NKAP (NF-kappa-B-activating protein) is a ubiquitously expressed nuclear protein involved in multiple biological processes. Males with missense NKAP mutations have been reported to present with marfanoid features and behavioral and musculoskeletal abnormalities. We have previously reported that a disruptive NKAP mutation resulted in extremely skewed X chromosome inactivation (XCI), leading to phenotypic manifestation of hemophilia A (HA) in a HA carrier. In this study, with the aim of exploring the phenotypic manifestations of deleterious NKAP mutations in males, as well as their involvement in the mechanism of XCI regulation in females, we generated NKAP mutant mice using CRISPR/Cas9 technology. Gait analysis studies conducted in male mice hemizygous for mutant NKAP by the CatWalk XT system revealed significant alterations in gait parameters, consistent with hypotonia reported in human mutant NKAP patients. By breeding mutant NKAP mice with HA mice, we generated a double heterozygous mutant NKAP/HA mouse model, i.e., female mice carrying mutant NKAP with a WT F8 copy on one X chromosome, and WT NKAP with a mutant F8 copy on the other X chromosome. XCI pattern analysis using methylation-sensitive restriction enzymes demonstrated that mutant NKAP/HA females exhibited significant XCI skewing of the X chromosome bearing the mutant NKAP copy. Furthermore, these females exhibited significantly reduced F8 mRNA levels and FVIII (factor VIII) antigen levels, as demonstrated by quantitative RT-PCR and ELISA, respectively. Murine embryonic fibroblasts (MEFs) derived from a hemizygous mutant NKAP embryo exhibited markedly reduced proliferation rate and increased senescence compared to WT NKAP MEFs, suggesting that XCI skewing induced by mutant NKAP results from secondary selection against cells with an active X chromosome bearing the mutant NKAP copy.

Clostridioides difficile colonization of Twin Patients Recovering From Infant Botulism.

Methyltransferase (MTase)-based DNA labeling has become a powerful strategy for genomic and epigenetic analysis because of its unique ability to recognize and functionalize DNA sequences in a site-specific manner. Expanding this toolbox is essential to fully exploit MTases as programmable molecular guides. Here, we introduce an MTase-directed proximity labeling approach that enables sequence-specific DNA modification beyond the natural catalytic transfer site: GLOW, Guided Labeling Outside the natural site With MTases. Using newly designed S-adenosyl-L-methionine (SAM) analogues, we demonstrate sequence-specific DNA labeling revealed by single-molecule fluorescence imaging and gel-based restriction enzyme assays, confirming that labeling occurs adjacent to, rather than within, the canonical recognition site. Unlike conventional MTase-mediated methods, this strategy provides enhanced ligand stability and avoids interference from endogenous DNA methylation, thereby broadening its potential to complex genomic contexts. These findings establish MTase-guided proximity labeling as a conceptually new mode of enzymatic targeting that enriches the chemical biology toolkit for sequence-specific DNA modification.

Thermosensitive hydrogel-enhanced RPA-CRISPR/Cas12a biosensor for ultrasensitive detection of methylated loci in breast cancer ctDNA.

Natural products (NPs) produced by actinobacteria, particularly Streptomyces species, represent a rich source of bioactive compounds and have yielded many clinically important compounds. Actinobacterial genomes are characterized by high GC content and typically harbor 20-40 biosynthetic gene clusters (BGCs) per genome, which encode diverse NPs such as polyketides, peptides, and glycosides. CRISPR/Cas-based genome editing has emerged as a promising tool to activate silent BGCs and engineer NP biosynthesis. However, the efficiency of multiplex editing drastically decreases as the number of targeted sites increases. Here, we report a novel one-pot DNA assembly method, termed direct pathway synthesis and editing (DiPaSE), for the efficient synthesis and multiplex editing of long, high-GC BGCs. DiPaSE accurately assembles multiple high-GC DNA fragments up to 60 kb and enables simultaneous deletions and insertions within a target BGC without compromising the assembly efficiency. Using this approach, we identified functions of previously uncharacterized genes in the aureothin BGC and significantly enhanced the titer of the corresponding NP. The workflow employs conventional polymerase chain reaction, type IIP restriction enzymes, commercially available DNA assembly reagents, and Escherichia coli, providing a simple, cost-effective, and broadly applicable platform for genome mining, BGC refactoring, and rational design of artificial biosynthetic pathways.

