of Pathophysiology, Clinical Presentation, Diagnosis, and Management Strategies.

Physiological hemostasis is a balance between pro- and anticoagulant pathways, and in sepsis, this equilibrium is disturbed, resulting in systemic thrombin generation, impaired anticoagulant activity, and suppression of fibrinolysis, a condition termed sepsis-induced coagulopathy (SIC). SIC is a common complication, being present in 24% of patients with sepsis and 66% of patients with septic shock, and is often associated with poor clinical outcomes and high mortality. 1 , 2 Recent preclinical and clinical studies have generated new insights into the molecular pathogenesis of SIC. In this article, we analyze the complex pathophysiology of SIC with a focus on the role of procoagulant innate immune signaling in hemostatic activation--tissue factor production, thrombin generation, endotheliopathy, and impaired antithrombotic functions. We also review clinical presentations of SIC, the diagnostic scoring system and laboratory tests, the current standard of care, and clinical trials evaluating the efficacies of anticoagulant therapies.

Exploration of the Dynamic Variations of the Characteristic Constituents and the Degradation Products of Catalpol during the Process of Radix Rehmanniae.

Radix Rehmanniae (RR), a famous traditional Chinese medicine (TCM) widely employed in nourishing Yin and invigorating the kidney, has three common processing forms in clinical practice, including fresh Radix Rehmanniae (FRR), raw Radix Rehmanniae (RRR), and processed Radix Rehmanniae (PRR). However, until now, there has been less exploration of the dynamic variations in the characteristic constituents and degradation products of catalpol as a representative iridoid glycoside with the highest content in RR during the process from FRR to PRR. In this study, an ultra-performance liquid chromatography coupled with photodiode array detector (UPLC-PDA) method was successfully established for the simultaneous determination of ten characteristic components to explore their dynamic variations in different processed products of RR. Among them, iridoid glycosides, especially catalpol, exhibited a sharp decrease from RRR to PRR. Then, three degradation products of catalpol were detected under simulated processing conditions (100 °C, pH 4.8 acetate buffer solution), which were isolated and identified as jiofuraldehyde, cataldehyde, and norviburtinal, respectively. Cataldehyde was first reported as a new compound. Moreover, the specificity of norviburtinal in self-made PRR samples was discovered and validated, which was further confirmed by testing in commercially available PRR samples. In conclusion, our study revealed the decrease in iridoid glycosides and the production of new degradation substances during the process from FRR to PRR, which is critical for unveiling the processing mechanism of RR.

[Analysis of the results for genetic disease screening among 1 000 newborns from Huzhou].

OBJECTIVE: To analyze the types and distribution of pathogenic variants for neonatal genetic diseases in Huzhou, Zhejiang Province. METHODS: One thousand neonates (48 ~ 42 h after birth) born to Huzhou region were selected as the study subjects. Dry blood spot samples were collected from the newborns, and targeted capture high-throughput sequencing was carried out for pathogenic genes underlying 542 inherited diseases. Candidate variants were verified by Sanger sequencing. RESULTS: Among the 1 000 newborns, the male to female ratio was 1.02 : 1.00. No pathogenic variants were detected in 253 cases, whilst 747 cases were found to carry at least one pathogenic variant, which yielded a carrier rate of 74.7%. The most frequently involved pathogenic gene was FLG, followed by GJB2, UGT1A1, USH2A and DUOX2. The variants were classified as homozygous, compound heterozygous, and hemizygous variants. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), 213 neonates were verified to have carried pathogenic and/or likely pathogenic variants, with a positive rate of 21.3%. The most commonly involved genes had included UGT1A1, FLG, GJB2, MEFV and G6PD. CONCLUSION: Newborn screening based on high-throughput sequencing technology can expand the scope of screening and improve the positive predictive value. Genetic counseling based on the results can improve the patients' medical care and reduce neonatal mortality and childhood morbidity, while provide assistance to family members' health management and reproductive decisions.

Identification and characterization of an α-1,3 mannosidase from Elizabethkingia meningoseptica and its potential attenuation impact on allergy associated with cross-reactive carbohydrate determinant.

