Development of multiplex allele-specific RT-qPCR assays for differentiation of SARS-CoV-2 Omicron subvariants.

Quick differentiation of current circulating variants and the emerging recombinant variants of SARS-CoV-2 is essential to monitor their transmissions. However, the widely applied gene sequencing method is time-consuming and costly especially when facing recombinant variants, because a large part or whole genome sequencing is required. Allele-specific reverse transcriptase real time RT-PCR (RT-qPCR) represents a quick and cost-effective method for SNP (single nucleotide polymorphism) genotyping and has been successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 5 multiplex allele-specific RT-qPCR assays targeting 20 key mutations for quick differentiation of the Omicron subvariants (BA.1 to BA.5 and their descendants) and the recombinant variants (XBB.1 and XBB.1.5). Two parallel multiplex RT-qPCR reactions were designed to separately target the prototype allele and the mutated allele of each mutation in the allele-specific RT-qPCR assay. Optimal annealing temperatures, primer and probe dosage, and time for annealing/extension for each reaction were determined by multi-factor and multi-level orthogonal test. The variation of Cp (crossing point) values (ΔCp) between the two multiplex RT-qPCR reactions was applied to determine if a mutation occurs or not. SARS-CoV-2 subvariants and related recombinant variants were differentiated by their unique mutation patterns. The developed multiplex allele-specific RT-qPCR assays exhibited excellent analytical sensitivities (with limits of detection (LoDs) of 1.47-18.52 copies per reaction), wide linear detection ranges (109-100 copies per reaction), good amplification efficiencies (88.25 to 110.68%), excellent reproducibility (coefficient of variations (CVs) < 5% in both intra-assay and inter-assay tests), and good clinical performances (99.5-100% consistencies with Sanger sequencing). The developed multiplex allele-specific RT-qPCR assays in the present study provide an alternative tool for quick differentiation of the SARS-CoV-2 Omicron subvariants and their recombinant variants. KEY POINTS: • A panel of five multiplex allele-specific RT-qPCR assays for quick differentiation of 11 SARS-CoV-2 Omicron subvariants (BA.1, BA.2, BA.4, BA.5, and their descendants) and 2 recombinant variants (XBB.1 and XBB.1.5). • The developed assays exhibited good analytical sensitivities and reproducibility, wide linear detection ranges, and good clinical performances, providing an alternative tool for quick differentiation of the SARS-CoV-2 Omicron subvariants and their recombinant variants.

Lipid droplet accumulation in Wdr45-deficient cells caused by impairment of chaperone-mediated autophagic degradation of Fasn.

BACKGROUND: ß-Propeller protein-associated neurodegeneration (BPAN) is a genetic neurodegenerative disease caused by mutations in WDR45. The impairment of autophagy caused by WDR45 deficiency contributes to the pathogenesis of BPAN; however, the pathomechanism of this disease is largely unknown. Lipid dyshomeostasis is involved in neurogenerative diseases, but whether lipid metabolism is affected by Wdr45 deficiency and whether lipid dyshomeostasis contributes to the progression of BPAN are unclear. METHODS: We generated Wdr45 knockout SN4741 cell lines using CRISPR‒Cas9-mediated genome editing, then lipid droplets (LDs) were stained using BODIPY 493/503. Chaperone-mediated autophagy was determined by RT-qPCR and western blotting. The expression of fatty acid synthase (Fasn) was detected by western blot in the presence or absence of the lysosomal inhibitor NH4Cl and the CMA activator AR7. The interaction between Fasn and HSC70 was analyzed using coimmunoprecipitation (Co-IP) assay. Cell viability was measured by a CCK-8 kit after treatment with the Fasn inhibitor C75 or the CMA activator AR7. RESULTS: Deletion of Wdr45 impaired chaperone-mediated autophagy (CMA), thus leading to lipid droplet (LD) accumulation. Moreover, Fasn can be degraded via CMA, and that defective CMA leads to elevated Fasn, which promotes LD formation. LD accumulation is toxic to cells; however, cell viability was not rescued by Fasn inhibition or CMA activation. Inhibition of Fasn with a low concentration of C75 did not affect cell viability but decreases LD density. CONCLUSIONS: These results suggested that Fasn is essential for cell survival but that excessive Fasn leads to LD accumulation in Wdr45 knockout cells.

