Genetic mutation of Tas2r104/Tas2r105/Tas2r114 cluster leads to a loss of taste perception to denatonium benzoate and cucurbitacin B

Background:Bitter taste receptors(Tas2rs)are generally considered to sense various bitter compounds to escape the intake of toxic substances.Bitter taste receptors have been found to widely express in extraoral tissues and have important physiological functions outside the gustatory system in vivo.Methods:To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues,multiple Tas2rs mutant mice and Gnat3 were produced using CRISPR/Cas9 gene-editing technique.A mixture containing Cas9 and sgRNA mRNAs for Tas2rs and Gnat3 gene was microinjected into the cytoplasm of the zygotes.Then,T7EN1 assays and sequencing were used to screen genetic mutation at the target sites in founder mice.Quantitative real-time polymerase chain reaction(qRT-PCR)and immunostaining were used to study the expression level of taste signaling cascade and bitter taste receptor in taste buds.Perception to taste substance was also studied using twobottle preference tests.Results:We successfully produced several Tas2rs and Gnat3 mutant mice using the CRISPR/Cas9 technique.Immunostaining results showed that the expression of GNAT3 and PLCB2 was not altered in Tas2rs mutant mice.But qRT-PCR results revealed the changed expression profile of m Tas2rs gene in taste buds of these mutant mice.With two-bottle preference tests,these mutant mice eliminate responses to cycloheximide due to genetic mutation of Tas2r105.In addition,these mutant mice showed a loss of taste perception to quinine dihydrochloride,denatonium benzoate,and cucurbitacin B(CuB).Gnat3-mediated taste receptor and its signal pathway contribute to CuB perception.Conclusions:These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound-induced responses mediated by these TAS2Rs in many extraoral tissues.

Probiotic microorganisms affect the reproductive and nervous systems of male rats treated with acrylamide

Objective:To evaluate the protective effects of probiotic microorganisms on the reproductive and nervous systems of male rats treated with acrylamide.Methods:Thirty-two rats were randomly divided into 4 groups and received normal saline through gavage(control),acrylamide 20 mg/kg body weight,acrylamide plus probiotic microorganisms(Lactobacillus acidophilus,Lactobacillus casei,Lactobacillus bulgaricus,Lactobacillus rhamnosus,Bifidobacterium breve,Bifidobacterium infantis,Streptococcus thermophilus and fructooligosaccharides,all mixed in sachets)20 or 200 mg/kg body weight,respectively.After 30 days,the testis,prostate,seminal vesicle and cerebellum were removed,fixed and stained with hematoxylin-eosin(H&E).The Johnsen score was used to classify spermatogenesis.Cavalieri's principle method was used to evaluate the total volume(in mm3)of the testes.The number of each intratubular cell type as well as intertubular Leydig cells in whole samples was measured using the physical dissector counting techniques.Stereological analysis and the grids were used to determine the volume of cerebellar layers as well as the Purkinje cell number.Results:The testis weight decreased significantly in the acrylamide-treated group compared to the other groups(P<0.001).The number of spermatogonia,spermatocytes,spermatids and Leydig cells in the acrylamide-treated group were significantly less compared to the control group(P<0.05),while they were increased significantly in the acrylamide+200 mg/kg probiotic group(P<0.05;P<0.01).The mean Johnsen score in the acrylamide-treated group was lower than in the control group(P<0.001).Acrylamide-induced changes including congestion,vacuolization in the secretory epithelial cells,and epithelial rupture were observed in the prostate and seminal vesicle.The volumes of cerebellar layers were decreased in the acrylamide group compared to the control group while recovered in both probiotic treated groups.Conclusions:Probiotic microorganisms alleviate acrylamide-induced toxicities against the reproductive and cerebellar tissues in rats.

