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Introduction: The CRISPR/Cas9 (Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated Protein9) method can create a nucleotide sequence complementary to the target sequence in the desired gene by Guide-RNA (gRNA) together with the Cas9 protein, which is a cutting enzyme for cutting in the both DNA strands of the desired sequence accurately and clearly. The DAZL (Deleted in azoospermia-like) gene encodes potential RNA-binding proteins that are expressed in male and female germ cells before and after birth. DAZL, which acts by post-transcriptionally binding mRNA in 3' untranslated regions, regulates the germ cell cycle. DAZL initiates the sexual differentiation of embryonic germ cells. The transfer and transplantation of gene-edited germ cells into recipient males is an effective method for targeted mutagenesis engineering. In mice knocked out for the Dazl gene, the number of testicular stem cells was reduced and it was found that the DAZL gene plays an important role in the differentiation of spermatogonial cells. The purpose of the current research is to edit the DAZL gene by knocking it out using the CRISPR/Cas9 technique and transferring the somatic cell nucleus into the genome of the Bakhtiari goat embryo. The inactivation of the gene will be investigated both at the level of the embryonic cell and the resulting embryo. There are no reports on the production of Bakhtiari goat cells and embryos edited for the DAZL gene by CRISPR technique and somatic nuclear transfer to improve any traits, including reproductive ones. Materials and methods: To target the DAZL gene and explore (predict) potential off-target genomic sites, guide RNA (20 bp sequences) immediately upstream of each 5'-NGG in the DAZL gene was designed using the CHOOPCHOOP tool. Plasmid pX459 (9151 bp) was used to insert the gRNA sequence into the CAS9 vector and determine the characteristics and replication of the vector. This plasmid encodes the Cas9 protein along with the puromycin resistance gene under the promoter/enhancer, CAGGS, as well as the gRNA scaffold under the U6 promoter. Plasmid pX459 was digested by BbsI-HF (NEB #R3539) at 37°C for 10 min, followed by purification by NucleoSpin Gel and PCR Clean-up Midi kit (#740,986.20, Machery/Nagel). The purified piece was kept at - 20°C for later use. Ligation of oligoduplex carrying diluted gRNA sequence (1:20 ratio from 10 μM source) (1 ìL), digested pX459 vector (50 ng), 10x T4DNA ligase buffer (2 ìL), and T4DNA ligase (1 ìL) in the final concentration of 20 ìL of the reaction was carried out. Ligation mixture transformation was performed with NEB 5-alpha Competent E. coli (#C2987I) and then placed on agar culture medium with 100 ìg/mL ampicillin and incubated at 37°C overnight. From the cultured plate, five colonies were selected and each was cultured in LB culture medium, followed by miniprep plasmid extraction (Genejet Plasmid miniPrep kit, #K0502). Plasmids carrying the CRISPR system were transferred into cells through an electroporation system. Results and discussion: The present results proved the possibility of knocking out the DZAL gene using the CRISPR/Cas9 technique in both fibroblast cell lines and Bakhtiari goat embryos. We performed somatic cell nuclear transfer (SCNT) to examine the embryonic developmental capacity to transition to the cleavage and blastocyst stages. The embryos created by knocking out the DAZL gene could grow and develop. The sequencing analysis using DECODR (Deconvolution of Complex DNA Repair) software showed the knock-out rate of the DAZL gene in fibroblast cells was 83.3% and in cloned blastocysts, it was 55.3%. The rate of blastocyst formation resulting from the cloning process with knockout cells was not significantly different from the control group. Based on these studies, we decided to edit the DAZL gene to produce knockout goat embryos by SCNT. The embryos were examined and evaluated with the help of PCR-RFLP test and sequencing. It turned out that in the first iteration, a knockout of 60.66% (the first ten embryos were paid), and in the second iteration, a knockout of 49.99% (the second ten embryos were paid) were achieved. Such mutations have not been described or detected in Iranian Bakhtiari goats, although polymorphisms have been identified. Using genetic sequencing, the mutations were of the INDEL type and were all in the form of frameshift, which resulted in a change in the protein. Conclusions: The CRISPR/Cas9 system easily influences the desired gene and can be used as a strategy to produce livestock animals that are superior in milk and meat production, reproduction, quality, disease resistance, etc. This research demonstrated the possibility of gene editing using the CRISPR/Cas9 technique in fibroblast cell lines and Bakhtiari goat embryos. The blastocytes produced can be used for transfer to the recipient animal and production of DAZL gene knockout goats for further study and optimization of spermatogonial stem cell transplantation. [ABSTRACT FROM AUTHOR]

