Identification of novel genes associated with atherosclerosis in Bama miniature pig.

BACKGROUND: Atherosclerosis is a chronic cardiovascular disease of great concern. However, it is difficult to establish a direct connection between conventional small animal models and clinical practice. The pig's genome, physiology, and anatomy reflect human biology better than other laboratory animals, which is crucial for studying the pathogenesis of atherosclerosis. METHODS: We used whole-genome sequencing data from nine Bama minipigs to perform a genome-wide linkage analysis, and further used bioinformatic tools to filter and identify underlying candidate genes. Candidate gene function prediction was performed using the online prediction tool STRING 12.0. Immunohistochemistry and immunofluorescence were used to detect the expression of proteins encoded by candidate genes. RESULTS: We mapped differential single nucleotide polymorphisms (SNPs) to genes and obtained a total of 102 differential genes, then we used GO and KEGG pathway enrichment analysis to identify four candidate genes, including SLA-1, SLA-2, SLA-3, and TAP2. nsSNPs cause changes in the primary and tertiary structures of SLA-I and TAP2 proteins, the primary structures of these two proteins have undergone amino acid changes, and the tertiary structures also show slight changes. In addition, immunohistochemistry and immunofluorescence results showed that the expression changes of TAP2 protein in coronary arteries showed a trend of increasing from the middle layer to the inner layer. CONCLUSIONS: We have identified SLA-I and TAP2 as potential susceptibility genes of atherosclerosis, highlighting the importance of antigen processing and immune response in atherogenesis.

Roles of heat shock protein A12A in the development of diabetic cardiomyopathy.

Long-term hyperglycemia can lead to diabetic cardiomyopathy (DCM), a main lethal complication of diabetes. However, the mechanisms underlying DCM development have not been fully elucidated. Heat shock protein A12A (HSPA12A) is the atypic member of the Heat shock 70kDa protein family. In the present study, we found that the expression of HSPA12A was upregulated in the hearts of mice with streptozotocin-induced diabetes, while ablation of HSPA12A improved cardiac systolic and diastolic dysfunction and increased cumulative survival of diabetic mice. An increased expression of HSPA12A was also found in H9c2 cardiac cells following treatment with high glucose (HG), while overexpression of HSPA12A-enhanced the HG-induced cardiac cell death, as reflected by higher levels of propidium iodide cells, lactate dehydrogenase leakage, and caspase 3 cleavage. Moreover, the HG-induced increase of oxidative stress, as indicated by dihydroethidium staining, was exaggerated by HSPA12A overexpression. Further studies demonstrated that the HG-induced increases of protein kinase B and forkhead box transcription factors 1 phosphorylation were diminished by HSPA12A overexpression, while pharmacologically inhibition of protein kinase B further enhanced the HG-induced lactate dehydrogenase leakage in HSPA12A overexpressed cardiac cells. Together, the results suggest that hyperglycemia upregulated HSPA12A expression in cardiac cells, by which induced cell death to promote DCM development. Targeting HSPA12A may serve as a potential approach for DCM management.

A mathematical model to simulate the release of Fe and Mn from sediments in a drinking water reservoir.

Fe and Mn release from sediments promotes the release of other chemicals and jointly affects downstream water safety, especially in drinking water reservoirs. Quantitative research on release processes and flux estimation methods for endogenous Fe and Mn in reservoirs is still limited. Static incubation experiments were designed to systematically explore the effects of water temperature (WT), dissolved oxygen (DO), pH, carbon sources, and microbial activity on Fe and Mn release. The results showed that increased WT and carbon source addition promoted the release of acid-extractable Fe and Mn from the sediments; hypoxia and acidification promoted the dissolution of reducible sediment Fe and Mn; and microorganisms participated in the cycling of Fe and Mn. Based on the experimental results, first-order kinetic equations for sediment Fe and Mn release to overlying water were proposed, and the relationships between release rate and environmental factors were mathematically represented by a surface equation (R2 = 0.88 and 0.86, respectively). A diffusion gradients in thin films (DGT) device based on the diffusion model was used in situ to obtain the diffusion fluxes of Fe (JFe = 13.93 mg m-2 d-1) and Mn (JMn = 3.48 mg m-2 d-1). When environmental factors obtained in the field were introduced into the established mathematical model, the modeled release fluxes of Fe and Mn were RFe = 20.92 mg m-2 d-1 and RMn = 13.12 mg m-2 d-1, respectively. The established model filled gaps in the diffusion model, which does not account for differences in release fluxes under changing physicochemical water conditions. This work serves as a reference for studying the release fluxes of endogenous chemicals in sediments.

