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Background: The number of multigene‐modified donor pigs for xenotransplantation is increasing with the advent of gene‐editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation. Methods: In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four‐gene‐edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. Results: First, 107 cell colonies were obtained through drug selection, of which seven were 4‐GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4‐GEC pigs were markedly reduced. Moreover, 4‐GEC porcine PBMCs had greater survival rates than those from wild‐type pigs through complement‐mediated cytolysis assays. In pig‐to‐monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. Conclusion: These results indicate that the GTKO/hCD55/hTBM/hCD39 four‐gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life. [ABSTRACT FROM AUTHOR]

Interspecies somatic cell nuclear transfer (iSCNT) has an immense potential to rescue endangered animals and extinct species like mammoths. In this study, we successfully established an Asian elephant's fibroblast cell lines from ear tissues, performed iSCNT with porcine oocytes and evaluated the in vitro and in vivo development of reconstructed embryos. A total of 7780 elephant–pig iSCNT embryos were successfully reconstructed and showed in vitro development with cleavage rate, 4-cell, 8-cell and blastocyst rate of 73.01, 30.48, 5.64, and 4.73%, respectively. The total number of elephant–pig blastocyte cells and diameter of hatched blastocyte was 38.67 and 252.75 μm, respectively. Next, we designed species-specific markers targeting EDNRB, AGRP and TYR genes to verify the genome of reconstructed embryos with donor nucleus/species. The results indicated that 53.2, 60.8, and 60.8% of reconstructed embryos (n = 235) contained elephant genome at 1-cell, 2-cell and 4-cell stages, respectively. However, the percentages decreased to 32.3 and 32.7% at 8-cell and blastocyst stages, respectively. Furthermore, we also evaluated the in vivo development of elephant–pig iSCNT cloned embryos and transferred 2260 reconstructed embryos into two surrogate gilts that successfully became pregnant and a total of 11 (1 and 10) fetuses were surgically recovered after 17 and 19 days of gestation, respectively. The crown-rump length and width of elephant–pig cloned fetuses were smaller than the control group. Unfortunately, none of these fetuses contained elephant genomes, which suggested that elephant embryos failed to develop in vivo. In conclusion, we successfully obtained elephant–pig reconstructed embryos for the first time and these embryos are able to develop to blastocyst, but the in vivo developmental failure needs further investigated. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis (CL), the most widespread form of leishmaniasis, caused by Leishmania tropica and L. major (1,2), has emerged as an endemic disease in Khyber Pakhtunkhwa, Pakistan (2-5), owing to [...]

Human hepatocyte transplantation for liver disease treatment have been hampered by the lack of quality human hepatocytes. Pigs with their large body size, longevity and physiological similarities with human are appropriate animal models for the in vivo expansion of human hepatocytes. Here we report on the generation of RAG2-/-IL2Rg-/YFAH-/-(RGFKO) pigs via CRISPR/Cas9 system and somatic cell nuclear transfer. We showed that thymic and splenic development in RGFKO pigs was impaired. V(D)J recombination processes were also inactivated. Consequently, RGFKO pigs had significantly reduced numbers of porcine T, B and NK cells. Moreover, due to the loss of FAH, porcine hepatocytes continuously undergo apoptosis and consequently suffer hepatic damage. Thus, RGFKO pigs are both immune deficient and constantly suffer liver injury in the absence of NTBC supplementation. These results suggest that RGFKO pigs have the potential to be engrafted with human hepatocytes without immune rejection, thereby allowing for large scale expansion of human hepatocytes. [ABSTRACT FROM AUTHOR]

Zygote, the first step in the life establishment, is sensitive to environmental impact. Any detrimental effect to this unit may lead to major abnormalities in future development. Artificial activation of oocyte is a one of key step in cloning. Strontium chloride (SrCl2), a calcium oscillation inducing salt, is commonly used for the artificial activation mouse oocytes; however, limited reports are available to evaluate its negative impact. The objective of this study was to optimize the concentration of SrCl2 for artificial activation of mouse oocytes and to evaluate its toxic effect on early embryonic development. Our results indicated that higher percentage of activation of mouse oocyte was achieved with 10mM and 15mM for period of 3 h and 6 h compared to the control group (P<0.05). To examine the toxic effect, 10mM SrCl2 with 3 h exposure showed minimal detrimental effect on embryo development. Collectively, our findings revealed that 10mM concentration of SrCl2 for 3 h exposure was appropriate for oocyte activation with the least toxic impact for embryonic development. [ABSTRACT FROM AUTHOR]