Here we report a simultaneous construction of two C-C and two C-N bonds in a unified procedure that incorporates alkynoic acid trifunctionalization, ortho C-H functionalization, and amination cascade. In an ordered process, the regioselective alkyne insertion reaction is favored over the decarboxylation process. The presence of the carboxyl group in alkynoic acid ensures the high regioselectivity in the carbopalladation process, paving the way for a novel method to synthesize unsymmetrically 2,3-diaryl substituted indole scaffolds with excellent regioselectivity. The protocol is demonstrated to be suitable for gram-scale synthesis.

PDX1 is a crucial transcription factor in pancreas development and mature β-cell function. However, the regulation of PDX1 expression in larger animals mirroring human pancreas morphogenesis and endocrine maturation remains poorly understood. Therefore, we conducted a comparative analysis to characterize regulatory regions of goat PDX1 gene and assessed their transcriptional activity by transient transfection of several transgenic EGFP constructs in β- and non-β cell lines. We recognized several highly conserved regions encompassing the promoter and cis-regulatory elements (Area I-IV) at 5' flanking sequence of the genes. Within the promoter, we identified that a key E-box and nearby CAAT element synergistically drive transcription, constituting the basal promoter of goat PDX1 gene. Furthermore, each recognized regulatory area separately enhances this basal promoter activity in β-cells compared to non-β cells; however, cooperatively, they exhibit a bifunctional regulatory effect on transcription. Additionally, the intact ~ 3 kb upstream region (Area I-III) functions as the most efficient reporter transgene in vitro and shows islet-specific expression in native rat pancreas. Together, our findings suggest that the regulation of goat PDX1 gene is governed by conserved regions similar to other mammals, while both structurally and functionally, these regions exhibit a closer resemblance to those found in humans., (© 2024. The Author(s).)

Advanced glycation end products (AGEs) are the final products of the Maillard reaction, formed through the interaction of carbohydrates and proteins. Reactive dicarbonyl compounds such as methylglyoxal (MGO) serve as precursors for AGEs formation. Elevated levels of MGO/AGEs are observed in conditions like obesity, polycystic ovarian syndrome (PCOS), and diabetes, negatively impacting oocyte development. Previous studies have shown that hydrogen sulfide, a gasotransmitter with anti-AGEs effects, is produced in a process influenced by vitamin B6. R-α-lipoic acid (ALA) inhibits protein glycation and AGEs formation while stimulating glutathione (GSH) production. Taurine mitigates oxidative stress and acts as an anti-glycation compound, preventing in vitro glycation and AGEs accumulation. This study aimed to explore the ameliorative effects of a micronutrient support (Taurine, ALA and B6: TAB) on mouse oocytes challenged with MGO. Our results indicate that MGO reduces oocyte developmental competence, while TAB supplementation improves maturation, fertilization, and blastocyst formation rates. TAB also restores cell lineage allocation, redox balance and mitigates mitochondrial dysfunction in MGO-challenged oocytes. Furthermore, cumulus cells express key enzymes in the transsulfuration pathway, and TAB enhances their mRNA expression. However, TAB does not rescue MGO-induced damage in denuded oocytes, emphasizing the supportive role of cumulus cells. Overall, these findings suggest that TAB interventions may have significant implications for addressing reproductive dysfunctions associated with elevated MGO/AGEs levels. This study highlights the potential of TAB supplementation in preserving the developmental competence of COCs exposed to MGO stress, providing insights into mitigating the impact of dicarbonyl stress on oocyte quality and reproductive outcomes., (© 2024. The Author(s).)

