The main aim of this study was to investigate the prevalence of intramammary infection (IMI) in early-lactation of primiparous cows using milk recording cow composite somatic cell count (CSCC) categories (combining th...The main aim of this study was to investigate the prevalence of intramammary infection (IMI) in early-lactation of primiparous cows using milk recording cow composite somatic cell count (CSCC) categories (combining the first 2 milk recording results after calving). Another aim was to evaluate the milk urea (MU) content as a potential supplementary indicator to SCC or CSCC for the identification of IMI in primiparous cows after calving. This retrospective observational study was conducted on records of test-day of primiparous cows over a period of 6 years (January 2016 to December 2021. The SCC data for 158 Holstein Friesian primiparous cows, with their first milk recording 5 to 35 days after calving and their second milk recording 28 to 56 days in milk (DIM), were identified. Each primiparous cow was assigned a CSCC category (low-low, low-high, high-low or high-high) based on the CSCC at the first 2 milking recordings using the following cut-offs: ≤150,000 cells/ml (low), >150,000 cells/ml (high). The association between CSCC categories and MV content was analyzed using correlation models. At the first milk recording, a proportion of 63.29% was in the low SCC category, and the rest (36.71%) was in the high SCC category. At the second milk recording, a proportion of primiparous cows in CSCC categories was 59.49%, 3.80%, 27.85% and 8.86% in low-low, low-high, high-low and high-high, respectively. At the second milk recording, a proportion of 12.66% of primiparous cows was in the high CSCC category and a proportion of 87.34% of primiparous cows was in the low CSCC category, indicating a poor and a good udder health, respectively. The association of SCC with MU content in low and in high SCC categories at the first milk recording was positive and moderate (+0.49) and negative and strong (-0.97), respectively. The association of CSCC categories with MU contents at the second milk recording was inconclusive. We concluded that CSCC categories may be a useful tool for identifying success and problems regarding the udder health of primiparous cows in early lactation.展开更多
Background Subclinical intramammary infection(IMI)represents a significant problem in maintaining dairy cows’health.Disease severity and extent depend on the interaction between the causative agent,environment,and ho...Background Subclinical intramammary infection(IMI)represents a significant problem in maintaining dairy cows’health.Disease severity and extent depend on the interaction between the causative agent,environment,and host.To investigate the molecular mechanisms behind the host immune response,we used RNA-Seq for the milk somatic cells(SC)transcriptome profiling in healthy cows(n=9),and cows naturally affected by subclinical IMI from Proto-theca spp.(n=11)and Streptococcus agalactiae(S.agalactiae;n=11).Data Integration Analysis for Biomarker discov-ery using Latent Components(DIABLO)was used to integrate transcriptomic data and host phenotypic traits related to milk composition,SC composition,and udder health to identify hub variables for subclinical IMI detection.Results A total of 1,682 and 2,427 differentially expressed genes(DEGs)were identified when comparing Prototheca spp.and S.agalactiae to healthy animals,respectively.Pathogen-specific pathway analyses evidenced that Proto-theca’s infection upregulated antigen processing and lymphocyte proliferation pathways while S.agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle,and carbohydrate and lipid metabolism.The integrative analysis of commonly shared DEGs between the two pathogens(n=681)referred to the core-mastitis response genes,and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells(r2=0.72),followed by the udder health(r2=0.64)and milk quality parameters(r2=0.64).Variables with r≥0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cyto-hubba plug-in.The genes in common between DIABLO and cytohubba(n=10)were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals(sensitivity>0.89,specificity>0.81,accuracy>0.87,and precision>0.69).Among these genes,CIITA could play a key role in regulating the animals’response to subclinical IMI.Conclusions Despite some differences in the enriched pathways,the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response.The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.展开更多
Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other...Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.展开更多
PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked ...PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein (eGFP) and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer (SCNT), the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.展开更多
In the present study authors present some aspects regarding udder health as well as the udder teat morphometry.The udder health was defined on the basis of somatic cell count(SCC)in milk.The morphometry parameters inc...In the present study authors present some aspects regarding udder health as well as the udder teat morphometry.The udder health was defined on the basis of somatic cell count(SCC)in milk.The morphometry parameters included:distance from rear teat-tip to the floor,distance from fore teat-tip to the floor,udder depth,udder length,udder width,udder volume,fore teat length,rear teat length,fore teat diameter,and rear teat diameter.The present investigation involved 92 Holstein Friesian cows,from an experimental herd.Considering particular structural elements of a cow udder,three location groups were distinguished:1st,2nd,and 3rd(L_(1),L_(2) and L_(3) respectively).From the study result,primiparous