Characterization of CpCaM, a protein potentially involved in the growth of Cryptosporidium parvum.

Cryptosporidium parvum is an important apicomplexan parasite causing severe diarrhea in both humans and animals. Calmodulin (CaM), a multifunctional and universal calcium-binding protein, contributes to the growth and development of apicomplexan parasites, but the role of CaM in C. parvum remains unknown. In this study, the CaM of C. parvum encoded by the cgd2_810 gene was expressed in Escherichia coli, and the biological functions of CpCaM were preliminarily investigated. The transcriptional level of the cgd2_810 gene peaked at 36 h post infection (pi), and the CpCaM protein was mainly located around the nucleus of the whole oocysts, in the middle of sporozoites and around the nucleus of merozoites. Anti-CpCaM antibody reduced the invasion of C. parvum sporozoites by 30.69%. The present study indicates that CpCaM is potentially involved in the growth of C. parvum. Results of the study expand our knowledge on the interaction between host and Cryptosporidium.

Luteolin alleviates cognitive impairment in Alzheimer's disease mouse model via inhibiting endoplasmic reticulum stress-dependent neuroinflammation.

Luteolin is a flavonoid in a variety of fruits, vegetables, and herbs, which has shown anti-inflammatory, antioxidant, and anti-cancer neuroprotective activities. In this study, we investigated the potential beneficial effects of luteolin on memory deficits and neuroinflammation in a triple-transgenic mouse model of Alzheimer's disease (AD) (3 × Tg-AD). The mice were treated with luteolin (20, 40 mg · kg-1 · d-1, ip) for 3 weeks. We showed that luteolin treatment dose-dependently improved spatial learning, ameliorated memory deficits in 3 × Tg-AD mice, accompanied by inhibiting astrocyte overactivation (GFAP) and neuroinflammation (TNF-α, IL-1ß, IL-6, NO, COX-2, and iNOS protein), and decreasing the expression of endoplasmic reticulum (ER) stress markers GRP78 and IRE1α in brain tissues. In rat C6 glioma cells, treatment with luteolin (1, 10 µM) dose-dependently inhibited LPS-induced cell proliferation, excessive release of inflammatory cytokines, and increase of ER stress marker GRP78. In conclusion, luteolin is an effective agent in the treatment of learning and memory deficits in 3 × Tg-AD mice, which may be attributable to the inhibition of ER stress in astrocytes and subsequent neuroinflammation. These results provide the experimental basis for further research and development of luteolin as a therapeutic agent for AD.

Quercetin attenuates ischemia reperfusion injury by protecting the blood-brain barrier through Sirt1 in MCAO rats.

The purpose of the present study was to examine the protective action and mechanisms of quercetin on the blood-brain barrier (BBB) in rats subjected to transient middle cerebral artery occlusion (tMCAO) and reperfusion. Quercetin (10, 30, 50 mg/kg) was intraperitoneally administered at the onset of reperfusion. The results showed that quercetin significantly reduced cerebral infarct volume, neurological deficit, BBB permeability and ROS generation via Sirt1/Nrf2/HO-1 signaling pathway. Moreover, EX527, a selective inhibitor of Sirt1, reversed these neuroprotective effects. Our findings indicate that quercetin has neuroprotective effects against cerebral ischemia-reperfusion injury by protecting BBB through Sirt1 signaling pathway in MCAO rats.

Establishment and preliminary application of nanoparticle-assisted PCR assay for detection of Cryptosporidium spp.

Cryptosporidium is an important intestinal protozoan parasite that causes diarrhoea in humans and animals. To rapidly and specifically detect Cryptosporidium spp., we designed a pair of primers based on the small subunit ribosomal RNA (SSU rRNA) gene of Cryptosporidium spp. to be used in a new nanoparticle-assisted PCR (nano-PCR) assay. The minimum detectable concentration (1.02 pg) of this nano-PCR was 10 times more sensitive than conventional PCR using the same primer pair. The DNA samples of C. parvum, C. baileyi, C. xiaoi, C. ryanae, and C. andersoni were successfully detected by the nano-PCR. No amplifications were evident with DNA samples of some common intestinal pathogens, including Eimeria tenella, Blastocystis sp., Giardia lamblia, Enterocytozoon bieneusi, and Balantidium coli. To validate the clinical usefulness of the novel nano-PCR, a total of 40 faecal samples from goats, camels, calves, and chickens were examined. The positive rate of Cryptosporidium spp. was 27.5% (11/40), which was consistent with that of an established nested PCR. These results indicate that the novel nano-PCR assay enables the rapid, specific, and accurate detection of Cryptosporidium infection in animals. The findings provide a technical basis for the clinical diagnosis, prevention, and control of cryptosporidiosis.

