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C25H22OS, monoclinic, P1211 (no. 4), a = 9.679(2) Å, b = 13.663(2) Å, c = 15.348(2) Å, β = 106.233(5)°, V = 1948.7 Å3, Z = 4, Rgt(F) = 0.047, wRref(F2) = 0.119, T = 210 K.

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]

Byline: G. Karimi, F. Jafarpour, M. Binazadeh, X. Li Water management is one of the most critical issues for high-performance Polymer Electrolyte Membrane (PEM) fuel cells. A flow network model was developed to study the possibility of two-phase flow inception along the gas flow channels in the cathode side of a PEM fuel cell stack. The conventional Darcy-Weisbach equation and homogeneous flow model are used to estimate the pressure distributions in the network. The corresponding species concentrations as well as the thermodynamic quality distributions are also calculated. The simulation results have revealed that under certain operating conditions, the quality is lower than one for parts of the stack. However, the amount of the liquified water in the stack is not significant to cause a major pressure drop or an appreciable change in the O2 concentration. Effects of current density and channel diameter on the system quality are also investigated.

In this study, the half-metallic properties, thermodynamic stability and optical parameters of the full-Heusler Co2MnGa compound and its four different terminations of Co–Co, Co–Mn, Mn–Ga and Co–Ga from the surface of Co2MnGa (001) have been calculated based on the density functional theory (DFT). The results confirm the ferromagnetic half-metallic behavior with a magnetic moment of 4.08 μ B and a gap of 0.32 eV at the Fermi level of Co2MnGa bulk phase having a Cu2MnAl-type structure. The density of states curves showed that all possible terminations from the Co2MnGa (001) surface eliminate the half-metallic behavior except the termination of Mn–Ga case. Moreover, the results indicate that the termination of Mn–Ga with the lowest surface energy is the most stable termination for the application in spintronics. The optical coefficients such as real and imaginary dielectric function, refraction, extinction, energy loss function, optical conductivity and reflections of the bulk and Mn–Ga termination have been calculated and compared. [ABSTRACT FROM AUTHOR]

In this study, the effects of inoculation of two lactic acid bacteria including Pediococcus pentosaceu and Lactobacillus plantarum were evaluated on the fermented fish sausages produced from Common carp, as a model, at incubation temperature of 35˚C. Common carp fermented sausages were prepared from minced fish, salt (3%), glucose (3%) and the combination of two starter cultures of bacteria (6 log CFU/g) and fermentation occurred during the 48 h incubation. Soluble peptides, pH, phsycrophilic and lactic acid bacterial counts, proximate composition (protein, lipid, moisture and ash), texture profile analysis and color of fermented sausages were measured and compared to the non-inoculated sausages (the control group) at 0, 24 and 48 h after incubation. According to the results, lactic acid bacteria showed rapid growth after 48 h fermentation which led to the significant decrease in pH from initial values of around 6.9 to 4.4 (P

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).)