cows(L_(1) group)were characterized by a relatively low SCC in milk,as compared to multiparous cows(L_(2) group or L_(3) group).The cows in the 3rd lactation in relation to the first lactation cows,head SCC increased over 3 times,which indicates the increase of the chance of the cows to be susceptible to intramammary infections.All data showed a gradual increase in depth length,width and volume of the udder as the number of parity increases.The differences observed in udder morphometry in different parities were found significant(p<0.05)to highly significant(p<0.01).The values of the coefficients of correlation for the analyzed udder and teat conformation traits and SCC in milk,were statistically varied and ranged from-0.32 to+0.28.Accordingly,the udder and teat morphometry characteristics such as distance from rear are fore teat-tip to the floor,udder depth,volume,teat diameter,can be said to have some degree of association with the udder health in Holstein Friesian cows evaluated in this study.Hence their inclusion in breeding program as indicator traits may help reduce the incidence of intramammary infections.展开更多
The main objective of this study was to analyze the effect of bacterial content (CFU) and somatic cell count (SCC) in milk quality in sheep (Ovis aries) farms according to the existing standards for fresh milk quality...The main objective of this study was to analyze the effect of bacterial content (CFU) and somatic cell count (SCC) in milk quality in sheep (Ovis aries) farms according to the existing standards for fresh milk quality in Kosovo. A total of 2256 milk test day records from 379 Bardhoka (BAR), Sharri sheep (SHA), Kosovo sheep (KOS) and Balusha (BAL) ewes in eight herds across the country were collected and analyzed through a period April-October 2010. The general linear model and Duncan’s test were used to analyze the effect of different variables on presence of CFU and SCC in fresh milk. The effect of all variables was considered as a fixed. The overall results show that farm (P 0422), respectively, had a significant effect on presence of SCC. For CFU farm (P 0422) and breed (P 1211.17 to 6425.76 CFU/mL and 846.07 to 2043.15 SCC/mL milk, respectively. Although the rate of fresh milk contamination with SCC tends to be relatively low compare with CFU, by all means both variables should not be underestimated bearing in mind that a high rate of them on fresh milk are negatively correlated with farmer’s profit, consumer food safety and overall animal health.展开更多
The basic goal of this research was to determine the impact of the presence of bacterial (CFU) and somatic cells count content (SCC) in quality of fresh milk in some small cattle farms in Kosovo. The survey was based ...The basic goal of this research was to determine the impact of the presence of bacterial (CFU) and somatic cells count content (SCC) in quality of fresh milk in some small cattle farms in Kosovo. The survey was based on existing standards for milk quality in Kosovo placed under administrative guidance MA-no. 20/2006. The study was based on fresh milk analysis of 150 farms performed during the period September-December 2012, which was obtained in 9 different localities (collection points) of the Kosovo. Our study reveals that CFU and SCC in fresh milk were significantly affected by a number of factors, as: sampling period (repetition), locality, breed, and time of sampling (evening or/and morning). According to the results for CFU and SCC, there were big differences between the farms (milk collection points) included in the study (P < 0.0403) and (P < 0.0293). The results show that small size breed like Busha and its crosses tend to be less exposed to SCC/mL in milk (72.840) and (293.592), compared to Black Holstein (613.462), Simmental (521.519) and Brown Swiss (418.44). Milk produced in evening tended to be of better quality (259.854 CFU/mL) compared to the one from morning milking (576.689 CFU/mL). Fresh milk quality analyzed in the third repetition was better for about 33.3% compared with the repletion first. For CFU and SCC, the analyses show that about 74.7% and 64.7% of milk produced belongs to extra quality, while lower quality of milk of category three is 12.0% and 23.3%, respectively. Considering that about 85% of milk produced in Kosovo comes from small-scale dairy farms, the current study sets out that small-scale milk production system cannot be neglected by interest parties in dairy sector and needs permanent following up and improvement.展开更多
Chinese scientists have succeeded in cloning a colony of cattle from fully differentiated somatic cells. The news was announced jointly by the Chinese Academy of Sciences (CAS), National Natural Science Foundation... Chinese scientists have succeeded in cloning a colony of cattle from fully differentiated somatic cells. The news was announced jointly by the Chinese Academy of Sciences (CAS), National Natural Science Foundation of China (NSFC) and the government of Shandong Province at a press conference held on March 7, 2002.……展开更多
