Chain Promoter Research Articles

Hypoxia-induced CTCF mediates alternative splicing via coupling chromatin looping and RNA Pol II pause to promote EMT in breast cancer.

Hypoxia influences the epithelial-mesenchymal transition (EMT) through the remodeling of the chromatin structure, epigenetics, and alternative splicing. Hypoxia drives CCCTC-binding factor (CTCF) induction through hypoxia-inducible factor 1-alpha (HIF1α), which promotes EMT, although the underlying mechanisms remain unclear. We find that hypoxia significantly increases CTCF occupancy at various EMT-related genes. We present a CTCF-mediated intricate mechanism promoting EMT wherein CTCF binding at the collagen type V alpha 1 chain (COL5A1) promoter is crucial for COL5A1 upregulation under hypoxia. Additionally, hypoxia drives exon64A inclusion in a mutually exclusive alternative splicing event of COL5A1exon64 (exon64A/64B). Notably, CTCF mediates COL5A1 promoter-alternatively spliced exon upstream looping that regulates DNA demethylation at distal exon64A. This further regulates the CTCF-mediated RNA polymerase II pause at COL5A1exon64A, leading to its inclusion in promoting the EMT under hypoxia. Genome-wide study indicates the association of gained CTCF occupancy with the alternative splicing of many cancer-related genes, similar to the proposed model. Specifically, disrupting the HIF1α-CTCF-COL5A1exon64A axis through the dCas9-DNMT3A system alleviates the EMT in hypoxic cancer cells and may represent a novel therapeutic target in breast cancer.

Effect of cardiomyocyte-specific lipid phosphate phosphatase 3 overexpression on high-fat diet-induced cardiometabolic dysfunction in mice.

Lipid phosphate phosphatase 3 (LPP3) is a membrane-bound enzyme that hydrolyzes lipid phosphates including the bioactive lipid, lysophosphatidic acid (LPA). Elevated circulating LPA production and cellular LPA signaling are implicated in obesity-induced metabolic and cardiac dysfunction. Deletion of LPP3 in the cardiomyocyte increases circulating LPA levels and causes heart failure and mitochondrial dysfunction in mice. To examine the influence of LPP3 modulation in the cardiomyocyte on obesity-induced cardiomyopathy, we generated mice with cardiomyocyte-specific LPP3 overexpression (LPP3OE mice) driven by the α myosin heavy chain promoter. Female and male control (LPP3FL) and LPP3OE mice were fed low-fat diet (LFD) or high-fat diet (HFD) for up to 22-23 weeks, followed by the analysis of glucose homeostasis, cardiac function, plasma LPA levels, and mitochondrial respiration in cardiac myofibers. On LFD, both female and male LPP3OE mice had markedly reduced plasma LPA levels and increased pyruvate-linked respiration when compared to LPP3FL mice while body weight and global insulin sensitivity were similar between genotypes. Following HFD feeding, female LPP3OE mice were protected from an increase in plasma LPA levels, excess adiposity, systemic insulin resistance, and systolic and diastolic cardiac dysfunction compared to LPP3FL mice. Female LPP3OE mice also maintained elevated cardiac pyruvate-linked mitochondrial respiration following HFD feeding while mitochondrial respiration was similar between genotypes in HFD-fed male mice. This study suggests that cardiomyocyte-specific LPP3 upregulation protects particularly female mice from HFD-induced metabolic dysfunction and cardiomyopathy.

Dexamethasone Upregulates the Expression of the Human SLC26A3 (DRA, Down-Regulated in Adenoma) Transporter (an IBD Susceptibility Gene) in Intestinal Epithelial Cells and Attenuates Gut Inflammation.

