Antimicrobial Properties of Carboxymethyl Cellulose/Starch/N'N Methylenebisacrylamide Membranes Endowed by Ultrasound and Their Potential Application in Antimicrobial Packaging.

Cellulose is used widely in antimicrobial packaging due to its abundance in nature, biodegradability, renewability, non-toxicity, and low cost. However, how efficiently and rapidly it imparts high antimicrobial activity to cellulose-based packaging materials remains a challenge. In this work, Ag NPs were deposited on the surface of carboxymethyl cellulose/starch/N'N Methylenebisacrylamide film using ultrasonic radiation. Morphology and structure analysis of as-prepared films were conducted, and the antibacterial effects under different ultrasonic times and reductant contents were investigated. These results showed that Ag NPs were distributed uniformly on the film surface under an ultrasonic time of 45 min. The size of Ag NPs changes as the reducing agent content decreases. The composite film demonstrated a slightly better antibacterial effect against E. coli than against S. aureus. Therefore, this work can provide valuable insights for the research on antimicrobial packaging.

Glucagon-like peptide-1 receptor agonists: new strategies and therapeutic targets to treat atherosclerotic cardiovascular disease.

Atherosclerotic cardiovascular disease (ASCVD) is a leading cause of cardiovascular mortality and is increasingly prevalent in our population. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) can safely and effectively lower glucose levels while concurrently managing the full spectrum of ASCVD risk factors and improving patients' long-term prognosis. Several cardiovascular outcome trials (CVOTs) have been carried out to further investigate the cardiovascular benefits of GLP-1RAs. Analyzing data from CVOTs can provide insights into the pathophysiologic mechanisms by which GLP-1RAs are linked to ASCVD and define the use of GLP-1RAs in clinical practice. Here, we discussed various mechanisms hypothesized in previous animal and preclinical human studies, including blockade of the production of adhesion molecules and inflammatory factors, induction of endothelial cells' synthesis of nitric oxide, protection of mitochondrial function and restriction of oxidative stress, suppression of NOD-like receptor thermal protein domain associated protein three inflammasome, reduction of foam cell formation and macrophage inflammation, and amelioration of vascular smooth muscle cell dysfunction, to help explain the cardiovascular benefits of GLP-1RAs in CVOTs. This paper provides an overview of the clinical research, molecular processes, and possible therapeutic applications of GLP-1RAs in ASCVD, while also addressing current limitations in the literature and suggesting future research directions.

Unveiling Gene Expression Dynamics during Early Embryogenesis in Cynoglossus semilaevis: A Transcriptomic Perspective.

Commencing with sperm-egg fusion, the early stages of metazoan development include the cleavage and formation of blastula and gastrula. These early embryonic events play a crucial role in ontogeny and are accompanied by a dramatic remodeling of the gene network, particularly encompassing the maternal-to-zygotic transition. Nonetheless, the gene expression dynamics governing early embryogenesis remain unclear in most metazoan lineages. We conducted transcriptomic profiling on two types of gametes (oocytes and sperms) and early embryos (ranging from the four-cell to the gastrula stage) of an economically valuable flatfish-the Chinese tongue sole Cynoglossus semilaevis (Pleuronectiformes: Cynoglossidae). Comparative transcriptome analysis revealed that large-scale zygotic genome activation (ZGA) occurs in the blastula stage, aligning with previous findings in zebrafish. Through the comparison of the most abundant transcripts identified in each sample and the functional analysis of co-expression modules, we unveiled distinct functional enrichments across different gametes/developmental stages: actin- and immune-related functions in sperms; mitosis, transcription inhibition, and mitochondrial function in oocytes and in pre-ZGA embryos (four- to 1000-cell stage); and organ development in post-ZGA embryos (blastula and gastrula). These results provide insights into the intricate transcriptional regulation of early embryonic development in Cynoglossidae fish and expand our knowledge of developmental constraints in vertebrates.

How short peptides can disassemble ultra-stable tau fibrils extracted from Alzheimer's disease brain by a strain-relief mechanism.

