A risk model for the early diagnosis of acute myocardial infarction in patients with chronic kidney disease.

Introduction: Acute myocardial infarction (AMI) remains a critical disease, characterized by a high fatality rate in several countries. In clinical practice, the incidence of AMI is increased in patients with chronic kidney disease (CKD). However, the early diagnosis of AMI in the above group of patients is still poor. Methods: In the present study, a total of 829 patients with CKD, defined by an estimated glomerular filtration rate (eGFR) of <60 ml/min/1.73 m2 or 60-90 ml/min/1.73 m2 for patients with mildly reduced kidney function, who attended the Sichuan Provincial People's Hospital (SPPH) between January 2018 and November 2022 were enrolled. All patients underwent coronary angiography due to the presence of typical or atypical symptoms of AMI. Patients were divided into the following two groups: The training cohort, including 255 participants with AMI and 242 without AMI; and the testing cohort, including 165 and 167 subjects with and without AMI, respectively. Furthermore, a forward stepwise regression model and a multivariable logistic regression model, named SPPH-AMI-model, were constructed to select significant predictors and assist the diagnosis of AMI in patients with CKD, respectively. Results: The following factors were evaluated in the model: Smoking status, high sensitivity cardiac troponin I, serum creatinine and uric acid levels, history of percutaneous coronary intervention and electrocardiogram. Additionally, the area under the curve (AUC) of the receiver operating characteristic curve were determined in the risk model in the training set [AUC, 0.78; 95% confidence interval (CI), 0.74-0.82] vs. the testing set (AUC, 0.74; 95% CI, 0.69-0.79) vs. the combined set (AUC, 0.76; 95% CI, 0.73-0.80). Finally, the sensitivity and specificity rates were 71.12 and 71.21%, respectively, the percentage of cases correctly classified was 71.14%, while positive and negative predictive values of 71.63 and 70.70%, respectively, were also recorded. Discussion: The results of the current study suggested that the SPPH-AMI-model could be currently considered as the only risk scoring system for the early diagnosis of AMI in patients with CKD. This method could help clinicians and emergency physicians to quickly and accurately diagnose AMI in patients with CKD to promote the immediate and effective treatment of these patients.

Molecular Cloning and Expression Analysis of Enolase in the Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus is the main blooding-sucking ectoparasite of bovines and is regarded as important vectors of animal diseases such as Babesiosis. Mining protective antigens of R. microplus to develop antitick vaccine is the most potential tick control strategy. In this study, the specific primers were designed according to the conserved nucleotide sequence of enolase gene in Haemaphysalis flava, Ixodes ricinus, and Ornithodoros moubata. The fragment of enolase gene was obtained by PCR using cDNA template from fully engorged female R. microplus. The full-length cDNA of enolase gene was amplified using rapid amplification of cDNA ends (RACE). Expression pattern of enolase gene in different tissues of R. microplus was analyzed by real-time quantitative PCR (qRT-PCR). Results showed that the full-length enolase cDNA containing 2052 bp was obtained successfully. The complete cDNA included an ORF of 1305 nucleotides encoding a protein of 434 amino acids. The enolase exhibited 85.0% amino acid identity to the enolase of H. flava, 81.1% to I. ricinus enolase, and 81.3% to O. moubata enolase. qRT-PCR analysis indicated that the enolase had the highest expression in the salivary gland of R. microplus.

Fibroblast growth factor-2/platelet-derived growth factor enhances atherosclerotic plaque stability.

Increased immature neovessels contribute to plaque growth and instability. Here, we investigated a method to establish functional and stable neovessel networks to increase plaque stability. Rabbits underwent aortic balloon injury and were divided into six groups: sham, vector and lentiviral transfection with vascular endothelial growth factor-A (VEGF)-A, fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-BB and FGF-2 + PDGF-BB. Lentivirus was percutaneously injected into the media-adventitia of the abdominal aorta by intravascular ultrasound guidance, and plaque-rupture rate, plaque-vulnerability index and plaque neovessel density at the injection site were evaluated. Confocal microscopy, Prussian Blue assay, Evans Blue, immunofluorescence and transmission electron microscopy were used to assess neovessel function and pericyte coverage. To evaluate the effect of FGF-2/PDGF-BB on pericyte migration, we used the mesenchymal progenitor cell line 10T1/2 as an in vitro model. VEGF-A- and FGF-2-overexpression increased the number of immature neovessels, which caused intraplaque haemorrhage and inflammatory cell infiltration, eventually resulting in the plaque vulnerability; however, FGF-2/PDGF-BB induced mature and functional neovessels, through increased neovessel pericyte coverage. Additionally, in vitro analysis of 10T1/2 cells revealed that FGF-2/PDGF-BB induced epsin-2 expression and enhanced the VEGF receptor-2 degradation, which negatively regulated pericyte function consistent with the in vivo data. These results showed that the combination of FGF-2 and PDGF-BB promoted the function and maturation of plaque neovessels, thereby representing a novel potential treatment strategy for vulnerable plaques.

Genetic variation in mitochondrial genes of the tick Haemaphysalis flava collected from wild hedgehogs in China.

