[Research progress of haematopoietic stem cells to generate immune cells and its application].

Hematopoietic stem cells (HSCs) posses the potential for highly self-renewal, proliferation and multi-lineage differentiation. HSC transplantation has long been the primary method for treating hematologic disorders and autoimmune diseases, and the ability to rebuild the immune system after transplantation is a key indicator of success. To enhance the reconstruction ability of the immune system after transplantation, current research focuses on genetic engineering and the use of HSCs modified by clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology as a source of transplant cells. This article summaries the biological characteristics, regulatory mechanism, ability to differentiate into immune cells, as well as the application and advance in the treatment of blood disorders, immune deficiencies, cancers and other related diseases, aiming to provide references for the research on relevant diseases.

The effect of 1,25-dihydroxyvitamin D3 on the Wnt signaling pathway in bovine intestinal epithelial cells is mediated by the DKK2 (dickkopf2) Wnt antagonist.

The Wnt/ß-catenin signaling pathway is aberrantly activated in most colorectal cancers. High-dose 1,25(OH)2D3 has anticancer effect by regulating Wnt signal pathway. However, it is not clear whether high-dose of 1,25(OH)2D3 have an effect on normal cells. The aim of the present study was to investigate the mechanism of high-dose 1,25(OH)2D3 on the Wnt signaling pathway in bovine intestinal epithelial cells. The potential mechanism of action was investigated after knockdown and overexpression of the Wnt pathway inhibitor, DKK2, in intestinal epithelial cells by observing the effects of 1,25(OH)2D3 on proliferation, apoptosis, pluripotency and the expression of genes related to the Wnt/ß-catenin signaling pathway. In the present study, we introduced the method of isolation and culture of primary bovine intestinal epithelial cells. After cells were treated with 50 ng/mL 1,25(OH)2D3 or DMSO for 48 h, total RNA was extracted, and six differentially expressed genes, including SERPINF1, SFRP2, SFRP4, FZD2, WISP1 and DKK2 were identified by transcriptome sequencing, which were related to Wnt signaling pathway. To further explore the mechanism of 1,25(OH)2D3 on the Wnt/ß-catenin signaling pathway, we constructed knockdown and overexpression plasmids of DKK2. After transfecting these plasmids into bovine intestinal epithelial cells, we measured the expression of DKK2 mRNA and protein through GFP expression, qRT-PCR and western blot analyses to verify the transfection efficiency. In addition, the CCK-8 assay was used to detect the cell proliferation rate after transfection. Subsequently, the transfected cells were treated with 1,25(OH)2D3 for 48 h, and the proliferation- (Ki67 and PCNA), apoptosis- (Bcl-2, p53, casp3 and casp8), pluripotency- (Bmi-1, Lrig1, KRT19 and TUFT1) and Wnt/ ß-catenin signaling pathway- related genes (LGR5, DKK2, VDR, ß- Catenin, SFRP2, WISP1 and FZD2) were detected by qRT-PCR and western blot analyses. Our results showed that the expression trend of some genes in bovine intestinal epithelial cells under high-dose 1,25(OH)2D3 was consistent with the sequencing results, including SFRP2 (P < 0.001), SFRP4 (P < 0.05), FZD2 (P < 0.01), WISP1 (P < 0.001) and DKK2 (P < 0.001). In addition, knockdown of DKK2 inhibited cell proliferation (P < 0.01), but DKK2 overexpression promoted cell proliferation (P < 0.01). Compared to the control group, 1,25(OH)2D3 promoted the expression of Wnt/ß-catenin signaling pathway-related proteins in bovine intestinal epithelium, thus maintaining intestinal homeostasis in normal intestinal epithelium. In addition, knockdown and overexpression of DKK2 indicated that 1,25(OH)2D3 weakened the inhibitory effect of DKK2 on the Wnt/ß-catenin signaling pathway. Together, these results suggest that high-dose 1,25(OH)2D3 has no killing effect on normal intestinal epithelial cells and regulates Wnt/ß-catenin signaling pathway through DKK2.

Transcriptome profiling of bovine endometrial epithelial cells induced by lipopolysaccharides in vitro.