Reducing fibrous aggregates of protein tau is a possible strategy for halting progression of Alzheimer's disease (AD). Previously we found that in vitro the D-peptide D-TLKIVWC disassembles tau fibrils from AD brains (AD-tau) into benign segments with no energy source present beyond ambient thermal agitation. This disassembly by a short peptide was unexpected, given that AD-tau is sufficiently stable to withstand disassembly in boiling SDS detergent. To consider D peptide-mediated disassembly as a potential therapeutic for AD, it is essential to understand the mechanism and energy source of the disassembly action. We find assembly of D-peptides into amyloid-like fibrils is essential for tau fibril disassembly. Cryo-EM and atomic force microscopy reveal that these D-peptide fibrils have a right-handed twist and embrace tau fibrils which have a left-handed twist. In binding to the AD-tau fibril, the oppositely twisted D-peptide fibril produces a strain, which is relieved by disassembly of both fibrils. This strain-relief mechanism appears to operate in other examples of amyloid fibril disassembly and provides a new direction for the development of first-in-class therapeutics for amyloid diseases.

Novel Human Milk Fat Substitutes Based on Medium- and Long-Chain Triacylglycerol Regulate Thermogenesis, Lipid Metabolism, and Gut Microbiota Diversity in C57BL/6J Mice.

Human milk is naturally rich in medium- and long-chain triacylglycerols (MLCT), accounting for approximately 30% of the total fat. However, infant formula fat is prepared using a physical blend of vegetable oils, which rarely contains MLCT, similar to human milk. The differences in MLCT between human milk and infant formulas may cause different lipid metabolisms and physiological effects on infants, which are unknown. This study aimed to analyze the metabolic characteristics of formula lipid containing novel human milk fat substitutes based on MLCT (FL-MLCT) and compare their effects with those of the physical blend of vegetable oils (FL-PB) on lipid metabolism and gut microbiota in mice. Compared with the FL-PB group, the FL-MLCT group showed increased energy expenditure, decreased serum triacylglycerol level, and significantly lower aspartate aminotransferase level, epididymal and perirenal fat weight, and adipocyte size. Moreover, the abundances of Firmicutes/Bacteroidota, Actinobacteriota, and Desulfovibrionaceae were significantly decreased in the FL-MLCT group. Novel human milk fat substitutes MLCT could inhibit visceral fat accumulation, improve liver function, and modulate the mice gut microbiota composition, which may contribute to controlling obesity.

Sesamin alleviates lipid accumulation induced by oleic acid via PINK1/Parkin-mediated mitophagy in HepG2 cells.

Sesamin, a special compound present in sesame and sesame oil, has been reported a role in regulating lipid metabolism, while the underlying mechanisms remain unclear. Autophagy has been reported associated with lipid metabolism and regarded as a key modulator in liver steatosis. The present work aimed to investigate whether sesamin could exert its protective effects against lipid accumulation via modulating autophagy in HepG2 cells stimulated with oleic acid (OA). Cell viability was evaluated using the CCK-8 method, and triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein, cholesterol (LDL-C), alanine aminotransferase (ALT), along with aspartate aminotransferase (AST) were assessed by oil red O staining, transmission electron microscopy (TEM), and biochemical kits to investigate the lipid-lowering effects of sesamin. Differentially expressed genes were screened by RNA sequencing and validated using real-time quantitative PCR and Western blot. Autophagy and mitophagy related molecules were analyzed employing TEM, Western blot, and immunofluorescence. The data shows that in HepG2 cells stimulated by OA, sesamin reduces levels of TG, TC, LDL-C, ALT, and AST while elevating HDL-C, alleviates the lipid accumulation and improves fatty acid metabolism through modulating the levels of fat metabolism related genes including PCSK9, FABP1, CD36, and SOX4. Sesamin restores the suppressed autophagy in HepG2 cells caused by OA, which could be blocked by autophagy inhibitors. This indicates that sesamin improves fatty acid metabolism by enhancing autophagy levels, thereby mitigating the intracellular lipid accumulation. Furthermore, sesamin significantly enhances the mitophagy and improves mitochondrial homeostasis via activating the PINK/Parkin pathway. These data suggest that sesamin alleviates the excessive lipid accumulation in HepG2 caused by OA by restoring the impaired mitophagy via the PINK1/Parkin pathway, probably playing a preventive or therapeutic role in hepatic steatosis.

