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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.

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

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.

The present study aimed to compare two different insemination times (72 vs 84 h) associated with an ovulation induction (GnRH) in a 7-day CIDR Co-synch to improve the conception rate of Nili Ravi buffalo heifers. Forty Nili Ravi buffalo heifers were randomly separated into two treatments based on artificial insemination (AI) timing (72 vs 84 h). All heifers were subjected to controlled internal drug release (CIDR), containing 1.38 g of progesterone for 7 days. On CIDR removal, both treatments received 150 µg of prostaglandin intramuscularly. In 7-day CIDR Co-synch (n = 20), animals were injected 100 µg of GnRH administration intramuscularly and inseminated concurrently at 72 h after CIDR removal. The remaining half (n = 20) were injected and inseminated concurrently at 84 h of CIDR removal. Pregnancy diagnosis was performed on day 40 of timed artificial insemination (TAI) with ultrasound. The follicular growth rate between 72 h after PGF2α/CIDR removal to pre-ovulatory follicle in 7-day CIDR Co-synch was more (0.102 ± 0.005 mm vs 0.079 ± 0.003 mm; P = 0.01) at 84 than 72 h. The interval from GnRH administration/TAI to ovulation was high (26.8 ± 1.64 h vs. 15.1 ± 1.25 h, P = 0.01) in 72 than 84 h. Conception rates were considerably higher in buffalo heifers inseminated at 84 h (65%) than 72 h (25%) in 7-day CIDR Co-synch protocol. In conclusion that in Nili Ravi buffalo heifers, GnRH administration/TAI after 84 h of CIDR removal allows greater follicular growth rate and shortens interval from AI to ovulation compared to the GnRH administration/TAI after 72 h of CIDR removal in 7-day CIDR-Co-synch protocol.

Considerable advancements have recently been achieved in porcine somatic cell nuclear transfer (SCNT), but the efficiency remains low. Donor cell size might play an important role in SCNT, but its effects in pigs remain unclear. This study aimed to evaluate the efficiency of porcine SCNT by selecting donor cells of suitable size. Porcine fetal fibroblasts (PFFs) were divided into three groups, group S (small, d ≤ 13 μm), group M (medium, 13 μm 18 μm), and their biological characteristics were analyzed. Next, SCNT was performed using PFFs of different sizes to evaluate the developmental potential of reconstructed embryos. The data showed that PFFs in groups S, M and L accounted for 17.5%, 47.7% and 34.8% of cells, respectively. Morphologically, cells in group S exhibited clear and regular cell membranes and nuclei, whereas cells in groups M and L displayed varying degrees of cell membrane protuberance, karyo-pyknosis, autophagy and mitochondrial abnormalities. In addition, the growth status and proliferation capabilities of cells in group S were significantly better than those of group M and group L. The percentage of cells at G0/G1 in group S and M were significantly greater than group L. The senescence rate of group S was lower than group M and group L. The apoptosis rate of group S was significantly lower than that of group L but comparable to that of group M . The cleavage rate of group S was also significantly greater than that of group M but comparable to that of group L . The blastocyst rate of group S was significantly greater than that of group M and group L. The blastocyst cell numbers of group S were also significantly greater than those of group M and group L. These findings suggested that small PFFs with a diameter of less than 13 μm are more suitable donor cells for SCNT in pigs. Abbreviations: DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; PFFs: Porcine fetal fibroblast cells; SCNT: Somatic cell nuclear transfer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancers and often produces resistance to paclitaxel (PTX) therapy. Autophagy plays an important cytoprotective role in PTX-induced tumor cell death and targeting autophagy is promising to improve the efficacy of tumor chemotherapy in recent years. Here, we reported that PTX induced both apoptosis and autophagy of MDA-MB-231 cells, and inhibition of autophagy enable to promote apoptotic cell death. Furthermore, we found that FOXO1 enhanced PTX-induced autophagy by a transcriptional activation pattern in MDA-MB-231 cells, which was associated with its downstream target genes ATG5, VPS34, BECN1 and MAP1LC3B. The knockdown of FOXO1 attenuated the survival of MDA-MB-231 cells under the PTX treatment. These findings will be beneficial to improve the treatment efficacy of PTX and to develop the autophagic target therapy of TNBC.