Multilocus genotyping of Giardia duodenalis in Bactrian camels (Camelus bactrianus) in China.

The protozoan parasite Giardia duodenalis is known to infect humans and a wide range of animals globally. However, no studies on G. duodenalis infection in Bactrian camels have been reported. In the present study, in order to examine the prevalence and genetic diversity of G. duodenalis in Bactrian camels, 852 fecal samples were collected from 24 sampling sites in three geographical areas (Gansu province, Inner Mongolia, and Xinjiang Uygur autonomous regions) of northwestern China, and subjected to multilocus sequence typing (MLST) analysis targeting the 18S rRNA, ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. About 84 fecal samples tested positive for Giardia infection, with an overall prevalence of 9.8%, including three samples from camel calves with diarrhea. Significant differences (χ2 = 80.7, df = 2, P < 0.01) in the prevalence were found in Bactrian camels belonging to three geographical areas, with the highest (33.3%) in Gansu province and the lowest (4.2%) in Xinjiang Uygur autonomous region. Furthermore, significantly different prevalences (χ2 = 34.2, df = 2, P < 0.01) were revealed among age groups, with the highest (35.7%) in camels aged 3 to 6 years old, and the lowest (7.5%) in camels aged > 6 years old. Sequence analysis identified two assemblages, including zoonotic assemblage A and ungulate-adapted assemblage E, with the latter as the dominant G. duodenalis assemblage in each age group and at all sampling sites having positive samples except Hotan. Genetic variations were detected among G. duodenalis isolates in these camels, and eight, three, and seven haplotypes were identified at loci bg, gdh, and tpi, respectively, forming two multilocus genotypes (MLGs) of zoonotic assemblage A and one MLG of assemblage E. To the best of our knowledge, this is the first report on G. duodenalis infection in Bactrian camels, and the data indicate that G. duodenalis have a broad host range.

Molecular characterization of Balantioides coli in pigs from Shaanxi province, northwestern China.

Balantioides coli (syn. Balantidium coli) is an important zoonotic but usually neglected protozoa infecting human and a great number of animals, and the pig was considered to be the most important natural host and reservoir. However, no information about the infection of B. coli in pigs in northwestern China was available. In the present study, the prevalence and genetic diversity of B. coli in pigs in Shaanxi province were investigated. A total of 560 fecal samples were collected from pigs of four age groups in five different geographical regions and analyzed by using PCR targeting the ITS1-5.8S rRNA-ITS2 gene fragment. The infection of B. coli was detected in all age groups and regions, with the total prevalence of 16.8% (94/560). Significant differences (P < 0.01) in prevalence were found among four investigated age groups, with the highest in fatteners (38.8%) and the lowest in adults (5.7%). The prevalence was also significantly (P < 0.01) different among pigs from five sampling regions. Sequence analysis revealed two genetic variants, namely, A and B, in these investigated pigs, and both of them were detected in all age groups and regions, with the latter as the predominant one. Further, sixty-eight different haplotypes were found, with 19 and 49 belonged to genetic variants A and B, respectively. The findings in the present study indicated wide distribution and high diversity of B. coli in pigs in Shaanxi province and provided fundamental data for implementing control strategies on B. coli infection in pigs as well as other hosts in this province.

Diterpene ginkgolides protect against cerebral ischemia/reperfusion damage in rats by activating Nrf2 and CREB through PI3K/Akt signaling.

