Hesheng Jiang

Trancriptomic profiling revealed the signatures of acute immune response in tilapia (Oreochromis niloticus) following Streptococcus iniae challenge

Streptococcus iniae is the most significant bacterial disease of tilapia throughout the world, and commonly leads to tremendous economic losses. In contrast to other important fish species, our knowledge about the molecular mechanisms of tilapia in response to bacterial infection is still limited. Here, therefore, we utilized RNA-seq to first profiling of host responses in tilapia spleen following S. iniae infection at transcriptome level. A total of 223 million reads were obtained and assembled into 192,884 contigs with average length 844 bp. Gene expression analysis between control and infected samples at 5 h, 50 h, and 7 d revealed 1475 differentially expressed genes. In particular, the differentially expressed gene set was dramatically induced as early as 5 h, and rapidly declined to basal levels at 50 h. Enrichment and pathway analysis of the differentially expressed genes revealed the centrality of the pathogen attachment and recognition, cytoskeletal rearrangement and immune activation/inflammation in the pathogen entry and host inflammatory responses. Understanding of these responses can highlight mechanisms of tilapia host defense, and expand our knowledge of teleost immunology. Our findings will set a foundation of valuable biomarkers for future individual, strain, and family-level studies to evaluate immune effect of vaccine and individual response in host defense mechanisms to S. iniae infection, to select disease resistant families and strains.

In Vitro Culture and Differentiation of Buffalo (Bubalus bubalis) Spermatogonia

The objective of this study was to develop a culture system which could support buffalo spermatogonia differentiation into spermatids in vitro. Testes from 3- to 5-month-old buffaloes were decapsulated and seminiferous tubules were enzymatically dissociated to recover spermatogonia and sertoli cells. The cells were cultured in modified Dulbecco modified Eagle medium supplemented with different concentrations of foetal bovine serum, retinol, testosterone for 2 months at 37 degrees C. Spermatogonia and sertoli cells were identified with an antibody against c-kit or GATA4, respectively. The viability of spermatogonia in the media supplemented with different concentrations of serum was all significantly higher (p < 0.05) compared with that in the medium without serum. A-paired or A-aligned spermatogonia and spermatogonial colonies (AP-positive) were observed after 7-10 days of culture and spermatid-like cells with a flagellum (6-8 microm) appeared after 30 days of culture. For cultured conditions, retinol could not significantly promote the formation of spermatid-like cells (p > 0.05), whereas supplementation of testosterone could significantly promote (p < 0.05) the formation of spermatid-like cells after 41 days of culture. The expression of the spermatid-specific marker gene (PRM2) was identified after 30 days of culture by RT-PCR. Yet, the transition protein 1 (TP1, a haploid makers) was not detected. Meanwhile, spermatids developed in vitro were also confirmed by Raman spectroscopy. These results suggest that buffalo spermatogonia could differentiate into spermatids in vitro based on the analysis of their morphology, PRM2 expression and Raman spectroscopy. Yet, the normality of the spermatid-like cells was not supported by TP1 expression.

Transcriptomic profiling analysis of tilapia (Oreochromis niloticus) following Streptococcus agalactiae challenge

ABSTRACT Innate immune system is the primary defense mechanism against pathogen infection in teleost, which are living in pathogen‐rich aquatic environment. It has been long hypothesized that the disease resistance in teleost are strongly correlated to the activities of innate immune genes. Tilapia is an important economical fish around the world, especially in China, where the production accounts for nearly half of the global production. Recently, S. agalactiae has become one of the most serious bacterial diseases in southern China, resulted in high cumulative mortality and economic loss to tilapia industry. Therefore, we sought here to characterize the expression profiles of tilapia against S. agalactiae infection at whole transcriptome level by RNA‐seq technology. A total of 2822 genes were revealed significantly expressed in tilapia spleen with a general trend of induction. Notably, most of the genes were rapidly the most induced at the early timepoint. The significantly changed genes highlighted the function of pathogen attachment and recognition, antioxidant/apoptosis, cytoskeletal rearrangement, and immune activation. Collectively, the induced expression patterns suggested the strong ability of tilapia to rapidly recognize the invasive bacteria, and activation of downstream immune signaling pathways to clear the bacteria and prevent the tissue damage and bacteria triggered cell apoptosis. Our results heighted important roles of novel candidate genes which were often missed in previous tilapia studies. Further studies are needed to characterize the molecular relationships between key immune genes and disease resistance, and to identify the candidate genes for molecular‐assistant selection of disease‐resistant broodstock and evaluation of disease prevention and treatment measures. HighlightsWe constructed RNA‐seq analysis in tilapia spleen after S. agalactiae challenge.We obtained 192,884 contigs with average length 844 bp by de novo assembly.We observed 2822 differentially expressed genes after S. iniae challenge.The up‐regulation is abundant at 5 h dpi and drastically decreases to the basal level at 7 d dpi.

