CYP1A1 based on metabolism of xenobiotics by cytochrome P450 regulates chicken male germ cell differentiation

This study aimed to explore the regulatory mechanism of metabolism of xenobiotics by cytochrome P450 during the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) and consummate the induction differentiation system of chicken embryonic stem cells (cESCs) into SSCs in vitro. We performed RNA-Seq in highly purified male ESCs, male primordial germ cells (PGCs), and SSCs that are associated with the male germ cell differentiation. Thereinto, the metabolism of xenobiotics by cytochrome P450 was selected and analyzed with Venny among male ESC vs male PGC, male PGC vs SSC, and male ESC vs SSC groups and several candidates differentially expressed genes (DEGs) were excavated. Finally, quantitative real-time PCR (qRT-PCR) detected related DEGs under the condition of retinoic acid (RA) induction in vitro, and the expressions were compared with RNA-Seq. By knocking down CYP1A1, we detected the effect of CYP1A1-mediated metabolism of xenobiotics by cytochrome P450 on male germ cell differentiation by qRT-PCR and immunocytochemistry. Results showed that 17,742 DEGs were found during differentiation of ESCs into SSCs and enriched in 72 differently significant pathways. Thereinto, the metabolism of xenobiotics by cytochrome P450 was involved in the whole differentiation process of ESCs into SSCs and several candidate DEGs: CYP1A1, CYP3A4, CYP2D6, ALDH3B1, and ALDH1A3 were expressed with the same trend with RNA-Seq. Knockdown of CYP1A1 caused male germ cell differentiation under restrictions. Our findings showed that the metabolism of xenobiotics by cytochrome P450 was significantly different during the process of male germ cell differentiation and was persistently activated when we induced cESCs to differentiate into SSCs with RA in vitro, which illustrated that the metabolism of xenobiotics by cytochrome P450 played a crucial role in the differentiation process of ESCs into SSCs.     

Copper-induced liver fibrosis affects the behavior of bone marrow cells in primary culture

Background

The present study investigated the effects of low-molecular-weight components of bovine colostrum (LMCC), which were administered per os, on the differentiation, growth, and survival of cells obtained from the bone marrow of rats in primary culture.

Methods

Bone marrow cells (BMCs) were obtained from both the rat femurs and were cultured in medium 199 supplemented with antibiotics (8% streptomycin and 8% gentamycin) and 20% inactivated fetal calf serum. In addition, the number of BMCs was counted and their morphotypes were determined.

Results

Animals were treated with copper (Cu) sulfate to induce liver fibrosis. Subsequent treatment with LMCC eliminated growth inhibition and normalized the bodyweight and temperature of animals with Cu-induced liver fibrosis. The number of lymphocytes in the bone marrow of animals with Cu-induced liver fibrosis was significantly higher than that in the bone marrow of control animals. The number of neutrophils in untreated animals with liver fibrosis and LMCC-treated animals with liver fibrosis was lower than that in control animals. Neutrophils obtained from untreated animals with liver fibrosis and LMCC-treated animals with liver fibrosis underwent two-times faster degradation in vitro than neutrophils obtained from control animals.

Conclusions

Our results indicate that LMCC affects the distribution of different morphological types of BMCs but does not prevent their degradation in vitro, which was two-times faster than that of BMCs obtained from control animals.

     

Analysis of the potential pathways and target genes in spinal cord injury using bioinformatics methods

