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Pigs are pivotal in agriculture and biomedical research and hold promise for xenotransplantation. Specific-pathogen-free (SPF) herds are essential for commercial swine production and xenotransplantation research facilities. Commercial herds aim to safeguard animal health, welfare, and productivity, and research facilities require SPF status to protect immunocompromised patients. Somatic cell nuclear transfer (SCNT) embryos are the norm for producing cloned and genetically edited animals. Oocytes for embryo reconstruction are most conveniently sourced from commercial abattoirs with unclear disease statuses. However, research on viral clearance from donor oocytes during embryo reconstruction remains limited. SCNT has previously been shown to reduce the transmission of Porcine reproductive and respiratory syndrome virus, Bovine viral diarrhea virus, Porcine Circovirus type 2, and Porcine parvovirus. Still, it is lacking for other pathogens, including endogenous viruses. This project contains two preliminary studies investigating the polymerase chain reaction (PCR) assay detection of common swine viruses through the phases of producing parthenogenic and SCNT embryos. Exogenous pathogens detected in oocyte donor tissue or the oocyte maturation media were not detected in the produced embryos. Porcine endogenous retrovirus type C (PERVC) was not removed by parthenogenic embryo activation and was detected in 1 of the 2 tested SCNT embryos reconstructed using a PERVC-negative cell line. SCNT and parthenogenic embryo construction similarly reduced exogenous virus detection. SCNT embryo construction helped reduce endogenous virus detection. This project demonstrates the importance of screening embryos for endogenous viruses and shows the usefulness of parthenogenic embryos in future exogenous virus clearance studies. [ABSTRACT FROM AUTHOR]

Anthocyanin content determines fruit color, which serves as a significant indicator for assessing the commercial value of fruits. The biosynthesis of anthocyanin is regulated by MYB transcription factors, however, the precise mechanism underlying the negative regulation of MYB on anthocyanin synthesis in pear fruit remains to be fully elucidated. Based on the previous transcriptomic data, a member of the MYB gene family, PbrMYB19, was screened out from the differentially expressed genes, suggesting that it may be involved in the regulation of fruit anthocyanin biosynthesis. To clarify its structure and function, and lay the foundation for further elucidating the regulatory mechanism of PbrMYB19 on fruit anthocyanins, in this study, the full-length PbrMYB19 was cloned by using the cDNA of Yuluxiang pear peel as a template. The structure, genetic relationship, conserved domain, and cis-acting elements of this gene were predicted via bioinformatics software. The expression level of PbrMYB19 gene in peels with different colors was analyzed by fluorescence quantitative PCR, and its function was verified by the fruit transient expression system. The results showed that the open reading frame of PbrMYB19 gene was 714 bp, encoding 237 amino acids, with a relative molecular weight of 26.85 ku, and the subcellular localization was predicted in the nucleus. Phylogenetic tree analysis showed that PbrMYB19 was on the same evolutionary branch as the reported anthocyanin transcription inhibitors FaMYB1 and PbrMYB120, and there was an EAR-like inhibitory sequence at the C-terminal of its protein. In addition, the promoter sequence of PbrMYB19 gene contained light regulation elements and plant hormone response elements. QRT-PCR analysis showed that the expression level of PbrMYB19 gene in red and yellow peels was significantly higher than that in green peels. Overexpression vector of PbrMYB19 gene was constructed, and the fruit was transiently transformed. It was found that PbrMYB19 overexpression significantly inhibited anthocyanin accumulation in peels. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a powerful tool that post‐transcriptionally silences target genes in eukaryotic cells. However, silencing efficacy varies greatly among different insect species. Recently, we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA injection. The disappearance of double‐stranded RNA (dsRNA) could be a potential factor that restricts RNAi efficiency. Here, we found that dsRNA can be degraded in midgut fluids, and a dsRNase of A. lucorum (AldsRNase) was identified and characterized. Sequence alignment indicated that its 6 key amino acid residues and the Mg2+‐binding site were similar to those of other insects' dsRNases. The signal peptide and endonuclease non‐specific domain shared high sequence identity with the brown‐winged green stinkbug Plautia stali dsRNase. AldsRNase showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle, with peaks at the 4th instar ecdysis in the whole body. The purified AldsRNase protein obtained by heterologously expressed can rapidly degrade dsRNA. When comparing the substrate specificity of AldsRNase, 3 specific substrates (dsRNA, small interfering RNA, and dsDNA) were all degraded, and the most efficient degradation is dsRNA. Subsequently, immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut cells. Through cloning and functional study of AldsRNase, the enzyme activity and substrate specificity of the recombinant protein, as well as the subcellular localization of nuclease, the reason for the disappearance of dsRNA was explained, which was useful in improving RNAi efficiency in A. lucorum and related species. [ABSTRACT FROM AUTHOR]

