Increased gene dosage of RFWD2 causes autistic-like behaviors and aberrant synaptic formation and function in mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interactions, communication deficits and repetitive behaviors. A study of autistic human subjects has identified RFWD2 as a susceptibility gene for autism, and autistic patients have 3 copies of the RFWD2 gene. The role of RFWD2 as an E3 ligase in neuronal functions, and its contribution to the pathophysiology of ASD, remain unknown. We generated RFWD2 knockin mice to model the human autistic condition of high gene dosage of RFWD2. We found that heterozygous knockin (Rfwd2+/-) male mice exhibited the core symptoms of autism. Rfwd2+/- male mice showed deficits in social interaction and communication, increased repetitive and anxiety-like behavior, and spatial memory deficits, whereas Rfwd2+/- female mice showed subtle deficits in social communication and spatial memory but were normal in anxiety-like, repetitive, and social behaviors. These autistic-like behaviors in males were accompanied by a reduction in dendritic spine density and abnormal synaptic function on layer II/III pyramidal neurons in the prelimbic area of the medial prefrontal cortex (mPFC), as well as decreased expression of synaptic proteins. Impaired social behaviors in Rfwd2+/- male mice were rescued by the expression of ETV5, one of the major substrates of RFWD2, in the mPFC. These findings indicate an important role of RFWD2 in the pathogenesis of autism.

4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) alleviates lung injury by inhibiting SIRT6-HIF-1α signaling pathway activation through the upregulation of miR-212-5p expression.

OBJECTIVE: Obstructive sleep apnea is closely related to oxidative stress. 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) can scavenge reactive oxygen species (ROS) and ameliorate oxidative damage in the body. The mechanism by which Tempol alleviates chronic intermittent hypoxia-induced lung injury has rarely been reported. This study aimed to confirm the molecular mechanism by which Tempol alleviates lung injury. METHODS: The levels of miR-212-5p and Sirtuin 6 (SIRT6) in injured lungs were analyzed using bioinformatics. In vitro, intermittent hypoxia (IH) treatment induced hypoxia in BEAS-2B cells and we established a model of chronic intermittent hypoxia (CIH) in mouse using a programmed hypoxia chamber. We used HE staining to observe the morphology of lung tissue, and the changes in lung fibers were observed by Masson staining. The levels of inflammatory factors in mouse serum were detected by ELISA, and the levels of the oxidative stress indicators GSH, MDA, SOD and ROS were detected using commercially available kits. Moreover, a real-time qPCR assay was used to detect miR-212-5p expression, and Western blotting was used to detect the levels of SIRT6, HIF-1α and apoptosis-related proteins. CCK-8 was used to detect cell proliferation. Subsequently, we used flow cytometry to detect cell apoptosis. Dual-luciferase gene reporters determine the on-target binding relationship of miR-212-5p and SIRT6. RESULTS: SIRT6 was highly expressed in CIH-induced lung injury, as shown by bioinformatics analysis; however, miR-212-5p expression was decreased. Tempol promoted miR-212-5p expression, and the levels of SIRT6 and HIF-1α were inhibited. In BEAS-2B cells, Tempol also increased proliferation, inhibited apoptosis and inhibited oxidative stress in BEAS-2B cells under IH conditions. In BEAS-2B cells, these effects of Tempol were reversed after transfection with an miR-212-5p inhibitor. miR-212-5p targeted and negatively regulated the level of SIRT6 and overexpression of SIRT6 effectively reversed the enhanced influence of the miR-212-5p mimic on Tempol's antioxidant activity. Tempol effectively ameliorated lung injury in CIH mice and inhibited collagen deposition and inflammatory cell infiltration. Likewise, the therapeutic effect of Tempol could be effectively reversed by interference with the miR-212-5p inhibitor. CONCLUSION: Inhibition of the SIRT6-HIF-1α signaling pathway could promote the effect of Tempol by upregulating the level of miR-212-5p, thereby alleviating the occurrence of lung injury and providing a new underlying target for the treatment of lung injury.

Tempol alleviates acute lung injury by affecting glutathione synthesis through Nrf2 and inhibiting ferroptosis in lung epithelial cells.

