Causal relationship between gut microbiota and gastrointestinal diseases: a mendelian randomization study.

BACKGROUND: Recent research increasingly highlights a strong correlation between gut microbiota and the risk of gastrointestinal diseases. However, whether this relationship is causal or merely coincidental remains uncertain. To address this, a Mendelian randomization (MR) analysis was undertaken to explore the connections between gut microbiota and prevalent gastrointestinal diseases. METHODS: Genome-wide association study (GWAS) summary statistics for gut microbiota, encompassing a diverse range of 211 taxa (131 genera, 35 families, 20 orders, 16 classes, and 9 phyla), were sourced from the comprehensive MiBioGen study. Genetic associations with 22 gastrointestinal diseases were gathered from the UK Biobank, FinnGen study, and various extensive GWAS studies. MR analysis was meticulously conducted to assess the causal relationship between genetically predicted gut microbiota and these gastrointestinal diseases. To validate the reliability of our findings, sensitivity analyses and tests for heterogeneity were systematically performed. RESULTS: The MR analysis yielded significant evidence for 251 causal relationships between genetically predicted gut microbiota and the risk of gastrointestinal diseases. This included 98 associations with upper gastrointestinal diseases, 81 with lower gastrointestinal diseases, 54 with hepatobiliary diseases, and 18 with pancreatic diseases. Notably, these associations were particularly evident in taxa belonging to the genera Ruminococcus and Eubacterium. Further sensitivity analyses reinforced the robustness of these results. CONCLUSIONS: The findings of this study indicate a potential genetic predisposition linking gut microbiota to gastrointestinal diseases. These insights pave the way for designing future clinical trials focusing on microbiome-related interventions, including the use of microbiome-dependent metabolites, to potentially treat or manage gastrointestinal diseases and their associated risk factors.

Weighted Gene Co-expression Network Analysis and Machine Learning Validation for Identifying Major Genes Related to Sjogren's Syndrome.

Sjogren's syndrome (SS) is an autoimmune disorder characterized by dry mouth and dry eyes. Its pathogenic mechanism is currently unclear. This study aims to integrate weighted gene co-expression network analysis (WGCNA) and machine learning to identify key genes associated with SS. We downloaded 3 publicly available datasets from the GEO database comprising the gene expression data of 231 SS and 78 control cases, including GSE84844, GSE48378 and GSE51092, and carried out WGCNA to elucidate differences in the abundant genes. Candidate biomarkers for SS were then identified using a LASSO regression model. Totally 6 machine-learning models were subsequently utilized for validating the biological significance of major genes according to their expression. Finally, immune cell infiltration of the SS tissue was assessed using the CIBERSORT algorithm. A weighted gene co-expression network was built to divide genes into 10 modules. Among them, blue and red modules were most closely associated with SS, and showed significant enrichment in type I interferon signaling, cellular response to type I interferon and response to virus, etc. Combined machine learning identified 5 hub genes, including OAS1, EIF2AK2, IFITM3, TOP2A and STAT1. Immune cell infiltration analysis showed that SS was associated with CD8+ T cell, CD4+ T cell, gamma delta T cell, NK cell and dendritic cell activation. WGCNA was combined with machine learning to uncover genes that may be involved in SS pathogenesis, which can be utilized for developing SS biomarkers and appropriate therapeutic targets.

Comparison of Three Different Extraction Methods on Osmanthus Volatile Oil: Aroma and Biological Activity.

The osmanthus volatile oil was welcomed by consumers even if the high price since the unique and pleasant odor. Meanwhile, the low yield of osmanthus volatile oil restricts industrial production. In this work, an osmanthus volatile oil was obtained by means of a novel ultrasonic-assisted flash extraction method and was compared with the oil from hydrodistillation and supercritical fluid extraction on yield, aroma, and biological activities. The volatile oil obtained from the ultrasonic-assisted flash extraction was obtained with the petroleum ether and got a high yield at 3.51 % within a 40-min process, an increase of nearly 81 % from the single solvent extraction. This oil also showed a high aroma intensity and aroma compound concentration. Meanwhile, the oil also has the highest antioxidant ability but lower antibacterial activity against oil from hydrodistillation. It was considered that this work was helpful for the optimization of the extraction method of osmanthus volatile oil.