Endometritis is an inflammation of the surface of the endometrium that does not penetrate the submucosa and can cause infertility and increase the elimination rate in cows. Endometrial epithelial cells are the first barrier of the endometrium against foreign stimuli and bacterial infection. Understanding the genetic changes in stimulated endometrial epithelial cells will help in the efforts to prevent and treat endometritis. This study investigated changes in bovine endometrial epithelial (BEEC) gene expression induced by lipopolysaccharide (LPS)-induced inflammation and compared transcriptome-wide gene changes between LPS- and phosphate-buffered saline (PBS)- treated BEECs by RNA sequencing. Compared with the PBS group, the LPS group showed 60 differentially expressed genes (DEGs) (36 upregulated, 24 downregulated). Gene Ontology enrichment analysis revealed that most enrichment occurred during CXCR chemokine receptor binding, inflammatory response, and neutrophil migration. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed DEGs mainly concentrated in cytokine-cytokine receptor interactions; IL-17, tumor necrosis factor, NOD-like receptor, chemokine, Toll-like receptor, and nuclear factor-κB signaling pathways; and the cytoplasmic DNA sensing pathway. Moreover, results revealed that cytokines SAA3 and HP increased significantly after LPS treatment. These effects of LPS on BEECs transcriptome and the molecular mechanism of endometritis provide a basis for improved clinical treatment and novel drug development.

Effect of VD3 on cell proliferation and the Wnt signaling pathway in bovine endometrial epithelial cells treated with lipopolysaccharide.

Vitamin D (VD) deficiency plays an important role in the occurrence and development of various uterine diseases. At present, most studies on the mechanism of VD in the Wnt signaling pathway focus on cancer, while there are no relevant reports on its mechanism in endometritis. This study investigated the effect of vitamin D3 (VD3) on the Wnt signaling pathway in endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS). BEECs obtained from bovine uteri were treated with VD3 (0, 50 ng/mL) and LPS (0, 10, 100 ng/mL) separately or in combination, and treated with the Wnt signaling pathway inhibitor IWR-1 to study the mechanism of action. The proliferation of BEECs was evaluated by a CCK-8 assay. qRT-PCR was used to assess the gene expression of Wnt pathway-related factors, including MYC, PCNA, LGR5, GREM1, ß-catenin, FZD7, FZD2, Wnt4 and VDR. The results showed that VD3 had no significant effect on cell proliferation (P > 0.05); LPS inhibited BEEC proliferation in a time- and dose-dependent manner, and cells treated with LPS at different concentrations for 24-48 h in combination with VD3 promoted cell proliferation to varying degrees. IWR-1 inhibited cell proliferation in a time- and concentration-dependent manner, while LPS + IWR-1 treatment also significantly promoted cell proliferation after VD3 treatment (P < 0.01). The qRT-PCR results showed that the expression of Wnt4 and PCNA genes showed different trends with different LPS concentrations for stimulation, and the expression of the MYC and GREM1 genes was only stimulated by high-dose (100 ng/mL) LPS stimulation. The expression of FZD7, LGR5, FZD2 and ß-catenin was upregulated by LPS at both concentrations. LPS + VD3 significantly downregulated the expression of the Wnt pathway-related genes MYC, PCNA, LGR5, GREM1 and ß-catenin (P < 0.001), Wnt4 and FZD2 (P < 0.01), and significantly upregulated the expression of VDR (P < 0.05). After LPS + IWR-1 treatment, the expression of the ß-catenin (P < 0.01) and LGR5 (P < 0.05) genes was significantly downregulated, while the Wnt4 (P < 0.01) and VDR (P < 0.001) genes were significantly upregulated, MYC was downregulated but without a significant difference (P > 0.05). In conclusion, VD3 treatment can mitigate the LPS-induced abnormal expression of Wnt signaling pathway genes in BEECs, showing that the Wnt pathway may be a protective pathway of VD3 against LPS-induced gene overexpression in BEECs. The results suggest that VD3 may play a regulatory role in pathways other than the Wnt signaling pathway. Whether VD3 affects the Wnt signaling pathway by affecting Wnt4 gene expression requires further study.

In-depth analysis of the relationship between bovine intestinal organoids and enteroids based on morphology and transcriptome.