Sodium butyrate alleviates free fatty acid-induced steatosis in primary chicken hepatocytes via the AMPK/PPARα pathway.

Fatty liver hemorrhagic syndrome (FLHS) is a prevalent metabolic disorder observed in egg-laying hens, characterized by fatty deposits and cellular steatosis in the liver. Our preliminary investigations have revealed a marked decrease in the concentration of butyric acid in the FLHS strain of laying hens. It has been established that sodium butyrate (NaB) protects against metabolic disorders. However, the underlying mechanism by which butyrate modulates hepato-lipid metabolism to a great extent remains unexplored. In this study, we constructed an isolated in vitro model of chicken primary hepatocytes to induce hepatic steatosis by free fatty acids (FFA). Our results demonstrate that treatment with NaB effectively mitigated FFA-induced hepatic steatosis in chicken hepatocytes by inhibiting lipid accumulation, downregulating the mRNA expression of lipo-synthesis-related genes (sterol regulatory element binding transcription factor 1 (SREBF1), acetyl-CoA carboxylase 1(ACC1), fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), liver X receptor α (LXRα), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)) (P < 0.05), and upregulating the mRNA and protein expression of AMP-activated protein kinase α1 (AMPKα1), peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyl-transferase 1A (CPT1A) (P < 0.05). Moreover, AMPK and PPARα inhibitors (Compound C (Comp C) and GW6471, respectively) reversed the protective effects of NaB against FFA-induced hepatic steatosis by blocking the AMPK/PPARα pathway, leading to lipid droplet accumulation and triglyceride (TG) contents in chicken primary hepatocytes. With these findings, NaB can alleviate hepatocyte lipoatrophy injury by activating the AMPK/PPARα pathway, promoting fatty acid oxidation, and reducing lipid synthesis in chicken hepatocytes, potentially being able to provide new ideas for the treatment of FLHS.

Sodium Butyrate Alleviates Free Fatty Acid-Induced Steatosis in Primary Chicken Hepatocytes via Regulating the ROS/GPX4/Ferroptosis Pathway.

Fatty liver hemorrhagic syndrome (FLHS) in laying hens is a nutritional metabolic disease commonly observed in high-yielding laying hens. Sodium butyrate (NaB) and ferroptosis were reported to contribute to the pathogenesis of fatty liver-related diseases. However, the underlying mechanism of NaB in FLHS and whether it mediates ferroptosis remains unclear. A chicken primary hepatocyte induced by free fatty acids (FFAs, keeping the ratio of sodium oleate and sodium palmitate concentrations at 2:1) was established, which received treatments with NaB, the ferroptosis inducer RAS-selective lethal 3 (RSL3), and the inhibitor ferrostatin-1 (Fer-1). As a result, NaB increased biochemical and lipid metabolism indices, and the antioxidant level, while inhibiting intracellular ROS accumulation and the activation of the ferroptosis signaling pathway, as evidenced by a reduction in intracellular iron concentration, upregulated GPX4 and xCT expression, and inhibited NCOA4 and ACSL4 expression. Furthermore, treatment with Fer-1 reinforced the protective effects of NaB, while RSL3 reversed it by blocking the ROS/GPX4/ferroptosis pathway, leading to the accumulation of lipid droplets and oxidative stress. Collectively, our findings demonstrated that NaB protects hepatocytes by regulating the ROS/GPX4-mediated ferroptosis pathway, providing a new strategy and target for the treatment of FLHS.

Enzymatic Synthesis of Structured Lipids Enriched with Medium- and Long-Chain Triacylglycerols via Pickering Emulsion-Assisted Interfacial Catalysis: A Preliminary Exploration.