Identification of Candidate Genes for Red-Eyed (Albinism) Domestic Guppies Using Genomic and Transcriptomic Analyses.

Guppies are small tropical fish with brightly colored bodies and variable tail shapes. There are two phenotypes of domestic guppy eye color: red and black. The wild type is black-eyed. The main object of this study was to identify candidate genes for the red-eyed phenotype in domestic guppies. We hope to provide molecular genetic information for the development of new domestic guppy strains. Additionally, the results also contribute to basic research concerning guppies. In this study, 121 domestic guppies were used for genomic analysis (GWAS), and 44 genes were identified. Furthermore, 21 domestic guppies were used for transcriptomic analysis, and 874 differentially expressed genes (DEGs) were identified, including 357 upregulated and 517 downregulated genes. Through GO and KEGG enrichment, we identified some important terms or pathways mainly related to melanin biosynthesis and ion transport. qRT-PCR was also performed to verify the differential expression levels of four important candidate genes (TYR, OCA2, SLC45A2, and SLC24A5) between red-eyed and black-eyed guppies. Based on the results of genomic and transcriptomic analyses, we propose that OCA2 is the most important candidate gene for the red-eyed phenotype in guppies.

Regulation of Follicular Development in Chickens: WIF1 Modulates Granulosa Cell Proliferation and Progesterone Synthesis via Wnt/ß-Catenin Signaling Pathway.

Proliferation, apoptosis, and steroid hormone secretion by granulosa cells (GCs) and theca cells (TCs) are essential for maintaining the fate of chicken follicles. Our previous study showed that the Wnt inhibitor factor 1 (WIF1) plays a role in follicle selection. However, the significance of WIF1 in GC- and TC-associated follicular development was not explicitly investigated. This study found that WIF1 expression was strongly downregulated during follicle selection (p < 0.05) and was significantly higher in GCs than in TCs (p < 0.05). WIF1 inhibits proliferation and promotes apoptosis in GCs. Additionally, it promotes progesterone secretion in prehierarchal GCs (pre-GCs, 1.16 ± 0.05 ng/mg vs. 1.58 ng/mg ± 0.12, p < 0.05) and hierarchal GCs (hie-GCs, 395.00 ng/mg ± 34.73 vs. 527.77 ng/mg ± 27.19, p < 0.05) with the participation of the follicle-stimulating hormone (FSH). WIF1 affected canonical Wnt pathways and phosphorylated ß-catenin expression in GCs. Furthermore, 604 upregulated differentially expressed genes (DEGs) and 360 downregulated DEGs in WIF1-overexpressed GCs were found through RNA-seq analysis (criteria: |log2⁡(FoldChange)| > 1 and p_adj < 0.05). Cytokine-cytokine receptor interaction and the steroid hormone biosynthesis pathway were identified. In addition, the transcript of estrogen receptor 2 (ESR2) increased significantly (log2⁡(FoldChange) = 1.27, p_adj < 0.05). Furthermore, we found that WIF1 regulated progesterone synthesis by upregulating ESR2 expression in GCs. Additionally, WIF1 suppressed proliferation and promoted apoptosis in TCs. Taken together, these results reveal that WIF1 stimulates follicle development by promoting GC differentiation and progesterone synthesis, which provides an insight into the molecular mechanism of follicle selection and egg-laying performance in poultry.