Immuno-Southern blot assay: combined detection of DNA modifications with methylation-responsive restriction Enzymes and antibody screening

The wheat leaf spot complex is a globally pervasive foliar disease caused by multiple fungal pathogens: Pyrenophora tritici-repentis (tan spot), Parastagonospora nodorum and Parastagonospora pseudonodorum (septoria nodorum blotch), Zymoseptoria tritici (septoria tritici blotch), and Bipolaris sorokiniana (spot blotch). Diagnostic challenges arise from overlapping symptoms and similar morphologies. We evaluated previously released molecular diagnostic tools and found that they either lacked specificity or failed to detect all species in the complex. Existing methods target different multicopy genomic regions and lacks validation against other wheat-associated pathogens. To overcome these limitations, we developed a detection tool targeting a single copy, conserved gene (β-tubulin 1, tub1) across all species. Species-specific primers were designed for multiplex PCR (mPCR) and TaqMan-based real-time quantitative PCR (qPCR), enabling sensitive, specific, and simultaneous quantification. The qPCR accurately quantified pathogen biomass with detection limits down to 0.04 pg of fungal DNA. We further showed that assays were highly accurate and species-specific when tested on wheat tissues inoculated under controlled conditions with defined single-species or mixed infections, as well as on naturally infected samples. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis with selected restriction enzymes further distinguished species with unique cleavage patterns, providing an easy to use and clear identification system. Moreover, we confirmed that the assays do not cross detect barley-associated species such as the barley leaf spot pathogen Pyrenophora teres, ensuring robustness for use where host overlaps occurs. This comprehensive diagnostic provides a rapid and reliable detection, quantification of these pathogens, supporting improved disease diagnosis and enhance breeding for resistance.

The cashew apple is a juicy and sweet fruit that loses its nutritional quality during the first week after falling from the cashew tree. This degradation is strongly linked to microorganisms that may present beneficial biotechnological potential. Therefore, the objective of this study is to characterize lactic acid bacteria and fermentative yeasts from cashew apples. Cashew apples collected from three localities (Tioro, Morovine, and Waraniene) in the Korhogo department (Northern Côte d’Ivoire) were washed in the laboratory, crushed, and subjected to fermentation. Samples were taken every two days over one week. Morphological studies focused on macroscopic and microscopic analyses of the strains. Physiological and biochemical tests of lactic acid bacteria and yeast isolates were all carried out in tubes. Molecular typing of these isolates was performed using the restriction enzymes DDeI or HinfI, and HaeIII. Characterization of lactic isolates revealed that all lactic acid bacteria isolated were bacillary in shape with catalase activity. They are homofermentative, mesophilic, acidophilic, and do not tolerate salinity beyond 4%. Moreover, they fermented lactose, glucose, maltose, fructose, starch, sucrose, and cellulose. They also produced exopolysaccharides. At the molecular level, all bands were observed at 1500 base pairs. Furthermore, the biochemical profile and enzymatic digestion performed with restriction enzymes identified the genus <i>Lactobacillus</i>, specifically <i>Lactobacillus plantarum</i> as the presumptive species. Characterization revealed yeasts of oval, elongated shape with lateral budding. Sediments were observed in their growth broths. They degraded urea and produced acetoin. They also fermented various sugars, except cellulose, citrate, and mannitol. All bands were observed at 500 base pairs. Biochemical tests and PCR confirmed that all isolates belonged to the genus <i>Saccharomyces sp</i>. Digestion with restriction enzymes (HaeIII and HinfI) divided them into two groups of <i>Saccharomyces</i>, of which 93.75% were <i>Saccharomyces cerevisiae</i> and 6.25% were <i>Saccharomyces uvarum</i>. The use of these presumptive species could serve in the production of cashew apple wine.

