A duplex nested RT-PCR method for monitoring porcine epidemic diarrhea virus and porcine delta-coronavirus.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) and porcine delta-coronavirus (PDCoV) are economically important pathogens that cause diarrhea in sows and acute death of newborn piglets. Moreover, the emerging PDCoV was reported to infect children. The current situation is that vaccine prevention has not met expectations, and emergency containment strategies following outbreaks cannot prevent the damages and losses already incurred. Therefore, a more sensitive detection method, that is both convenient and enables accurate and effective sequencing, that will provide early warning of PEDV and PDCoV is necessary. This will enable active, effective, and comprehensive prevention and control, which will possibly reduce disease occurrences. RESULTS: Duplex nested RT-PCR (dnRT-PCR) is an ideal method to achieve early warning and monitoring of PEDV and PDCoV diseases, and to additionally investigate any molecular epidemiological characteristics. In this study, two pairs of primers were designed for each virus based upon the highly conserved N protein sequences of both PEDV and PDCoV strains retrieved from the NCBI Genbank. After optimization of the reaction conditions, the dnRT-PCR assay amplified a 749-bp fragment specific to PEDV and a 344-bp fragment specific to PDCoV. Meanwhile, the specificity and sensitivity of the primers and clinical samples were tested to verify and establish this dnRT-PCR method. The limit of detection (LoD)for both PEDV and PDCoV was 10 copies/µL. The results showed that among 251 samples, 1 sample contained PEDV infection, 19 samples contained a PDCoV infection, and 8 samples were infected with both viruses, following the use of dnRT-PCR. Subsequently, the positive samples were sent for sequencing, and the sequencing results confirmed that they were all positive for the viruses detected using dnRT-PCR, and conventional RT-PCR detection was conducted again after the onset of disease. As these results were consistent with previous results, a detection method for PEDV and PDCoV using dnRT-PCR was successfully established. In conclusion, the dnRT-PCR method established in this study was able to detect both PEDV and PDCoV, concomitantly. CONCLUSIONS: The duplex nested RT-PCR method represents a convenient, reliable, specific, sensitive and anti-interference technique for detecting PEDV and PDCoV, and can additionally be used to simultaneously determine the molecular epidemiological background.

Beneficial effects of andrographolide in a rat model of autoimmune myocarditis and its effects on PI3K/Akt pathway.

The study is to investigate effects of andrographolide on experimental autoimmune myocarditis (EAM). Lewis rats were immunized on day 0 with porcine cardiac myosin to establish EAM. The EAM rats were treated with either andrographolide (25, 50, 100 mg/kg/day) or vehicle for 21 days. An antigen-specific splenocytes proliferation assay was performed by using the cells from control rats immunized with cardiac myosin. Survival rates, myocardial pathology and myocardial functional parameters (left ventricle end-diastolic pressure, ± dP/dt and left ventricular internal dimension) of EAM rats received andrographolide were significantly improved. Andrographolide treatment caused an decrease in the infiltration of CD3+ and CD14+ positive cells in myocardial tissue. Moreover, andrographolide treatment caused a reduction in the plasma levels of tumor necrosis factor-alpha, interleukin-17 (IL-17) and myosin-antibody, and an increase in the level of IL-10 in EAM rats. Oral administration of andrographolide resulted in the decreased expression of p-PI3K, p-Akt without any change of PI3K and Akt. Further results indicate andrographolide significantly inhibited myosin-induced proliferation in splenocytes, and this effect was inhibited by co-treatment of SC79 (Akt activator). Our data indicate andrographolide inhibits development of EAM, and this beneficial effect may be due to powerful anti-inflammatory activity and inhibitory effect on PI3K/Akt pathway.

Camphyl-based α-diimine palladium complexes: highly efficient precatalysts for direct arylation of thiazoles in open-air.

Based on the strategy of the development of phosphine-free palladium-catalyzed direct C-H arylation, a series of camphyl-based α-diimine palladium complexes bearing sterically bulky substituents were synthesized and characterized. The palladium complexes were applied for the cross-coupling of thiazole derivatives with aryl bromides. The effect of the sterically bulky substituent on the N-aryl moiety as well as the reaction conditions was screened. Under the optimal protocols, a wide range of aryl bromides can be smoothly coupled with thiazoles in good to excellent yields in the presence of a low palladium loading of 0.2 mol% under open-air conditions.

[Application of Extreme-Ultraviolet Ar Spectra Analysis in the Study of Divertor Impurity Screening in EAST Tokamak].