The tick Haemaphysalis flava (Acari: Ixodidae) is an important ectoparasite, which causes direct damage to their hosts and also acts as a vector of various infectious disease agents in China. Despite its significance, the epidemiology, genetics and biology of H. flava has not been studied in detail. In the present study, the genetic variation in three mitochondrial (mt) DNA regions, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 and 4 (nad1 and nad4), was examined in H. flava ticks collected from wild hedgehogs in China. A portion of cox1 (pcox1), nad1 (pnad1) and nad4 (pnad4) genes were PCR amplified from individual H. flava ticks and the amplicons were sequenced. The length of the sequences of pcox1, pnad1 and pnad4 were 849, 285 and 626 bp, respectively. The intra-specific sequence variation within H. flava was 0-0.4% for pcox1, 0-0.4% for pnad1 and 0-0.3% for pnad4. However, the inter-specific variation was significantly higher, 12.5-14.3%, 13.6-24.8% and 14.8-19% for pcox1, pnad1 and pnad4, respectively. Phylogenetic analysis based on Maximum likelihood (ML) method using the combined target mt gene sequences confirmed that all isolates of Haemaphysalis were H. flava. The molecular approach employed in this study provides a tool for further elucidating the molecular diversity of H. flava in China and elsewhere in Asia.

Integrative analysis of transcriptomics and proteomics of skeletal muscles of the Chinese indigenous Shaziling pig compared with the Yorkshire breed.

BACKGROUND: The Shaziling pig (Sus scrofa) is a well-known indigenous breed in China. One of its main advantages over European breeds is its high meat quality. However, little genetic information is available for the Shaziling pig. To screen for differentially expressed genes and proteins that might be responsible for the meat quality, the longissimus dorsi muscles from Shaziling and Yorkshire pig breeds were investigated using an integrative analysis of transcriptomics and proteomics, involving high-throughput sequencing, the two-dimensional gel electrophoresis, and mass spectrometry. RESULTS: Sequencing produced 79,320 unigenes by de novo assembly, and 488 differentially expressed genes in the longissimus dorsi muscle of Shaziling pig compared with the Yorkshire breed were identified. Gene Ontology term enrichment of biological functions and Kyoto Encyclopedia of Genes and Genomes analysis showed that the gene products were mainly involved in metabolism, protein binding, and regulation of skeletal muscle development. At the protein level, 23 differentially expressed proteins were identified, which were potentially associated with fatty acid metabolism, the glycolytic pathway, and skeletal muscle growth. Eight differentially expressed genes were confirmed by real-time PCR. These results give an insight into the mechanisms underlying the formation of skeletal muscle in the Shaziling pig. CONCLUSIONS: Certain differentially expressed genes and proteins are involved in fatty acid metabolism, intramuscular fat deposition, and skeletal muscle growth in the Shaziling pig. These results provide candidate genes for improving meat quality and will promote further transcriptomic research in Shaziling pigs.

Enolase, a plasminogen receptor isolated from salivary gland transcriptome of the ixodid tick Haemaphysalis flava.

Enolase, a multifunctional protein, is shown to act as a plasminogen receptor that contributes to fibrinolysis, which plays an important role in preventing the formation of blood clots during tick feeding. The study of enolase genes provides opportunities to develop a potential antigen target for tick control. So far, enolase has been identified in only a few species of ticks. Knowledge of the exact mechanisms of plasminogen activation and fibrinolysis by enolase as a plasminogen receptor is limited. Here, we cloned the enolase full-length complementary DNA (cDNA) from the salivary glands of Haemaphysalis flava, expressed it, and analyzed the function of the recombinant H. flava enolase. The enolase cDNA was 1988 bp in length and encoded 433 amino acid residues. It contained two domains and some highly conserved functional motifs including an assumed membrane re-association region "AAVPSGASTGI." The enolase exhibited 83.3 % amino acid similarity to that of the putative enolase of Ixodes ricinus, and 85 % to that of Ornithodoros moubata enolase. After eukaryotic expression in insect cells, Western blot analysis showed that the mouse antiserum against the hexahistidine-tagged recombinant enolase protein recognized a band of approximately 48 kDa. The recombinant enolase bound human plasminogen in a dose-dependent manner and enhanced plasminogen activation in the presence of host tissue plasminogen activator (t-PA), most probably to promote fibrinolysis and maintain blood flow at the host-tick interface. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that the expression level of enolase in salivary glands was significantly higher than in other tested tissues. Although the enolase was expressed in all developmental stages, it had the highest expression in the rapid blood feeding period of ticks. These findings indicate that the enolase might play an important role in blood feeding of H. flava.

De novo assembly and analysis of midgut transcriptome of Haemaphysalis flava and identification of genes involved in blood digestion, feeding and defending from pathogens.