A survey on the status of semen analysis in 118 laboratories in China

Collecting baseline information on how laboratories perform testing is a reasonable first step towards establishing intra- and inter-laboratory standardization and quality control for semen analysis. We carried out a survey of the laboratories performing the testing in China's Mainland. A questionnaire, composed of 36 questions covering all aspects of semen analysis, was designed, and a copy was distributed to each of the 145 laboratories. Of these, 118 laboratories completed the questionnaires. The survey results showed that semen volume was measured visually in 53.6% （59/110） of the responding laboratories, and 70.9% （73/103） of laboratories analysed incompletely liquefied semen without any treatment. In addition, both manual-microscopic and computer-assisted semen-analysis systems were applied to analyse sperm concentration, motility and morphology. However, more than five methods were employed in routine sperm staining. An enzyme-linked immunosorbent assay was commonly used for determining whether antisperm antibodies were present. Several seminal biochemical markers were analysed in only 27.1% （32/118） of the responding laboratories. Generally, there was a lack of intra- and inter-laboratory quality control measures for semen analysis in all laboratories responding to this survey. In conclusion, the methods of semen analysis and the interpretation of test results in the surveyed laboratories differed markedly. In particular, many laboratories employed methods other than those recommended by the World Health Organization Laboratory Manual for the Examination of Human Semen and Sperm- cervical Mucus Interaction （1999）. These findings suggest an urgent need for the standardization of semen analysis with acceptable quality controls for each parameter to make the results repeatable and meaningful.

Development of a humanized HLA-A30 transgenic mouse model

Background:There are remarkable genetic differences between animal major histocompatibility complex(MHC)systems and the human leukocyte antigen(HLA)system.HLA transgenic humanized mouse model systems offer a much better method to study the HLA-A-related principal mechanisms for vaccine development and HLA-Arestricted responses against infection in human.Methods:A recombinant gene encoding the chimeric HLA-A30 monochain was constructed.This HHD molecule contains the following:α1-α2 domains of HLA-A30,α3 and cytoplasmic domains of H-2D~b,linked at its N-terminus to the C-terminus of humanβ2m by a 15-amino-acid peptide linker.The recombinant gene encoding the chimeric HLA-A30 monochain cassette was introduced into bacterial artificial chromosome(BAC)CH502-67J3 containing the HLA-A01 gene locus by Red-mediated homologous recombination.Modified BAC CH502-67J3 was microinjected into the pronuclei of wild-type mouse oocytes.This humanized mouse model was further used to assess the immune responses against influenza A virus(H1N1)pdm09 clinically isolated from human patients.Immune cell population,cytokine production,and histopathology in the lung were analyzed.Results:We describe a novel humanβ2m-HLA-A30(α1α2)-H-2D~b(α3 transmembrane cytoplasmic)(HHD)monochain transgenic mouse strain,which contains the intact HLA-A01 gene locus including 49 kb 5’-UTR and 74 kb 3’-UTR of HLA-A01*01.Five transgenic lines integrated into the large genomic region of HLA-A gene locus were obtained,and the robust expression of exogenous transgene was detected in various tissues from A30-18#and A30-19#lines encompassing the intact flanking sequences.Flow cytometry revealed that the introduction of a large genomic region in HLA-A gene locus can influence the immune cell constitution in humanized mice.Pdm09 infection caused a similar immune response among HLA-A30 Tg humanized mice and wild-type mice,and induced the rapid increase of cytokines,including IFN-γ,TNF-α,and IL-6,in both HLA-A30 humanized Tg mice and wild-type mice.The expression of HLA-A30 transgene was dramatically promoted in tissues from A30-9#line at 3 days post-infection(dpi).Conclusions:We established a promising preclinical research animal model of HLA-A30 Tg humanized mouse,which could accelerate the identification of novel HLA-A30-restricted epitopes and vaccine development,and support the study of HLA-A-restricted responses against infection in humans.

