Immune escape of avian oncogenic Marek's disease herpesvirus and antagonistic host immune responses.

Marek's disease virus (MDV) is a highly pathogenic and oncogenic alpha herpesvirus that causes Marek's disease (MD), which is one of the most important immunosuppressive and rapid-onset neoplastic diseases in poultry. The onset of MD lymphomas and other clinical diseases can be efficiently prevented by vaccination; these vaccines are heralded as the first demonstration of a successful vaccination strategy against a cancer. However, the persistent evolution of epidemic MDV strains towards greater virulence has recently resulted in frequent outbreaks of MD in vaccinated chicken flocks worldwide. Herein, we provide an overall review focusing on the discovery and identification of the strategies by which MDV evades host immunity and attacks the immune system. We have also highlighted the decrease in the immune efficacy of current MD vaccines. The prospects, strategies and new techniques for the development of efficient MD vaccines, together with the possibilities of antiviral therapy in MD, are also discussed.

Evaluation of Antifungal Phenolics from Helianthus tuberosus L. Leaves against Phytophthora capsici Leonian by Chemometric Analysis.

Phytophthora capsici Leonian causes destructive economical losses in pepper production, and a promising source of natural fungicides- Helianthus tuberosus leaves was reported. The antifungal activities of different extracts and compounds from H. tuberosus leaves against the phytopathogen, P. capsici Leonian, were examined by chemometric analysis, including HPLC-MS/MS and multivariate data analyses. Principal component analysis and orthogonal partial least squares-discriminate analysis were applied to examine the four groups of H. tuberosus leaves samples, including crude extracts obtained by different methods, including refluxing, macerating, and refluxing under vacuum; four fractions, namely, petroleum ether (PE), chloroform (Chl), ethyl acetate (EA), and n-butanol (NB) fractions; the samples of three H. tuberosus cultivars; and the samples at three growth stages of cultivar Nan Yu. The phenolics contents were categorized based on 3,5-Dicaffeoylquinic acid (3,5-DiCQA), 1,5-Dicaffeoylquinic acid (1,5-DiCQA), 3-O-Caffeoylquinic acid (3-CQA), and 4,5-Dicaffeoylquinic acid (4,5-DiCQA), which were predominant in all the samples. Antifungal activity assay revealed that Chl and NB fractions were more active against P. capsici Leonian with lower IC50(half of maximal inhibitory concentration) values, whereas partial least squares-discriminate analysis suggested caffeoylquinic acid isomer(4-CQA), methyl-quercetin glycoside(MQG), and caffeic acid(CA) might be the main active components in H. tuberosus leaves against P. capsici Leonian. Furthermore, microscopic evaluation demonstrated structural deformities in P. capsici Leonian treated with Chl and NB fractions, indicating the antifungal effects of H. tuberosus leaves. These results imply that H. tuberosus leaves with a high concentration of phenolics might be a promising source of natural fungicides.

Identification, expression analysis, and antibacterial activity of NK-lysin from common carp Cyprinus carpio.

Natural killer lysin (NK-lysin), produced by cytotoxic T lymphocytes and natural killer cells, is a cationic antimicrobial peptide that has a broad antimicrobial spectrum, including bacteria, viruses, and parasites. Nevertheless, the implication of NK-lysin in the protection against bacterial infection is not aware in common carp. In this study, six different NK-lysin genes (nkl1, nkl2, nkl3, nkl4, nkl5 and nkl6) were identified in the common carp genome. Each of the mature peptides of common carp NK-lysin has six well-conserved cysteine residues, and shares a Saposin B domain, characteristic of saposin-like protein (SALIP) family. The gene nkl1 contains 5 extrons and 4 introns, and nkl2, nkl3, nkl4 or nkl5 contains 4 extrons and 3 introns, however, the nkl6 has 3 extrons and 2 introns. By quantitative real-time PCR, nkl2 transcripts were predominantly expressed in spleen of healthy common carp, while elevated mainly in gill and spleen upon Aeromonas hydrophila infection. The recombinant NK-lysin-2 purified from Pichia pastoris shows antibacterial activity against Staphylococcus aureus (Gram-positive), and Escherichia coli M15, Aeromonas hydrophila, as well as Edwardsiella tarda (Gram-negative), the latter two are important pathogens of aquaculture. Our results indicate that NK-lysin in common carp might play an important role in fish immune response by enhancing antibacterial defense against bacterial pathogens.