Down-Regulated in Adenoma (DRA) plays a critical role in intestinal chloride absorption and a decrease in its expression is a key event in diarrheal disorders. Recently, DRA has emerged as an Inflammatory Bowel Disease(IBD) susceptibility gene. Therefore, the strategies to upregulate DRA expression are potentially novel approaches to not only treat IBD-associated diarrhea but also gut inflammation. In this study, the effect of dexamethasone (DEX), an anti-inflammatory corticosteroid on DRA expression was investigated. GR (glucocorticoid receptor) overexpressed Caco-2 cells and C57BL/6/J mice and anti-αIL-10R mAb model of IBD were used. Protein expression was assessed by immunoblotting and immunofluorescence. Transcript levels were assessed by quantative-real-timepolymerase chain reaction (qRT-PCR) and promoter activity was measured by luciferase assays. Our results showed that DEX significantly increased DRA mRNA and protein expression in GR overexpressing Caco-2 cells. DEX-induced upregulation of DRA was GR dependent and appeared at least in part to occur via a transcriptional mechanism, as promoter activity of the DRA construct (-1183/+114bp) was significantly increased in response to DEX. The increase in DRA mRNA was abrogated in the presence of MKP-1 inhibitor, triptolide. Administration of DEX (2mg/kg body weight) to mice for 24 and 48 hours significantly increased the DRA expression in mouse colon. DEX treatment to mice for 7 days in the αIL-10R mAb model of colitis was able to significantly attenuate the gut inflammation and associated decrease in DRA expression. We demonstrate that DEX stimulates DRA expression via transcriptional mechanisms and suggest that upregulation of DRA may contribute to both anti-inflammatory and pro-absorptive effects of DEX.

The industrial production and application of historical building component heritage from the perspective of digital technology module chain

The protection of historical architectural heritage is the key research object of the ancient architecture industry, and the research on the partial construction of buildings is gradually deepening. Nowadays, the use of digital means to research and protect architectural heritage has become an important way for many scholars. However, there are many types of digital software on the market with complex functions, and common digital means include X-spectrum, Jingdiao JDP, point cloud data, 3D printing, etc. Based on the simple construction of dougong, this paper analyzes the method of digital technology module chain, and applies it to the field of architectural construction such as murals and sculptures. This digital technology module chain can not only study and analyze the construction of historical buildings, but also meet the needs of cultural relics preservation, industrial production, education and communication. Finally, it is expected that through the promotion of this digital technology module chain, the protection of historical architectural heritage and cultural communication will be promoted in a more optimized direction.

Abstract 4147616: Targeting Skeletal Muscle Growth Improves Diastolic Dysfunction and Left Ventricular Hypertrophy in Obesity-Related Diabetic Cardiomyopathy

Background and Significance: Mice fed a high-fat, high-sucrose (HFHS) “Western” diet develop a cardiac phenotype similar to patients with type 2 diabetes mellitus-related heart failure with preserved ejection fraction (HFpEF). This includes obesity, insulin resistance, left ventricular hypertrophy (LVH), and diastolic dysfunction. Skeletal muscle is responsible for up to 80% of whole-body glucose disposal, making the relationship between muscle mass and insulin resistance crucial for identifying new therapeutic targets in HFpEF. Myostatin is an evolutionally conserved skeletal muscle growth-limiting signaling molecule. Although whole-body deletion of myostatin/activin signaling (MAS) promotes skeletal muscle hypertrophy (SH) and provides metabolic benefits, overall benefits are complicated by direct inhibition in the myocardium. Hypothesis: Sedentary mice with skeletal muscle-specific deletion of MAS will be protected from the adverse cardiac effects of HFHS diet. Methods and Results: BL6 mice with floxed alleles for myostatin Acvr2 and Acvr2b receptors were crossbred with mice expressing Cre recombinase from a myosin light chain promoter, rendering the MAS downregulation skeletal muscle specific. Four months of HFHS feeding resulted in similar increase in body weight between Cre+ and Cre- (34% and 37%, respectively). SH was present in Cre+ mice as the muscle weight/tibia length ratio of m. pectoralis, triceps and quadriceps was elevated by 44, 22 and 25%, respectively. Oral glucose tolerance was improved in Cre+ vs Cre-. Echocardiography showed improved LV hypertrophy and diastolic function (increased E/A ratio and Em by 27 and 21%) in the Cre+ group. Moreover, exercise capacity was improved in Cre+ mice by 59%. Conclusion: Our novel study demonstrates that targeting skeletal muscle growth through the inhibition of MAS, without direct cardiac effects, can ameliorate the cardiac dysfunction associated with obesity-related diabetic cardiomyopathy. Increased muscle mass improves glucose tolerance, LVH, and diastolic function, demonstrating skeletal muscle-to-myocardium cross-talk. This highlights the potential for muscle-targeted therapies in managing HFpEF, particularly in insulin-resistant conditions.