Core α-1,3 mannose is structurally near the core xylose and core fucose on core pentasaccharide from plant and insect glycoproteins. Mannosidase is a useful tool for characterization the role of core α-1,3 mannose in the composition of glycan related epitope, especially for those epitopes in which core xylose and core fucose are involved. Through functional genomic analysis, we identified a glycoprotein α-1,3 mannosidase and named it MA3. We used MA3 to treat allergen horseradish peroxidase (HRP) and phospholipase A2 (PLA2) separately. The results showed that after MA3 removed α-1,3 mannose on HRP, the reactivity of HRP with anti-core xylose polyclonal antibody almost disappeared. And the reactivity of MA3-treated PLA2 with anti-core fucose polyclonal antibody decreased partially. In addition, when PLA2 was conducted enzyme digestion by MA3, the reactivity between PLA2 and allergic patients' sera diminished. These results demonstrated that α-1,3 mannose was an critical component of glycan related epitope.

LncRNA AC142119.1 facilitates the progression of neuroblastoma by epigenetically initiating the transcription of MYCN.

BACKGROUND: Oncogene MYCN is closely related with malignant progression and poor prognosis of neuroblastoma (NB). Recently, long non-coding RNAs (lncRNAs) have been recognized as crucial regulators in various cancers. However, whether lncRNAs contribute to the overexpression of MYCN in NB is unclear. METHODS: Microarray analysis were applied to analyze the differentially expressed lncRNAs between MYCN-amplified and MYCN-non-amplified NB cell lines. Bioinformatic analyses were utilized to identify lncRNAs nearby MYCN locus. qRT-PCR was used to detect the expression level of lncRNA AC142119.1 in NB cell lines and tissues. Gain- and loss-of-function assays were conducted to investigate the biological effect of AC142119.1 in NB. Fluorescence in situ hybridization, RNA pull-down, RNA immunoprecipitation, mass spectrometry, RNA electrophoretic mobility shift, chromatin immunoprecipitation and chromatin isolation by RNA purification assays were performed to validate the interaction between AC142119.1 and WDR5 protein as well as MYCN promoter. RESULTS: AC142119.1 was significantly elevated in NB tissues with MYCN amplification, advanced INSS stage and high risk, and associated with poor survival of NB patients. Moreover, enforced expression of AC142119.1 reinforced the proliferation of NB cells in vitro and in vivo. Additionally, AC142119.1 specifically recruited WDR5 protein to interact with MYCN promoter, further initiating the transcription of MYCN and accelerating NB progression. CONCLUSIONS: We identified a novel lncRNA AC142119.1, which promoted the progression of NB through epigenetically initiating the transcription of MYCN via interacting with both WDR5 protein and the promoter of MYCN, indicating that AC142119.1 might be a potential diagnostic biomarker and therapeutic target for NB.

Community Mitigation Strategies, Mobility, and COVID-19 Incidence Across Three Waves in the United States in 2020.

BACKGROUND: Summarizing the impact of community-based mitigation strategies and mobility on COVID-19 infections throughout the pandemic is critical for informing responses and future infectious disease outbreaks. Here, we employed time-series analyses to empirically investigate the relationships between mitigation strategies and mobility on COVID-19 incident cases across US states during the first three waves of infections. METHODS: We linked data on daily COVID-19 incidence by US state from March to December 2020 with the stringency index, a well-known index capturing the strictness of mitigation strategies, and the trip ratio, which measures the ratio of the number of trips taken per day compared with the same day in 2019. We utilized multilevel models to determine the relative impacts of policy stringency and the trip ratio on COVID-19 cumulative incidence and the effective reproduction number. We stratified analyses by three waves of infections. RESULTS: Every five-point increase in the stringency index was associated with 2.89% (95% confidence interval = 1.52, 4.26%) and 5.01% (3.02, 6.95%) reductions in COVID-19 incidence for the first and third waves, respectively. Reducing the number of trips taken by 50% compared with the same time in 2019 was associated with a 16.2% (-0.07, 35.2%) decline in COVID-19 incidence at the state level during the second wave and 19.3% (2.30, 39.0%) during the third wave. CONCLUSIONS: Mitigation strategies and reductions in mobility are associated with marked health gains through the reduction of COVID-19 infections, but we estimate variable impacts depending on policy stringency and levels of adherence.

Research progress on the mechanism of Treponema pallidum breaking through placental barrier.