The objective of this study was to analyze the relationship of somatic cell count(SCC)with milk yield,fat and protein percentage,fat and protein yield using analysis of variance and correlation analysis in Chinese Hol...The objective of this study was to analyze the relationship of somatic cell count(SCC)with milk yield,fat and protein percentage,fat and protein yield using analysis of variance and correlation analysis in Chinese Holstein population.The 10 524 test-day records of 568 Chinese Holstein Cattle were obtained from 2 commercial herds in Xi'an region of China during February 2002 to March 2009.Milk yield,fat percentage,fat and protein yield initially increased and then dropped down with parity,whereas protein percentage decreased and SCC increased.Analysis of variance showed highly significant effects of different subclasses SCC on milk yield and composition(P < 0.01).Compared with milk yield with SCC 200 000 cells mL-1,milk yield losses with SCC of 200 000-500 000 cells mL-1,501 000-1 000 000 cells mL-1,1 000 000 cells mL-1 were 0.387,0.961 and 2.351 kg,respectively.The highly significant negative correlation coefficient between somatic cell score(SCS)and milk and protein yield,milk yield and fat and protein percentage,protein percentage and fat yield were-0.084,-0.037,-0.061,-0.168,and-0.088,respectively(P < 0.01).The highly significant positive correlation coefficients between SCS and fat yield and fat and protein percentage,milk yield and fat and protein yield,fat percentage and protein percentage and fat yield,protein yield and protein percentage and fat yield were 0.041,0.177,0.105,0.771,0.865,0.122,0.568,0.318,and 0.695,respectively(P < 0.01).There was no significant relationship between fat percentage and protein yield(P > 0.05).The results of the present study first time provide the relevant base-line data for assessing milk production at Xi'an region of China.展开更多
This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (St...This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.展开更多
Based on the coherent feedforward transcription regulation loops in somatic cell reprogramming process, a stochastic kinetic model is proposed to study the intrinsic fluctuations in the somatic cell reprogramming. The...Based on the coherent feedforward transcription regulation loops in somatic cell reprogramming process, a stochastic kinetic model is proposed to study the intrinsic fluctuations in the somatic cell reprogramming. The Fano factor formulas of key genes expression level in the coherent feedforward transcription regulation loops are derived by using of Langevin theory. It is found that the internal fluctuations of gene expression levels mainly depend on itself activation ratio and degradation ratio. When the self-activation ratio(or self-degradation ratio) is increased, the Fano factor increases reaches a maximum and then decreases. The susceptibility is used to measure the sensitivity of steady-state response to the variation in systemic parameters. It is found that with the increase of the self-activation ratio(or self-degradation ratio), the susceptibility of steady-state increases at first, it reaches a maximum, and it then decreases. The magnitude of the maximum is increased with the increase of activated ratio by the upstream transcription factor.展开更多
Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover,...Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.展开更多
Cell transplantation is an attractive potential therapy for heart diseases. For example, myocardial infarction(MI) is a leading cause of mortality in many countries. Numerous medical interventions have been developed ...Cell transplantation is an attractive potential therapy for heart diseases. For example, myocardial infarction(MI) is a leading cause of mortality in many countries. Numerous medical interventions have been developed to stabilize patients with MI and, although this has increased survival rates, there is currently no clinically approved method to reverse the loss of cardiac muscle cells(cardiomyocytes) that accompanies this disease. Cell transplantation has been proposed as a method to replace cardiomyocytes, but a safe and reliable source of cardiogenic cells is required. An ideal source would be the patients' own somatic tissue cells, which could be converted into cardiogenic cells and transplanted into the site of MI. However, these are difficult to produce in large quantities and standardized protocols to produce cardiac cells would be advantageous for the research community. To achieve these research goals, small molecules represent attractive tools to control cell behavior. In this editorial, we introduce the use of small molecules in stem cell research and summarize their application to the induction of cardiogenesis in noncardiac cells. Exciting new developments in this field are discussed, which we hope will encourage cardiac stem cell biologists to further consider employing small molecules in their culture protocols.展开更多
The porcine microsatellite SW943 was regionally localized on 12p11-(2/3p13) by the two methods: the Primed in situ (PRINS) labelling on the pachytene bivalents of pigs using the Dig-11-dUTP as the report molecule and ...The porcine microsatellite SW943 was regionally localized on 12p11-(2/3p13) by the two methods: the Primed in situ (PRINS) labelling on the pachytene bivalents of pigs using the Dig-11-dUTP as the report molecule and pig × rodent Somatic Cell Hybrid PaneI(SCHP) which contains 27 cell lines through PCR amplification. Advantages and disadvantages of the two methods for physical mapping of microsatellites were also discussed.展开更多
In the field of regenerative medicine,generating numerous transplantable functional cells in the laboratory setting on a large scale is a major challenge.However,the in vitro maintenance and expansion of terminally di...In the field of regenerative medicine,generating numerous transplantable functional cells in the laboratory setting on a large scale is a major challenge.However,the in vitro maintenance and expansion of terminally differentiated cells are challenging because of the lack of specific environmental and intercellular signal stimulations,markedly hindering their therapeutic application.Remarkably,the generation of stem/progenitor cells or functional cells with effective proliferative potential is markedly in demand for disease modeling,cell-based transplantation,and drug discovery.Despite the potent genetic manipulation of transcription factors,integration-free chemically defined approaches for the