Genome editing tools have revolutionized the generation of genetically modified animals including livestock. In particular, the domestic pig is a proven model of human physiology and an agriculturally important species. In this study, we utilized the CRISPR/Cas9 system to edit the NANOS2 gene in pig embryos to generate offspring with mono-allelic and bi-allelic mutations. We found that NANOS2 knockout pigs phenocopy knockout mice with male specific germline ablation but other aspects of testicular development are normal. Moreover, male pigs with one intact NANOS2 allele and female knockout pigs are fertile. From an agriculture perspective, NANOS2 knockout male pigs are expected to serve as an ideal surrogate for transplantation of donor spermatogonial stem cells to expand the availability of gametes from genetically desirable sires.

Spermatogonial stem cell transplantation (SSCT) is an experimental technique for transfer of germline between donor and recipient males that could be used as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics in food animal populations. To fully realize these potentials, recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis. Here we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal. In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis. With prepubertal mice, allogeneic SSCT resulted in attainment of natural fertility. Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species.

Continual and robust spermatogenesis relies on the actions of an undifferentiated spermatogonial population that contains stem cells. A remarkable feature of spermatogonial stem cells (SSCs) is the capacity to regenerate spermatogenesis following isolation from a donor testis and transplantation into a permissive recipient testis. This capacity has enormous potential as a tool for enhancing the reproductive capacity of livestock, which can improve production efficiency. Because SSCs are a rare subset of the undifferentiated spermatogonial population, a period of in vitro amplification in number following isolation from donor testicular tissue is essential. Here, we describe methodology for isolation of a cell fraction from prepubertal bull testes that is enriched for undifferentiated spermatogonia and long-term maintenance of the cells in both the feeder cell coculture and the feeder-free format. To achieve this method, we derived bovine fetal fibroblasts (BFF) to serve as feeders for optimizing medium conditions that promote maintenance of bovine undifferentiated spermatogonia for at least 2 mo. In addition, we devised a feeder-free system with BFF-conditioned medium that sustained bovine undifferentiated spermatogonia for at least 1 mo in vitro. The methodologies described could be optimized to provide platforms for exponential expansion of bovine SSCs that will provide the numbers needed for transplantation into recipient testes.

BackgroundThe success of the Global Polio Eradication Initiative was remarkable, but four countries - Afghanistan, Pakistan, India and Nigeria - never interrupted polio transmission. Pakistan reportedly achieved all milestones except interrupting virus transmission. The aim of the study was to establish valid and reliable estimate for: routine oral polio vaccine (OPV) coverage, logistics management and the quality of monitoring systems in health facilities, NIDs OPV coverage, the quality of NIDs service delivery in static centers and mobile teams, and to ultimately provide scientific evidence for tailoring future interventions.MethodsA cross-sectional study using lot quality assessment sampling was conducted in the District Nankana Sahib of Pakistan's Punjab province. Twenty primary health centers and their catchment areas were selected randomly as 'lots'. The study involved the evaluation of 1080 children aged 12-23 months for routine OPV coverage, 20 health centers for logistics management and quality of monitoring systems, 420 households for NIDs OPV coverage, 20 static centers and 20 mobile teams for quality of NIDs service delivery. Study instruments were designed according to WHO guidelines.ResultsFive out of twenty lots were rejected for unacceptably low routine immunization coverage. The validity of coverage was questionable to extent that all lots were rejected. Among the 54.1% who were able to present immunization cards, only 74.0% had valid immunization. Routine coverage was significantly associated with card availability and socioeconomic factors. The main reasons for routine immunization failure were absence of a vaccinator and unawareness of need for immunization. Health workers (96.9%) were a major source of information. All of the 20 lots were rejected for poor compliance in logistics management and quality of monitoring systems. Mean compliance score and compliance percentage for logistics management were 5.4 ± 2.0 (scale 0-9) and 59.4% while those for quality of monitoring systems were 3.3 ± 1.2 (scale 0-6) and 54.2%. The 15 out of 20 lots were rejected for unacceptably low NIDs coverage by finger-mark. All of the 20 lots were rejected for poor NIDs service delivery (mean compliance score = 11.7 ± 2.1 [scale 0-16]; compliance percentage = 72.8%).ConclusionLow coverage, both routine and during NIDs, and poor quality of logistics management, monitoring systems and NIDs service delivery were highlighted as major constraints in polio eradication and these should be considered in prioritizing future strategies.

