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CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.

Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries.

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

Glutaminases are a unique group of hydrolytic enzymes with potential applications in cancer therapy and the food industry. This study focused on the biological characterisation and computational conformation dynamics of YLaM, a potential L-glutaminase from a halothermotolerant Bacillus licheniformis. The research integrated experimental and computational methods to identify the YLaM sequence and predict its structural and functional properties. YLaM was amplified using a polymerase chain reaction, cloned into a pET-22b(+) vector, sequenced, and submitted to BankIt/NCBI. This experimental work was complemented by in silico analysis, including structural modelling via the I-TASSER web server, molecular docking using AutoDockTools 1.5.6, and molecular dynamic (MD) simulations using GROMACS v5.1.4. The study resulted in the development of a high-quality 3D model of YLaM through homology modelling and structural refinement, enabling a detailed exploration of its binding affinity with L-glutamine (L-Gln). By identifying active site residues using the homotetramer structure of human glutaminase, crucial interactions with L-Gln were validated, confirming its catalytic function. Finally, MD simulations confirmed the stability of the complex under physiological conditions. This combined molecular identification and functional simulations provide comprehensive insights into the physicochemical properties of the enzyme, offering valuable information for subsequent assessments, including recombinant production and biological impact evaluations. [ABSTRACT FROM AUTHOR]

This study aimed to develop a nano-biosystem by covalently binding recombinant laccase from Bacillus licheniformis (rLAC BL) to PEGylated silica-coated magnetic nanoparticles (EMNPs-PEG) and evaluate its cytotoxicity and anti-inflammatory properties on a pro-monocytic cell line. The active rLAC BL was purified from the bacterial culture with a yield of approximately 14 mg/L and purity of about 91 %. The physicochemical characteristics of EMNP-PEG-rLAC BL were confirmed using DLS, UV-Vis spectrum, FTIR, EDS, and SEM. The immobilized rLAC BL exhibited improved kinetic parameters compared to the bare enzyme. The IC 50 value for EMNP-PEG-rLAC BL (58.7 μg/mL) was lower than that of the bare enzyme after 48 hours of treatment on U937 cells. Both EMNP-PEG-rLAC BL and rLAC BL significantly decreased the expression levels of key cytokines (TNF-α, IL-6, IL-17, and IL-23) in LPS-stimulated cells, indicating promising anti-inflammatory properties of rLAC BL. Given the association between chronic inflammation and cancer, this newly developed enzyme-based nanobiomedicine holds potential as a therapeutic option for inflammatory conditions and malignant diseases. However, further research is needed to validate these findings and fully assess the therapeutic potential of LACs before clinical translation can be considered. [Display omitted] • Recombinant laccase (rLAC BL) was produced from Bacillus licheniformis. • The rLAC B was immobilized on PEGylated silica-coated magnetic nanoparticles. • Immobilization of rLAC BL notably enhanced enzyme affinity for its substrate. • Immobilized rLAC BL exhibited anti-inflammatory effects on the U937 cell line. [ABSTRACT FROM AUTHOR]

Aluminium (Al3+) toxicity in acidic soils poses a significant challenge for crop cultivation and reduces crop productivity. The primary defense mechanism against Al3+ toxicity involves the activation of organic acid secretion. In this study, responses of 9 Vigna mungo cultivars to Al3+ toxicity were investigated, with a particular emphasis on the root system and crucial genes involved in Al3+ tolerance using molecular cloning and expression analysis. Sensitive blackgram-KM2 cultivars exposed to 100-µM Al3+ toxicity for 72 h exhibited a root-growth inhibition of approximately 66.17%. Significant loss of membrane integrity and structural deformative roots were found to be the primary symptoms of Al3+ toxicity in blackgram. MATE (Multidrug and Toxic Compound Extrusion) and ALS3 (Aluminium Sensitive 3) genes were successfully cloned from a sensitive blackgram cv KM2 with phylogenetic analysis revealing their evolutionary relationship to Vigna radiata and Glycine max. The MATE gene is mainly localized in the plasma membrane, and highly expressed under Al3+, thus suggesting its role in transports of citrate-Al3+ complexes, and detoxifying Al3+ within plant cells. In addition, ALS3 was also induced under Al3+ toxicity, which codes the UDP-glucose transporter and is required for the maintenance of ions homeostasis. In summary, this study highlights the understanding of Al3+ toxicity and underlying molecular mechanisms linked to the efflux of organic acid in blackgram, ultimately aiding the framework for the development of strategies to enhance the resilience of blackgram and other pulse crops in Al-rich soils. [ABSTRACT FROM AUTHOR]

Introduction: The CDO1 gene is an important gene in the taurine synthesis pathway and has been observed to have high expression in ovaries of female mammals. This study aims to explore the conservation of CDO1 gene in domestic yaks, as well as to examine the fundamental characteristics of CDO1 gene and its expression in female yaks. Methods: Ovarian samples were collected from yaks in the follicular phase, luteal phase and gestation period in this experiment, and their total RNA and protein were extracted. Then Polymerase Chain Reaction (PCR) and bioinformatics online software were used to clone and analyze the CDO1 gene. The relative expression of CDO1 in yak ovaries was detected by Quantitative Real-time PCR (RT-qPCR) and Western blotting. The distribution and localization of CDO1 protein in ovary were detected by immunohistochemistry. Results: We have successfully cloned the coding region of CDO1 gene in yak. The results showed that the CDS region of CDO1 gene was 603 bp, encoding 200 amino acids, and was a relatively stable hydrophilic protein. CDO1 is relatively conservative in species evolution. The protein encoded by CDO1 gene does not have a signaling peptide or a transmembrane structure. It is a protein that is not involved in transmembrane transport and is mainly located in the cytoplasm. The secondary structure of the protein is dominated by the random coil. CDO1 is estimated to interact with 10 proteins. The results of RTqPCR and Western blotting showed that the CDO1 gene exhibited the highest expression in the ovary during the luteal phase and the lowest expression in the ovary during the follicular phase (P < 0.01). The results of immunohistochemistry showed that CDO1 was mainly expressed in granular cells, theca cells and lutein cells of ovarian tissue. Conclusion: These results suggest that the CDO1 gene has undergone minimal evolutionary changes during the course of animal evolution. The results provide a reference for further investigation of the function of CDO1 gene in reproduction and production in yaks. [ABSTRACT FROM AUTHOR]

Phage display is widely used in biomedical research. One of the great advantages of phage display is the specificity of the connection of a foreign peptide exposed outside the capsid to the intended target. Secondary detection systems, which are often laborious and costly, are required to identify and quantify the peptide/target interaction. In this study, we generated a novel dual-display phage to facilitate the detection and quantification of the peptide/target interaction. First, we generated a biotin-tagged phage by adding a small biotin-accepting peptide (sBT) to gene-3 of the M13K07 helper phage. Subsequently, we enhanced the M13K07 biotin-tagged phage by incorporating a selective peptide on gene-8, which is then exposed to the phage capsid. The exposed peptide acts as a probe to bind to a selective molecular target, whose interaction can be readily visualized thanks to the biotinylated phage. Our versatile dual-display phage exhibits high flexibility; by swapping the displayed peptide/probe, one can change the phage target while retaining the sBT gene in-frame with the pIII. We expect the generated biotin-tagged dual phages to be used as a multifunctional probe to couple with several streptavidin-biotin-based systems. [ABSTRACT FROM AUTHOR]

Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don't tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization. [ABSTRACT FROM AUTHOR]

Interspecies somatic cell nuclear transfer (iSCNT) has the capacity to rescue endangered species, ethically produce human embryonic stem cells, and allow for more specific analysis of pre‐implantation embryos. However, several obstacles negatively affect iSCNT efficiency.Over 100 iSCNT studies have been published between 1999 and the spring of 2023. This review details the possible benefits of iSCNT, describes the hypothesized issues that prevented the development of some embryos and/or live offspring from developing; and analyzes the cleavage, blastocyst, pregnancy, live birth, and iSCNT efficiency rates based on published data.Embryos produced by iSCNT may help with the rescue of endangered species and the production of human embryonic stem cells with the use of oocytes from domestic species, as well as in‐depth analyses of embryonic genome activation (EGA) and mRNA transcripts. But the combination of the cells of two species in a single embryo presents several challenges: lack of EGA synchronisation, presence of mitochondrial DNA (mtDNA) from both species in the embryo, requirement of cell and embryo culture optimization for each species combination, lack of embryo transfer data, and an extremely low overall efficiency in terms of producing live offspring. Additional research on the aforementioned topics is warranted to increase iSCNT efficiency. [ABSTRACT FROM AUTHOR]

Monika Piotrowska has defended a new account of reproduction. Her account seems able to answer the question of whether reproduction takes place, and who reproduces, when we employ biotechnologies that bear little to no resemblance to naturally occurring human sexual reproduction. Piotrowska's account also seems to increase our understanding of biological individuality and seems to be compatible with the theory of evolution via natural selection. In this paper, I do two things. First, I show that Piotrowska's account is found wanting, because it is extensionally inadequate. Second, I show that her criticism of James Grisemer's account of reproduction is mistaken. [ABSTRACT FROM AUTHOR]

Chitinases have vast applications due to their chitin degrading potential, as chitin is the main component of the cell-wall and exoskeleton of fungi and insects, respectively. In the late 20th century, wide research was conducted to explore their antifungal and insecticidal potential. The unavailability of complete sequences of genes, and less enzyme production from natural sources have restricted their applications at the industrial scale. In the present day, the availability of gene sequences may help to accomplish the long-awaited targets. In previous studies, Streptomyces sp. strain 130 was studied for chitinase production, where ten chitinase-producing genes were observed. In the current study, chitinases from strain 130 were validated using proteomics, where eight chitinases were found. Out of all chitinases, Chi65_1884 was selected for protein structure prediction, molecular docking and cloning of the chitinase-producing gene. Protein structure prediction was performed using homology modeling (Modeller 9.21). Molecular docking was conducted using Autodock 4.2 and MGL tools to examine bond interactions. The binding efficiency of Chi65_1884 with ligand was found to be -6.3 kcal/mol and the inhibition constant was 24.18 μM. Based on the molecular docking results, the chi65 gene was cloned into the vector pET32a and transformed into E. coli DH5α. Subsequent transformation into E.coli BL21 was facilitated for protein expression. The size of the expressed protein was observed to be ~85kDa (65kDa chitinase and ~20kDa carrier proteins of the vector). In future studies, chitinase produced by the cloned gene may tested against a range of pathogenic fungi to check its antifungal potential. [ABSTRACT FROM AUTHOR]

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the deamination of phenylalanine, which is the initial step in the biosynthesis of phenylpropanoids. It serves as a crucial enzyme that facilitates the transfer of carbon from primary to secondary metabolism in plants. Duckweed is regarded as a promising chassis plant in synthetic biology research and application, due to its being rich in secondary metabolites and other advantages. The genes encoding PAL in Spirodela polyrhiza (L.) Schleid, the giant duckweed, were investigated in this study. Three SpPAL genes (SpPAL1–SpPAL3) were identified and cloned. All of them were successfully expressed in E. coli, and their recombinant proteins all showed PAL activity. In addition, SpPAL1 and SpPAL2 proteins could also utilize tyrosine as substrate, although the activity was low. A qRT-PCR analysis demonstrated that the expression of SpPAL3 was most pronounced in young fronds. It was found that the expression of SpPAL1 and SpPAL3 was significantly induced by MeJA treatment. Overexpression of SpPAL3 in Lemna turionifera inhibited the growth of fronds and adventitious roots in the transgenic plants, indicating the importance of SpPAL3 in duckweed besides its involvement in the secondary metabolism. [ABSTRACT FROM AUTHOR]

Yellow mosaic disease (YMD) with typical symptoms of alternating bright yellow to green patches associated with stunting, downward cupping, and wrinkling has been observed in mung bean on agricultural farms in Coimbatore, Tamil Nadu, India. PCR using gene-specific primers indicated the presence of the yellow mosaic virus in symptomatic plants. Rolling circle amplification (RCA) followed by restriction digestion detected ~2.7 kb of DNA-A and DNA-B, allowing the identification of a bipartite genome. The full-length genome sequences were deposited in NCBI GenBank with the accession numbers MK317961 (DNA-A) and MK317962 (DNA-B). Sequence analysis of DNA-A showed the highest sequence identity of 98.39% to the DNA-A of mungbean yellow mosaic virus (MYMV)-Vigna radiata (MW736047), while DNA-B exhibited the highest level of identity (98.21%) to the MYMV-Vigna aconitifolia isolate (DQ865203) reported from Tamil Nadu. Recombinant analysis revealed distinct evidence of recombinant breakpoints of DNA-A within the region encoding the open reading frame (ORF) AC2 (transcription activation protein), with the major parent identified as MYMV-PA1 (KC9111717) and the potential minor parent as MYMV-Namakkal (DQ86520.1). Interestingly, a recombination event in the common region (CR) of DNA-B, which encodes the nuclear shuttle protein and the movement protein, was detected. MYMIV-M120 (FM202447) and MYMV-Vigna (AJ132574) were identified as the event's major and minor parents, respectively. This large variation in DNA-B led us to suspect a recombination in DNA-B. Dimeric MYMV infectious clones were constructed, and the infectivity was confirmed through agroinoculation. In future prospects, unless relying on screening using whiteflies, breeders and plant pathologists can readily use this agroinoculation procedure to identify resistant and susceptible cultivars to YMD. [ABSTRACT FROM AUTHOR]