Latrophilin-2 Deletion in Cardiomyocyte Disrupts Cell Junction, Leading to D-CMP.

Latrophilin-2 (Lphn2), an adhesive GPCR (G protein-coupled receptor), was found to be a specific marker of cardiac progenitors during the differentiation of pluripotent stem cells into cardiomyocytes or during embryonic heart development in our previous studies. Its role in adult heart physiology, however, remains unclear. The embryonic lethality resulting from Lphn2 deletion necessitates the establishment of cardiomyocyte-specific, tamoxifen-inducible Lphn2 knockout mice, which was achieved by crossing Lphn2 flox/flox mice with mice having MerCreMer (tamoxifen-inducible Cre [Cyclization recombinase] recombinase) under the α-myosin heavy chain promoter. Tamoxifen treatment for several days completely suppressed Lphn2 expression, specifically in the myocardium, and induced the dilated cardiomyopathy (D-CMP) phenotype with serious arrhythmia and sudden death in a short period of time. Transmission electron microscopy showed mitochondrial abnormalities, blurred Z-discs, and dehiscent myofibrils. The D-CMP phenotype, or heart failure, worsened during myocardial infarction. In a mechanistic study of D-CMP, Lphn2 knockout suppressed PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and mitochondrial dysfunction, leading to the accumulation of reactive oxygen species and the global suppression of junctional molecules, such as N-cadherin (adherens junction), DSC-2 (desmocollin-2; desmosome), and connexin-43 (gap junction), leading to the dehiscence of cardiac myofibers and serious arrhythmia. In an experimental therapeutic trial, activators of p38-MAPK (p38 mitogen-activated protein kinases), which is a downstream signaling molecule of Lphn2, remarkably rescued the D-CMP phenotype of Lphn2 knockout in the heart by restoring PGC-1α and mitochondrial function and recovering global junctional proteins. Lphn2 is a critical regulator of heart integrity by controlling mitochondrial functions and cell-to-cell junctions in cardiomyocytes. Its deficiency leads to D-CMP, which can be rescued by activators of the p38-MAPK pathway.

LncRNA DCRT Protects Against Dilated Cardiomyopathy by Preventing NDUFS2 Alternative Splicing by Binding to PTBP1.

Dilated cardiomyopathy is characterized by left ventricular dilation and continuous systolic dysfunction. Mitochondrial impairment is critical in dilated cardiomyopathy; however, the underlying mechanisms remain unclear. Here, we explored the cardioprotective role of a heart-enriched long noncoding RNA, the dilated cardiomyopathy repressive transcript (DCRT), in maintaining mitochondrial function. The DCRT knockout (DCRT-/-) mice and DCRT knockout cells were developed using CRISPR-Cas9 technology. Cardiac-specific DCRT transgenic mice were generated using α-myosin heavy chain promoter. Chromatin coimmunoprecipitation, RNA immunoprecipitation, Western blot, and isoform sequencing were performed to investigate the underlying mechanisms. We found that the long noncoding RNA DCRT was highly enriched in the normal heart tissues and that its expression was significantly downregulated in the myocardium of patients with dilated cardiomyopathy. DCRT-/- mice spontaneously developed cardiac dysfunction and enlargement with mitochondrial impairment. DCRT transgene or overexpression with the recombinant adeno-associated virus system in mice attenuated cardiac dysfunction induced by transverse aortic constriction treatment. Mechanistically, DCRT inhibited the third exon skipping of NDUFS2 (NADH dehydrogenase ubiquinone iron-sulfur protein 2) by directly binding to PTBP1 (polypyrimidine tract binding protein 1) in the nucleus of cardiomyocytes. Skipping of the third exon of NDUFS2 induced mitochondrial dysfunction by competitively inhibiting mitochondrial complex I activity and binding to PRDX5 (peroxiredoxin 5) and suppressing its antioxidant activity. Furthermore, coenzyme Q10 partially alleviated mitochondrial dysfunction in cardiomyocytes caused by DCRT reduction. Our study revealed that the loss of DCRT contributed to PTBP1-mediated exon skipping of NDUFS2, thereby inducing cardiac mitochondrial dysfunction during dilated cardiomyopathy development, which could be partially treated with coenzyme Q10 supplementation.

