Template-independent synthesis and 3'-end labelling of 2'-modified oligonucleotides with terminal deoxynucleotidyl transferases.

Xenobiotic nucleic acids (XNAs) are artificial genetic polymers with altered structural moieties and useful features, such as enhanced biological and chemical stability. Enzymatic synthesis and efficient labelling of XNAs are crucial for their broader application. Terminal deoxynucleotidyl transferases (TdTs) have been exploited for the de novo synthesis and labelling of DNA and demonstrated the capability of recognizing various substrates. However, the activities of TdTs for the synthesis and labelling of commonly used XNAs with 2' modifications have not been systematically explored. In this work, we explored and demonstrated the varied activities of three TdTs (bovine TdT, MTdT-evo and murine TdT) for the template-independent incorporation of 2'-methoxy NTPs, 2'-fluoro NTPs and 2'-fluoroarabino NTPs into the 3' ends of single- and double-stranded DNAs and the extension of 2'-modified XNAs with (d)NTPs containing a natural or unnatural nucleobase. Taking advantages of these activities, we established a strategy for protecting single-stranded DNAs from exonuclease I degradation by TdT-synthesized 2'-modified XNA tails and methods for 3'-end labelling of 2'-modified XNAs by TdT-mediated synthesis of G-quadruplex-containing tails or incorporation of nucleotides with a functionalized nucleobase. A DNA-2'-fluoroarabino nucleic acid (FANA) chimeric hydrogel was also successfully constructed based on the extraordinary activity of MTdT-evo for template-independent FANA synthesis.

Linking performance to dynamic migration of biofilm ecosystem reveals the role of voltage in the start-up of hybrid microbial electrolysis cell-anaerobic digestion.

Applied voltage is a crucial parameter in hybrid microbial electrolysis cells-anaerobic digestion (MEC-AD) systems for enhancing methane production from waste activated sludge (WAS). This study explored the impact of applied voltage on the initial biofilm formation on electrodes during the MEC-AD startup using raw WAS (Rr) and heat-pretreated WAS (Rh). The findings indicated that the maximum methane productivity for Rr and Rh were 3.4 ±â€¯0.5 and 3.4 ±â€¯0.2 mL/gVSS/d, respectively, increasing 1.5 times and 2.6 times over the productivity at 0 V. The biomass on electrode biofilms for Rr and Rh at 0.8 V increased by 70 % and 100 % compared to 0 V. The core functional microorganisms in the cathode biofilm were Methanobacterium and Syntrophomonas, and Geobacter in the anode biofilm, enhancing methane production through syntrophism and direct interspecies electron transfer, respectively. These results offer academic insights into optimizing AD functional electrode biofilms by applying voltage.

Does rAj-Tspin, a novel peptide from A. japonicus, exert antihepatocellular carcinoma effects via the ITGB1/ZYX/FAK/AKT signaling pathway?

rAj-Tspin, a soluble recombinant peptide from Apostichopus japonicus, can inhibit the integrin ß1 (ITGB1)/FAK/AKT signaling pathway in hepatocellular carcinoma (HCC) via cell epithelial-mesenchymal transition (EMT) and apoptosis. Zyxin (ZYX) is a focal adhesion protein that is considered a novel mediator of EMT and apoptosis. However, the inhibitory mechanisms of rAj-Tspin in HCC and whether it is related to ZYX are unclear. We examined the antitumor effect of rAj-Tspin on the Huh7 human HCC cell line and on a nude mouse model generated via subcutaneous injection or orthotopic intrahepatic transplantation of Huh7 cells. Our results revealed that rAj-Tspin strikingly reduced the viability and promoted the apoptosis of Huh7 cells and inhibited HCC tumor growth in nude mice. rAj-Tspin inhibited ITGB1 and ZYX protein expression in vivo and in vitro in a dose-dependent manner. Mechanistically, the FAK/AKT signaling pathway and the proliferation and invasion of HCC cells were suppressed upon ITGB1 and ZYX knockdown. Moreover, the effect of ITGB1 overexpression on the growth of HCC cells was inhibited by rAj-Tspin. In contrast, the promoting effect of ITGB1 overexpression could be inhibited by ZYX knockdown. ZYX knockdown had no effect on ITGB1 expression. These findings suggest that ZYX is required for the indispensable role of ITGB1 in rAj-Tspin-alleviated HCC and provide an important therapeutic target for HCC. In summary, the anti-HCC effect of rAj-Tspin potentially involves the regulation of the ITGB1/ZYX/FAK/AKT pathway, which in turn impacts EMT and apoptosis.