Hydrogen bond-driven assembly of coral-like soy protein isolate-tannic acid microcomplex for encapsulation of limonene.

BACKGROUND: The encapsulation of flavor and aroma compounds has great potential in foods, while effective preparation in the food industry is still a great challenge. Inspired by leather tanning, tannic acid (TA) was used for deep crosslinking through hydrogen bond-driven assembly on soy protein isolate for encapsulating limonene with a high loading ratio. RESULTS: The added TA changed the protein structure and formed a limonene-loaded microcomplex. The morphology of these microcomplexes changed from smooth to rough, followed by the formation of smooth nanoparticle aggregates, by changing the amount of TA. The encapsulation efficiency and loading ratio were increased from 0.78% and 4.30% to 59.32% and 45.78% after increasing TA from 1.875 to 60 mg mL-1 . The result of confocal laser scanning microscopy indicated that limonene is evenly distributed in microcomplexes. Additionally, the results of thermal stability demonstrated protection of limonene by soy protein-tannic acid microcomplex. CONCLUSION: It is suggested that the added TA improved the encapsulation efficiency and loading ratio. Limonene is loaded in the complex in two ways. The present research provides a new and easy path for the preparation of the non-thermal soy protein aroma carrier. © 2022 Society of Chemical Industry.

Study on the Effect of Mentha × piperita L. Essential Oil on Electroencephalography upon Stimulation with Different Visual Effects.

Essential oils have long been used to fight infections and treat various diseases. Peppermint (Mentha × piperita L.) is an herbal medicine that has been widely used in daily life since ancient times, and it has a wide range of applications in food, cosmetics, and medicine. Mint oil is refreshing because of its cool and comfortable smell; therefore, it is often used in ethnopharmacological studies. The present study investigated the effects of peppermint essential oil in electroencephalographic activity response to various visual stimuli. The electroencephalographic changes of participants during peppermint essential oil inhalation under white, red, and blue colour stimulations were recorded. A rapid Fourier transform analysis was used to examine the electroencephalograph power spectra of the various microstates induced by inhaling the oils. Peppermint essential oil had various effects on the brain when subjected to different visual stimuli. Alpha waves increased in the prefrontal area in the white-sniffing group, which facilitated learning and thinking. In the blue-sniffing group, the changes were less pronounced than those in the red group, and the increased alpha wave activity in the occipital area was more controlled, indicating that the participants' visual function increased in this state. Based on EEG investigations, this is the first study to indicate that vision influences the effects of peppermint essential oils. Hence, the results of this study support the use of essential oils in a broader context to serve as a resource for future studies on the effects of different types of essential oils.

A novel strategy to identify candidate diagnostic and prognostic biomarkers for gastric cancer.

BACKGROUND: Gastric cancer (GC) is one of the most common cancer worldwide. It is essential to identify non-invasive diagnostic and prognostic biomarkers of GC. The aim of the present study was to screen candidate biomarkers associated with the pathogenesis and prognosis of GC by a novel strategy. METHODS: The expression level of gene higher in cancer than in adjacent non-cancer tissue was defined as "positive", and the top 5% genes with "positive rate" were filtered out as candidate diagnostic biomarkers in three Gene Expression Omnibus (GEO) datasets. Further, a prognostic risk model was constructed by multivariate Cox regression analysis in GEO dataset and validated in The Cancer Genome Atlas (TCGA). The expression level of candidate biomarkers was determined in serum and serum-derived exosomes of GC patients. Moreover, the effect of biomarkers in exosomes on migration of GC cells was analyzed by transwell assay. RESULTS: Ten candidate biomarkers (AGT, SERPINH1, WNT2, LIPG, PLAU, COL1A1, MMP7, MXRA5, CXCL1 and COL11A1) were identified with efficient diagnostic value in GC. A prognostic gene signature consisted of AGT, SERPINH1 and MMP7 was constructed and showed a good performance in predicting overall survivals in TCGA. Consistently, serum levels of the three biomarkers also showed high sensitivity and specificity in distinguishing GC patients from controls. In addition, the expression level of the three biomarkers were associated with malignant degree and decreased after surgery in GC patients. Moreover, the expression level of AGT and MMP7 in exosomes correlated positively with serum level. The exosomes derived from serum of GC patients can promote migration of SGC-7901 cells. After neutralized the expression level of three proteins in exosomes with antibodies, the migration of GC cells was obviously suppressed. CONCLUSIONS: Our findings provided a novel strategy to identify diagnostic biomarkers based on public datasets, and suggested that the three-gene signature was a candidate diagnostic and prognostic biomarker for patients with GC.