This article will critically appraise the extent to which new developments in the fields of reproductive technology are shown to impact female bodily autonomy and reproductive choice in Eva Hoffman's novel The Secret. The Secret pushes its readers towards the more pressing and urgent questions arising from ongoing developments within the field of NRT and human cloning in a neoliberal climate. The novel cautions that, ultimately, the individual right to reproductive choice is never completely free; an awareness of external influences and a consideration of possible repercussions is integral to responsible decision-making in the context of NRT and cloning. However, the novel moves towards a possible reconceptualization of NRTs as part of the evolutionary progress of humankind. In returning to the body and biopolitical figurations, this article sees the novel's protagonist, Iris, and her emergent cyborg identity as a manifestation of Haraway's monstrous cyborg replete with possibility. [ABSTRACT FROM AUTHOR]

In this study, we developed a method for the expression of the antimicrobial peptide SE-33-A2P in E. coli bacterial cells. The SE-33-A2P peptide consists of A2P and SE-33 peptides and is a retro analog of cathelicidin possessing antimicrobial activity against both Gram-positive and Gram-negative bacteria. Furthermore, the A2P peptide is a self-cleaving peptide. For an efficient expression of the SE-33-A2P peptide, a gene encoding several repetitive sequences of the SE-33 peptide separated by A2P sequences was created. The gene was cloned into a plasmid, with which E. coli cells were transformed. An induction of the product expression was carried out by IPTG after the cell culture gained high density. The inducible expression product, due to the properties of the A2P peptide, was cleaved in the cell into SE-33-A2P peptides. As the next step, the SE-33-A2P peptide was purified using filtration and chromatography. Its activity against both Gram-positive and Gram-negative bacteria, including antibiotic-resistant bacteria, was proved. The developed approach for obtaining a prokaryotic system for the expression of a highly active antimicrobial peptide expands the opportunities for producing antimicrobial peptides via industrial methods. [ABSTRACT FROM AUTHOR]

Viewers' responses to Orphan Black (2013–2017), a popular, genetics-themed sci-fi television series, reveal much about public understanding of the ethical challenges associated with genetic science. In this article, we assess how fans of Orphan Black process the bioethical themes that are prominent in the show through an analysis of 182 viewer-created blog posts. Using a mixed methods approach, our findings reveal that Orphan Black 's fans distill the essence of the show down to its characters' fight for autonomy. Furthermore, fan blogs reveal two notable pathways through which this bioethical principle is explored: gender and reproduction. Viewers draw striking connections between the moral problems they observe on screen in Orphan Black and those they see in the real world—both today and in a possible future—particularly as those problems affect women. While existing scholarship acknowledges these themes in the show itself, our approach demonstrates science fiction fans' active participation in meaning-making and bioethical reasoning and offers a novel approach to studying fan-generated content for public understanding of science research. [ABSTRACT FROM AUTHOR]

The genus Eucalyptus has shown great potential for the establishment of commercial forest plantations, it is one of the most widely planted worldwide, it is planted in different regions of the planet, which indicates its ability to adapt to various environmental conditions. Eucalyptus urophylla is a species of this genus that adapts to tropical and subtropical regions, in suitable sites it can achieve high growth rates. It is one of the species with the most research in breeding programs in the world, mainly due to its capacity to hybridize naturally, since it generates populations with high genetic diversity, coupled with its ability to reproduce asexually. In Mexico, the breeding programs for Eucalyptus urophylla are in the initial phase, although plantations are already managed under clonal silviculture, controlled crossing are just beginning, so pollen management and crossing techniques are the most important activities in these programs. Since there is currently no information about the management of pollen for Eucalyptus urophylla in Mexico, the aim of this paper is to show a methodology based on the information documented in other parts of the world and the recent experience acquired in the collection and management of pollen, as well as the controlled pollination techniques recently employed in the breeding programs of Eucalyptus urophylla in southeastern Mexico. [ABSTRACT FROM AUTHOR]

Characterization of T-cell receptors (TCRs) repertoire was revolutionized by next-generation sequencing technologies; however, standardization using biological controls to facilitate precision of current alignment and assembly tools remains a challenge. Additionally, availability of TCR libraries for off-the-shelf cloning and engineering TCR-specific T cells is a valuable resource for TCR-based immunotherapies. We established nine human TCR α and β clones that were evaluated using the 5′-rapid amplification of cDNA ends-like RNA-based TCR sequencing on the Illumina platform. TCR sequences were extracted and aligned using MiXCR, TRUST4, and CATT to validate their sensitivity and specificity and to validate library preparation methods. The correlation between actual and expected TCR ratios within libraries confirmed accuracy of the approach. Our findings established the development of biological standards and library of TCR clones to be leveraged in TCR sequencing and engineering. The remaining human TCR clones' libraries for a more diverse biological control will be generated. [ABSTRACT FROM AUTHOR]