Evolutionary and Phylogenetic Dynamics of SARS-CoV-2 Variants: A Genetic Comparative Study of Taiyuan and Wuhan Cities of China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA genome-containing virus which has infected millions of people all over the world. The virus has been mutating rapidly enough, resulting in the emergence of new variants and sub-variants which have reportedly been spread from Wuhan city in China, the epicenter of the virus, to the rest of China and all over the world. The occurrence of mutations in the viral genome, especially in the viral spike protein region, has resulted in the evolution of multiple variants and sub-variants which gives the virus the benefit of host immune evasion and thus renders modern-day vaccines and therapeutics ineffective. Therefore, there is a continuous need to study the genetic characteristics and evolutionary dynamics of the SARS-CoV-2 variants. Hence, in this study, a total of 832 complete genomes of SARS-CoV-2 variants from the cities of Taiyuan and Wuhan in China was genetically characterized and their phylogenetic and evolutionary dynamics studied using phylogenetics, genetic similarity, and phylogenetic network analyses. This study shows that the four most prevalent lineages in Taiyuan and Wuhan are as follows: the Omicron lineages EG.5.1.1, followed by HK.3, FY.3, and XBB.1.16 (Pangolin classification), and clades 23F (EG.5.1), followed by 23H (HK.3), 22F (XBB), and 23D (XBB.1.9) (Nextclade classification), and lineage B followed by the Omicron FY.3, lineage A, and Omicron FL.2.3 (Pangolin classification), and the clades 19A, followed by 22F (XBB), 23F (EG.5.1), and 23H (HK.3) (Nextclade classification), respectively. Furthermore, our genetic similarity analysis show that the SARS-CoV-2 clade 19A-B.4 from Wuhan (name starting with 412981) has the least genetic similarity of about 95.5% in the spike region of the genome as compared to the query sequence of Omicron XBB.2.3.2 from Taiyuan (name starting with 18495234), followed by the Omicron FR.1.4 from Taiyuan (name starting with 18495199) with ~97.2% similarity and Omicron DY.3 (name starting with 17485740) with ~97.9% similarity. The rest of the variants showed ≥98% similarity with the query sequence of Omicron XBB.2.3.2 from Taiyuan (name starting with 18495234). In addition, our recombination analysis results show that the SARS-CoV-2 variants have three statistically significant recombinant events which could have possibly resulted in the emergence of Omicron XBB.1.16 (recombination event 3), FY.3 (recombination event 5), and FL.2.4 (recombination event 7), suggesting some very important information regarding viral evolution. Also, our phylogenetic tree and network analyses show that there are a total of 14 clusters and more than 10,000 mutations which may have probably resulted in the emergence of cluster-I, followed by 47 mutations resulting in the emergence of cluster-II and so on. The clustering of the viral variants of both cities reveals significant information regarding the phylodynamics of the virus among them. The results of our temporal phylogenetic analysis suggest that the variants of Taiyuan have likely emerged as independent variants separate from the variants of Wuhan. This study, to the best of our knowledge, is the first ever genetic comparative study between Taiyuan and Wuhan cities in China. This study will help us better understand the virus and cope with the emergence and spread of new variants at a local as well as an international level, and keep the public health authorities informed for them to make better decisions in designing new viral vaccines and therapeutics. It will also help the outbreak investigators to better examine any future outbreak.

Dietary, addictive and habitual factors, and risk of colorectal cancer.

BACKGROUND: In Pakistan, the incidence of colorectal cancer (CRC) has sharply increased in recent years. Although several studies have reported global risk factors for CRC, no study has been conducted in Khyber Pakhtunkhwa (KPK), Pakistan, to investigate the risk factors associated with the increased CRC burden in this population. OBJECTIVES: Therefore, we conducted a clinical survey using a case-control study design to explore the risk factors associatd with CRC. METHODS: In the present study, one control was enrolled for each case. Both cases and controls were asked to complete a questionnaire to gather data. We analyzed all data using SPSS. RESULTS: Our study found that certain dietary factors, such as consuming fast food (OR: 3.0; P = 0.0001) and reusing ghee (OR: 2.45; P = 0.0001) and oil (OR: 4.30; P = 0.0001), increase the risk of CRC. Additionally, use of tobacco products like smoking cigarettes (OR: 1.91; P = 0.0001) and using snuff (OR: 3.72; P = 0.0001) significantly increases the risk of CRC. Certain habitual factors, including binge eating (OR: 2.42; P = 0.0001) and spending excessive time watching TV (OR: 1.98; P = 0.0001), also increase the odds of developing CRC. However, our study also identified some protective factors against CRC, such as consuming vegetables (OR: .41; P = 0.0001), developing healthy eating habits (OR: .61; P = 0.0001), and maintaining regular sleeping patterns (OR: .45; P = 0.0001). CONCLUSION: Given these findings, targeted health education is necessary to prevent the increase in CRC in this area. We also recommend developing and enforcing appropriate control guidelines for cancer risk factors to curb the incidence of CRC.