As a life-threatening disease, acute lung injury (ALI) may progress to chronic pulmonary fibrosis. For the treatment of lung injury, Tempol is a superoxide dismutase mimetic and intracellular redox agent that can be a potential drug. This study investigated the regulatory mechanism of Tempol in the treatment of ALI. A mouse model of ALI was established, and HE staining was used to examine histomorphology. The CCK-8 assay was used to measure cell viability, and oxidative stress was assessed by corresponding kits. Flow cytometry and dichlorodihydrofluorescein diacetate staining assays were used to detect reactive oxygen species (ROS) levels. Protein expression levels were measured by Western blot analysis and ELISA. Pulmonary vascular permeability was used to measure the lung wet/dry weight ratio. The level of oxidative stress was increased in ALI mice, and the level of ferroptosis was upregulated. Tempol inhibited this effect and alleviated ALI. The administration of Tempol alleviated the pathological changes in ALI, inhibited pulmonary vascular permeability, and improved lung injury in ALI mice. The upregulation of genes essential for glutathione (GSH) metabolism induced by lipopolysaccharide (LPS) was inhibited by Tempol. In addition, nuclear factor-related factor 2 (Nrf2) is activated by Tempol therapy to regulate the de novo synthesis pathway of GSH, thereby alleviating LPS-induced lung epithelial cell damage. The results showed that Tempol alleviated ALI by activating the Nrf2 pathway to inhibit oxidative stress and ferroptosis in lung epithelial cells. In conclusion, this study demonstrates that Tempol alleviates ALI by inhibiting ferroptosis in lung epithelial cells through the effect of Nrf2 on GSH synthesis.

Chronic intermittent hypoxia-induced oxidative stress activates TRB3 and phosphorylated JNK to mediate insulin resistance and cell apoptosis in the pancreas.

This study explores the potential mechanisms of obstructive sleep apnoea (OSA) complicates type 2 diabetes mellitus (T2DM) by which chronic intermittent hypoxia (CIH) induces insulin resistance and cell apoptosis in the pancreas through oxidative stress. Four- and eight-week CIH rat models were established, and Tempol (100 mg/kg/d), was used as an oxidative stress inhibitor. This study included five groups: 4-week CIH, 4-week CIH-Tempol, 8-week CIH, 8-week CIH-Tempol and normal control (NC) groups. Fasting blood glucose and insulin levels were measured in the serum. The expression levels of 8-hidroxy-2-deoxyguanosine (8-OHdG), tribbles homologue 3 (TRB3), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), insulin receptor substrate-1 (IRS-1), phosphorylated IRS-1 (Ser307) (p-IRS-1ser307 ), protein kinase B (AKT), phosphorylated AKT (Ser473) (p-AKTser473 ), B cell lymphoma protein-2 (Bcl-2), cleaved-caspase-3 (Cl-caspase-3), and the islet cell apoptosis were detected in the pancreas. CIH induced oxidative stress in the pancreas. Compared with that in the NC group and CIH-Tempol groups individually, the homeostasis model assessment of insulin resistance (HOMA-IR) and apoptosis of islet cells was increased in the CIH groups. CIH-induced oxidative stress increased the expression of p-IRS-1Ser307 and decreased the expression of p-AKTSer473 . The expression levels of TRB3 and p-JNK were higher in the CIH groups than in both the CIH-Tempol and NC groups. Meanwhile, the expressions of Cl-caspase-3 and Bcl-2 were upregulated and downregulated, respectively, in the CIH groups. Hence, the present study demonstrated that CIH-induced oxidative stress might not only induce insulin resistance but also islet cell apoptosis in the pancreas through TRB3 and p-JNK.

Effect of Preoperative Nutritional Status on Postoperative Functional Recovery of Hip Joint in Elderly Patients with Intertrochanteric Fractures.