This study examines the shifting meaning of subjectivity and the blurred boundaries between humans and nonhumans. Additionally, it explores the impact of technology intervention on gender, drawing from the principles of posthumanism philosophy. Cloning is one of the issues highlighted in the posthuman realm. This study seeks to examine the incorporation of posthumanism and technofeminism in the character Tomie in the anime Junji Ito Collection (2018, Itô Junji "Korekushon"), with the intention of challenging the sexualization of shôjo while remaining skeptical using the narrative analysis method. The results and findings show that Tomie presents a posthuman idea centered on her super regeneration ability and cloned creations to defy the limits of humanity. This idea also promotes elements of technofeminism that lead to the use of biotechnology related to reproduction. Nevertheless, in the end, Ito tries to invite the audience to remain critical in responding to the issue of technofeminism, both in terms of ethics and objectivity. [ABSTRACT FROM AUTHOR]

This paper covers developments from International Embryo Technology Society (IETS) meetings over the past 50 years. The IETS was officially 'born' at a meeting in Denver, Colorado on Sunday 26 May 1974. There have been 51 IETS meetings (the first was in May 1974), and the first conference at which papers were presented was in 1975. The name of the IETS was changed from International Embryo Transfer Society to International Embryo Technology Society in 2016. The annual IETS conferences are held once a year in January. Embryo technology advanced from the laborious and slow techniques of surgical recovery and transfer of bovine embryos to non-surgical recovery and transfer in many species. Cryopreservation of embryos was initially a slow process that met with only moderate technical success before the development of technology that resulted in high embryo survival rates. The polymerase chain reaction was used to determine the sex of embryos following laminar flow cytology, which came into use to separate X- and Y-bearing spermatozoa. IVF has grown to become widely used, and several factors make it more useful than superovulation and embryo recovery/transfer. Perhaps the most important tool of all was the system that allowed the genetic 'mapping' of the genomic sequence of Bos taurus cattle. Charting key DNA differences by scientists, 'haplotypes' are now used routinely to identify animals with desirable traits of economic importance, discover new genetic disorders, and track carrier status of genotyped animals. Future technology is discussed. This paper deals with the 50-year history of embryo transfer technology. It covers the formation of the International Embryo Technology Society, a group of scientists and veterinarians who study or work in the field of embryos and the animals that produce them. These embryos can be frozen, moved from animal to animal, maintained outside the female, and modified by a number of procedures. Photograph by Jenson. [ABSTRACT FROM AUTHOR]

Objective Most of the garlic clones are non-bolting and sterile and propagate asexually; however, some of the clones are bolting, and there are some fertile clones among the bolting ones. A correct understanding of the MADS-BOX genes family expression pattern and structure, including AGL6, is a valuable project and will improve garlic breeding programs. So far there are no reports relating AGL6 identification as a flowering and fertility integrator gene in garlic. So the aim of this study is to investigate the relative expression of AGL6 in Iranian bolting, semi- and non)bolting garlic clones and to identify its structure and nucleotide sequence. Materials and methods At first, the relative expression of the AGL6 was investigated in the different organs of non)bolting, semi-bolting and bolting Iranian garlic clones using quantitative Real time- PCR. Then the isolation and cloning of the AGL6 partial sequences from the garlic floret was carried out using RT-PCR. The sequence of the recombinant plasmid was evaluated using BLAST, Vector NTI, ORF finder and MEGA6 software. Results According to the obtained results, AGL6 was expressed in the meristem, inflorescence, tepal and carpel of green and purple flowers of the bolting garlic. But there was no sign of its expression in the stamen. AGL6 expression level in the meristem of bolting garlic was higher than the meristem of the semi-bolting and non-bolting garlic in all sampling stages, especially in the flowering induction stage. AGL6 was also expressed in the inflorescence of the semi-bolting and bolting garlic, but its expression in the inflorescence of the semi- bolting garlic was 4 times less than the inflorescence of the bolting garlic. Sequence analysis of the colonized fragment showed the coding region with a 728 bp length, which was registered in the NCBI database with the accession number of OK086759.1. This nucleotide sequence encodes a protein with 243 amino acids and has MADS-box and K-box domains. Results showed the high similarity of the sequence obtained in this research with other AGL6 gene homologues in other plant species, especially petaloid monocots such as onion, lily, narcissus and saffron in NCBI. Hence, the resulting sequence was named AsAGL6. Conclusions In this research, for the first time, the expression pattern of a key gene of the flowering and fertility pathway (AsAGL6) and its nucleotide structure were determined in garlic. The results of this research can be effectively used in the design of classical and molecular garlic breeding programs. [ABSTRACT FROM AUTHOR]