Advanced glycation end products (AGEs) can disrupt antioxidant system and steroidogenesis, resulting in detrimental effects on assisted reproductive technology (ART) outcomes. This study aimed to investigate the association of AGEs in follicular fluid (FF) with morphokinetic parameters of embryos and ART outcomes. Fifty women undergoing ART treatment were studied. AGEs, glucose, 25(OH) vitamin D, malondialdehyde (MDA) levels and catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were evaluated in FF. The expression of 3βHSD, CYP11A1, and CYP19A1 genes were analyzed in granulosa cells (GCs) by qRT-PCR technique. Morphokinetic parameters were evaluated using time-lapse technology. The FF level of AGEs was reversely associated with CAT, SOD, and GPX activities, and total and mature oocytes number, blastocyst formation rate, and high-grade embryos number, while it showed positive correlations with the FF MDA levels, the expression of steroidogenesis genes, number of immature oocytes, morphokinetic parameters, and number of low-grade embryos. Furthermore, the level of vitamin D in FF had an inverse association with AGEs and positive correlations with ART outcomes and morphokinetic parameters. Comparison between the those with positive and negative biochemical pregnancy showed no significant differences in terms of FF factors and just the expression of 3βHSD, CYP11A1, and CYP19A1 genes were higher in pregnant women (p < 0.05). AGEs could delay blastomere division and lead to an increase in the number of low-quality embryos, while vitamin D have an adverse effect on AGEs and a protective function against AGEs negative effects., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Cryopreservation of sperm can cause oxidative stress and damage, leading to decreased different functional parameters and fertilization potential. In this study, we evaluated two types of H 2 S donors: NaHS, a fast-releasing donor, and GYY4137, a slow-releasing donor during cryopreservation of goat sperm. Initially, we determined that 1.5 and 3 μM NaHS, and 15 and 30 μM GYY4137 are optimal concentrations that improved different sperm functional parameters including motility, viability, membrane integrity, lipid peroxidation, and ROS production during incubation at 38.5 °C for 90 min. We subsequently evaluated the impact of the optimal concentration of NaHS and GYY4137 supplementation on various functional parameters following thawing during cryopreservation. Our data revealed that supplementation of extender improved different parameters including post-thaw sperm motility, viability, membrane integrity, and reduced DNA damage compared to the frozen-thawed control group. The supplementation also restored the redox state, decreased lipid peroxidation, and improved mitochondrial membrane potential in the thawed sperm. Finally, we found that supplementation of the extender with NaHS and GYY4137 enhanced IVF outcomes in terms of blastocyst rate and quality of blastocysts. Our results suggest that both donors can be applied for cryopreservation as antioxidants to improve sperm quality and IVF outcomes of frozen-thawed goat sperm., (© 2024. The Author(s).)

This study involved the synthesis of a new biocompatible slow-release hydrogel named poly (acrylic acid) grafted onto sodium alginate (poly (AA-g-SA)) double network hydrogel (DNH). The hydrogel was created by polymerization of acrylic acid grafted onto sodium alginate polysaccharide using crosslinkers N,N'-methylenebisacrylamide and calcium chloride via free radical polymerization. The water absorbency of the poly (AA-g-SA) double network hydrogel was improved by optimizing the quantities of ammonium persulfate initiator, pH-sensitive monomer of acrylic acid, and crosslinkers. Various analytical techniques including attenuated total reflection Fourier-transformed infrared (ATR-FTIR), X-ray diffraction analysis (XRD), X-ray photoelectron spectroscopy (XPS), thermal gravimetric analysis (TGA), field emission scanning electron microscopy (FESEM), high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), and Brunauer-Emmett-Teller specific surface area analysis (BET) were used to characterize the synthesized hydrogels. The swelling and on-off switching behaviors of the hydrogels were investigated in deionized (DI) water at different temperatures and pH values. The optimum poly (AA-g-SA) DNH was tested for in vitro release of a hydrophilic chemotherapeutic drug, doxorubicin hydrochloride (DOX). The eco-friendly hydrogel favorably optimized the DOX slow release owing to its swelling rate, high absorption and regeneration capabilities. The findings of this study may have significant implications for medical and scientific research., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023. Published by Elsevier B.V.)

