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The genus Vigna (Leguminosae) comprises about 150 species grouped into five subgenera. The present study aimed to improve the understanding of karyotype diversity and evolution in Vigna, using new and previously published data through different cytogenetic and DNA content approaches. In the Vigna subgenera, we observed a random distribution of rDNA patterns. The 35S rDNA varied in position, from terminal to proximal, and in number, ranging from one (V. aconitifolia, V. subg. Ceratotropis) to seven pairs (V. unguiculata subsp. unguiculata, V. subg. Vigna). On the other hand, the number of 5S rDNA was conserved (one or two pairs), except for V. radiata (V. subg. Ceratotropis), which had three pairs. Genome size was relatively conserved within the genus, ranging from 1C = 0.43 to 0.70 pg in V. oblongifolia and V. unguiculata subsp. unguiculata, respectively, both belonging to V. subg. Vigna. However, we observed a positive correlation between DNA content and the number of 35S rDNA sites. In addition, data from chromosome-specific BAC-FISH suggest that the ancestral 35S rDNA locus is conserved on chromosome 6 within Vigna. Considering the rapid diversification in the number and position of rDNA sites, such conservation is surprising and suggests that additional sites may have spread out from this ancestral locus. [ABSTRACT FROM AUTHOR]

Nematodes of the Onchocercidae family, such as Pelecitus spp., are filarial parasites of medical and veterinary importance. Although infections are widely distributed among avian species, only 2 cases of human Pelecitus ocular infection, both in South America, have been reported. We describe a 61-year-old man in northeast Thailand diagnosed with an ocular infection. Morphologic characteristics suggested the causative agent was a female Pelecitus nematode: coiled body, rounded anterior and posterior extremities, a distinct preesophageal cuticular ring, lateral alae, a postdeirid, and a protuberant vulva. Sequences of the 12S rDNA gene indicated 95%–96% identity and cox1 gene 92%–96% identity with published P. copsychi sequences. P-distance for cox1 sequences between the causative agent and P. copsychi was 6.71%. Phylogenetic trees of 12S rDNA and cox1 genes indicated the species differed from but is closely associated with P. copsychi. Healthcare providers should be aware of the threat of ocular infection from Pelecitus spp. nematodes. [ABSTRACT FROM AUTHOR]

Sweet cherry (Prunus avium) is a commercially important species in China, experiencing a rapid increase in production. In June 2022, a severe infection, affecting more than 8% of sweet cherry trees, was observed on the Tetian cultivar in a 2–3‐year‐old orchard in Dalian City, northern China. Initially, spindle‐shaped brown disease spots formed on the surfaces of the branches. These spots continued to spread and merge, and the middle portion of the cankers sunk inward and gradually dried. Small black particles were found on the surface of the stems. The disease spread was more prevalent during the rainy season, leading to the withering of numerous branches and the death of the whole plant. Seven isolates, named lncy1‐1, lncy4‐1, lncy5‐1, lncy6‐1, lncy7‐1, lncy8‐1 and lncy9‐1, were obtained from 24 disease samples. Phylogenetic analysis based on three loci (rDNA ITS, TEF1 and TUB2) coupled with morphological identification confirmed that these seven isolates belong to Diaporthe eres. A representative isolate, lncy1‐1, was inoculated onto sweet cherry branches in a controlled environment, and showed that the isolate lncy1‐1 was pathogenic. The fungus isolated from diseased tissues was identified as D. eres based on morphological and molecular criteria. To the best of our knowledge, this is the first report of stem canker in sweet cherry caused by D. eres in China, which will promote disease management and expand the known host range of D. eres. [ABSTRACT FROM AUTHOR]

During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs. Fanconi anemia proteins are required for repair of DNA interstrand crosslinks. Here, the authors show that these proteins are also recruited to DNA double-strand breaks to promote repair by homologous recombination. [ABSTRACT FROM AUTHOR]