[Aerobic exercise improves spermatogenesis of male rats: Results of iTRAQ-based proteomic analysis of the testis tissue].

OBJECTIVE: To investigate the effect of aerobic exercise on the spermatogenic function of male rats and screen out differentially expressed proteins related to spermatonesis-regulation by proteomic analysis. METHODS: We randomly divided 24 SD male rats into groups A (non-exercise control), B (exercise), and C (weight-bearing exercise), those in the latter two groups made to swim for 60 minutes a day and those in group C bearing a load 3% of the body weight, both 6 times a week for 9 weeks. At 24 hours after the last exercise, we obtained the sperm count, measured the levels of such serum reproductive hormones as testosterone (T), luteotrophic hormone (LH), follicle-stimulating hormone (FSH), and gonadotrophin-releasing hormone (GnRH), and employed isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of the testicular tissue. RESULTS: Compared with group A, group C showed significant increases in sperm concentration (ï¼»2.12 ± 0.43ï¼½ vs ï¼»3.54 ± 0.52ï¼½ ×106/ml, P <0.01) and the levels of serum LH (ï¼»35.99 ± 2.90ï¼½ vs ï¼»38.96 ± 1.34ï¼½ IU/L, P <0.01) and T (ï¼»19.99 ± 0.25ï¼½ vs ï¼»21.36 ± 0.53ï¼½ nmol/L, P <0.01), but no statistically significant differences in GnRH (ï¼»623.95 ± 41.44ï¼½ vs ï¼»641.82 ± 42.78ï¼½ ng/L, P >0.05) and FSH (ï¼»20.49 ± 2.44ï¼½ vs ï¼»22.29 ± 2.31ï¼½ IU/L, P >0.05). No significant changes were observed in sperm concentration or reproductive hormone levels in group B as compared with A. Group B exhibited obviously more mature sperm and cell layers in the seminiferous epithelium than group A. A total of 47 differentially expressed proteins were identified, of which 37 were up-regulated and the other 10 down-regulated. In addition, another 5 significantly differentially expressed proteins closely related to reproductive function were identified, including up-regulated Anx A1, GPX3, Rimbp3, and Dpy19l2 and down-regulated CYP17. Enrichment analysis showed that the differentially expressed proteins were mainly involved in extracellular matrix-receptor interaction, protein digestion and absorption, and focal adhesion pathways. CONCLUSIONS: Proper-intensity exercise can improve the spermatogenic function of rats. Aerobic exercise promotes spermatogenesis mainly by up-regulating the expressions of the proteins related to the production and differentiation of spermatozoa.

Proper heat shock pretreatment reduces acute liver injury induced by carbon tetrachloride and accelerates liver repair in mice.

Whether proper heat shock preconditioning can reduce liver injury and accelerate liver repair after acute liver injury is worth study. So mice received heat shock preconditioning at 40°C for 10 minutes (min), 20 min or 30 min and recovered at room temperature for 8 hours (h) under normal feeding conditions. Then acute liver injury was induced in the heat shock-pretreated mice and unheated control mice by intraperitoneal (i.p.) injection of carbon tetrachloride (CCl4). Hematoxylin and eosin (H&E) staining, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and the expression levels of heat shock protein 70 (HSP70), cytochrome P450 1A2 (CYP1A2) and proliferating cell nuclear antigen (PCNA) were detected in the unheated control mice and heat shock-pretreated mice after CCl4 administration. Our results showed that heat shock preconditioning at 40°C for 20 min remarkably improved the mice's survival rate (P<0.05), lowered the levels of serum AST and ALT (P<0.05), induced HSP70 (P<0.01), CYP1A2 (P<0.01) and PCNA (P<0.05) expression, effectively reduced liver injury (P<0.05) and accelerated the liver repair (P<0.05) compared with heat shock preconditioning at 40°C for 10 min or 30 min in the mice after acute liver injury induced by CCl4 when compared with the control mice. Our results may be helpful in further investigation of heat shock pretreatment as a potential clinical approach to target liver injury.