The continued emergence of antibiotic resistance among bacterial pathogens remains a significant challenge. Indeed, the enhanced antibiotic resistance profiles of contemporary pathogens often restrict the number of suitable molecular tools that are available. We have constructed a series of plasmids that confer resistance to two infrequently used antibiotics with variants of each plasmid backbone incorporating several regulatory control systems. The regulatory systems include both commonly used systems based on the lac- and arabinose-controlled promoters found in Escherichia coli, as well as less frequently used systems that respond to tetracycline/anhydrotetracycline and toluic acid. As a test case, we demonstrate the utility of these plasmids for regulated and tunable gene expression in a multidrug-resistant (MDR) isolate of Acinetobacter baumannii, strain AB5075-UW. The plasmids include derivatives of a freely replicating, broad-host-range plasmid allowing for inducible gene expression as well as a set of vectors for introducing genetic material at the highly conserved Tn7-attachment site. We also modified a set of CRISPR-interference plasmids for use in MDR organisms, thus allowing researchers to more readily interrogate essential genes in currently circulating clinical isolates. These tools will enhance molecular genetic analyses of bacterial pathogens in situations where existing plasmids cannot be used due to their antibiotic resistance determinants or lack of suitable regulatory control systems. [ABSTRACT FROM AUTHOR]

During early development of biopharmaceuticals, suboptimal producing clones and production conditions can result in limited quantities of high-purity products. Here we describe a systematic approach, which requires minimal amounts of protein (~10 mg) to assess critical quality attributes of a monoclonal antibody (mAb). A commercial anti-PCSK9 IgG2 (evolocumab, Repatha®) and an early-stage biosimilar candidate were compared head-to-head using a range of high-throughput physicochemical and in-vitro binding analytical methods. Overall, both mAbs were shown to be highly pure and primarily monomeric, to share an identical primary structure, and to have similar higher-order structural integrity, apparent solubility, aggregation propensity, and physical stability profiles under temperature and pH stress conditions. Low levels of dimers were detected for the innovator (1.2%) and the biosimilar candidate mAb (0.3%), which also presented fragments (1.2%). Regarding charge heterogeneity, the amount of the main charge isoform was 53.6% for the innovator and 61.6% for the biosimilar candidate mAb. Acidic species were 38% for the innovator and 30% for the biosimilar candidate. Variations in the relative content of a few N-glycan species were found. The in-vitro binding affinity to PCSK9 was monitored, and no differences were detected. The mathematical approach called "error spectral difference" (ESD), proposed herein, enabled a quantitative comparison of the biophysical datasets. The workflow used in the present work to characterize CQAs at early stages is helpful in supporting the development of biosimilar mAb candidates. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunist pathogen most commonly related to nosocomial infections. P. aeruginosa infection therapy poses a significant challenge due to its ability to resist various antibiotics currently available. As a result, excessive use of antibiotics during therapy expedites the development of multidrug resistant P. aeruginosa. Hence, this study aimed to identify novel genes involved in multiple antibiotic resistance using plasposon mutagenesis technique. One hundred and ten P. aeruginosa isolates were collected from various clinical sources involving urine, burns and wound's pus. An antimicrobial susceptibility test was performed to detect their resistance to 18 antibiotics. Results showed that all isolates were resistant to ampicillin and tetracycline, and the highest resistance ras were detected for nitrofurantoin and sulfamethoxazole (99%), followed by amoxiclav, cefotaxime, cefoxitin, ceftriaxone, and kanamycin (98%). While the lowest resistance rate was towards imipenem (49%). Plasposon mutagenesis was used to detect the genes involved in multi-antibiotic resistance. The pTnMod-Gm was introduced to the recipient P. aeruginosa PA4 isolate via triparental mating using E. coli HB101/pRK2013 as a helper strain. Mutants were screened for resistance defects by plating them on nutrient agar supplemented with different antibiotics. Two mutants were identified; one (M1) exhibited susceptibility to tetracycline, cefotaxime, and ceftazidime, and the other (M9) to ceftazidime and ceftriaxone. The analysis of these mutants revealed the insertion of the plasposon into an open reading frame for the ABC transporter in P. aeruginosa, which plays a distinctive role in extruding antibiotics out of cells. [ABSTRACT FROM AUTHOR]

New technologies, as well as their accessibility, contribute significantly to the change in counterfeiting methods used by criminals, raising many questions among forensic scientists. Knowing the operating parameters of these technologies provides the answers that forensic scientists need to adapt the examination methods and be able to provide correct conclusions. Cloning rubber stamps using a laser engraver, based on a genuine rubber stamp impression, is the method that removes the shortcomings of the rubber stamp impressions process counterfeited with the help of computing and printers. The objectives of the study are to construct clone rubber stamps starting from a genuine rubber stamp impression, using different processing possibilities offered by CorelDRAW graphic editing/processing software, as well as to establish the parameters that allow distinguishing between genuine rubber stamp impressions and counterfeit impressions made with cloned rubber stamps. In the content of the study, it is demonstrated that it is possible to clone rubber stamps using this method. The quality of the result of the cloning process is directly influenced by the type and quality of the image used. The study demonstrates that clone rubber stamps made based on a bitmap image or a vectorized bitmap image within the software, produce impressions that present certain specific features of character construction that allow them to be distinguished from the impressions of authentic rubber stamps. In the case of rubber stamps made by scanning an impression, loading it into CorelDraw software and using it as a pattern to create next to it a graphic design that imitates it as accurate as possible, there is the possibility of both discriminating or impossibility to discriminate between impressions made with a clone rubber stamps and those made with an authentic rubber stamp. [ABSTRACT FROM AUTHOR]

There is a growing need for applications capable of handling large synthesis biology experiments. At the core of synthetic biology is the process of cloning and manipulating DNA as plasmids. Here, we report the development of an application named DNAda capable of writing automation instructions for any given DNA construct design generated by the J5 DNA assembly program. We also describe the automation pipeline and several useful features. The pipeline is particularly useful for the construction of combinatorial DNA assemblies. Furthermore, we demonstrate the platform by constructing a library of polyketide synthase parts, which includes 120 plasmids ranging in size from 7 to 14 kb from 4 to 7 DNA fragments.

Phosphoinositide 3-kinases (PI3Ks) are a class of key regulatory factors in eukaryotes that can inhibit viral replication by influencing autophagy. Currently, cyprinid herpesvirus 3 (CyHV-3) poses a serious threat to common carp culture. However, PI3K has not yet been identified in common carp. In this study, full-length PI3KC3 from common carp (CcPI3KC3), consisting of an open reading frame (ORF) of 2664 bp encoding a polypeptide of 887 amino acids, with a predicted molecular mass of 101.19 kDa and a theoretical isoelectric point (pI) of 5.97, was cloned. The amino acid and nucleotide sequences of CcPI3KC3 displayed high similarity to yellow catfish’s (Tachysurus fulvidraco) PI3KC3. The tissue expression profile revealed that the mRNA levels of CcPI3KC3 in the liver, spleen, and head kidney were significantly greater than those in the brain, heart, intestines, gills, eyes, testes, and ovaries of common carp. We compared the expression patterns of CcPI3KC3 between “Longke-11” mirror carp (CyHV-3-resistant carp) and German mirror carp (non-resistant to CyHV-3) at different times (0, 48, 96, 144 h, 192, 240, 288 h post-infection (hpi)) after CyHV-3 infection. The results revealed that CcPI3KC3 mRNA expression significantly increased in the early infection stage. In the CyHV-3-resistant mirror carp variety, the relative expression of CcPI3KC3 was significantly greater at 48, 96, and 144 hpi compared with the nonbreeding strain groups after infection (p < 0.001). These results indicate that the full-length CcPI3KC3 sequence was successfully cloned from common carp for the first time, and it might play an important role in the immune system of common carp against CyHV-3 infection. This study provides a theoretical basis for the molecular mechanism of CyHV-3 resistance.

