Uspostava sendvič ELISE testa za goveđi plazma PON-1 i njegova prediktivna vrijednost za masnu jetru u mliječnih krava

The aim of this study to improve the clinical diagnosis of fatty livers (FL) in dairy cows by using the paraoxonase-1 (PON-1) enzyme as a detection index. Prokaryotic expression technology was used to generate recombinant bovine PON-1 protein. Mice were immunized with this protein to generate hybridoma cells, stably secreting anti-PON-1. Cells were injected into the peritoneal cavity of mice, and ascites were purified to generate bovine PON-1 monoclonal antibody. Rabbits were then immunized with this antigen, and a polyclonal antibody against bovine PON-1 was obtained. Using monoclonal and polyclonal antibodies, a double-antibody sandwich ELISA for plasma PON-1 was constructed. Plasma samples were collected from healthy (n = 13) and FL (n = 13) cows, and plasma PON-1 levels were detected using the PON-1 ELISA. Receiver operating characteristic curve (ROC) analysis was used to analyze correlations between PON-1 levels and FL. Results showed that the ideal working concentration of the monoclonal antibody was 0.8 mg/mL, and the quantitative detection limit was 90 ng/mL. Plasma PON-1 levels were significantly lower in FL cows, when compared with healthy animals. It is concluded that PON-1 ELISA predicts risk factors for dairy cows with FL. PON-1 levels in plasma can be used as an early warning indicator for FL and concentration of 61.87 nmol/L was identified as warning index.Cilj ovog rada bio je unaprijediti kliničku dijagnostiku masne jetre (FL) u mliječnih krava upotrebom enzima paraoksonaze-1 (PON-1) kao indeksa za otkrivanje bolesti. Primijenjena je metoda prokariotske ekspresije kako bi se proizveo rekombinantni goveđi protein PON-1. Miševi su imunizirani ovim proteinom kako bi porizveli stanice hibridoma sa stabilnim izlučivanjem anti-PON-1. Stanice su injektirane u peritonealnu šupljinu miševa te je ascites pročišćen kako bi proizveo goveđa monoklonska protutijela PON-1. Tim su antigenom imunizirani kunići te je dobiveno poliklonsko antitijelo na goveđi PON-1. Upotrebom monoklonskih i poliklonskih antitijela uspostavljen je sendvič ELISA test dva sloja antitijela za plazmatski PON-1. Uzorci plazme prikupljeni su iz zdravih krava (n = 13) i krava s masnom jetrom (n = 13), dok su razine plazmatskog PON-1 detektirane upotrebom PON-1 ELISA. Analizom ROC krivulje analizirane su korelacije između razina PON-1 i FL-a. Rezultati pokazuju da je idealna radna koncentracija monoklonskih protutijela bila 0,8 mg/mL, a kvantitativno ograničenje detekcije 90 ng/mL. Razine plazmatskog PON-1 bile su znakovito niže u krava s masnom jetrom u usporedbi sa zdravim životinjama. Zaključeno je da je PON-1 dobiven ELISA-om prediktor rizičnih čimbenika za masnu jetru u krava. Plazmatske razine PON-1 mogu poslužiti kao rani pokazatelj masne jetre, a kao upozoravajući indeks pokazala se koncentracija od 61,87 nmol/L

Vitamin D status in tuberculosis patients with diabetes, prediabetes and normal blood glucose in China: a cross-sectional study.

OBJECTIVE: The association between tuberculosis (TB), diabetes mellitus (DM) and vitamin D status is poorly characterised. We therefore: (1) determined vitamin D status in patients with TB in relation to whether they had normal fasting blood glucose (FBG), pre-DM or DM and (2) assessed whether baseline characteristics in patients with TB, including their DM status, were associated with vitamin D deficiency. METHODS: In patients with TB consecutively attending six clinics or hospitals in China, we measured 25-hydroxycholecalciferol (25-(OH)D3) at the time of registration using electrochemiluminescence in a COBASE 601 Roche analyser by chemiluminescence immunoassay. Data analysis was performed using the χ2 test, ORs and multivariate logistic regression. RESULTS: There were 306 eligible patients with TB, including 96 with smear positive pulmonary TB, 187 with smear negative pulmonary TB and 23 with extrapulmonary TB. Of these, 95 (31%) had normal blood glucose, 83 (27%) had pre-DM and 128 (42%) had DM. Median serum vitamin D levels were 16.1 ng/mL in patients with TB with normal FBG, 12.6 ng/mL in patients with TB with pre-DM and 12.1 ng/mL in patients with TB with DM (p<0.001). The study highlighted certain baseline characteristics associated with vitamin D deficiency (25-(OH)D3<20 ng/mL). After adjusting for confounders, serum vitamin D deficiency was significantly more common in patients being registered in the cold season (November to April) (p=0.006) and in those with DM (p=0.003). CONCLUSION: Vitamin D levels are lower in patients with TB with pre-DM and DM and are also affected by certain baseline characteristics that include being registered in the cold season and having DM. TB programmes need to pay more attention to vitamin D status in their patients, especially if there is coexisting pre-DM or DM