A manuscript published in the scientific journal "Nature" in 1996 reported on the cloning of sheep by somatic cell nuclear transfer (SCNT) into enucleated oocytes. The donor nuclei came from an established cell line, which in turn were derived from a sheep fetus. This method provided fundamental insights into understanding the molecular basis of cellular totipotency, pluripotency and the mechanisms underlying nuclear genome reprogramming. However, it was not until the birth of the cloned sheep "Dolly" that cloning came to the attention of the general public. However, new technologies often only unfold their impact in combination with other findings. In the case of cloning by somatic nuclear transfer, this is the combination with genome editing, as the combination of these two techniques enables the efficient generation of animals with edited genomes. This summary therefore aims to provide a comprehensive overview of the animals that have already been cloned in the past and the current status of edited farm animals in the context of commercial and pre-commercial applications. This overview will explicitly cover applications in the agricultural and food sector and does not claim to be exhaustive with regard to applications that serve biomedical purposes. In terms of content, the opportunities and risks associated with these technologies are addressed, with particular reference to the current legal framework in various countries around the world. [ABSTRACT FROM AUTHOR]

Enzymes used as feed additives increase the ability of animals to benefit from feed. From these enzymes, animals fed with lichenase enzyme can produce animal foods with high immunity and performance. Therefore, it is possible to obtain more healthy, high quality products in a shorter time. In this study, the recombinant vector pNW33N carrying the lichenase gene of Streptococcus bovis genome was transferred into Bacillus subtilis RSKK245 strain via electroporation technique. Besides, the DNA band of lichenase obtained from recombinant vector pNW33N/Lichenase after restriction endonucleases was observed on agarose gel. Enzymatic activity sites around B. subtilis RSKK245 colonies are shown by staining with Congo-red. The molecular weight of the enzyme was determined as 26 kDa via SDS-PAGE and zymogram analysis. This study has successfully demonstrated expression of lichenase gene in B. subtilis RSKK245 strain. [ABSTRACT FROM AUTHOR]

Background: The digital era has witnessed an escalating dependence on digital platforms for news and information, coupled with the advent of "deepfake" technology. Deepfakes, leveraging deep learning models on extensive data sets of voice recordings and images, pose substantial threats to media authenticity, potentially leading to unethical misuse such as impersonation and the dissemination of false information. Objective: To counteract this challenge, this study aims to introduce the concept of innate biological processes to discern between authentic human voices and cloned voices. We propose that the presence or absence of certain perceptual features, such as pauses in speech, can effectively distinguish between cloned and authentic audio. Methods: A total of 49 adult participants representing diverse ethnic backgrounds and accents were recruited. Each participant contributed voice samples for the training of up to 3 distinct voice cloning text-to-speech models and 3 control paragraphs. Subsequently, the cloning models generated synthetic versions of the control paragraphs, resulting in a data set consisting of up to 9 cloned audio samples and 3 control samples per participant. We analyzed the speech pauses caused by biological actions such as respiration, swallowing, and cognitive processes. Five audio features corresponding to speech pause profiles were calculated. Differences between authentic and cloned audio for these features were assessed, and 5 classical machine learning algorithms were implemented using these features to create a prediction model. The generalization capability of the optimal model was evaluated through testing on unseen data, incorporating a model-naive generator, a model-naive paragraph, and model-naive participants. Results: Cloned audio exhibited significantly increased time between pauses (P <.001), decreased variation in speech segment length (P =.003), increased overall proportion of time speaking (P =.04), and decreased rates of micro- and macropauses in speech (both P =.01). Five machine learning models were implemented using these features, with the AdaBoost model demonstrating the highest performance, achieving a 5-fold cross-validation balanced accuracy of 0.81 (SD 0.05). Other models included support vector machine (balanced accuracy 0.79, SD 0.03), random forest (balanced accuracy 0.78, SD 0.04), logistic regression, and decision tree (balanced accuracies 0.76, SD 0.10 and 0.72, SD 0.06). When evaluating the optimal AdaBoost model, it achieved an overall test accuracy of 0.79 when predicting unseen data. Conclusions: The incorporation of perceptual, biological features into machine learning models demonstrates promising results in distinguishing between authentic human voices and cloned audio. [ABSTRACT FROM AUTHOR]