conversion of somatic cell fate have garnered considerable attention in recent years.This review aims to summarize the progress thus far and discuss the advantages,limitations,and challenges of the impact of full chemicals on the stepwise reprogramming of pluripotency,direct lineage conversion,and direct lineage expansion on somatic cells.Owing to the current chemical-mediated induction,reprogrammed pluripotent stem cells with reproducibility difficulties,and direct lineage converted cells with marked functional deficiency,it is imperative to generate the desired cell types directly by chemically inducing their potent proliferation ability through a lineagecommitted progenitor state,while upholding the maturation and engraftment capacity posttransplantation in vivo.Together with the comprehensive understanding of the mechanism of chemical drives,as well as the elucidation of specificity and commonalities,the precise manipulation of the expansion for diverse functional cell types could broaden the available cell sources and enhance the cellular function for clinical application in future.展开更多
In animals the prion disease includes sheep and goat scrapie and the bovine spongiform encephalopathy (BSE). While several polymorphisms of the prion (PRNP) gene have been identified in sheep and some of them have bee...In animals the prion disease includes sheep and goat scrapie and the bovine spongiform encephalopathy (BSE). While several polymorphisms of the prion (PRNP) gene have been identified in sheep and some of them have been associated with susceptibility to scrapie, few mutations are reported in cattle and no correlation with BSE have been demonstrated. Genetic screening for mutants in the PRNP gene of 21 BSE positive animals by direct sequencing of the amplified gene, using DNA extracted from brain as template, confirmed that only few polymorphisms are present. However DNA molecules cloned and sequenced from the population of fragments considering a total of 90 clones from 9 BSE positive and 70 clones from 7 BSE negative animals, gave a highly significant differences in the frequency of mutations (p = 0.01). The high frequency and type of variants found cannot be explained only with misincorporation error of the Taq polymerase. Interestingly one of the mutations found in the BSE positive animals (F209S) corresponds to a mutant that causes a familiar form of prion disease in humans (F198S). These data can be explained with the presence of somatic mutations modifying the PRNP gene in single brain cells.展开更多
The transcription activator-like effector nuclease(TALEN) technique combined with the somatic cell nuclear transfer(SCNT) method has been successfully applied for creating genetically modified pigs. However, methods f...The transcription activator-like effector nuclease(TALEN) technique combined with the somatic cell nuclear transfer(SCNT) method has been successfully applied for creating genetically modified pigs. However, methods for isolating cells with biallelic indels requires further improvement because of the relatively low enrichment efficiency of mutated somatic cells. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modified pigs. In this study, an efficient method to increase the enrichment efficiency of TALEN-mediated biallelic knockout(KO) cells was established, and corresponding genetically modified pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase(GGTA1) gene locus. TALEN m RNA was transfected into the ear fibroblasts followed by the enrichment of α-Gal null cells of minipigs using isolectin B4(IB4) lectin and m agnetic beads. A total of 115 cell c olonies were formed and validated to be GGTA1 KO cells by sequencing and 10 biallelic KO cell colonies were used as nuclear donors for SCNT. Thirty GGTA1 biallelic KO piglets were successfully delivered and grew normally. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine t he fertility and heredity characteristics of TALEN-modified pigs, 10 mature founders w ere mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing genetically modified pig lines with expected genotypes for agricultural breeding and biomedical application.展开更多
Cancer tissues contain cancer stem cells (CSCs), which play important roles in cancer metastasis. However, the mechanisms through which cancer cells dedifferentiate into stem cells have not yet been elucidated. In thi...Cancer tissues contain cancer stem cells (CSCs), which play important roles in cancer metastasis. However, the mechanisms through which cancer cells dedifferentiate into stem cells have not yet been elucidated. In this study, the effects of high concentrations of polyamines produced in cancer cells on dedifferentiation were examined. The results showed that when normal human fibroblasts were cultured with high concentrations of spermine, the obtained polyamine-induced cells expressed alkaline phosphatase and marker proteins of pluripotent stem cells, although apoptosis occurred in most cells. In contrast, another polyamine-induced stem (PIS) cell line (Spe-2 PIS cells), obtained by culture in medium containing Rock, p53, and Bax inhibitors plus spermine, did not show signs of apoptosis. These Spe-2 PIS cells expressed marker proteins of pluripotent stem cells and differentiated into cardiomyocytes, brown adipocytes, and nerve cells. These results suggest that a high concentration of spermine, which often induces apoptosis in normal cells, has the capacity to dedifferentiate somatic cells into pluripotent stem cells and may be associated with the dedifferentiation of cancer cells, which continuously produce high concentrations of spermine. Moreover, the procedure to obtain Spe-2 PIS cells, which is simple and efficient, may have potential applications in regenerative medicine.展开更多
