Isolation of Mouse Pancreatic Endothelial Cells.

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

Role of long noncoding RNAs in diabetes-associated peripheral arterial disease.

Diabetes mellitus (DM) is a metabolic disease that heightens the risks of many vascular complications, including peripheral arterial disease (PAD). Various types of cells, including but not limited to endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and macrophages (MΦs), play crucial roles in the pathogenesis of DM-PAD. Long non-coding RNAs (lncRNAs) are epigenetic regulators that play important roles in cellular function, and their dysregulation in DM can contribute to PAD. This review focuses on the developing field of lncRNAs and their emerging roles in linking DM and PAD. We review the studies investigating the role of lncRNAs in crucial cellular processes contributing to DM-PAD, including those in ECs, VSMCs, and MΦ. By examining the intricate molecular landscape governed by lncRNAs in these relevant cell types, we hope to shed light on the roles of lncRNAs in EC dysfunction, inflammatory responses, and vascular remodeling contributing to DM-PAD. Additionally, we provide an overview of the research approach and methodologies, from identifying disease-relevant lncRNAs to characterizing their molecular and cellular functions in the context of DM-PAD. We also discuss the potential of leveraging lncRNAs in the diagnosis and therapeutics for DM-PAD. Collectively, this review provides a summary of lncRNA-regulated cell functions contributing to DM-PAD and highlights the translational potential of leveraging lncRNA biology to tackle this increasingly prevalent and complex disease.

[Examination of the Thermal Stability and Swallowing Safety of a Self-Developed Enteral Nutrition Thickening Agent]. / è‡ªåˆ¶è‚ å† è¥å »æ¶²å¢žç¨ å‰‚çš„çƒ­ç¨³å®šæ€§åŠåžå’½å®‰å ¨æ€§æŽ¢ç´¢.

Objective: To experimentally validate the effects of a self-developed heat-stable thickening agent on the textual characteristics of enteral nutrition solutions of standard concentration and its applicability in improving dysphagia. Methods: A gradient of different doses of the self-developed thickening agent (1.0 g, 1.5 g, 2.0 g, 2.5 g, and3.0 g) and three commonly used commercial thickeners were mixed with 23.391 g of a complete nutrition formula powder dissolved in 85 mL of purified water to prepare 100 mL standard concentration nutrition solutions. The textual parameters (cohesiveness, viscosity, thickness, and hardness) of these nutrition solutions were measured using a texture analyzer at various temperature gradients (20 ℃, 40 ℃, 60 ℃, and 80 ℃) to compare their thermal stability. A dysphagia rat model was created via epiglottectomy to explore the effects of the thickener on lung tissue damage scores and levels of inflammatory markers. The rats were divided into a test intervention group, a positive control group, a negative control group, and a blank control group (no surgery and normal feeding after fasting for one day), with 15 rats in each group. After fasting for one day post-surgery, the test intervention group was fed with the standard concentration nutrition solution thickened with the self-developed thickener, while the positive control group was given a standard concentration nutrition solution thickened with product 3, and the negative control group was fed a normal diet. All groups were fed for two weeks with food dyed with food-grade green dye. General conditions, body mass, and food intake were observed and recorded. After two weeks, abdominal aorta blood was collected, and heart, liver, spleen, lung, and kidney tissues were harvested and weighed to calculate the lung tissue organ coefficient. The organ conditions were evaluated using routine H&E staining, and lung damage was semi-quantitatively analyzed based on the Mikawa scoring criteria. Blood supernatants were collected to measure the total serum protein and albumin levels to determine the nutritional status of the rats. The expression of IL-6 and TNF-α genes in lung tissues was measured by RT-qPCR. IL-6 and TNF-α protein expression levels in lung tissues, lung tissue homogenate, and serum were measured by ELISA. The aspiration incidence rate was calculated. Results: Within the dosage range of 1.0 g to 3.0 g, the self-developed thickener in the test samples exhibited superior thermal stability in cohesiveness compared to the three commercially available thickeners, with a statistically significant difference (P<0.01). The differences in the thermal stability of viscosity and hardness between the self-developed thickener and the three commercially available thickeners were not statistically significant. The viscosity stability was optimal for the self-developed thickener, followed by the commercially available thickeners 1 and 3, with thickeners 2 being the least stable, though the differences were not statistically significant (P>0.05). Product 1 showed the best thermal stability in thickness, followed by the self-developed thickener and product 2, while the product 3 exhibited the worst performance, with the difference being statistically significant (P<0.01). The self-developed thickener had the best thermal stability in hardness at temperatures ranging from 20℃ to 80 ℃, followed by products 1 and 2, with product 3 being the least stable. However, the differences were not statistically significant (P>0.05). Animal experiment results indicated that the body weight gain in the positive control group and the test intervention group was lower than that in the blank and negative control groups (P<0.01). The spleen coefficient of the intervention group was lower than that of the positive control group and the blank control group (P<0.01), while the heart, liver, and kidney coefficients were lower than those of the blank control group (P<0.01). The differences in the lung coefficient of the intervention group and those of the other three groups were no statistically significant. Levels of TP and ALB in the test intervention group, the positive control group, and the negative control group were all lower than those in the blank control group, with statistically significant differences (P<0.01). ELISA results showed that serum IL-6 levels in the blank and test intervention groups were lower than those in the negative and positive control groups (P<0.05), while the difference in the other indicators across the four groups were not statistically significant (P>0.05). There were no statistically significant differences among the four groups in terms of lung tissue damage pathology scores, or in the levels of IL-6 and TNF-α gene expression in lung tissues. The aspiration incidence rate was 0% in all groups. Conclusion: The self-developed enteral nutrition thickening agent demonstrated excellent thermal stability and swallowing safety. Further research to explore its application in patients with dysphagia is warranted.

Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response.

Vasculopathies occur 15 years earlier in individuals with diabetes mellitus (DM) as compared to those without, but the underlying mechanisms driving diabetic vasculopathy remain incompletely understood. Endothelial cells (ECs) and macrophages (MΦ) are critical players in vascular wall and their crosstalk is crucial in diabetic vasculopathy. In diabetes, EC activation enables monocyte recruitment, which transmigrate into the intima and differentiate into macrophages (MΦ). Beyond this established model of diapedesis, EC-MΦ interplay is highly intricate and heterogenous. To capture these highly context dependent EC-MΦ interactions, we leveraged single-cell (sc)RNA-seq in conjunction with spatial transcriptome (ST)-seq profiling to analyze human mesenteric arteries from non-diabetic (ND) and type 2 diabetic (T2D) donors. We provide in this study a transcriptomic map encompassing major arterial vascular cells, e.g., EC, mononuclear phagocyte (MP), and T cells, and their interactions associated with human T2D. Furthermore, we identified Triggering Receptor Expressed on Myeloid Cells 2 ( TREM2) as a top T2D-induced gene in MP, with concomitant increase of TREM2 ligands in ECs. TREM2 induction was confirmed in mouse models of T2D and monocyte/MΦ subjected to DM-mimicking stimuli. Perturbing TREM2 with either an antibody or silencing RNA in MPs led to decreased pro-inflammatory responses in MPs and ECs and increased EC migration in vitro . In a mouse model of diabetes, TREM2 expression and its interaction with ECs are increased in the ischemic, as compared to non-ischemic muscles. Importantly, neutralization of TREM2 using a neutralizing antibody enhanced ischemic recovery and flow reperfusion in the diabetic mice, suggesting a role of TREM2 in promoting diabetic PAD. Finally, we verified that both TREM2 expression and the TREM2-EC-interaction are increased in human patients with DM-PAD. Collectively, our study presents the first atlas of human diabetic vessels with a focus on EC-MP interactions. Exemplified by TREM2, our study provides valuable insights into EC-MΦ interactions, key processes contributing to diabetic vasculopathies and the potential of targeting these interactions for therapeutic development.

Identification and characterization analysis of candidate genes controlling mushroom leaf development in Chinese kale by BSA-seq.