Effects of Ligustrum robustum on gut microbes and obesity in rats

AIM: To investigate the anti-obesity and antibacterial effects of Ligustrum robustum(L. robustum) in vivoand in vitro and its possible mechanisms. METHODS: The effects of L. robustum aqueous extract(LR) on various gut bacteria in vitro were evaluated. The effects of LR on high-fat diet-fed(HFD) rats in vivo were also assessed. Culture methods,quantitative polymerase chain reaction,and terminalrestriction fragment length polymorphism were used to analyze the effects of LR on gut bacteria. Biochemical tests were also performed to detect the changes in obesity-related indicators after LR treatment. RESULTS: LR treatment lowered adipose weight and decreased Lee's index,blood glucose,total cholesterol,and lipid in the tested groups relative to control(P < 0.05). To determine the reasons for these changes,we assessed the potential bacteriostatic and bactericidal effects of LR on specific bacterial species in vitro. LR affected the richness,diversity,and evenness of gut bacteria,increased fecal Lactobacillus,and decreased Enterococci in HFD rats(P < 0.05). CONCLUSION: L. robustum may be a safe and effective food for weight loss and obesity control,and the effects of L. robustum might be mediated by the regulation of gut bacteria.

A more consistent intraluminal rhesus monkey model of ischemic stroke

Endovascular surgery is advantageous in experimentally induced ischemic stroke because it causes fewer cranial traumatic lesions than invasive surgery and can closely mimic the pathophysiology in stroke patients. However, the outcomes are highly variable, which limits the accuracy of evaluations of ischemic stroke studies. In this study, eight healthy adult rhesus monkeys were randomized into two groups with four monkeys in each group： middle cerebral artery occlusion at origin segment （M1） and middle cerebral artery occlusion at M2 segment. The blood flow in the middle cerebral artery was blocked completely for 2 hours using the endovascular microcoil placement technique （1 mm × 10 cm） （undetachable）, to establish a model of cerebral ischemia. The microcoil was withdrawn and the middle cerebral artery blood flow was restored. A reversible middle cerebral artery occlusion model was identified by hematoxylin-eosin staining, digital subtraction angiography, magnetic resonance angiography, magnetic resonance imaging, and neurological evaluation. The results showed that the middle cerebral artery occlusion model was successfully established in eight adult healthy rhesus monkeys, and ischemic lesions were apparent in the brain tissue of rhesus monkeys at 24 hours after occlusion. The rhesus monkeys had symptoms of neurological deficits. Compared with the M1 occlusion group, the M2 occlusion group had lower infarction volume and higher neurological scores. These experimental findings indicate that reversible middle cerebral artery occlusion can be produced with the endovascular microcoil technique in rhesus monkeys. The M2 occluded model had less infarction and less neurological impairment, which offers the potential for application in the field of brain injury research.

Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin

AIM： To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin （ADFMChR） on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved.METHODS： HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry （FCM） using propidium iodide （PI） fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ （PPARγ）, NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting.RESULTS： MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose- dependent manner, with little effect on growth of L-02 cells, and when ICs0 was measured as 8.45 μmol/L and 191.55 μmol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil （5-FU, ICso was 9.27 μmol/L）. The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 （191.55/8.45）, higher than 5-FU （SI was 7.05 （65.37/9.27）. FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 μmol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 μmol/L ADFMChR than when treated with 30.0 μmol/L ChR （16.0%） （P 〈 0.05） and were similar to those obtained with 30.0 μmol/L 5-FU（41.0%）. DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 μmol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 pmol/1 GW9662, a blocker of PPARy. Western blotting analysis revealed that aEer 24 h of treatment with 3.0, 10.0, 30.0 μmol/L ADFMChR, PPARy and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 μmol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 μmol/L ADFMChR on PPARy and NF-KB protein expression in HepG2 cells.CONCLUSION： ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF-κB, and increasing Bax expression.