Congenital syphilis, a significant cause of fetal mortality worldwide, is a congenital infectious disease instigated by the vertical transmission of Treponema pallidum during pregnancy. Clinical manifestations include preterm delivery, stillbirth, neonatal skin lesions, skeletal abnormalities, and central nervous system aberrations. The ongoing increase in the incidence of congenital syphilis, coupled with complexities in diagnosis, necessitates a detailed understanding of its pathogenesis for the development of improved diagnostic approaches, and to interrupt the route of vertical transmission. Drawing from the broader body of research associated with vertical transmission pathogens, we aim to clarify the potential mechanisms by which Treponema pallidum breaches the placental barrier to infect the fetus.

TRAF7 inhibits glycolysis to potentiate growth inhibition and apoptosis of myeloid leukemia cells via regulating the KLF2-PFKFB3 axis.

Tumor necrosis factor receptor-related factor 7 (TRAF7) can regulate cell differentiation and apoptosis, but its specific functional mechanism in the pathological process of acute myeloid leukemia (AML) closely related to differentiation and apoptosis disorders is largely unclear. In this study, TRAF7 was found to be lowly expressed in AML patients and a variety of myeloid leukemia cells. TRAF7 was overexpressed in AML Molm-13 and chronic myeloid leukemia (CML) K562 cells by transfection with pcDNA3.1-TRAF7. CCK-8 assay and flow cytometry analysis showed that TRAF7 overexpression induced growth inhibition and apoptosis in K562 and Molm-13 cells. Measurements of glucose and lactate suggested that TRAF7 overexpression impaired glycolysis of K562 and Molm-13 cells. Cell cycle analysis indicated that most of K562 and Molm-13 cells were captured in G0/G1 phase by TRAF7 overexpression. PCR and western blot assay revealed that TRAF7 increased Kruppel-like factor 2 (KLF2) expression but decreased 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression in AML cells. KLF2 knockdown can counteract TRAF7-triggered PFKFB3 inhibition, and abolish TRAF7-mediated glycolysis inhibition and cell cycle arrest. KLF2 knockdown or PFKFB3 overexpression both can partially neutralize TRAF7-induced growth inhibition and apoptosis of K562 and Molm-13 cells. Moreover, Lv-TRAF7 decreased human CD45+ cells in mouse peripheral blood in the xenograft mice established by NOD/SCID mice. Taken together, TRAF7 exerts anti-leukemia effects by impairing glycolysis and cell cycle progression of myeloid leukemia cells via modulating the KLF2-PFKFB3 axis.

A reliable and high throughput HPLC-HRMS method for the rapid screening of ß-thalassemia and hemoglobinopathy in dried blood spots.

OBJECTIVES: Traditional methods for ß-thalassemia screening usually rely on the structural integrity of hemoglobin (Hb), which can be affected by the hemolysis of red blood cells and Hb degradation. Here, we aim to develop a reliable and high throughput method for rapid detection of ß-thalassemia using dried blood spots (DBS). METHODS: Hb components were extracted from a disc (3.2 mm diameter) punched from the DBS samples and digested by trypsin to produce a series of Hb-specific peptides. An analytical system combining high-resolution mass spectrometry and high-performance liquid chromatography was used for biomarker selection. The selected marker peptides were used to calculate delta/beta (δ/ß) and beta-mutated/beta (ßM/ß) globin ratios for disease evaluation. RESULTS: Totally, 699 patients and 629 normal individuals, aged 3 days to 89 years, were recruited for method construction. Method assessment showed both the inter-assay and intra-assay relative standard deviation values were less than 10.8%, and the limits of quantitation for the proteo-specific peptides were quite low (1.0-5.0 µg/L). No appreciable matrix effects or carryover rates were observed. The extraction recoveries ranged from 93.8 to 128.7%, and the method was shown to be stable even when the samples were stored for 24 days. Prospective applications of this method in 909 participants also indicated good performance with a sensitivity of 100% and a specificity of 99.6%. CONCLUSIONS: We have developed a fast, high throughput and reliable method for screening of ß-thalassemia and hemoglobinopathy in children and adults, which is expected to be used as a first-line screening assay.

Identification of linear scaling relationships in polysulfide conversion on α-In2Se3-supported single-atom catalysts.