Heat stress is a significant factor affecting the fertility of dairy cattle due to the generation of free radicals. In assisted reproductive techniques, the inclusion of protective antioxidants becomes crucial to mitigate potential cellular damage. This study aimed to explore the impact of supplementing vitamins E, C, and coenzyme Q10 into the oocyte culture medium, with the goal of ameliorating the adverse effects of heat stress on oocyte maturation and embryo development in dairy cattle. A group of fifty Holstein dairy cows were synchronized, and their oocytes were harvested using the ovum pick-up method. High-quality oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF) procedures, utilizing a culture medium containing, no supplements (Group 1), 100 μM of vitamins E (Group 2) and C (Group 3), along with 50 μM of coenzyme Q10 (Group 4). The ensuing zygotes were cultured, and the ensuing embryos were evaluated for blastocyst formation by the seventh day. An analysis of the blastocysts' inner cell mass (ICM) and trophectoderm (TE) cells was also conducted. The findings revealed that the group receiving supplementation of vitamin E and coenzyme Q10 exhibited significantly higher maturation and cleavage rates in comparison to both the control and the vitamin C groups. Furthermore, the count of ICM, TE, and blastocyst cells was notably elevated in the vitamin E supplemented group when compared to the control group. In summary, the effectiveness of vitamin E in enhancing IVM, IVF, and embryo development under conditions of heat stress surpassed that of vitamin C and coenzyme Q10., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

3-Aryl-4-aminomaleimides have well-demonstrated applications, such as being used as fluorophores and inhibitors. However, their previous synthesis methods have involved tedious multi-step procedures or methods that need pre-functionalized maleimides and toxic or unstable reagents. Here, a feasible method is developed to synthesize these useful compounds. This includes the one-pot preparation of 3-aminomaleimides, followed by their direct arylation through a palladium-catalyzed Heck reaction with various aryl iodides regioselectively at the β-position of their amine substituents. The results show that this method efficiently exhibits a broad scope.

An imbalance between omega-6 and omega-3 fatty acids in sperm has been linked with lipid peroxidation and DNA damage in sperm, indicating a possible correlation to fertility potential. This cross-sectional study involved 56 infertile men (aged 25-45), and assessed the relationship between the omega-6 to omega-3 fatty acid ratio in sperm and seminal plasma with sperm DNA fragmentation. Individuals were categorized based on high or low levels of sperm DNA fragmentation according to two tests (TUNEL and SCSA assay less or greater than 10 and 30%, respectively), and their fatty acid composition, as well as sperm functional tests, were analyzed. Results showed that men with high DNA fragmentation exhibited higher percentages of total saturated, monounsaturated, and omega-6 to omega-3 fatty acid ratios in both sperm (P < 0.001) and seminal plasma (P < 0.001) compared to men with low DNA fragmentation. The percentage of sperm lipid peroxidation, and residual histone (P < 0.05) were higher, while the percentage of sperm motility (P < 0.001) was lower in the former compared to the latter group. Moreover, Pearson's correlation revealed positive associations between the omega-6 to omega-3 fatty acid ratio with sperm lipid peroxidation, DNA fragmentation, and residual histones in both sperm and seminal plasma. Overall, these observations suggest that consumption of omega-3 fatty acids may be related to male fertility potential, as it appears that individuals with a high percentage of omega-3 fatty acids have better sperm quality compared to men with a lower omega-3 fatty acid., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Generating stable livestock pluripotent stem cells (PSCs) can be used for complex genome editing, cellular agriculture, gamete generation, regenerative medicine and in vitro breeding schemes. Over the past decade, significant progress has been made in characterizing pluripotency markers for livestock species. In this study, we investigated embryo development and gene expression of the core pluripotency triad (OCT4, NANOG, SOX2) and cell lineage commitment markers (REX1, CDX2, GATA4) in the presence of three small molecules and their combination [PD0325901 (FGF inhibitor), SB431542 (TGFβ inhibitor), and CHIR99021 (GSK3B inhibitor)] from day 2-7 post-insemination in goat. Significant reduction in rate of blastocyst formation was observed when SB was used along with PD or CHIR and their three combinations had more sever effect. SB and CHIR decreased the expression of SOX2 while increasing the GATA4 expression. PD decrease the relative expression of NANOG, OCT4 and GATA4, while increased the expression of REX1. Among the combination of two molecules, only SB + CHIR combination significantly decreased the expression of GATA4, while the combination of the three molecules significantly decreases the expression of NANOG, SOX2 and CDX2. According to these results, the inhibition of the FGF signaling pathway, by PD may lead to blocking the hypoblast formation as observed by reduction of GATA4. OCT4 and NANOG expressions did not show signs of maintenance pluripotency. GATA4, NANOG and OCT4 in the PD group were downregulated and REX1 as EPI-marker was upregulated thus REX1 may be considered as a marker of EPI/ICM in goat., Competing Interests: Declaration of competing interest We confirm this manuscript is currently not being submitted elsewhere and all author is in complete agreement with the contents of this manuscript. In addition, Authors have no conflict of interest to disclose., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A base/solvent controlled divergent synthesis for the construction of polycyclic hydrocarbons has been developed. In this process, norbornene through a reagent role leads to the synthesis of norbornane-fused dihydrophenanthrenes, which are essential due to their biological activities. Amazingly, by switching solvent and base, the role of norbornene becomes limited to a mediator/catalyst; therefore, it is removed from the final scaffold, and triphenylenes are regioselectively synthesized. Additionally, by removing norbornene from the reaction conditions, a different path leading to synthesis of unsymmetrically substituted triphenylenes with exceptional regioselectivity is established. This reaction includes a rare domino decarboxylation/C-H activation/annulation in a chemo- and regioselective manner.