Noggin versus basic fibroblast growth factor on the differentiation of human embryonic stem cells

The difference between Noggin and basic fibroblast growth factor for the neural precursor differen- tiation from human embryonic stem cells has not been studied. In this study, 100 tJg/L Noggin or 20 IJg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen- tiate human embryonic stem cells H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro- scope. Immunofluorescence staining revealed expression levels of Nestin, [3-111 Tubulin and Sox-1 were higher in the induced cells and reverse-transcription PCR showed induced cells expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cell differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in- creases the differentiation of neural precursors.

Nucleus Transfer Efficiency of Ear Fibroblast Cells Isolated from Bama Miniature Pigs at Various Ages

Summary： Somatic cell nucleus transfer （SCNT） has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a pan- city of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer （NT）. The present study examined the NT efficiency of ear fibroblasts from fetal, new- born, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells ini- tially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were ＂S-shaped＂ in different age groups. The cell proliferation of postnatal ear fi- broblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts （P〈0.05 or P〈0.01）. Two-month- and 4-month-old ear fibroblasts had a sig- nificantly higher proportion of G1 stage cells （85% to 91%） than those at 6 and 12 months （66% to 74%, P〈0.01）. The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group （8.06%）. It was concluded that 〈4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

Efficacy of Gold Nanoparticles against Nephrotoxicity Induced by Schistosoma mansoni Infection in Mice

Objective In this study, the ameliorative effects of gold nanoparticles （gold NP） on the renal tissue damage in Schistosoma mansoni （S. mansoni）-infected mice was investigated. Methods High-resolution transmission electron microscopy was used for the characterization of NP. The gold NP at concentrations of 250, 500, and 1000 μg/kg body weight were inoculated into 5. mansoni-infected mice. Results The parasite caused alterations in the histological architecture. Furthermore, it induced a significant reduction in the renal glutathione levels; however, the levels of nitric oxide and malondialdehyde were significantly elevated. The parasite also managed to downregulate KIM-I, NGAL, MCP-1, and TGF-8 mRNA expression in infected animals. Notably, gold NP treatment in mice reduced the extent of histological impairment and renal oxidative damage. Gold NP were able to regulate gene expression impaired by 5. Mansoni infection. Conclusion The curative effect of gold NP against renal toxicity in 5. mansoni-infected mice is associated with their role as free radical scavengers.

Generation and expression analysis of BAC humanized mice carrying HLA-DP401 haplotype

Background:Human leukocyte antigen(HLA)-DP is much less studied than other HLA class Ⅱ antigens,that is,HLA-DR and HLA-DQ,etc.However,the accumulating data have suggested the important roles of DP-restricted responses in the context of cancer,allergy,and infectious disease.Lack of animal models expressing these genes as authentic cis-haplotypes blocks our understanding for the role of HLA-DP haplotypes in immunity.Methods:To explore the potential cis-acting control elements involved in the tran-scriptional regulation of the HLA-DPA1/DPB1 gene,we performed the expression analysis using bacterial artificial chromosome(BAC)-based transgenic humanized mice in the C57BL/6 background,which carried the entire HLA-DP401 gene locus.We further developed a mouse model of Staphylococcus aureus pneumonia in HLA-DP401 humanized transgenic mice,and performed the analysis on the expres-sion pattern of HLA-DP401 and immunological responses in the model.Results:In this study,we screened and identified a BAC clone spanning the entire HLA-DP gene locus.DNA from this clone was analyzed for integrity by pulsed-field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals.Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA-DPA1/DPB1 gene.Transgene copy numbers were determined by real-time PCR analysis.HLA-DP401 gene expression was analyzed at the mRNA and protein level.Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA-DP401-H2-Aβ1 humanized mice.Increased expression of HLA-DP401 was observed in the thymus of the humanized mice infected by S aureus.Conclusions:We generated several BAC transgenic mice,and analyzed the expres-sion of HLA-DPA1/DPB1 in those mice.A model of S aureus-induced pneumonia in the HLA-DP401-H2-Aβ1^(-/-)humanized mice was further developed,and S aureus in-fection upregulated the HLA-DP401 expression in thymus of those humanized mice.These findings demonstrate the potential of those HLA-DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC-associated immunological diseases.