Direct reprogramming of somatic cells into neurons or neural stem cells is one of the most important frontier fields in current neuroscience research. Without undergoing the pluripotency stage, induced neurons or indu...Direct reprogramming of somatic cells into neurons or neural stem cells is one of the most important frontier fields in current neuroscience research. Without undergoing the pluripotency stage, induced neurons or induced neural stem cells are a safer and timelier manner resource in comparison to those derived from induced pluripotent stem cells. In this prospective, we review the recent advances in generation of induced neurons and induced neural stem cells in vitro and in vivo and their potential treatments of neurological disorders.展开更多
文摘The main aim of this study was to investigate the prevalence of intramammary infection (IMI) in early-lactation of primiparous cows using milk recording cow composite somatic cell count (CSCC) categories (combining the first 2 milk recording results after calving). Another aim was to evaluate the milk urea (MU) content as a potential supplementary indicator to SCC or CSCC for the identification of IMI in primiparous cows after calving. This retrospective observational study was conducted on records of test-day of primiparous cows over a period of 6 years (January 2016 to December 2021. The SCC data for 158 Holstein Friesian primiparous cows, with their first milk recording 5 to 35 days after calving and their second milk recording 28 to 56 days in milk (DIM), were identified. Each primiparous cow was assigned a CSCC category (low-low, low-high, high-low or high-high) based on the CSCC at the first 2 milking recordings using the following cut-offs: ≤150,000 cells/ml (low), >150,000 cells/ml (high). The association between CSCC categories and MV content was analyzed using correlation models. At the first milk recording, a proportion of 63.29% was in the low SCC category, and the rest (36.71%) was in the high SCC category. At the second milk recording, a proportion of primiparous cows in CSCC categories was 59.49%, 3.80%, 27.85% and 8.86% in low-low, low-high, high-low and high-high, respectively. At the second milk recording, a proportion of 12.66% of primiparous cows was in the high CSCC category and a proportion of 87.34% of primiparous cows was in the low CSCC category, indicating a poor and a good udder health, respectively. The association of SCC with MU content in low and in high SCC categories at the first milk recording was positive and moderate (+0.49) and negative and strong (-0.97), respectively. The association of CSCC categories with MU contents at the second milk recording was inconclusive. We concluded that CSCC categories may be a useful tool for identifying success and problems regarding the udder health of primiparous cows in early lactation.
基金the Ministero delle politiche agricole alimentari,forestali e del turismo(MIPAAF),Rome,Italy.Moreover,the study was conducted within the Agritech National Research Center and received funding from the European Union Next-GenerationEU(PIANO NAZIONALE DI RIPRESA E RESILIENZA(PNRR)-MISSIONE 4 COMPONENTE 2,INVESTIMENTO 1.4-D.D.103217/06/2022,CN00000022).
文摘Background Subclinical intramammary infection(IMI)represents a significant problem in maintaining dairy cows’health.Disease severity and extent depend on the interaction between the causative agent,environment,and host.To investigate the molecular mechanisms behind the host immune response,we used RNA-Seq for the milk somatic cells(SC)transcriptome profiling in healthy cows(n=9),and cows naturally affected by subclinical IMI from Proto-theca spp.(n=11)and Streptococcus agalactiae(S.agalactiae;n=11).Data Integration Analysis for Biomarker discov-ery using Latent Components(DIABLO)was used to integrate transcriptomic data and host phenotypic traits related to milk composition,SC composition,and udder health to identify hub variables for subclinical IMI detection.Results A total of 1,682 and 2,427 differentially expressed genes(DEGs)were identified when comparing Prototheca spp.and S.agalactiae to healthy animals,respectively.Pathogen-specific pathway analyses evidenced that Proto-theca’s infection upregulated antigen processing and lymphocyte proliferation pathways while S.agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle,and carbohydrate and lipid metabolism.The integrative analysis of commonly shared DEGs between the two pathogens(n=681)referred to the core-mastitis response genes,and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells(r2=0.72),followed by the udder health(r2=0.64)and milk quality parameters(r2=0.64).Variables with r≥0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cyto-hubba plug-in.The genes in common between DIABLO and cytohubba(n=10)were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals(sensitivity>0.89,specificity>0.81,accuracy>0.87,and precision>0.69).Among these genes,CIITA could play a key role in regulating the animals’response to subclinical IMI.Conclusions Despite some differences in the enriched pathways,the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response.The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.
基金Key Research and Development Project of Hainan Province(ZDYF2021XDNY174)Science and Technology Major Project of Inner Mongolia(2021ZD0023–1)National Transgenic Key Project of the Ministry of Agriculture of China(2018ZX0800801B)。
文摘Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.
基金Supported by the National Projects of Genetic Modified Organism Breeding Technology (2008ZX08006-002)the State Transgenic Research Programme of China (2008ZX08006-002)
文摘PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein (eGFP) and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer (SCNT), the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.