Developing highly active single-atom catalysts (SACs) for suppressing the shuttle effect and enhancing the kinetics of polysulfide conversion is regarded as an important approach to improve the performance of Li-S batteries. However, the adsorption behaviors of polysulfides and the catalytic properties of host materials remain obscure due to the lack of mechanistic understanding of the structure-performance relationship. Here, we identify that the adsorption energies of polysulfides on 3d transition-metal atoms supported by two-dimensional α-In2Se3 with downward polarization (TM@In2Se3) are highly correlated with the d-band centers of the TM atoms. Introduction of the TM atoms on the α-In2Se3 surface improves the electrical conductivity and meanwhile, significantly enhances the adsorption strength of polysulfides and suppresses the shuttle effect. A mechanistic study of polysulfide conversion on TM@In2Se3 shows that the Li2S2 dissociation is the potential-determining step with low activation energies, indicating that TM@In2Se3 can accelerate the kinetics of polysulfide conversion. Electronic structure analysis shows that the kinetics of the potential-determining step on TM@In2Se3 is related to the TM-S interaction in Li2S2-adsorbed TM@In2Se3. A linear scaling relationship between activation energy and the integrated crystal orbital Hamilton population of TM-S in the potential-determining step on TM@In2Se3 is identified. Based on the evaluation of stability, conductivity and activity, we concluded that Ti@In2Se3, V@In2Se3, and Fe@In2Se3 are the promising cathode materials for Li-S batteries. Our findings provide a fundamental understanding of the intrinsic link between the electronic structure and catalytic activity for polysulfide conversion and pave a way for the rational design of SAC-based cathodes for Li-S batteries.

Diagnostic value of SHOX2, RASSF1A gene methylation combined with CEA level detection in malignant pleural effusion.

AIM: To investigate the diagnostic value of combined detection of SHOX2 and RASSF1A gene methylation with carcinoembryonic antigen (CEA) level in diagnosing malignant pleural effusion. METHODS: Between March 2020 and December 2021, we enrolled 68 patients with pleural effusion admitted to the Department of Respiratory and critical care medicine of Foshan Second People's Hospital. The study group included 35 cases of malignant pleural effusion and 33 cases of benign pleural effusion. Methylation of the short homeobox 2 genes (SHOX2) and RAS-related region family 1A gene (RASSF1A) in pleural effusion samples were detected by real-time fluorescence quantitative PCR, and the level of carcinoembryonic antigen (CEA) in pleural effusion samples was detected by immune flow cytometry fluorescence quantitative chemiluminescence. RESULTS: SHOX2 or RASSF1A gene methylation was detected in 5 cases in the benign pleural effusion group and 25 patients in the malignant pleural effusion group. The positive rate of SHOX2 or RASSF1A gene methylation in the malignant pleural effusion group was significantly higher than in the benign pleural effusion group (71.4% vs. 15.2%, P < 0.01). Positive CEA (CEA > 5 ng/m) was detected in 1 case in the benign pleural effusion group and 26 patients in the malignant pleural effusion group. The CEA-positive rate in the malignant pleural effusion group was significantly higher than in the benign pleural effusion group (74.3% vs. 3%, P < 0.01). When SHOX2 and RASSF1A gene methylation was combined with CEA detection, 6 cases were positive in the benign pleural effusion group, and 31 patients were positive in the malignant pleural effusion group. The positive rate of combined detection in the malignant pleural effusion group was significantly higher than in the benign pleural effusion group (88.6% vs. 18.2%, P < 0.01). The sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and Youden's index of SHOX2, RASSF1A gene methylation combined with CEA in diagnosing malignant pleural effusion were 88.6%, 81.8%, 85.3%, 83.8%, 87.1% and 0.7 respectively. CONCLUSION: The combined detection of SHOX2 and RASSF1A gene methylation with CEA level in pleural effusion has a high diagnostic value for malignant pleural effusion.

Quantitative muscle ultrasound in children with Duchenne muscular dystrophy: Comparing to magnetic resonance imaging.