Lipopolysaccharides Improve Mesenchymal Stem Cell-Mediated Cardioprotection by MyD88 and stat

Bone marrow-derived mesenchymal stem cells (MSCs) improve cardiac function after ischemia/reperfusion injury, in part, due to the release of cytoprotective paracrine factors. Toll-like receptor 4 (TLR4) is expressed in MSCs and regulates the expression of cytoprotective factors, cytokines, and chemokines. Lipopolysaccharide (LPS) stimulation of TLR4 activates two distinct signaling pathways that are either MyD88 dependent or MyD88 independent/TIR-domain-containing adapter-inducing interferon-β (TRIF) dependent. While it was reported previously that LPS treatment improved MSC-mediated cardioprotection, the mechanism underlying such improved effect remains unknown. To study the role of MyD88 signaling in MSC cardioprotective activity, wild type (WT) and MyD88-/- MSCs were treated with LPS (200 ng/mL) for 24 h. WT and MyD88-/- MSCs with or without LPS pretreatment were infused into the coronary circulation of isolated mouse hearts (Langendorff model) and then subjected to ischemia (25 min) and reperfusion (50 min). Saline served as a negative control. Both untreated and LPS-pretreated WT MSCs significantly improved postischemic recovery of myocardial function of isolated mouse hearts, as evidenced by improved left ventricular developed pressure and ventricular contractility assessment (ie, the rate of left ventricle pressure change over time, ± dp/dt). LPS-pretreated WT MSCs conferred better cardiac function recovery than untreated MSCs; however, such effect of LPS was abolished when using MyD88-/- MSCs. In addition, LPS stimulated stat3 activity in WT MSCs, but not MyD88-/- MSCs. stat3 small interfering RNA abolished the effect of LPS in improving the cardioprotection of WT MSCs. In conclusion, this study demonstrates that LPS improves MSC-mediated cardioprotection by MyD88-dependent activation of stat3

Research on the coordination degree of ecological protection and high-quality development of urban agglomerations in the Yellow River Basin

The Yellow River Basin is ecologically fragile, the relationship between water and sand is unbalanced and water resources are scarce. This paper takes the urban agglomerations in the Yellow River Basin as the research object, based on the guidelines for the coordinated promotion of ecological protection and high-quality development in the basin, and uses the entropy weight method and the coefficient of variation method as the means to establish a quantitative evaluation system for coordinated development. The degree of synergy is between ecological protection and high-quality development of the urban agglomerations from the time dimension and the space dimension, and we give the optimization directions. The results show that the degree of synergy among urban agglomerations in the river basin is steadily increasing, but there are significant spatial differences in the degree of coordination of ecological protection and high-quality development of the urban agglomerations and the development index of its subsystems. The degree of synergy generally presents a cascade distribution pattern that gradually decreases from east to west and from downstream to upstream. The ecological destruction, economic fluctuations and water waste of the river basin urban agglomeration are the main factors hindering the improvement of synergy

Povezanost negativne energetske bilance (NEB) s energetskim metabolizmom, proizvodnjom mlijeka i reprodukcijskom sposobnošću mliječnih krava tijekom rane laktacije u pokrajini Heilongjiang, Kina

Negative energy balance (NEB) causes economic losses to dairy farms around the globe. The aim of this study was to investigate the effect of NEB on energy metabolism, reproduction, etc. during early lactation in dairy cows on intensive farms in Heilongjiang, China. According to β-hydroxybutyric acid (BHBA), glucose (GLU), Non-esterified fatty acid (NEFA) levels, and clinical manifestations 14-21 days postpartum, 118 cows were divided into a positive energy balance (PEB) group (BHBA2.8, NEFA1.2, GLU0.7 mmol/L, n=51). These indicators were analyzed by cross-sectional research methods combined with Pearson correlation analysis and a prospective cohort study. The results showed that at 14-21 days postpartum, compared with the PEB, the body condition score, body condition loss (BCL), milk urea nitrogen, BHBA, NEFA, the interval from calving to first estrus (ICFE), pregnancy per artificial insemination (P/AI), and calving interval were higher (P2,8, NEFA1,2, GLU0,7 mmol/L; n=51). Navedeni pokazatelji analizirani su kombinacijom metoda presječnog istraživanja, Pearson-ovog koeficijenta korelacije i prospektivnog kohortnog istraživanja. Rezultati su pokazali da je 14-21 dan nakon porođaja skupina NEB u usporedbi sa skupinom PEB imala višu ocjenu tjelesne kondicije, veći gubitak tjelesne kondicije (BCL), te veće vrijednosti za dušik iz ureje mlijeka, BHBA, NEFA, interval od teljenja do prvog estrusa (ICFE), graviditete po umjetnoj oplodnji (P/AI) i interval između teljenja (P<0,05), a niže vrijednosti zadnevnu proizvodnju mlijeka (DL), mliječni protein, GLU, stopu pojave estrusa i stopu koncepcije. Gubitak tjelesne kondicije (BCL) je bio pozitivno povezan s ICFE i P/AI (P<0,05) i negativno povezan sa stopom pojave estrusa te stopom koncepcije (P<0,05). Dnevna proizvodnja mlijeka (DL) pokazala je negativnu povezanost s P/AI (P<0,05). Uočena je pozitivna povezanost NEB-a i anestrusa (2M-H = 12,63, P = 0,0004), a rizik od anestrusa uzrokovanih NEB povećan je 3,67 puta u odnosu na PEB. Zaključci su pokazali da je NEB usko povezan s BCL, što je čimbenik koji utječe na snižavanje proizvodnje mlijeka i reprodukciju mliječnih krava. Osim toga, NEB se pokazao i kao čimbenik rizika za anestrus u mliječnih krava