Hepatocyte HSPA12A inhibits macrophage chemotaxis and activation to attenuate liver ischemia/reperfusion injury via suppressing glycolysis-mediated HMGB1 lactylation and secretion of hepatocytes.

Rationale: Liver ischemia-reperfusion (LI/R) injury is characterized by two interconnected phases: local ischemia that causes hepatic cell damage to release damage-associated molecular pattern (DAMPs), and DAMPs that recruit immune cells to elicit inflammatory cascade for further injury of hepatocytes. High-mobility group box 1 (HMGB1) is a representative DAMP. Studies in macrophages demonstrated that HMGB1 is secreted after lactylation during sepsis. However, whether lactylation mediates HMGB1 secretion from hepatocytes after LI/R is known. Heat shock protein A12A (HSPA12A) is an atypical member of HSP70 family. Methods: Gene expression was examined by microarray analysis and immunoblotting. The hepatic injury was analyzed using released ALT and AST activities assays. Hepatic macrophage chemotaxis was evaluated by Transwell chemotaxis assays. Inflammatory mediators were evaluated by immunoblotting. HMGB1 secretion was examined in exosomes or serum. HMGB1 lactylation was determined using immunoprecipitation and immunoblotting. Results: Here, we report that LI/R decreased HSPA12A expression in hepatocytes, while hepatocyte-specific HSPA12A overexpression attenuated LI/R-induced hepatic dysfunction and mortality of mice. We also noticed that hepatocyte HSPA12A overexpression suppressed macrophage chemotaxis to LI/R-exposed livers in vivo and to hypoxia/reoxygenation (H/R)-exposed hepatocytes in vitro. The LI/R-increased serum HMGB1 levels of mice and the H/R-increased HMGB1 lactylation and secretion levels of hepatocytes were also inhibited by hepatocyte HSPA12A overexpression. By contrast, HSPA12A knockout in hepatocytes promoted not only H/R-induced HMGB1 lactylation and secretion of hepatocytes but also the effects of H/R-hepatocytes on macrophage chemotaxis and inflammatory activation, while all these deleterious effects of HSPA12A knockout were reversed following hepatocyte HMGB1 knockdown. Further molecular analyses showed that HSPA12A overexpression reduced glycolysis-generated lactate, thus decreasing HMGB1 lactylation and secretion from hepatocytes, thereby inhibiting not only macrophage chemotaxis but also the subsequent inflammatory cascade, which ultimately protecting against LI/R injury. Conclusion: Taken together, these findings suggest that hepatocyte HSPA12A is a novel regulator that protects livers from LI/R injury by suppressing glycolysis-mediated HMGB1 lactylation and secretion from hepatocytes to inhibit macrophage chemotaxis and inflammatory activation. Therefore, targeting hepatocyte HSPA12A may have therapeutic potential in the management of LI/R injury in patients.

Identification of RNA-Binding Protein Targets with HyperTRIBE in Saccharomyces cerevisiae.

As a master regulator in cells, RNA-binding protein (RBP) plays critical roles in organismal development, metabolism and various diseases. It regulates gene expression at various levels mostly by specific recognition of target RNA. The traditional CLIP-seq method to detect transcriptome-wide RNA targets of RBP is less efficient in yeast due to the low UV transmissivity of their cell walls. Here, we established an efficient HyperTRIBE (Targets of RNA-binding proteins Identified By Editing) in yeast, by fusing an RBP to the hyper-active catalytic domain of human RNA editing enzyme ADAR2 and expressing the fusion protein in yeast cells. The target transcripts of RBP were marked with new RNA editing events and identified by high-throughput sequencing. We successfully applied HyperTRIBE to identifying the RNA targets of two yeast RBPs, KHD1 and BFR1. The antibody-free HyperTRIBE has competitive advantages including a low background, high sensitivity and reproducibility, as well as a simple library preparation procedure, providing a reliable strategy for RBP target identification in Saccharomyces cerevisiae.