Human-induced pluripotent stem cells (hiPSCs) grow as colonies of interconnected cells that undergo apoptosis after being enzymatically dissociated into single cells. This makes it challenging to generate single-cell hiPSC clones during genetic modification procedures. We hypothesized that activation of NOTCH via DLK-1 ligand might provide a survival signal for hiPSCs, and may promote the growth and survival of hiPSCs during single-cell cloning procedures. In the first experiment, we transfected plasmids containing the DLK-1 gene into hiPSCs. Through fluorescence microscopy, we found the DLK-1 plasmids did not express well, possibly because the DLK-1 gene was driven by a cytomegalovirus (CMV) promoter, which tends to undergo silencing in hiPSCs. We are working to address this issue by transferring the DLK-1 expression cassettes to a CAG expression vector which will not undergo silencing. In the second experiment, we treated hiPSCs with recombinant rsDLK-1 protein. We observed that rsDLK-1 led to a slight reduction in cell number, which was not caused by an increase in apoptosis but the results were not statistically significant. These results do not support or disprove our hypothesis that DLK-1 would enhance the growth and survival of hiPSCs. [ABSTRACT FROM AUTHOR]

The Forkhead box factor 1 (FoxO1) gene plays a vital role in the growth and development of skeletal muscle. In the present study, expression analysis of the bovine FoxO1 gene exhibited the highest expression in longissimus dorsi muscle followed by its expression in adipose tissue. Moreover, high mRNA expression of FoxO1 gene was found in differentiated bovine myoblasts and adipocytes at day 6 of induced differentiation (p < 0.05). The regulatory pattern of the bovine FoxO1 gene was investigated through screening and dual-luciferase activity of the 1.7 kb 5′UTR (untranslated region) within pGL3-basic vector and a core promoter region was explored at (-285/-27) upstream of the transcription start site. The transcription factors (TFs) MEF2A and HOXA5 within the core promoter region (-285/-27) were found as the regulatory cis-acting element. The siRNA interference of the TFs, chromatin immunoprecipitation (ChIP) assay, and site-directed mutation validated that MEF2A and HOXA5 binding occurs in the region -285/-27 bp and performs an essential role in the transcriptional regulation of bovine FoxO1 gene. These findings explored the regulatory network mechanism of the FoxO1 gene in skeletal muscle development and adipogenesis for the bovine breed improvement program. [ABSTRACT FROM AUTHOR]

Cloning and expression of the full-length endo-processive-type xyloglucanase from the Trichoderma reesei (TrXeg74A) fungus, as well as its catalytic domain TrXeg74A-CD, in the Penicillium verruculosum B1-537 recipient strain have been carried out. P. verruculosum is a highly effective producer of cellulases. The levels of protein secretion after culturing the obtained recombinant strains in a laboratory fermenter were 35.4 and 31.4 g/L, respectively. TrXeg74A accounted for at least 30% of the total protein, while TrXeg74A-CD was expressed to a much lesser extent. Both forms of the recombinant enzyme were isolated in purified state and their properties were studied. TrXeg74A and TrXeg74A-CD were characterized by a similar degree of processivity when exposed to tamarind xyloglucan and the same Michaelis constant (0.35-0.38 g/L), close to that for the native enzyme (0.30 g/L), while the catalytic constant for TrXeg74A-CD was 1.5 times higher than the corresponding parameter for full-length xyloglucanase. The obtained new recombinant P. verruculosum strains can be useful in the development of composite enzyme preparations for efficient hydrolysis of renewable lignocellulosic raw materials. [ABSTRACT FROM AUTHOR]