Plasmonic Enhanced Gold Nanoclusters-Based Photoelectrochemical Biosensor for Sensitive Alkaline Phosphatase Activity Analysis.

Low-toxicity gold nanoclusters-decorated Ag@SiO2 (Au NCs-Ag@SiO2) nanocomposites modified plasmonic photoelectrodes were first fabricated to improve the photoelectric properties of Au NCs and practical application in biological detection. Through adjusting distance between Au NCs and plasmonic silver nanoparticles (Ag NPs), the photocurrent intensity of Au NCs enhanced by 3.8 times attributed to strong competition between enhancement functions of hot electron transfer, local electric field, light scattering effects, and quenching functions of nonradiative energy transfer. Further comparison between experimental results and theoretical simulations were conducted to gain a deeper understanding toward the photoelectric enhancement mechanism. Moreover, Au NCs-Ag@SiO2 nanocomposites was successfully applied to the construction of photoelectrochemical (PEC) biosensors for sensitively detecting alkaline phosphatase activity. This proposed PEC biosensor showed a wide linear range from 0.04 to 400 U·L-1, and a low detection limit of 0.022 U·L-1.

Formation rate of secondary anal fistula after incision and drainage of perianal Sepsis and analysis of risk factors.

BACKGROUND: The choice of surgery for perianal sepsis is currently controversial. Some people advocate one-time radical surgery for perianal sepsis, while others advocate incision and drainage. The objective of this study is to observe the formation probability of secondary anal fistula after incision and drainage in patients with perianal sepsis and determine factors that contribute to secondary anal fistula after incision and drainage. METHODS: A retrospective descriptive analysis was conducted in 288 patients with perianal sepsis who were treated with anorectal surgery in the Suzhou Hospital of Traditional Chinese Medicine from January 2016 to June 2018. The patients were followed by telephone, physical examination, and pelvic MRI examination for at least 1 year after surgery. RESULTS: Three patients were not followed, 98 patients did not receive surgical treatment or one-time radical surgery for perianal sepsis, and 187 patients were ultimately identified for the study. Anal fistula was present in 105 patients, and the rate of formation of secondary anal fistula was 56.15%. There was no statistically significant difference in the fistula formation rate between different types of sepsis (P>0.05). And, in patients with secondary anal fistula, there was no significant correlation between the location of sepsis and the type of secondary anal fistula (P>0.05). CONCLUSIONS: The incidence of secondary anal fistula after incision and drainage of perianal sepsis is 56.15%, which is lower than the incidence found in previous study. Young is a risk factor for secondary anal fistula after incision and drainage of perianal sepsis. There is no significant correlation between the location of sepsis and the type of secondary anal fistula. Simple incision and drainage is a suitable choice for patients with acute perianal sepsis.

Ellagic acid-loaded soy protein isolate self-assembled particles: Characterization, stability, and antioxidant activity.

The limited water solubility and bioactivity of lipophilic phytochemicals may be enhanced by delivery systems. Ellagic acid (EA) has antioxidant and anti-inflammatory properties, but low solubility and instability limit its use in the food industry. In this study, the pH-shift method was applied to encapsulate EA with soy protein isolate (SPI). The interaction, encapsulation, and protective potential of the EA-loaded soy SPI complexes (SPI-EA) were investigated. The fluorescence spectra results suggest that the reaction between SPI and EA is spontaneous, with hydrophobic interactions predominating. Binding of EA molecules quenches the intrinsic fluorescence of SPI, mainly static quenching, with a binding site involved in the binding process. The ultraviolet (UV)-visible spectroscopy of the SPI-EA complexes included the characteristic absorption peaks of both SPI and EA, and the scanning electron microscopy images further indicated that the EA had been successfully embedded in SPI. Fourier transform infrared spectroscopy illustrates that EA has significantly changed the secondary structure of the SPI, primarily in the form of a decreased content of α-helix structures and an increased content of ß-sheet and random coil structures. The encapsulation efficiency of EA was concentration-dependent, up to 81.08%. The addition of EA reduces the size of SPI particles (d < 155 nm). In addition, the SPI-EA complex showed up to 81.05% and 96.46% 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity. TGA showed that the degradation temperature of SPI-EA complex could be extended up to 300°C. And by encapsulation of EA, the loss of EA under the action of UV light, heat treatment, and high concentration of salt ion sensitive environment can be reduced. PRACTICAL APPLICATION: Ellagic acid (EA), a natural bioactive with low water solubility and stability, can be enhanced by forming an inclusion complex with soy protein isolate (SPI). SPI-EA complex has broad potential applications in the food, beverage, and pharmaceutical industries. Multiple spectral analyses have contributed to our understanding of the formation and interaction mechanisms of the SPI-EA complex under pH-driven conditions. Stability assays have also aided in the development of dietary resources for EA.