Objective: The state of nutrition of senior patients with intertrochanteric fracture of femur before operation affects the patients' tolerance to the operation,the body recovery, healing of the wound and clinical prognosis. For these patients, the poor state of nutrition may lengthen the time of being in hospital, leading to poor hip recovery and clinical outcome. But currently, the relationship between functional recovery of hip joint after operation of intertrochanteric fracture in elderly patients and camp condition has not been reported. To investigate the effect of preoperative nutritional status on postoperative recovery of hip joint function in elderly patients with intertrochanteric fractures. Methods: Retrospective analysis was performed on the data of 96 elderly patients with intertrochanteric fracture of the femur treated with closed reduction PFNAfrom January 2021 to January 2022 in Dongying People's Hospital Trauma Orthopedics Department. There were 36 male patients and 60 female patients aged from 65 to 92.The patients were divided into the normal nutrition group (GNRI ≥ 92, n = 46) and the malnutrition group (GNRI < 92, n = 50). The general clinical data, time from injury to operation, intraoperative and postoperative allogeneic blood transfusion rate, postoperative complication rate, postoperative mortality 1 year, and Harris hip function score at 3, 6, 9 months and the last follow-up were compared between the two groups. Results: All patients were followed up for 9 to 15 months(mean,13.9 months) after surgery. The preoperative hemoglobin levels in the normal and malnutrition groups were 8.6-13.2 and 7.4-11.2 g/dL, respectively (P < .05). The time from injury to surgery in the normal nutrition group was significantly shorter than that in the malnutrition group (P < .01). The preoperative hemoglobin level in the normal nutrition group was significantly higher than that in the malnutrition group. The time from injury to operation in normal nutrition group and malnutrition group are respectively (1.1-5.2), (4.3-6.6)d; the intraoperative and postoperative allogeneic blood transfusion rates are respectively 47.8%(22/46), 92%(46/50);the incidence of postoperative complications are respectively 6.52%(3/46), 32%(16/50); the mortality rates within 1 year after operation are respectively 2.17%(1/46), 12%(6/50). In contrast, the postoperative allogeneic blood transfusion, postoperative complication, and postoperative complication rates in the normal nutrition group were significantly lower than those in the malnutrition group (P < .05). 3 months after surgery, the Harris hip function scores of patients in normal nutrition group and malnutrition group are respectively (75.26±4.02), (64.28±3.82); 6 months after surgery, the Harris hip function scores of them are respectively (80.42±3.86), (70.14±5.06).During the last follow-up, scores are (82.23±2.98), (72.12±4.62). At the 3, 6, and last follow-up after surgery, the Harris hip function score in the normal nutrition group was significantly higher than in the malnutrition group (P < .05). Conclusion: Preoperative malnutrition in elderly patients with intertrochanteric fracture has adverse effects on postoperative hip function recovery, and 1-year postoperative survival rate.GNRI can be used for simple screening. Early assessment of patients' nutritional status.

Isolation and Characterization of Antimicrobial Metabolites from the Sophora tonkinensis-Associated Fungus Penicillium sp. GDGJ-N37.

Chemical investigation of Penicillium sp. GDGJ-N37, a Sophora tonkinensis-associated fungus, yielded two new azaphilone derivatives, N-isoamylsclerotiorinamine (1) and 7-methoxyl-N-isoamylsclerotiorinamine (2), and four known azaphilones (3-6), together with two new chromone derivatives, penithochromones X and Y (7 and 8). Their structures were elucidated based on spectroscopic data, CD spectrum, and semi-synthesis. Sclerotioramine (3) showed significant antibacterial activities against B. subtilis and S. dysentery, and it also showed most potent anti-plant pathogenic fungi activities against P. theae, C. miyabeanus, and E. turcicum.

DNA methylation on C5-Cytosine and N6-Adenine in the Bursaphelenchus xylophilus genome.

BACKGROUND: The pinewood nematode is the causal agent of the pine wilt disease, which causes severe ecological and economic losses in coniferous forests. The invasion of pine wood nematode has undergone various rapid adaptations to a wide range of temperatures and to new hosts and vector insects. DNA methylation may play crucial roles in the rapid adaptation of PWN during invasion. However, whether the PWN genome contins functional DNA modifications remains elusive. RESULTS: Here, we detected the extensive presence of 5-methylcytosine (5mC) and N6-methyladenine (6mA) in the B. xylophilus genome, with low methylation levels at most positions. Cytosines were methylated in the CpG, CHG. and CHH sequence contexts, with the lowest methylation levels at CpG sites. The methylation levels of CpG and 6mA in gene regions showed opposite trends. The changes in the abundance of 5mC and 6mA showed the same trends in response to temperature change, but opposite trends during development. Sequence and phylogenetic analyses showed that the proteins BxDAMT and BxNMAD have typical characteristics of a methylase and demethylase, respectively, and are conserved among species. CONCLUSIONS: These findings shed light on the epigenetic modifications present in the genome of PWN, and will improve our understanding of its invasiveness and evolution.

Analysis of DNA Methylation Differences during the JIII Formation of Bursaphelenchus xylophilus.