Intestinal organoids and enteroids as excellent models are miniaturized and simplified for studying intestinal physiological and pathological functions, drug screening, and regenerative medicine. Recently, the application demands for organoids and enteroids in organ development and nutrition metabolism, immune and cancer research increased. But there are few comparative studies on both of them, especially in immunity and metabolism, which is also conducive to further clarifying the role of crypt stem cells and stromal cells. In our study, "natural" organoids were obtained by tissue culture from fetal bovine jejunum and enteroids were successfully isolated and cultured from organoids without supplementing exogenous factors and Matrigel. These mini-guts displayed similar features to the intestine through immunohistochemistry and transmission electron microscopy. Organoid and enteroid were systematically compared based on the transcriptome. And some of the results were verified by qRT-PCR. Our results showed KDGs (Key driver genes) (e.g., SLC13A1, HOXA7, HOXA6, HOXA5, and HOXD4) of organoids enriched in signaling pathways related to organ development and morphology and metabolism. KDGs (e.g., IL-6, PTGS2, CDH1, JUN, and EGFR) of enteroid were involved in cancer, MAPK, and immune-related signaling pathways. To the Wnt signaling pathway, highly expressed genes in organoids, including RSPO2, NOTUM, WNT6, and RSPO3, supported the homeostasis of crypt stem cells. Enteroids highly expressed CTNNB1 and WNTs. In addition, we found that organoids and enteroids carried out different functions in immunity and metabolism due to different cell compositions. Therefore, it suggested organoid is more compatible and comprehensive, and enteroid is qualified for the research of immunity and cancer.

Role of MicroRNAs in Protective Effects of Forsythoside A Against Lipopolysaccharide-Induced Inflammation in Bovine Endometrial Stromal Cells.

Bovine endometrial stromal cells (bESCs) are exposed to a complex environment of bacteria and viruses due to the rupture of epithelial cells after delivery. Inflammatory responses are elicited by the activation of host pattern recognition receptors through pathogen-related molecules such as lipopolysaccharides (LPS) on the cell membrane. Forsythoside A (FTA) is a major active constituent of Forsythia suspensa (Thunb.) Vahl. is a flowering plant widely employed as a traditional Chinese herbal medicine to treat various inflammatory diseases such as nephritis, eye swelling, scabies, ulcers, and mastitis; however, the molecular mechanisms underlying its therapeutic effects on bovine endometritis are still unclear. The aim of this study was to explore the role of miRNA and the mechanisms underlying the protective activity of FTA on the inflammation of bovine endometrial stromal cells induced by LPS. Based on previous research, we isolated and cultured bESCs in vitro and categorized them into LPS and LPS+FTA groups with three replicates. Upon reaching 80% confluence, the bESCs were treated with 0.5 µg/mL of LPS or 0.5 µg/mL of LPS + 100 µg/mL of FTA. We, then, performed high-throughput sequencing (RNA-Seq) to investigate the effects of FTA on LPS-stimulated primary bESCs and their underlying mechanisms. We identified 167 miRNAs differentially expressed in the LPS groups; 72 miRNAs were up-regulated, and 95 were down-regulated. Gene ontology enrichment analysis revealed that differentially expressed microRNA (DEGs) were most enriched during the cellular metabolic process; they were mostly located intracellularly and participated in protein, enzyme, and ion binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were most enriched in the mitogen-activated protein kinase, tumor necrosis factor, and Interleukin-17 signaling pathways. These results reveal the complex molecular mechanism involved in the FTA and provide a basis for future studies of bovine endometritis treatment with traditional Chinese medicine monomer.

Analysis of Transcriptomic Changes in Bovine Endometrial Stromal Cells Treated With Lipopolysaccharide.

Endometritis adversely affects the ability of cattle to reproduce and significantly reduces milk production. The is mainly composed of epithelial and stromal cells, and they produce the first immune response to invading pathogens. However, most of the epithelial cells are disrupted, and stromal cells are exposed to an inflammatory environment when endometritis occurs, especially postpartum. Many bacteria and toxins start attacking stromal cell due to loss of epithelium, which stimulates Toll-like receptor (TLRs) on stromal cells and causes upregulated expression of cytokines. Understanding the genome-wide characterization of bovine endometritis will be beneficial for prevention and treatment of endometritis. In this study, whole-transcriptomic gene changes in bovine endometrial stromal cells (BESCs) treated with LPS were compared with those treated with PBS (control group) and were analyzed by RNA sequencing. Compared with the control group, a total of 366 differentially expressed genes (DEGs) were identified in the LPS-induced group (234 upregulated and 132 downregulated genes), with an adjusted P < 0.05 by DESeq. Gene Ontology (GO) enrichment analysis revealed that DEGs were most enriched in interleukin-1 receptor binding, regulation of cell activation, and lymphocyte-activated interleukin-12 production. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed DEGs were most enriched in the TNF signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction, NF-κB signaling pathway, and chemokine signaling pathway. The results of this study unraveled BESCs affected with LPS transcriptome profile alterations, which may have a significant effect on treatment inflammation by comprehending molecular mechanisms and authenticating unique genes related to endometritis.