Deleted in azoospermia-like (DAZL) is essential for mammalian testicular function and spermatogenesis. To explore the molecular characterization, expression patterns, and cellular localization of the DAZL in Hezuo pig testes, testicular tissue was isolated from Hezuo pig at five development stages including 30 days old (30 d), 90 days old (90 d), 120 days old (120 d), 180 days old (180 d), and 240 days old (240 d). DAZL cDNA was first cloned using the RT-PCR method, and its molecular characterization was analyzed using relevant bioinformatics software. Subsequently, the expression patterns and cellular localization of DAZL were evaluated using quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry. The cloning and sequence analysis showed that the Hezuo pig DAZL cDNA fragment contained 888 bp open reading frame (ORF) capable of encoding 295 amino acid residues and exhibited high identities with some other mammals. The qRT-PCR and Western blot results indicated that DAZL was specifically expressed in Hezuo pig testes, and DAZL levels of both mRNA and protein were expressed at all five reproductive stages of Hezuo pig testes, with extremely significant higher expression levels in 90 d, 120 d, 180 d, and 240 d than those in 30 d (p < 0.01). Additionally, immunohistochemistry results revealed that DAZL protein was mainly localized in gonocytes at 30 d testes, primary spermatocytes, and spermatozoon at other developmental stages, and Leydig cells throughout five development stages. Together, these results suggested that DAZL may play an important role by regulating the proliferation or differentiation of gonocytes, development of primary spermatocytes and spermatozoon, and functional maintenance of Leydig cells in testicular development and spermatogenesis of Hezuo pig. Nevertheless, the specific regulatory mechanisms underlying these phenomena still requires further investigated and verified. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The objective of this study was to clone and express the hepatitis B surface antigen gene (HBsAg) in Escherichia coli (E. coli), thereby aiming to develop potential local therapeutics for combating Hepatitis B virus (HBV) infection in the Pakistani community by producing HBsAg in E. coli. MATERIALS AND METHODS: Blood serum samples were collected from hepatitis B-infected patients, and their genomic DNA was extracted. Real-time and nested polymerase chain reaction (PCR) was performed to amplify the HBsAg gene. The gene of interest was cloned into the pET20b expression vector and transformed into E. coli BL21 (DE3) using Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The gene’s precise size was confirmed with gene-specific external and internal primers (681 bp and 400 bp, respectively). RESULTS: The HBsAg gene was successfully sequenced and submitted to GenBank, exhibiting 98% homology with targeted HBV sequences worldwide. The expression of HBsAg protein was confirmed through silver staining, Coomassie staining, western blot, and dot blot analysis. CONCLUSIONS: The expressed protein clones are now available for further development as a local recombinant DNA vaccine to prevent hepatitis B viral infection in the local community. [ABSTRACT FROM AUTHOR]

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Mammalian cell entry (mce) operons play a vital role in cell invasion and survival of M. tuberculosis. Of the mce genes, the function of Rv0590A is still unknown. The present study was performed to investigate the function and immunogenic properties of the protein Rv0590A. Human leukemia monocytic cell line (THP-1) derived macrophages were infected with M. tuberculosis H37Rv at 3, 6, and 24 h of infection. The maximum colony forming units (CFU) were observed at 6 h (p < 0.005), followed by 3 h after infection. M. tuberculosis H37Rv and clinical isolates representative of Delhi/CAS, EAI, Beijing, Haarlem and Euro-American-superlineage were included in the study for expression analysis of mce1A, mce2A, mce3A, mce4A, and Rv0590A genes. Maximum upregulation of all mce genes was observed at 3 h of infection. All the five clinical isolates and H37Rv upregulated Rv0590A at various time points. Macrophage infection with M. tuberculosis H37Rv-overexpressing Rv0590A gene showed higher intracellular CFU as compared to that of wild-type H37Rv. Further, purified Rv0590A protein stimulated the production of TNFα, IFNγ, and IL-10 in macrophages. Thus, Rv0590A was found to be involved in cell invasion and showed good immunological response. [ABSTRACT FROM AUTHOR]

The article focuses on the process of growth in Kazuo Ishiguro's "Never Let Me Go," a novel set in an alternative Britain in the late 1990s, narrating the coming-of-age story of Kathy H., a thirty-one-year-old clone. Topics include the revelation of the clones' roles as donors, the critique of Kathy's passive acceptance of her predetermined fate, and the novel's departure from the traditional bildungsroman by linking Kathy's anti-Bildung to remote geographies.

PDZK1-interacting protein 1(PDZK1IP1), also known as MAP17, is encoded by the PDZK1IP1 gene and is a membrane-associated protein. PDZK1IP1 have been proven to be a potent regulator of cancer cell proliferation. However, the role of PDZK1IP1 in regulating goat subcutaneous preadipocyte proliferation is unknown. Here, we cloned the full-length coding sequence of PDZK1IP1 gene, investigated the potential functional of PDZK1IP1 in goat subcutaneous preadipocyte proliferation by gaining or losing function in vitro. Our results indicated that goat PDZK1IP1 gene consists of 345 bp, encoding a protein of 114 amino acids containing a typical PDZK1IP1 (MAP17) super family domain. Overexpression of PDZK1IP1 significantly increased the number of EdU-positive cells and cell viability, and also upregulated mRNA expression of cell proliferation-associated genes including CCND1 and CDK2 in vitro cultured cells. Conversely, knockdown of PDZK1IP1 mediated by siRNA technique significantly inhibited subcutaneous preadipocyte proliferation and downregulated mRNA expression of cell proliferation-associated genes including CCNE1, CCND1 and CDK2. Collectively, these results suggested that PDZK1IP1 can promote proliferation of goat subcutaneous preadipocyte. [ABSTRACT FROM AUTHOR]