A new genus is proposed to accommodate an undescribed dorylaimid form collected in Iran. Persianema gen. n. is characterised by its three-layered cuticle, oral field appreciably sunken but its perioral area visibly protruding, odontostyle less than one-half of lip region diam. and with large aperture, guiding ring simple and always located in front of odontostyle, odontophore 4-5 times longer than odontostyle, dorsal gland nucleus (DN) very posteriorly located, female genital system di-ovarian with well-developed pars refringens vaginae, tail short and rounded in both sexes, spicules dorylaimid and 3-4 spaced ventromedian supplements with hiatus. Its type and only species, P. enigmaticum sp. n., is described and illustrated, including SEM observations. It is distiguished by its 1.45-2.03 mm long body, lip region offset by shallow depression and 15.5-17.5 μm wide, odontostyle 6-7 μm long with aperture occupying more than half of its length, neck 285-340 μm long, pharyngeal expansion 38-43% of the total neck length, three gland cells present at pharyngo-intestinal junction, simple uterus 81-113 μm or 1.7-3.1 body diam. long, transverse vulva (V = 50-54), tail 15-25 μm long (c = 69-106, c′ = 0.6-0.8) and spicules 38-48 μm long. Molecular analysis of D2-D3 28S rDNA sequences revealed its closest similarity with Pungentus sequences, and an integrative comparison supports its belonging to Pungentinae. [ABSTRACT FROM AUTHOR]

During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1's role during meiotic recombination in mammals remains unknown. Here, we decipher the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1ΔN alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates. Successful meiotic homologous recombination relies on the tight regulation of RAD51 and DMC1 strand exchange proteins. This study shows that the conserved FIGNL1-FIRRM complex plays a critical role for fertility by restraining uncontrolled assembly of RAD51 and DMC1 filament during meiosis. [ABSTRACT FROM AUTHOR]

Bioflavor and fermented foods in Indonesian cuisine were interesting for studying the relationship between fermentation products, microbial aspects, functional implications and biotechnological applications. The methodology employed in the literature review, including the sources used and inclusion criteria, demonstrates a meticulous approach to gathering and synthesizing information. Additionally, the factors influencing the perception of flavors on the tongue provide valuable insights into the complexities of taste perception, encompassing the role of specific amino acids and alkaloid compounds. The discussions on flavor production through microbial fermentation and the application of recombinant DNA technology in microbial flavor production showcase the strides made in biotechnology and their profound impact on flavor development. The escalating significance of natural ingredients and biocatalyst processes in producing flavor compounds aligns with consumer preferences for natural and sustainable options. Moreover, safety considerations for bioflavor products derived from biotechnology underscore the critical importance of ensuring consumer-friendly and safe products in this field. Functional bioflavor constraints provide practical considerations for developing and applying functional flavors, emphasizing the necessity for natural, safe and stable alternatives to conventional food additives. Overall, it offers a comprehensive and in-depth exploration of the multifaceted realm of flavor, integrating scientific, cultural and technological perspectives. It is an invaluable resource for researchers, industry professionals and enthusiasts engaged in flavor science and technology. [ABSTRACT FROM AUTHOR]

Although previous studies have reported that pre-mRNA splicing factors (SFs) are involved in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR), their exact role in promoting HR remains poorly understood. Here, we showed that SART1, an SF upregulated in several types of cancer, promotes DSB end resection, an essential first step of HR. The resection-promoting function of SART1 requires phosphorylation at threonine 430 and 695 by ATM/ATR. SART1 is recruited to DSB sites in a manner dependent on transcription and its RS domain. SART1 is epistatic with BRCA1, a major HR factor, in the promotion of resection, especially transcription-associated resection in the G2 phase. SART1 and BRCA1 accumulate at DSB sites in an interdependent manner, and epistatically counteract the resection blockade posed by 53BP1 and RIF1. Furthermore, chromosome analysis demonstrated that SART1 and BRCA1 epistatically suppressed genomic alterations caused by DSB misrepair in the G2 phase. Collectively, these results indicate that SART1 and BRCA1 cooperatively facilitate resection of DSBs arising in transcriptionally active genomic regions in the G2 phase, thereby promoting faithful repair by HR, and suppressing genome instability. [ABSTRACT FROM AUTHOR]