Diterpene ginkgolides meglumine injection (DGMI) is a therapeutic extract of Ginkgo biloba L, which has been used for the treatment of cerebral ischemic stroke in China. Ginkgolides A, B and C are the main components of DGMI. This study was designed to investigate the neuroprotective effects of DGMI components against ischemic stroke in vivo and in vitro. Acute cerebral ischemic injury was induced in rats by occlusion of the middle cerebral artery (MCA) for 1.5 h followed by 24 h reperfusion. The rats were treated with DGMI (1, 3 and 10 mg/kg, iv) at the onset of reperfusion and 12 h after reperfusion. Administration of DGMI significantly decreased rat neurological deficit scores, reduced brain infarct volume, and induced protein kinase B (Akt) phosphorylation, which prompted the nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and phosphorylation of the survival regulatory protein cyclic AMP-responsive element binding protein (CREB). Nrf2 activation led to expression of the downstream protein heme oxygenase-1 (HO-1). In addition, PC12 cells were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) in vitro, treatment with DGMI (1, 10 and 20 µg/mL) or ginkgolides A, B or C (10 µmol/L for each) significantly reduced PC12 cell death and increased phosphorylation of Akt, nuclear translocation of Nrf2 and activation of CREB. Activation of Nrf2 and CREB could be reversed by co-treatment with a phosphoinositide-3-kinase (PI3K) inhibitor LY294002. These observations suggest that ginkgolides act as novel extrinsic regulators activating both Akt/Nrf2 and Akt/CREB signaling pathways, protecting against cerebral ischemia/reperfusion (I/R) damage in vivo and in vitro.

Effects of the Nrf2 Protein Modulator Salvianolic Acid A Alone or Combined with Metformin on Diabetes-associated Macrovascular and Renal Injury.

Nuclear factor E2-related factor 2 (Nrf2) is considered a promising target against diabetic complications such as cardiovascular diseases and diabetic nephropathy. Herein, we investigated the effects of a potential Nrf2 modulator, salvianolic acid A (SAA), which is a natural polyphenol, on diabetes-associated macrovascular and renal injuries in streptozotocin-induced diabetic mice. Given that lowering glucose is the first objective of diabetic patients, we also examined the effects of SAA combined with metformin (MET) on both complications. Our results showed that SAA significantly increased the macrovascular relaxation response to acetylcholine and sodium nitroprusside in diabetic mice. Interestingly, treatment with SAA alone only provided minor protection against renal injury, as reflected by minor improvements in impaired renal function and structure, despite significantly reduced oxidative stress observed in the diabetic kidney. We demonstrated that decreased oxidative stress and NF-κB p65 expression were associated with SAA-induced expression of Nrf2-responsive antioxidant enzymes heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase (quinone) 1 (NQO-1), and glutathione peroxidase-1 (GPx-1) in vivo or in vitro, which suggested that SAA was a potential Nrf2 modulator. More significantly, compared with treatment with either SAA or MET alone, we found that their combination provided further protection against the macrovascular and renal injury, which was at least partly due to therapeutic activation of both MET-mediated AMP-activated protein kinase and SAA-mediated Nrf2/antioxidant-response element pathways. These findings suggested that polyphenol Nrf2 modulators, especially combined with drugs activating AMP-activated protein kinase, including hypoglycemic drugs, are worthy of further investigation to combat diabetic complications.

DL0410, a novel dual cholinesterase inhibitor, protects mouse brains against Aß-induced neuronal damage via the Akt/JNK signaling pathway.