Whole-genome sequencing study to identify candidate markers indicating susceptibility to type 2 diabetes in Bama miniature pigs.

BACKGROUND: Hundreds of single-nucleotide polymorphism (SNP) sites have been found to be potential genetic markers of type 2 diabetes mellitus (T2DM). However, SNPs related to T2DM in minipigs have been less reported. This study aimed to screen the T2DM-susceptible candidate SNP loci in Bama minipigs so as to improve the success rate of the minipig T2DM model. METHODS: The genomic DNAs of three Bama minipigs with T2DM, six sibling low-susceptibility minipigs with T2DM, and three normal control minipigs were compared by whole-genome sequencing. The T2DM Bama minipig-specific loci were obtained, and their functions were annotated. Meanwhile, the Biomart software was used to perform homology alignment with T2DM-related loci obtained from the human genome-wide association study to screen candidate SNP markers for T2DM in Bama miniature pigs. RESULTS: Whole-genome resequencing detected 6960 specific loci in the minipigs with T2DM, and 13 loci corresponding to 9 diabetes-related genes were selected. Further, a set of 122 specific loci in 69 orthologous genes of human T2DM candidate genes were obtained in the pigs. Collectively, a batch of T2DM-susceptible candidate SNP markers in Bama minipigs, covering 16 genes and 135 loci, was established. CONCLUSIONS: Whole-genome sequencing and comparative genomics analysis of the orthologous genes in pigs that corresponded to the human T2DM-related variant loci successfully screened out T2DM-susceptible candidate markers in Bama miniature pigs. Using these loci to predict the susceptibility of the pigs before constructing an animal model of T2DM may help to establish an ideal animal model.

The trends in sports-related spinal cord injury in China.

STUDY DESIGN: Retrospective epidemiological study. OBJECTIVES: To determine the characteristics of sports-related spinal cord injury (SCI) in China and assess changes in the trend of these injuries that may impact policy making. SETTING: China Rehabilitation Research Center (CRRC), Beijing. METHODS: Of the 2448 SCI cases reviewed, 6.7% (n = 164) were caused by sport- and recreation-related accidents. They were admitted to the CRRC between January 1, 2013 and December 31, 2019. We collected data on age, sex, etiology, the neurological level of injury, the American Spinal Injury Association (ASIA) Impairment Scale (AIS) scores on admission, and the neurological recovery results at discharge. RESULTS: Dancing (58.6%), followed by water sports (14.7%) and taekwondo (4.2%) were the leading etiologies. Of the SCIs caused by dancing, 27.1% of the individuals had incomplete injury, and of these, 57.7% showed improved neurological function. However, 72.9% had complete injury, and these individuals did not show any improvement in neurological function. Individuals with dance-related SCIs graded as A and D according to the AIS, showed no significant improvement in their motor function scores at the time of discharge. While the scores of those graded B and C increased significantly, there were no significant differences in the light touch and pin touch scores. CONCLUSIONS: The etiology of sports-related SCI in China has changed dramatically, with dancing replacing water sports as the primary cause of SCIs. Individuals with dance-related SCIs have a poor prognosis. In China, prevention of dance-related SCIs has become a priority.

Establishment of type 2 diabetes mellitus models using streptozotocin after 3 months high-fat diet in Bama minipigs.