Ginsenosides and Tumors: A Comprehensive and Visualized Analysis of Research Hotspots and Antitumor Mechanisms.

Background: Ginsenoside, the main active constituent of traditional Chinese medicine Ginseng, has been shown to play an important role in the prevention and treatment of cancer. However, the literature as well as the antitumor mechanisms of ginsenosides has not yet been systematically studied. Methods: We screened all relevant literature on ginsenosides and tumors from Web of Science during 2001-2021 and analyzed the extracted terms of these publications by VOSviewer and CiteSpace. DAVID online tool was used to perform Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathways analysis of ginsenoside-related genes. Cytoscape and String software were used to construct the interaction networks of ginsenoside-related genes and corresponding proteins. Results: A total of 919 publications were included in the study. A total of 122 identified keywords were mainly divided into 3 clusters: "pharmacological function research", "functional validation in animal models" and "anti-tumor efficacy and mechanism". The keywords of "oxidative stress" had the strongest citation burst in the past 5 years. A total of 50 genes were identified as ginsenoside-related genes in tumors. They have the function of regulating gene expression and apoptosis, and they are closely related to signaling pathways in cancers. Ginsenoside-related genes form a complex interactional network, in which TP53 and IL-6 are centrally located. Conclusions: We explored and revealed research hotspots related to the ginsenosides and tumors. More precise anti-tumor mechanism research will be promising in the future. TP53 and IL-6 may be the key points to comprehending the anti-tumor mechanism of ginsenosides.

Genetic correction of induced pluripotent stem cells from a DFNA36 patient results in morphologic and functional recovery of derived hair cell-like cells.

BACKGROUND: TMC1 is one of the most common deafness genes causing DFNA36. Patient-derived human induced pluripotent stem cells (iPSCs) provide an opportunity to modelling diseases. TMC1 p.M418K mutation in human is orthologous to Beethoven mice. Here, we investigated the differentiation, morphology and electrophysiological properties of hair cell-like cells (HC-like cells) derived from DFNA36 patient. METHODS: Inner ear HC-like cells were induced from iPSCs derived from DFNA36 (TMC1 p.M418K) patient (M+/-), normal control (M+/+) and genetic corrected iPSCs (M+/C). Immunofluorescence, scanning electron microscopy and whole-cell patch-clamp were used to study the mechanism and influence of TMC1 p.M418K mutation. RESULTS: In this study we successfully generated HC-like cells from iPSCs with three different genotypes. HC-like cells from M+/- showed defected morphology of microvilli and physiological properties compared to M+/+. HC-like cells from M+/C showed recovery in morphology of microvilli and physiological properties. CONCLUSIONS: Our results indicate that TMC1 p.M418K mutation didn't influence inner ear hair cell differentiation but the morphology of microvilli and electrophysiological properties and gene correction induced recovery. CRISPR/Cas9 gene therapy is feasible in human patient with TMC1 p.M418K mutation.

The Effect of Scanning Strategy on the Thermal Behavior and Residual Stress Distribution of Damping Alloys during Selective Laser Melting.