Corporate social responsibility and circular economy integration framework within sustainable supply chain management: Building blocks for industry 5.0

AbstractThe emergence of Industry 5.0 (I5.0) underscores the importance of integrating sustainability and human‐centricity into organizations. This requires combining corporate social responsibility (CSR) and circular economy (CE) within sustainable supply chain management (SSCM). However, the interplay between CSR, CE, and SSCM remains underexplored, especially in the context of I5.0. Therefore, this article proposes a framework for integrating CSR and CE principles within SSCM, serving as a transition to I5.0. The framework is derived from a comprehensive scoping review and experts' input, with a backdrop of the business legitimacy theory. It comprises several key constructs, including the supply chain structure and promoters, organized around four pillars representing the components of CE and embracing the triple bottom line dimensions. With 12 guidelines, the framework stands as a practical tool for organizations aiming to seamlessly integrate CSR and CE within SSCM, facilitating the transition to I5.0 and promoting a sustainable, socially responsible ethos.

Comparison of SNCG and NEFH Promoter-Driven Expression of Human SIRT1 Expression in a Mouse Model of Glaucoma.

Adeno-associated virus (AAV) demonstrates promise in delivering therapeutic genes to retinal ganglion cells (RGCs). Delivery of neuroprotective genes is constrained by packaging size and/or cell selectivity. This study compares the ability of the RGC-selective gamma-synuclein (SNCG) promoter and the smaller RGC-selective neurofilament heavy chain (NEFH) promoter, as well as portions of the RGC-selective atonal bHLH transcription factor 7 (ATOH7) enhancer, to drive gene expression in RGCs. AAV2 constructs with green fluorescent protein (GFP) or human sirtuin 1 (hSIRT1) driven by cytomegalovirus (CMV) enhancer and NEFH promoter (AAV2-eCMV-NEFH) or distal active sequences of the ATOH7 enhancer (DiATOH7) with the SNCG promoter (AAV2-DiATOH7-SNCG) were intravitreally injected into C57BL/6J mice. RGCs were immunolabeled with Brn3a antibodies and counted. AAV constructs with the utmost transduction efficiency were used to test the therapeutic efficacy of the hSIRT1 gene in 12-week-old C57BL/6J mice subjected to microbead (MB)-induced intraocular pressure (IOP) elevation. Visual function was measured using optokinetic responses (OKRs). The eGFP transduction efficiency of AAV2-eCMV-NEFH was similar to that of AAV2-eCMV-SNCG and AAV2-DiATOH7-SNCG. When combined with the SNCG promoter, a larger ATOH7 enhancer was less efficient than the shorter DiATOH7 enhancer. Similarly, the hSIRT1 efficiency of AAV2-eCMV-NEFH was similar to that of AAV2-eCMV-SNCG. The latter two vectors were equally efficient in increasing RGC survival and improving visual function in the mouse model of MB-induced IOP elevation. SNCG and NEFH promoters represent two equally efficient and comparable RGC selective promoter sequences; however, the NEFH promoter offers a smaller packaging size. Smaller enhancer-promoter combinations can be used to deliver larger genes in human cells and for treatment in optic neuropathies including glaucoma.

Diacylglycerol kinase delta overexpression improves glucose clearance and protects against the development of obesity

Background and aimDiacylglycerol kinase (DGK) isoforms catalyze an enzymatic reaction that removes diacylglycerol (DAG) and thereby terminates protein kinase C signaling by converting DAG to phosphatidic acid. DGKδ (type II isozyme) downregulation causes insulin resistance, metabolic inflexibility, and obesity. Here we determined whether DGKδ overexpression prevents these metabolic impairments. MethodsWe generated a transgenic mouse model overexpressing human DGKδ2 under the myosin light chain promoter (DGKδ TG). We performed deep metabolic phenotyping of DGKδ TG mice and wild-type littermates fed chow or high-fat diet (HFD). Mice were also provided free access to running wheels to examine the effects of DGKδ overexpression on exercise-induced metabolic outcomes. ResultsDGKδ TG mice were leaner than wild-type littermates, with improved glucose tolerance and increased skeletal muscle glycogen content. DGKδ TG mice were protected against HFD-induced glucose intolerance and obesity. DGKδ TG mice had reduced epididymal fat and enhanced lipolysis. Strikingly, DGKδ overexpression recapitulated the beneficial effects of exercise on metabolic outcomes. DGKδ overexpression and exercise had a synergistic effect on body weight reduction. Microarray analysis of skeletal muscle revealed common gene ontology signatures of exercise and DGKδ overexpression that were related to lipid storage, extracellular matrix, and glycerophospholipids biosynthesis pathways. ConclusionOverexpression of DGKδ induces adaptive changes in both skeletal muscle and adipose tissue, resulting in protection against HFD-induced obesity. DGKδ overexpression recapitulates exercise-induced adaptations on energy homeostasis and skeletal muscle gene expression profiles.

