Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus.

An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/µL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health.

Vitexin protects against ischemia/reperfusion-induced brain endothelial permeability.

Brain endothelial permeability plays a crucial role in blood-brain barrier (BBB), but the permeability enhancement in cerebral ischemia reperfusion (I/R). Vitexin has certain neuroprotective effects, but the effect brain endothelial permeability in I/R injury was unknown. In this study, the effects of Vitexin on endothelial permeability and the underlying mechanisms in human brain microvascular endothelial cells (HBMEc) I/R injury model were investigated. Cell viability, lactate dehydrogenase (LDH), inflammation and apoptosis were detected. The effects of Vitexin on BBB integrity tight junction, matrix metalloproteinases (MMP) were also investigated. The mechanism was confirmed by PI3K inhibitor and NOS inhibitor in normal or eNOS siRNA transfection HBMEc. Vitexin significantly reduced LDH, Caspase 3 level, alleviated inflammation, also could maintain BBB integrity, increased tight junction proteins expression and inhibited MMP. The mechanism is related to reduction of intracellular NO and ONOO-, regulated eNOS, iNOS activity. Vitexin significantly preserved eNOS phosphorylation in response to the activated Akt. Moreover, combined with PI3K inhibitor or low dosage of NOS inhibitor, totally abolished Vitexin-induced eNOS phosphorylation, the protected effect was also attenuated, but still significantly between model cells. However, combined with high dosage NOS inhibitor which both inhibited the eNOS phosphorylation and iNOS, the protected effect of Vitexin was abrogated. In addition, eNOS silencing cells were used to further clarify the regulatory role of Vitexin on iNOS. Our findings showed that Vitexin could play a protective role in I/R-induced brain endothelial permeability by simultaneously increase eNOS phosphorylation and inhibit iNOS.

Toosendanin mediates cisplatin sensitization through targeting Annexin A4/ATP7A in non-small cell lung cancer cells.

Cisplatin (CDDP) is used in the treatment of non-small cell lung cancer (NSCLC), but due to the development of resistance, the benefit has been limited. Toosendanin (TSN) has shown therapeutic effects on NSCLC; however, the role of TSN on CDDP sensitization in NSCLC remains unknown. The antitumor effects of TSN and CDDP sensitization mediated by TSN were explored. TSN was added in various amounts to measure dose- and time-dependent cytotoxicity. Intracellular CDDP was detected by high-performance liquid chromatography. The protein levels of ATP7A, ATP7B, hCTR1, MRP-2, P-gp and Annexin A4 (Anxa4) were analyzed. The tests were conducted using normal NSCLC (A549 cell line) and CDDP-resistant cells (A549/DDP cell line). Anxa4 promotes CDDP resistance by regulating ATP7A, so Anxa4 was overexpressed and silenced and also transfected with pcMV6 or siRNA/ATP7A, respectively. Mechanistic investigations revealed that TSN decreased relative viability in NSCLC cells. Remarkably, TSN significantly enhanced CDDPsensitization in invalid doses. TSN downregulated Anxa4 expression, enhanced intracellular CDDP, and had no effect on MRP-2, P-gp, ATP7A, ATP7B or hCTR1. Subsequently, overexpression of Anxa4 led to a significant decrease in intracellular CDDP concentration. The adjustment of CDDP concentration regulated by TSN disappeared in Anxa4 or ATP7A-silenced cells. TSN also enhanced CDDP sensitization in single ATP7A-overexpressing cells, but had no effect on cells with simultaneous ATP7A overexpression and Anxa4 silencing. The present study suggests that TSN can mediate CDDP sensitization in NSCLC through downregulation of Anxa4.

6-Hydroxydopamine promotes iron traffic in primary cultured astrocytes.