Spinal cord injury (SCI) remains to be the most devastating type of trauma for patients because of long lasting disability and limited response to the acute drug administration and efforts at rehabilitation. With the purpose to identify potential targets for SCI treatment and to gain more insights into the mechanisms of SCI, the microarray data of GSE2270, including 119 raphe magnus (RM) samples and 125 sensorimotor cortex (SMTC) samples, was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were screened in RM group and SMTC group compared with their corresponding controls, respectively. A protein–protein interaction (PPI) network was constructed based on the common DEGs identified in both RM group and SMTC group. Gene ontology (GO) and pathway enrichment analyses of the overlapping DEGs were performed. Furthermore, the common DEGs enriched in each pathway were analyzed to identify significant regulatory elements. Totally, 173 overlapping DEGs (130 up-regulated and 43 down-regulated) were identified in both RM and SMTC samples. These overlapping DEGs were enriched in different GO terms. Pathway enrichment analysis revealed that DEGs were mainly related to inflammation and immunity. CD68 molecule (CD68) was a hub protein in the PPI network. Moreover, the regulatory network showed that ras-related C3 botulinum toxin substrate 2 (RAC2), CD44 molecule (CD44), and actin related protein 2/3 complex (ARPC1B) were hub genes. RAC2, CD44, and ARPC1B may be significantly involved in the pathogenesis of SCI by participating significant pathways such as extracellular matrix-receptor signaling pathway and Toll-like receptor signaling pathway.     

Genome-wide association study of body weight in Wenshang Barred chicken based on the SLAF-seq technology

Chicken body weight (BW) is an economically important trait, and many studies have been conducted on genetic selection for BW. However, previous studies have detected functional chromosome mutations or regions using gene chips. The present study used the specific-locus amplified fragment sequencing (SLAF-seq) technology to perform a genome-wide association study (GWAS) on purebred Wengshang Barred chicken. A total of 1,286,715 single-nucleotide polymorphisms (SNPs) were detected, and 175,211 SNPs were selected as candidate SNPs for genome-wide association analysis using TASSEL general linear models. Six SNP markers reached genome-wide significance. Of these, rs732048524, rs735522839, rs738991545, and rs15837818 were significantly associated with body weight at 28 days (BW28), while rs314086457 and rs315694878 were significantly associated with BW120. These SNPs are close to seven genes (PRSS23, ME3, FAM181B, NABP1, SDPR, TSSK6L2, and RBBP8). Moreover, 24 BW-associated SNPs reached “suggestive” genome-wide significance. Of these, 6, 13, 1, and 4 SNPs were associated with BW28, BW56, BW80, and BW120, respectively. These results would enrich the studies on BW and promote the use of Chinese chicken, especially the Wenshang Barred chicken.     

Evaluation of polymorphism loci associated with viral diseases in spangled Orloff chicken breed

The indigenous Russian Spangled Orloff chicken breed (Large Fowl) was for the first time studied by analyzing polymorphism in loci associated with viral diseases caused by Marek’s disease viruses (MDV), avian leukosis viruses (ALV), and avian influenza viruses. Samples for the analysis were collected at the farms of the All-Russian Poultry Research and Technological Institute (VNITIP), the All-Russian Institute of Farm Animal Genetics and Breeding (VNIIGRZh), and the Moscow Zoo. Assessed populations have a distinction in the frequency and range of allelic variants and as a consequence the frequencies of genotypes LEI0258. The highest level of polymorphism was found in microsatellite loci LEI0258 and Mx1 gene (VNIIGRZh population). The evaluation of polymorphism in genes Mxl (interferon-induced guanosine triphosphatase) and TVB (Tumor Virus В locus, cell receptors) showed, that the Mxl and TVB genotypes associated with resistance to avian influenza viruses and ALV, respectively, were not found in studied populations of Spangled Orloff chicken breed. The most homogeneous group for all studied loci is from the VNITIP population. These data are in good agreement with the results from the study of mtDNA polymorphism in the same samples. The observed polymorphism in nuclear and mitochondrial markers demonstrates that most representative population (about 700 individuals) of Spangled Orloff chicken breed from VNITIP during its long conservation breeding program has remained almost unchanged in the investigated molecular markers and is a source of valuable alleles 357 from microsatellite loci LEI0258 associated with Marek’s disease resistance. The observed frequency of the genotype 357/357 was 48%. The obtained data on the genetic homogeneity of Spangled Orloff chicken breed in all analyzed loci show the need for measures to maintain and increase the number of local groups of Orloffs with the aim of its further preservation.     