Effectiveness of Saccharomyces boulardii in a rat model of colitis

AIM:To investigate the effects of Saccharomyces boulardii(S.boulardii) in an experimental rat model of trinitrobenzene sulfonic acid(TNBS)-induced colitis.METHODS:Thirty-two Wistar albino female rats were categorized into five groups.On the first day of the study,50 mg TNBS was administered via a rectal catheter in order to induce colitis in all rats,except those in the control group.For 14 d,the rats were fed a standard diet,without the administration of any additional supplements to either the control or TNBS groups,in addition to 1 mg/kg per day S.boulardii to the S.boulardii group,1 mg/kg per day methyl prednisolone(MP) to the MP group.The animals in the S.boulardii + MP group were coadministered these doses of S.boulardii and MP.During the study,weight loss,stool consistency,and the presence of obvious blood in the stool were evaluated,and the disease activity index(DAI) for colitis was recorded.The intestines were examined and colitis was macro-and microscopically scored.The serum and tissue levels of tumor necrosis factor-α(TNF-α) and nitric oxide(NO) were determined,and fungemia was evaluated in the blood samples.RESULTS:The mean DAI scores for the MP and S.boulardii + MP groups was significantly lower than the TNBS group(3.69 ± 0.61 vs 4.46 ± 0.34,P = 0.018 and 3.77 ± 0.73 vs 4.46 ± 0.34,P = 0.025,respectively).While no significant differences between the TNBS and the S.boulardii or MP groups could be determined in terms of serum NO levels,the level of serum NO in the S.boulardii + MP group was significantly higher than in the TNBS and S.boulardii groups(8.12 ± 4.25 μmol/L vs 3.18 ± 1.19 μmol/L,P = 0.013;8.12 ± 4.25 μmol/L vs 3.47 ± 1.66 μmol/L,P = 0.012,respectively).The tissue NO levels in the S.boulardii,MP and S.boulardii + MP groups were significantly lower than the TNBS group(16.62 ± 2.27 μmol/L vs 29.72 ± 6.10 μmol/L,P = 0.002;14.66 ± 5.18 μmol/Lvs 29.72 ± 6.10 μmol/L,P = 0.003;11.95 ± 2.34 μmol/Lvs 29.72 ± 6.10 μmol/L,P = 0.002,respectively).The tissue NO levels in the S.boulardii,MP and S.boulardii + MP groups were similar.The mean serum and tissue TNF-α levels were determined to be 12.97 ± 18.90 pg/mL and 21.75 ± 15.04 pg/mL in the control group,18.25 ± 15.44 pg/mL and 25.27 ± 11.95 pg/mL in the TNBS group,20.59 ± 16.15 pg/mL and 24.39 ± 13.06 pg/mL in the S.boulardii group,9.05 ± 5.13 pg/mL and 24.46 ± 10.85 pg/mL in the MP group,and 13.95 ± 10.17 pg/mL and 24.26 ± 10.37 pg/mL in the S.boulardii + MP group.Significant differences in terms of the levels of serum and tissue TNF-α and the macroscopic and microscopic scores were not found between the groups.S.boulardii fungemia was not observed in any of the rats.However,Candida fungemia was detected in one rat(14%) in the TNBS group,two rats(28%) in the S.boulardii group,three rats(50%) in the MP group,and three rats(42%) in S.boulardii + MP group.CONCLUSION:S.boulardii does not demonstrate considerable effects on the DAI,pathological scores,or cytokine levels but does decrease the tissue NO levels.