Exosomal Small RNA Sequencing Profiles in Plasma from Subjects with Latent Mycobacterium tuberculosis Infection.

Despite huge efforts, tuberculosis (TB) is still a major public health threat worldwide, with approximately 23% of the human population harboring a latent TB infection (LTBI). LTBI can reactivate and progress to active and transmissible TB disease, contributing to its spread within the population. The challenges in diagnosing and treating LTBI patients have been major factors contributing to this phenomenon. Exosomes offer a novel avenue for investigating the process of TB infection. In this study, we conducted small RNA sequencing to investigate the small RNA profiles of plasma exosomes derived from individuals with LTBI and healthy controls. Our findings revealed distinct miRNA profiles in the exosomes between the two groups. We identified 12 differentially expressed miRNAs through this analysis, which were further validated via qRT-PCR using the same exosomes. Notably, six miRNAs (hsa-miR-7850-5p, hsa-miR-1306-5p, hsa-miR-363-5p, hsa-miR-374a-5p, hsa-miR-4654, has-miR-6529-5p, and hsa-miR-140-5p) exhibited specifically elevated expression in individuals with LTBI. Gene ontology and KEGG pathway analyses revealed that the targets of these miRNAs were enriched in functions associated with ferroptosis and fatty acid metabolism, underscoring the critical role of these miRNAs in regulating the intracellular survival of Mycobacterium tuberculosis (Mtb). Furthermore, our results indicated that the overexpression of miR-7850-5p downregulated the expression of the SLC11A1 protein in both Mtb-infected and Mtb-uninfected THP1 cells. Additionally, we observed that miR-7850-5p promoted the intracellular survival of Mtb by suppressing the expression of the SLC11A1 protein. Overall, our findings provide valuable insights into the role of miRNAs and repetitive region-derived small RNAs in exosomes during the infectious process of Mtb and contribute to the identification of potential molecular targets for the detection and diagnosis of latent tuberculosis.

Syringaldehyde Exhibits Antibacterial and Antioxidant Activities against Mycobacterium marinum Infection.

Tuberculosis (TB) is caused by infection with Mycobacterium tuberculosis (Mtb), which has a unique resistance to many antimicrobial agents. TB has emerged as a significant worldwide health issue because of the rise of multidrug-resistant strains causing drug-resistant TB (DR-TB). As a result, the development of new drugs or effective strategies is crucial for patients with TB. Mycobacterium marinum (Mm) and Mtb are both species of mycobacteria. In zebrafish, Mm proliferates and forms chronic granulomatous infections, which are similar to Mtb infections in lung tissue. Syringaldehyde (SA) is a member of the phenolic aldehyde family found in various plants. Here, we investigated its antioxidative and antibacterial properties in Mm-infected cells and zebrafish. Our results demonstrated that SA inhibits Mm-infected pulmonary epithelial cells and inhibits the proliferation of Mm in Mm-infected zebrafish, suggesting that SA provides an antibacterial effect during Mm infection. Further study demonstrated that supplementation with SA inhibits the production of malondialdehyde (MDA) and reactive oxygen species (ROS) and increases the levels of reduced glutathione (GSH) in Mm-infection-induced macrophages. SA inhibits the levels of MDA in Mm-infected zebrafish, suggesting that SA exerts antioxidative effects in vivo. Additionally, we found that SA promotes the expression of NRF2/HO-1/NQO-1 and the activation of the AMPK-α1/AKT/GSK-3ß signaling pathway. In summary, our data demonstrated that SA exerts antioxidative and antibacterial effects during Mm infection both in vivo and in vitro and that the antioxidative effects of SA may be due to the regulation of NRF2/HO-1/NQO-1 and the AMPK-α1/AKT/GSK-3ß signaling pathway.