Medium- and long-chain triacylglycerol (MLCT), as a novel functional lipid, is valuable due to its special nutritional properties. Its low content in natural resources and inefficient synthesis during preparation have limited its practical applications. In this study, we developed an effective Pickering emulsion interfacial catalysis system (PE system) for the enzymatic synthesis of MLCT by trans-esterification. Lipase NS 40086 served simultaneously as a catalyst and a solid emulsifier to stabilize the Pickering emulsion. Benefitting from the sufficient oil-water interface, the obtained PE system exhibited outstanding catalytic efficiency, achieving 77.5% of MLCT content within 30 min, 26% higher than that of a water-free system. The Km value (0.259 mM) and activation energy (14.45 kJ mol-1) were 6.8-fold and 1.6-fold lower than those of the water-free system, respectively. The kinetic parameters as well as the molecular dynamics simulation and the tunnel analysis implied that the oil-water interface enhanced the binding between substrate and lipase and thus boosted catalytic efficiency. The conformational changes in the lipase were further explored by FT-IR. This method could give a novel strategy for enhancing lipase activity and the design of efficient catalytic systems to produce added-value lipids. This work will open a new methodology for the enzymatic synthesis of structured lipids.

Medium- and Long-Chain Triacylglycerol: Preparation, Health Benefits, and Food Utilization.

Medium- and long-chain triacylglycerol (MLCT) is a structured lipid with both medium- and long-chain fatty acids in one triacylglycerol molecule. Compared with long-chain triacylglycerol (LCT), which is mainly present in common edible oils, and the physical blend of medium-chain triacylglycerol with LCT (MCT/LCT), MLCT has different physicochemical properties, metabolic characteristics, and nutritional values. In this article, the recent advances in the use of MLCT in food formulations are reviewed. The natural sources and preparation of MLCT are discussed. A comprehensive summary of MLCT digestion, absorption, transport, and oxidation is provided as well as its health benefits, including reducing the risk of overweight, hypolipidemic and hypoglycemic effects, etc. The potential MLCT uses in food formulations, such as infant formulas, healthy foods for weight loss, and sports foods, are summarized. Finally, the current safety assessment and regulatory status of MLCT in food formulations are reviewed. Expected final online publication date for the Annual Review of Food Science and Technology, Volume 15 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Disrupting the gut microbiota/metabolites axis by Di-(2-ethylhexyl) phthalate drives intestinal inflammation via AhR/NF-κB pathway in mice.

Di-(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer known for its environmental endocrine-disrupting properties, posing potential risks to various organs. However, the precise impact of DEHP on intestinal health and its contribution to the initiation of intestinal inflammation remains elucidated. This study aims to investigate the underlying mechanisms of DEHP-induced intestinal inflammation in mice, specifically focusing on the complex interplay between the gut microbiota-metabolite axis and associated pathophysiological alterations. Our findings showed that DEHP-induced damage of multiple organs systemically, as indicated by abnormal liver and kidney biochemical markers, along with a disrupted ileum morphology. Additionally, DEHP exposure disrupted gut barrier function, causing intestinal inflammation characterized by bacterial translocation and alterations in defense and inflammation-related gene expressions. Moreover, 16S rRNA analysis suggested that DEHP-induced gut microbial remodeling is characterized by an upregulation of detrimental bacteria (Erysipelotrichaceae) and a downregulation of beneficial bacteria (Muribaculaceae, Ruminococcaceae, and Lachnospiraceae). Metabolomics analysis revealed DEHP perturbed gut metabolic homeostasis, particularly affecting the degradation of aromatic compounds, which generated an aberrant activation of the AhR and NF-κB, subsequently causing intestinal inflammation. Consequently, our results elucidate the mechanistic link between disrupted gut microbiota and metabolome and the initiation of DEHP-induced intestinal inflammation, mediated through the AhR/NF-κB signaling pathway.

The relevance between abnormally elevated serum ceramide and cognitive impairment in Alzheimer's disease model mice and its mechanism.