The present study aimed to compare the pregnancy rates at the optimal time of artificial insemination in a 7-day CIDR Co-synch in Nili Ravi buffalo heifers. Forty Nili Ravi buffalo heifers were randomly separated into two treatments based on artificial insemination (A.I.) timing (72 vs 84 hours). All heifers were subjected to controlled internal drug release (CIDR), containing 1.38 grams of progesterone for 7 days. On CIDR removal, both treatments received 150 µg of prostaglandin intramuscularly. In 7-day CIDR Co-synch (n = 20), animals were injected 100 µg of GnRH intramuscularly and inseminated concurrently at 72 hours after CIDRs removal. The remaining half (n=20) were injected and inseminated concurrently at 84 hours of CIDRs removal. Pregnancy diagnosis was performed after 40 days of timed artificial insemination (TAI). The follicular growth rate in 7-day CIDR Co-synch was more significant at 84 than 72 hours. The interval from GnRH/A.I. to ovulation in 7-day CIDR Co-synch was significantly (P < 0.05) differ between 72 and 84 hours. Pregnancy rates were considerably higher in buffalo heifers inseminated at 84 hours (65%) than 72 hours (25%) in 7-day CIDR Co-synch treatment. In conclusion, the buffalo heifers treated with CIDR Co-synch based protocols in relation to TAI at 84 hours improves follicular growth rate, interval from GnRH/A.I. to ovulation, and pregnancy rate.

Leptin is a well-known adipokine that plays critical role in adiposity. To further investigate the role of leptin in adiposity, we utilized leptin overexpressing transgenic pigs and evaluated the effect of leptin on growth and development, fat deposition, and lipid metabolism at tissue and cell level. Leptin transgenic pigs were produced and divided into two groups: elevated leptin expression (leptin ( +)) and normal leptin expression group (control). Results indicated that leptin ( +) pigs had elevated leptin protein and mRNA expression levels and exhibited sluggish growth and development followed by decreased subcutaneous fat thickness, low serum triglycerides, saturated, unsaturated fatty acids and high cholesterol esters (p

The antidotal efficacy of aqueous garlic extract, methylene blue, and velenium (Vitamin E + sodium selenite) was compared against experimental nitrate intoxication in rabbits. Forty-two, albino rabbits of identical age, gender, and body weight were randomly divided into 7 groups (A to G) and subjected to experimental treatments for a period of 40 days. Rabbits of group A were offered only normal feed and served as negative control, while, rabbits of group B constituted the positive control group and received feed supplemented with sodium nitrate at 400 mg/kg body weight. Sodium nitrate-containing feed and intraperitoneal injection of 1% methylene blue solution at 2 mg/kg body weight were administered to group C. Rabbits of group D were given sodium nitrate-supplemented feed and aqueous garlic extract at 500 mg/kg body weight through oral route. Group E was treated with sodium nitrate-added feed, intraperitoneal injection of 1% methylene blue solution at 2 mg/kg body weight, and oral administration of garlic extract at 500 mg/kg. Velenium (25 mg of Vitamin E + 2.2 mg of sodium selenite per ml) was intraperitoneally injected at 1 ml/kg body weight to rabbits of group F along with the provision of sodium nitrate-supplemented feed. In addition to being fed with sodium nitrate-containing feed, group G obtained intraperitoneal injection of velenium at 1 ml/kg body weight and oral administration of garlic extract at 500 mg/kg body weight. The efficacy of antidotes was assessed on the basis of changes in blood nitrite level, biochemical profile, and gross pathological lesions manifested by the treated rabbits. The combination of methylene blue and garlic extract was highly effective in treating nitrate toxicity followed by methylene blue, garlic extract, and velenium, respectively. Whereas, the concurrent administration of garlic extract and velenium was least efficacious in terms of antidotal efficacy. In conclusion, aqueous garlic extract can be effectively used either alone or in combination with methylene blue when treating rabbits diagnosed with nitrate toxicity.

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis (CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1 (ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica (L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major (L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents (Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.

Objective: To evaluate prevalence of Anaplasma marginale (A. marginale) and associated risk factors in sheep and goat. Methods: A total of 500 blood samples (250 from sheep and 250 from goat) were collected from three different tehsils of Karak District and analyzed for presence of A. marginale using microscopy, indirect ELISA and real-time PCR. Results: The overall prevalence rate was 33.87%. The infection rate was significantly higher (P < 0.05) in Karak and Banda Daud Shah compared to Takht-e-Nasrati. It was observed that prevalence rate was higher by real-time PCR compared to indirect ELISA and microscopy. In tehsil Karak, the infection rate by microscopy, indirect ELISA and real-time PCR was 24.38%, 40.62% and 56.25%, respectively. Similar findings were observed in other two tehsils that real- time PCR results were more reliable. Conclusions: The overall prevalence rate was higher in sheep (47.25%) compared to goat (34.85%). Furthermore, among all the risk factors associated, presence of tick and unhygienic condition were highly associated with infection as shown by coefficient of correlation. This is the first report regarding A. marginale prevalence in sheep and goat in Karak District, Pakistan.