Milk fat globule is an alternative to mammary epithelial cells for gene expression analysis in buffalo

Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells.

Basal polarization of the immune responses to Streptococcus agalactiae susceptible and resistant tilapia (Oreochromis niloticus)

ABSTRACT One of the highest priority areas for improvement is the development of effective strategies for decreasing disease mortality levels in aquaculture production, a better understanding of the components of the fish immune system and their functions in the context of pathogen invasion is needed. Tilapia is the most common fish in South China, and Streptococcus agalactiae has become the most serious disease problem for tilapia industry in China. Here, we profiled gene expression differences between tilapia differing in their susceptibility to S. agalactiae both basally (before infection) and at three early timepoints post‐infection (5 h, 50 h, and 7 d). Between group comparisons revealed 5756 unique genes differentially expressed greater than 2‐fold at one or more timepoints. And the resistant fish showed much more strong ability in pathogen recognition, antigen presentation, immune activation, while the susceptible fish showed fast activation of apoptosis. Taken together, the immune profiles expand our knowledge for molecular mechanisms for disease resistance, as well as provide solid molecular resources for further identification of the candidate markers for disease‐resistant selection and evaluation of disease prevention and treatment options for tilapia industry. HighlightsGene expression differences between tilapia with different susceptibility to S. agalactiae were profiled.Between group comparison revealed 5,756 unique genes differentially expressed at one or more timepoints.The resistant fish showed strong ability in pathogen recognition, antigen presentation, immune activation.

Qualitative Analyses of Protein Phosphorylation in Bovine Pluripotent Stem Cells Generated from Embryonic Fibroblasts.

Pluripotent stem cells (PSCs) generated from somatic cells via ectopic expression of specific transcription factors provide an unlimited cell resource for regenerative medicine and transgenic breeding. Here, we describe the successful generation of bovine induced PSCs (biPSCs) from foetal fibroblasts by lentivirus-mediated delivery of bovine pluripotency reprogramming factors (PRFs) OCT3/4, SOX2, KLF4, c-MYC, NANOG and LIN28. The generated biPSCs resembled embryonic stem cells (ESCs) in their gene expression profiles, self-renewal capabilities and proliferation, as well as maintenance of a normal karyotype and differentiation into diverse cell types of all three germ layers both in vitro and in vivo. Qualitative phosphoproteomics of biPSCs revealed a large number of phosphorylated proteins, which might be related to the control of biPSCs status. The successful generation of biPSCs and the analysis of their phosphoproteome would further our understanding of the epigenetic mechanisms underlying iPSC pluripotency, thus promoting their application in bovine transgenic breeding and marking avenues for future research.

Assessment of M. longissimus fibre types and metabolic enzymes in Bama miniature pigs and Landrace swine

Abstract Overt differences exist between Chinese local pigs and exotic pig breeds, especially in muscle growth rate and meat quality. However, the underlying molecular mechanisms remain unclear. This study aimed to assess muscle fibre types and metabolic enzymes in Bama miniature pigs and Landrace swine. Meat quality traits, including intramuscular fat content, and muscle colour, conductivity, and tenderness, were assessed in these pig breeds. Then, muscle fibre types were classified, and mRNA amounts and activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) assessed, in M. longissimus from the two pig breeds, at various ages. Our data showed significantly higher back fat thickness, muscle conductivity, and intramuscular fat content in samples from Bama miniature pigs compared with the values obtained for Landrace pigs (p < .05). In addition, SDH activity was significantly higher, and LDH activity overtly lower in Bama pigs compared with Landrace swine (p < .05). Furthermore, myosin heavy-chain (MyHC) II A, II B, and II X mRNA levels in Bama miniature pigs at 180 were significantly higher than values obtained for Landrace pigs of the same age. Although MyHC I gene expression levels were similar in Bama miniature and Landrace pigs at 180 days of age, significantly higher amounts were obtained in 300 day old Bama miniature pigs compared with 180 day old Landrace pigs (p < .05). Collectively, these preliminary findings indicated that skeletal muscles from Bama miniature pigs may contain more oxidative fibres compared with those from Landrace pigs, which might explain the meat quality differences between the two pig breeds.