Viperin, also known as radical S-adenosyl methionine domain-containing protein (RSAD2) is a multifunctional interferon-stimulated gene (ISG) that is activated during the viral infections. Viperin belongs to S-adenosyl methionine (SAM) superfamily of enzymes known to catalyze radical-mediated reactions and viperin inhibits a wide range of DNA and RNA viruses through its broad range of activity. The present study reports cloning and expression of bovine viperin in a bacterial expression system. PCR-based site-directed mutagenesis was carried out for deletion of N-terminal 1–70 amino acid containing amphipathic helix of viperin that interferes in protein expression and purification. The resultant truncated viperin protein was expressed in Escherichia coli, BL-21(DE3) competent cells and purified using nickel charged affinity column. The truncated 54 kDa protein was confirmed by western blot using human RSAD2 as a probe. Further, in house, hyperimmune serum was raised against the truncated viperin in the rabbit and the reactivity was confirmed by western blot using mammalian expression vector construct of viperin transfected in Baby Hamster kidney (BHK) cells and in MDBK cells infected with Foot and Mouth disease Asia I virus. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) holds a pivotal role within the glycolytic pathway of higher plants. It has garnered attention as a significant target protein in instances of oxidative stress, where it can engage in thiolation reactions within its active site. Numerous genes encoding cytosolic iterations of GAPDH have been identified and analyzed in specific plant species. This investigation was conducted to gain insights into GAPDH's function amidst drought-induced stress. Within this framework, the basil plant (Ocimum basilicum) was chosen for focused exploration, encompassing the cloning of the comprehensive cDNA of basil GAPDH (ObGAPDH) and scrutinizing its patterns of expression. The complete sequence of Ob-GAPDH spanned 1315 base pairs. The resultant protein derived from this sequence comprised 399 amino acids, projecting a molecular weight of approximately 42.54 kDa and an isoelectric point (pI) of 6.01. An examination of the evolutionary connections among various GAPDH proteins unveiled ObGAPDH's shared lineage with GAPDH proteins sourced from other plants, such as Salvia splendens and Sesamum indicum. Furthermore, computational methodologies were harnessed to predict the potential oxidative role of ObGAPDH in response to external signals. Molecular docking simulations illuminated the interaction between ObGAPDH and hydrogen peroxide (H2O2) as a ligand. Scrutinizing the expression patterns of the ObGAPDH gene under conditions of water scarcity stress brought to light diverse levels of transcriptional activity. Collectively, these findings underscore the notion that the regulation of ObGAPDH expression is contingent upon both the specific plant cultivar and the presence of stress stemming from drought conditions. [ABSTRACT FROM AUTHOR]

Maintaining optimal oral hygiene is essential for the long-term success of implant-supported overdentures. The Denture Cleanliness Index (DCI) is a reliable tool to assess denture hygiene and bacterial plaque accumulation. In this study, we evaluated the hygiene status of implant-supported overdentures in 30 patients using the DCI. The findings showed that majority of patients had plaque accumulation on the impression surface of dentures within one year of using the dentures. Patients were given the denture cleaning instructions according to the DCI scores. [ABSTRACT FROM AUTHOR]

Aims: LysM containing-protein is widely distributed in all domains of life and this kind of protein is essential for various biological activities in living organisms. Rv1288, a LysM containing-protein with esterase, was found in Mycobacterium tuberculosis. Biophysical studies revealed that the protein is responsible for modulating lipid metabolism that enables pathogens to survive under extreme conditions and decrease the permeability of the pathogen's cell wall to drug therapeutic agents. However, recognition and interaction between the protein, lipid and carbohydrate moieties at the molecular level remains largely unknown and must be investigated. Therefore, a production of recombinant protein Rv1288 should be performed to aid the study. Methodology and results: In this study, we cloned the full-length cDNA of Rv1288 from M. tuberculosis strain H37Rv and expressed it in pET-24d- Escherichia coli BL21(DE3) cells. Affinity and size exclusion chromatography methods purified the protein, and its preliminary oligomerisation state was determined based on a calculated apparent molecular weight of the protein. Rv1288 was expressed as a soluble protein at 20 °C, induced with 1 mM of isopropyl β-D-1- thiogalactopyranoside (IPTG). The calculated apparent molecular weight suggested that the Rv1288 protein formed a hexamer in solution. Conclusion, significance and impact of study: All the methods involved in this study to produce the recombinant Rv1288-pET24d and its soluble protein in E. coli cells have been described. Hence, it can be implemented for future studies. [ABSTRACT FROM AUTHOR]