Follicular fluid (FF) advanced glycation end products (AGEs) have been associated with low oocyte quality and number, low fertilization rate, impaired embryonic development and low pregnancy rate. These findings are especially relevant in women undergoing in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), ie, assisted reproductive technology (ART). A systematic literature search was conducted to examine various AGEs including pentosidine, carboxymethyl-lysine (CML), methylglyoxal 5-hydro-5-methylimidazolones (MG-H1), toxic AGE (TAGE), and soluble receptor for AGE (sRAGE) with ART outcomes. Studies showed that total AGEs and sRAGE in FF were associated with the ovarian response, follicle number, retrieved oocyte number, mature (MII) oocyte number, fertilization rate, embryo number, embryo quality, and successful pregnancy. Although FF AGEs could be considered predictive biomarkers, population heterogeneity and differences in ovulation induction protocols make the findings less clear. This review highlights important role of AGEs in ART and necessity of evaluating AGEs in serum vs with FF to better predict ART outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In the past decade, great strides have been made to quantify the dynamics of single-cell growth and division in microbes. In order to make sense of the evolutionary history of these organisms, we must understand how features of single-cell growth and division influence evolutionary dynamics. This requires us to connect processes on the single-cell scale to population dynamics. Here, we consider a model of microbial growth in finite populations which explicitly incorporates the single-cell dynamics. We study the behavior of a mutant population in such a model and ask: can the evolutionary dynamics be coarse-grained so that the forces of natural selection and genetic drift can be expressed in terms of the long-term fitness? We show that it is in fact not possible, as there is no way to define a single fitness parameter (or reproductive rate) that defines the fate of an organism even in a constant environment. This is due to fluctuations in the population averaged division rate. As a result, various details of the single-cell dynamics affect the fate of a new mutant independently from how they affect the long-term growth rate of the mutant population. In particular, we show that in the case of neutral mutations, variability in generation times increases the rate of genetic drift, and in the case of beneficial mutations, variability decreases its fixation probability. Furthermore, we explain the source of the persistent division rate fluctuations and provide analytic solutions for the fixation probability as a multispecies generalization of the Euler-Lotka equation.

Three-dimensional (3D) printing technology has become an advanced approach for fabricating patient-specific scaffolds with complex geometric shapes to replace damaged or diseased tissue. Herein, polylactic acid (PLA)-Baghdadite (Bgh) scaffold were made through the fused deposition modeling (FDM) 3D printing method and subjected to alkaline treatment. Following fabrication, the scaffolds were coated with either chitosan (Cs)-vascular endothelial growth factor (VEGF) or lyophilized Cs-VEGF known as PLA-Bgh/Cs-VEGF and PLA-Bgh/L.(Cs-VEGF), respectively. Based on the results, it was found that the coated scaffolds had higher porosity, compressive strength and elastic modulus than PLA and PLA-Bgh samples. Also, the osteogenic differentiation potential of scaffolds following culture with rat bone marrow-derived mesenchymal stem cells (rMSCs) was evaluated through crystal violet and Alizarin-red staining, alkaline phosphatase (ALP) activity and calcium content assays, osteocalcin measurements, and gene expression analysis. The release of VEGF from the coated scaffolds was assessed and also the angiogenic potential of scaffolds was evaluated. The sum of results presented in the current study strongly suggests that the PLA-Bgh/L.(Cs-VEGF) scaffold can be a proper candidate for bone healing applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

A palladium (II)-catalyzed borono- ortho -CH activation/amination cascade for the construction of two C-N bonds and one C-C bond in a single synthetic sequence is reported. This method proceeds through a formal syn -carbopalladation of alkynes with organoboron compounds, forming alkenyl palladium species, which are trapped by simple amines to provide highly substituted indoles. Remarkably, with an electron-rich arylboronic acid, the reaction proceeds through an unexpected anti -carbopalladation terminated by ortho -CH activation of diarylalkyne/amination reaction to provide an unsymmetrically substituted 2,3-diaryl indole instead. In the follow-up chemistry, we demonstrate that urea participates in this cascade to offer a variety of free NH-indoles.