RATIONALE: The plasma ceramide levels in Alzheimer's disease (AD) patients are found abnormally elevated, which is related to cognitive decline. OBJECTIVES: This research was aimed to investigate the mechanisms of aberrant elevated ceramides in the pathogenesis of AD. RESULTS: The ICR mice intracerebroventricularly injected with Aß1-42 and APP/PS1 transgenic mice were employed as AD mice. The cognitive deficiency, impaired episodic and spatial memory were observed without altered spontaneous ability. The serum levels of p-tau and ceramide were evidently elevated. The modified expressions and activities of glycogen synthase kinase-3ß (GSK-3ß) and protein phosphatase 2A (PP2A) influenced the serum content of p-tau. The levels of ceramide synthesis-related genes including sptlc1, sptlc2, cers2, and cers6 in the liver of AD mice were increased, while the ceramide degradation-related gene asah2 did not significantly change. The regulations of these genes were conducted by activated nuclear factor kappa-B (NF-κB) signaling. NF-κB, promoted by free fatty acid (FFA), also increased the hepatic concentrations of proinflammatory cytokines. The FFA amount was modulated by fatty acid synthesis-related genes acc1 and srebp-1c. Besides, the decreased levels of pre-proopiomelanocortin (pomc) mRNA and increased agouti-related protein (agrp) mRNA were found in the hypothalamus without significant alteration of melanocortin receptor 4 (MC4R) mRNA. The bioinformatic analyses proved the results using GEO datasets and AlzData. CONCLUSIONS: Ceramide was positively related to the increased p-tau and impaired cognitive function. The increased generation of ceramide and endoplasmic reticulum stress in the hypothalamus was positively related to fatty acid synthesis and NF-κB signaling via brain-liver axis.

Corydalis tomentella Franch. Exerts anti-inflammatory and analgesic effects by regulating the calcium signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis tomentella Franch. is a perennial cespitose plant commonly used to treat stomachaches as a folk medicine. The C. tomentella total alkaloids have good protective effects against acute liver injury and potential anti-hepatoma and anti-Alzheimer's disease activities. AIM OF THE STUDY: To establish an effective purification process for total alkaloids from C. tomentella and investigate the mechanism of their anti-inflammatory effects. MATERIALS AND METHODS: Corydalis tomentella were purified using macroporous resin. Then the crude and purified C. tomentella extracts (cCTE and pCTE) were qualitatively analyzed using UPLC-Triple-TOF-MS/MS. The cCTE and pCTE were used to investigate and compare their anti-inflammatory effects on lipopolysaccharide (LPS)-induced RAW264.7 cells. Doses at 100, 200 and 400 mg/kg/d of pCTE were used to study their anti-inflammatory and analgesic activities in mice with xylene-induced ear swelling and acetic acid-induced writhing tests. Content of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) were determined both in RAW264.7 cells and mice. Network pharmacology was used to predict the anti-inflammatory mechanism of C. tomentella, and the key enzymes were validated using qPCR and Western Blot analysis. Concentration of intracellular Ca2+ was detected using flow cytometric analysis. RESULTS: The C. tomentella total alkaloid purity increased from 6.29% to 47.34% under optimal purification conditions. A total of 54 alkaloids were identified from CTE. Both cCTE and pCTE could suppress the LPS-induced production of NO, IL-6, IL-1ß, and TNF-α in RAW264.7 cells. The pCTE exhibited a more potent anti-inflammatory effect; it also inhibited pain induced by xylene and acetic acid in mice. The calcium signaling pathway is associated with the anti-inflammatory and analgesic activities of C. tomentella. The mRNA expression of nitric oxide synthase (NOS) 2, NOS3 and calmodulin1 (CALM1) was regulated by C. tomentella through the reduction of inflammation-induced Ca2+ influx, and it also exhibited a more pronounced effect than the positive control (L-NG-nitro arginine methyl ester). CONCLUSIONS: Purified C. tomentella extract shows anti-inflammatory effect both in vitro and in vivo. It exerts anti-inflammatory and analgesic effects through the calcium signaling pathway by down-regulating NOS2 and CALM1 expression and up-regulating NOS3 expression in LPS-induced RAW264.7 cells, and decreasing intracellular Ca2+ concentration.

TIL-Derived CAR T Cells Improve Immune Cell Infiltration and Survival in the Treatment of CD19-Humanized Mouse Colorectal Cancer.