[Cloning of gdnf in the mouse testis and its expression in sertoli cells].

OBJECTIVE: To clone the glial cell line-derived neurotrophic factor (GDNF) from the mouse testis, construct the eukaryotic expression vector and transfect this vector into Sertoli cells in order to use the gdnf-transfected Sertoli cells as the feeder layer to cultivate spermatogonial stem cells (SSCs). METHODS: Total RNA was extracted from the testes of normal mature mice and gdnf was cloned and amplified using RT-PCR, inserted into the eukaryotic expression vector and transfected into sertoli cells (TM4 cell line). Immunofluorescence with anti-GDNF antibodies was performed at 40 h following the transfection. RESULTS: gdnf cDNA was cloned successfully, and GDNF expressed after transfected into Sertoli cells. CONCLUSION: This study provides a basis for culturing SSCs with gdnf-transfected Sertoli cells as the feeder layer.

17beta-estradiol stimulates proliferation of spermatogonia in experimental cryptorchid mice.

AIM: To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice. METHODS: Mice were surgically rendered cryptorchid, then treated with different doses of 17beta-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured. RESULTS: Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia. CONCLUSION: E2 has a dose-related mitogenic effect on spermatogonia.

[Differential analysis of proteomic profiles between cryptorchid and normal mouse testes].

To screen factors related to spermatogonial stem cell (SSC) proliferation, and to investigate the mechanism of infertility caused by cryptorchidism, ten-day-old Kunming (KM) mice were used and experimental cryptorchidism was conducted. On the 35th day after cryptorchid operation, the left testes were fixed in Bouin's fluid and used for histological analysis. The testes of 45-day-old mice were subjected to the same histological analysis, and it was found that they contained germ cells at every stage of development, from SSCs to sperm, indicating that the animals were fully sexually mature at this age. While in experimental cryptorchid mice, the spermatogenesis was arrested at the stage of spermatocytes, and only spermatogonia and primary spermatocytes were present in cryptorchid testes. The proportion of spermatogonia to other types of germ cells was much higher than that in sexually mature mice. On the other hand, the right testes were used for proteomic analysis. The total protein in testes was extracted on the 35th day after cryptorchid operation. The differentially expressed proteins in cryptorchid mice and sexually mature mice were screened and compared by the proteomic techniques. Through the separation of two-dimensional gel electrophoresis (2-DE), 20 differential protein spots were found, and 9 of them were digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. In cryptorchid mice, 6 out of 9 proteins were down-regulated, and 3 were up-regulated. Among these proteins, 4 proteins were identified, and they were Stathmin, phosphatidylethanolamine-binding protein1 (PEBP1), HES-related basic helix-loop-helix protein (HERP), and one unnamed protein (we temporarily named it Px). More Stathmin, PEBP1 and Px were expressed in sexually mature mice than in experimental cryptorchid mice. But HERP1 was the other way round. In the present study, we have screened 4 proteins related to cryptorchidism. It is helpful to study the mechanism of SSC proliferation and infertility caused by cryptorchidism.

[Cloning of Bmi1 cDNA from mouse testis and its expression in E. coli BL21].

OBJECTIVE: To produce BMI1 polyclonal antibody, mouse Bmi1 cDNA was cloned from mouse testis and expressed in E. coli BL21. METHODS: Bmi1 gene was amplified from mouse testis by RT-PCR and inserted into the prokaryotic expression vector pET-28c(+). Subsequently the recombined vector was transformed and expressed in E. coli BL21 (DE3) and the immunogenicity of recombined protein BMI1 (rBMI1) was tested by Western blot. RESULTS: Mouse Bmi1 cDNA of 975 bp was successfully cloned and recombined. E. coli BL21 strains expressed rBMI1 were screened. The expression protein amounted to 12% of the total bacterial protein after induced with IPTG, which included inclusion body and soluble protein. Inclusion body was the major pattern of the expression that amounted to 71% of the insoluble protein. Western blot analysis showed that rBMI1 could be specially recognized by mouse monoclonal IgG1 anti-BMI1 and His-tag antibody. CONCLUSION: There was expression of Bmi1 gene in mouse testis. Mouse Bmi1 cDNA was successfully cloned and expressed prokaryoticly.