Pseudohydnum, commonly known as cat’s tongue mushrooms, is a monophyletic assemblage within Auriculariales, which encompasses species with gelatinous basidiomata, spathulate, flabellate, or shell-shaped pileus, hydnoid hymenophore, globose to ellipsoidal basidiospores, and longitudinally cruciate-septate basidia. According to the available literature, 16 species have been described in Pseudohydnum, mostly represented in temperate-boreal forests of the Northern Hemisphere. However, the limited morphological, molecular, and ecological information, especially from the Southern Hemisphere ecosystems, does not presently allow a reliable assessment of its taxonomic boundaries nor provide a complete picture of the species diversity in the genus. In an ongoing effort to examine specimens collected in dense and mixed ombrophilous forest fragments (Atlantic Rainforest domain) from Southeastern and Southern Brazil, additional taxa assigned to Pseudohydnum were identified. Four new species are recognized based mostly on characters of the pileus surface, stipe, hymenium, and basidiospores. Molecular phylogenetic analyses based on nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode), partial nuc rDNA 28S, and partial RNA polymerase II largest subunit (RPB1) sequences supported the description of these new taxa. Here, we propose Pseudohydnum brasiliense, P. brunneovelutinum, P. cupulisnymphae, and P. viridimontanum as new species. Morphological descriptions, line drawings, habitat photos, and comparisons with closely related taxa are provided. A dichotomous key for identification of currently known Southern Hemisphere Pseudohydnum species is presented. [ABSTRACT FROM AUTHOR]

Introduction: Cultured fish species are increasingly exposed to fungi and bacteria in the rearing environment, often causing disease and mortality causing aquaculture producers to rely on the use of chemical therapeutants which may have negative consequences for both human and environmental health. This study investigated the effect of humic acid as a treatment to reduce fungal and bacterial infections to increase survival during the incubation of rainbow trout (Oncorhynchus mykiss) eggs. Methods: Humic acid, an aqueous extract of oxidized lignite was added to water entering both stacked tray and jar type incubators at a dosage rate of 5 mg/L from fertilization until hatching. Results: The water treatment eliminated observable fungus and significantly improved survival until hatching in the treated incubator trays (77.2%) compared to the untreated controls (55.5%) (p = 0.001). Egg survival was not improved in the incubator jars. The humic acid reduced bacterial diversity, but only in the stacked tray incubators and altered bacterial composition of the water after 20 days of treatment. Discussion: The treatment increased the bacterial abundance of Burkholderiales, positively associated with healthy fish eggs and decreased the abundance of Flavobacterium and Aeromonas, known fish pathogens. These findings support the topical treatment of humic acid as a potential alternative treatment to prevent fungal infections and reduce mortality during incubation of rainbow trout eggs. [ABSTRACT FROM AUTHOR]

PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations. [ABSTRACT FROM AUTHOR]

The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111-bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into 4 independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3′ fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila , with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes. [ABSTRACT FROM AUTHOR]

Epilithic green algae play crucial roles in aquatic ecosystems. While traditional morphological approaches have provided valuable insights into these algae, their limitations in resolving cryptic species and fully capturing the extent of diversity have impeded a comprehensive understanding. The diversity and phylogenetic relationships of these algae largely remain unexplored. In the present study, single molecular real-time (SMRT) sequencing of the full-length 18S rDNA was employed to investigate the molecular diversity and phylogenetic relationships of epilithic green algae in biodiversity conservation hotspots. A total of 79 709 algal reads were generated, yielding 169 amplicon sequence variants (ASVs) from 21 epilithic riverine green algal assemblages classified in the Ulvophyceae, Chlorophyceae and Trebouxiophyceae. The 10 families in which the greatest number of unique ASVs (diversity) were observed were the Ulvellaceae, Protosiphonaceae, Chlorococcaceae, Watanabeaceae, Chlorellaceae, Neochloridaceae, Chaetophoraceae, Hazeniaceae, Tupiellaceae and Scenedesmaceae. Unique clades formed by a substantial number of sequences of green algae were detected within the Ulvales/Ulotrichales (Ulvophyceae) and the Chlorococcaceae/Protosiphonaceae (Chlorophyceae). This study has unveiled hidden diversity within epilithic green algae, expanding our knowledge of their evolutionary relationships and providing fundamental data for taxonomic investigations within this group, as well as contributing to the assessment of algal diversity in biodiversity conservation hotspots. This study suggests that these epilithic green algal assemblages may be significantly more diverse than previously recognized, but we acknowledge the limited ecological and temporal scope of our sampling effort and the resulting effects on which taxa are identified, warranting further research. Molecular analysis shows high hidden diversity of epilithic green algae The families Ulvellaceae and Protosiphonaceae had the greatest number of unique ASVs Many unique clades within the Ulvales/Ulotrichales and Chlorococcaceae/Protosiphonaceae [ABSTRACT FROM AUTHOR]