Divertor impurity injection on Tokamak is the most important means to achieve divertor impurity screening efficiency. In this paper, a fast-response extreme-ultraviolet (EUV) spectrometer is used to monitor the Ar emission lines during the EAST(Experimental Advanced Superconducting Tokamak)divertor Ar injection experiment. Based on the NIST(National Institute of Standards and Technology)atomic spectrum database, the emission lines from different ionized Ar ions in 2～50 nm wavelength range, e.g. Ar Ⅳ, Ar Ⅳ-Ⅺ and Ar ⅩⅣ-ⅩⅥ, are being identified. Ar ⅩⅥ 35.39 nm and Ar Ⅳ 44.22 nm with the ionization energy of 918.4 and 59.6 eV respectively are being monitored during the experiment with Ar puffing to observe the behavior of Ar impurities in different regions in plasmasimultaneously. The preliminary analysis on divertor impurity screening efficiency is carried outwith the time evolution of intensities of two Ar emission lines. The results of experiment puffing from the same gas puffing inlet (e. g. from lower outer target inlet) and withdifferent plasma configurations (e. g. lower single null, upper single null) show that the screening effect on the impurity injected from the divertor region is better thanfrom the main plasma region; the screening effect of lower divertor and particle pumping by internal cryopump installed in lower divertor is stronger than upper divertor.

Catechin ameliorates cardiac dysfunction in rats with chronic heart failure by regulating the balance between Th17 and Treg cells.

OBJECTIVE: Disequilibrium of the cytokine network was reported to play an important role in the progression of chronic heart failure (CHF). Catechin exerts cardioprotection through treating many kinds of angiocardiopathy. However, the effects of catechin on CHF are currently unclear. Therefore, the main aim of this study was to investigate the efficacy of catechin on CHF rats as well as its relationship to immunoregulation. METHODS: CHF was induced in rats by ligation of the abdominal aorta. Myocardial function was evaluated by left ventricular systolic pressure and left ventricular end-diastolic pressure. The cytokine level was measured by enzyme-linked immunosorbent assay. Th17 and Treg levels in peripheral blood and spleen were analyzed by flow cytometry. RESULTS: The results showed that catechin treatment (50, 100 mg/kg/day) markedly improved myocardial function in rats treated with abdominal aortic coarctation. Severity of myocardial dysfunction in CHF rats significantly correlated with serum values of interleukin-17 (IL-17)/IL-10. Further results indicated catechin obviously inhibited immune activation, regulated unbalanced levels of IL-17/IL-10, and reversed abnormal polarization of TH17 as well as Treg in peripheral blood and spleen. CONCLUSIONS: Taken together, oral administration of catechin effectively suppressed abdominal aorta ligation-induced CHF in rats, which was closely associated with its modulation on Th17 and Treg.

[Experiment study on the treatment of SCI based on HSV carried BDNF transgene technique].

OBJECTIVE: To explore the effect of brain derived neurotrophic factor (BDNF) transgenic treatment in rats following spinal cord injury (SCI). METHODS: BDNF gene was cloned into plasmid then enveloped with human single herpes virus (HSV) to construct HSV carried BDNF transgenic recombinant. BDNF recombinant was injected into sciatic nerve to last label in motorneurous in the caudal cords, then ventral motor neurons were counted and the area of cell body was measured. The BBB scores representing motor function in hindlimbs was also recorded. RESULTS: Five days were needed for the GFP-HSV to arrive motorneurons from sciatic nerve. BDNF release could increase the number of motroneurons and inhibit neuronal atrophy in injured spinal cord. BDNF administration also improves motor function in hindlimbs. CONCLUSION: BDNF transgene carried by HSV is a useful strategy for the treatment of SCI, indicating its clinic implication in future treatment.

Roles of BDNF in spinal neuroplasticity in cats subjected to partial dorsal ganglionectomy.

This study investigated the role of brain-derived neurotrophic factor (BDNF) in neuroplasticity in cats subjected to the removal of dorsal root ganglia (DRG). Following partial ganglionectomy, the number of BDNF-positive varicosities from spared L6 DRG decreased significantly. This reduction was observed at 3 days post operation (dpo) in spinal lamina II of L3 and L5. Whereas the percentages of positive neurons for BDNF and its mRNA in spared L6 DRG at 10 dpo were significantly increased, and accumulated BDNF was seen on the DRG side of the ligated axons. Importantly, BDNF antibody neutralization in vivo results in a significant reduction in the number of varicosities in spinal lamina II, evidenced by BDNF and calcitonin gene-related peptide immunohistochemical staining. These findings suggested that peripheral-derived BDNF could play a critical role in spinal neuroplasticity in cats subjected to partial ganglionectomy. This may underlie the basis of molecular therapy depending on gene drug-like BDNF release.