文摘In the present study authors present some aspects regarding udder health as well as the udder teat morphometry.The udder health was defined on the basis of somatic cell count(SCC)in milk.The morphometry parameters included:distance from rear teat-tip to the floor,distance from fore teat-tip to the floor,udder depth,udder length,udder width,udder volume,fore teat length,rear teat length,fore teat diameter,and rear teat diameter.The present investigation involved 92 Holstein Friesian cows,from an experimental herd.Considering particular structural elements of a cow udder,three location groups were distinguished:1st,2nd,and 3rd(L_(1),L_(2) and L_(3) respectively).From the study result,primiparous cows(L_(1) group)were characterized by a relatively low SCC in milk,as compared to multiparous cows(L_(2) group or L_(3) group).The cows in the 3rd lactation in relation to the first lactation cows,head SCC increased over 3 times,which indicates the increase of the chance of the cows to be susceptible to intramammary infections.All data showed a gradual increase in depth length,width and volume of the udder as the number of parity increases.The differences observed in udder morphometry in different parities were found significant(p<0.05)to highly significant(p<0.01).The values of the coefficients of correlation for the analyzed udder and teat conformation traits and SCC in milk,were statistically varied and ranged from-0.32 to+0.28.Accordingly,the udder and teat morphometry characteristics such as distance from rear are fore teat-tip to the floor,udder depth,volume,teat diameter,can be said to have some degree of association with the udder health in Holstein Friesian cows evaluated in this study.Hence their inclusion in breeding program as indicator traits may help reduce the incidence of intramammary infections.
文摘The main objective of this study was to analyze the effect of bacterial content (CFU) and somatic cell count (SCC) in milk quality in sheep (Ovis aries) farms according to the existing standards for fresh milk quality in Kosovo. A total of 2256 milk test day records from 379 Bardhoka (BAR), Sharri sheep (SHA), Kosovo sheep (KOS) and Balusha (BAL) ewes in eight herds across the country were collected and analyzed through a period April-October 2010. The general linear model and Duncan’s test were used to analyze the effect of different variables on presence of CFU and SCC in fresh milk. The effect of all variables was considered as a fixed. The overall results show that farm (P 0422), respectively, had a significant effect on presence of SCC. For CFU farm (P 0422) and breed (P 1211.17 to 6425.76 CFU/mL and 846.07 to 2043.15 SCC/mL milk, respectively. Although the rate of fresh milk contamination with SCC tends to be relatively low compare with CFU, by all means both variables should not be underestimated bearing in mind that a high rate of them on fresh milk are negatively correlated with farmer’s profit, consumer food safety and overall animal health.
基金the support for the Ministry of Education Science and Technology of Republic of Kos-ovo for granting the study through the small grant project“Study of Autochthon Busha Cattle in Kosovo”.
文摘The basic goal of this research was to determine the impact of the presence of bacterial (CFU) and somatic cells count content (SCC) in quality of fresh milk in some small cattle farms in Kosovo. The survey was based on existing standards for milk quality in Kosovo placed under administrative guidance MA-no. 20/2006. The study was based on fresh milk analysis of 150 farms performed during the period September-December 2012, which was obtained in 9 different localities (collection points) of the Kosovo. Our study reveals that CFU and SCC in fresh milk were significantly affected by a number of factors, as: sampling period (repetition), locality, breed, and time of sampling (evening or/and morning). According to the results for CFU and SCC, there were big differences between the farms (milk collection points) included in the study (P < 0.0403) and (P < 0.0293). The results show that small size breed like Busha and its crosses tend to be less exposed to SCC/mL in milk (72.840) and (293.592), compared to Black Holstein (613.462), Simmental (521.519) and Brown Swiss (418.44). Milk produced in evening tended to be of better quality (259.854 CFU/mL) compared to the one from morning milking (576.689 CFU/mL). Fresh milk quality analyzed in the third repetition was better for about 33.3% compared with the repletion first. For CFU and SCC, the analyses show that about 74.7% and 64.7% of milk produced belongs to extra quality, while lower quality of milk of category three is 12.0% and 23.3%, respectively. Considering that about 85% of milk produced in Kosovo comes from small-scale dairy farms, the current study sets out that small-scale milk production system cannot be neglected by interest parties in dairy sector and needs permanent following up and improvement.
文摘 Chinese scientists have succeeded in cloning a colony of cattle from fully differentiated somatic cells. The news was announced jointly by the Chinese Academy of Sciences (CAS), National Natural Science Foundation of China (NSFC) and the government of Shandong Province at a press conference held on March 7, 2002.……
基金supported by the National 863 Program of China(2008AA10Z144)"13115"Sci-Tech Innovation Program of Shaanxi Province(2008ZDKG-11)
文摘The objective of this study was to analyze the relationship of somatic cell count(SCC)with milk yield,fat and protein percentage,fat and protein yield using analysis of variance and correlation analysis in Chinese Holstein population.The 10 524 test-day records of 568 Chinese Holstein Cattle were obtained from 2 commercial herds in Xi'an region of China during February 2002 to March 2009.Milk yield,fat percentage,fat and protein yield initially increased and then dropped down with parity,whereas protein percentage decreased and SCC increased.Analysis of variance showed highly significant effects of different subclasses SCC on milk yield and composition(P < 0.01).Compared with milk yield with SCC 200 000 cells mL-1,milk yield losses with SCC of 200 000-500 000 cells mL-1,501 000-1 000 000 cells mL-1,1 000 000 cells mL-1 were 0.387,0.961 and 2.351 kg,respectively.The highly significant negative correlation coefficient between somatic cell score(SCS)and milk and protein yield,milk yield and fat and protein percentage,protein percentage and fat yield were-0.084,-0.037,-0.061,-0.168,and-0.088,respectively(P < 0.01).The highly significant positive correlation coefficients between SCS and fat yield and fat and protein percentage,milk yield and fat and protein yield,fat percentage and protein percentage and fat yield,protein yield and protein percentage and fat yield were 0.041,0.177,0.105,0.771,0.865,0.122,0.568,0.318,and 0.695,respectively(P < 0.01).There was no significant relationship between fat percentage and protein yield(P > 0.05).The results of the present study first time provide the relevant base-line data for assessing milk production at Xi'an region of China.