Conventional gene cloning methods always require tedious processes confined by PCR conditions and restriction sites, which might result in a large number of redundant fragments in the constructs. These shortages can be overcome by using DNA recombination methods. Homologous recombination in Escherichia coli cells was used to develop positive/negative cassette for phage λ red operon recombination. In the bi-functional cassette, the Ala294Gly α-subunit mutant of phenylalanyl-tRNA synthetase (mPheS) was fused with the kanamycin-resistant (KanR) cassette under the control of the promoter of the E. coli gene NEOKAN as a single open reading frame (ORF) by PCR. KanR was utilized as a positive selection marker, while the mutant gene mPheS served as a negative selection marker. Two-step red-mediated recombination was used to replace the third exon of TMEM18 in the BAC clone RP23-25C13 with eGFP. Initially, the third TMEM18 exon in the BAC was replaced with the NEOKAN-mPheS-KanR cassette, and 40% of the clones on the positive selection plate (LB plate with kanamycin) were positive. The second step involved replacing the NEOKAN-mPheS-KanR cassette with eGFP in the modified BAC, and 60% of the clones on the negative plate (p-Cl-phe plate) were positive. Following the two steps, eGFP was used to replace the third exon of TMEM18 in the BAC. The NEOKAN-mPheS-KanR could be a promising choice as a positive/negative selection cassette for red recombination to establish constructs and make gene manipulation on BAC more applicable and effortless. [ABSTRACT FROM AUTHOR]

Aporcelaimellus obtusicaudatus was recovered from mustard and sugarcane fields of Uttar Pradesh and Uttarakhand, India. The present species was identified through morphology, molecular and phylogenetic analysis. The Indian populations are characterized by having females with a body length (1.7–3.4 mm), lip region (4.9–8.8 µm), Amphids 6–9 µm, odontostyle length (15.7–26.4 µm), neck length (374.6–745.1 µm), pharyngeal expansion occupying 45–60% of neck length, simple uterus (67.4–124.3 µm), V = 49.1–58.7, female tail (32–44.9 µm, c = 41.1–105, c' = 0.5–1.3) with short, conoid, rounded terminus to convex conical. The molecular phylogenetic analysis based on 18S and 28S rDNA revealed A. obtusicaudatus as a paraphyly. The present populations of Indian A. obtusicaudatus (OM269524; OP020675) placed close to deposited Indian sequence (OP048816; OP048823) and Slovakian populations of A. obtusicaudatus (MH206119). The result of 28S rDNA phylogeny showed that the present population (OM258706) placed close to other populations of the same species from India (OM418794) with 0.55 posterior probability supports. The haplotype analysis based on 28S rDNA revealed 6 haplotypes, which displayed low nucleotide (π = 0.013) diversity. In contrast, haplotype diversity (Hd) was 1.00, and the number of segregating sites (S) was 14. The phylogenetic analysis grouped the various populations into two clades, and the result showed German populations placed into two clades, which showed high variability in haplotype supported by the haplotype network. Tajima (D) and Fu's Fs were found to be negative (P > 0.05), whereas the overall FST value was 0.31040 (P = 0.118). In conclusion, genetic analysis revealed 31.04% variation among groups/populations and 68.95 within populations, indicating a low genetic variation between the same populations of A. obtusicaudatus belonging to the same country. [ABSTRACT FROM AUTHOR]

The inception of recombinant DNA technology and live cell genomic alteration have paved the path for the excellence of cell and gene therapies and often provided the first curative treatment for many indications. The approval of the first Chimeric Antigen Receptor (CAR) T-cell therapy was one of the breakthrough innovations that became the headline in 2017. Currently, the therapy is primarily restricted to a few nations, and the market is growing at a CAGR (current annual growth rate) of 11.6% (2022–2032), as opposed to the established bio-therapeutic market at a CAGR of 15.9% (2023–2030). The limited technology democratization is attributed to its autologous nature, lack of awareness, therapy inclusion criteria, high infrastructure cost, trained personnel, complex manufacturing processes, regulatory challenges, recurrence of the disease, and long-term follow-ups. This review discusses the vision and strategies focusing on the CAR T-cell therapy democratization with mitigation plans. Further, it also covers the strategies to leverage the mRNA-based CAR T platform for building an ecosystem to ensure availability, accessibility, and affordability to the community. mRNA-guided CAR T cell therapy is a rapidly growing area wherein a collaborative approach among the stakeholders is needed for its success. [ABSTRACT FROM AUTHOR]