Mushroom leaves (MLs) are malformed leaves that develop from the leaf veins in some of Chinese kale genotypes. To study the genetic model and molecular mechanism of ML development in Chinese kale, the F2 segregation population was constructed by two inbred lines, genotype Boc52 with ML and genotype Boc55 with normal leaves (NL). In the present study, we have identified for the first time that the development of mushroom leaves may be affected by the change of adaxial-abaxial polarity of leaves. Examination of the phenotypes of F1 and F2 segregation populations suggested that ML development is controlled by two dominant major genes inherited independently. BSA-seq analysis showed that a major quantitative trait locus (QTL) qML4.1 that controls ML development is located within 7.4 Mb on chromosome kC4. The candidate region was further narrowed to 255 kb by linkage analysis combined with insertion/deletion (InDel) markers, and 37 genes were predicted in this region. According to the expression and annotation analysis, a B3 domain-containing transcription factor NGA1-like gene, BocNGA1, was identified as a key candidate gene for controlling ML development in Chinese kale. Fifteen single nucleotide polymorphisms (SNPs) were found in coding sequences and 21 SNPs and 3 InDels found in the promoter sequences of BocNGA1 from the genotype Boc52 with ML. The expression levels of BocNGA1 in ML genotypes are significantly lower than in the NL genotypes, which suggests that BocNGA1 may act as a negative regulator for ML genesis in Chinese kale. This study provides a new foundation for Chinese kale breeding and for the study of the molecular mechanism of plant leaf differentiation. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01364-6.

Analysis of the Complete Mitochondrial Genome of the Bitter Gourd (Momordica charantia).

Bitter gourd (Momordica charantia L.) is a significant vegetable. Although it has a special bitter taste, it is still popular with the public. The industrialization of bitter gourd could be hampered by a lack of genetic resources. The bitter gourd's mitochondrial and chloroplast genomes have not been extensively studied. In the present study, the mitochondrial genome of bitter gourd was sequenced and assembled, and its substructure was investigated. The mitochondrial genome of bitter gourd is 331,440 bp with 24 unique core genes, 16 variable genes, 3 rRNAs, and 23 tRNAs. We identified 134 SSRs and 15 tandem repeats in the entire mitochondrial genome of bitter gourd. Moreover, 402 pairs of repeats with a length greater than or equal to 30 were observed in total. The longest palindromic repeat was 523 bp, and the longest forward repeat was 342 bp. We found 20 homologous DNA fragments in bitter gourd, and the summary insert length was 19,427 bp, accounting for 5.86% of the mitochondrial genome. We predicted a total of 447 potential RNA editing sites in 39 unique PCGs and also discovered that the ccmFN gene has been edited the most often, at 38 times. This study provides a basis for a better understanding and analysis of differences in the evolution and inheritance patterns of cucurbit mitochondrial genomes.

Development of core outcome sets of Food for Special Medical Purposes designed for type 2 diabetes mellitus: a study protocol.

BACKGROUND: The Chinese government stipulates all food for special medical purposes (FSMP) designed for specific diseases to be tested in clinical trials before approving it for registration. The process of developing core outcome sets (COSs), the minimum sets of outcomes supposed to be measured and reported, provides an economical and practical option for stakeholders to communicate and cooperate in conducting clinical trials as well as in reporting FSMP outcomes. This study uses type 2 diabetes mellitus (T2DM) as an example to develop COS for clinical trials of FSMP. METHODS: The COS for FSMP-T2DM will be divided into 3 phases and developed following COS-STAP and COS-STAD: (1) Generate a list of relevant outcomes identified from a systematic review, in which information sources will mainly include published studies, regulatory documentation, and qualitative interviews of stakeholders. The identified outcomes will be categorized using a conceptual framework and formatted into the first round of the Delphi survey questionnaire items. (2) At least 2 rounds of Delphi surveys will be performed among stakeholders to create the COS for FSMP-T2DM. Patients, clinical dietitians, physicians, COS researchers, journal editors, FSMP manufacturers, and regulatory representatives will be invited to score each outcome from aspects of importance. (3) Hold a face-to-face or online consensus meeting to refine the content of the COS for FSMP-T2DM. Key stakeholders will be invited to attend the meeting to discuss and agree on the final COS. DISCUSSION: We have prepared an alternative solution of the Likert scale selection, Delphi survey rounds, scoring group, and consensus definitions in case of an unexpected situation. TRIAL REGISTRATION: COMET (1547). Registered on March 23, 2020.