The manufacture of damping alloy parts with stable damping properties and high mechanical performances in the selective laser melting (SLM) process is influenced by temperature evolution and residual stress distribution. Choosing an appropriate scanning strategy, namely the specific trajectory along which the laser head scans powders within given area, is crucial, but clearly defined criteria for scanning strategy design are lacking. In this study, a three-dimensional finite element model (FEM) of the SLM process for manufacturing a WE43 alloy component was established and validated against the published experimental data. Eleven different scanning strategies were designed and simulated, considering variables such as scanning track length, direction, Out-In or In-Out strategy, start point, and interlayer variation. The results showed that scanning strategy, geometry, and layer number collectively affect temperature, melt pool, and stress outputs. For instance, starting scanning at a colder part of the powder layer could lead to a high peak temperature and low melt pool depth. A higher layer number generally results in lower cooling rate, a lower temperature gradient, a longer melt pool life, and larger melt pool dimensions. Changing the start point between scanning circulations helps mitigate detrimental residual stress. This work highlights the potential of analyzing various scanning strategy-related variables, which contributes to reducing trial-and-error tests and selecting optimal scanning strategies under different product quality requirements. This article can assist in the design of appropriate scanning strategies to prevent defects such as element loss due to evaporation, poor bonding, and deformation or cracking from high residual stress. Additionally, identifying stress concentration locations and understanding the effects of geometry and layer number on thermal and mechanical behaviors can assist in geometry design.

Inflammatory Bowel Disease and Skin Cancer: A Two-Sample Mendelian Randomization Analysis.

Background: At present, numerous clinical studies suggest a correlation between inflammatory bowel disease (IBD) and skin cancer. However, some articles present differing views that IBD does not increase the risk of skin cancer. The presence of potential reverse causality and residual confounding is inherent in conventional observational studies. Thus, this study used a two-sample Mendelian randomization (MR) study design to estimate the causal effect of IBD on the risk of skin cancer, including cutaneous malignant melanoma (CMM, also named melanoma skin cancer) and nonmelanoma skin cancer (NMSC). Design: In this study, a two-sample MR analysis was used to estimate the causal effect of IBD on skin cancer outcomes. The inverse-variance weighted (IVW) method was used as the main MR analysis, with multiple sensitivity analyses conducted to assess the robustness of findings. Results: In examining the association between IBD and NMSC, all p-values of the IVW methods were found to be <0.05, providing evidence for a causal effect of IBD on an increased risk of NMSC. However, IVW for IBD on CMM yielded p-values >0.05, indicating no causal relationship between IBD and CMM. These findings were consistent across other MR methods, with no evidence of pleiotropy or heterogeneity. Sensitivity analyses confirmed the robustness of our results. Conclusion: Using MR analysis, we found evidence for a causal effect of genetic liability for IBD on an increased risk of NMSC. However, our study did not find sufficient evidence to support a significant impact of IBD on CMM outcomes.

NADH improves AIF dimerization and inhibits apoptosis in iPSCs-derived neurons from patients with auditory neuropathy spectrum disorder.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment involving disruptions to inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself. The outcomes of cochlear implants (CI) for ANSD are variable and dependent on the location of lesion sites. Discovering a potential therapeutic agent for ANSD remains an urgent requirement. Here, 293T stable transfection cell lines and patient induced pluripotent stem cells (iPSCs)-derived auditory neurons carrying the apoptosis inducing factor (AIF) p.R422Q variant were used to pursue a therapeutic regent for ANSD. Nicotinamide adenine dinucleotide (NADH) is a main electron donor in the electron transport chain (ETC). In 293T stable transfection cells with the p.R422Q variant, NADH treatment improved AIF dimerization, rescued mitochondrial dysfunctions, and decreased cell apoptosis. The effects of NADH were further confirmed in patient iPSCs-derived neurons. The relative level of AIF dimers was increased to 150.7 % (P = 0.026) from 59.2 % in patient-neurons upon NADH treatment. Such increased AIF dimerization promoted the mitochondrial import of coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), which further restored mitochondrial functions. Similarly, the content of mitochondrial calcium (mCa2+) was downregulated from 136.7 % to 102.3 % (P = 0.0024) in patient-neurons upon NADH treatment. Such decreased mCa2+ levels inhibited calpain activity, ultimately reducing the percentage of apoptotic cells from 30.5 % to 21.1 % (P = 0.021). We also compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD. NADH treatment had comparable restorative effects on functions of ANSD patient-specific cells to that of gene correction. Our findings offer evidence of the molecular mechanisms of ANSD and introduce NADH as a potential therapeutic agent for ANSD therapy.