In the past twenty years, the number of adults with diabetes has tripled. Most studies have been conducted using rodent models of type 2 diabetes mellitus (T2DM), and the developed drugs have low clinical conversion efficiency. Therefore, it is urgent to establish a more human-like large animal model to explore T2DM pathogenesis and formulate new disease prevention and control strategies. This study was designed to establish and validate a T2DM model using minipigs fed a high-fat or high-cholesterol/high-fat diet and injected with low-dose streptozotocin (STZ). We examined the influence of the STZ injection timing with a diet high in fat (HFD) compared with one high in cholesterol and fat (HCFD) on the atherosclerotic lesions accelerated by T2DM. Male Bama minipigs (n = 24) were randomly divided into five groups. The control group was fed a normal diet for 9 months. The STZ + HFD and STZ + HCFD groups were infused with 90 mg/kg STZ and then fed a high-fat diet or high-cholesterol and high-fat diet for 9 months, respectively. The HFD + STZ and HCFD + STZ groups were fed a high-fat diet or a high-cholesterol and high-fat diet, respectively, for 9 months (after 3 months, these pigs were injected intravenously with 90 mg/kg STZ). During the induction period, animal body weight, BMI, and serum GLU, INS, TG, TC, HDL-C, LDL-C, FFA, ALT, AST, CRE, and BUN were detected monthly intervals. IVGTT and insulin release tests were performed at 3-month intervals. At the end of the test, the coronary artery and abdominal aorta were examined by computed tomography and pathological observations, and the thickness of the basement membrane of the capillary of the retina and kidney glomerulus was measured under a transmission electron microscope. The serum glucose concentrations were normal in all groups except the HFD + STZ and HCFD + STZ groups. Animals fed an HFD for 9 months did not develop apparent atherosclerotic lesions, but atherosclerotic lesions were seen in the animals fed an HCFD. Hyperglycemia accelerated the formation of atherosclerotic lesions on the intimal surface of the abdominal aorta. Low-dose STZ after 3 months of HFD or HCFD successfully established a T2DM model in minipigs. The HFD did not induce apparent atherosclerotic lesions, but these were seen with the HCFD. Hyperglycemia accelerated atherosclerosis in the minipigs.

Evaluation of a new developed disposable and portable bronchoscopy system.

BACKGROUND: Bronchoscopy is critical in the treatment of patients with coronavirus disease (COVID-19), and its use is associated with the challenges of stringent sterilization and virus transmission risk. We developed a disposable and portable bronchoscope (YunSendo-R) and compared its safety and function with those of current reusable and single-use bronchoscopes using an animal model. METHODS: We compared the YunSendo-R system with a commercially available reusable bronchoscope (Olympus, BF-H290) and single-use bronchoscope (Ambu, Ambu® aScope3™). Eight physicians used the three types of bronchoscopes to operate on Guangxi Bama mini pigs. Each operator performed bronchoscopy and completed a 10-point Likert scale questionnaire for evaluating visual ability and manoeuvrability. Operation time and scores were collected. RESULTS: Operation time had no significant differences among the three bronchoscopes. In visual ability, the YunSendo-R bronchoscope showed superior performance to the Ambu bronchoscope in image clarity, colour contrast, and illumination (P < 0.05) and no significant difference in performance compared with the Olympus bronchoscope (P > 0.05). The YunSendo-R bronchoscope had similar manoeuvrability to the Olympus bronchoscope and better scope tip flexibility than the Ambu bronchoscope (P > 0.05). No relevant complications were reported. CONCLUSION: We have developed a new bronchoscopy system with the advantages of disposability and portability, which was effective and safe in an animal model. It has better visual ability than the Ambu bronchoscope and similar visual ability and manoeuvrability to the Olympus bronchoscope. The YunSendo-R bronchoscope is a promising device for clinical practice, especially in reusable-endoscope-transmitted infectious diseases such as COVID-19.

16S rRNA gene sequencing analysis of gut microbiome in a mini-pig diabetes model.