The specific role of the canonical WNT/β-catenin signaling pathway during the preimplantation development of goat remains unclear. Our objective was to investigate the expression of β-CATENIN, one of the critical components of Wnt signaling pathway, in IVF embryos and compare it with SCNT embryos in goat. In addition, we evaluated the consequence of inhibition of β-catenin using IWR1. Initially, we observed cytoplasmic expression of β-CATENIN in 2 and 8-16 cell stage embryos and membranous expression of β-CATENIN in compact morula and blastocyst stages. Furthermore, while we observed exclusively membranous localization of β-catenin in IVF blastocysts, we observed both membranous and cytoplasmic localization in SCNT blastocysts. We observed that Inhibition of WNT signaling by IWR1 during compact morula to blastocyst transition (from day 4 till day 7 of in vitro culture) increased blastocyst formation rate in both IVF and SCNT embryos. In conclusion, it seems that WNT signaling system has functional role in the preimplantation goat embryos, and inhibition of this pathway during the period of compact morula to blastocyst transition (D4-D7) can improve preimplantation embryonic development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sadeghi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Previous studies described aberrant nuclear reprogramming in somatic cell nuclear transfer (SCNT) embryos that is distinctly different from fertilized embryos. This abnormal nuclear reprogramming hampers the proper pre- and/or post-implantation development. It has been demonstrated that SCNT blastocysts aberrantly expressed POU5F1 and POU5F1-related genes. With regard to this, it has been postulated that promoting the expression of POU5F1 in SCNT embryos may enhance reprogramming in SCNT embryos. In this study, we treated either fibroblast donor cells or SCNT embryos with OAC1 as a novel small molecule that has been reported to induce POU5F1 expression. Quantitative results from the MTS assay revealed that lower concentrations of OAC1 (1, 1.5, and 3 μM) are non-toxic after 2, 4, and 6 days, but higher concentrations (6, 8, 10, and 12 μM) are toxic and reduced the proliferation of cells after 6 days. No enhancement in the expression of endogenous POU5F1 was observed when both mouse and bovine fibroblast cells were treated with 1.5 and 3 μM OAC1 for up to 6 consecutive days. Subsequently, we treated either fibroblast as donor cells in the SCNT procedure (BFF-OAC1 group) or SCNT embryos [for 4 days (IVC-OAC1: D4-D7 group) or 7 days (IVC-OAC1: D0-D7 group)] with 1.5 μM OAC1. We observed that neither treatment of fibroblast donor cells nor SCNT embryos improved the cleavage and blastocyst rates. Interestingly, we observed that treatment of SCNT embryos all throughout the in vitro culture (IVC) (IVC-OAC1: D0-D7) with 1.5 μM OAC1 improves the quality of derived blastocyst which was indexed by morphological grading, blastomere allocation, epigenetic marks and mRNA expression of target genes. In conclusion, our results showed that supplementation of IVC medium with 1.5 μM OAC1 (D0-D7) accelerates SCNT reprogramming in bovine species., Competing Interests: Declaration of competing interest There is no conflict of interest in this study., (Copyright © 2022. Published by Elsevier Inc.)

The one carbon metabolism (OCM) has a primary role in the process of oocyte maturation. In this study bovine oocytes were cultured for 24 h, up to MII stage, with standard medium supplemented or not with 8 metabolic enhancers of the OCM and the MII and blastocyst rate were compared. Additional analyses were performed on matured oocytes, cumulus cells, zygotes and blastocysts. The OCM supplementation increased the blastocyst rate derived from in vitro fertilization. The mitochondrial mass and DNMT3a protein expression were increased whereas DNA fragmentation decreased in matured oocytes. DNA methylation in female pronucleus of zygotes was increased. The supplementation did not directly affect the redox balance as ROS and GSH in matured oocytes and homocysteine in the spent medium were unchanged. The supplementation of the oocytes with metabolic enhancers of the OCM may increase the yield from the culture, likely due to improved DNA methylation and epigenetic programming. The lack of effects on MII rate with huge differences appearing at the blastocyst stage suggest the existence of a OCM metabolic check point that hampers oocytes progression to blastocyst post-fertilization, if they were not properly primed at the time of maturation., (© 2022. The Author(s).)