Osteoarthritis and risk of cardiovascular diseases: A Mendelian randomization study.

BACKGROUND: Observational studies have suggested that osteoarthritis may increase the risk of cardiovascular diseases. However, there is still no high-quality evidence to explain this causal relationship. We conducted a two-sample Mendelian randomization (MR) study to assess the associations of hip arthritis and knee arthritis with 14 types of cardiovascular diseases in the general population. METHOD: Genome-wide association studies for hip arthritis and knee arthritis were obtained from the UK Biobank. Genome-wide association studies of the 14 types of cardiovascular diseases we studied were extracted from the genetic consortia and the FinnGen consortium. Inverse variance weighted (IVW), maximum likelihood, weighted medium, penalized weighted median, and IVW (fixed effects) of MR were applied to a two-sample MR analysis. The mean pleiotropy of genetic variation and sensitivity analysis were used to evaluate the reliability of the results, and the MR-Egger test and leave-one-out method are the core evaluation methods. RESULT: Genetically predicted knee arthritis was causally associated with vein thromboembolism (IVW Odds Ratio (OR): 1.005, 95% Confidence Interval (CI): 0.842-1.199, P = 0.020) and pulmonary embolism (IVW OR: 1.003, 95% CI: 0.841-1.197, P = 0.025). Furthermore, hip arthritis also has a significant impact on cardiovascular diseases and is positively correlated with ischemic stroke (IVW OR: 1.086, 95% CI: 0.910-1.295, P = 0.024), atrial fibrillation (IVW OR: 1.093, 95% CI: 0.917-1.304, P = 0.019), and coronary artery disease (IVW OR: 1.061, 95% CI: 0.890-1.266, P = 0. 0.002). CONCLUSION: Our study suggested that osteoarthritis may increase the risk of vein thromboembolism, pulmonary embolism, ischemic stroke, atrial fibrillation, and coronary artery disease. However, the findings provided no evidence to support that osteoarthritis has a large effect on the risk of cardiovascular diseases that we studied. Further research is needed to clarify the results.

Multi-object detection for crowded road scene based on ML-AFP of YOLOv5.

Aiming at the problem of multi-object detection such as target occlusion and tiny targets in road scenes, this paper proposes an improved YOLOv5 multi-object detection model based on ML-AFP (multi-level aggregation feature perception) mechanism. Since tiny targets such as non-motor vehicle and pedestrians are not easily detected, this paper adds a micro target detection layer and a double head mechanism to improve the detection ability of tiny targets. Varifocal loss is used to achieve a more accurate ranking in the process of non-maximum suppression to solve the problem of target occlusion, and this paper also proposes a ML-AFP mechanism. The adaptive fusion of spatial feature information at different scales improves the expression ability of network model features, and improves the detection accuracy of the model as a whole. Our experimental results on multiple challenging datasets such as KITTI, BDD100K, and show that the accuracy, recall rate and mAP value of the proposed model are greatly improved, which solves the problem of multi-object detection in crowded road scenes.

Comparison of proteomic landscape of extracellular vesicles in pleural effusions isolated by three strategies.

Extracellular vesicles (EVs) derived from pleural effusion (PE) is emerging as disease biomarkers. However, the methods for isolation of EVs from PE (pEVs) were rarely studied. In our study, three methods for isolating pEVs of lung cancer patients were compared, including ultracentrifugation (UC), a combination of UC and size exclusion chromatography (UC-SEC) and a combination of UC and density gradient ultracentrifugation (UC-DGU). The subpopulation of pEVs was identified by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western blotting (WB) and nano-flow cytometry (nFCM). Additionally, the proteomic landscape of pEVs was analyzed by Label-free proteomics. The results showed that, compared with UC and UC-DGU, the UC-SEC method separated pEVs with the highest purity. In the proteomic analysis, on average, 1595 proteins were identified in the pEVs isolated by UC-SEC, much more than pEVs isolated by UC (1222) or UC-DGU (807). Furthermore, approximately 90% of identified proteins in each method were found in the EVs public database ExoCarta. Consistent with this, GO annotation indicated that the core proteins identified in each method were mainly enriched in "extracellular exosome." Many of the top 100 proteins with high expression in each method were suggested as protein markers to validate the presence of EVs in the MISEV2018 guidelines. In addition, combined with lung tissue-specific proteins and vesicular membrane proteins, we screened out and validated several novel protein markers (CD11C, HLA DPA1 and HLA DRB1), which were enriched in pEVs rather than in plasma EVs. In conclusion, our study shows that the method of UC-SEC could significantly improve the purity of EVs and the performance of mass spectrometry-based proteomic profiling in analyzing pEVs. The exosomal proteins CD11C, HLA DPA1 and HLA DRB1 may act as potential markers of pEVs. The proteomic analysis of pEVs provides important information and new ideas for studying diseases complicated with PE.

AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization. / å¬ç¥žç»ç— ç›¸å ³AIFM1åŸºå› çªå˜å¼•èµ·å‡‹äº¡è¯±å¯¼å› å­äºŒèšä½“å½¢æˆéšœç¢å¹¶å¯¼è‡´ç»†èƒžå‡‹äº¡å¢žåŠ .

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.|T260A, p.|R422W, and p.|R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%|‒|49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%|‒|17.9%, which was significantly higher than that (6.9%|‒|7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.

Impaired AIF-CHCHD4 interaction and mitochondrial calcium overload contribute to auditory neuropathy spectrum disorder in patient-iPSC-derived neurons with AIFM1 variant.

Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment caused by dysfunction of inner hair cells, ribbon synapses, spiral ganglion neurons and/or the auditory nerve itself. Approximately 1/7000 newborns have abnormal auditory nerve function, accounting for 10%-14% of cases of permanent hearing loss in children. Although we previously identified the AIFM1 c.1265 G > A variant to be associated with ANSD, the mechanism by which ANSD is associated with AIFM1 is poorly understood. We generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) via nucleofection with episomal plasmids. The patient-specific iPSCs were edited via CRISPR/Cas9 technology to generate gene-corrected isogenic iPSCs. These iPSCs were further differentiated into neurons via neural stem cells (NSCs). The pathogenic mechanism was explored in these neurons. In patient cells (PBMCs, iPSCs, and neurons), the AIFM1 c.1265 G > A variant caused a novel splicing variant (c.1267-1305del), resulting in AIF p.R422Q and p.423-435del proteins, which impaired AIF dimerization. Such impaired AIF dimerization then weakened the interaction between AIF and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). On the one hand, the mitochondrial import of ETC complex subunits was inhibited, subsequently leading to an increased ADP/ATP ratio and elevated ROS levels. On the other hand, MICU1-MICU2 heterodimerization was impaired, leading to mCa2+ overload. Calpain was activated by mCa2+ and subsequently cleaved AIF for its translocation into the nucleus, ultimately resulting in caspase-independent apoptosis. Interestingly, correction of the AIFM1 variant significantly restored the structure and function of AIF, further improving the physiological state of patient-specific iPSC-derived neurons. This study demonstrates that the AIFM1 variant is one of the molecular bases of ANSD. Mitochondrial dysfunction, especially mCa2+ overload, plays a prominent role in ANSD associated with AIFM1. Our findings help elucidate the mechanism of ANSD and may lead to the provision of novel therapies.

Role of hypoxia in the tumor microenvironment and targeted therapy.

Tumor microenvironment (TME), which is characterized by hypoxia, widely exists in solid tumors. As a current research hotspot in the TME, hypoxia is expected to become a key element to break through the bottleneck of tumor treatment. More and more research results show that a variety of biological behaviors of tumor cells are affected by many factors in TME which are closely related to hypoxia. In order to inhibiting the immune response in TME, hypoxia plays an important role in tumor cell metabolism and anti-apoptosis. Therefore, exploring the molecular mechanism of hypoxia mediated malignant tumor behavior and therapeutic targets is expected to provide new ideas for anti-tumor therapy. In this review, we discussed the effects of hypoxia on tumor behavior and its interaction with TME from the perspectives of immune cells, cell metabolism, oxidative stress and hypoxia inducible factor (HIF), and listed the therapeutic targets or signal pathways found so far. Finally, we summarize the current therapies targeting hypoxia, such as glycolysis inhibitors, anti-angiogenesis drugs, HIF inhibitors, hypoxia-activated prodrugs, and hyperbaric medicine.