Prediction of non-dipper blood pressure pattern in Chinese patients with hypertension using a nomogram model.

Non-dipper blood pressure has been shown to affect cardiovascular outcomes and cognitive function in patients with hypertension. Although some studies have explored the influencing factors of non-dipper blood pressure, there is still relatively little research on constructing a prediction model. This study aimed to develop and validate a simple and practical nomogram prediction model and explore relevant elements that could affect the dipper blood pressure relationship in patients with hypertension. A convenient sampling method was used to select 356 inpatients with hypertension who visited the Affiliated Hospital of Jining Medical College from January 2022 to September 2022. All patients were randomly assigned to the training cohort (75%, n = 267) and the validation cohort (25%, n = 89). Univariate and multivariate logistic regression were utilized to identify influencing factors. The nomogram was developed and evaluated based on the receiver operating characteristic (ROC) curve, the area under the ROC curve (AUC), and decision curve analyses. The optimal cutoff values for the prevalence of dipper blood pressure were estimated. The nomogram was established using six variables, including age, sex, hemoglobin (Hb), estimated glomerular filtration rate (eGFR), ejection fraction (EF), and heart rate. The AUC was 0.860 in the training cohort. The cutoff values for optimally predicting the prevalence of dipper blood pressure were 41.50 years, 151.00 g/L, 117.53 mL/min/1.73 m2, 64.50%, and 75 beats per minute for age, Hb, eGFR, ejection fraction, and heart rate, respectively. In summary, our nomogram can be used as a simple, plausible, affordable, and widely implementable tool to predict the blood pressure pattern of Chinese patients with hypertension.

Carex meyeriana Kunth Extract Is a Novel Natural Drug against Candida albicans.

As a widely distributed plant in Northeast China, Carex meyeriana Kunth (CMK) is generally considered to have antibacterial properties; however, there is a lack of scientific evidence for this. Therefore, we investigated the chemical composition of CMK extract and its effect against C. albicans. A total of 105 compounds were identified in the alcohol extracts of CMK by UPLC-Q-TOF-MS. Most were flavonoids, with Luteolin being the most represented. Among them, 19 compounds are found in the C. albicans lysates. After treatment with CMK ethanol extract, a significant reduction in the number of C. albicans colonies was observed in a vaginal douche solution from day 5 (p < 0.05). Furthermore, the CMK extract can reduce the number of C. albicans spores. The levels of IL-4, IL-6, IL-10, IL-1ß, and TNF-α in vaginal tissues all exhibited a significant decrease (p < 0.05) compared to those in the model group as determined by ELISA. The results of HE staining showed that CMK extract can eliminate vaginal mucosa inflammation. CMK adjusts the vaginal mucosa cells by targeting twenty-six different metabolites and five specific metabolic pathways in order to effectively eliminate inflammation. Simultaneously, the CMK regulates twenty-three types of metabolites and six metabolic pathways against C. albicans infection. So, CMK strongly inhibits the growth of C. albicans and significantly reduces vaginal inflammation, making it a promising candidate for treating C. albicans infection.

Intermittent hypoxia exacerbates anxiety in high-fat diet-induced diabetic mice by inhibiting TREM2-regulated IFNAR1 signaling.