Proteo-transcriptomic profiles reveal key regulatory pathways and functions of LDHA in the ovulation of domestic chickens (Gallus gallus).

BACKGROUND: In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear. RESULTS: Critical biochemical events involved in ovulation in domestic chickens (Gallus gallus) were evaluated by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory follicle (F1) and postovulatory follicle (POF1) in continuous laying (CL) and intermittent laying (IL) chickens indicated the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes (DEGs), and the smallest difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin A, and lactate dehydrogenase A (LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro. CONCLUSIONS: This study reveals several important biochemical events involved in the process of ovulation, as well as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian species.

The New Delhi metallo-ß-lactamase-1 biosensor rapidly and accurately detected antibiotic plasma concentrations in cefuroxime-treated patients.

OBJECTIVES: Therapeutic drug monitoring (TDM) of ß-lactam antibiotics in critically ill patients may benefit dose optimisation, thus improving therapeutic outcomes. However, rapidly and accurately detecting these antibiotics in blood remains a challenge. This research group recently developed a thermometric biosensor called the New Delhi metallo-ß-lactamase-1 (NDM-1) biosensor, which detects multiple classes of ß-lactam antibiotics in spiked plasma samples. METHODS: This study assessed the NDM-1 biosensor's effectiveness in detecting plasma concentrations of ß-lactam antibiotics in treated patients. Seven patients receiving cefuroxime were studied. Plasma samples collected pre- and post-antibiotic treatment were analysed using the NDM-1 biosensor and compared with liquid chromatography coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: The biosensor detected plasma samples without dilution, and a brief pre-treatment using a polyvinylidene fluoride filter significantly lowered matrix effects, reducing the running time to 5-8 minutes per sample. The assay's linear range for cefuroxime (6.25-200 mg/L) covered target concentrations during the trough phase of pharmacokinetics in critically ill patients. The pharmacokinetic properties of cefuroxime in treated patients determined by the NDM-1 biosensor and the UPLC-MS/MS were comparable, and the cefuroxime plasma concentrations measured by the two methods showed statistically good consistency. CONCLUSION: These data demonstrate that the NDM-1 biosensor assay is a fast, sensitive, and accurate method for detecting cefuroxime plasma concentrations in treated patients and highlights the NDM-1 biosensor as a promising tool for on-site TDM of ß-lactam antibiotics in critically ill patients.

GLA Mutations Suppress Autophagy and Stimulate Lysosome Generation in Fabry Disease.

Fabry disease (FD) is an X-linked recessive inheritance lysosomal storage disorder caused by pathogenic mutations in the GLA gene leading to a deficiency of the enzyme alpha-galactosidase A (α-Gal A). Multiple organ systems are implicated in FD, most notably the kidney, heart, and central nervous system. In our previous study, we identified four GLA mutations from four independent Fabry disease families with kidney disease or neuropathic pain: c.119C>A (p.P40H), c.280T>C (C94R), c.680G>C (p.R227P) and c.801+1G>A (p.L268fsX3). To reveal the molecular mechanism underlying the predisposition to Fabry disease caused by GLA mutations, we analyzed the effects of these four GLA mutations on the protein structure of α-galactosidase A using bioinformatics methods. The results showed that these mutations have a significant impact on the internal dynamics and structures of GLA, and all these altered amino acids are close to the enzyme activity center and lead to significantly reduced enzyme activity. Furthermore, these mutations led to the accumulation of autophagosomes and impairment of autophagy in the cells, which may in turn negatively regulate autophagy by slightly increasing the phosphorylation of mTOR. Moreover, the overexpression of these GLA mutants promoted the expression of lysosome-associated membrane protein 2 (LAMP2), resulting in an increased number of lysosomes. Our study reveals the pathogenesis of these four GLA mutations in FD and provides a scientific foundation for accurate diagnosis and precise medical intervention for FD.