It is well known that disrupted brain iron homeostasis was involved in Parkinson's disease. We previously reported 6-hydroxydopamine (6-OHDA) could enhance iron influx and attenuate iron efflux process, thus promote iron accumulation in neurons. Astrocytes, the major glial cell type in the central nervous system, are largely responsible for iron distribution in the brain. However, how iron metabolism changes in astrocytes with 6-OHDA treatment are not fully elucidated. In the present study, we first observed that both iron influx and efflux were enhanced with 10 µM 6-OHDA treatment for 24 h in primary cultured astrocytes. In accordance with these iron traffic modulations, both mRNA and protein levels of iron importer divalent metal transporter 1 with iron responsive element (DMT1+IRE) and exporter ferroportin 1 (FPN1) were up-regulated in these cells. L-ferritin mRNA levels were increased. Iron regulatory protein 1 (IRP1) showed a dynamic regulation with 6-OHDA treatment, as indicated by a moderate up-regulation at 12 h, however, down-regulation at 24 h. We further demonstrated that 6-OHDA treatment could induce activation of nuclear factor-kappaB (NF-κB) p65. IκBα activation inhibitor BAY11-7082 fully blocked 6-OHDA induced NF-κB p65 phosphorylation and DMT1 + IRE up-regulation. These results suggest that 6-OHDA might promote iron transport rate in astrocytes by regulating iron transporters, IRP1 expression and NF-κB p65 activation, indicating a different response between neurons and astrocytes.

Transmission of porcine circovirus type 2b (PCV2b) in Kunming mice.

To investigate porcine circovirus type 2b (PCV2b) transmission by contact and vertical infection in Kunming mice (an outbred mouse stock deriving from Swiss albino mice with a high ratio of gene heterozygosis), four mice in cage 6 were inoculated with PCV2b and 25 mice without any treatment were placed into cages 1 to 5 (five mice in each cage). Seven days after being infected, the PCV2-binoculated mice were co-mingled with non-inoculated mice from cages 1 to 5 successively at 7, 14, 21, 28 and 35 days post infection (dpi), respectively, for 3 days. In addition, eleven pregnant mice were injected with PCV2b. Samples were collected from non-inoculated mice and three newborn mice from each litter for PCV2b detection by polymerase chain reaction (PCR) and immunohistochemistry (IHC). The PCR results showed that PCV2b transmission rate among mice in cages 1, 2, 3, 4 and 5 was 0/5, 2/5, 5/5, 5/5 and 1/5, respectively. PCV2b antigen signals generally appeared in most organs of the non-inoculated mice in which viruses were detected by PCR. PCV2b DNA was also detected in newborn mice of PCV2b-infected litters, and viral antigen signals were observed in their organs as well. PCV2b was transmitted in Kunming mice by contact, and it also caused vertical infection through the placenta.

Implication of IL-33 gene polymorphism in Chinese patients with Alzheimer's disease.

Interleukin-33 (IL-33), a newly described member of the IL-1 family, is located on chromosome 9p24, a chromosomal region of interest in Alzheimer's disease (AD) defined by many genome-wide studies. Three intronic rs1157505, rs11792633, and rs7044343 single nucleotide polymorphisms (SNPs) within IL-33 have recently been reported to be associated with risk of AD in Caucasian populations. In order to assess the involvement of the IL-33 polymorphisms in the risk of developing late onset AD (LOAD), we analyzed the genotype and allele distributions of these 3 polymorphisms in 704 Han Chinese subjects. The minor alleles of the rs11792633 polymorphism within IL-33 was significantly associated with a reduced risk of LOAD (odds ratio [OR] = 0.73, p = 0.005). Furthermore, rs11792633 polymorphism was still strongly associated with LOAD (dominant model: OR = 0.67, p = 0.015; recessive model: OR 0.57, p = 0.021; additive model: OR = 0.71, p = 0.004) after adjusting for age, gender, and the apolipoprotein E (APOE) ε4 status. Our results support the evidence that genetic variants of IL-33 affect susceptibility to LOAD in Han Chinese.

Low-frequency stimulation of bilateral anterior nucleus of thalamus inhibits amygdale-kindled seizures in rats.