Double sperm cloning (DSC) is a promising strategy in mammalian genetic engineering and stem cell research.

Embryonic stem cells (ESCs) derived from somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) are promising tools for meeting the personalized requirements of regenerative medicine. However, some obstacles need to be overcome before clinical trials can be undertaken. First, donor cells vary, and the reprogramming procedures are diverse, so standardization is a great obstacle regarding SCNT and iPSCs. Second, somatic cells derived from a patient may carry mitochondrial DNA mutations and exhibit telomere instability with aging or disease, and SCNT-ESCs and iPSCs retain the epigenetic memory or epigenetic modification errors. Third, reprogramming efficiency has remained low. Therefore, in addition to improving their success rate, other alternatives for producing ESCs should be explored. Producing androgenetic diploid embryos could be an outstanding strategy; androgenic diploid embryos are produced through double sperm cloning (DSC), in which two capacitated sperms (XY or XX, sorted by flow cytometer) are injected into a denucleated oocyte by intracytoplasmic sperm injection (ICSI) to reconstruct embryo and derive DSC-ESCs. This process could avoid some potential issues, such as mitochondrial interference, telomere shortening, and somatic epigenetic memory, all of which accompany somatic donor cells. Oocytes are naturally activated by sperm, which is unlike the artificial activation that occurs in SCNT. The procedure is simple and practical and can be easily standardized. In addition, DSC-ESCs can overcome ethical concerns and resolve immunological response matching with sperm providers. Certainly, some challenges must be faced regarding imprinted genes, epigenetics, X chromosome inactivation, and dosage compensation. In mice, DSC-ESCs have been produced and have shown excellent differentiation ability. Therefore, the many advantages of DSC make the study of this process worthwhile for regenerative medicine and animal breeding.

Ground transport stress affects bacteria in the rumen of beef cattle: A real-time PCR analysis.

Transport stress syndrome often appears in beef cattle during ground transportation, leading to changes in their capacity to digest food due to changes in rumen microbiota. The present study aimed to analyze bacteria before and after cattle transport. Eight Xianan beef cattle were transported over 1000 km. Rumen fluid and blood were sampled before and after transport. Real-time PCR was used to quantify rumen bacteria. Cortisol and adrenocorticotrophic hormone (ACTH) were measured. Cortisol and ACTH were increased on day 1 after transportation and decreased by day 3. Cellulolytic bacteria (Fibrobacter succinogenes and Ruminococcus flavefaciens), Ruminococcus amylophilus and Prevotella albensis were increased at 6 h and declined by 15 days after transport. There was a significant reduction in Succinivibrio dextrinosolvens, Prevotella bryantii, Prevotella ruminicola and Anaerovibrio lipolytica after transport. Rumen concentration of acetic acid increased after transport, while rumen pH and concentrations of propionic and butyric acids were decreased. Body weight decreased by 3 days and increased by 15 days after transportation. Using real-time PCR analysis, we detected changes in bacteria in the rumen of beef cattle after transport, which might affect the growth of cattle after transport.

[Determination of Arsenic in Food Package Aluminum by Ultrasound Assisted Solid Phase Extraction/ICP-AES].

Determination of arsenic in pure aluminum by inductively coupled plasma atomic emission spectrometry was interfered by aluminum matrix. The experiment showed that when the mass concentration of Al was greater than or equal to 5 000 times the As in the test solution, the measurement error was greater than 5%. In order to eliminate the interference, strong acid cation exchange fiber (SACEF) was used as solid phase extraction agent to adsorb Al(3+). The extraction conditions included amount of SACEF, extraction time, temperature and pH were investigated. The optimal extraction conditions were that 0.9000 g SACEF was used to extract the aluminum from the sample solution of pH 2.0 at 55 °C for 5 min with the ultrasonic assist, and in this case, the arsenic in the form of arsenic acid was not extracted and left in the solution for the determination. The results showed that after treating 10. 00 mL test solution containing 1.00 µg arsenic and 20.0 mg aluminum, arsenic did not lose. The mass concentration of residual aluminum in the raffinate was about 2,000 times the As, which had not interfered the determination of arsenic. The detection limit (3 s) was 0.027 µg · mL(-1) and quantification limit (10 s) was 0.0091 µg · mL(-1). The proposed method was successfully applied to the separation and determination of arsenic in the synthetic samples, the aluminum cans and the barbecue aluminum foil. Recovery was in the range of 98.3%-105% and RSD (n = 3) was in the range of 0.1%-4.3%. The results showed that the content of arsenic in the aluminum cans and the aluminum barbecue foil was below the limited value of national standard (GB/T 3190-2008).