DNA methylation is a pivotal process that regulates gene expression and facilitates rapid adaptation to challenging environments. The pinewood nematode (PWN; Bursaphelenchus xylophilus), the causative agent of pine wilt disease, survives at low temperatures through third-stage dispersal juvenile, making it a major pathogen for pines in Asia. To comprehend the impact of DNA methylation on the formation and environmental adaptation of third-stage dispersal juvenile, we conducted whole-genome bisulfite sequencing and transcriptional sequencing on both the third-stage dispersal juvenile and three other propagative juvenile stages of PWN. Our findings revealed that the average methylation rate of cytosine in the samples ranged from 0.89% to 0.99%. Moreover, we observed significant DNA methylation changes in the third-stage dispersal juvenile and the second-stage propagative juvenile of PWN, including differentially methylated cytosine (DMCs, n = 435) and regions (DMRs, n = 72). In the joint analysis of methylation-associated transcription, we observed that 23 genes exhibited overlap between differentially methylated regions and differential gene expression during the formation of the third-stage dispersal juvenile of PWN. Further functional analysis of these genes revealed enrichment in processes related to lipid metabolism and fatty acid synthesis. These findings emphasize the significance of DNA methylation in the development of third-stage dispersal juvenile of PWN, as it regulates transcription to enhance the probability of rapid expansion in PWN.

Tempol attenuates chronic intermittent hypoxia-induced lung injury through the miR-145-5p/Nrf2 signaling pathway.

This study mainly explored the effect of Tempol on OSA-induced lung injury and the specific molecular mechanism. A hypoxia/reoxygenation (H/R) cell model and an IH-induced lung injury model in rats were constructed. The expression of miRNAs and related proteins was detected by RT‒qPCR and Western blotting. HE and Masson staining were used to observe the pathological changes in lung tissues. The expression levels of inflammatory cytokines were detected by ELISA. Apoptotic cells were observed by TUNEL. The ROS levels were detected by a DCFH-DA probe. Tempol administration effectively reduced the pathological changes in lung tissue and the progression of pulmonary fibrosis in rats with lung injury and reduced the expression of inflammatory factors in lung tissue. miR-145-5p was significantly upregulated in rats with IH-induced lung injury, and Tempol treatment inhibited the expression of miR-145-5p. Transfection with the miR-145-5p inhibitor effectively inhibited H/R cell apoptosis and autophagy, while transfection with the miR-145-5p mimic had the opposite effect. Targeting miR-145-5p negatively regulates the expression of Nrf2. Transfection of the miR-145-5p mimic weakened the inhibitory effects of Tempol on apoptosis and autophagy in H/R cells. Overexpression of the Nrf2 mimic reversed the effects of the miR-145-5p mimic on Tempol to a certain extent. It was also confirmed in animal experiments that overexpression of Nrf2 reversed the inhibitory effect of the miR-145-5p mimic on Tempol's lung injury relief effect. Tempol alleviates lung injury induced by chronic interstitial hypoxia by regulating the miR-145-5p/Nrf2 molecular axis and inhibiting autophagy.

Thoracic empyema due to nontuberculous mycobacteria in an immunocompetent patient without pulmonary disease: a case report.

BACKGROUND: Pleural involvement by non-tuberculous mycobacteria (NTM), especially NTM empyema in the immunocompetent patient without pulmonary diseases is a rare disease. It is difficult to diagnose with only a few cases of immunodeficient patients in the literature. CASE PRESENTATION: We describe a 63-year-old male with empyema due to NTM and highlight the challenges of diagnosis. CONCLUSIONS: Non-tuberculous mycobacterial infection should be considered as a cause of pleuritis or empyema without pulmonary disease, however it is a real diagnostic dilemma.

Comparative Analysis of Therapeutic Outcomes and Prognoses Among Osteoporosis Patients with Varied Bone Mineral Density T-Values Following Percutaneous Kyphoplasty.