AIM: 1,1'-([1,1'-Biphenyl]-4,4'-diyl)bis(3-(piperidin-1-yl)propan-1-one)dihydrochloride (DL0410) is a novel synthetic dual acetylcholinesterase (AChE)/butyrocholinesterase (BuChE) inhibitor, which has shown a potential therapeutic effect on Alzheimer's disease (AD). In this study we examined whether DL0410 produced neuroprotective effects in an AD cellular model and an Aß1-42-induced amnesia mouse model. METHODS: The in vitro inhibitory activities against AChE and BuChE were estimated using Ellman's assay. Copper-induced toxicity in APPsw-SY5Y cells was used as AD cellular model, the cell viability was assessed using MTS assay, and cell apoptosis was evaluated based on mitochondrial membrane potential detection. Aß1-42-induced amnesia mouse model was made in male mice by injecting aggregated Aß1-42 (2 µg in 2 µL 0.1% DMSO) into the right cerebral ventricle. Before and after Aß1-42 injection, the mice were orally administered DL0410 (1, 3, 9 mg·kg-1·d-1) or rivastigmine (2 mg·kg-1·d-1) for 3 and 11 d, respectively. Memory impairments were examined using Morris water maze (MWM) test and passive avoidance test. The expression levels of APP, CREB, BDNF, JNK and Akt in the mouse brains were measured with either immunohistochemistry or Western blotting. RESULTS: DL0410 exhibited in vitro inhibitory abilities against AChE and BuChE with IC50 values of 0.286±0.004 and 3.962±0.099 µmol/L, respectively, which were comparable to those of donepezil and rivastigmine. In APPsw-SY5Y cells, pretreatment with DL0410 (1, 3, and 10 µmol/L) decreased the phosphorylation of JNK and increased the phosphorylation of Akt, markedly decreased copper-stimulated Aß1-42 production, reversed the loss of mitochondrial membrane potential, and dose-dependently increased the cell viability. In Aß1-42-treated mice, DL0410 administration significantly ameliorated learning and memory deficits in MWM test and passive avoidance test. Furthermore, DL0410 administration markedly decreased Aß1-40/42 deposits in mouse cerebral cortices, and significantly up-regulated neurotrophic CREB/BDNF. Meanwhile, Akt/JNK signaling pathway may play a key role in the neuroprotective effect of DL0410. CONCLUSION: DL0410 ameliorates cognitive deficit and exerts neuronal protection in AD models, implicating this compound as a candidate drug for the prevention and therapy of AD.

[Salvianolic acid A alleviate the brain damage in rats after cerebral ischemia-reperfusion through Nrf2/HO-1 pathway].

The aim of present study is to investigate the protective effects and mechanism of salvianolic acid A (SAA) on cerebral ischemia-reperfusion injury in rats. The model was established with middle cerebral artery occlusion and reperfusion (MCAO/R) with ischemia for 1.5 h and reperfusion for 24 h in adult male SD rats. After the behavior assessment, TTC assay was used to calculate the infarct volume of rat brain; the distribution of Nrf2 in nuclear and cytoplasm and expression of HO-1 were detected by Western blot. The PC12 cells injury model was established with oxygen-glucose deprivation for 6 h and reintroduction for 24 h. Cell viability was determined with MTT assay, and the expression of Nrf2 and HO-1 were detected through immunofluorescence staining. The mechanisms were investigated in PC12 cells with Nrf2 knocking down by siRNA. SAA (10 and 20 mg·kg(-1)) significantly reduced the neuronal damage in MCAO/R model, and SAA(0.5 and 5 µmol·L(-1)) increased cell viability in PC12 cells injury model. Meanwhile, the nuclear translocation of Nrf-2 and the expression of HO-1 were increased in PC12 cell and rats brain. SAA exhibited anti-cerebral ischemia- reperfusion effects. The mechanism may be related to activation of Nrf2/HO-1 signaling pathway, which promotes the synthesis and nuclear translocation of Nrf2 to enhance the expression of the antioxidant protein HO-1.

Preclinical studies of the potent and selective nicotinic α4ß2 receptor ligand VMY-2-95.

The discovery and development of small molecules that antagonize neuronal nicotinic acetylcholine receptors may provide new ligands for evaluation in models of depression or addiction. We discovered a small molecule, VMY-2-95, a nAChR ligand with picomolar affinity and high selectivity for α4ß2 receptors. In this study, we investigated its preclinical profile in regards to solubility, lipophilicity, metabolic stability, intestinal permeability, bioavailability, and drug delivery to the rat brain. Metabolic stability of VMY-2-95·2HCl was monitored on human liver microsomes, and specific activity of VMY-2-95·2HCl on substrate metabolism by CYP1A2, 2C9, 2C19, 2D6, and 3A4 was tested in a high-throughput manner. The intestinal transport of VMY-2-95·2HCl was studied through Caco-2 cell monolayer permeability. VMY-2-95·2HCl was soluble in water and chemically stable, and the apparent partition coefficient was 0.682. VMY-2-95·2HCl showed significant inhibition of CYP2C9 and 2C19, but weak or no effect on 1A2, 2D6, and 3A4. The Caco-2 cell model studies revealed that VMY-2-95·2HCl was highly permeable with efflux ratio of 1.11. VMY-2-95·2HCl achieved a maximum serum concentration of 0.56 mg/mL at 0.9 h and was orally available with a half-life of ∼9 h. Furthermore, VMY-2-95·2HCl was detected in the rat brain after 3 mg/kg oral administration and achieved a maximal brain tissue concentration of 2.3 µg/g within 60 min. Overall, the results demonstrate that VMY-2-95·2HCl has good drug like properties and can penetrate the blood-brain barrier with oral administration.