BACKGROUND: Type 2 diabetes mellitus (T2DM) and obstructive sleep apnea (OSA) are mutual risk factors, with both conditions inducing cognitive impairment and anxiety. However, whether OSA exacerbates cognitive impairment and anxiety in patients with T2DM remains unclear. Moreover, TREM2 upregulation has been suggested to play a protective role in attenuating microglia activation and improving synaptic function in T2DM mice. The aim of this study was to explore the regulatory mechanisms of TREM2 and the cognitive and anxiety-like behavioral changes in mice with OSA combined with T2DM. METHODS: A T2DM with OSA model was developed by treating mice with a 60% kcal high-fat diet (HFD) combined with intermittent hypoxia (IH). Spatial learning memory capacity and anxiety in mice were investigated. Neuronal damage in the brain was determined by the quantity of synapses density, the number and morphology of brain microglia, and pro-inflammatory factors. For mechanism exploration, an in vitro model of T2DM combined with OSA was generated by co-treating microglia with high glucose (HG) and IH. Regulation of TREM2 on IFNAR1-STAT1 pathway was determined by RNA sequencing and qRT-PCR. RESULTS: Our results showed that HFD mice exhibited significant cognitive dysfunction and anxiety-like behavior, accompanied by significant synaptic loss. Furthermore, significant activation of brain microglia and enhanced microglial phagocytosis of synapses were observed. Moreover, IH was found to significantly aggravate anxiety in the HFD mice. The mechanism of HG treatment may potentially involve the promotion of TREM2 upregulation, which in turn attenuates the proinflammatory microglia by inhibiting the IFNAR1-STAT1 pathway. Conversely, a significant reduction in TREM2 in IH-co-treated HFD mice and HG-treated microglia resulted in the further activation of the IFNAR1-STAT1 pathway and consequently increased proinflammatory microglial activation. CONCLUSIONS: HFD upregulated the IFNAR1-STAT1 pathway and induced proinflammatory microglia, leading to synaptic damage and causing anxiety and cognitive deficits. The upregulated TREM2 inT2DM mice brain exerted a negative regulation of the IFNAR1-STAT1 pathway. Mice with T2DM combined with OSA exacerbated anxiety via the downregulation of TREM2, causing heightened IFNAR1-STAT1 pathway activation and consequently increasing proinflammatory microglia.

Systematic review of the mechanism and assessment of liver fibrosis in biliary atresia.

PURPOSE: This study systematically reviewed our team's research on the mechanism and assessment of liver fibrosis in BA, summarized our experience, and discussed the future development direction. METHODS: In this study, Pubmed and Wanfang databases were searched to collect the literature published by our team on the mechanisms of liver fibrosis in BA and the assessment of liver fibrosis in BA, and the above research results were systematically reviewed. RESULTS: A total of 58 articles were retrieved. Among the included articles, 25 articles related to the mechanism of liver fibrosis in BA, and five articles evaluated liver fibrosis in BA. This article introduces the key pathways and molecules of liver fibrosis in BA and proposes a new grading system for liver fibrosis in BA. CONCLUSIONS: The new BA liver fibrosis grading method is expected to assess children's conditions, guide treatment, and improve prognosis more accurately. In addition, we believe that the TGF-ß1 signaling pathway is the most important in the study of liver fibrosis in BA, and at the same time, the study of EMT occurrence in BA should also be deepened to resolve the controversy on this issue.

The generation and transformation mechanisms of reactive oxygen species in the environment and their implications for pollution control processes: A review.

Reactive oxygen species (ROS), substances with strong activity generated by oxygen during electron transfer, play a significant role in the decomposition of organic matter in various environmental settings, including soil, water and atmosphere. Although ROS has a short lifespan (ranging from a few nanoseconds to a few days), it continuously generated during the interaction between microorganisms and their environment, especially in environments characterized by strong ultraviolet radiation, fluctuating oxygen concentration or redox conditions, and the abundance of metal minerals. A comprehensive understanding of the fate of ROS in nature can provide new ideas for pollutant degradation and is of great significance for the development of green degradation technologies for organic pollutants. At present, the review of ROS generally revolves around various advanced oxidation processes, but lacks a description and summary of the fate of ROS in nature, this article starts with the definition of reactive oxidants species and reviews the production, migration, and transformation mechanisms of ROS in soil, water and atmospheric environments, focusing on recent developments. In addition, the stimulating effects of ROS on organisms were reviewed. Conclusively, the article summarizes the classic processes, possible improvements, and future directions for ROS-mediated degradation of pollutants. This review offers suggestions for future research directions in this field and provides the possible ROS technology application in pollutants treatment.

Identification of an alternative ligand-binding pocket in peroxisome proliferator-activated receptor gamma and its correlated selective agonist for promoting beige adipocyte differentiation.