Natural maternal transmission of H pylori in Mongolian gerbils

AIM: To investigate maternal H pylori infection status to determine the potential of maternal transmission. METHODS: In the present study, we examined these issues in an experimental murine model, which is a Mongolian gerbil model that has been reported as an optimal laboratory animal model to study H pylori . Pregnant Mongolian gerbils, infected experimentally with H pylori, were divided into as four groups. Following the experimental design, the stomachs of the mother and litters were isolated and assessed for transmission of H pylori at the prenatal period, parturition day, 1-wk old and 3-wk old respectively. Bacterial culture and polymerase chain reaction (PCR) were used to examine the presence of transmitted H pylori. RESULTS: All litters showed no transmission of H pylori during pregnancy and at parturition day. However, they revealed 33.3% and 69.6% at 1-wk and 3-wk of age respectively by PCR. CONCLUSION: These results suggested that vertical infection during the prenatal period or delivery procedure is unlikely as a route of mother-to-child H pylori infection. It may be that H pylori is acquired through breast- feeding, contaminated saliva and fecal-oral transmission during co-habitation.

Characterization of genetic humanized mice with transgenic HLA DP401 or DRA but deficient in endogenous murine MHC classⅡgenes upon Staphylococcus aureus pneumonia

Background:Staphylococcus aureus can cause serious infections by secreting many superantigen exotoxins in“carrier”or“pathogenic”states.HLA DQ and HLA DR humanized mice have been used as a small animal model to study the role of two molecules during S.aureus infection.However,the contribution of HLA DP to S.aureus infection is unknown yet.Methods:In this study,we have produced HLA DP401 and HLA DRA0101 humanized mice by microinjection of C57BL/6J zygotes.Neo-floxed IAβ+/-mice were crossbred with Ella-Cre and further crossbred with HLA DP401 or HLA-DRA0101 humanized mice.After several rounds of traditional crossbreeding,we finally obtained HLA DP401-IAβ-/-and HLA DRA-IAβ-/-humanized mice,in which human DP401 or DRA0101 molecule was introduced into IAβ-/-mice deficient in endogenous murine MHC classⅡmolecules.A transnasal infection murine model of S.aureus pneumonia was induced in the humanized mice by administering 2×108CFU of S.aureus Newman dropwise into the nasal cavity.The immune responses and histopathology changes were further assessed in lungs in these infected mice.Results:We evaluated the local and systemic effects of S.aureus delivered intranasally in HLA DP401-IAβ-/-and HLA DRA-IAβ-/-transgenic mice.S.aureus Newman infection significantly increased the m RNA level of IL 12p40 in lungs in humanized mice.An increase in IFN-γand IL-6 protein was observed in HLA DRA-IAβ-/-mice.We observed a declining trend in the percentage of F4/80+macrophages in lungs in HLA DP401-IAβ-/-mice and a decreasing ratio of CD4+to CD8+T cells in lungs in IAβ-/-mice and HLA DP401-IAβ-/-mice.A decreasing ratio of Vβ3+to Vβ8+T cells was also found in the lymph node of IAβ-/-mice and HLA DP401-IAβ-/-mice.S.aureus Newman infection resulted in a weaker pathological injury in lungs in IAβ-/-genetic background mice.Conclusion:These humanized mice will be an invaluable mouse model to resolve the pathological mechanism of S.aureus pneumonia and study what role DP molecule plays in S.aureus infection.

Targeting EZH1/2 induces cell cycle arrest and inhibits cell proliferation through reactivation of p57CDKN1C and TP53INP1 in mantle cell lymphoma