Background: Duck is very high in protein and fat content, and could be required in Africa to meet the shortfall in animal protein; due to its potentials like high feed efficiency, early maturity, high disease resistance and adaptability among others. Aims: To identify polymorphic forms of exon 7 of Lipoprotein Lipase (LPL) gene in locally adapted Mallard and Muscovy duck breeds in Nigeria. Methodology: Case-control study with candidate gene approach could not be used due to monomorphic results obtained from gel electrophoresis analysis of the extracted DNAs from both breeds. Samples collection was carried out at some markets in Ogun State while the laboratory work was carried out at the Animal Science Laboratory of University of Ibadan between May and June, 2023. 80 ducks; 40 Mallard and 40 Muscovy ducks were used to carry out the study. Blood samples of 3 ml each, were collected from 80 ducks. Blood samples were collected through the jugular vein; of 40 mallard breed and 40 Muscovy breed. The DNA was extracted and amplified using exon 7 LPL gene primer and HinfI digestive enzyme. The products obtained from PCR-RFLP were analyzed with 2% agarose gel electrophoresis. Results: 33 of all the samples in both Mallard and Muscovy were amplified. The percentage of which was 37.50% for both breeds. While 18 of Mallard samples were amplified, 15 of Muscovy were amplified and their percentages were 22.50% and 18.75% respectively. Considering the percentage of each of the breeds that were amplified, Mallard percentage of amplified sample was 45% while that of Muscovy was 37.5%. Similar gel patterns was obtained in all amplified samples. Therefore, there was no polymorphism detected at exon 7 of LPL gene but instead the breeds were monomorphic. The result obtained could be due to fewer population of selected samples, non polymorphic of the gene at exon 7 and it may also be as a result of closeness in relationship due to proximity of birds sampled from one another. Conclusion: Further studies can be carried out using larger sample size or other restriction enzymes in order to validate the results obtained in this study, and provide insight into the variations within each breed and many other Nigerian locally adapted ducks at the LPL gene locus.

Abstract Rice ( Oryza sativa L.) agriculture of the southern United States is plagued by strong biotic competition with several species in the Echinochloa genus. Despite clear genomic differences between barnyardgrass ( Echinochloa crus-galli (L.) P. Beauv.] and junglerice ( Echinochloa colona (L.) Link], the two major Echinochloa agricultural weeds are nearly indistinguishable phenotypically. This inability to reliably differentiate the species has led farmers to treat the group as a single species, often resulting in ineffective weed control efforts. In this study, we first develop a simple chloroplast-anchored PCR-based restriction enzyme assay to differentiate between E. colona and the other Echinochloa species of agricultural concern. Applying this assay, we identify a strong bias towards E. colona in 2024 rice field collections from eastern Arkansas. Finally, we evaluate anecdotal reports of interspecific hybridization between species and find no evidence. Despite the drawbacks of the maternally inherited nature of the chloroplast, the availability of this species determinant assay will help USDA and academia extension agents and stakeholders to make educated, species-specific decisions about precision chemical weed control and field management.

Abstract. This study investigated the association between growth hormone (GH) gene polymorphism and postnatal growth and placental traits of Tuj sheep over a 3-year period (2020–2022) at the Atatürk University Sheep Research Farm, Erzurum. The genotype–phenotype association analyses comprised Tuj ewes and their progeny. The polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method using the Hae-III restriction enzyme was applied to identify GH gene variants. Three genotypes were detected: LL (49.0 %), LV (39.0 %), and VV (11.0 %). The mean birth weights were 4.45 kg (LL), 4.52 kg (LV), and 4.36 kg (VV); the weaning weights were 14.80, 15.55, and 14.43 kg, respectively; and the average daily gains were 0.213, 0.226, and 0.209 kg d−1, respectively. Although the GH genotype had no significant association with birth weight (p>0.05), it was significantly associated with weaning weight (p<0.05) and daily live weight gain (p<0.01). The present study involves an analysis of the placental traits (placental area, placental weight, and placental efficiency). The results indicated that there were no statistically significant relationships between the GH genotype and placental traits (p>0.05). The study revealed a significant effect of parity on placental area (p<0.05). Twin lambs demonstrated a higher placental weight than single lambs (p<0.05). No significant differences were identified between lamb sex and placental traits. The LV genotype in Tuj ewes was found to be associated with slightly higher growth performance and had favorable indicators for placental traits in comparison with the LL and VV genotypes. These findings suggest that GH gene polymorphism could serve as a potential genetic marker for improving growth performance in Tuj sheep populations.

Restriction-modification (RM) systems contribute to genome plasticity in Mycoplasma hominis, a facultative pathogen with an extremely small but highly heterogeneous genome. The MhoVII RM system, which contains a fusion of two methyltransferases (MTases), M1 and M2, was recently identified within a family of Type II RM systems, but its specificity and biological function remained unknown. Phylogenetic analysis revealed that M1 and M2 belong to distinct MTase classes clustering within the YhdJ and MTaseD12 branches, respectively. In this study, the dissemination, expression and function of the MhoVII system was analyzed in detail using Oxford Nanopore-based methylation analysis, recombinant expression of the individual RM components in Escherichia coli, and methylation-sensitive restriction assays. It was thus possible to demonstrate that M1 and M2 methylate the complementary non-palindromic motifs GATG and CATC, and that the associated restriction endonuclease cleaves only DNA lacking 6mA methylation at these sites. The transcriptional analysis of mid-to-late logarithmic cultures indicated a polycistronic organization of the MhoVII genes, and GATG/CATC-driven methylation analysis revealed culture-dependent methylation differences, suggesting a post-transcriptional regulation, whereas in the infection of HeLa cells, MhoVII transcription was highest at the beginning and was then gradually downregulated in the later stages of infection. These findings establish MhoVII as a previously uncharacterized Type II RM system.