The pharmacological activation of peroxisome proliferator-activated receptor gamma (PPARγ) is a convenient and promising strategy for promoting beige adipocyte biogenesis to combat obesity-related metabolic disorders. However, thiazolidinediones (TZDs), the full agonists of PPARγ exhibit severe side effects in animal models and in clinical settings. Therefore, the development of efficient and safe PPARγ modulators for the treatment of metabolic diseases is emerging. In this study, using comprehensive methods, we report a previously unidentified ligand-binding pocket (LBP) in PPARγ and link it to beige adipocyte differentiation. Further virtual screening of 4097 natural compounds based on this novel LBP revealed that saikosaponin A (NJT-2), a terpenoid compound, can bind to PPARγ to induce coactivator recruitment and effectively activate PPARγ-mediated transcription of the beige adipocyte program. In a mouse model, NJT-2 administration efficiently promoted beige adipocyte biogenesis and improved obesity-associated metabolic dysfunction, with significantly fewer adverse effects than those observed with TZD. Our results not only provide an advanced molecular insight into the structural ligand-binding details in PPARγ, but also develop a linked selective and safe agonist for obesity treatment.

Genome-wide identification of cold shock proteins (CSPs) in sweet cherry (Prunus avium L.) and exploring the differential responses of PavCSP1 and PavCSP3 to low temperature and salt stress.

BACKGROUND: Cold shock proteins (CSPs) are ubiquitous nucleic acid-binding proteins involved in growth, development, and stress response across various organisms. While extensively studied in many species, their regulatory roles in sweet cherry (Prunus avium L.) remain unclear. OBJECTIVE: To identify and analyze CSP genes (PavCSPs) in sweet cherry genome, and explore the differential responses of PavCSP1 and PavCSP3 to low temperature and salt stress. METHODS: Three methods were employed to identify and characterize CSP in sweet cherry genomes. To explore the potential functions and evolutionary relationships of sweet cherry CSP proteins, sequence alignment and phylogenetic tree incorporating genes from five species were conducted and constructed, respectively. To investigate the responses to abiotic stresses, cis-acting elements analysis and gene expression patterns to low-temperature and salt stress were examined. Moreover, transgenic yeasts overexpressing PavCSP1 or PavCSP3 were generated and their growth under stress conditions were observed. RESULTS: In this study, three CSP genes (PavCSPs) were identified and comprehensively analyzed. The quantitative real-time PCR revealed diverse expression patterns, with PavCSP1-3 demonstrating a particular activity in the upper stem and all members were responsive to low-temperature and salt stress. Further investigation demonstrated that transgenic yeasts overexpressing PavCSP1 or PavCSP3 exhibited improved growth states following high-salt and low-temperature stress. CONCLUSION: These findings elucidated the responses of PavCSP1 and PavCSP3 to salt and low-temperature stresses, laying the groundwork for further functional studies of PavCSPs in response to abiotic stresses.

Association between volatile organic compounds exposure and periodontitis: A representative cross-sectional study.

AIM: Periodontitis is one of the most common oral diseases and a major cause of tooth loss in adults. Environmental pollution is closely associated with the prevalence of periodontitis. However, few studies have focused on the association between volatile organic compounds (VOCs) and periodontitis. This cross-sectional study aims to examine whether exposure to VOCs is associated with periodontitis, based on data from the National Health and Nutrition Examination Survey (NHANES, 2011-2014). MATERIALS AND METHODS: We analysed data on blood VOC levels, periodontitis and related covariates from 2772 participants of the NHANES. The association between the blood VOCs and periodontitis was analysed using weighted logistic regression analysis, the restricted cubic spline (RCS) model and the weighted quantile sum (WQS) regression model. Interaction tests and mediation analysis were also conducted. RESULTS: After adjusting for covariates, for each natural constant-fold increase in 1,4-dichlorobenzene, the odds of having periodontitis increased by 16% (odds ratio = 1.16; 95% confidence interval: 1.08-1.24, p < .001). WQS regression model indicated that 1,4-dichlorobenzene contributed the most to the association between VOC co-exposure and periodontitis. Mediation analysis further revealed that total bilirubin levels mediated the association between 1,4-dichlorobenzene and the prevalence of periodontitis, accounting for 4.32%. In addition, the positive association between o-xylene and periodontitis was more pronounced in the <65-year-old group. CONCLUSIONS: This study has provided relatively little evidence to demonstrate a specific link between VOCs and periodontitis. Nonetheless, exposure to VOCs remains a non-negligible public health concern, and further research is required to investigate the association and potential mechanisms of action between VOCs and periodontitis.