SCNT embryos suffer from poor developmental competence (both in vitro and in vivo) due to various defects such as oxidative stress, incomplete epigenetic reprogramming, and flaws in telomere rejuvenation. It is very promising to ameliorate all these defects in SCNT embryos by supplementing the culture medium with a single compound. It has been demonstrated that melatonin, as a multitasking molecule, can improve the development of SCNT embryos, but its function during ovine SCNT embryos is unclear. We observed that supplementation of embryonic culture medium with 10 nM melatonin for 7 days accelerated the rate of blastocyst formation in ovine SCNT embryos. In addition, the quality of blastocysts increased in the melatonin-treated group compared with the SCNT control groups in terms of ICM, TE, total cell number, and mRNA expression of NANOG. Mechanistic studies in this study revealed that the melatonin-treated group had significantly lower ROS level, apoptotic cell ratio, and mRNA expression of CASPASE-3 and BAX/BCL2 ratio. In addition, melatonin promotes mitochondrial membrane potential and autophagy status (higher number of LC3B dots). Our results indicate that melatonin decreased the global level of 5mC and increased the level of H3K9ac in the treated blastocyst group compared with the blastocysts in the control group. More importantly, we demonstrated for the first time that melatonin treatment promoted telomere elongation in ovine SCNT embryos. This result offers the possibility of better development of ovine SCNT embryos after implantation. We concluded that melatonin can accelerate the reprogramming of telomere length in sheep SCNT embryos, in addition to its various beneficial effects such as increasing antioxidant capacity, reducing DNA damage, and improving the quality of derived blastocysts, all of which led to a higher in vitro development rate., Competing Interests: The authors have declared that no competing interests exist.

Despite significant advances in surgery and postoperative care, there are still challenges in the treatment of wounds. In the current study, a freeze-dried chitosan (Cs)/polyvinylpyrrolidone (PVP) sponges containing platelet-rich fibrin (PRF at 1, 1.5 and 2% w/v) for wound dressing application is fabricated and fully characterized. Addition of 1% w/v of PRF to Cs/PVP (CS/PVP/1PRF) sample significantly increased the tensile strength (from 0.147 ± 0.005 to 0.242 ± 0.001 MPa), elastic modulus (from 0.414 ± 0.014 to 0.611 ± 0.022 MPa) and strain at break (from 53.4 ± 0.9 to 61.83 ± 1.17%) compared to Cs sample, and was hence selected as the optimal sample. The antibacterial activity of Cs/PVP/1PRF sponge wound dressing against E. coli and S. aureus was confirmed to be effective. Enzyme-linked immunosorbent assays revealed that the release of both VEGF and PDGF-AB from PRF powder, as well as PDGF-AB from Cs/PVP/1PRF sample was time-independent, but the release of VEGF from Cs/PVP/1PRF sample increased significantly with time. According to MTT and CAM assays, the Cs/PVP/1PRF sample significantly increased proliferation and angiogenic potential, respectively. Furthermore, in vivo studies demonstrated a 97.16 ± 1.55% wound closure for Cs/PVP/1PRF group after 14 days., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Developmental competence of in vitro matured cumulus oocyte complexes (COCs) in conventional IVM (C.IVM) is lower than in vivo maturated COCs and is related to unsynchronized nuclear and cytoplasmic maturation. To overcome this dearth, COCs can be exposed to granulosa secreted factors in a two-step system. Therefore, in the first experiment, 1000 nM of C-type natriuretic peptide for 8 h was determined (CAPA), as the best time and concentration to retain oocytes in germinal vesicle stage. This condition, also reduces lipid droplets and increases the expression of ATGL and PLIN2 involved in lipolysis and lipogenesis, respectively. In the second experiment, maturation was stimulated with prostaglandin E2 and amphiregulin for 18 h (CAPA-IVM), and their optimal concentrations based on blastocyst formation rates through in vitro fertilization (IVF) were determined as 1 and 600 nM, respectively. In the third experiment, the in vitro and in vivo developmental competency of SCNT embryos in CAPA-IVM group were determined. Despite similar blastocyst formation rates in IVF and SCNT between CAPA-IVM and C.IVM, the quality of blastocysts were quality was higher in CAPA-IVM, which reflected itself, as higher ICM/TE ratio and also expression of NANOG in SCNT blastocysts. Pregnancy rate, live births rate and SCNT efficiency were not significant between CAPA-IVM and C.IVM groups. Therefore, CAPA-IVM can improve the developmental competency of SCNT derived embryos., (© 2022. The Author(s).)