Abstract Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don’t tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization.

Leaf rust, caused by Puccinia triticina Eriksson (Pt), is one of the most severe foliar diseases of wheat. Breeding for leaf rust resistance is a practical and sustainable method to control this devastating disease. Here, we report the identification of Lr47, a broadly effective leaf rust resistance gene introgressed into wheat from Aegilops speltoides. Lr47 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that is both necessary and sufficient to confer Pt resistance, as demonstrated by loss-of-function mutations and transgenic complementation. Lr47 introgression lines with no or reduced linkage drag are generated using the Pairing homoeologous1 mutation, and a diagnostic molecular marker for Lr47 is developed. The coiled-coil domain of the Lr47 protein is unable to induce cell death, nor does it have self-protein interaction. The cloning of Lr47 expands the number of leaf rust resistance genes that can be incorporated into multigene transgenic cassettes to control this devastating disease.

Chaperonin-containing T-complex polypeptide 1 (CCT) is a molecular chaperone in the cytoplasm of eukaryotic cells involved in the correct folding and assembly of proteins, which plays an important role in cold-stress response. This study cloned two transcripts of Pinctada maxima CCT-beta composed of 1,829 bp (CCT-betal) and 2,289 bp (CCT-beta2), respectively, using rapid amplification of cDNA ends. Both transcripts possess 105 bp of 5' noncoding region and the same coding region, bul contain different 3' noncoding regions of 591 bp (CCT-betal) and 131 bp (CCT-beta2), respectively. The open reading framework (ORF) consisted of 1,593 bp that encodes 530 amino acids. The CCT-beta protein has three conserved functional domains (equatorial, apical, and intermediate domains), three CCT-family signature sequences, nine conserved ATP-binding sites, one DEAD box motif, and three protein-binding sites. The CCT-beta is highly conserved, with the cDNA homologies between P. maxima and Crassostrea gigas, Danio rerio, and Homo sapiens being 90.4%. 76.8%, and 75.8%, respectively. Real-time quantitative PCR showed that CCT-beta was differentially expressed in different organs of P. maxima, with mRNA contents declining from the gill, adductor muscle, foot, labial palps, mantle, and heart to hepatopancreas. Under cold stress, CCT-beta mRNA expression in various organs increased, but the patterns of increase varied among organs. The transcriptional level of CCT-beta peaked at 6h in the heart and hepatopancreas, and decreased significantly at 12h. In the adductor muscle, gill, and mantle, CCT-beta mRNA increased significantly at 12h and declined markedly at 24h in cold stress-exposed animals. These results indicate that CCT-beta is a cold-stress response gene, but the response pattern varies among organs. Organs with low CCT-beta basal expression may be more sensitive to cold stress than the organs with relatively high basal expression content. KEY WORDS: Pinctada maxima, CCT-beta, cDNA, cold stress, INTRODUCTION Chaperonin-containing T-complex polypeptide 1 (CCT) is a kind of type II chaperonin that is localized in the cytoplasm of eukaryotes (Frydman et al. 1992, Gao et al. 1992, Lewis [...]

The efficacy of the BCG vaccine, a widely recognized tuberculosis vaccine, has shown varying degrees of effectiveness, ranging from 0% to 80%. Sirohydrochlorin cobaltochelatase (CbiX), found in Mycobacterium tuberculosis, plays a crucial role in the bacteria metabolism, making it a promising target for future vaccine and drug development. Although several studies had been published regarding its role in M. tuberculosis vitamin B-12 metabolism, the potentials of CbiX protein as a vaccine candidate had not been widely discussed nor explored. This study focuses on the cloning and expression of the Rv0259c gene obtained from a clinical isolate of M. tuberculosis, as well as the exploration of CbiX protein epitopes. The Rv0259c gene was isolated by PCR, cloned into the pGEM®-TEasy vector, and subsequently sub-cloned into the pTrcHisA expression vector. Sanger sequencing, followed by BLASTN and BLASTX analyses, confirmed the presence of the CbiX protein-encoding gene. The amino acid sequence was predicted using BioEdit v.7.0.11, and a three-dimensional (3D) model was generated using SwissModel. Exploration for both B and T-cell epitopes was conducted using IEDB Ellipro, MHCI, and MHCII tools, revealing highly immunogenic epitopes, indicating the potential of CbiX as a vaccine candidate. Alignment using MAFFT between the putative amino acid sequence and CbiX proteins available in the NCBI database identified an amino acid variation (A182), situated outside the B-cell epitopes but within the T-cell epitopes. In silicoanalysis of HLA allele frequency predicted vaccine coverage of 86.14%±10.77%, with two significant epitope cores identified: AASAHPHVT and RRVAVASFL (both highly antigenic and showing high-frequency HLA allele binding), suggesting the protein might be a potential antigen for a future vaccine candidate. Doi: 10.28991/ESJ-2024-08-04-07 Full Text: PDF

Objective Parathyroid hormone like hormone (PTHLH), as an essential factor for bone growth, is involved in a variety of physiological processes. The aim of this study was to explore the role of PTHLH gene in the growth of antlers. Methods The coding sequence (CDS) of PTHLH gene cDNA was obtained by cloning in sika deer (Cervus nippon), and the bioinformatics was analyzed. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the differences expression of PTHLH mRNA in different tissues of the antler tip at different growth periods (early period, EP; middle period, MP; late period, LP). Results The CDS of PTHLH gene was 534 bp in length and encoded 177 amino acids. Predictive analysis results revealed that the PTHLH protein was a hydrophilic protein without transmembrane structure, with its secondary structure consisting mainly of random coil. The PTHLH protein of sika deer had the identity of 98.31%, 96.82%, 96.05%, and 94.92% with Cervus canadensis, Bos mutus, Oryx dammah and Budorcas taxicolor, which were highly conserved among the artiodactyls. The qRT-PCR results showed that PTHLH mRNA had a unique spatio-temporal expression pattern in antlers. In the dermis, precartilage, and cartilage tissues, the expression of PTHLH mRNA was extremely significantly higher in MP than in EP, LP (p