Objective: To evaluate the prevalence of extended spectrum beta-lactamases (ESBL) in clinical isolates from burn patients using phenotypic and genotypic analyses. Methods: During 2015–2016, a total of 126 samples were collected at a tertiary care hospital, Islamabad. Antibiotic sensitivity and ESBL prevalence were evaluated according to the Clinical Laboratory and Standards Institute, and molecular analysis of the CTX-M type ESBL gene was performed in 225 bacterial isolates from these samples. Results: The most prevalent bacterial species were Escherichia coli (28.4%), Pseudomonas aeruginosa (22.2%), Staphylococcus aureus (19.6%), Klebsiella pneumoniae (16.4%), and coagulase-negative staphylococci (13.3%). Of the 225 bacterial isolates, 89 (39.5%) were found to be ESBL producers. The isolates were highly susceptible to meropenem (88%) and imipenem (84%), followed by the aminoglycoside amikacin (81%). Molecular epidemiology of the ESBL isolates indicated 19% prevalence of CTX-M. Resistance to antibiotics was exhibited by 28% isolates. Conclusions: In the present study, bacteria such as P. aeruginosa, K. pneumoniae, S. aureus, and E. coli isolated from burn patients exhibited resistance to one or more antibiotics and produced large amounts of ESBL. Further studies are needed to investigate the virulence and epidemiology of CTX-M type ESBL in clinical isolates from burn patients.

An epidemiological and molecular study was carried out for the first time in Kohat District of Khyber Pakhtunkhwa (KP) province, Pakistan from April 2015 to May 2016 to determine the prevalence of Cutaneous Leishmaniasis (CL) in local population and Internally Displaced People (IDPs). In 13 different villages, a total of 1359 (out of 26,250 individuals belonging to local population) and 140 (out of 3615 IDPs residing in these villages) cases were recorded and 300 samples were collected. The total prevalence of CL in local population was 5.17% with active lesions and scar prevalence of 3.91% and 1.26% respectively. Similarly a prevalence of 3.86% for CL was recorded in IDPs. Highest number of IDPs having CL active lesions and scars were recorded in villages Sherkot, Surgul, and Jarma and their presence was positively correlated with CL in local population. Age wise prevalence was highest in young children of age group 1-15 years. The microscopic examination showed 64.33% (193/300) positive samples while kinteoplastic PCR showed 84.66% (254/300) positive. For the first time in KP province, 2/784 sandflies trapped from the study villages was found positive for Leishmania by PCR. Restriction Fragment Length Polymorphism of patients and sandflies samples revealed L. tropica as the prevalent Leishmania species in this district. The results of sequencing and RFLP identified L. tropica in Phlebotomus sergenti. This is the first ever report of molecular identification of L. tropica from sandflies of genus P. sergenti in Khyber Pakhtunkhwa province. This data can be helpful for health authorities in finding out new CL foci and to plan effective strategies for the provision of health facilities to poor people of this area.

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.

This study aimed to evaluate the efficacy of Eucalyptus globulus and Calotropis procera against MDRSA and their potential role in modulating resistance to beta-lactam drugs. The study used a total of n = 263 camel milk samples as well as aqueous and ethanolic extracts of both plants from the Cholistan Desert and the Suleman Mountain Range in Pakistan. Different concentrations of the two plants (25, 12.6, 6.25, and 3.125 mg/mL), either alone or in combination with each other, were tested against MDRSA by well diffusion and broth dilution methods. Further, the efficacy of both plant extracts in combination with cefotaxime (30 μg) and ampicillin (1 μg) was also tested. The results indicate that the zone of inhibition (ZOI) of E. globulus alone and in combination with C. procera was nearly 3 times as large as that of C. procera. Ethanolic extracts of the two plants plants, alone or in combination with either of the two antibiotics, had a significantly (P

Objective To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis (CL) is endemic and was thought to be caused by Leishmania tropica only. Methods Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011–2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. Results Leishmania amastigotes were detected by microscopy in 308 of 432 samples (71.3%) while 374 out of 432 samples (86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed L. tropica in 351 and L. major in 6 biopsy samples. Conclusions This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of L. major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.