IntroductionThe current monkeypox (MPX) outbreak, caused by the monkeypox virus (MPXV), has turned into a global concern, with over 59,000 infection cases and 23 deaths worldwide.ObjectivesHerein, we aimed to exploit robust immunoinformatics approach, targeting membrane-bound, enveloped, and extracellular proteins of MPXV to formulate a chimeric antigen. Such a strategy could similarly be applied for identifying immunodominant epitopes and designing multi-epitope vaccine ensembles in other pathogens responsible for chronic pathologies that are difficult to intervene against.MethodsA reverse vaccinology pipeline was used to select 11 potential vaccine candidates, which were screened and mapped to predict immunodominant B-cell and T-cell epitopes. The finalized epitopes were merged with the aid of suitable linkers, an adjuvant (Resuscitation-promoting factor), a PADRE sequence (13 aa), and an HIV TAT sequence (11 aa) to formulate a multivalent epitope vaccine. Bioinformatics tools were employed to carry out codon adaptation and computational cloning. The tertiary structure of the chimeric vaccine construct was modeled via I-TASSER, and its interaction with Toll-like receptor 4 (TLR4) was evaluated using molecular docking and molecular dynamics simulation. C-ImmSim server was implemented to examine the immune response against the designed multi-epitope antigen.Results and discussionThe designed chimeric vaccine construct included 21 immunodominant epitopes (six B-cell, eight cytotoxic T lymphocyte, and seven helper T-lymphocyte) and is predicted non-allergen, antigenic, soluble, with suitable physicochemical features, that can promote cross-protection among the MPXV strains. The selected epitopes indicated a wide global population coverage (93.62%). Most finalized epitopes have 70%–100% sequence similarity with the experimentally validated immune epitopes of the vaccinia virus, which can be helpful in the speedy progression of vaccine design. Lastly, molecular docking and molecular dynamics simulation computed stable and energetically favourable interaction between the putative antigen and TLR4.ConclusionOur results show that the multi-epitope vaccine might elicit cellular and humoral immune responses and could be a potential vaccine candidate against the MPXV infection. Further experimental testing of the proposed vaccine is warranted to validate its safety and efficacy profile.

The molecular mechanism regulating embryo development under reduced oxygen tension remains elusive. This study aimed to identify the molecular mechanism impacting embryo development under low oxygen conditions. Buffalo embryos were cultured under 5% or 20% oxygen and were evaluated according to their morphological parameters related to embryo development. The protein profiles of these embryos were compared using iTRAQ-based quantitative proteomics. Physiological O2 (5%) significantly promoted blastocyst yield, hatching rate, embryo quality and cell count as compared to atmospheric O2 (20%). The embryos in the 5% O2 group had an improved hatching rate of cryopreserved blastocysts post-warming (p < 0.05). Comparative proteome profiles of hatched blastocysts cultured under 5% vs. 20% O2 levels identified 43 differentially expressed proteins (DEPs). Functional analysis indicated that DEPs were mainly associated with glycolysis, fatty acid degradation, inositol phosphate metabolism and terpenoid backbone synthesis. Our results suggest that embryos under physiological oxygen had greater developmental potential due to the pronounced Warburg Effect (aerobic glycolysis). Moreover, our proteomic data suggested that higher lipid degradation, an elevated cholesterol level and a higher unsaturated to saturated fatty acid ratio might be involved in the better cryo-survival ability reported in embryos cultured under low oxygen. These data provide new information on the early embryo protein repertoire and general molecular mechanisms of embryo development under varying oxygen levels.

The current global outbreak of monkeypox (MPX) disease, caused by Monkeypox virus (MPXV), has resulted in 16 thousand infection cases, five deaths, and has been declared a global health emergency of international concern by the World Health Organization. Given current challenges in the safety of existing vaccines, a vaccine to prevent MPX infection and/or onset of symptoms would significantly advance disease management. In this context, a multi-epitope-based vaccine could be a well-suited approach. Herein, we searched a publicly accessible database (Virus Pathogen Database and Analysis Resource) for MPXV immune epitopes from various antigens. We prioritized a group of epitopes (10 CD8+ T cells and four B-cell epitopes) using a computer-aided technique based on desirable immunological and physicochemical properties, sequence conservation criteria, and non-human homology. Three multi-epitope vaccines were constructed (MPXV-1–3) by fusing finalized epitopes with the aid of appropriate linkers and adjuvant (beta-defensin 3, 50S ribosomal protein L7/L12, and Heparin-binding hemagglutinin). Codon optimization and in silico cloning in the pET28a (+) expression vector ensure the optimal expression of each construct in the Escherichia Coli system. Two and three-dimensional structures of the constructed vaccines were predicted and refined. The optimal binding mode of the construct with immune receptors [Toll-like receptors (TLR2, TLR3, and TLR4)] was explored by molecular docking, which revealed high docking energies of MPXV-1–TLR3 (–99.09 kcal/mol), MPXV-2–TLR3 (–98.68 kcal/mol), and MPXV-3–TLR2 (–85.22 kcal/mol). Conformational stability and energetically favourable binding of the vaccine-TLR2/3 complexes were assessed by performing molecular dynamics simulations and free energy calculations (Molecular Mechanics/Generalized Born Surface Area method). In silico immune simulation suggested that innate, adaptive, and humoral responses will be elicited upon administration of such potent multi-epitope vaccine constructs. The vaccine constructs are antigenic, non-allergen, non-toxic, soluble, topographically exposed, and possess favourable physicochemical characteristics. These results may help experimental vaccinologists design a potent MPX vaccine.

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