Currently, artificial oocyte activation has attracted wide attention in assisted reproduction due to extensive range of applications, particularly in somatic cell nuclear transfer and deriving pluripotent stem cell lines and it is the unique model to determine the role of paternal genome. Numbers of artificial activating agents have been used extensively to induce the oocytes activation; however, embryos developmental competency of artificially activated oocytes is still very low. In the present study, we determined the functional impact of strontium chloride supplementation with gold nanoparticles (AuNPs) in artificial oocytes activation and subsequent embryonic development. Oocytes were activated artificially in the culture medium containing 250 nM AuNPs with constant concentration of strontium chloride 10.00 mM. We found that adding 250 nM AuNPs with constant concentration of strontium chloride (10.00 mM for 3 hr) in culture medium improves the proportion of embryos reaching to the morula and blastocyst stages from 61.00% and 42.00% (controls) to 75.00% and 58.00% (250 nM AuNPs), respectively. In addition, foster mothers receiving AuNPs-treated embryos showed more implantation percentage and pregnancy rate relative to females received control embryos. Finally, embryos treated with 250 nM AuNPs concentration showed no toxic effect in term of blastocyst development. Collectively, our findings suggest the potential role of AuNPs in early embryonic development for mouse oocytes activated artificially and provide new insights in the field of animal biotechnology and assisted reproduction in humans. [ABSTRACT FROM AUTHOR]

Although, the water buffalo (Bubalus bubalis) is a backbone of the Pakistani dairy industry but a very limited knowledge at molecular and genetic level is available. Hence, the objective of current study was to analyze the genetic diversity of Pakistani buffaloes. Seventy D-loop partial sequence (880-bp) haplotypes with 71 polymorphic sites were detected among 212 individuals, collected form Punjab and Sindh provinces of Pakistan, mtDNA d-loop region was first amplified and sequenced. The UPGMA phylogenetic tree revealed that, the Ancient Pakistani buffalo clade was more old and ancient as compare to Indian Riverine buffalo clade. Whereas, the haplotype and nucleotide diversity (0.9561±0.00010 and 0.00988±0.00060) was higher in the Nili-Ravi, followed by Kundhi (0.9386±0.00020 and 0.01043±0.00083), Ravi (0.9130±0.016 and 0.01012±0.00112) and Nili (0.8874±0.054 and 0.00999±0.00164), respectively. Median Joining Network also disclosed two different clades in only Pakistani buffaloes, Ancient Pakistani buffalo and Sister Pakistani buffalo clade same as in phylogenetic tree. However, Pakistani and Indian Riverine buffaloes did not share any haplotype with each other. The buffalo populations were separated from each other through of (>50 mutational steps). Further, the Ancient Pakistani clade showed high time depth in phylogenetic tree by residing close to the base root and highest Ancestry in Median Joining network by having the most numbers of mutational steps as compared to other two Riverine buffalo clades. Our findings indicated that the Pakistani Riverine buffaloes diverged earlier. The genetic information obtained from the mtDNA d-loop is of utmost importance for the future genetic studies and improvements. [ABSTRACT FROM AUTHOR]

The current study investigates the incidence of subclinical mastitis (SCM), associated risk factors, involvement and antimicrobial susceptibility of Staphylococcus aureus in the development of SCM in District Faisalabad of Pakistan. For this purpose, a total of 384 goat milk samples were screened for SCM through surf field mastitis test (SFMT) and mastitis-positives cases were further investigated for isolation of S. aureus using standard procedures. Coagulase gene was PCR amplified from the clinical isolates to categorize them into Coagulase positive Staphylococci (CPS) and coagulase negative Staphylococci (CNS). A questionnaire was used to record risk factors associated with occurrence of SCM and results were analyzed using non-probability statistical analysis. Results indicated that an overall 63.28% (243/384) of goats were found positive for SCM, of which 58.85% (143/243) revealed Staphylococcal growth among them 69.93% (100/143) were CPS, while 30.07% (43/143) were found as CNS. Drug susceptibility against penicillins, cephalosporins, macrolides and other drugs showed that 50% of CPS and 30% of CNS were found multi-drug resistant-exhibiting resistance against more than two or more than two classes of drugs. None of the CPS while 20 % from CNS isolates were 100% susceptible to all kinds of drugs tested. Finally, age, grazing system, use of beta-lactam antibiotics, parity, and poor hygiene were potential risk factors. Altogether, the study concluded high incidence of SCM and isolation rate of staphylococci that were found resistant against most of the commonly used antibiotics. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it often becomes resistant to paclitaxel (PTX) therapy. Autophagy plays an important cytoprotective role in PTX-induced tumor cell death, and targeting autophagy has been promising for improving the efficacy of tumor chemotherapy in recent years. The aim of the present study was to clarify the mechanism of PTX inducing autophagy in TNBC cells to provide a potential clinical chemotherapy strategy of PTX for TNBC. The present study reported that PTX induced both apoptosis and autophagy in MDA-MB-231 cells and that inhibition of autophagy promoted apoptotic cell death. Furthermore, it was found that forkhead box transcription factor O1 (FOXO1) enhanced PTX-induced autophagy through a transcriptional activation pattern in MDA-MB-231 cells, which was associated with the downstream target genes autophagy related 5, class III phosphoinositide 3-kinase vacuolar protein sorting 34, autophagy related 4B cysteine peptidase, beclin 1 and microtubule associated protein 1 light chain 3β. Knocking down FOXO1 attenuated the survival of MDA-MB-231 cells in response to PTX treatment. These findings may be beneficial for improving the treatment efficacy of PTX and to develop autophagic targeted therapy for TNBC. [ABSTRACT FROM AUTHOR]