Objective: This study aims to evaluate and compare the therapeutic outcomes and prognoses of osteoporotic fracture (OSF) patients undergoing percutaneous kyphoplasty (PKP), emphasizing the role of bone mineral density T-values (BMD-T) as a guiding factor in the surgical intervention for OSF. Methods: An observational cohort study was conducted, and 162 OSF patients admitted to our hospital from March 2021 to December 2021 were selected. Patients were categorized based on BMD-T into mild (-2.5 ≤ BMD-T ≤ -4, n=40), moderate (-4 < BMD-T ≤ -5, n = 78), and severe groups (BMD-T < -5, n = 44). All patients underwent PKP treatment, and vertebral body (VB) lavage fluid was analyzed for calcium (Ca), magnesium (Mg), and zinc (Zn) levels. X-ray assessments were performed before and after surgery to examine changes in wedge and kyphosis angles and VB height. Additionally, the Oswestry Disability Index (ODI), Visual Analogue Scale (VAS), and Barthel Index (BI) scores were recorded. Results: The mild group exhibited the highest Ca, Mg, and Zn contents in VB lavage fluid, while the severe group had the lowest (P < .05). A positive correlation was observed between patients' Ca, Mg, and Zn levels and BMD-T (P < .05). The severe group, characterized by lower BMD-T, required a higher amount of bone cement injection, resulting in more significant differences in wedge angle, kyphosis angle, and VB height before and after surgery (P < .05). Moreover, the severe group demonstrated a higher incidence of postoperative adverse reactions (P < .05). Age, bone cement leakage, BMD-T, Ca, Mg, and Zn were identified as independent factors influencing post-PKP re-fracture in OSF patients (P < .05). Conclusion: In PKP for OSF, lower BMD-T correlates with improved correction but is also associated with a higher likelihood of cement leakage.

Klotho Regulated by Estrogen Plays a Key Role in Sex Differences in Stress Resilience in Rats.

Klotho (KL) is a glycosyl hydrolase and aging-suppressor gene. Stress is a risk factor for depression and anxiety, which are highly comorbid with each other. The aim of this study is to determine whether KL is regulated by estrogen and plays an important role in sex differences in stress resilience. Our results showed that KL is regulated by estrogen in rat hippocampal neurons in vivo and in vitro and is essential for the estrogen-mediated increase in the number of presynaptic vesicular glutamate transporter 1 (Vglut1)-positive clusters on the dendrites of hippocampal neurons. The role of KL in sex differences in stress response was examined in rats using 3-week chronic unpredictable mild stress (CUMS). CUMS produced a deficit in spatial learning and memory, anhedonic-like behaviors, and anxiety-like behaviors in male but not female rats, which was accompanied by a reduction in KL protein levels in the hippocampus of male but not female rats. This demonstrated the resilience of female rats to CUMS. Interestingly, the knockdown of KL protein levels in the rat hippocampus of both sexes caused a decrease in stress resilience in both sexes, especially in female rats. These results suggest that the regulation of KL by estrogen plays an important role in estrogen-mediated synapse formation and that KL plays a critical role in the sex differences in cognitive deficit, anhedonic-like behaviors, and anxiety-like behaviors induced by chronic stress in rats, highlighting an important role of KL in sex differences in stress resilience.

A critical review of the central role of microbial regulation in the nitrogen biogeochemical process: New insights for controlling groundwater nitrogen contamination.

With the increase of nitrogen (N) input in vadose zones-groundwater systems, N contamination in groundwater has become a global environmental and geological issue that has a profound impact on the ecological environment and human health. N migration in the vadose zone is the most significant means of contaminating the groundwater aquifer. However, the current research on the control of groundwater N contamination focuses solely on the content change of certain indicators and is unable to comprehend the cause and subsequent development of groundwater N contamination. These factors pose significant environmental management challenges in areas where groundwater is contaminated with nitrate. In recent years, research on the migration and transformation behavior of various N forms in vadose zones-groundwater systems has yielded some breakthroughs but also encountered some roadblocks. The biogeochemical behavior of nitrogen consists of a series of intricate chain reaction cycles (called N-cycle). The crucial role of microorganisms in the N biogeochemical process has attracted the interest of soil carbon- and N-cycle researchers and become a hot topic of study. Nonetheless, the role of microbial regulation in groundwater systems has been largely neglected and needs to be summarized immediately. Consequently, this review summarizes recent advancements, mechanisms, and challenges, and proposes a dynamic perspective on microbial regulation. On the basis of these findings, we propose a dynamic and comprehensive groundwater N system centered on microbial regulation. In addition, we critically summarized the migration and transformation behavior of the most recent N indicators, the impact of global environmental change on each N component, and the non-negligible effects of these factors on the control of groundwater N contamination. Future research must focus on the migration and transformation behavior of nitrogen in the deep vadose zone, based on the dynamic regulation of microorganisms, and complete the missing pieces of the developed N-cycle index system. These are essential for providing scientific guidance for global N management and effectively mitigating N contamination in groundwater.