Green Tea Catechin EGCG Ameliorates Thioacetamide-Induced Hepatic Encephalopathy in Rats via Modulation of the Microbiota-Gut-Liver Axis.

SCOPE: Existing research suggests that (-)-epigallocatechin-3-gallate (EGCG), which is a natural tea catechin active substance, can protect against liver injury. However, its mechanism for hepatic encephalopathy (HE) treatment is still unclear. In this study, the role of EGCG in the amelioration of HE rats and the effect on the microbiota-gut-liver axis are mainly analyzed. METHODS AND RESULTS: Thioacetamide (TAA) is employed to induce the HE model in rats. The results of open field test show that EGCG restores locomotor activity and exploratory behavior. Histological and biochemical results demonstrate that EGCG ameliorates brain and liver damage, decreases the expression of pro-inflammatory cytokines, and increases the activity of antioxidant enzymes. Meanwhile, EGCG modulates the Nrf2 pathway and TLR4/NF-κB pathway to mitigate TAA-induced oxidative stress and inflammatory responses. Immunohistochemistry reveals protection of the intestinal barrier by EGCG upregulating the expression of occludin and zonula occludens-1. Furthermore, serum levels of ammonia and LPS are reduced. 16S rRNA analysis shows that EGCG treatment increases the abundance of beneficial bacteria (e.g., Bifidobacterium, Lactobacillus, and Limosilactobacillus). CONCLUSION: The above results reveal that EGCG has anti-oxidative stress and anti-inflammatory effects, and ameliorates the condition through the microbiota-gut-liver axis, with potential for the treatment of HE.

BocODD1 and BocODD2 Regulate the Biosynthesis of Progoitrin Glucosinolate in Chinese Kale.

Progoitrin (2-hydroxy-3-butenyl glucosinolate, PRO) is the main source of bitterness of Brassica plants. Research on the biosynthesis of PRO glucosinolate can aid the understanding of the nutritional value in Brassica plants. In this study, four ODD genes likely involved in PRO biosynthesis were cloned from Chinese kale. These four genes, designated as BocODD1-4, shared 75-82% similarities with the ODD sequence of Arabidopsis. The sequences of these four BocODDs were analyzed, and BocODD1 and BocODD2 were chosen for further study. The gene BocODD1,2 showed the highest expression levels in the roots, followed by the leaves, flowers, and stems, which is in accordance with the trend of the PRO content in the same tissues. Both the expression levels of BocODD1,2 and the content of PRO were significantly induced by high- and low-temperature treatments. The function of BocODDs involved in PRO biosynthesis was identified. Compared with the wild type, the content of PRO was increased twofold in the over-expressing BocODD1 or BocODD2 plants. Meanwhile, the content of PRO was decreased in the BocODD1 or BocODD2 RNAi lines more than twofold compared to the wildtype plants. These results suggested that BocODD1 and BocODD2 may play important roles in the biosynthesis of PRO glucosinolate in Chinese kale.

Genome-wide analysis of histone acetyltransferase and histone deacetylase families and their expression in fruit development and ripening stage of pepper (Capsicum annuum).