Bioactive components in the midgut of ticks play a key role in tick blood digestion, feeding and pathogen transmission. The study of protein and gene targets in midgut provides opportunities to explore novel tick control strategies. Only a few nucleotide sequences are available in public databases for Haemaphysalis flava, an important disease vector for humans and animals. Knowledge of the process of blood digestion by the ticks and protein expression in the digestive tract is limited. Here, we utilize high-throughput sequencing to characterize the midgut transcriptome of fully engorged (FE, average length of 10mm) and partially engorged (PE, average length of 5mm) female H. flava. 6.8GB and 8.3GB of high-quality sequence data were obtained using Illumina sequencing technology. 54,357,490 and 66,116,050 reads were finally assembled into 76,556 unigenes with mean length of 704bp. The transcripts involved in blood meal digestion were classified into eight large categories, including peptidase inhibitor, peptidase (serine-, metallo-, cysteine-, aspartic-peptidase), phospholipase, carbohydrate digestion/hydrolases, lipid binding, immunity-related proteins, iron/heme metabolism and secreted proteins. A total of 5508 differentially expressed genes (DEGs) were identified between FE and PE. To confirm the DEG results, ten genes involved in blood digestion, feeding and defending from pathogens, were validated using qPCR. Our results not only contribute to better understanding of the changes in midgut transcript expression during different blood feeding stages, but also provide a valuable resource for identifying targets for future tick control studies.

The complete sequence of mitochondrial genome of Laiwu Black pig (Sus Scrofa).

In the present study, the ear tissue of an adult Laiwu Black pig is from the Shandong province of China. The complete mitochondrial genome of Laiwu Black pig was determined by polymerase chain reaction (PCR). The complete mitochondrial genome is 16,710 bp, and it contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, a control region (D-loop), with the genome organization and gene order being identical to that of the typical vertebrates.

The complete sequence of mitochondrial genome of Bama miniature pig (Sus scrofa).

The complete mitochondrial nucleotide sequence of the Bama miniature pig was sequenced in this study. This sequence is homologous to that of the other vertebrate mitochondrial genome. The mitochondrial genome of the Bama miniature pig is 16,711 bp and contains 37 genes including 22 tRNA genes, 13 protein-coding genes (PCGs), 2 ribosomal RNA genes and 1 control region (D-loop). The 13 PCGs initiated with ATN as start codon and terminated with TAA, TA or T as stop codon. The DNA sequence in the Bama miniature pig D-loop region contains the repeat motif (-TACACGTGCG-) and 5'nucleotide of the first repeat is at the position 796 bp.

Identification of intestinal bacterial flora in Rhipicephalus microplus ticks by conventional methods and PCR-DGGE analysis.

In this study, we have analyzed the intestinal microbial flora associated with Rhipicephalus microplus ticks using both culture-dependent and independent methods based on PCR and denaturing gradient gel electrophoresis (PCR-DGGE). The R. microplus ticks were collected from cattle and goats in Jiangxi, Hunan and Guizhou Provinces of China. Three distinct strains of bacteria were isolated using culture-dependent methods: Staphylococcus simulans, Bacillus subtilis and Bacillus flexus strain. Nineteen distinct DGGE bands were found using PCR-DGGE analysis, and their search for identity shows that they belonged to Rickettsiaceae, Xanthomonadaceae, Coxiella sp., Ehrlichia sp., Pseudomonas sp., Ehrlichia sp., Orphnebius sp., Rickettsia peacockii, Bacillus flexus. Rickettsia peacockii and Coxiella genus were the dominant strain of the R. microplus ticks from cattle, Pseudomonas sp. and B. flexus strain were the most common species in all tick samples from goats. Ehrlichia canis were detected only in R. microplus ticks from Yongshun area in Hunan Province. The results indicate that the intestinal microbial diversity of R. microplus ticks was influenced by tick hosts and local differences in the sampling location and these two aspects may affect transmission of pathogen to humans and animals.

De novo sequencing, assembly and analysis of salivary gland transcriptome of Haemaphysalis flava and identification of sialoprotein genes.

Saliva plays an important role in feeding and pathogen transmission, identification and analysis of tick salivary gland (SG) proteins is considered as a hot spot in anti-tick researching area. Herein, we present the first description of SG transcriptome of Haemaphysalis flava using next-generation sequencing (NGS). A total of over 143 million high-quality reads were assembled into 54,357 unigenes, of which 20,145 (37.06%) had significant similarities to proteins in the Swiss-Prot database. 13,513 annotated sequences were associated with GO terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 14,280 unigenes were assigned to 279 KEGG pathways in total. Reads per kb per million reads (RPKM) analysis showed that there were 3035 down-regulated unigenes and 2260 up-regulated unigenes in the engorged ticks (ET) compared with the semi-engorged one (SET). Several important genes are associated with blood feeding and ingestion as secreted salivary proteins, concluding cysteine, longipain, 4D8, calreticulin, metalloproteases, serine protease inhibitor, enolase, heat shock protein and AV422 in SG, were identified. The qRT-PCR results confirmed that patterns of these genes (except for the longipain gene) expression were consistent with RNA-seq results. This de novo assembly of SG transcriptome of H. flava not only provides more chance for screening and cloning functional genes, but also forms a solid basis for further insight into the changes of salivary proteins during blood-feeding.