OBJECTIVE: There is a need today for favorable biomarkers to follow up on the disease progression and therapeutic response in patients with Duchenne muscular dystrophy (DMD). This study evaluates whether quantitative muscle ultrasound (QMUS) or magnetic resonance imaging (MRI) is more suitable for the assessment of DMD in China. METHODS: Thirty-six boys with DMD, who were treated with prednisone from baseline to month 12, were enrolled in this longitudinal, observational cohort study. Muscle thickness and echo intensity on QMUS and T1-weighted MRI grading were measured in the right rectus femoris. RESULTS: Scores for muscle thickness and echo intensity in QMUS and T1-weighted MRI grading showed significant correlations with the clinical characteristics of muscle strength, timed testing, and quality of life (p < 0.05). Scores for muscle thickness and echo intensity on QMUS also showed good correlations with T1-weighted MRI grading (p < 0.05). However, 15 of 36 boys with DMD did not undergo MRI examinations for various reasons. CONCLUSIONS: QMUS and MRI can be used as biomarkers for tracking DMD to some extent. Both have strengths and weaknesses and the specific needs and goals of the clinical or research project are what make one preferable to the other.

An anatomical study of the tibial nerve branches innervating the posterior tibial artery.

The arteries of the lower limbs are innervated by vascular branches (VBs) originating from the lumbar sympathetic trunk and branches of the spinal nerve. Although lumbar sympathectomy is used to treat nonreconstructive critical lower limb ischemia (CLLI), it has limited long-term effects. In addition, the anatomical structure of tibial nerve (TN) VBs remain incompletely understood. This study aimed to clarify their anatomy and better inform the surgical approach for nonreconstructive CLLI. Thirty-six adult cadavers were dissected under surgical microscopy to observe the patterns and origin points of VBs under direct vision. The calves were anatomically divided into five equal segments, and the number of VB origin points found in each was expressed as a proportion of the total found in the whole calf. Immunofluorescence staining was used to identify the sympathetic nerve fibers of the VBs. Our results showed that the TN gave off 3-4 VBs to innervate the posterior tibial artery (PTA), and the distances between VBs origin points and the medial tibial condyle were: 24.7 ± 16.3 mm, 91.7 ± 66.1 mm, 199.6 ± 52.0 mm, 231.7 ± 38.5 mm, respectively. They were mainly located in the first (40.46%) and fourth (31.68%) calf segments, and immunofluorescence staining showed that they contained tyrosine hydroxylase-positive sympathetic nerve fibers. These findings indicate that the TN gives off VBs to innervate the PTA and that these contain sympathetic nerve fibers. Therefore, these VBs may need to be cut to surgically treat nonreconstructable CLLI.

TLR7 Mediates Acute Respiratory Distress Syndrome in Sepsis by Sensing Extracellular miR-146a.

TLR7 (Toll-like receptor 7), the sensor for single-stranded RNA, contributes to systemic inflammation and mortality in murine polymicrobial sepsis. Recent studies show that extracellular miR-146a-5p serves as a TLR7 ligand and plays an important role in regulating host innate immunity. However, the role of miR-146a-5p and TLR7 signaling in pulmonary inflammation, endothelial activation, and sepsis-associated acute respiratory distress syndrome remains unclear. Here, we show that intratracheal administration of exogenous miR-146a-5p in mice evokes lung inflammation, activates endothelium, and increases endothelial permeability via TLR7-dependent mechanisms. TLR7 deficiency attenuates pulmonary barrier dysfunction and reduces lung inflammatory response in a murine sepsis model. Moreover, the impact of miR-146a-5p-TLR7 signaling on endothelial activation appears to be a secondary effect because TLR7 is undetectable in the human pulmonary artery and microvascular endothelial cells (ECs), which show no response to direct miR-146a-5p treatment in vitro. Both conditioned media of miR-146a-5p-treated macrophages (Mϕ) and septic sera of wild-type mice induce a marked EC barrier disruption in vitro, whereas Mϕ conditioned media or septic sera of TLR7-/- mice do not exhibit such effect. Cytokine array and pathway enrichment analysis of the Mϕ conditioned media and septic sera identify TNFα (tumor necrosis factor α) as the main downstream effector of miR-146a-5p-TLR7 signaling responsible for the EC barrier dysfunction, which is further supported by neutralizing anti-TNFα antibody intervention. Together, these data demonstrate that TLR7 activation elicits pulmonary inflammation and endothelial barrier disruption by sensing extracellular miR-146a-5p and contributes to sepsis-associated acute respiratory distress syndrome.

Effect of Advanced Oxidation Protein Products (AOPPs) and aging on the osteoclast differentiation of Myeloid-Derived Suppressor Cells (MDSCs) and its preliminary mechanism.