Ternary XBe4H5- (X = Si, Ge, Sn, Pb) Clusters: Planar Tetracoordinate Si/Ge/Sn/Pb Species with 18 Valence Electrons.

As one of the important probes of chemical bonding, planar tetracoordinate carbon (ptC) compounds have been receiving much attention. Compared with ptC clusters, the heavier planar tetracoordinate silicon, germanium, tin, lead (ptSi/Ge/Sn/Pb) systems are scarcer and more exotic. The 18-valence-electron (ve)-counting is one important guide, though not the only rule, for the design of planar tetra-, penta-coordinate carbon and silicon clusters. The 18ve ptSi/Ge system is very scarce and needs to be expanded. Based on the isoelectronic principle and bonding similarity between the Al atom and the BeH unit, inspired by the previously reported ptSi global minimum (GM) SiAl42-, a series of ternary 18 ve XBe4H5- (X = Si, Ge, Sn, Pb) clusters were predicted with the ptSi/Ge/Sn/Pb centers. Extensive density functional theory (DFT) global minimum searches and high-level CCSD(T) calculations performed herein indicated that these ptSi/Ge/Sn/Pb XBe4H5- (X = Si, Ge, Sn, Pb) clusters were all true GMs on their potential energy surfaces. These GMs of XBe4H5- (X = Si, Ge, Sn, Pb) species possessed the beautiful fan-shaped structures: XBe4 unit can be stabilized by three peripheries bridging H and two terminal H atoms. It should be noted that XBe4H5- (X = Si, Ge, Sn, Pb) were the first ternary 18 ve ptSi/Ge/Sn/Pb species. The natural bond orbital (NBO), canonical molecular orbitals (CMOs) and adaptive natural densitpartitioning (AdNDP) analyses indicated that 18ve are ideal for these ptX clusters: delocalized one π and three σ bonds for the XBe4 core, three Be-H-Be 3c-2e and two Be-H σ bonds for the periphery. Additionally, 2π plus 6σ double aromaticity was found to be crucial for the stability of the ptX XBe4H5- (X = Si, Ge, Sn, Pb) clusters. The simulated photoelectron spectra of XBe4H5- (X = Si, Ge, Sn, Pb) clusters will provide theoretical basis for further experimental characterization.

miR-146a-5p Attenuates Allergic Airway Inflammation by Inhibiting the NLRP3 Inflammasome Activation in Macrophages.

OBJECTIVE: Asthma is a common inflammatory respiratory disease with increasing incidence worldwide. This study aimed to investigate the mechanism of miR-146a-5p in reducing allergic airway inflammation by inhibiting NLRP3 inflammasome activation in macrophages. METHODS: Allergic mouse models were established by ovalbumin stimulation, and mice were treated with miR-146a-5p agomir and oe-TIRAP 3 h before OVA stimulation. The pathological changes of lung tissues were observed by hematoxylin-eosin staining. The airway hyperresponsiveness of mice were examined. The miR-146a-5p level was detected by RT-qPCR. The inflammatory cytokines (IL-18/TNF-α) and anti-inflammatory cytokine IL-10 levels in bronchoalveolar lavage fluid and serum IgE levels were examined by ELISA. Airway inflammation in mice was detected after miR-146a-5p overexpression. The levels of NLRP3/ASC/caspase1 proteins and macrophage M1/M2 surface markers in mouse lung tissues were examined using immunohistochemistry, Western blot, and flow cytometry. The targeting relationship between miR-146a-5p and TIRAP was verified by dual-luciferase assay. The p65 levels in the cytoplasm/nucleus of mouse lung tissue were measured. RESULTS: miR-146a-5p was downregulated in the lung tissues of allergic mice, and miR-146a-5p overexpression alleviated airway inflammation in asthmatic mice. miR-146a-5p suppressed NLRP3 inflammasome activation in macrophages of allergic mice, reduced NLRP3/ASC/caspase1 protein levels in lung tissues, blocked M1 polarization, and promoted M2 polarization. miR-146a-5p targeted TIRAP. TIRAP overexpression partially reversed the promoting effect of miR-146a-5p on M2 polarization. miR-146a-5p can inhibit the activation of the TIRAP/NF-κB pathway. CONCLUSION: miR-146a-5p inhibited NLRP3 inflammasome activation in macrophages in the lung tissue of allergic mice, prevented pro-inflammatory phenotype M1 polarization, and promoted anti-inflammatory phenotype M2 polarization by targeting the TIRAP/NF-κB pathway, thus alleviating airway inflammation in allergic asthma.

