Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5％,with no significant differences observed(P＞0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100％agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57％(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.

Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5％,with no significant differences observed(P＞0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100％agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57％(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.

OBJECTIVE: To investigate the role and mechanism of the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of interferon gene (cGAS/STING) pathway in oleic acid-induced acute lung injury (ALI) in mice. METHODS: Male wild-type C57BL/6J mice were randomly divided into five groups (each n = 10): normal control group, ALI model group, and 5, 50, 500 μg/kg inhibitor pretreatment groups. The ALI model was established by tail vein injection of oleic acid (7 mL/kg), while the normal control group received no intervention. The inhibitor pretreatment groups were intraperitoneally injected with the corresponding doses of cGAS inhibitor RU.521 respectively 1 hour before modeling. At 24 hours post-modeling, blood was collected, and mice were sacrificed. Lung tissue pathological changes were observed under light microscopy after hematoxylin-eosin (HE) staining, and pathological scores were assessed. Western blotting was used to detect the protein expressions of cGAS, STING, phosphorylated TANK-binding kinase 1 (p-TBK1), phosphorylated interferon regulatory factor 3 (p-IRF3), and phosphorylated nuclear factor-κB p65 (p-NF-κB p65) in lung tissue. Immunohistochemistry was performed to observe STING and p-NF-κB positive expressions in lung tissue. Serum interferon-β (IFN-β) levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with the normal control group, the ALI model group exhibited significant focal alveolar thickening, intra-alveolar hemorrhage, pulmonary capillary congestion, and neutrophil infiltration in the pulmonary interstitium and alveoli, along with markedly increased pathological scores (10.33±0.58 vs. 1.33±0.58, P < 0.05). Protein expressions of cGAS, STING, p-TBK1, p-IRF3, and p-NF-κB p65 in lung tissue significantly increased [cGAS protein (cGAS/β-actin): 1.24±0.02 vs. 0.56±0.02, STING protein (STING/β-actin): 1.27±0.01 vs. 0.55±0.01, p-TBK1 protin (p-TBK1/β-actin): 1.34±0.03 vs. 0.22±0.01, p-IRF3 protein (p-IRF3/β-actin): 1.23±0.02 vs. 0.36±0.01, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 1.30±0.02 vs. 0.53±0.02, all P < 0.05], positive expressions of STING and p-NF-κB in lung tissue were significantly elevated [STING (A value): 0.51±0.03 vs. 0.30±0.07, p-NF-κB (A value): 0.57±0.05 vs. 0.31±0.03, both P < 0.05], and serum IFN-β levels were also significantly higher (ng/L: 256.02±3.84 vs. 64.15±1.17, P < 0.05). The cGAS inhibitor pretreatment groups showed restored alveolar structural integrity, reduced inflammatory cell infiltration, and decreased hemorrhage area, along with dose-dependent lower pathological scores as well as the protein expressions of cGAS, STING, p-TBK1, p-IRF3 and p-NF-κB p65 in lung tissue, with significant differences between the 500 μg/kg inhibitor group and ALI model group [pathological score: 2.67±0.58 vs. 10.33±0.58, cGAS protein (cGAS/β-actin): 0.56±0.03 vs. 1.24±0.02, STING protein (STING/β-actin): 0.67±0.03 vs. 1.27±0.01, p-TBK1 protein (p-TBK1/β-actin): 0.28±0.01 vs. 1.34±0.03, p-IRF3 protein (p-IRF3/β-actin): 0.32±0.01 vs. 1.23±0.02, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 0.63±0.01 vs. 1.30±0.02, all P < 0.05]. Compared with the ALI model group, positive expressions of STING and p-NF-κB in lung tissue were significantly reduced in the 500 μg/kg inhibitor group [STING (A value): 0.40±0.01 vs. 0.51±0.03, p-NF-κB (A value): 0.43±0.02 vs. 0.57±0.05, both P < 0.05], and serum IFN-β levels were also markedly reduced (ng/L: 150.03±6.19 vs. 256.02±3.84, P < 0.05). CONCLUSIONS The cGAS/STING pathway is activated in oleic acid-induced ALI, leading to exacerbated inflammatory responses and increased lung damage. RU.521 can inhibit cGAS, thereby down-regulating the expression of pathway proteins and cytokines, and providing protection to lung tissue.
Animals ; Acute Lung Injury/chemically induced* ; Male ; Nucleotidyltransferases/metabolism* ; Mice ; Signal Transduction ; Mice, Inbred C57BL ; Membrane Proteins/metabolism* ; Oleic Acid/adverse effects* ; Transcription Factor RelA/metabolism* ; Lung/pathology* ; Interferon Regulatory Factor-3/metabolism* ; Disease Models, Animal