The fruit development and ripening process involve a series of changes regulated by fine-tune gene expression at the transcriptional level. Acetylation levels of histones on lysine residues are dynamically regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), which play an essential role in the control of gene expression. However, their role in regulating fruit development and ripening process, especially in pepper (Capsicum annuum), a typical non-climacteric fruit, remains to understand. Herein, we performed genome-wide analyses of the HDAC and HAT family in the pepper, including phylogenetic analysis, gene structure, encoding protein conserved domain, and expression assays. A total of 30 HAT and 15 HDAC were identified from the pepper genome and the number of gene differentiation among species. The sequence and phylogenetic analysis of CaHDACs and CaHATs compared with other plant HDAC and HAT proteins revealed gene conserved and potential genus-specialized genes. Furthermore, fruit developmental trajectory expression profiles showed that CaHDAC and CaHAT genes were differentially expressed, suggesting that some are functionally divergent. The integrative analysis allowed us to propose CaHDAC and CaHAT candidates to be regulating fruit development and ripening-related phytohormone metabolism and signaling, which also accompanied capsaicinoid and carotenoid biosynthesis. This study provides new insights into the role of histone modification mediate development and ripening in non-climacteric fruits.

Coexpression network analysis reveals an MYB transcriptional activator involved in capsaicinoid biosynthesis in hot peppers.

Plant biosynthesis involves numerous specialized metabolites with diverse chemical natures and biological activities. The biosynthesis of metabolites often exclusively occurs in response to tissue-specific combinatorial developmental cues that are controlled at the transcriptional level. Capsaicinoids are a group of specialized metabolites that confer a pungent flavor to pepper fruits. Capsaicinoid biosynthesis occurs in the fruit placenta and combines its developmental cues. Although the capsaicinoid biosynthetic pathway has been largely characterized, the regulatory mechanisms that control capsaicinoid metabolism have not been fully elucidated. In this study, we combined fruit placenta transcriptome data with weighted gene coexpression network analysis (WGCNA) to generate coexpression networks. A capsaicinoid-related gene module was identified in which the MYB transcription factor CaMYB48 plays a critical role in regulating capsaicinoid in pepper. Capsaicinoid biosynthetic gene (CBG) and CaMYB48 expression primarily occurs in the placenta and is consistent with capsaicinoid biosynthesis. CaMYB48 encodes a nucleus-localized protein that primarily functions as a transcriptional activator through its C-terminal activation motif. CaMYB48 regulates capsaicinoid biosynthesis by directly regulating the expression of CBGs, including AT3a and KasIa. Taken together, the results of this study indicate ways to generate robust networks optimized for the mining of CBG-related regulators, establishing a foundation for future research elucidating capsaicinoid regulation.

Systematic analysis of the Capsicum ERF transcription factor family: identification of regulatory factors involved in the regulation of species-specific metabolites.

BACKGROUND: ERF transcription factors (TFs) belong to the Apetala2/Ethylene responsive Factor (AP2/ERF) TF family and play a vital role in plant growth and development processes. Capsorubin and capsaicinoids have relatively high economic and nutritional value, and they are specifically found in Capsicum. However, there is little understanding of how ERFs participate in the regulatory networks of capsorubin and capsaicinoids biosynthesis. RESULTS: In this study, a total of 142 ERFs were identified in the Capsicum annuum genome. Subsequent phylogenetic analysis allowed us to divide ERFs into DREB (dehydration responsive element binding proteins) and ERF subfamilies, and further classify them into 11 groups with several subgroups. Expression analysis of biosynthetic pathway genes and CaERFs facilitated the identification of candidate genes related to the regulation of capsorubin and capsaicinoids biosynthesis; the candidates were focused in cluster C9 and cluster C10, as well as cluster L3 and cluster L4, respectively. The expression patterns of CaERF82, CaERF97, CaERF66, CaERF107 and CaERF101, which were found in cluster C9 and cluster C10, were consistent with those of accumulating of carotenoids (ß-carotene, zeaxanthin and capsorubin) in the pericarp. In cluster L3 and cluster L4, the expression patterns of CaERF102, CaERF53, CaERF111 and CaERF92 were similar to those of the accumulating capsaicinoids. Furthermore, CaERF92, CaERF102 and CaERF111 were found to be potentially involved in temperature-mediated capsaicinoids biosynthesis. CONCLUSION: This study will provide an extremely useful foundation for the study of candidate ERFs in the regulation of carotenoids and capsaicinoids biosynthesis in peppers.