[Pharmacokinetics of salvianolic acid A after single intravenous administration in Rhesus monkey].

Salvianolic acid A (Sal A) is one of the most effective compounds isolated from the root of Salvia miltiorrhiza. Up to now, several studies regarding the pharmacokinetic profiles of Sal A have been reported, however there is no such study reported in monkeys, the species which is more similar to human. The aim of this study is to develop a LC-MS method for the determination of Sal A in monkey plasma and apply it to the pharmacokinetic studies of monkeys. After single intravenous administration of Sal A, the plasma concentration-time curves were observed and the main pharmacokinetic parameters were calculated. The plasma concentration at 2 min (C2 (min)) values were (28.343 ± 6.426), (45.679 ± 12.301) and (113.293 ± 24.360) mg x L(-1) for Rhesus monkeys treated with Sal A at 2.5, 5 and 10 mg x kg(-1). The area under the concentration-time curve (AUC(0-∞)) values were (3.316 ± 0.871), (5.754 ± 2.150) and (13.761 ± 2.825) µg x L(-1) x h, respectively. Furthermore, this method was improved and applied to the simultaneous determination of Sal A, Sal B and Sal C, which provided useful information for preclinical studies and clinical trials of Sal A, Sal B and Sal C.

[Pharmacokinetic comparison of two ozagrel polymorph forms in SD rats].

To enhance the quality and efficiency of ozagrel by investigating the differences between the ozagrel polymorphs in bioavailability. Solid ozagrel in different polymorph forms were orally administered to SD rats. An HPLC method was established to determinate plasma level of ozagrel. The bioavailabilities of two polymorph forms were calculated and compared. The pharmacokinetic parameters of ozagrel, were as follows: Cmax was 32.72 ± 17.04 and 34.01 ± 19.13 mg · L(-1), respectively; AUC0-t was 61.14 ± 14.76 and 85.56 ± 18.08 mg · L(-1) · h, respectively; t½ was 1.53 ± 0.51 and 4.73 ± 3.00 h, respectively. There was no significant difference in pharmacokinetic parameters between form I and II polymorphs of ozagrel while the t½ of form II is longer, which indicates that the use of form II polymorph as pharmaceutical product may prolong the effective action time in clinics. This would help the polymorph quality control in drug production.

Protective effects of the novel adenosine derivative WS0701 in a mouse model of posttraumatic stress disorder.

AIM: To investigate the effects of the novel N6-substituted adenosine derivative {(2R,3S,4R,5R)-3,4-dihydroxy-5-[6-[(4-hydroxy-3-methoxybenzyl)amino]-9H-purin-9-yl]tetrahydrofuran-2-yl} methyl decanoate (WS0701) on stress-induced excessive fear, anxiety, and cognitive deficits in a mouse model of posttraumatic stress disorder (PTSD). METHODS: Male mice underwent a conditioned foot shock and single prolonged stress procedure to induce PTSD. Contextual/cued fear, elevated plus-maze, open field and novel object recognition tests were conduced to assess PTSD-like behaviors. From d 1, the mice were orally administered WS0701 (7.5, 15, or 30 mg·kg(-1)·d(-1)) or paroxetine (10 mg·kg(-1)·d(-1)) for two weeks. Apoptosis of hippocampal neurons was detected using flow cytometry and TUNEL staining, and expression of Bcl-2 and Bax in the hippocampus was measured with Western boltting and qPCR assays. RESULTS: WS0701 administration significantly alleviated fear, anxious behaviors and memory deficits in the mouse model of PTSD. Furthermore, WS0701 administration significantly reduced the stress-induced apoptosis of hippocampal neurons, and increased the Bcl-2/Bax ratio in the hippocampus. The positive control drug paroxetine exerted similar effects on PTSD-like behaviors and hippocampal neuron apoptosis in the mouse model of PTSD, which were comparable to those caused by the high dose of WS0701. CONCLUSION: WS0701 effectively mitigates stress-induced PTSD-like behaviors in mice, partly via inhibition of neuronal apoptosis in the hippocampus.