Objective: To explore the effect of dysregulation of epigenetic regulator EZH1 and EZH2 on the proliferation in MCL and the underlying mechanisms.Methods: In this study, we elucidated the role of EZH1 and EZH2 overexpression by immunohistochemistry and correlated them to clinical outcome in 41 MCL patients.Quantitative real-time PCR and Western blot were applied to confirm the level of EZH1 and EZH2 in well-characterized MCL cell lines which were compared to those of na?ve B cells.Then we manipulated the expression of EZH1 and EZH2 in MCL cells using CRISPR/Cas9 system to directly investigate their functional roles in MCL.We also evaluated the effect of two small molecule selective inhibitors, EPZ005687 and UNC1999, on MCL cell proliferation, cell cycle distribution and apoptosis in vitro.Finally, we performed RNA-sequencing(RNA-Seq) and Chromatin immunoprecipitation(ChIP) assay to further gain insight into the underlying molecular mechanisms.Results: We found that EZH2 protein is overexpressed in approximately half of this cohort of MCL cases.More importantly, the overexpression of EZH2 is associated with poor OS in the patients.Nevertheless, simple EZH2 depletion in vitro has little impact on the viability of MCL cells, predominantly because of the consequent up-regulation of EZH1.Consistently, UNC1999, a dual EZH1/2 inhibitor, unlike the EZH2 selective inhibitor EPZ005687, exerts a potent inhibitory effect on MCL cells.Furthermore, we discover CDKN1C and TP53 INP1 as the two important cell cycle regulators, the expression of which are repressed by EZH1/2 mediated epigenetic regulation and are restored by EZH1/2 dual inhibition.Conclusions: Our study suggests that EZH2 participates in the pathogenesis of MCL which may serve as a potential biomarker for prognosis prediction.The dual inhibition of EZH1/2 is a promising therapeutic strategy for MCL.

Prevalence of West Nile virus in Mashhad,Iran:A population-based study

Objective:To evaluate the prevalence of West Nile virus seropositivity in the general population of Mashhad.Northeast of Iran.Methods:One hundred and eighty two individuals living in the city of Mashhad were studied using cluster sampling method.Both IgM and IgG antibodies against WNV were detected by ELISA method.Results:In this study,the overall IgG seroprevalence of positive West Nile virus was 11%;however.IgM antibody was not found in the participants.Conclusions:Our study suggested that the prevalence rate of West virus is considerable in Mashhad city.It seems necessary for clinicians and health care workers to be aware of WNV infection in the Northeast Iran.

Trigonella foenum-graecum seed extract modulates expression of lipid metabolism-related genes in HepG2 cells

Objective:To investigate anti-dyslipidemic effects of hydroalcoholic fenugreek seed extracts,diosgenin,and 4-OH-Ile on HepG2 cell line.Methods:HepG2 cells were treated with hydroalcoholic fenugreek seed extracts,diosgenin,4-OH-Ile,and orlistat.IC20 was calculated using the MTT method.The cells were then pretreated with IC20 concentrations for 24 and 48 h.Real time PCR was employed to measure expression of liver X receptor alpha(LXR a),sterol regulatory element-binding protein-1 C(SREBP-1 C),acetyl-CoA carboxylase(ACC),fatty acid synthase(FAS),fibroblast growth factor 21(FGF21),peroxisome proliferator-activated receptor gamma(PPAR γ),and lowdensity lipoprotein receptor(LDLR).Results:The results showed that LXR α(P=0.003,P<0.001).,SREBP-1 C(P<0.001,P<0.001),ACC(P=0.002,P=0.006),and FAS(P<0.001,P<0.001)were downregulated significantly,while FGF21(P<0.001,P<0.001),PPAR γ(P=0.004,P<0.001),and LDLR(P<0.001,P<0.001)were upregulated significantly in HepG2 cells treated with the IC20 of hydroalcoholic fenugreek seed extracts,diosgenin,4-OH-Ile,and orlistat in 24 and 48 h,respectively.Conclusions:Hydroalcoholic fenugreek seed extracts,diosgenin,and 4-OH-Ile significantly modulate the expression of some important lipid metabolism related genes,which is similar to orlistat.Trigonella foenum.-graecum seed extract or its derivatives should be further investigated for their dyslipidemia effects and its complications.