RNA guanine quadruplexes (rG4) assume important roles in post-transcriptional regulations of gene expression, which are often modulated by rG4-binding proteins. Hence, understanding the biological functions of rG4s requires the identification and functional characterizations of rG4-recognition proteins. By employing a bioinformatic approach based on the analysis of overlap between peaks obtained from rG4-seq analysis and those detected in >230 eCLIP-seq datasets for RNA-binding proteins generated from the ENCODE project, we identified a large number of candidate rG4-binding proteins. We showed that one of these proteins, G3BP1, is able to bind directly to rG4 structures with high affinity and selectivity, where the binding entails its C-terminal RGG domain and is further enhanced by its RRM domain. Additionally, our seCLIP-Seq data revealed that pyridostatin, a small-molecule rG4 ligand, could displace G3BP1 from mRNA in cells, with the most pronounced effects being observed for the 3-untranslated regions (3-UTR) of mRNAs. Moreover, luciferase reporter assay results showed that G3BP1 positively regulates mRNA stability through its binding with rG4 structures. Together, we identified a number of candidate rG4-binding proteins and validated that G3BP1 can bind directly with rG4 structures and regulate the stabilities of mRNAs.

Marine pollutants bioaccumulate at high trophic levels of marine food webs and are transferred to humans through consumption of apex species. Yellowfin tuna (Thunnus albacares) are marine predators, and one of largest commercial fisheries in the world. Previous studies have shown that yellowfin tuna can accumulate high levels of persistent organic pollutants, including Transporter Interfering Chemicals (TICs), which are chemicals shown to bind to mammalian xenobiotic transporters and interfere with their function. Here, we examined the extent to which these same compounds might interfere with the activity of the yellowfin tuna (Thunnus albacares) ortholog of this transporter. To accomplish this goal we identified, expressed, and functionally assayed tuna ABCB1. The results demonstrated a common mode of vertebrate ABCB1 interaction with TICs that predicts effects across these species, based on high conservation of specific interacting residues. Importantly several TICs showed potent inhibition of Ta-ABCB1, such as the organochlorine pesticides Endrin (EC50 = 1.2 ± 0.2 μM) and Mirex (EC50 = 2.3 ± 0.9 μM). However, unlike the effects observed on mouse ABCB1, low concentrations of the organochlorine pesticide TICs p,p'-DDT and its metabolite p,p'-DDD co-stimulated verapamil-induced Ta-ABCB1 ATPase activity possibly suggesting a low transport activity for these ligands in tuna. These results provide a mechanistic basis for understanding the potential vulnerability of tuna to these ubquitous pollutants.

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.

La-related proteins 7 (LARP7) are a class of RNA chaperones that bind the 3' ends of RNA and are constitutively associated with their specific target RNAs. In metazoa, Larp7 binds to the long non-coding 7SK RNA as a core component of the 7SK RNP, a major regulator of eukaryotic transcription. In the ciliate Tetrahymena the LARP7 protein p65 is a component of telomerase, an essential ribonucleoprotein complex that maintains the telomeric DNA at eukaryotic chromosome ends. p65 is important for the ordered assembly of telomerase RNA (TER) with telomerase reverse transcriptase. Unexpectedly, Schizosaccharomyces pombe Pof8 was recently identified as a LARP7 protein and a core component of fission yeast telomerase essential for biogenesis. LARP7 proteins have a conserved N-terminal La motif and RRM1 (La module) and C-terminal RRM2 with specific RNA substrate recognition attributed to RRM2, first structurally characterized in p65 as an atypical RRM named xRRM. Here we present the X-ray crystal structure and NMR studies of S. pombe Pof8 RRM2. Sequence and structure comparison of Pof8 RRM2 to p65 and human Larp7 xRRMs reveals conserved features for RNA binding with the main variability in the length of the non-canonical helix α3. This study shows that Pof8 has conserved xRRM features, providing insight into TER recognition and the defining characteristics of the xRRM.

Here, we describe a protocol combining functional metrics with genomic data to elucidate drivers of within-cell-type heterogeneity via the phenotype-to-genotype link. This technique involves using fluorescence tagging to label and isolate cells grown in 3D culture, enabling high-throughput enrichment of phenotypically defined cell subpopulations by fluorescence-activated cell sorting. We then perform a validated phenotypically supervised single-cell analysis pipeline to reveal unique functional cell states, including genes and pathways that contribute to cellular heterogeneity and were undetectable by unsupervised analysis. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).