BACKGROUND: Currently, increasing attention is being paid to the important role of intestinal microbiome in diabetes. However, few studies have evaluated the characteristics of gut microbiome in diabetic miniature pigs, despite it being a good model animal for assessing diabetes. METHODS: In this study, a mini-pig diabetes model (DM) was established by 9-month high-fat diet (HFD) combined with low-dose streptozotocin, while the animals fed standard chow diet constituted the control group. 16S ribosomal RNA (rRNA) gene sequencing was performed to assess the characteristics of the intestinal microbiome in diabetic mini-pigs. RESULTS: The results showed that microbial structure in diabetic mini-pigs was altered, reflected by increases in levels of Coprococcus_3 and Clostridium_sensu_stricto_1, which were positively correlated with diabetes, and decreases in levels of the bacteria Rikenellaceae, Clostridiales_vadinBB60_group, and Bacteroidales_RF16_group, which were inversely correlated with blood glucose and insulin resistance. Moreover, PICRUSt-predicted pathways related to the glycolysis and Entner-Doudoroff superpathway, enterobactin biosynthesis, and the l-tryptophan biosynthesis were significantly elevated in the DM group. CONCLUSION: These results reveal the composition and predictive functions of the intestinal microbiome in the mini-pig diabetes model, further verifying the relationship between HFD, gut microbiome, and diabetes, and providing novel insights into the application of the mini-pig diabetes model in gut microbiome research.

Induced pluripotent stem cells as natural biofactories for exosomes carrying miR-199b-5p in the treatment of spinal cord injury.

Background: Induced pluripotent stem cells-derived exosomes (iPSCs-Exo) can effectively treat spinal cord injury (SCI) in mice. But the role of iPSCs-Exo in SCI mice and its molecular mechanisms remain unclear. This research intended to study the effects and molecular mechanism of iPSCs-Exo in SCI mice models. Methods: The feature of iPSCs-Exo was determined by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), and western blot. The effects of iPSCs-Exo in the SCI mice model were evaluated by Basso Mouse Scale (BMS) scores and H&E staining. The roles of iPSCs-Exo and miR-199b-5p in LPS-treated BMDM were verified by immunofluorescence, RT-qPCR, and Cytokine assays. The target genes of miR-199b-5p were identified, and the function of miR-199b-5p and its target genes on LPS-treated BMDM was explored by recuse experiment. Results: iPSCs-Exo improved motor function in SCI mice model in vivo, shifted the polarization from M1 macrophage to M2 phenotype, and regulated related inflammatory factors expression to accelerate the SCI recovery in LPS-treated BMDM in vitro. Meanwhile, miR-199b-5p was a functional player of iPSCs-Exo, which could target hepatocyte growth factor (Hgf). Moreover, miR-199b-5p overexpression polarized M1 macrophage into M2 phenotype and promoted neural regeneration in SCI. The rescue experiments confirmed that miR-199b-5p induced macrophage polarization and SCI recovery by regulating Hgf and Phosphoinositide 3-kinase (PI3K) signaling pathways. Conclusion: The miR-199b-5p-bearing iPSCs-Exo might become an effective method to treat SCI.

Long-term case study of a Wuzhishan miniature pig with diabetes.

BACKGROUND: Miniature pigs are attractive animal models for exploring diabetes because they are similar to humans in terms of physiological structure and metabolism. However, little is known about the complications of diabetes in pigs. METHODS: In this study, a 28-month observation of a Wuzhishan miniature pig with streptozotocin (STZ)-induced (120 mg/kg) diabetes was conducted, to investigate diabetes-related complications and the possibility of self-recovery in miniature pigs. Blood glucose, serum and urinary biochemistry was measured, and histopathologic examinations of eyes, kidney and pancreas were made. RESULTS: During the observation, diabetic complications of eyes and kidney were observed. The eye complications included bilateral cataracts in the 15th month and degeneration of inner retina and microaneurysm in the 28th month. Kidney complications included glomerular mesangial expansion, focal segmental glomerular sclerosis, and renal tubular epithelial degeneration, but no proteinuria was observed. By 28 months after the application of STZ, with no treatment given, blood glucose had recovered and the number of pancreatic islet beta-cells had increased significantly. CONCLUSIONS: We showed that the STZ-induced diabetes model in miniature pigs could accurately mimic the pathological changes of human diabetes, and that pancreatic islet beta-cell regeneration did occur in an adult miniature pig, providing a new means for exploring diabetic complications and pancreatic islet beta-cell regeneration.

Green, fast, and large-scale synthesis of highly fluorescent Au nanoclusters for Cu2+ detection and temperature sensing.