MiR-4521 affects the propagation of Cryptosporidium parvum in HCT-8 cells through targeting foxm1 by regulating cell apoptosis.

Cryptosporidium parvum could regulate the expression of microRNAs of epithelial cells to facilitate its intracellular propagation. MiR-4521 has been reported to play an important role during the development and progression of tumors and infectious diseases by regulating cell proliferation, apoptosis, and autophagy. However, the implication of miR-4521 during C. parvum infection was still unknown. In this study, the expression of miR-4521 was found to be upregulated in HCT-8 cells infected with C. parvum from 8 h post-infection (pi) to 48 hpi, and its upregulation would be related with the TLR/NF-κB signal pathway during C. parvum infection. One potential target of miR-4521, foxm1, was down-regulated in HCT-8 cells from 24 hpi to 48 hpi, and the expression of foxm1 was negatively regulated by miR-4521. The target relationship between miR-4521 and foxm1 was further validated by using dual luciferase reporter assay. Further studies showed that miR-4521 promoted the propagation of C. parvum in HCT-8 cells through targeting foxm1 by regulating BCL2-mediating cell apoptosis. These results contribute to further understanding of the regulatory mechanisms of host miRNAs during Cryptosporidium infection.

Circular RNA ciRS-7 affects the propagation of Cryptosporidium parvum in HCT-8 cells via regulating miR-135a-5p/stat1 axis.

Cryptosporidium spp. are protozoan parasites that mainly inhabit intestinal epithelial cells, causing diarrheal diseases in humans and a great number of animals. Cryptosporidium parvum is the most common zoonotic species, responsible for nearly 45% of human cryptosporidiosis worldwide. Understanding the interaction mechanisms between C. parvum and host gastrointestinal epithelial cells has significant implications to control cryptosporidiosis. One up-regulated circRNA ciRS-7 was found previously by our group to promote in vitro propagation of C. parvum in HCT-8 cells. In the present study, miR-135a-5p, was found to be a miRNA target of ciRS-7. Cryptosporidium parvum infection induced significantly down-regulation of miR-135a-5p and dramatic up-regulation of its potential target stat1 gene at mRNA and protein levels. Dual luciferase reporter assays validated the physical interactions between miR-135a-5p and stat1, and between ciRS-7 and miR-135a-5p. Further study revealed that ciRS-7 could sponge miR-135a-5p to positively regulate the protein levels of STAT1 and phosphorylated STAT1 (p-STAT1) and thus promote C. parvum propagation in HCT-8 cells. Our findings further reveal the mystery of regulatory roles of host circRNAs during Cryptosporidium infection, and provide a novel insight to develop strategies to control cryptosporidiosis.

Temporal transcriptomic changes in microRNAs involved in the host immune response and metabolism during Neospora caninum infection.