To verify the osteoclast differentiation ability of MDSCs from mice of different ages and explore the effect of AOPPs on the osteoclast differentiation of bone marrow MDSCs. Bone marrow cells from C57BL/6 (a.k.a C57) mice of different ages were subjected to flow cytometry, and CD11b+Ly6C+Ly6G+ MDSCs were sorted out. After induction of osteoclast differentiation, these cells were subjected to tartrate-resistant acid phosphatase (TRAP) and F-actin. MDSCs from bone marrows of old mice were injected into the tibial medullary cavity of young mice. One week later, the bone marrows were subjected to histological examination, TRAP, and cell count. MDSCs from bone marrows of old mice were sorted for induction of osteoclast differentiation, intervened with reactive oxygen species (ROS) scavenger, inducible nitric oxide synthase (iNOS) inhibitor, and nitric oxide (NO) scavenger, and then subjected to TRAP. 8-weeks-old C57 mice were injected with the same concentrations of either AOPPs or mouse serum albumin (MSA). Four weeks later, MDSCs from bone marrows were sorted and subjected to induction of osteoclast differentiation, followed by IHC staining and TRAP. MDSCs of 8-weeks-old C57 mice were extracted and subjected to in vitro induction of osteoclast differentiation with different concentrations of AOPPs, followed by TRAP training. The number of MDSCs in the bone marrows of old mice was significantly higher than that in young mice. MDSCs from bone marrows of old mice differentiated into large multinucleated TRAP+ osteoclasts, which were significantly different from those in the middle-aged and young mice in terms of cell quantity and morphology. The actin rings formed in the differentiated osteoclasts from MDSCs of bone marrows were densely distributed in the whole field of view, which were significantly denser than those in the middle-aged and young mice. After injection of MDSCs of old mice, the number of TRAP + osteoclasts in the tibial medullary cavity of young mice was significantly increased. NO inhibitor can significantly inhibit the osteoclast differentiation capacity of MDSCs from bone marrows of old mice. In vivo treatment with AOPPs significantly increased the proportion of MDSCs in the bone marrow, which is up to 55.2%. After injection of AOPPs in 8-week-old mice and induction of osteoclast differentiation from the MDSCs, the ratios of CD11b+ and Gr1+ cells were significantly higher than that in the control and MSA groups but was not significantly different from that in the 15-month-old mice. Upon in vitro treatment with different concentrations of AOPPs, the MDSCs did not show any sign of osteoclast differentiation. MDSCs can directly undergo osteoclast differentiation, the capacity of which is stronger in MDSCs of bone marrows of old mice; the NO pathway is a potential mechanism underlying this phenomenon. In vivo but not in vitro AOPPs treatment can induce osteoclast differentiation of MDSCs, indicating there might be other factors in the body that can interact with AOPPs to induce osteoclast differentiation of MDSCs.

Hypoxic preconditioning improves the survival and pro-angiogenic capacity of transplanted human umbilical cord mesenchymal stem cells via HIF-1α signaling in a rat model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a serious chronic respiratory disease that predominates in the neonatal period. Currently, efficacious and effective specific treatments are lacking. Mesenchymal stem cells (MSCs) transplantation has emerged as a promising option for treating BPD. However, the lower cell survival rate limits its therapeutic efficacy. Hypoxic preconditioning is a direct and effective strategy for promoting MSCs survival, proliferation, and paracrine secretion in the recipient after transplantation, which is greatly important to tissue engineering. We investigated whether hypoxia-pretreated MSCs (HPMSCs) confer superior benefit in an experimental BPD rat model. Neonatal Sprague-Dawley rats were exposed to 80-85% O2 for 14 days. Before tracheal transplantation, the MSCs were pretreated for 48 h with deferoxamine, a chemical hypoxia-mimicking agent. In vitro, the HPMSCs reduced the apoptosis rare, caspase-3 expression, and reactive oxygen species (ROS) generation and promoted proliferation, hypoxia inducible factor-1α (HIF-1α) expression, VEGF secretion, and human umbilical vein endothelial cell tube formation (p < 0.05). In vivo, the HPMSCs restored alveolar structure and lung function, ameliorated pulmonary hypertension, increased vessel density in the BPD rat model (p < 0.05). This work demonstrates for the first time that HPMSCs could have a markedly improved therapeutic effect in BPD, presenting a new potential strategy for the clinical implementation of stem cell biotechnology.