[Objective] Trypanosoma cruzi, a causal agent of Chagas’ disease, is a parasite whose life cycle alternates between an invertebrate (triatomine) and a vertebrate (mammal) host. Various studies have shown that in T. cruzi, HSP83 (a homolog of HSP90) is essential for cell division and control of the response to thermal stress. This investigation focused on studying the cloning, bioinformatic characterization, and expression of the heat shock protein HSP83 T. cruzi gene for further cell signaling studies. [Methodology] RNA was extracted from T. cruzi epimastigotes (EPm6 clone, MHOM/VE/2007/ 6c) using a commercial kit. The cDNA encoding HSP83 was determined using RT-PCR on extracted mRNA, for which the primers were designed based on the HSP83 sequence of T. cruzi strain CL Brener. Cloning was performed using pGEM®T-Easy and subcloned into the expression vector pQE30. Sequence and bioinformatic characterization were performed. The gene was expressed, and the recombinant protein was purified using affinity chromatography and identified through immunoblotting. [Results] Sequence analysis showed similarity to the gene encoding HSP83 from Trypanosoma cruzi, and HSP domains and B epitopes in the sequence were also observed. After 3 hours of induction with IPTG, a recombinant protein with an approximate weight of 83 kDa was obtained. The immunoblotting reaction with hyperimmune anti-T. cruzi epimastigote serum helped detect a single band with a molecular weight of nearly 83 kDa. [Conclusions] All results indicate that the cloning and characterization of HSP83 from Trypanosoma cruzi was achieved.

Aim. The purpose of the study was to generate and characterize anti-hERα polyclonal antibodies for elucidation of functional relationships between isoforms of estrogen receptor ERα and isoforms of ribosomal protein S6 kinase — S6K1. Methods. cDNA cloning. Expression of recombinant proteins in bacterial system. Affinity purification of His-tag fused recombinant proteins using Ni-NTA chromatography from bacterial lysates. Generation of polyclonal sera by mice immunization. Western blot analysis and immunoprecipitation. Results. cDNA coding for full length hERα was cloned into expression vector pET28a in frame with His-tag sequence. Recombinant hERα-His protein was expressed in E.Coli and purified by Ni-NTA chromatography. Purified hERα-His was used as antigen for mice immunization and generation of polyclonal antibodies. Specificity of polyclonal antibodies was analyzed by Western blot and immunoprecipitation of hERα from MCf-7 cell lysates. Conclusions. Generated anti-hERα polyclonal antibodies are of conformational type since specifically recognized hERα only in immunoprecipitation but not in Western blot. Created polyclonal antibodies a suitable for detection and analysis of hERα protein complexes.

Transhumanism sees the next step of the evolution of humanity in technology, together with the developments in the twentieth century and the influence of positivism. As a modern repetition of the golem myth, it desires to use technology as o tool for a stronger, smarter, non-sick, even immortal humanity. Transhumanists developed in an environment where the influence of humanism was reduced due to materialism and they aim to create a super human through technologies such as biotechnology, nanotechnology, artificial intelligence and cloning, and tries to bring humans to a higher position. In literature, which is a close follower mirror of social developments, transhumanist works are frequently encountered as they create a suitable environment for the development and discussion of transhumanist ideas. Therefor Transhumanism has become an important subject in world literature and has attracted the attention of French authors, especially with the twenty-first century. Michel Houellebecq, one of the leading figures of contemporary French literature, is famous for deeply processing existential themes and bringing striking subjects such as religion, nudity and the human condition into his novels by refence. His novel Elementary Particles, published in 1998, problematizes the potential return of transhumanist ideas, human desires and technological advances, while depicting a modern society where life has lost its meaning with the advancement of scientific developments. This article aims to reveal the author’s ideas for the future of humanity in a world driven by the increasing use of technology while critically investigating the traces of transhumanism in Houellebecq’s Elementary Particles. After providing an overview of the foundations of the theory by creating a framework for understanding the concept of transhumanism, the study tries to reveal the author’s transhumanist ideas and the potential consequences of transcending human boundaries by making use of important themes and passages in the novel.

Proteins are essential for the proper functioning of cells. The techniques of cloning and protein production have facilitated the advancement of various fields and the creation of specific proteins for industrial and therapeutic uses. The bacterium Aeribacillus pallidus, which is able to survive in extreme conditions, is being studied with a view to identifying its robust enzymes. The objective of this study was to clone the protease gene from the A. pallidus P18 strain into the SUMO vector and produce recombinant protein in Escherichia coli BL21 for protein production. The protease enzyme gene from the A. pallidus P18 strain was isolated and amplified by using PCR. The PCR product was transferred into the SUMO expression vector and amplified in One Shot® Mach1TM-T1R bacteria, followed by colony PCR. Plasmid isolation was performed after positive colony selection. Gene integration was confirmed by cross-PCR using the gene forward, and vector reverse primers. For expression, the plasmid was transferred to E. coli BL21 cells. Two cultures were induced with different IPTG concentrations (0.5 mM and 1 mM) to optimize protein production. Bacterial cells were lysed, and SDS-PAGE analysis was conducted. Purification involved cell lysate preparation and purification using a ProbondTM column. SDS-PAGE and Coomassie Brilliant Blue G-250 staining confirmed successful purification. The results of this study indicate that the optimal product for protein production is that derived from a culture induced with 1 mM IPTG. Upon completion of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure, the weight mass of the produced protein was determined to be 37 kDa, as indicated by the result of the gel stained with Coomassie brilliant blue G-250. This research successfully cloned the protease enzyme gene from the A. pallidus P18 strain using the pET-SUMO vector, performed purification and achieved the targeted result of protein production.

Abstract In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg−1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg−1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg−1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5ʹ-dithio-bis-[2-nitrobenzoic acid (DNTB), β-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme’s activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (K m ) and maximum reaction rate (V max ), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL−1 and 1580.3 ± 183.7 μmol mg−1 protein min−1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with β-glucosidase for 24 h.

The present study outlines the approach that was employed for cloning, expression, and characterization of the recombinant pullulanase enzyme from Bacillus cereus ATCC 14579 into Escherichia coli BL21(DE3) using pET‐25b (+) expression vector. The recombinant pullulanase enzyme was purified using ammonium sulfate precipitation and immobilized metal ion affinity chromatography (IMAC). The molecular mass of the purified pullulanase enzyme was measured using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as 95 kDa. The purified recombinant pullulanase enzyme demonstrated significant thermal stability, maintaining its structural integrity and functionality at temperatures as high as 90°C over a period of 4 h. The inclusion of divalent metal ions, specifically Ca2+ and Mg2+, had a positive effect on the activity of the pullulanase enzyme. Conversely, the presence of Co2+ and EDTA (Ethylene Diamine Tetra Acetic acid) resulted in suppression of the enzyme activity. The purified pullulanase enzyme demonstrated remarkable resistance when exposed to organic solvents. The enzyme activity was notably decreased in the presence of SDS (Sodium Dodecyl Sulfate) while β‐mercaptoethanol and tween‐60 did not substantially affect the enzyme activity and stability which suggest its potential applicability in the detergent sector. This discovery indicates a potential approach to improve the effectiveness of currently available detergents in the marketplace. [ABSTRACT FROM AUTHOR]