Differences Between Microbial Communities of Pinus Species Having Differing Level of Resistance to the Pine Wood Nematode.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a destructive invasive species that exerts devastating effects on most native pines in invaded regions, while many of the non-native pines have resistance to PWN. Recently, increasingly more research is focused on how microbial communities can improve host resistance against pathogens. However, the relationship between the microbial community structures and varying levels of pathogen resistance observed in different pine tree species remains unclear. Here, the bacterial and fungal communities of introduced resistant pines Pinus elliottii, P. caribaea, and P. taeda and native susceptible pines healthy and wilted P. massoniana infected by PWN were analyzed. The results showed that 6057 bacterial and 3931 fungal OTUs were annotated. The pine samples shared 944 bacterial OTUs primarily in the phyla Proteobacteria, Acidobacteria, Firmicutes, Bacteroidetes, and Chloroflexi and 111 fungal OTUs primarily in phyla Ascomycota and Basidiomycota, though different pines had unique OTUs. There were significant differences in microbial community diversity between different pines, especially between the bacterial communities of resistant and susceptible pines, and fungal communities between healthy pines (resistant pines included) and the wilted P. massoniana. Resistant pines had a greater abundance of bacteria in the genera Acidothermus (class unidentified_Actinobacteria) and Prevotellaceae (class Alphaproteobacteria), but a lower abundance of Erwinia (class Gammaproteobacteria). Healthy pines had a higher fungal abundance of Cladosporium (class Dothideomycetes) and class Eurotiomycetes, but a lower abundance of Graphilbum, Sporothrix, Geosmithia (class Sordariomycetes), and Cryptoporus (classes Agaricomycetes and Saccharomycetes). These differences in microbial abundance between resistant and healthy pines might be associated with pathogen resistance of the pines, and the results of this study contribute to the studies exploring microbial-based control of PWN.

Expression of the Thaumatin-Like Protein-1 Gene (Bx-tlp-1) from Pine Wood Nematode Bursaphelenchus xylophilus Affects Terpene Metabolism in Pine Trees.

Pine wilt disease is a major forest disease worldwide, including in China, where it has severely damaged pine forest ecosystems, and the pathogen is pine wood nematode (Bursaphelenchus xylophilus). The thaumatin-like protein-1 gene (Bx-tlp-1) is a key gene associated with B. xylophilus pathogenicity, which is also responsive to α-pinene. In this study, an examination of Pinus massoniana seedlings infected by B. xylophilus revealed that monoterpene (sesquiterpene) levels peaked on days 15 and 27 (days 18 and 27). Meanwhile, P. massoniana Pm-tlp expression levels were high on days 3, 12, and 27, which were consistent with the expression of key enzymes genes in the terpene biosynthesis pathway. The functional similarity of B. xylophilus Bx-TLP-1 and P. massoniana Pm-TLP suggests Bx-TLP-1 and Pm-TLP may have similar roles in P. massoniana. There was also no secondary accumulation of terpenes in P. massoniana seedlings during B. xylophilus treated with dsRNA targeting Bx-tlp-1 (dsTLP1) infections, reflecting the decreased pathogenicity of B. xylophilus and the delayed disease progression in pine trees. And the results of micro-CT showed that the degree of cavitation for the trees inoculated with Bx-TLP-1 (0.3811 mm3) was greater than that for the trees inoculated with dsTLP1 PWNs (0.1204 mm3) on day 15 after inoculation. Results from this study indicated that B. xylophilus Bx-tlp-1 gene may induce the upregulated expression of related genes encoding enzymes in the terpene synthesis pathway of P. massoniana, resulting in the accumulation of terpenes, which also provided an insight to investigate the B. xylophilus pathogenicity in the future.

Network Pharmacology Analysis and Experimental Validation to Explore the Anti-inflammatory Mechanism of Asiatic Acid on Alcoholic Steatohepatitis.