Expose to volatile organic compounds is associated with increased risk of depression: A cross-sectional study.

With increasing prevalence rate of depression by years, more attention has been paid to the influence of environmental pollutants on depression, but relationship between exposure to volatile organic compounds (VOCs) and depression is rarely studied. Therefore, this cross-sectional study use the National Center for Health Statistics (NHANES) database (2013-2016 years) to explore association between exposure to multiple VOCs and depression in general population. Multiple linear and logistic regression models were used to analyze the association between urinary VOC metabolism (mVOCs) and depression. To further analyze effect of multiple mVOCs mixed exposure, Bayesian kernel machine regression (BKMR) models were performed. A total of 3240 participants and 16 mVOCs were included in the analysis. Results showed that 10 mVOCs exposure were positively correlated with depression by multiple linear and logistic regression models, especially CYMA and MHBMA3, which also showed significant positive association with depression in BKMR model. Mixed exposure of multiple mVOCs was significantly positively correlated with depression. Gender differences were existed in effects of some VOCs concentrations on depression. AAMA, CYMA and MA had significant positive correlations with depression by women, and DHBMA had significant positive correlations with depression by men. Hence, this study showed that exposing to VOCs might have negative impacts on depression, and impact of CYMA and MHBMA3 on depression may be more evident, which provide new ideas for prevention and control of depression. But further research and exploration are needed to clarify the mechanism and influence factors of this relationship, to demonstrate the reliability of these relationship.

[Value of modified ROX index in predicting the outcome of patients with acute respiratory distress syndrome due to SARS-CoV-2 infection treated with high-flow nasal cannula oxygen therapy].

OBJECTIVE: To investigate the value of the modified ROX (mROX) index in predicting the outcome of patients with acute respiratory distress syndrome (ARDS) due to SARS-CoV-2 infection treated with high-flow nasal cannula oxygen therapy (HFNC). METHODS: A retrospective observational study was conducted, including 57 patients with ARDS caused by SARS-CoV-2 infection who required HFNC treatment in the intensive care unit (ICU) of the Lanzhou University Second Hospital from December 2022 to June 2023. The patients were divided into HFNC failure group and HFNC success group according to whether they were successfully weaned from HFNC. Laboratory tests, acute physiology and chronic health evaluation II (APACHE II), and sequential organ failure assessment (SOFA) in the first 24 hours of ICU admission were recorded in both groups, vital signs and arterial blood gas analysis immediately and after 6 hours of HFNC treatment, treatment regimen, length of ICU stay, and total length of hospital stay were recorded in both groups, and patients' outcomes at 28 days and 90 days were followed up by telephone. Univariate analysis was used to analyze the above indexes, and the significant indexes were included in the binary multivariate Logistic regression analysis to analyze the influencing factors of HFNC failure in patients. Kaplan-Meier survival curves were plotted to analyze the 28-day and 90-day outcomes of patients in both groups. Receiver operator characteristic curve (ROC curve) was plotted to analyze the value of treatment 6-hour mROX index and 6-hour ROX index in predicting the success of HFNC. RESULTS: A total of 57 patients with ARDS due to SARS-CoV-2 infection were enrolled, including 34 patients in the HFNC success group and 23 patients in the HFNC failure group. Procalcitonin (PCT), C-reactive protein (CRP), interleukin-6 (IL-6), lactic acid (Lac) and the proportion of vasopressors, the proportion of continuous renal replacement therapy (CRRT), the APACHE II score and the SOFA score, the respiratory rate (RR) immediately and 6 hours after treatment were significantly higher in the HFNC failure group compared with the HFNC success group. The length of ICU stay was significantly longer, and oxygenation index (PaO2/FiO2) at the time of treatment, and pulse oxygen saturation (SpO2), arterial partial pressure of oxygen (PaO2), ROX index, and mROX index at the time of treatment and at 6 hours after treatment were significantly lower in the HFNC failure group compared with the HFNC success group (all P < 0.05). Kaplan-Meier survival curves showed that the 28-day cumulative survival rates (100% vs. 26.1%) and 90-day cumulative survival rates (85.3% vs. 21.7%) of patients in the HFNC success group were significantly higher than those in the HFNC failure group (both P < 0.001). On binary multivariate Logistic regression analysis, Lac [odds ratio (OR) = 0.129, 95% confidence interval (95%CI) was 0.020-0.824], SOFA score (OR = 0.382, 95%CI was 0.158-0.925), 6-hour ROX index (OR = 0.099, 95%CI was 0.011-0.920), and 6-hour mROX index (OR = 23.703, 95%CI was 1.415-396.947) were associated with HFNC treatment outcome (all P < 0.05). ROC curve analysis showed that the area under the ROC curve (AUC) of the 6-hour mROX index and the 6-hour ROX index for predicting the success of HFNC were both higher (0.809 and 0.714, respectively), and the AUC of 6-hour mROX index was significantly higher than that of 6-hour ROX index (P < 0.01), and the sensitivity was 88.2% and the specificity was 52.2% when the cut-off value of 6-hour mROX index was 4.5. CONCLUSIONS: The predictive value of the 6-hour mROX index in the treatment of patients with ARDS caused by SARS-CoV-2 infection is higher than that of the 6-hour ROX index, and the 6-hour mROX index is greater than 4.5, which is more likely to predict the success of HFNC treatment.