In the current digital era, images have become one of the key channels for communication and information. There are multiple platforms where digital images are used as an essential identity, like social media platforms, chat applications, electronic and print media, medical science, forensics and criminal investigation, the court of law, and many more. Alternation of digital images becomes easy because multiple image editing software applications are accessible freely on the internet. These modified images can create severe problems in the field where the correctness of the image is essential. In such situations, the authenticity of the digital images from the bare eye is almost impossible. To prove the validity of the digital images, we have only one option: Digital Image Forensics (DIF). This study reviewed various image forgery and image forgery detection methods based on blind forgery detection techniques mainly. We describe the essential components of these approaches, as well as the datasets used to train and verify them. Performance analysis of these methods on various metrics is also discussed here. [ABSTRACT FROM AUTHOR]

Pig skeletal muscle-derived stem cells (SK-MSCs) were transplanted onto the common peroneal nerve with a collagen tube as a preclinical large animal experiment designed to address long nerve gaps. In terms of therapeutic usefulness, a human family case was simulated by adjusting the major histocompatibility complex to 50% and 100% correspondences. Swine leukocyte antigen (SLA) class I haplotypes were analyzed and clarified, as well as cell transplantation. Skeletal muscle-derived CD34+/45− (Sk-34) cells were injected into bridged tubes in two groups (50% and 100%) and with non-cell groups. Therapeutic effects were evaluated using sedentary/general behavior-based functional recovery score, muscle atrophy ratio, and immunohistochemistry. The results indicated that a two-Sk-34-cell-transplantation group showed clearly and significantly favorable functional recovery compared to a non-cell bridging-only group. Supporting functional recovery, the morphological reconstitution of the axons, endoneurium, and perineurium was predominantly evident in the transplanted groups. Thus, Sk-34 cell transplantation is effective for the regeneration of peripheral nerve gap injury. Additionally, 50% and 100% SLA correspondences were therapeutically similar and not problematic, and no adverse reaction was found in the 50% group. Therefore, the immunological response to Sk-MSCs is considered relatively low. The possibility of the Sk-MSC transplantation therapy may extend to the family members beyond the autologous transplantation. [ABSTRACT FROM AUTHOR]

Simple Summary: Proteases are vital enzymes that break down proteins into smaller units, peptides, or amino acids, playing a role in several biological processes. Due to various industrial applications, including detergents, laundry, leather, and pharmaceutical industries, the production of thermostable proteases is of great concern. Thermophilic bacterium Streptomyces thermovulgaris produced the metalloprotease in a bacterial expression system. Overexpression was achieved using an inducer in 4 h, yielding a soluble protease. Protein was purified by precipitation and an affinity chromatography system and was stable. It retained 80% activity at a wide range of pH and temperature with a half-life of 4 h, which is highly efficient for its use in various industries. Proteases hydrolyze proteins and reduce them to smaller peptides or amino acids. Besides many biological processes, proteases play a crucial in different industrial applications. A 792 bp protease gene (nprB) from the thermophilic bacterium Streptomyces thermovulgaris was cloned and expressed in E. coli BL21 using pET 50b (+). Optimal recombinant protease expression was observed at 1 mM IPTG, 37 °C for 4 h. The resulting protease was observed in soluble form. The molecular mass estimated by SDS-PAGE and Western blot analysis of the protease (NprB) fused with His and Nus tag is ~70 KDa. The protease protein was purified by Ammonium sulfate precipitation and immobilized metal ion affinity chromatography. The optimum pH and temperature for protease activity using casein as substrate were 7.2 and 70 °C, respectively. The mature protease was active and retained 80% of its activity in a broad spectrum of pH 6–8 after 4 h of incubation. Also, the half-life of the protease at 70 °C was 4 h. EDTA (5 mM) completely inhibited the enzyme, proving the isolated protease was a metalloprotease. NprB activity was enhanced in the presence of Zn2+, Mn2+, Fe2+ and Ca2+, while Hg2+ and Ni2+ decreased its activity. Exposure to organic solvents did not affect the protease activity. The recombinant protease was stable in the presence of 10% organic solvents and surfactants. Further characterization showed that zinc-metalloprotease is promising for the detergent, laundry, leather, and pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Plant WRKY transcription factors are responsible for biotic and abiotic stresses and play an important role in enhancing their adaptability. The AtWRKY33 is a gene that functions in response to abiotic stresses such as low temperature, drought, salinity, etc. In this study, a recombinant vector YG8198-ZmWRKY53 carrying the ZmWRKY53, an interspecific homolog of the dicotyledonous AtWRKY33, was transferred to rice plants by Agrobacterium mediated transformation. The ectopic expression of the ZmWRKY53 in transgenic rice plants conferred cold tolerance with a higher accumulation of free proline and water-soluble sugars, an increase in chlorophyll content, a decrease in electrolyte leakage rate and MDA levels compared to control plants. This result suggests that ZmWRKY53 may confer cold tolerance in rice. [ABSTRACT FROM AUTHOR]

Ophibolin A, a fungal sesterterpene, exerts a pivotal influence in a diverse array of biological processes, encompassing herbicidal, bactericidal, fungicidal, and cytotoxic activities. Sixty genes associated with sesterterpene compound biosynthesis were obtained from Bipolaris eleusines via transcriptome sequencing, and those closely linked to ophiobolin A biosynthesis were subsequently filtered. A gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) that catalyzes the first committed step of ophiobolin biosynthesis in the mevalonic acid (MVA) pathway was isolated and characterized using RACE (Rapid Amplification of cDNA Ends) technology from ophiobolin A-producing fungus, B. eleusines. The full-length cDNA of the B. eleusines HMGR gene (BeHMGR) was 3906 bp and contained a 3474 bp open reading frame (ORF) encoding 1157 amino acids. Sequence analysis revealed that deduced BeHMGR had high homology to the known HMGRs from Pyrenophora tritici-repentis and Leptosphaeria maculans. It had a calculated molecular mass of about 124.65 kDa and an isoelectric point (pI) of 6.90. It contained two putative HMG-CoA-binding motifs and two NADP(H)-binding motifs. Induced expression analysis of the BeHMGR gene by methyl jasmonate treatment using quantitative fluorescence PCR showed that it significantly elevated after 3 h of methyl jasmonate treatment, peaked at 6 h, and then gradually decreased. This demonstrates that BeHMGR gene expression is induced by methyl jasmonate. [ABSTRACT FROM AUTHOR]