Objective: The mechanism of action of asiatic acid (AA) on alcoholic steatohepatitis (ASH) was investigated using network pharmacology and experiments. Methods: Through data retrieval, network construction, and enrichment analysis, the potential mechanism of AA in the treatment of alcoholic steatohepatitis was explored. Animal and cell models were established in this study. Animal Model. The mouse model was divided into six groups: normal group; model group; low, medium, and high AA group; and silibinin-positive group. Cell Model. An in vitro inflammatory model of RAW264.7 cells was established by alcohol stimulation. Results: Compared with the model group, the low, medium, and high AA group showed decreased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), and total cholesterol (T-CHO). The inflammatory factor tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), and interleukin 6 (IL-6) in a dose-dependent manner were decreased. In addition, hematoxylin-eosin staining showed that liver tissue damage and inflammatory cell infiltration in mice were significantly reduced with increasing doses. Further, oil red staining showed that lipid accumulation in hepatocytes in the low, medium, and high AA group was significantly reduced, with increasing dose. In addition, in the cellular model, real-time reverse transcriptase-polymerase chain reaction (Real-Time RT-PCR) and enzyme-linked immunosorbent assay (ELISA) results showed that AA could alleviate alcohol-induced cellular inflammation, while western blot and immunofluorescence results showed that AA might alleviate alcohol-induced cellular inflammation by inhibiting the nuclear factor-κB (NF-κB) pathway. Conclusion: This study provides multiple lines of evidence that asiatic acid may alleviate alcoholic hepatitis in mice by modulating the NF-κB pathway.

Identification of the Extracellular Nuclease Influencing Soaking RNA Interference Efficiency in Bursaphelenchus xylophilus.

RNA interference (RNAi) efficiency dramatically varies among different nematodes, which impacts research on their gene function and pest control. Bursaphelenchus xylophilus is a pine wood nematode in which RNAi-mediated gene silencing has unstable interference efficiency through soaking in dsRNA solutions, the factors of which remain unknown. Using agarose gel electrophoresis, we found that dsRNA can be degraded by nematode secretions in the soaking system which is responsible for the low RNAi efficiency. Based on the previously published genome and secretome data of B. xylophilus, 154 nucleases were screened including 11 extracellular nucleases which are potential factors reducing RNAi efficacy. To confirm the function of nucleases in RNAi efficiency, eight extracellular nuclease genes (BxyNuc1-8) were cloned in the genome. BxyNuc4, BxyNuc6 and BxyNuc7 can be upregulated in response to dsGFP, considered as the major nuclease performing dsRNA degradation. After soaking with the dsRNA of nucleases BxyNuc4/BxyNuc6/BxyNuc7 and Pat10 gene (ineffective in RNAi) simultaneously for 24 h, the expression of Pat10 gene decreased by 23.25%, 26.05% and 11.29%, respectively. With soaking for 36 h, the expression of Pat10 gene decreased by 43.25% and 33.25% in dsBxyNuc6+dsPat10 and dsBxyNuc7+dsPat10 groups, respectively. However, without dsPat10, dsBxyNuc7 alone could cause downregulation of Pat10 gene expression, while dsBxyNuc6 could not disturb this gene. In conclusion, the nuclease BxyNuc6 might be a major barrier to the RNAi efficiency in B. xylophilus.

Pinewood Nematode Alters the Endophytic and Rhizospheric Microbial Communities of Pinus massoniana.

Pinewood nematode, Bursaphelenchus xylophilus, is one of the greatest threats to pine trees and is spreading all over the world. During the nematode's pathogenesis, plant microorganisms play important roles. However, many microbial communities, such as that in Pinus massoniana, a major host of B. xylophilus that is widely distributed in China, are not well studied, especially the fungal communities. Here, the endophytic and rhizospheric bacterial and fungal communities associated with healthy and B. xylophilus-infected P. massoniana were analyzed. The results showed that 7639 bacterial and 3108 fungal OTUs were annotated from samples of P. massoniana, the rhizosphere, and B. xylophilus. There were significant diversity differences of endophytic microbes between healthy and infected P. massoniana. The abundances of endophytic bacteria Paenibacillus, unidentified_Burkholderiaceae, Serratia, Erwinia, and Pseudoxanthomonas and fungi Penicillifer, Zygoascus, Kirschsteiniothelia, Cyberlindnera, and Sporothrix in infected pines were greater than those in healthy pines, suggesting an association of particular microbial abundances with the pathogenesis of B. xylophilus in pines. Meanwhile, the abundances of microbes of unidentified_Burkholderiaceae, Saitozyma, and Pestalotiopsis were greater and Acidothermus and Trichoderma were lower in the rhizosphere under infected pines than those under healthy pines and the differences might be caused by B. xylophilus-induced weakening of the health of pines. Our study explored the endophytic and rhizospheric microbial community changes potentially caused by B. xylophilus infection of pines.