Genome and metabolome analysis of Bacillus sp. Hex-HIT36: A newly screened functional microorganism for the degradation of 1-hexadecene in industrial wastewater.

1-Hexadecene has been detected at a level of mg/L in both influent and effluent of wastewater treatment plants situated in chemical/pharmaceutical industrial parks, which poses a potential threat to the environment. However, few reports are available on aerobic metabolic pathways and microorganisms involved in 1-Hexadecene degradation. In this study, a new strain of 1-Hexadecene-degrading bacteria, Bacillus sp. Hex-HIT36 (HIT36), was isolated from the activated sludge of a wastewater treatment plants located in an industrial park. The physicochemical properties and degradation efficacy of HIT36 were investigated. HIT36 was cultured on a medium containing 1-Hexadecene as a sole carbon source; it was found to remove ∼67% of total organic carbon as confirmed by mass spectrometric analysis of intermediate metabolites. Metabolomic and genomic analysis showed that HIT36 possesses various enzymes, namely, pyruvate dehydrogenase, dihydropolyhydroxyl dehydrogenase, and 2-oxoglutarate-2-oxoiron oxidoreductase (subunit alpha), which assist in the metabolization of readily available carbon source or long chain hydrocarbons present in the growth medium/vicinity. This suggests that HIT36 has efficient long-chain alkane degradation efficacy, and understanding the alkane degradation mechanism of this strain can help in developing technologies for the degradation of long-chain alkanes present in wastewater, thereby assisting in the bioremediation of environment.

Intermittent hypoxia training enhances Aß endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer's disease.

BACKGROUND: Beta-amyloid (Aß) deposition in the brain parenchyma is a crucial initiating step in the amyloid cascade hypothesis of Alzheimer's disease (AD) pathology. Furthermore, dysfunction of plaque-associated microglia, also known as disease-associated microglia (DAM) has been reported to accelerate Aß deposition and cognitive impairment. Our previous research demonstrated that intermittent hypoxia training (IHT) improved AD pathology by upregulating autophagy in DAM, thereby enhancing oligomeric Aß (oAß) clearance. Considering that oAß internalization is the initial stage of oAß clearance, this study focused on the IHT mechanism involved in upregulating Aß uptake by DAM. METHODS: IHT was administered to 8-month-old APP/PS1 mice or 6-month-old microglial vacuolar protein sorting 35 (VPS35) knockout mice in APP/PS1 background (MG VPS35 KO: APP/PS1) for 28 days. After the IHT, the spatial learning-memory capacity of the mice was assessed. Additionally, AD pathology was determined by estimating the nerve fiber and synapse density, Aß plaque deposition, and Aß load in the brain. A model of Aß-exposed microglia was constructed and treated with IHT to explore the related mechanism. Finally, triggering receptor expressed on myeloid cells 2 (TREM2) intracellular recycling and Aß internalization were measured using a fluorescence tracing technique. RESULTS: Our results showed that IHT ameliorated cognitive function and Aß pathology. In particular, IHT enhanced Aß endocytosis by augmenting the intracellular transport function of microglial TREM2, thereby contributing to Aß clearance. Furthermore, IHT specifically upregulated VPS35 in DAM, the primary cause for the enhanced intracellular recycling of TREM2. IHT lost ameliorative effect on Aß pathology in MG VPS35 KO: APP/PS1 mice brain. Lastly, the IHT mechanism of VPS35 upregulation in DAM was mediated by the transcriptional regulation of VPS35 by transcription factor EB (TFEB). CONCLUSION: IHT enhances Aß endocytosis in DAM by upregulating VPS35-dependent TREM2 recycling, thereby facilitating oAß clearance and mitigation of Aß pathology. Moreover, the transcriptional regulation of VPS35 by TFEB demonstrates a close link between endocytosis and autophagy in microglia. Our study further elucidates the IHT mechanism in improving AD pathology and provides evidence supporting the potential application of IHT as a complementary therapy for AD.