Recombinant DNA vaccine against inhibition of neurite outgrowth promotes functional recovery associated with endogeous NGF expression in spinal cord hemisected adult rats.

Axonal regeneration across the site of spinal cord lesion is often aborted in adult mammalian species. The use of DNA vaccine to nullify the inhibitory molecules has been shown to be effective in promoting axonal regeneration in injured spinal cord. The possible molecular mechanisms, however, remain to be elucidated. The present study showed that the administration of recombinant DNA vaccine encoding multiple domains, Nogo-66, Nogo-N, TnR, and MAG, significantly improved hindlimb locomotor functions in rats subjected to ablation of the dorsal halves of the cord. Western blot analysis demonstrated that nerve growth factor (NGF) levels in the spinal cord of immunized rats were significantly upregulated than those of control rats. Immunohistochemistry as well as in situ hybridization confirmed that NGF was expressed in neurons of the spinal cord. These findings indicated that functional recovery in immunized rats could be correlated with endogeous NGF expression in hemisected rat spinal cords.

Effects of electro-acupuncture on NT-4 expression in spinal dorsal root ganglion and associated segments of the spinal dorsal horn in cats subjected to adjacent dorsal root ganglionectomy.

It is well known that neuroplasticity occurs in the central nervous system in response to injury. Electro-acupuncture (EA) may also promote neuroplasticity. But little is known about the underlying molecular mechanisms for the beneficial effects of EA. This study investigated the effects of EA on neurotrophin-4 (NT-4) expression in L(6) spinal dorsal root ganglion (DRG) and associated segments of the spinal dorsal horn in cats subjected to unilateral removal of L(1)-L(5) and L(7)-S(2) DRG. NT-4 protein was normally present in the cytoplasm of the L(6) DRG neurons and L(3) and L(6) spinal dorsal horn neurons and glia. Adjacent ganglionectomy leads to a significant decrease in NT-4 expression in the L(6) DRG, but no change in the spinal dorsal horn. Following EA treatment a significant increase occurred in the L(6) DRG at 14 days post-operation (dpo) as well as the L(6) cord segment at 7 and 14 dpo. These findings pointed to a possible association between NT-4 expression and EA promoted spinal cord plasticity in adult cats subjected to partial ganglionectomy.

[Effects of auditory integrative training on autistic children].

OBJECTIVE: To explore the short-term treatment effect of the auditory integrative training on autistic children and provide them with clinical support for rehabilitative treatment. METHODS: A total of 81 cases of autistic children were selected through the standard of DSM-4 and clinical case study was used. They were divided randomly into experimental group and control one, and respectively received auditory integrative training and no training based on the multiple therapies. The patients were investigated using clinical manifestation and Autism Behavior Checklist (ABC) and intelligence quotient (IQ) before and after six months of treatment. The effect was evaluated through the changes of clinical manifestations and scores of ABC and IQ. The changes of scores of IQ were determined with Gesell and WPPSI or WISC-R. RESULTS: Compared with 40 patients of the control group after the six months of the auditory integrative training, 41 of the experimental group had greatly improved in many aspects, such as the disorders of their language, social interactions and typical behavior symptoms while they had not changed in their abnormal behaviors. The scores of IQ or DQ had increased and scores of ABC had dropped. The differences between the two groups were greatly significant in statistics (P < 0.01). The decreasing level of both ABC scores and the increasing level of the IQ scores were negatively correlated with age, and the decreasing level of ABC scores was in line regression(positive correlation) with base IQ. CONCLUSION: The treatment of auditory integrative training (AIT) could greatly improve on language disorders, the difficulties of social interactions, typical behavior symptoms and developmental levels,therefore it is positive to the autistic children in its short-term treatment effect.

Obestatin attenuated doxorubicin-induced cardiomyopathy via enhancing long noncoding Mhrt RNA expression.