Telomeres are part of chromatin structures containing repeated DNA sequences, which function as protective caps at the ends of chromosomes and prevent DNA degradation and recombination, thus ensuring the integrity of the genome. While telomere length (TL) can be genetically inherited, TL shortening has been associated with ageing and multiple xenobiotics and bioactive substances. TL has been characterised as a reliable biomarker for the predisposition to developing chronic pathologies and their progression. This narrative review aims to provide arguments in favour of including TL measurements in a complex prognostic and diagnostic panel of chronic pathologies and the importance of assessing the effect of different pharmacologically active molecules on the biology of telomeres. Medicines used in the management of cardiovascular diseases, diabetes, schizophrenia, hormone replacement therapy at menopause, danazol, melatonin, and probiotics have been studied for their positive protective effects against TL shortening. All these classes of drugs are analysed in the present review, with a particular focus on the molecular mechanisms involved. [ABSTRACT FROM AUTHOR]

Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states. [ABSTRACT FROM AUTHOR]

AbstractPlant-parasitic nematodes (PPNs) pose a significant threat to global agriculture, leading to substantial yield losses. Managing nematode populations remains a challenging option due to financial, technical and environmental constraints. In Ontario, Canada, the presence of Xiphinema nematodes, a virus vector species, is of particular concern. This study reports the discovery of X. diversicaudatum in a peach field in Southern Ontario, Canada. The description includes morphological and molecular characterization using the 18S and 28S rDNA sequences. Morphological and molecular characterization of Ontario’s population matched those from Europe. While X. diversicaudatum was presumed eradicated from Canada, the present finding showed that the nematode could have spread beyond greenhouses. The study highlights the importance of intensive soil sampling for early nematode detection, even as strict reporting requirements may deter such efforts. We report for the first time the presence of X. diversicaudatum on peaches in Canada and in North America. [ABSTRACT FROM AUTHOR]

Summary: Forests make immense contributions to societies in the form of ecological services and sustainable industrial products. However, they face major challenges to their viability and economic use due to climate change and growing biotic and economic threats, for which recombinant DNA (rDNA) technology can sometimes provide solutions. But the application of rDNA technologies to forest trees faces major social and biological obstacles that make its societal acceptance a 'wicked' problem without straightforward solutions. We discuss the nature of these problems, and the social and biological innovations that we consider essential for progress. As case studies of biological challenges, we focus on studies of modifications in wood chemistry and transformation efficiency. We call for major innovations in regulations, and the dissolution of method‐based market barriers, that together could lead to greater research investments, enable wide use of field studies, and facilitate the integration of rDNA‐modified trees into conventional breeding programs. Without near‐term adoption of such innovations, rDNA‐based solutions will be largely unavailable to help forests adapt to the growing stresses from climate change and the proliferation of forest pests, nor will they be available to provide economic and environmental benefits from expanded use of wood and related bioproducts as part of an expanding bioeconomy. [ABSTRACT FROM AUTHOR]

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology—which we refer to as spatial organization—of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions. Author summary: Cells devote significant energy and resources to produce ribosomes, large protein complexes that carry out protein synthesis. To ensure that ribosome production matches cellular needs, activation of ribosome assembly is regulated by many inputs. One key regulatory target is transcription of the tandemly repeated rRNA genes in the ribosomal DNA arrays. A broad understanding of how cells regulate the organization of the rDNA under different cellular conditions and transcriptional states has been difficult to assess with current tools and model systems. We present a new imaging reporter, a fluorescently tagged DNA-binding protein called GapR, that highlights rDNA spatial organization in fission yeast, a model system that shares similar regulatory networks with mammals. Using GapR, we found that the 3D organization of the rDNA arrays changes with the cell cycle, glucose starvation, RNA pol I inhibition, and TOR signaling. We surveyed hundreds of yeast mutants to identify factors that regulate rDNA spatial organization, discovering that the deletion of ribosome protein genes causes rDNA array expansion. Further experiments indicated that ribosome protein insufficiency activates conserved regulatory signaling pathways as part of a compensatory response. Our work presents the first broad investigation for regulators of rDNA spatial organization in any organism. Importantly, we identify a compensatory response to compromised ribosome biogenesis that could be conserved in mammals. [ABSTRACT FROM AUTHOR]