CD133 and BMI1 expressions and its prognostic role in primary glioblastoma

Glioblastoma is the most common malignant brain tumour, generated by bulk of malignant cancer stem cells, which express various stem cell factors like CD133, BMI1 and nestin. There are several studies which show the importance of CD133 in cancer, but the function and interaction with other major oncogenes and tumour suppressor genes is still not understood. This study aimed to analyse the expression of CD133 mRNA and its correlations with BMI1 protein expression and TP53 mutations in newly diagnosed glioblastoma patients and its role in prognosis. Overexpression of CD133 mRNA and BMI1 protein was found in 47.6 and 76.2% patients respectively and TP53 mutations was seen in 57.1% of patients in our study. There was no correlation among TP53 mutations and expressions of CD133 and BMI1. We found that high level of BMI1 expression was favourable for the patient survival (P= 0.0075) and high CD133 mRNA expression was unfavourable for the patient survival (P= 0.0226). CD133 mRNA and BMI1 protein expression could independently predict the glioblastoma patient survival in multivariate analysis. In conclusion, the overexpression of these stem cell markers is a common event in glioblastoma progression and could be used as potential prognostic markers.     

Genome-wide association study on chicken carcass traits using sequence data imputed from SNP array

Chicken carcass traits are economically important for the chicken industry. Detecting which genes affect chicken carcass traits is of great benefit to the genetic improvement of this important agricultural species. To investigate the genetic mechanism of carcass traits in chickens, we carried out a genome-wide association study (GWAS). A total of 435 Chinese indigenous chickens were phenotyped for carcass weight (CW), eviscerated weight with giblets (EWG), and eviscerated weight (EW) after slaughter at 91 days and were genotyped using a 600-K single nucleotide polymorphism (SNP) genotyping array. Twenty-four birds were selected for sequencing, and the 600 K SNP panel data were imputed to sequence data with the 24 birds as the reference. Univariate GWASs were performed with GEMMA software using the whole genome sequence data imputed from SNP chip data. Finally, 3, 25, and 63 suggestively significant SNPs were identified to be associated with carcass weight (CW), eviscerated weight with giblets (EWG), and eviscerated weight (EW), respectively. Six candidate genes, RNF219, SCEL, MYCBP2, ETS1, APLP2, and PRDM10 were detected. SCEL and MYCBP2 were potentially associated with these three traits, RNF219 and APLP2 were potentially associated with EWG and EW, and ETS1 and PRDM10 were only potentially associated with EWG and EW, respectively. Compared with forefathers’ research, 10 reported QTLs associated with CW were located within a 5-Mb distance near the SNPs with P value lower than 1×10^?5. This study enriched the knowledge of the genetic mechanisms of chicken carcass traits.     

<Emphasis Type="Italic">In vitro</Emphasis> study of HAX1 gene therapy by retro viral transduction as a therapeutic target in severe congenital neutropenia

Severe congenital neutropenia (SCN) is a primary immunodeficiency disease in which a number of underlying gene defects are responsible for abnormalities in neutrophil development. The HCLS1-associated protein X1 (HAX1) mutation is associated with an autosomal-recessive form of SCN. Considering the potential of gene therapy approaches for the treatment of monogenic disorders, in this study we aimed to develop retroviral vectors expressing coding sequences (CDS) to be used for the removal of the genetic blockade in deficient hematopoietic cells. Following amplification of CDS with primers containing appropriate restriction sites, HAX1 CDS was cloned into an intermediate vector using TA-cloning. The sequence was transferred into a retroviral vector, followed by retroviral packaging in Plat-A cells. To show HAX1 protein expression, HEK293T cells were exposed to 10 multiplicity of infection (MOI) of retroviral particles and HAX1 expression was confirmed in these cells, using indirect intracellular flow cytometry. This vector was applied for in vitro transduction of hematopoietic stem cell with HAX1 mutation; after 11 days, cultured cells were analyzed for CD66acde and CD177 (neutrophil surface markers) expression. Increased neutrophil production in HAX1 viral vector-expressing hematopoietic cells was observed as compared to control vector transduced cells. Hence, according to the results, this type of therapy could be considered a potential treatment protocol for the disease.     