基金Project (No. R-174-000-065-112/303) supported by the NationalUniversity of Singapore
文摘This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.11775091 and 11474117)
文摘Based on the coherent feedforward transcription regulation loops in somatic cell reprogramming process, a stochastic kinetic model is proposed to study the intrinsic fluctuations in the somatic cell reprogramming. The Fano factor formulas of key genes expression level in the coherent feedforward transcription regulation loops are derived by using of Langevin theory. It is found that the internal fluctuations of gene expression levels mainly depend on itself activation ratio and degradation ratio. When the self-activation ratio(or self-degradation ratio) is increased, the Fano factor increases reaches a maximum and then decreases. The susceptibility is used to measure the sensitivity of steady-state response to the variation in systemic parameters. It is found that with the increase of the self-activation ratio(or self-degradation ratio), the susceptibility of steady-state increases at first, it reaches a maximum, and it then decreases. The magnitude of the maximum is increased with the increase of activated ratio by the upstream transcription factor.
基金supported by the National Major Special Project on New Varieties Cultivation for Transgenic Organisms,China(2014ZX08006-005 and 2014ZX0800950B)
文摘Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.
基金Supported by the National Research Foundation(NRF)funded by the Korean Government,(MEST Basic Science Research Program grant,No.NRF-2012R1A1B5000462 to D.-W.J+1 种基金the Korean Health Technology R&D Project,Ministry of Health and Welfare,South Korea,No.HI12C0275the Bioimaging Center,Gwangju Institute of Science and Technology
文摘Cell transplantation is an attractive potential therapy for heart diseases. For example, myocardial infarction(MI) is a leading cause of mortality in many countries. Numerous medical interventions have been developed to stabilize patients with MI and, although this has increased survival rates, there is currently no clinically approved method to reverse the loss of cardiac muscle cells(cardiomyocytes) that accompanies this disease. Cell transplantation has been proposed as a method to replace cardiomyocytes, but a safe and reliable source of cardiogenic cells is required. An ideal source would be the patients' own somatic tissue cells, which could be converted into cardiogenic cells and transplanted into the site of MI. However, these are difficult to produce in large quantities and standardized protocols to produce cardiac cells would be advantageous for the research community. To achieve these research goals, small molecules represent attractive tools to control cell behavior. In this editorial, we introduce the use of small molecules in stem cell research and summarize their application to the induction of cardiogenesis in noncardiac cells. Exciting new developments in this field are discussed, which we hope will encourage cardiac stem cell biologists to further consider employing small molecules in their culture protocols.
基金supported by the National Key Projects of Basic Research and Development Plan(G2000016103)the National Natural Science Foundation of China(39970541)+1 种基金the National Outstanding Youth Science Foundation(3992507)the Doctorate Foundation of the Minister of Education of China(1999004004).
文摘The porcine microsatellite SW943 was regionally localized on 12p11-(2/3p13) by the two methods: the Primed in situ (PRINS) labelling on the pachytene bivalents of pigs using the Dig-11-dUTP as the report molecule and pig × rodent Somatic Cell Hybrid PaneI(SCHP) which contains 27 cell lines through PCR amplification. Advantages and disadvantages of the two methods for physical mapping of microsatellites were also discussed.