We describe a new species of Discostella from Guiyang based on light and scanning electron microscopic observations, as well as molecular analysis. This species is morphologically distinguished from other species by a relatively smooth and slightly recessed central area with marginal striae of unequal lengths forked near the margin. Marginal fultoportulae and a single rimoportula are situated between every two costae or on the costae. Both morphological characters and the concatenated data of rbcL and SSU rDNA indicate that Discostella guiyangensis is a unique and new species. According to divergence time estimation analysis, Discostella guiyangensis is the oldest species among the Discostella species sequenced currently. In addition, we complement molecular data of Discostella asterocostata and Discostella stelligera. [ABSTRACT FROM AUTHOR]

Summary: A member of the genus Pellioditis Dougherty, 1953 (syn. Phasmarhabditis Andrássy, 1976) was detected in three introduced gastropod species in Moscow for the first time. Both the nematode and its gastropod hosts were found to have originated from the Caucasus region. No native gastropod species as well as the alien invasive slug Arion vulgaris were infected by P. akhaldaba. The Moscow strain of P. akhaldaba is very close to P. akhaldaba from Georgia in the ITS rDNA (7 bp difference) but they differ significantly in the sequences of COI mtDNA (50 bp difference). However, phylogenetic analysis of both loci showed that these two strains were the closest relative to all other species and strains of the genus Pellioditis studied. [ABSTRACT FROM AUTHOR]

This study was aimed to identify major species of Streptomyces that are able to cause potato common scab in Egypt. Potato tubers showing typical symptoms of common scab disease were collected from three different regions of Upper Egypt. The causal pathogens were isolated, and morphological, biochemical and pathogenicity tests were conducted in vitro to preliminary identify the isolated Streptomyces isolates. Moreover, the partial 16S rDNA gene sequencing was used to genetically identification of the isolates. Four new pathogenic species that have not previously been reported in Egypt were identified. Four species of Streptomyces rochei, S. rutgersensis, S. lateritius, and S. bottropensis were identified. Isolates S1, S10, S14, S17, S18, S20, and S21 were identified as Streptomyces rochei, isolates S11 was identified as S. rutgersensis, isolates S13 identified as S. lateritius and isolates S15 was identified as S. bottropensis. To the obtained data, this is the first report that S. rochei, S. rutgersensis, S. lateritius, and S. bottropensis cause of potato common scab disease in Egypt. The different of obtained 16S rDNA sequences were deposited at NCBI GenBank with the following accession numbers: (S1) MZ267260, (S10) MZ267264, (S14) MZ267268, (S17) MZ267271, (S18) MZ267272, (S20) MZ267274, (S21) MZ267275, (S11) MZ267265, (S13) MZ267267, and (S15) MZ267269. The four newly identified isolates showed different pathogenicity, morphological and biochemical characteristics. [ABSTRACT FROM AUTHOR]

Four new species of jelly fungi were described from northeastern China based on morphological and molecular evidence. These new species were classified into the four genera Sirobasidium (Sirobasidium jilinense), Calocera (Calocera velutina), Dacrymyces (Dacrymyces jauensis), and Dacryopinax (Dacryopinax manghanensis). Maximum likelihood and Bayesian analyses were performed using a combined nuc rDNA internal transcribed spacer region (ITS) and nuc 28S rDNA (nrLSU) dataset for the construction of phylogenetic trees. Morphological descriptions, line illustrations, and the ecological habits of these new species are provided. [ABSTRACT FROM AUTHOR]

Short‐Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single‐strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single‐strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single‐strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR‐avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non‐transformable bacteria. [ABSTRACT FROM AUTHOR]