Chimeric antigen receptor-engineered T cells (CAR Ts) targeting CD19 have shown unprecedented prognosis in treating hematological cancers. However, the lack of a tumor-specific antigen as the target and an inhospitable tumor environment limit the clinical application of CAR T in solid tumors. Tumor-infiltrating T lymphocytes (TIL) exhibit diverse T cell receptor clonality and superior tumor-homing abilities. Therefore, in our study, human CD19-target TIL CAR-Ts armed with CD3ζ and 4-1BB signaling domains were constructed. Mouse colorectal cancer CT26 cells expressing human CD19 (hCD19+-CT26) were developed to assess the anti-tumor activity of TIL CAR-T cells, both in vitro and in vivo. Compared with splenic CAR T adoptive transfer, TIL CAR-T administration showed superior tumor suppression ability in hCD19+-CT26 tumor-bearing mice. Furthermore, more T cells were found at the tumor site and had lower exhaustion-related inhibitory receptor (T cell immunoglobulin and mucin domain-containing protein 3, Tim3) expression and higher immune memory molecule (CD62L) expression. Overall, we provided an artificial tumor-specific antigen in solid tumors and demonstrated that combined CAR-expressing TIL-Ts (TIL CAR-Ts) exhibited strong anti-tumor activity, with improved T cell infiltration and immune memory. Our humanized tumor antigen presented platform of mice suggests that TIL CAR-T-based adoptive therapy could be a promising strategy for solid cancer treatment.

Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

Purtscher-Like Retinopathy in a Patient of Atopic Dermatitis Associated with Dupilumab Use: A Case Report.

PURPOSE: Purtscher-like retinopathy is a rare and vision-threatening eye disease, which is usually associated with acute pancreatitis, autoimmune diseases, and renal failure. We here reported a case of Purtscher-like retinopathy after dupilumab treatment in atopic dermatitis. CASE REPORT: A 48-year-old woman with a history of severe atopic dermatitis developed ocular manifestations after second exposure to dupilumab. Best-corrected visual acuity (BCVA) was count fingers at 2 feet in the right eye and 20/133 in the left eye when she referred to our hospital. Fundus examination demonstrated bilateral serous macular off, thickening of papillomacular bundle, intraretinal hemorrhage, cotton-wool spots and Purtscher flecken surrounding the optic disc. Optical coherence tomography (OCT) angiography showed flow void in the superficial and deep capillary plexuses. And early hypofluorescence corresponding to grayish-white retinal thickening was observed in fundus fluorescein angiography. Laboratory tests revealed hypereosinophilia after symptoms appeared. A diagnosis of Purtscher-like retinopathy was made, which may associate with dupilumab. The patient accepted the treatment with glucocorticoids. At six months follow up, BCVA was 20/40 in the right eye and 20/25 in the left eye. CONCLUSION: The Purtscher-like retinopathy we report may be the adverse event of dupilumab. Rare adverse reactions to biologics deserve the attention of ophthalmologists.

Baicalin Attenuates H2O2-Induced Oxidative Stress by Regulating the AMPK/Nrf2 Signaling Pathway in IPEC-J2 Cells.

Oxidative stress can adversely affect the health status of the body, more specifically by causing intestinal damage by disrupting the permeability of the intestinal barrier. This is closely related to intestinal epithelial cell apoptosis caused by the mass production of reactive oxygen species (ROS). Baicalin (Bai) is a major active ingredient in Chinese traditional herbal medicine that has antioxidant, anti-inflammatory, and anti-cancer properties. The purpose of this study was to explore the underlying mechanisms by which Bai protects against hydrogen peroxide (H2O2)-induced intestinal injury in vitro. Our results indicated that H2O2 treatment caused injury to IPEC-J2 cells, resulting in their apoptosis. However, Bai treatment attenuated H2O2-induced IPEC-J2 cell damage by up-regulating the mRNA and protein expression of ZO-1, Occludin, and Claudin1. Besides, Bai treatment prevented H2O2-induced ROS and MDA production and increased the activities of antioxidant enzymes (SOD, CAT, and GSH-PX). Moreover, Bai treatment also attenuated H2O2-induced apoptosis in IPEC-J2 cells by down-regulating the mRNA expression of Caspase-3 and Caspase-9 and up-regulating the mRNA expression of FAS and Bax, which are involved in the inhibition of mitochondrial pathways. The expression of Nrf2 increased after treatment with H2O2, and Bai can alleviate this phenomenon. Meanwhile, Bai down-regulated the ratio of phosphorylated AMPK to unphosphorylated AMPK, which is indicative of the mRNA abundance of antioxidant-related genes. In addition, knockdown of AMPK by short-hairpin RNA (shRNA) significantly reduced the protein levels of AMPK and Nrf2, increased the percentage of apoptotic cells, and abrogated Bai-mediated protection against oxidative stress. Collectively, our results indicated that Bai attenuated H2O2-induced cell injury and apoptosis in IPEC-J2 cells through improving the antioxidant capacity through the inhibition of the oxidative stress-mediated AMPK/Nrf2 signaling pathway.