Brain stimulation with low-frequency is emerging as an alternative treatment for refractory epilepsy. The anterior nucleus thalamus (ANT) is thought to be a key structure in the circuits of seizure generation and propagation. The present study aimed to investigate the effects of low frequency stimulation (LFS) targeting ANT on amygdala-kindled seizures in Sprague-Dawley rats. Electrodes were implanted into the right basolateral amygdala and the right or bilateral ANT of Sprague-Dawley rats. When fully kindled seizures were achieved by daily electrical stimulation of the amygdala, LFS (15 min train of 0.1 ms pulses at 1 Hz and 200-500 µA) was applied to the unilateral or bilateral ANT immediately before the kindling stimulation (pre-treatment). Our study showed that LFS of the bilateral ANT significantly decreased the incidence of generalized seizures (GS) and seizure stage, as well as shortened duration of afterdischarge and GS demonstrating an inhibition of the severity of seizures. Moreover, LFS elevated the afterdischarge threshold (ADT) and GS threshold indicating an inhibition of susceptibility to seizures. On the other hand, LFS of the unilateral ANT failed to show any significance in inhibiting seizures. Our study demonstrated that bilateral LFS in ANT could significantly inhibit amygdala-kindled seizures by preventing both afterdischarge generation and propagation. It provided further evidence for clinical use of LFS in ANT.

Deep brain stimulation for epilepsy in clinical practice and in animal models.

Given the tremendous success of deep brain stimulation (DBS) for the treatment of movement and neuropsychiatric disorders, clinicians have begun to open up to the possible use of electrical stimulation for the treatment of patients with uncontrolled seizures. DBS of various neural targets has been investigated in clinical studies and animal studies, including the anterior nucleus of thalamus (ANT), cerebellum, hippocampus, subthalamic nucleus (STN), centromedian nucleus of the thalamus (CMT), caudate nucleus (CN). Recently, a large and multicenter trial (SANTE: Stimulation of the Anterior Nucleus of the Thalamus for Epilepsy) was conducted and subsequently with encouraging results, making ANT the most well-established target for DBS in the treatment of epilepsy to date. Here, we endeavor to review mainly the animal studies and clinical studies of ANT DBS to further explore the more reliable target.

Viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b.

The viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b (PCV-2b) were investigated. Seventy special pathogen free mice were divided into 2 groups with 35 mice in each group. The test group (TG) was infected with PCV-2b, the control group (CG) was inoculated with sterile cell cultures. Five mice in each group were sacrificed at 3, 7, 14, 21, 28, 35 and 42 dpi (day post infection), respectively. Necropsies were performed on all mice and tissues were collected for testing by histopathology, immunohistochemistry, transmission electron microscope (TEM) and polymerase chain reaction (PCR). Apoptosis and necrosis in lymphoid organs were observed in virus-infected mice, and became severe from 14 to 28 dpi. The proportion of PCV-2b antigen-positive cells was moderate in lung, heart, thymus, liver or kidney, and low in brain from TG. In spleen and cervical lymph node, the proportions of PCV-2b antigen-positive cells were low to high from 7 to 28 dpi, and moderate from 35 to 42 dpi. PCV-2b DNA was detected in all tissues examined in TG from 7 to 42 dpi. Viral inclusion bodies presented in the cytoplasm of lymphocytes, macrophages, hepatocytes, podocytes, neurocytes, spermatids and uterine epithelial cells in TG. In CG, no viruses and viral lesions were detected. PCV-2b could replicate in mice, and PCV-2b associated lesions in mice were similar to those observed in pigs. The present results indicate that it is possible to use Kunming mouse as an animal model for PMWS research.

Roles of ß-adrenergic receptors in Alzheimer's disease: implications for novel therapeutics.

Alzheimer's disease (AD), the most common cause of age-related dementia, is a progressive neurodegenerative disorder with an enormous unmet medical need. In recent years, several unexpected longitudinal and cross-sectional epidemiological studies reveal that beta-blockers treatment reduces the prevalence of AD in patients suffering from hypertension. Now, a newly population-based study of individuals with incident AD demonstrates that beta-blockers are also associated with delay of functional decline. Furthermore, accumulated convincing evidences from cell culture experiments and animal studies have also suggested that ß-adrenergic receptors (ß-ARs) may involve in the AD pathogenesis through effects on amyloid-ß (Aß) production or inflammation. This review explores clinical and experimental studies that might help to explain the roles of ß-ARs in the AD pathogenesis and the potential underlying mechanisms and whether treatment with ß-ARs antagonists provides a new therapeutic option for AD.