IMAC (Mato Grosso Meat Institute): sustainable development and promotion of beef chain in Brazil

Abstract Founded in 2016, the Mato Grosso Meat Institute is an autonomous social service formed by representatives of the farm production sector, of the industry, of society and of the state government to promote the meat produced in the state of Mato Grosso, one of the most important states for Brazilian agriculture and livestock production. Despite several promising projects in the pipeline on the topics of information sharing, sustainable insertion of smallholders and other, IMAC still has not reached a protagonist role in Mato Grosso beef chain. With several changes and trends emerging in consumer food behavior, its president (Caio Penido), is still looking for the best strategies to improve Mato Grosso’s competitive advantage and align all the agents involved to fulfill IMAC’s “vision for the chain”. The Teaching Note to this Case Study is available on request via e-mail to ifamr@ifama.org

Custom-designed, mass silk production in genetically engineered silkworms.

Genetically engineered silkworms have been widely used to obtain silk with modified characteristics especially by introducing spider silk genes. However, these attempts are still challenging due to limitations in transformation strategies and difficulties in integration of the large DNA fragments. Here, we describe three different transformation strategies in genetically engineered silkworms, including transcription-activator-like effector nuclease (TALEN)-mediated fibroin light chain (FibL) fusion (BmFibL-F), TALEN-mediated FibH replacement (BmFibH-R), and transposon-mediated genetic transformation with the silk gland-specific fibroin heavy chain (FibH) promoter (BmFibH-T). As the result, the yields of exogenous silk proteins, a 160 kDa major ampullate spidroin 2 (MaSp2) from the orb-weaving spider Nephila clavipes and a 226 kDa fibroin heavy chain protein (EvFibH) from the bagworm Eumeta variegate, reach 51.02 and 64.13% in BmFibH-R transformed cocoon shells, respectively. Moreover, the presence of MaSp2 or EvFibH significantly enhances the toughness of genetically engineered silk fibers by ∼86% in BmFibH-T and ∼80% in BmFibH-R silkworms, respectively. Structural analysis reveals a substantial ∼40% increase in fiber crystallinity, primarily attributed to the presence of unique polyalanines in the repetitive sequences of MaSp2 or EvFibH. In addition, RNA-seq analysis reveals that BmFibH-R system only causes minor impact on the expression of endogenous genes. Our study thus provides insights into developing custom-designed silk production using the genetically engineered silkworm as the bioreactor.

Exploring Italian Wine Companies: A Study of Supply Chain Dynamics, Quality, and Promotion Strategies through Semi-Structured Interviews.

In Italy, the wine supply chain is a cornerstone of the national agri-food system and a driving force for the entire economy. The aim of this study is to map the profile of Italian wine companies through the analysis of multiple case studies. The study focuses specifically on companies in the Marche region, investigating their features and supply chain, with a specific focus on product and system certification adopted, marketing policies implemented, and the businesses' relationship with institutions. A total of 18 companies participated in the study. The primary data for the research was collected through semi-structured interviews lasting 1 h, based on an interview protocol. The results show that the supply chain for the wine sector is configured to be short, even when cooperatives act as an aggregator. The small size and limited financial resources make it particularly difficult to promote the wine outside its reference context, and there is a lack of a network strategy at the local level. Many companies consider organic certification a disadvantage since it tends to homologate production to the large wine industries, making the product's naturalness, typicality, and craftsmanship disappear.

Reduction in Insulin Uncovers a Novel Effect of VEGFB on Cardiac Substrate Utilization.