Gastroprotective Effect of Isoferulic Acid Derived from Foxtail Millet Bran against Ethanol-Induced Gastric Mucosal Injury by Enhancing GALNT2 Enzyme Activity.

Excessive alcohol consumption has led to the prevalence of gastrointestinal ailments. Alleviating gastric disorders attributed to alcohol-induced thinning of the mucus layer has centered on enhancing mucin secretion as a pivotal approach. In this study, foxtail millet bran polyphenol BPIS was divided into two components with MW < 200 D and MW > 200 D by molecular interception technology. Combined with MTT, cell morphology observation, and trypan blue staining, isoferulic acid (IFA) within the MW < 200 D fraction was determined as the effective constituent to mitigate ethanol-induced damage of gastric epithelial cells. Furthermore, a Wistar rat model with similar clinical features to alcohol-induced gastric mucosal injury was established. Then, gastric morphological observation, H&E staining, and assessments of changes in gastric hexosamine content and gastric wall binding mucus levels were carried out, and the results revealed that IFA (10 mg/Kg) significantly ameliorated alcohol-induced gastric mucosal damage. Finally, we applied techniques including Co-IP, molecular docking, and fluorescence spectroscopy and found that IFA inhibited the alcohol-induced downregulation of N-acetylgalactosamintransferase 2 (GALNT2) activity related to mucus synthesis through direct interaction with GALNT2 in gastric epithelial cells, thus promoting mucin synthesis. Our study lays a foundation for whole grain dietary intervention tailored to individuals suffering from alcoholic gastric mucosal injury.

Brain-Type Glycogen Phosphorylase (PYGB) in the Pathologies of Diseases: A Systematic Review.

Glycogen metabolism is a form of crucial metabolic reprogramming in cells. PYGB, the brain-type glycogen phosphorylase (GP), serves as the rate-limiting enzyme of glycogen catabolism. Evidence is mounting for the association of PYGB with diverse human diseases. This review covers the advancements in PYGB research across a range of diseases, including cancer, cardiovascular diseases, metabolic diseases, nervous system diseases, and other diseases, providing a succinct overview of how PYGB functions as a critical factor in both physiological and pathological processes. We present the latest progress in PYGB in the diagnosis and treatment of various diseases and discuss the current limitations and future prospects of this novel and promising target.

miR-4687-5p Affects Intracellular Survival of Mycobacterium tuberculosis through Its Regulation of NRAMP1 Expression in A549 Cells.

Tuberculosis (TB), as one of the leading causes of death, poses a serious predicament to the world. MicroRNAs (miRNAs) play a role in the post-transcriptional regulation of gene expression. It has been reported that the expression of miRNAs changes upon mycobacterial infection; the screening and identification of miRNAs regulating the expression of genes could benefit our understanding of TB pathogenesis and generate effective strategies for its control and prevention. In this study, luciferase assays showed that miR-4687-5p is bound to the 3'-untranslated region of natural resistance-associated macrophage protein 1 (NRAMP1). Additionally, we found a significant increase in miR-4687-5p expression in Mycobacterium tuberculosis (Mtb)-infected A549 cells. Concomitantly, we detected a reduced level of NRAMP1 expression, suggesting that NRAMP1 is one of the targets of miR-4687-5p. Infection experiments evidenced that the transfection of miR-4687-5p induced a decrease in NRAMP1 expression and increased intracellular Mtb loads post-infection, indicating that miR-4687-5p promotes the intracellular survival of Mtb through its downregulation of the NRAMP1 protein level. We also found that the transfection of miR-4687-5p induced increased apoptosis and decreased cell proliferation post-infection with Mtb. The results presented in our study suggest that miR-4687-5p may be indicative of the susceptibility of Mtb infection to humans and could act as a potential therapeutic target for tuberculosis treatment.