TCM proposes that the core pathological mechanism of depression is"deficient qi with stagnation and heat",with the following pathogenic characteristics and evolution patterns:"deficient qi"as the nature,and deficiency in nature is in spleen,and deficiency in superficiality is in brain;"stagnation"is the superficiality,and qi stagnation,phlegm stagnation,and blood stagnation are in the brain collaterals;"heat"fires the brain collaterals,depression raised the heat,and excessive heat accumulated to stagnation.Based on the understanding of the pathogenesis of depression caused by stress sensitization in modern medicine,this article explored the potential association between this mechanism and the core pathogenesis of"deficient qi with stagnation and heat".It proposed that tonifying deficiency,promoting circulation,and clearing heat are the basic treatment principles for depression.By inhibiting inflammatory reactions and improving the stress sensitization state of neurons and glial cells,TCM compound formulas can exert multi-target and multi-dimensional therapeutic characteristics.

Oxalate is an organic dicarboxylic acid that is a common component of plant foods.The kidneys are essential organs for oxalate excretion,but excessive oxalates may induce kidney stones.Jupiter micro-tubule associated homolog 2(JPT2)is a critical molecule in Ca2+mobilization,and its intrinsic mecha-nism in oxalate exposure and kidney stones remains unclear.This study aimed to reveal the mechanism of JPT2 in oxalate exposure and kidney stones.Genetic approaches were used to control JPT2 expression in cells and mice,and theJPT2 mechanism of action was analyzed using transcriptomics and untargeted metabolomics.The results showed that oxalate exposure triggered the upregulation of JPT2,which is involved in nicotinic acid adenine dinucleotide phosphate(NAADP)-mediated Ca2+mobilization.Tran-scriptomic analysis revealed that cell adhesion and macrophage inflammatory polarization were inhibited by JPT2 knockdown,and these were dominated by phosphatidylinositol 3-kinase(PI3K)/AKT signaling,respectively.Untargeted metabolomics indicated that JPT2 knockdown inhibited the produc-tion of succinic acid semialdehyde(SSA)in macrophages.Furthermore,JPT2 deficiency in mice inhibited kidney stones mineralization.In conclusion,this study demonstrates that oxalate exposure facilitates kidney stones by promoting crystal-cell adhesion,and modulating macrophage metabolism and in-flammatory polarization via JPT2/PI3K/AKT signaling.

Kidney stones are mixed by various inorganic salts and organic matter according to certain rules. The process of crystal nucleation, growth and aggregation is the key step of kidney stone formation. The different crystal structures will bring about the different formation process and physicochemical properties of kidney stones. It is of great significance to study the crystal structures and formation characteristics of kidney stone to clarify the causes of it and prevent the recurrence of it. In this paper, based on the microstructure and crystal structure of kidney stones, the distribution of different crystals and components in kidney stones, the nucleation and growth process of crystal forming kidney stones, and the different treatment methods based on crystal structure are reviewed in recent years.

The formation mechanism of kidney stones is complex. It is generally recognized that abnormal urine conditions or renal tubular epithelial cell damage, together with other factors cause the formation of renal papillary subepithelial calcium plaques (Randall’s plaques) or stone crystals that block the renal tubules (Randall’s plugs), and then oversaturated crystals gathering on Randall's plaque or plug and forming stones. However, there are many pathophysiological changes and manifestations, such as renal papillary anchoring stones, renal papillary crypts, renal papillary tip erosion, and exogenous renal papilla Renal papillary lesions, which may be an early manifestations before the formation of kidney stones. The study of renal papillary calcium plaque is very important for the pathogenesis of kidney stones, as well as the prevention and treatment of patients with stones. By focusing on the development process of Randall plaque theory, the formation and transformation mechanism of Randall plaque, as well as the manifestations and clinical treatment of the above mentioned different types of renal papillary calcium plaque lesions, this article reviewed three aspects of stone formation, including Randall’s plaque, renal papillary lesions with stones, and renal papillary lesions related to stone.