The heart relies heavily on external fatty acid (FA) for energy production. VEGFB (vascular endothelial growth factor B) has been shown to promote endothelial FA uptake by upregulating FA transporters. However, its impact on LPL (lipoprotein lipase)-mediated lipolysis of lipoproteins, a major source of FA for cardiac use, is unknown. VEGFB transgenic (Tg) rats were generated by using the α-myosin heavy chain promoter to drive cardiomyocyte-specific overexpression. To measure coronary LPL activity, Langendorff hearts were perfused with heparin. In vivo positron emission tomography imaging with [18F]-triglyceride-fluoro-6-thia-heptadecanoic acid and [11C]-palmitate was used to determine cardiac FA uptake. Mitochondrial FA oxidation was evaluated by high-resolution respirometry. Streptozotocin was used to induce diabetes, and cardiac function was monitored using echocardiography. In Tg hearts, the vectorial transfer of LPL to the vascular lumen is obstructed, resulting in LPL buildup within cardiomyocytes, an effect likely due to coronary vascular development with its associated augmentation of insulin action. With insulin insufficiency following fasting, VEGFB acted unimpeded to facilitate LPL movement and increase its activity at the coronary lumen. In vivo PET imaging following fasting confirmed that VEGFB induced a greater FA uptake to the heart from circulating lipoproteins as compared with plasma-free FAs. As this was associated with augmented mitochondrial oxidation, lipid accumulation in the heart was prevented. We further examined whether this property of VEGFB on cardiac metabolism could be useful following diabetes and its associated cardiac dysfunction, with attendant loss of metabolic flexibility. In Tg hearts, diabetes inhibited myocyte VEGFB gene expression and protein secretion together with its downstream receptor signaling, effects that could explain its lack of cardioprotection. Our study highlights the novel role of VEGFB in LPL-derived FA supply and utilization. In diabetes, loss of VEGFB action may contribute toward metabolic inflexibility, lipotoxicity, and development of diabetic cardiomyopathy.

The role of latrophilin-2 in maintaining cardiac cell-to-cell junctions and its implications for dilated cardiomyopathy and sudden death

Abstract Background Identifying genes that are important for maintaining heart function and characterizing their roles and pathways are essential for developing therapeutic approaches. Latrophilin-2 (LPHN2), a relatively unknown GPCR, critically contributes to differentiation from pluripotent stem cells (PSC) to the cardiac lineage, but it is not known whether LPHN2 is active in the adult heart. Purpose In this study, we explored the functions of LPHN2 in adult hearts. Methods and Results To determine whether there is an association between variation in LPHN2 and cardiac phenotypes in humans, we used gnomAD and UK Biobank data. The data suggested that LPHN2 variants could be fatal. Accordingly, we developed a mouse model for further analysis of Lphn2. Deletion of Lphn2 causes embryonic lethality, so we generated a cardiac-specific, tamoxifen-inducible Lphn2 conditional knockout mouse (Lphn2icKO mouse) by cross-breeding Lphn2flox/flox mice with the TMX-inducible Cre recombinase (MerCreMer) under control of the α-Myh6 (α-myosin heavy chain) promoter mice. After administering tamoxifen, we found that the left ventricle became dilated and the systolic function was decreased, and gaps were created between cardiomyocytes. We also observed ventricular tachycardia and atrioventricular block associated with the dilated cardiomyopathy and subsequent sudden cardiac death. Cell-to-cell junctions (adherence, gap, and desmosomes,) were found to be dysregulated in the Lphn2icKO adult mouse heart and the heart at embryonic day 13.5 of Lphn2−/− (homozygous Lphn2 deficiency). To investigate Lphn2 function in a disease model, we generated a myocardial infarction (MI) in Lphn2icKO mice. MI with tamoxifen group showed wide QRS tachycardia and various types of block, and a survival reduction of 40% compared with that of the MI group. Regarding underlying mechanisms, we found that PGC-1α, a factor downstream of p38-MAPK that contributes to arrhythmia and maturation of PSC-derived cardiomyocytes, was downregulated (Figure). Conclusions LPHN2 is a key molecule that maintain adult heart integrity via its effects on cell-to-cell junctions, and the deficiency of Lphn2 exacerbates heart failure. Our findings provide evidence in support of LPHN2 being a pivotal regulator of heart function through the p38-MAPK-PGC-1α pathway.Schematic illustration

MHC II-EGFP Knock-in Mouse Model.