Micranthes nelsoniana possesses multiple different variants and numerous chromosomes. Based on the internal transcribed spacer (ITS) and chloroplast (cp)DNA sequences, the phylogeography of M. nelsoniana and its relatives in Northeast Asia was investigated, with extensive sampling around the Kuril Islands. The Arctic–Asian continent and a clade of marginal islands were the two main groupings that comprised the ITS phylogenetic tree. The island clade was separated into five well-supported clades: Kamchatka and Hokkaido highlands, Kuril–Aleutian Islands, southern Kuril Islands, Japanese archipelago, and Primorye region. Micranthes fusca was found in Japan and in the southern Kuril Islands. It is a separate species that created several types of hybrids between M. nelsoniana in the centre of the Kuril Islands based on a comparison of the ITS and cpDNA networks. Micranthes nelsoniana and M. ohwii appear to have hybridized in the northern Kuril Islands. Cytological investigation on the local species of M. nelsoniana showed that the chromosomal numbers are: 2 n  = 24, 26, 28, 30, 50, and 80. Among them, two usual numbers to this area, 2 n  = 24 and 50, appear to encourage interspecific gene exchange. The genomes of Hokkaido plants with high chromosome counts were cloned, revealing that they contained genes of both continental and marginal origins. This study revealed the crucial role of marginal islands along Northeast Asia in the genetic diversity of M. nelsoniana and related species. [ABSTRACT FROM AUTHOR]

Genetic transformation is helpful in enhancing crops, utilising promoters that can be constitutive, inducible, or tissue-specific. However, the use of constitutive promoters may hinder plant growth due to energy consumption during cellular processes. To optimise transgene effects, tissue-specific promoters like root-specific ones prove valuable in addressing root-related issues and enhancing productivity. Yet, identified root-specific promoters in crop are limited. To address this gap, the expression pattern of the root-specific SlREO promoter was examined across various crops. Sequencing confirmed its identity and high homology (99%) with the NCBI database, distinct from other plants tested. Using the PLACE database, six motifs associated with root expression were identified, along with several other important elements. The 2.4 kb SlREO promoter was linked to a ß-glucuronidase (GUS) reporter gene alongside the CaMV35S promoter in pRI 201-AN- GUS vectors to study its expression. Histochemistry revealed strong root-specific expression in tomato (Solanum lycopersicum) root tissues and limited expression in stems. However, the SlREO promoter did not consistently maintain its root-specific expression in other plants. Conversely, the CaMV35S promoter exhibited constitutive expression across all tissues in various plants. This study underscores the potential of the SlREO promoter as a root-specific regulatory element, offering avenues for improving crops, particularly against environmental stresses. The improvement of tomato (Solanum lycopersicum) for various biotic and abiotic traits that affect the roots functions is vital, and root-specific promoters are tools to improve the gene expression of transgenes. SlREO is one tissue-specific promoters in tomato. This study cloned and characterised SlREO in Indian tomato cultivar, and evaluated its activity in various crops. Transgenic studies suggest that SlREO is a crop-dependent tissue-specific promoter that cannot be used to improve the expression of genes in other crops. This article belongs to the Collection Functional Genomics for Developing Climate Resilient Crops. [ABSTRACT FROM AUTHOR]

Salvia guaranitica is considered one of the most significant medicinal and aromatic herbs in terms of nutritional and medical benefits due to its wealth of important active components. Among these compounds, terpenoids are the most prominent and abundant, particularly monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20). They are biologically advantageous to plants and perform a multitude of functions. The current study aimed to clone the S. guaranitica gene that encodes for geranyllinalool synthases (SgGES, EC: 4.2.3.144), with consideration for these features. The open reading frame of the 867-amino-acid protein encoded by SgGES consists of 2.721 base pairs. In addition, the SgGES protein has five domains that belong to the terpene synthase family, which are related to the terpene and terpenoid synthase domains. We manipulated and overexpressed the SgGES gene in Nicotiana tabacum to explore its function. When compared to the GUS control, the transgenic N. tabacum plants displayed an increase in leaf production and diameter when compared with the wild-type plants. Finally, analysis of transgenic plants using gas chromatography/mass spectrometry (GC-MS) showed that SgGES is responsible for producing various terpene species, especially diterpenes. [ABSTRACT FROM AUTHOR]

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The research aims to study the genetic cloning objectively. It is found, through what is mentioned in the Holy Quran, that people is unable to create, as challenged by the Almighty Creator, glory is to Him. In fact, cloning is not creation, but rather a kind of changing Allah’s creation, but it is forbidden. Cloning is a process in which a genetically identical copy of a cell, tissue or living organism is produced from genetic material. This happens naturally in organisms such as bacteria, insects, or plants, where reproduction happens without mating. The newly created copy is termed as a “clone”. The Scottish sheep “Dolly” is considered the most famous example. Cloning also refers to the process of applying a nuclear transfer technique to somatic cells. The research comes to the following findings: 1) Cloning is not considered creation; creation and creativity belong to Almighty Allah alone. 2) Admitting the falsehood of the suspicion of the creation. 3. Genetic cloning is viewed as a part of Allah’s creation and is considered to make significant risks across various aspects, thus doing it is religiously prohibited. Research methodology: a definitional approach and an inductive analysis. [ABSTRACT FROM AUTHOR]

In this study, acetovanillone derivative compounds (1–7) were synthesized, and their structures were analyzed using nuclear magnetic resonance (1H NMR—13C NMR)and Fourier-transform infrared (FTIR) methods. The xylanase gene within the scope of the study was cloned using recombinant DNA techniques. The purified recombinant protein was subjected to comprehensive characterization, including assessment of pH stability, temperature dependency, and substrate specificity. The optimal temperature and pH for the enzyme were determined to be 72 ºC and 6.0, respectively. Investigations were done into how the synthetic compounds affected the xylanase enzyme. Especially compound 2 increased the activity at concentrations of 0.1, 0.3, 0.5 and 0.7 mM by 176.9, 153.25, 146.22 and 115.41%, respectively, compared to the positive control. It has also been observed that the effect on activity decreases with increasing concentration. Compound 3, on the contrary, increased the activity at high concentrations (0.7 and 1 mM) by 147.30 and 153.93%, respectively. Considering the positive sample (100%), CaCI2 shows 70.38% activity at 0.1 mM, 68.04% at 0.3 mM, 79.96% at 0.5 mM, 61.99% at 0.7 mM, and 85.59% at 1 mM.These results show that CaCI2 causes a slight decrease in enzyme activity.ZnCI2, the zinc compound, and CuCI2, the copper compound, showed a similar decrease in activity. It was determined that these different reactions showed different relative activity of the metal ions at varying concentrations for the recombinant xylanase enzyme. [ABSTRACT FROM AUTHOR]