基金Supported by National Natural Science Foundation of China,No.81770621Ministry of Education,Culture,Sports,Science,and Technology of Japan,KAKENHI,No.16K15604 and No.18H02866Japan Science and Technology Agency-Japan International Cooperation Agency’s(JST-JICA)Science and Technology Research Partnership for Sustainable Development(SATREPS)Project
文摘In the field of regenerative medicine,generating numerous transplantable functional cells in the laboratory setting on a large scale is a major challenge.However,the in vitro maintenance and expansion of terminally differentiated cells are challenging because of the lack of specific environmental and intercellular signal stimulations,markedly hindering their therapeutic application.Remarkably,the generation of stem/progenitor cells or functional cells with effective proliferative potential is markedly in demand for disease modeling,cell-based transplantation,and drug discovery.Despite the potent genetic manipulation of transcription factors,integration-free chemically defined approaches for the conversion of somatic cell fate have garnered considerable attention in recent years.This review aims to summarize the progress thus far and discuss the advantages,limitations,and challenges of the impact of full chemicals on the stepwise reprogramming of pluripotency,direct lineage conversion,and direct lineage expansion on somatic cells.Owing to the current chemical-mediated induction,reprogrammed pluripotent stem cells with reproducibility difficulties,and direct lineage converted cells with marked functional deficiency,it is imperative to generate the desired cell types directly by chemically inducing their potent proliferation ability through a lineagecommitted progenitor state,while upholding the maturation and engraftment capacity posttransplantation in vivo.Together with the comprehensive understanding of the mechanism of chemical drives,as well as the elucidation of specificity and commonalities,the precise manipulation of the expansion for diverse functional cell types could broaden the available cell sources and enhance the cellular function for clinical application in future.
文摘In animals the prion disease includes sheep and goat scrapie and the bovine spongiform encephalopathy (BSE). While several polymorphisms of the prion (PRNP) gene have been identified in sheep and some of them have been associated with susceptibility to scrapie, few mutations are reported in cattle and no correlation with BSE have been demonstrated. Genetic screening for mutants in the PRNP gene of 21 BSE positive animals by direct sequencing of the amplified gene, using DNA extracted from brain as template, confirmed that only few polymorphisms are present. However DNA molecules cloned and sequenced from the population of fragments considering a total of 90 clones from 9 BSE positive and 70 clones from 7 BSE negative animals, gave a highly significant differences in the frequency of mutations (p = 0.01). The high frequency and type of variants found cannot be explained only with misincorporation error of the Taq polymerase. Interestingly one of the mutations found in the BSE positive animals (F209S) corresponds to a mutant that causes a familiar form of prion disease in humans (F198S). These data can be explained with the presence of somatic mutations modifying the PRNP gene in single brain cells.
基金supported by the National Basic Research Program of China(973 Program)(2015CB554103 and 2011CBA01004)
文摘The transcription activator-like effector nuclease(TALEN) technique combined with the somatic cell nuclear transfer(SCNT) method has been successfully applied for creating genetically modified pigs. However, methods for isolating cells with biallelic indels requires further improvement because of the relatively low enrichment efficiency of mutated somatic cells. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modified pigs. In this study, an efficient method to increase the enrichment efficiency of TALEN-mediated biallelic knockout(KO) cells was established, and corresponding genetically modified pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase(GGTA1) gene locus. TALEN m RNA was transfected into the ear fibroblasts followed by the enrichment of α-Gal null cells of minipigs using isolectin B4(IB4) lectin and m agnetic beads. A total of 115 cell c olonies were formed and validated to be GGTA1 KO cells by sequencing and 10 biallelic KO cell colonies were used as nuclear donors for SCNT. Thirty GGTA1 biallelic KO piglets were successfully delivered and grew normally. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine t he fertility and heredity characteristics of TALEN-modified pigs, 10 mature founders w ere mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing genetically modified pig lines with expected genotypes for agricultural breeding and biomedical application.
文摘Cancer tissues contain cancer stem cells (CSCs), which play important roles in cancer metastasis. However, the mechanisms through which cancer cells dedifferentiate into stem cells have not yet been elucidated. In this study, the effects of high concentrations of polyamines produced in cancer cells on dedifferentiation were examined. The results showed that when normal human fibroblasts were cultured with high concentrations of spermine, the obtained polyamine-induced cells expressed alkaline phosphatase and marker proteins of pluripotent stem cells, although apoptosis occurred in most cells. In contrast, another polyamine-induced stem (PIS) cell line (Spe-2 PIS cells), obtained by culture in medium containing Rock, p53, and Bax inhibitors plus spermine, did not show signs of apoptosis. These Spe-2 PIS cells expressed marker proteins of pluripotent stem cells and differentiated into cardiomyocytes, brown adipocytes, and nerve cells. These results suggest that a high concentration of spermine, which often induces apoptosis in normal cells, has the capacity to dedifferentiate somatic cells into pluripotent stem cells and may be associated with the dedifferentiation of cancer cells, which continuously produce high concentrations of spermine. Moreover, the procedure to obtain Spe-2 PIS cells, which is simple and efficient, may have potential applications in regenerative medicine.
基金supported by the Veterans Administrationthe Craig H.Neilsen Foundation(280072 to SH)the California Institute for Regenerative Medicine
文摘Direct reprogramming of somatic cells into neurons or neural stem cells is one of the most important frontier fields in current neuroscience research. Without undergoing the pluripotency stage, induced neurons or induced neural stem cells are a safer and timelier manner resource in comparison to those derived from induced pluripotent stem cells. In this prospective, we review the recent advances in generation of induced neurons and induced neural stem cells in vitro and in vivo and their potential treatments of neurological disorders.