Cloning, expression, and characterization of para-aminobenzoic acid (PABA) synthase from Agaricus bisporus 02, a thermotolerant mushroom strain.

The pabS gene of Agaricus bisporus 02 encoding a putative PABA synthase was cloned, and then the recombinant protein was expressed in Escherichia coli BL21 under the control of the T7 promoter. The enzyme with an N-terminal GST tag or His tag, designated GST-AbADCS or His-AbADCS, was purified with glutathione Sepharose 4B or Ni Sepharose 6 Fast Flow. The enzyme was an aminodeoxychorismate synthase, and it was necessary to add with an aminodeoxychorismate lyase for synthesizing PABA. AbADCS has maximum activity at a temperature of approximately 25°C and pH 8.0. Magnesium or manganese ions were necessary for the enzymatic activity. The Michaelis-Menten constant for chorismate was 0.12 mM, and 2.55 mM for glutamine. H2O2 did distinct damage on the activity of the enzyme, which could be slightly recovered by Hsp20. Sulfydryl reagents could remarkably promote its activity, suggesting that cysteine residues are essential for catalytic function.

Hypovitaminosis A coupled to secondary bacterial infection in beef cattle.

BACKGROUND: Vitamin A is essential for normal growth, development, reproduction, cell proliferation, cell differentiation, immune function and vision. Hypovitaminosis A can lead to a series of pathological damage in animals. This report describes the case of hypovitaminosis A associated with secondary complications in calves. CASE PRESENTATION: From February to March in 2011, 2-and 3-month old beef calves presented with decreased eyesight, apparent blindness and persistent diarrhea occurred in a cattle farm of Hubei province, China. Based on history inspection and clinical observation, we made a tentative diagnosis of hypovitaminosis A. The disease was confirmed as a congenital vitamin A deficiency by determination of the concentrations of vitamin A in serum and feed samples. Furthermore, pathological and microbiological examination showed that the disease was associated with pathogenic Escherichia coli (E. coli) infection and mucosal barriers damage in intestines. The corresponding treatments were taken immediately, and the disease was finally under control for a month. CONCLUSIONS: To our knowledge, this is the first report of hypovitaminosis A coupled to secondary infection of E. coli in beef cattle, advancing our knowledge of how vitamin A affects infection and immunity in animals. This study could also be contributed to scientific diagnosis and treatments of complex hypovitaminosis A in cattle.

Phenotypic and molecular characterization of TEM-116 extended-spectrum beta-lactamase produced by a Shigella flexneri clinical isolate from chickens.

Extended-spectrum beta-lactamases (ESBLs) produced by a clinical isolate of Shigella flexneri from chickens were detected with confirmatory phenotypic tests of the Clinical and Laboratory Standards Institute, and minimum inhibitory concentrations of several antibacterial drugs against the isolate were determined by the twofold dilution method. The genotype and subtype of the ESBL-producing S. flexneri isolate were identified by PCR amplifying of ESBL genes and DNA sequencing analysis. The results revealed that the isolate was able to produce ESBLs. They were resistant to third-generation cephalosporins such as ceftiofur and ceftriaxone and showed characteristics of multidrug resistance. The ESBL gene from the S. flexneri isolate was of the TEM type. Sequence analysis indicated that the TEM-type gene had 99.1% and 99.2% identity to TEM-1D ESBL and TEM-1 beta-lactamase, respectively, at the nucleotide level. The amino acid sequence inferred from the TEM-type gene revealed three substitutions compared with the TEM-1 and TEM-1D enzymes: Ser51Gly, Val82Ila and Ala182Val. When it was compared with TEM-116 (99.8% identity), there were only two mutations (A(151)G and T(403)C) in the TEM-type gene, resulting in the substitution of Ser to Gly at position 51 in the amino acid sequence. The TEM type was a TEM-116 derivative.