Lack of association between PCDH11X genetic variation and late-onset Alzheimer's disease in a Han Chinese population.

Protocadherin 11X (Pcdh11X) has been suspected to be associated with Alzheimer's disease through participating in the metabolism of PP1α and ß-catenin or by altering the synaptic functions. A recent genome-wide association study reported that a common single nucleotide polymorphism (SNP, rs5984894) in the gene encoding Pcdh11X was associated with susceptibility to late-onset Alzheimer's disease (LOAD) in Caucasians. In order to assess the involvement of the PCDH11X polymorphism in the risk of developing AD in Chinese, we analyzed the genotype and allele distributions of the PCDH11X rs5984894 polymorphism in a Han Chinese population (355 LOAD cases and 399 healthy controls). Our results failed to find any significant association between the tested SNP and LOAD, indicating that PCDH11X gene polymorphism does not play a major role in the genetic predisposition to LOAD in this Han Chinese population.

Blocking beta2-adrenergic receptor attenuates acute stress-induced amyloid beta peptides production.

Environmental factors play an important role in the Alzheimer's disease (AD) development and stress may accelerate the progression of AD. Beta-adrenergic receptors are activated by stress and may influence different aspects of cognitive function. So, it was hypothesized that stress may accelerate the pathological progression of AD by the activation of beta(2)-adrenergic receptor (beta(2)-AR). We have investigated the role of acute stress and activation of beta(2)-AR in amyloid beta (Abeta) peptides production in a mouse model of acute restraint stress. Injections of the beta(2)-AR-selective agonist clenbuterol hydrochloride enhanced the production of acute stress-induced Abeta peptides production; the beta(2)-AR-selective antagonist ICI 118,551 reduced Abeta peptides production. It is suggested that acute stress induces abnormal activation of beta(2)-AR which subsequently enhances Abeta peptides (the main neuropathological hallmarks of AD) production possibly resulting in the onset of AD. The findings indicate that new therapeutic strategies designed to blocking beta(2)-AR might be valuable for the prevention and treatment of AD.

Interleukin-18 promoter polymorphisms and risk of ischemic stroke.

Ischemic stroke (IS) is a major cause of morbidity and mortality around the world. Interleukin-18 (IL-18) plays an important role in the pathogenesis of IS and IL-18 promoter polymorphisms have been shown to be associated with levels of expression of IL-18. We investigated the association of two functional polymorphisms in IL-18 promoter, -607C/A (rs1946518) and -137G/C (rs187238), with the risk of ischemic stroke in a Han Chinese population of 423 patients and 384 healthy controls matched for sex and age. The results revealed that the -607C allele was associated with an increased risk of IS with an odds ratios (OR) of 1.358 (P = 0.002, power = 100%) and the presence of the -137G allele was correlated with increased the risk of IS in the subtype of patients with large artery atherosclerosis (LAA) (OR = 1.583, P = 0.02, power = 94%). Patients with the -607C/-137G haplotype also had significantly increased risk of IS compared to controls (OR = 1.341, P = 0.005, power = 100%). Our findings suggest that these functional polymorphisms in the IL-18 promoter are involved in development of IS in the Han Chinese population.

Neuropeptide Y polymorphisms and ischemic stroke in Chinese population.

BACKGROUND: Stroke is the second most common cause of death in developed countries and a major cause of adult disability and mortality worldwide. New data strongly suggest that neuropeptide Y (NPY) may be a candidate gene for ischemic stroke. METHODS: We investigated 450 ischemic stroke patients and 423 healthy controls matched for sex and age in a Han Chinese population. Three functional polymorphisms (-883TGins/del, -602G/T and -399 T/C) located in NPY gene promoter were genotyped using DNA sequencing methods. RESULTS: Of 3 NPY polymorphisms investigated in our study, the -399CC genotype (OR: 1.699, 95% CI: 1.124-2.567, P=0.011) and the -399C allele (OR: 1.254, 95% CI: 1.031-1.524, P=0.023) were more frequent among ischemic stroke patients than in controls, especially in the small vessel disease (SVD) subtype patients. The similar results were observed in multivariable logistic regression analysis. Haplotype analysis revealed that the -883ins/-399C haplotype was a risk marker for ischemic stroke (P=0.008). CONCLUSIONS: The C allele of -399 T/C polymorphism in the promoter regions of NPY is an independent risk factor for ischemic stroke, suggesting that NYP system may involve in the mechanisms of stroke pathology.