BACKGROUND: Neospora caninum infection is a major cause of abortion in cattle, which results in serious economic losses to the cattle industry. However, there are no effective drugs or vaccines for the control of N. caninum infections. There is increasing evidence that microRNAs (miRNAs) are involved in many physiological and pathological processes, and dysregulated expression of host miRNAs and the biological implications of this have been reported for infections by various protozoan parasites. However, to our knowledge, there is presently no published information on host miRNA expression during N. caninum infection. METHODS: The expression profiles of miRNAs were investigated by RNA sequencing (RNA-seq) in caprine endometrial epithelial cells (EECs) infected with N. caninum at 24 h post infection (pi) and 48 hpi, and the functions of differentially expressed (DE) miRNAs were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The transcriptome data were validated by using quantitative real-time polymerase chain reaction. One of the upregulated DEmiRNAs, namely chi-miR-146a, was selected to study the effect of DEmiRNAs on the propagation of N. caninum tachyzoites in caprine EECs. RESULTS: RNA-seq showed 18 (17 up- and one downregulated) and 79 (54 up- and 25 downregulated) DEmiRNAs at 24 hpi and 48 hpi, respectively. Quantitative real-time polymerase chain reaction analysis of 13 randomly selected DEmiRNAs (10 up- and three downregulated miRNAs) confirmed the validity of the RNA-seq data. A total of 7835 messenger RNAs were predicted to be potential targets for 66 DEmiRNAs, and GO and KEGG enrichment analysis of these predicted targets revealed that DEmiRNAs altered by N. caninum infection may be involved in host immune responses (e.g. Fc gamma R-mediated phagocytosis, Toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, transforming growth factor-ß signaling pathway, mitogen-activated protein kinase signaling pathway) and metabolic pathways (e.g. lysine degradation, insulin signaling pathway, AMP-activated protein kinase signaling pathway, Rap1 signaling pathway, calcium signaling pathway). Upregulated chi-miR-146a was found to promote N. caninum propagation in caprine EECs. CONCLUSIONS: This is, to our knowledge, the first report on the expression profiles of host miRNAs during infection with N. caninum, and shows that chi-miR-146a may promote N. caninum propagation in host cells. The novel findings of the present study should help to elucidate the interactions between host cells and N. caninum.

High genotype diversity and zoonotic potential of Enterocytozoon bieneusi in yaks (Bos grunniens) from Ganzi Tibetan Autonomous Prefecture, Sichuan Province.

Enterocytozoon bieneusi is a common pathogen in humans and various animals, threatening the breeding industry and public health. However, there is limited information on the molecular characteristics of E. bieneusi in yaks, an economically important animal mainly domesticated in the Qinghai Tibet Plateau in China. In the present study, nested PCR targeting the ITS gene region was applied to investigate the positive rates and genetic diversity of E. bieneusi in 223 faecal samples of yaks from three locations in Ganzi Tibetan Autonomous Prefecture, Sichuan Province. The total positive rate of E. bieneusi was 23.8% (53/223). Significant differences in positive rates were identified among yaks from three locations (χ2 = 8.535, p = 0.014) and four age groups (χ2 = 17.259, p = 0.001), with the highest positive rates in yaks from Yajiang and aged < 6 months, respectively. Sequence analysis identified seven known (EbpC, LW1, LQ10, PigEBITS5, ESH-01, J and BEB4) and five novel (Ganzi1-5) ITS genotypes. Phylogenetic analysis showed eight genotypes (EbpC, LW1, LQ10, PigEBITS5, ESH-01, Ganzi1, Ganzi2 and Ganzi4) in group 1 and three genotypes (J, BEB4 and Ganzi3) in group 2, indicating high genotype diversity and zoonotic potential of E. bieneusi in yaks from Ganzi. Considering the increasing zoonotic genotypes in yaks in the present study compared with previous findings, interventions should be developed to reduce the potential transmission of E. bieneusi between humans and animals.