Clock genes are essential for the daily functions of vertebrates and invertebrates and participate in a wide variety of biological functions including biochemical, physiological, and behavioral processes. Circadian clock disorders are detrimental to both humans and fish, and therefore research on the regulation of genes involved in the circadian clock has attracted increasing attention. Period 2 (per2) is an important gene whose expression is closely related to melatonin secretion, in addition to being affected by the photoperiod length. To gain more insights into the potential biological function of per2, we have cloned and expressed per2 in Amur minnow (Phoxinus lagowskii). We also analyzed melatonin content and AANAT2 gene expression under different short or long-term photoperiod conditions. In the present study, the secretion of melatonin in P. lagowskii was highest in the dark phase under short-term experiments. In the pituitary, the expression of the per2 gene was highest during the dark phase in both the 16L: 8D and 8L: 16D groups. The expression of the per2 gene in the brain was also highest during the dark phase in the 12L: 12D group. Though the expression of the per2 gene was not the highest in the dark phase for the other groups, there were no significant differences compared to the highest value. In addition, in the brain and pituitary, AANAT2 expression was highest during the light phase, except in the 8L: 16D group. Our findings suggested that Amper2 may participate in the feedback regulation of melatonin by regulating the expression of AANAT2. Fluorescence in situ hybridization analyses confirmed that per2 mRNA was expressed during the dark phase, especially in 16L: 8D group. Immunohistochemistry analyses confirmed that PER2 protein was expressed higher in 8L: 16D group. The dual luciferase experiment first validated the promotion of Wnt/β-Catenin expression by the per2 gene. Our results provide novel insights into the expression and location of per2, which contributes to a better understanding of the role of clock genes in the circadian rhythm and seasonal responses of high-latitude fish and might provide useful information of practical interest for high-latitude aquaculture. [ABSTRACT FROM AUTHOR]

β-Phenylethanol is the primary flavor compound in Sichuan industrial paocai. Metatranscriptomics analysis revealed that the gene encoding aryl-alcohol dehydrogenase (AADh1), which is responsible for β-phenylethanol synthesis, had the highest expression level during the late stage of fermentation (180 days). In the present study, we explored the potential AADh1 in Sichuan industrial qingcai paocai by combining metatranscriptomics results and AADh1 analysis in NCBI. AADh1 was successfully cloned and expressed in Escherichia coli BL21, and the enzymatic properties of AADh1 were characterized. AADh1 exhibited the highest activity at pH 7.0 and 50°C, with the highest stability at pH 8.0 and 25°C. Furthermore, the enzyme exhibited low tolerance to metal ions, and only 1 mM Mn2+ could slightly activate the enzyme. Moreover, AADh1 was successfully used to preliminarily enhance the β-phenylethanol of industrial paocai, which could improve the flavor of paocai to a certain extent in practical production. [ABSTRACT FROM AUTHOR]

Bell non-locality and steering are archetypal characteristics of quantum mechanics that mark a significant departure from conventional classical notions. They basically refer to the presence of quantum correlations between separated systems which violate a non-local inequality, the violation otherwise is not possible if we restrict ourselves only to classical correlations. In view of the importance of such unique correlations, one may be interested to generate more states exhibiting non-locality starting from a few, a protocol which is termed as broadcasting. However, in the present submission, we show using universal Buzek–Hillary (BH) quantum cloning machine that, if one restricts to broadcasting through local quantum cloning, then such non-locality cannot be broadcasted. Our study is done in the purview of the Bell-CHSH inequality and the CJWR [E G Cavalcanti et al, Phys. Rev. A80, 032112 (2009)] steering inequality. It is observed that when the number of measurement settings is greater than 6, some of the output states are steerable after the application of local optimal BH cloners. We found that, under some restrictions, if the Werner and Bell diagonal states are subjected to such procedures, then the resultant state is rendered unsteerable. We extended this study to three qubit systems and found that genuine tripartite non-locality cannot be broadcasted using universal BH local quantum cloners, when the Svetlichny's inequality was considered. For two qubit systems, we considered 10 5 simulated general local unitaries over Werner and Bell-diagonal states and found that for none of these states, broadcasting on non-locality and 3-steerability is possible. [ABSTRACT FROM AUTHOR]

The article aims to examine bioethical concerns presented in Kazuo Ishiguro's 2005 novel Never Let Me Go, focusing on the lives of cloned beings who become organ donors for non-cloned humans. The analysis addresses such ethical implications of cloning and organ donation as dignity, healing, care, and virtue. Through the lens of utilitarian and virtue ethics, the analysis focuses on the novel's portrayal of these characters, examining how these models function in the narrative and enhance its literary effect. Ishiguro's text highlights some of the bioethical concerns surrounding clone characters in fiction. The novel questions whether clone characters are part of a social transformation or if they are part of the existing distinction between nature and artifact. The bioethical understanding of human dignity is emphasized, as it is intrinsic to every human being. [ABSTRACT FROM AUTHOR]

Proteins are essential for the proper functioning of cells. The techniques of cloning and protein production have facilitated the advancement of various fields and the creation of specific proteins for industrial and therapeutic uses. The bacterium Aeribacillus pallidus, which is able to survive in extreme conditions, is being studied with a view to identifying its robust enzymes. The objective of this study was to clone the protease gene from the A. pallidus P18 strain into the SUMO vector and produce recombinant protein in Escherichia coli BL21 for protein production. The protease enzyme gene from the A. pallidus P18 strain was isolated and amplified by using PCR. The PCR product was transferred into the SUMO expression vector and amplified in One Shot® Mach1TM-T1R bacteria, followed by colony PCR. Plasmid isolation was performed after positive colony selection. Gene integration was confirmed by cross-PCR using the gene forward, and vector reverse primers. For expression, the plasmid was transferred to E. coli BL21 cells. Two cultures were induced with different IPTG concentrations (0.5 mM and 1 mM) to optimize protein production. Bacterial cells were lysed, and SDS-PAGE analysis was conducted. Purification involved cell lysate preparation and purification using a ProbondTM column. SDSPAGE and Coomassie Brilliant Blue G-250 staining confirmed successful purification. The results of this study indicate that the optimal product for protein production is that derived from a culture induced with 1 mM IPTG. Upon completion of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure, the weight mass of the produced protein was determined to be 37 kDa, as indicated by the result of the gel stained with Coomassie brilliant blue G-250. This research successfully cloned the protease enzyme gene from the A. pallidus P18 strain using the pET-SUMO vector, performed purification and achieved the targeted result of protein production. [ABSTRACT FROM AUTHOR]

Transhumanism sees the next step of the evolution of humanity in technology, together with the developments in the twentieth century and the influence of positivism. It desires to use technology as o tool for a stronger, smarter, non-sick, even immortal humanity. Transhumanists developed in an environment where the influence of humanism was reduced due to materialism and they aim to create a superhuman through technologies such as biotechnology, nanotechnology, artificial intelligence and cloning. In literature, which is a close follower mirror of social developments, transhumanist works are frequently encountered as they create a suitable environment for the development and discussion of transhumanist ideas. Therefor Transhumanism has become an important subject in world literature and has attracted the attention of French authors. Michel Houellebecq, one of the leading figures of contemporary French literature, is famous for deeply processing existential themes and bringing striking subjects such as religion, nudity and the human condition into his novels by refence. His novel Elementary Particles, published in 1998, problematizes the potential return of transhumanist ideas, human desires and technological advances, while depicting a modern society where life has lost its meaning with the advancement of scientific developments. This article aims to reveal the author’s ideas for the future of humanity in a world driven by the increasing use of technology while critically investigating the traces of transhumanism in Houellebecq’s Elementary Particles. After providing an overview of the theory, the study tries to reveal the author’s transhumanist ideas. [ABSTRACT FROM AUTHOR]