BioGlue: a protective barrier after pericardiotomy.

BACKGROUND: Repeat operation on the heart composes about 20% of procedures in contemporary practice of cardiac surgery. A sheet of material providing a barrier against cardiac adhesion to the sternum would be desirable. METHODS: Anterior pericardiectomy was performed in rats. BioGlue milled to a 0.4 mm sheet was applied to the anterior surface of the heart in 16 rats; Surgicel plus liquid BioGlue in seven; Surgicel alone in three; and nothing (control) in eight. The operative site was reexamined for gross evidence of adhesion, scarring, and residual BioGlue 1, 3, and 6 months later. RESULTS: There was formation of a loose connective tissue barrier containing blood vessels without scar formation in all animals treated with milled BioGlue. Surgicel plus BioGlue resulted in a barrier containing more denser connective tissue with collagen fibers. Surgicel alone resulted in a similar barrier. No barrier formed in the control experiments. CONCLUSIONS: A sheet of milled BioGlue applied over the surface of the heart but not attached to it after partial pericardiectomy has been shown to stimulate formation of a loose connective tissue barrier containing blood vessels. This barrier is unique compared to dense fibrous scar which usually forms after opening the pericardium for cardiac operations.

[Cloning and analysis of phage Fab antibodies of mouse male specific antigen].

To clone mouse phage antibodies against H-Y antigen from a phage antibody library, three cycles of affinity enrichment of the mouse phage antibody library with male spleen cells and two cycles of nonspecific absorption with female spleen cells were performed. The presence of mouse Fab on the phage surface was determined by ELISA and sequence analysis. 9 of 15 strains can bind to male spleen cells with the specific activity. Recombination rate of the phage antibody library clones is 60%. Sequence analysis of the PCR products of plasmid DNA of E5 clones show VH and Vkappa had common characteristics shared by other known variable region of antibodies. The mouse phage Fab antibody could be used for identifying H-Y antigen, and for the development of sex determination of early embryos in mammals.

Blunt chest trauma: an experimental model for heart and lung contusion.

BACKGROUND: Blunt chest trauma is an important clinical problem leading to injury of the heart and lungs that may be fatal. Experimental models in large animals have been developed previously. This study was aimed at developing a small-animal (rat) model for the purpose of evaluating blunt chest trauma. METHODS: Blunt trauma was delivered to the left side of the chest in rats by a captive bolt handgun. The gun was modified so that the amount of energy delivered to the chest wall could be adjusted. The injury energy varied from 1.7 to 6.8 J. Thirty-eight experiments in adult rats were performed. Electrocardiographic monitoring was performed continuously to determine cardiac rhythm. Gross and histologic examination of lungs and heart was performed at the time of death resulting from injury or euthanasia up to 13 days after injury. RESULTS: Some form of cardiac arrhythmia accompanied blunt chest trauma in every case. Serious ventricular arrhythmia (tachycardia or fibrillation) was nearly always fatal (15 of 16 cases), but gross or histologic evidence of cardiac injury was present in only 31% of fatal cases. Lung injury (often bilateral) as shown by atelectasis and hemorrhage into the parenchyma or airway was found in 93% of the experiments when medium range energy force was applied. CONCLUSION: This study has established a useful model for the study of blunt chest trauma in a small animal (rat). Blunt chest trauma is associated with cardiac arrhythmia, which may be fatal. Injury to the heart may not correlate with serious cardiac arrhythmia resulting in death, lending credence to the concept of cardiac concussion or commotio cordis. Lung contusion is always more obvious than morphologic injury to the heart.