Objective:To study the effect of Pterostilbene on endoplasmic reticulum stress and apoptosis in human renal tubular epithelial cells (HK-2 cells) induced by oxalate.Methods:From January 2019 to January 2020, HK-2 cells were divided into a control group (cultured with normal medium), an oxalate group (cultured with a medium containing 4 mmol/L of oxalate), and an intervention group of Pterostilbene (containing 4 mmol/L of oxalate + Pterostilbene 5, 10, and 20 μmol/L mixed medium were cultured at the same time), and the following tests were performed after 12 hours of treatment. Pterostilbene (5, 10, and 20 μmol/L) intervention group for cell viability test, lactate dehydrogenase cytotoxicity test, reduced glutathione, superoxide dismutase, malondialdehyde, hydrogen peroxide enzyme, total antioxidant capacity detection experiments to explore the degree of oxidative damage, and Western blotting experiments to explore the protein expression of ATF6, GRP78, DDIT3, caspase12, Clevead caspase 3/9; Pterostilbene (10 μmol/L) intervention group to detect mitochondrial membrane potential, caspase 3 enzyme activity, apoptosis rate, reactive oxygen detection to detect the apoptosis, reactive oxygen level, and qRT-PCR to detect ATF6, GRP78, DDIT3 of cells mRNA expression.Results:CCK-8 and lactate dehydrogenase toxicity test results showed that the cell activity of the oxalate group was significantly lower than that of the control group [(45.6±3.1)% vs. 100.0%, P<0.001]; the lactate dehydrogenase [(330.2±11.1)U/L vs. (2.6±6.7) U/L, P<0.001] of the oxalate group was higher than that of the control group increased obviously; the cell viability[ (57.2±1.7)%, (67.2±3.4)%, (78.9±1.8)%] of Pterostilbene intervention group (5, 10, 20 μmol/L) significantly increased compared with oxalate group ( P<0.05); lactate dehydrogenase [(288.1±4.3)U/L, (260.9±5.5)U, (202.7±10.2)U/L] in Pterostilbene intervention group (5, 10, 20 μmol/L ) was significantly lower than oxalate group ( P<0.05). The results of the five biochemical indexes of malondialdehyde, reduced glutathione, total superoxide dismutase, catalase, and total antioxidant capacity showed that the cell damage state was consistent with the experimental results of CCK-8 and lactate dehydrogenase. The active oxygen test results showed that the oxalate group had a significantly higher active oxygen level (76.3±4.9 vs. 6.2±1.7, P<0.01); the active oxygen level (39.5±5.4) of the Pterostilbene intervention group(10 μmol/L) was significantly lower than oxalate group ( P<0.01). The flow cytometry and caspase3 enzyme activity showed an increase in apoptosis rate and caspase3 activity in line with the trend of reactive oxygen levels. Mitochondrial membrane potential results showed that the oxalate group had a significantly lower mitochondrial membrane potential (0.76±0.15 vs. 7.84±0.26, P<0.01), and the mitochondrial membrane potential (2.26±0.27) of the Pterostilbene intervention group (10 μmol/L) was significantly higher than oxalate group( P<0.01). Western blot analysis showed that the relative expression of ATF6, DDIT3, GRP78, caspase12 and Cleaved caspase3/9 protein in the oxalate group was significantly higher than that in the control group. The relative expression of ATF6, DDIT3, GRP78, caspase12, Cleaved caspase3/9 protein in the Pterostilbene intervention group was significantly lower than that in the oxalate group ( P<0.05). qRT-PCR results showed that the mRNA expression trends of ATF6, DDIT3 and GRP78 in the three groups were consistent with the results of Western blotting. Conclusion:Pterostilbene can effectively inhibit the endoplasmic reticulum stress and apoptosis of HK-2 cells induced by oxalate.

Objective To investigate the problems during using, standard and quality management of the POCT glucose meters in hospital, to analyze the solutions, and to provide reference data for improving the test level of POCT in hospital.Methods The amount, brand and application of portable glucose meters in the hospital were obtained by 3 rounds of surveillance from May to July in 2013.All of those glucose meters were taken part in external quality assessment of Clinical Laboratory Center of National Health and Family Planning Commission.The test results of those glucose meters were compared with that of automatic biochemical analyzer, the comparison results were then analyzed.Results The POCT glucose meters possessed 5 brands in our hospital, and the amount and type of glucose meters in some clinical departments were often changed.When 4 brands which were detected as quality control samples by ministry of health, the accuracy of detection results of 3 concentration of brand Ⅲ were substandard, the CV% of two levels were 11.9%and 10.1%respectively, the remaining 3 brands were in line with the requirements.The qualified percentages of 3 times of comparison were 85.0%, 92.0%and 97.4%.Conclusions The hospital should select the brand of portable glucose meters reasonably, correct use of glucose meters, and it is very necessary to build indoor and interstitial quality evaluation system, at the same time, suggesting the hospital to establish the POCT quality management team, to carry out the instrument comparison periodically, so to guarantee the accurate, reliable results of POCT in hospital.