Structure of LARP7 Protein p65-telomerase RNA Complex in Telomerase Revealed by Cryo-EM and NMR.

La-related protein 7 (LARP7) are a family of RNA chaperones that protect the 3'-end of RNA and are components of specific ribonucleoprotein complexes (RNP). In Tetrahymena thermophila telomerase, LARP7 protein p65 together with telomerase reverse transcriptase (TERT) and telomerase RNA (TER) form the core RNP. p65 has four known domains-N-terminal domain (NTD), La motif (LaM), RNA recognition motif 1 (RRM1), and C-terminal xRRM2. To date, only the xRRM2 and LaM and their interactions with TER have been structurally characterized. Conformational dynamics leading to low resolution in cryo-EM density maps have limited our understanding of how full-length p65 specifically recognizes and remodels TER for telomerase assembly. Here, we combined focused classification of Tetrahymena telomerase cryo-EM maps with NMR spectroscopy to determine the structure of p65-TER. Three previously unknown helices are identified, one in the otherwise intrinsically disordered NTD that binds the La module, one that extends RRM1, and another preceding xRRM2, that stabilize p65-TER interactions. The extended La module (αN, LaM and RRM1) interacts with the four 3' terminal U nucleotides, while LaM and αN additionally interact with TER pseudoknot, and LaM with stem 1 and 5' end. Our results reveal the extensive p65-TER interactions that promote TER 3'-end protection, TER folding, and core RNP assembly and stabilization. The structure of full-length p65 with TER also sheds light on the biological roles of genuine La and LARP7 proteins as RNA chaperones and core RNP components.

Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils.

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP's propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

Blunting ROS/TRPML1 pathway protects AFB1-induced porcine intestinal epithelial cells apoptosis by restoring impaired autophagic flux.

Aflatoxin B1 (AFB1) is a stable mycotoxin that contaminates animal feed on a large scale and causes severe damage to intestinal cells, induces inflammation and stimulates autophagy. Transient receptor potential mucolipin subfamily 1 (TRPML1) is a regulatory factor of autophagy, but the underlying mechanisms of TRPML1-mediated autophagy in AFB1 intestine toxicity remain elucidated. In the present study, AFB1 (0, 5, 10 µg/mL) was shown to reduce cell viability, increase reactive oxygen species (ROS) accumulation and apoptosis rate. Additionally, AFB1 caused structural damage to mitochondria and lysosomes and increased autophagosomes numbers. Furthermore, AFB1 promoted Ca2+ release by activating the TRPML1 channel, stimulated the expression of autophagy-related proteins, and induced autophagic flux blockade. Moreover, pharmacological inhibition of autophagosome formation by 3-methyladenine attenuated AFB1-induced apoptosis by downregulating the levels of TRPML1 and ROS, whereas blockade of autophagosome-lysosomal fusion by chloroquine alleviated AFB1-induced apoptosis by upregulating TRPML1 expression and exacerbating ROS accumulation. Intriguingly, blocking AFB1-induced autophagic flux generated ROS- and TRPML1-dependent cell death, as shown by the decreased apoptosis in the presence the free radical scavenger N-Acetyl-L-cysteine and the TRPML1 inhibitor ML-SI1. Overall, these results showed that AFB1 promoted apoptosis of IPEC-J2 cells by disrupting autophagic flux through activation of the ROS/TRPML1 pathway.