Concave microwell plate facilitates chondrogenesis from mesenchymal stem cells

Objectives

To compare in vitro chondrogenesis from bone marrow-derived mesenchymal stem cells using concave microwell plates with those obtained using culture tubes.

Results

Pellets cultured in concave microwell plates had a significantly higher level of GAG per DNA content and greater proteoglycan content than those cultured in tubes at day 7 and 14. Three chondrogenic markers, SOX-9, COL2A1 and aggrecan, showed significantly higher expression in pellets cultured in concave microwell plates than those cultured in tubes at day 7 and 14. At day 21, there was not a significant difference in the expression of these markers. COL10A1, the typical hypertrophy marker, was significantly lower in concave microwell plates during the whole culture period. Runx-2, a marker of hypertrophy and osteogenesis, was significantly lower at day 7 in pellets cultured in concave microwell plates than those cultured in tubes.

Conclusion

Concave microwell plates provide a convenient and effective tool for the study of in vitro chondrogenesis and may replace the use of propylene culture tube.

     

RNA binding protein QKI contributes to WT1 mRNA and suppresses apoptosis in ST cells

The RNA binding protein quaking (QKI), a key member of the STAR family, as an upstream gene could involve in much process including cell proliferation, apoptosis, differentiation and so on. However, the roles of QKI in germ cell, especially in swine testis (ST) cells, was not clear currently. And apoptosis plays important roles in the growth and development. The purpose of the present study was to clarify the relationship between QKI and apoptosis in ST cells. Firstly, our results showed that pEF1α-QKI and shQKI3 have clear effects on expression levels of QKI. Secondly, we established that QKI directly binds to WT1 3′UTR by binding with QRE-1 (2046–2052 bp, ACTAAC) only. Furthermore, QKI overexpression significantly increased the expression levels of WT1 and Bcl-2. QKI also has the effect on delaying the degradation of WT1 mRNA. In addition, we verified that QKI had a significantly suppressed apoptosis in ST cells. Finally, pBI-WT1 could make up for shQKI3-induced decrease in WT1, Bcl-2 mRNA levels and suppress apoptosis in ST cells. The results demonstrated that QKI was an important regulatory factor that affects apoptosis by targeting WT1 gene.     

Blastema cells derived from New Zealand white rabbit’s pinna carry stemness properties as shown by differentiation into insulin producing,neural, and osteogenic lineages representing three embryonic germ layers

Stem cells (SCs) are known as undifferentiated cells with self-renewal and differentiation capacities. Regeneration is a phenomenon that occurs in a limited number of animals after injury, during which blastema tissue is formed. It has been hypothesized that upon injury, the dedifferentiation of surrounding tissues leads into the appearance of cells with SC characteristics. In present study, stem-like cells (SLCs) were obtained from regenerating tissue of New Zealand white rabbit’s pinna and their stemness properties were examined by their capacity to differentiate toward insulin producing cells (IPCs), as well as neural and osteogenic lineages. Differentiation was induced by culture of SLCs in defined medium, and cell fates were monitored by specific staining, RT-PCR and flow cytometry assays. Our results revealed that dithizone positive cells, which represent IPCs, and islet-like structures appeared 1 week after induction of SLCs, and this observation was confirmed by the elevated expression of Ins, Pax6 and Glut4 at mRNA level. Furthermore, SLCs were able to express neural markers as early as 1 week after retinoic acid treatment. Finally, SLCs were able to differentiate into osteogenic lineage, as confirmed by Alizarin Red S staining and RT-PCR studies. In conclusion, SLCs, which could successfully differentiate into cells derived from all three germ layers, can be considered as a valuable model to study developmental biology and regenerative medicine.