[TFEB activator 1 enhances autophagic degradation of oligomeric amyloid-ß in microglia].

The purpose of the study was to investigate the mechanism of TFEB activator 1 (TA1) improving the autophagic degradation of oligomeric amyloid-ß (oAß) in microglia, and to explore the therapeutic effect of TA1 on an in vitro model of microglia in Alzheimer's disease (AD). Primary microglia were exposed to 1 µmol/L oAß for 0, 3, 12, and 24 h respectively to construct the in vitro model of microglia in AD. In order to explore the therapeutic effect of TA1, primary microglia were co-treated with 1 µmol/L oAß and 1 µmol/L TA1 for 12 h. To determine the autophagy flux, the above cells were further treated with 100 nmol/L Bafilomycin A1 for 1 h before fixation. Fluorescent probes were used to detect the endocytosis or degradation of oAß1-42 by microglia. The autophagic flux was determined by infection of lentivirus mCherry-EGFP-LC3. The nuclear TFEB intensity, the autophagosomes number, and the colocalization ratio of oAß1-42 with lysosome-associated membrane protein 1 (LAMP1) or microtubule-associated protein light chain 3 (LC3), were detected by immunofluorescence assay. Expressions of autophagy-related-genes, including Lamp1, Atg5, and Map1lc3b, were detected by qRT-PCR. Results showed that prolonged oAß exposure inhibited the endocytosis and degradation of oAß by microglia. Meanwhile, the number of autophagosomes and autophagy flux in microglia decreased after 12 h of oAß treatment. We further found that the nuclear expression of autophagy regulator TFEB decreased after 12 h of oAß exposure, resulting in the decrease of autophagy genes, thus leading to the damage of autophagic degradation of oAß. Therefore, long-term oAß exposure was considered to construct the in vitro model of microglia in AD. After TA1 treatment, the nuclear expression of TFEB in cells was obviously upregulated. TA1 treatment upregulated the expressions of autophagy-related genes, leading to the recovery of autophagy flux. TA1 also recovered the endocytosis and degradation of oAß by microglia. In conclusion, TA1 could improve oAß clearance by microglia in AD by upregulating microglial TFEB-mediated autophagy, suggesting TA1 as a potential therapeutic drug for AD.

Small extracellular vesicles-derived from 3d cultured human nasal mucosal mesenchymal stem cells during differentiation to dopaminergic progenitors promote neural damage repair via miR-494-3p after manganese exposed mice.