Gold nanoclusters have attracted widespread attention because of their unique optical and physical properties. However, traditional synthesis methods are complicated and require additional reducing agents, while the yield is often very low. Such resource and time-consuming synthesis processes limit their further application. Herein, a rapid sonochemical route is used to synthesize fluorescent Au nanoclusters in large quantities using glutathione (GSH) both as a capping and reducing agent. These Au nanoclusters are synthesized quickly (∼40 min) due to the presence of ultrasonic waves, and show orange red photoluminescence (Em = 598 nm), small size (∼2 nm) and good dispersion in aqueous solution. Moreover, GSH, as a protecting agent on the surface of resultant Au nanoclusters, has many functional groups including carboxyl and amino groups because of which the nanoclusters show high photo-, storage-, metal- and pH-stability. A stable Au nanoclusters-based nano-sensor is designed for highly sensitive and selective label-free detection of Cu2+ with a low limit of detection of 7 ppb (based on S/N = 3). The fluorescent probe can be used in versatile nanothermometry devices, because their photoluminescence intensity correlates strongly with temperature and varies considerably over a wide temperature range (20-80 °C). Therefore, the novel fluorescent sensing probe has great application prospects in Cu2+ detection and temperature sensing.

Regeneration of islet ß-cells in tree shrews and rats.

BACKGROUD: Current understanding of injury and regeneration of islet ß-cells in diabetes is mainly based on rodent studies. The tree shrew is now generally accepted as being among the closest living relatives of primates, and has been widely used in animal experimentation. However, there are few reports on islet cell composition and regeneration of ß-cells in tree shrews. METHODS: In this study, we examined the changes in islet cell composition and regeneration of ß-cells after streptozotocin (STZ) treatment in tree shrews compared with Sprague-Dawley rats. Injury and regeneration of islet ß-cells were observed using hematoxylin and eosin (HE) staining and immunohistochemical staining for insulin, glucagon, somatostatin and PDX-1. RESULTS: Our data showed that in rats islet injury was most obvious on day 3 after injection, and islet morphologies were significantly restored by day 21. Regeneration of islet ß-cells was very pronounced in rats, and mainly involved regeneration of centro-acinar cells and transformation of extra-islet ductal cells. In tree shrews, the regeneration of islet ß-cells was not as significant. On days 3 and 7, only scattered regenerated cells were observed in the remaining islets. Further, no regeneration of centro-acinar cells was observed. CONCLUSION: The results suggest that the repair mechanism of islet ß-cells in tree shrews is similar to that of humans.

Intercellular adhesion molecule-1 expression in activated eosinophils is associated with mucosal remodeling in nasal polyps.

Nasal polyposis (NP) is characterized by chronic mucosal inflammation with infiltrating eosinophils. Eosinophil­mediated tissue remodeling may be involved in NP pathogenesis; therefore, improved understanding of tissue remodeling may result the identification of novel pathways and therapeutic strategies. The present study aimed to investigate the pathological changes occurring during tissue remodeling in NP, in order to assess the role of intercellular adhesion molecule­1 (ICAM­1) in localized tissue remodeling and the potential association between ICAM­1 expression and markers of eosinophil activation. A total of 28 eligible patients and 10 healthy controls participated in the current study. Nasal mucosal tissues of these subjects were retrospectively evaluated for mucosal remodeling using histopathological staining. ICAM­1 and eosinophil cationic protein (ECP) expression levels were determined by immunohistochemical analysis. Compared with the healthy controls, all the specimens from NP patients presented substantial epithelial damage, skewed cellular distribution with a reduced density of goblet cells, an increased density of subepithelial gland and increased subepithelial collagen deposition. In addition, the NP specimens exhibited significantly higher eosinophil infiltration and ICAM­1 expression compared with the controls. Positive correlations were observed between ICAM­1 and ECP expression levels (P=0.010), as well as between extracellular collagen deposition and ICAM­1 (P=0.010) and ECP (P=0.012) expression levels in the NP specimens, but not in the control specimens. Morphological evidence demonstrated eosinophil­mediated tissue remodeling in NP tissues. ICAM­1 expression in activated eosinophils was associated with NP remodeling, indicating the possibility that ICAM­1 may regulate NP remodeling.