All genomic data were deposited in the European Nucleotide Archive and are available in NCBI under the project accession number PRJEB2705. Supporting data are provided in supplementary data files. Accession numbers are given in Table 1. Staphylococcus haemolyticus is a multidrug-resistant opportunistic pathogen which causes disease in vulnerable patients. Previously, genetic modification of this pathogen was prevented due to the prevalence of strain-specific restriction modification systems, which use DNA methylation to identify and foreign DNA (including plasmids) when it enters the cells, and then destroy it using restriction enzymes. We analysed the methylomes of a selection of S. haemolyticus strains and used this data to tailor molecular tools to evade the RM systems. This enabled us to improve transformation efficiency and perform genome editing, including large-scale chromosomal modifications and targeted deletion of suspected virulence genes (a first for S. haemolyticus ). Beyond its immediate relevance to researchers studying this pathogen, the tools and approaches developed here have broader utility for genetic engineering of other coagulase-negative staphylococci and bacteria with similar RM barriers. Additionally, we found that two RM systems were enriched in clinical strains, suggesting they may have a function in virulence, in addition to their roles as bacterial phage defence systems.

Mitochondrial genome editing tools: prospects in animal breeding.

ABSTRACTReduced‐representation sequencing methods, such as Restriction‐site Associated DNA sequencing (RAD‐seq), use restriction enzymes to achieve a cost‐effective approach for generating genome‐wide SNP data. However, a major limitation of these methods is their inability to directly assay specific loci of interest unless located near restriction sites. Here, we present ampliRAD, a novel method combining targeted (i.e., amplicon) and reduced‐representation sequencing. AmpliRAD uses an initial multiplex PCR step to amplify target loci and append restriction enzyme recognition sites onto them. The PCR product is then combined with genomic DNA and used as input for a traditional RAD library preparation protocol, enabling the incorporation of virtually any target loci into a standard RAD dataset. We also introduce updates to an existing RAD protocol, including enzymatic shearing, that enhance its accessibility and efficiency. To demonstrate ampliRAD's utility, we investigate genetic associations with adult migration timing in Dean River Chinook salmon, revealing a clear link between the GREB1L locus and migration timing that extends previous findings from southern populations to this northern river. AmpliRAD provides a powerful new tool for genomic analyses, offering the combined benefits of both reduced representation and targeted sequencing approaches.

Episomal maintenance of introduced plasmid with or without AMA1 sequence in the basidiomycete fungus Pleurotus ostreatus.

Hierarchical spindle-structured carbon nanotube fiber as efficient enzyme-enriched reservoir for robust Biosensing textiles.

Interaction of plant genomic DNA with arsenic and cadmium: Double trouble or protective pair.

The coupling of methylation-sensitive restriction enzyme with recombinant polymerase amplification and lateral flow dipstick for the detection of (menstrual) blood stains.

The genetic stability of recombinant CHO cell lines producing therapeutic proteins is critical for ensuring consistent quality in biopharma-ceutical products. Southern blotting remains the gold standard for evaluating transgene integrity and stability in these cell lines. In the biopharmaceutical industry, transposon-based expression systems are widely utilized to generate highly productive and genetically stable CHO cell lines. However, evaluating transgene integration sites and integrity in such cell lines is challenging with standard Southern blotting protocols. This difficulty arises because transposon-mediated transfection often results in multiple independent integration sites in the host genome, each typically harboring a single transgene copy. Upon restriction enzyme digestion, similar-sized DNA fragments are generated, reducing resolution and complicating the separation and detection of the transgenes using standard blotting protocols. Here, we present a modified Southern blotting protocol that significantly improves the resolution of integration banding patterns by refining key steps, including purification of digested DNA prior to electrophoresis and an enhanced DNA transfer method. This protocol was successfully applied to analyze multiple transposon-derived CHO cell lines with high transgene copy numbers, enabling more precise and efficient detection of transgene integration.