Manganese (Mn) exposure is a common environmental risk factor for Parkinson's disease (PD), with pathogenic mechanisms associated with dopaminergic neuron damage and neuroinflammation. Mesenchymal stem cells (MSCs)-derived small extracellular vesicles (sEVs) have emerged as a novel therapeutic approach for neural damage repair. The functional sEVs released from MSCs when they are induced into dopaminergic progenitors may have a better repair effect on neural injury. Therefore, we collected sEVs obtained from primary human nasal mucosal mesenchymal stem cells (hnmMSC-sEVs) or cells in the process of dopaminergic progenitor cell differentiation (da-hnmMSC-sEVs), which were cultured in a 3D dynamic system, and observed their repair effects and mechanisms of Mn-induced neural damage by intranasal administration of sEVs. In Mn-exposed mice, sEVs could reach the site of brain injury after intranasal administration, da-hnmMSC enhanced the repair effects of sEVs in neural damage and behavioral competence, as evidenced by restoration of motor dysfunction, enhanced neurogenesis, decreased microglia activation, up-regulation of anti-inflammatory factors, and down-regulation of pro-inflammatory factors. The transcriptomics of hnmMSC-sEVs and da-hnmMSC-sEVs revealed that miRNAs, especially miR-494-3p in sEVs were involved in neuroprotective and anti-inflammatory effects. Overexpression of miR-494-3p in sEVs inhibited Mn-induced inflammation and neural injury, and its repair mechanism might be related to the down-regulation of CMPK2 and NLRP3 in vitro experiments. Thus, intranasal delivery of da-hnmMSC-sEVs is an effective strategy for the treatment of neural injury repair.

The two-step strategy for enhancing the specific activity and thermostability of alginate lyase AlyG2 with mechanism for improved thermostability.

To overcome the trade-off challenge encountered in the engineering of alginate lyase AlyG2 from Seonamhaeicola algicola Gy8T and to expand its potential industrial applications, we devised a two-step strategy encompassing activity enhancement followed by thermal stability engineering. To enhance the specific activity of efficient AlyG2, we strategically substituted residues with bulky steric hindrance proximal to the active pocket with glycine or alanine. This led to the generation of three promising positive mutants, with particular emphasis on the T91S mutant, exhibiting a 1.91-fold specific activity compared to the wild type. To mitigate the poor thermal stability of T91S, mutants with negative ΔΔG values in the thermal flexibility region were screened out. Notably, the S72Ya mutant not only displayed 17.96 % further increase in specific activity but also exhibited improved stability compared to T91S, manifesting as a remarkable 30.97 % increase in relative activity following a 1-hour incubation at 42 °C. Furthermore, enhanced kinetic stability was observed. To gain deeper insights into the mechanism underlying the enhanced thermostability of the S72Ya mutant, we conducted molecular dynamics simulations, principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. The results unveiled a reduction in the flexibility of the surface loop, a stronger correlation dynamic and a narrower motion subspace in S72Ya system, along with the formation of more stable hydrogen bonds. Collectively, our findings suggest amino acids substitutions resulting in smaller side chains proximate to the active site can positively impact enzyme activity, while reducing the flexibility of surface loops emerges as a pivotal factor in conferring thermal stability. These insights offer valuable guidance and a framework for the engineering of other enzyme types.

A Broad-specificity Neutralizing Nanobody against Hepatitis E Virus Capsid Protein.

Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were ∼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology.

Ln3+ Induced Thermally Activated Delayed Fluorescence of Chiral Heterometallic Clusters Ln2Ag28.

A series of TADF-active compounds: 0D chiral Ln-Ag(I) clusters L-/D-Ln2Ag28-0D (Ln=Eu/Gd) and 2D chiral Ln-Ag(I) cluster-based frameworks L-/D-Ln2Ag28-2D (Ln=Gd) has been synthesized. Atomic-level structural analysis showed that the chiral Ag(I) cluster units {Ag14S12} in L-/D-Ln2Ag28-0D and L-/D-Ln2Ag28-2D exhibited similar configurations, linked by varying numbers of [Ln(H2O)x]3+ (x=6 for 0D, x=3 for 2D) to form the final target compounds. Temperature-dependent emission spectra and decay lifetimes measurement demonstrated the presence of TADF in L-Ln2Ag28-0D (Ln=Eu/Gd) and L-Gd2Ag28-2D. Experimentally, the remarkable TADF properties primarily originated from {Ag14S12} moieties in these compounds. Notably, {Ag14S12} in L-Eu2Ag28-0D and L-Gd2Ag28-2D displayed higher promote fluorescence rate and shorter TADF decay times than L-Gd2Ag28-0D. Combined with theoretical calculations, it was determined that the TADF behaviors of {Ag14S12} cluster units were induced by 4 f perturbation of Ln3+ ions. Specially, while maintaining ΔE(S1-T1) small enough, it can significantly increase k(S1→S0) and reduce TADF decay time by adjusting the type or number of Ln3+ ions, thus achieving the purpose of improving TADF for cluster-based luminescent materials.