OBJECTIVE: The emergence of side-effect of doxorubicin in cardiomyopathy and heart failure has led to the search for diverse strategies to prevent its cytotoxic effects. This study was to determine the role of obestatin on doxorubicin-induced cardiomyocytes apoptosis and possible underlying mechanism. METHODS: Sprague Dawley rats were divided into 3 groups and received treatment for a total of 6 weeks: group1, untreated normal rats; group2, Doxorubicin-induced heart cardiomyopathy (DC) rats; and group3, obestatin treated HC rats. Doxorubicin (2.5mg/kg) or obestatin (100µg/kg/d) were discontinuously administered via intraperitoneal injection. Primary cardiomyocytes and H9C2 cell line were used for in vitro experiments. Mhrt and Nrf2 (nuclear factor erythroid 2 -related factor 2) mRNA expressions were determined using qRT-PCR. Expression of Nrf2 protein was determined using western blotting. TUNEL assay was performed to evaluate cell apoptosis. RESULTS: Administration of obestatin significantly improved doxorubicin-induced dysfunction of left ventricular contractility function, moreover, resulted in upregulation of Mhrt and Nrf2 in failing myocardial tissue. Co-incubation of obestatin and doxorubicin in primary cardiomyocytes also enhanced Mhrt and Nrf2 expression as well as prevented cell apoptosis in comparison with doxorubicin only. Manipulation of cellular Mhrt by pcDNA-Mhrt or si-Mhrt transfection positively regulated Nrf2 expression in doxorubicin-incubated cardiomyocytes. Silencing Mhrt reversed cardioprotective effects of obestatin both in vivo and in vitro. CONCLUSION: Administration of obestatin attenuates doxorubicin-induced cardiac dysfunction via preservation of cardiomyocytes apoptosis in a Mhrt-Nrf2 dependent pathway.

Captopril inhibits maturation of dendritic cells and maintains their tolerogenic property in atherosclerotic rats.

Atherosclerosis (AS) is a systemic disease of the immune system featuring hyperactive dendritic cells (DCs) in atherosclerotic plaques and organs. Captopril, a representative medicine of angiotensin-converting enzyme inhibitors, has been demonstrated to be effective in treating AS. However, captopril's anti-atherosclerotic mechanism is still poorly understood. Therefore, this study was primarily performed to investigate the effects of captopril on the function of DCs in vivo. AS in rats was induced by feeding them with atherogenic diets, and it was evaluated by the levels of plasma lipids and aortic cholesterol. DCs' activity was appraised by endocytic activity, mixed lymphocyte reactions and cytokine secretion. The markers of DCs (CD103, CD80, CD86 and MHC-II) and Treg (CD4(+), CD25(+) and Foxp3(+)) were assayed by western blotting analysis and flow cytometry. Cytokine level was measured by an enzyme-linked immunosorbent assay. The results showed that captopril treatment (10, 20mg/kg/d) obviously improved dyslipidemia and reduced the levels of aortic cholesterol. Captopril significantly reduced CD103, CD80, CD86 and MHC-II protein expression while increasing that of Foxp3 in aortic tissue. Further study indicated oral administration of captopril up-regulated endocytic activity and reduced the immunostimulatory function of splenic DCs. Captopril treatment also promoted IL-10 & TGF-ß production while decreasing that of IL-6 & IL-12 in splenic DCs. Finally, the results of flow cytometry indicated that captopril obviously inhibited DC maturation and promoted Treg polarization. Captopril treatment was able to inhibit DC maturation and maintain their tolerogenic property, which is closely associated with DC anti-atherosclerosis activity.

Role of endogenous PDGF-BB in cultured cardiomyocytes exposed to hypoxia.

Platelet-derived growth factor-BB (PDGF-BB) plays a critical role in cell proliferation, angiogenesis and fibrosis. However, its exact role in cardiomyocytes exposed to hypoxia is not well known. This study was therefore designed to detect whether PDGF-BB expression was changed in a hypoxic condition, then the possible role of endogenous PDGF-BB in cardiomyocytes was explored, with interference RNA in a lentiviral vector ex vivo. The results showed that cultured cardiomyocytes exhibited an optimal proliferation from 3 to 10 days. However, LDH level was significantly increased but the heart rhythm was not altered in cardiomyocytes exposed to hypoxia for 24 hours. PDGF-BB expression was substantially upregulated in hypoxic cardiomyocytes. In order to know the role of PDGF-BB, we performed PDGF-BB knockdown in cultured cardiomyocytes. The number of apoptotic cells and the level of LDH were significantly increased but the beat rhythm was reduced in cardiomyocytes with PDGF-BB knockdown. These findings suggest that endogenous PDGF-BB exerts a crucial protective effect to cultured cardiomyocytes exposed to hypoxia.