Title: Grande diversité génotypique et potentiel zoonotique d'Enterocytozoon bieneusi chez les yaks (Bos grunniens) de la préfecture autonome tibétaine de Ganzi, province du Sichuan. Abstract: Enterocytozoon bieneusi est un agent pathogène courant chez l'homme et chez divers animaux, menaçant l'industrie de l'élevage et la santé publique. Cependant, il existe peu d'informations sur les caractéristiques moléculaires d'E. bieneusi chez les yaks, un animal important pour l'économie, principalement domestiqué sur le plateau du Qinghai au Tibet en Chine. Dans la présente étude, une PCR imbriquée ciblant la région du gène ITS a été appliquée pour étudier la positivité et la diversité génétique d'E. bieneusi dans 223 échantillons fécaux de yaks provenant de trois sites de la préfecture autonome tibétaine de Ganzi, province du Sichuan. Le taux total de positivité pour E. bieneusi était de 23,8 % (53/223). Des différences significatives dans les taux positifs ont été identifiées parmi les yaks de trois emplacements (χ2 = 8,535, P = 0,014) et de quatre groupes d'âge (χ2 = 17,259, P = 0,001), avec les taux positifs les plus élevés respectivement chez les yaks de Yajiang et ceux âgés de moins de 6 mois. L'analyse de séquence a identifié sept génotypes ITS connus (EbpC, LW1, LQ10, PigEBITS5, ESH-01, J et BEB4) et cinq nouveaux (Ganzi1­5). L'analyse phylogénétique a montré huit génotypes (EbpC, LW1, LQ10, PigEBITS5, ESH-01, Ganzi1, Ganzi2 et Ganzi4) dans le groupe 1 et trois génotypes (J, BEB4 et Ganzi3) dans le groupe 2, indiquant une diversité génotypique élevée et un potentiel zoonotique d'E. bieneusi chez les yaks de Ganzi. Compte tenu de l'augmentation des génotypes zoonotiques chez les yaks dans la présente étude par rapport aux résultats précédents, des interventions devraient être développées pour réduire la transmission potentielle d'E. bieneusi entre les humains et les animaux.

Baicalein ameliorates Alzheimer's disease via orchestration of CX3CR1/NF-κB pathway in a triple transgenic mouse model.

Alzheimer's disease (AD) is a common chronic neurodegenerative disease. Some studies have suggested that dysregulation of microglia activation and the resulting neuroinflammation play an important role in the development of AD pathology. Activated microglia have both M1 and M2 phenotypes and inhibition of M1 phenotype while stimulating M2 phenotype has been considered as a potential treatment for neuroinflammation-related diseases. Baicalein is a class of flavonoids with anti-inflammatory, antioxidant and other biological activities, but its role in AD and the regulation of microglia are limited. The purpose of this study was to investigate the effect of baicalein on the activation of microglia in AD model mice and the related molecular mechanism. Our results showed that baicalein significantly improved the learning and memory ability and AD-related pathology of 3 × Tg-AD mice, inhibited the level of pro-inflammatory factors TNF-α, IL-1ß and IL-6, promoted the production of anti-inflammatory factors IL-4 and IL-10, and regulated the microglia phenotype through CX3CR1/NF-κB signaling pathway. In conclusion, baicalein can regulate the phenotypic transformation of activated microglia and reduce neuroinflammation through CX3CR1/NF-κB pathway, thereby improving the learning and memory ability of 3 × Tg-AD mice.

Hydroxyl radical (·OH) played a pivotal role in oridonin-induced apoptosis and autophagy in human epidermoid carcinoma A431 cells.

Oridonin, a diterpenoid compound extracted and purified from Rabdosia rubescen, has been reported to induce tumor cell apoptosis through tyrosine kinase pathway. To further examine the mechanism of oridonin, we selected human epidermoid carcinoma A431 cell as a test object. Besides apoptosis, oridonin also induced A431 cell autophagy, and this autophagy antagonized apoptosis and played a protective role for A431 cells. Reactive oxygen species (ROS) played a pivotal role in induction of cytotoxicity. Therefore, a ROS scavenger, N-acetylcysteine (NAC) combined with oridonin was appiled. Results of morphologic observation, flow cytometric analysis and Western blot analysis showed that NAC could significantly reverse both ROS generation and down-regulation of mitochondrial membrane potential in oridonin treated cells. NAC inhibited oridonin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. NAC effectively inhibited both oridonin-induced apoptosis and autophagy by reducing intracellular oxidative stress. To further examine the mechanism of ROS, exogenous enzyme antioxidants (superoxide dismutase (SOD), catalase (CAT)) and non-enzyme antioxidants (glutathione (GSH)) were applied to detect the effect of oridonin on ROS generation. Only GSH exerted a similar role with NAC, suggesting that hydroxyl radical (·OH) played the major role in oridonin-induced cell death. Oridonin could decrease the GSH level in A431 cells in a dose-dependent manner. In addition, after treatment with ·OH donor, Fenton reagent, the changes in A431cells were similar to the results of oridonin treatment. All the results proved that ·OH played the pivotal role in oridonin induced apoptosis and autophagy in A431 cells.