A facile and efficient synthetic approach to various valuable 3-aryl- and 3-aroylcoumarins by the direct arylation and aroylation of coumarins with glyoxals in a metal-free manner is presented. The aryl glyoxal is for the first time recognized to serve as an aryl surrogate in addition to its role as an aroyl transfer reagent via a simple switch in reaction conditions. The approach accommodates a broad substrate scope and high yields of the two types of cross-coupling reactions starting from identical starting materials.

Recently, nanofibrous structures have shown great potential for a wide range of medical applications. The aim of the current study was to evaluate the wound healing process using Polycaprolactone/Keratin/Platelet-rich fibrin (PCL/Kr/PRF) fibrous scaffold fabricated through electrospinning process. A range of techniques were utilized to fully characterize the chemical, physical and biological properties of the resultant structure. Results revealed that by the addition of only 0.5%w/v PRF to PCL/Kr (PCL/Kr/0.5PRF) sample, the fibers diameter decreased from 193.93 ± 64.80 nm to 65.98 ± 14.03 nm, and the stress at break demonstrated a 18.27% increase in comparison to the PCL sample (from 2.90 ± 0.80 MPa to 3.43 ± 0.90 MPa). The PCL/Kr/0.5PRF scaffold showed more antibacterial activity against gram-positive and gram-negative bacteria than PCL/Kr sample. Based on enzyme-linked immunosorbent assays, the PCL/Kr/0.5PRF sample revealed an independent release of VEGF and PDGF for 7 days. Cell viability studies demonstrated non-cytotoxic nature of PRF-containing dressings. Also, chorioallantoic membrane (CAM) assay was performed to evaluate the angiogenic potential of the wound dressings. The in vivo assessments also showed that PCL/Kr/0.5PRF accelerated the wound healing process in terms of collagen deposition and the formation of skin appendages which was comparable to the normal skin. Overall, the data presented in this study greatly suggest that the PCL/Kr/0.5PRF wound dressing could be a suitable candidate for wound healing and skin regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Background: Somatic cell nuclear transfer (SCNT) is an approach for the propagation of elite animals. In vitro condition, especially the composition of culture media has a profound effect on the developmental competency of in vitro derived e mbryos. There are limited studies evaluating the effect of culture media on SCNT outcomes. To address this gap, we compare the effect of two culture media synthetic oviduct fluid (SOF) vs. commercial bracket-oliphant (BO) on developmental comptenecy., Materials and Methods: In this experimental study, embryos derived from in vitro fertilized (IVF) and SCNT were cultured in both BO and SOF media for 7 days. In addition to the assessment of cleavage and blastocyst on day 3, and 7, the quantitative expression of 16 genes in theresultant blastocysts were assessed. The resultant SCNT blastocysts from SOF and BO groups were also transferred to the synchronized recipient for developmental competency to term., Results: The blastocyst rate in the BO medium was significantly higher than that of the SOF medium in the SCNT group (P<0.05). All of the examined genes showed increased expression levels in SCNT blastocyst in both media compared to IVF Blastocyst. In the IVF group, Oct4, Bmpr1 , and Gcn5 showed significantly higher expression in the SOF medium compared to the BO medium while Akt, Fgfr4, Sox2 showed significantly lower expression in the SOF medium compared to the BO medium. In the SCNT group, Fgfr4, Gcn5, Fzd, Ctnnb, Bmpr1 , and Fgfr4 showed significantly higher expression in SOF compared to BO derived blastocyst., Conclusion: It appears that in SCNT blastocysts, gene regulation is less controlled compared to IVF ones, irrespective of the type of medium. In addition, there are differences regarding certain genes expressions between IVF and SCNT derived blastocysts between SOF and BO, reiterating that culture composition affects developmental competency and gene expression., Competing Interests: The authors declare no conflicts of interest., (Copyright© by Royan Institute. All rights reserved.)