Reduction in Migratory Phenotype in a Metastasized Breast Cancer Cell Line via Downregulation of S100A4 and GRM3

To investigate phenotypic and genotypic alterations before and after bone metastasis, we conducted genome-wide mRNA profiling and DNA exon sequencing of two cell lines (TMD and BMD) derived from a mouse xenograft model. TMD cells were harvested from the mammary fat pad after transfecting MDA-MB-231 breast cancer cells, while BMD cells were isolated from the metastasized bone. Compared to BMD cells, TMD cells exhibited higher cellular motility. In contrast, BMD cells formed a spheroid with a smoother and more circular surface when co-cultured with osteoblasts. In characterizing mRNA expression using principal component analysis, S100 calcium-binding protein A4 (S100A4) was aligned to a principal axis associated with metastasis. Partial silencing of S100A4 suppressed migratory capabilities of TMD cells, while Paclitaxel decreased the S100A4 level and reduced TMD's cellular motility. DNA mutation analysis revealed that the glutamate metabotropic receptor 3 (GRM3) gene gained a premature stop codon in BMD cells, and silencing GRM3 in TMD cells altered their spheroid shape closer to that of BMD cells. Collectively, this study demonstrates that metastasized cells are less migratory due in part to the post-metastatic downregulation of S100A4 and GRM3. Targeting S100A4 and GRM3 may help prevent bone metastasis

Regulation of autophagy by AMP-activated protein kinase/ sirtuin 1 pathway reduces spinal cord neurons damage

Objective(s): AMP-activated protein kinase/sirtuin 1 (AMPK/SIRT1) signaling pathway has been proved to be involved in the regulation of autophagy in various models. The aim of this study was to evaluate the effect of AMPK/SIRT1 pathway on autophagy after spinal cord injury (SCI). Materials and Methods:The SCI model was established in rats in vivo and the primary spinal cord neurons were subjected to mechanical injury (MI) in vitro. The apoptosis in spinal cord tissue and neurons was assessed by TUNEL staining and Hoechst 33342 staining, respectively. The autophagy-related proteins levels were detected by Western blot. The activation of AMPK/SIRT1 pathway was determined by Western blot and immunohistochemical staining. Results: We found that the apoptosis of spinal cord tissue and cell damage of spinal cord neurons was obvious after the trauma. The ratio of LC3II/LC3I and level of p62 were first increased significantly and then decreased after the trauma in vivo and in vitro, indicating the defect in autophagy. The levels of p-AMPK and SIRT1 were increased obviously after the trauma in vivo and in vitro. Further activation of the AMPK/SIRT1 pathway by pretreatment with resveratrol, a confirmed activator of the AMPK/SIRT1 pathway, alleviated the cell damage and promoted the autophagy flux via downregulation of p62 in spinal cord neurons at 24 hr after MI. Conclusion: Our results demonstrate that regulation of autophagy by AMPK/SIRT1 pathway can restrain spinal cord neurons damage, which may be a potential intervention of SCI

Effects of rhythm control on left atrial structure remodeling in atrial fibrillation and heart failure with preserved ejection fraction

BackgroundThe benefits of rhythm control for atrial fibrillation (AF) in heart failure with preserved ejection fraction (HFpEF) have not been conclusively determined. We assessed the effects of rhythm control on left atrial (LA) structure remodeling and prognosis in patients with AF and HFpEF.MethodsThis was a retrospective, real-world, observational study involving patients diagnosed with AF and HFpEF. The cohort was divided into rhythm-control and rate-control groups depending on their treatment strategies. The primary outcomes were all-cause mortality, rehospitalization for any cause, HF-related rehospitalization, and stroke. Differences in follow-up LA structure parameters were also analyzed.ResultsCompared to the rate-control group, patients in the rhythm-control group had a lower risk of HF-related rehospitalization even after adjusting for potential confounders (adjusted HR 0.605, 95% CI 0.413–0.887, p = 0.010). Moreover, rhythm-control therapy led to marked reductions in LA echocardiographic indicators and a higher proportion of LA reverse remodeling (LARR).ConclusionsRhythm-control therapy reverses LA structure remodeling and is associated with improved clinical outcomes; therefore, it is an optimal treatment approach for AF in HFpEF patients