The MHC II-EGFP knock-in mouse model enables us to visualize and track MHC-II-expressing cells in vivo by expressing enhanced green fluorescent protein (EGFP) fused to the MHC class II molecule under the MHC II beta chain promoter. Using this model, we can easily identify MHC-II-expressing cells, including dendritic cells, B cells, macrophages, and ILC3s, which play a key role as antigen-presenting cells (APCs) for CD4+ T cells. In addition, we can also precisely identify and analyze APC-containing tissues and organs. Even after fixation, EGFP retains its fluorescence, so this model is suitable for immunofluorescence studies, facilitating an unbiased characterization of the histological context, especially with techniques such as light-sheet fluorescence microscopy. Furthermore, the MHC II-EGFP knock-in mouse model is valuable for studying the molecular mechanisms of MHC II gene regulation and expression by making it possible to correlate MHC II expression (MHC II-EGFP) with surface fraction through antibody detection, thereby shedding light on the intricate regulation of MHC II expression. Overall, this model is an essential asset for quantitative and systems immunological research, providing insights into immune cell dynamics and localization, with a tool for precise cell identification and with the ability to study MHC II gene regulation, thus furthering the understanding of immune responses and underlying mechanisms © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Characterization of antigen-specific MHC II loading compartment tubulation toward the immunological synapse Basic Protocol 2: Characterization of overall versus surface MHC II expression Basic Protocol 3: Identification and preparation of the lymphoid organs Basic Protocol 4: Quantification of APC content in lymphoid organs by fluorescence stereomicroscopy Basic Protocol 5: Quantification and measurement of intestinal lymphoid tissue by light-sheet fluorescence stereomicroscopy Basic Protocol 6: Visualization of corneal APCs Basic Protocol 7: Quantification of MHC II+ cells in maternal milk by flow cytometry Support Protocol 1: Cell surface staining and flow cytometry analysis of spleen mononuclear cells.

Quantitative proteomics reveals the neurotoxicity of trimethyltin chloride on mitochondria in the hippocampus of mice

Trimethyltin chloride (TMT) is a potent neurotoxin widely used as a constituent of polyvinyl chloride plastic in the industrial and agricultural fields. However, the underlying mechanisms by which TMT leads to neurotoxicity remain elusive. In the present study, we constructed a dose and time dependent neurotoxic mouse model of TMT exposure to explore the molecular mechanisms involved in TMT-induced neurological damage. Based on this model, the cognitive ability of TMT exposed mice was assessed by the Morris water maze test and a passive avoidance task. The ultrastructure of hippocampus was analyzed by the transmission electron microscope. Subsequently, proteomics integrated with bioinformatics and experimental verification were employed to reveal potential mechanisms of TMT-induced neurotoxicity. Gene ontology (GO) and pathway enrichment analysis were done by using Metascape and GeneCards database respectively. Our results demonstrated that TMT-exposed mice exhibited cognitive disorder, and mitochondrial respiratory chain abnormality of the hippocampus. Proteomics data showed that a total of 7303 proteins were identified in hippocampus of mice of which 224 ones displayed a 1.5-fold increase or decrease in TMT exposed mice compared with controls. Further analysis indicated that these proteins were mainly involved in tricarboxylic acid (TCA) cycle and respiratory electron transport, proteasome degradation, and multiple metabolic pathways as well as inflammatory signaling pathways. Some proteins, including succinate-CoA ligase subunit (Suclg1), NADH dehydrogenase subunit 5 (Nd5), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (Ndufa4l2) and cytochrome c oxidase assembly factor 7 (Coa7), which were closely related to mitochondrial respiratory electron transport, showed TMT dose and time dependent changes in the hippocampus of mice. Moreover, apoptotic molecules Bax and cleaved caspase-3 were up-regulated, while anti-apoptotic Bcl-2 was down-regulated compared with controls. In conclusion, our findings suggest that impairment of mitochondrial respiratory chain transport and promotion of apoptosis are the potential mechanisms of TMT induced hippocampus toxicity in mice.