Sulforaphane Inhibits Oxidative Stress and May Exert Anti-Pyroptotic Effects by Modulating NRF2/NLRP3 Signaling Pathway in Mycobacterium tuberculosis-Infected Macrophages.

Sulforaphane (SFN) is a natural isothiocyanate derived from cruciferous vegetables such as broccoli, Brussels sprouts, and cabbage. SFN plays a crucial role in maintaining redox homeostasis by interacting with the active cysteine residues of Keap1, leading to the dissociation and activation of NRF2 in various diseases. In this study, our objective was to investigate the impact of SFN on oxidative stress and pyroptosis in Mycobacterium tuberculosis (Mtb)-infected macrophages. Our findings demonstrated that Mtb infection significantly increased the production of iNOS and ROS, indicating the induction of oxidative stress in macrophages. However, treatment with SFN effectively suppressed the expression of iNOS and COX-2 and reduced MDA and ROS levels, while enhancing GSH content as well as upregulating NRF2, HO-1, and NQO-1 expression in Mtb-infected RAW264.7 macrophages and primary peritoneal macrophages from WT mice. These results suggest that SFN mitigates oxidative stress by activating the NRF2 signaling pathway in Mtb-infected macrophages. Furthermore, excessive ROS production activates the NLRP3 signaling pathway, thereby promoting pyroptosis onset. Further investigations revealed that SFN effectively suppressed the expression of NLRP3, Caspase-1, and GSDMD, IL-1ß, and IL-18 levels, as well as the production of LDH, suggesting that it may exhibit anti-pyroptotic effects through activation of the NRF2 signaling pathway and reductions in ROS production during Mtb infection. Moreover, we observed that SFN also inhibited the expression of NLRP3, ASC, Caspase1, and IL-1ß along with LDH production in Mtb-infected primary peritoneal macrophages from NFR2-/- mice. This indicates that SFN can directly suppress NLRP3 activation and possibly inhibit pyroptosis initiation in an NRF2-independent manner. In summary, our findings demonstrate that SFN exerts its inhibitory effects on oxidative stress by activating the NRF2 signaling pathway in Mtb-infected macrophages, while it may simultaneously exert anti-pyroptotic properties through both NRF2-dependent and independent mechanisms targeting the NLRP3 signaling pathway.

Transcriptomic profiling of Dip2a in the neural differentiation of mouse embryonic stem cells.

Introduction: The disconnected-interacting protein 2 homolog A (DIP2A), a member of disconnected-interacting 2 protein family, has been shown to be involved in human nervous system-related mental illness. This protein is highly expressed in the nervous system of mouse. Mutation of mouse DIP2A causes defects in spine morphology and synaptic transmission, autism-like behaviors, and defective social novelty [5], [27], indicating that DIP2A is critical to the maintenance of neural development. However, the role of DIP2A in neural differentiation has yet to be investigated. Objective: To determine the role of DIP2A in neural differentiation, a neural differentiation model was established using mouse embryonic stem cells (mESCs) and studied by using gene-knockout technology and RNA-sequencing-based transcriptome analysis. Results: We found that DIP2A is not required for mESCs pluripotency maintenance, but loss of DIP2A causes the neural differentiation abnormalities in both N2B27 and KSR medium. Functional knockout of Dip2a gene also decreased proliferation of mESCs by perturbation of the cell cycle and profoundly inhibited the expression of a large number of neural development-associated genes which mainly enriched in spinal cord development and postsynapse assembly. Conclusions: The results of this report demonstrate that DIP2A plays an essential role in regulating differentiation of mESCs towards the neural fate.