Effects of Walnut on Lipid Profile as Well as the Expression of Sterol-Regulatory Element Binding Protein-1c(SREBP-1c) and Peroxisome Proliferator Activated Receptors <i>α</i>(PPAR<i>α</i>) in Diabetic Rat

Diabetes Mellitus has appeared as a universal burden. Studies have reported that mortality from Coronary Heart Disease (CHD) in diabetic patients is 2 - 4 times higher than nondiabetics. In this respect, walnut is a treatment which has beneficial effects on CHD risk factors. PPARα and SREBP-1c play an important role in the regulation of lipid metabolism. This study was aimed to evaluate the effects of walnut on lipid profile as well as SREBP-1c and PPARα protein levels in rats. Animals were randomly divided into 3 groups (n = 6);Group 1: Received chow only (control), Group 2: Diabetic rats + chow, Group 3: Diabetic rats + chow supplemented with 4% of whole walnuts. After four weeks rats were sacrificed, blood was collected;lipid profiles as well as SREBP-1c and PPARα protein levels were determined. Compared with diabetic rats walnut significantly decreased serum cholesterol (P < 0.01), LDL-c (P < 0.01), triglyceride (P < 0.001) and VLDL-c (P < 0.001) and also increased HDL-c (P < 0.05) compared with diabetic. Moreover, SREBP-1c protein level significantly decreased (P < 0.05) and PPARα significantly increased in walnut group compared with diabetic group (P < 0.05). The findings showed that walnut administration in diet clinically decreases atherosclerosis risk factors. Lipid profile reduction might be due to the rise of PPARα and the reduction of SREBP-1c by this medical treatment in liver.

Functional Microvascular Anatomy of the Horse Eye: A Scanning Electron Microscopic Study of Corrosion Casts

Objective: This study presents the microvasculature of the horse iris, ciliary process, retina, and choroid and discusses the functional significance of the vasculature. Procedure: Seven horses were used for this study. The ocular vascular system was injected with methylmethacrylate resin via the carotid artery, and the vascular corrosion casts were observed using a scanning electron microscope. Results: The iridial vessels showed a wavy course. The ciliary process was supplied by 2 arterial routes: the iridial and ciliary arterial circles. The subjects displayed a paurangiotic retina with retinal vessels extending only a short distance around the disc. The retinal arterioles and venules ran in closely related pairs, and the capillaries formed hairpin loops. No central retinal artery was seen in the equine eyes examined. The choriocapillaris in the avascular retina was arranged in honeycomb hexagon lobules and formed a more densely packed network than that in the vascular retina. There were 2 distinct venous drainage systems in the horse choroid: the vortex veins and the posterior ciliary veins. The vortex vein ampulla was flattened and showed a slit-like lumen at the merge site with the ophthalmic vein. The vortex veins demonstrated a marked constriction before leaving the eye. Discussion: The 2 choroidal drainage systems may compensate each other in event of occlusion. The ampulla and the constriction in the vortex veins may act as a valve regulating the blood flow to keep the eye at an optimum size and the intraocular pressure within the normal physiological range.

Vibration in mice: A review of comparative effects and use in translational research

Sound pressure waves surround individuals in everyday life and are perceived by animals and humans primarily through sound or vibration. When sound pressure waves traverse through a solid medium, vibration will result. Vibration has long been considered an unwanted variable in animal research and may confound scientific endeavors using animals. Understanding the characteristics of vibration is required to determine whether effects in animals are likely to be therapeutic or result in adverse biological effects. The eighth edition of the "Guide for the Care and Use of Laboratory Animals" highlights the importance of considering vibration and its effects on animals in the research setting, but knowledge of the level of vibration for eliciting these effects was unknown. The literature provides information regarding therapeutic use of vibration in humans, but the range of conditions to be of therapeutic benefit is varied and without clarity. Understanding the characteristics of vibration(eg, frequency and magnitude) necessary to cause various effects will ultimately assist in the evaluation of this environmental factor and its role on a number of potential therapeutic regimens for use in humans. This paper will review the principles of vibration, sources within a research setting, comparative physiological effects in various species, and the relative potential use of vibration in the mouse as a translational research model.