Phosphorylation of AMPKα at Ser485/491 Is Dependent on Muscle Contraction and Not Muscle-Specific IGF-I Overexpression.

Glucose is an important fuel for highly active skeletal muscles. Increased adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratios during repetitive contractions trigger AMP-activated protein kinase (AMPK), indicated by phosphorylation of AMPKαThr172, which promotes glucose uptake to support heightened energy needs, but it also suppresses anabolic processes. Inhibition of AMPK can occur by protein kinase B (AKT)-mediated phosphorylation of AMPKαSer485/491, releasing its brake on growth. The influence of insulin-like growth factor I (IGF-I) on glucose uptake and its interplay with AMPK activation is not well understood. Thus, the goal of this study was to determine if increased muscle IGF-I altered AMPKα phosphorylation and activity during muscle contraction. Adult male mice harboring the rat Igf1a cDNA regulated by the fast myosin light chain promoter (mIgf1+/+) and wildtype littermates (WT) were used in the study. mIgf1+/+ mice had enhanced glucose tolerance and insulin-stimulated glucose uptake, but similar exercise capacity. Fatiguing stimulations of extensor digitorum longus (EDL) muscles resulted in upregulated AMPKα phosphorylation at both Thr172 and Ser485/491 in WT and mIgf1+/+ muscles. No differences in the phosphorylation response of the downstream AMPK target TBC1D1 were observed, but phosphorylation of raptor was significantly higher only in WT muscles. Further, total raptor content was elevated in mIgf1+/+ muscles. The results show that high muscle IGF-I can enhance glucose uptake under resting conditions; however, in contracting muscle, it is not sufficient to inhibit AMPK activity.

Knowledge management for supply chain resilience in pharmaceutical industry: evidence from the Middle East region

PurposeThis study investigates the role of supply chain knowledge management in enhancing pharmaceutical supply chain resilience.Design/methodology/approachThis study focusses on the Middle East region, where semi-structured online interviews were conducted with 38 professionals from the pharmaceutical supply chain to collect empirical data.FindingsThe study reveals that supply chain knowledge management is a crucial value-adding practice that improves pharmaceutical supply chain resilience. Effective supply chain knowledge management enables organisations to develop agility, change, adaptability, problem-solving, response and innovation capabilities that support supply chain resilience. However, challenges related to supply chain management practices, people, processes and technology hinder the effective promotion of supply chain knowledge.Practical implicationsThis study reminds managers that knowledge management is critical for building resilience in supply chains.Social implicationsThe study highlights the importance of a resilient pharmaceutical supply chain for organisations and society. The study advocates that effective supply chain knowledge management can help ensure a sustained supply of high-quality pharmaceutical products and services during crises.Originality/valueThe study offers novel insights by examining pharmaceutical supply chain resilience from a knowledge management perspective and highlighting the potential of knowledge capabilities to enable supply chains to recover from crises and adapt to the new normal. This study also highlights the key strategic considerations for managing knowledge effectively throughout the supply chain.

A study on the adoption intention of cold chain prepared Dishes based on consumer orientation mentality

Background: Cold chain prepared dishes, as a new type of food with low temperature and dual attributes, have attracted more and more attention from consumers. From the perspective of consumers' brand positioning mentality, this study integrated the brand, quality and other attributes of cold chain prepared vegetables, and studied the positioning mentality and adoption behavior of Chinese consumers In this study, three cold-chain prefabricated dishes, "pickled cabbage fish", "tomato Braised beef brisket" and "Huangpi fishball", were selected as investigation objects. Contribution: The contribution of this study is that different food value attributes explain the law of consumers' willingness to adopt cold chain prepared dishes through brand positioning mentality, and find out how different degrees of vulnerability and quality warranty Method: Based on ZMET survey method, product effect data were used to analyze and verify structural equation model. Results: Different value evaluation directly affects consumers' brand positioning mentality, brand positioning mind as an intermediate variable has a significant correlation with adoption intention. The degree of vulnerability moderates the relationship between value evaluation and brand positioning mentality, and the degree of warranty period regulates the relationship between quality value, brand positioning mentality and adoption intention respectively. Conclusion: It also provides new marketing tools and theories for entrepreneurs and marketers in the cold chain food industry, which contributes to the promotion and diffusion of cold chain prepared food for consumers