The planktonic dinoflagellate Prorocentrum compressum is widespread in warm and temperate seas. A strain identified as P. cf. compressum BEA 0681B isolated from the island of Gran Canaria, NE Atlantic Ocean, showed a divergence in rDNA/ITS phylogenies with respect to P. compressum. The Canarian strain was oval, with an average length‐to‐width ratio of 1.35, smooth thecal surface with less than 150 thecal pores, including oblique pores, sometimes with a bifurcated opening. In contrast, P. compressum was rounder, with a length‐to‐width ratio < 1.2, with reticulate‐foveate ornamentation and 200–300 pores per valve. We propose Prorocentrum canariense sp. nov. These species clustered as the most early‐branching lineage in the clade Prorocentrum sensu stricto. Although this clade mainly contains planktonic species, the closer relatives were the benthic species P. tsawwassenense and P. elegans. Interestingly, P. compressum and P. canariense sp. nov. are widely distributed in temperate and warm seas without an apparent morphological adaptation to planktonic life. The formation of two concentric hyaline mucilaginous walls could contribute to this success. We discuss the use of Prorocentrum bidens to solve the nomenclature issue of P. compressum that was described citing a diatom as basionym. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are garnering attention as a safe and efficient biomolecule delivery system. EVs intrinsically play a crucial role in intercellular communication and pathophysiology by transporting functionally active DNA molecules. The internalized DNA pleiotropically affects the recipient cells. Considering these salient features, an intentional incorporation of specific DNA gene cassettes into EVs and their subsequent delivery to the target cells has potential applications in genetic engineering. Moreover, efficient ways to insert the DNA into EVs during their biogenesis is valuable. Our current research is a step in the development of this technology. As such, cancer cells are known to secrete exosomes containing increased amounts of double-stranded DNA than normal cells. The clonal analysis in our previously published data revealed that exosomes released from various cancer cells contained a significantly larger population of NANOGP8 DNA with a 22-base pair insertion in the 3′-untranslated region (UTR) compared to those secreted by normal cells. This finding led us to hypothesize that the 22-base pair insertion may act as a signal to facilitate the incorporation of NANOGP8 DNA into the exosomes. To test this hypothesis, we compared the EV localization of an Enhanced Green Fluorescent Protein (EGFP) gene fused with the NANOGP8 3′-UTR, with and without the 22-base pair insertion. The quantitative PCR analysis showed a significantly higher EGFP DNA accumulation in exosomes released from cells transfected with the gene cassette containing the 3′-UTR with the 22-base pair insertion. The discovery of a DNA localization signal in exosomal DNA's 3'-UTR could pave the way for the development of an EV-based DNA delivery system. This technology will open new possibilities in genetic engineering and innovative therapies using nucleic acid medicine. [ABSTRACT FROM AUTHOR]

Ribosomal DNA (rDNA) has a vital role in ribosome biogenesis as it contains the genes that encode ribosomal RNA (rRNA) separated by intergenic spacers (IGSs). The rRNA genes occur in hundreds to tens of thousands of copies per haploid genome in eukaryotes and are generally highly conserved with low variation within species. Due to the repetitive nature and large size of rDNA arrays, detecting intraindividual variation can be difficult. In this study, we use whole-genome sequences of 169 Daphnia pulex individuals from 10 natural populations to measure the copy number and sequence variation in rDNA. This revealed that variation in rDNA copy number between individuals spans an order of magnitude. We further observed a substantial level of sequence variation within individual genomes. As expected, single-nucleotide polymorphisms occurred in regions of lower functional constraint such as the IGS and expansion segments of the rRNA genes. The presence of strong linkage disequilibrium among variants facilitated identification of haplotypes within each population. Although there was evidence of recombination among haplotypes from different populations, it is insufficient to eliminate linkage disequilibrium within populations. Estimating copy number and haplotype diversity within individuals revealed that the level of intraindividual sequence variation is not strongly correlated with copy number. The observed patterns of variation highlight a complex evolutionary history of rDNA in D. pulex. Future research should explore the functional implications of rDNA copy number and sequence variation on organismal phenotypes. [ABSTRACT FROM AUTHOR]

Rice-fish symbiosis is an ecological farming model that can improve fish's muscle nutrient profile and intestinal flora. We investigated the effect of rice-fish symbiosis on tilapia's intestinal flora and muscle nutrients. We used 16S rDNA technology to analyze the intestinal flora of tilapia in pond culture and rice-fish symbiosis. Furthermore, spearman correlation analysis was performed on tilapia intestinal flora and muscle nutrients. The results showed that the contents of Valine, Phenylalanine, and Docosahexaenoic acid were significantly higher in the rice-fish symbiosis than those in the pond culture (p<0.05). Intestinal flora analysis showed that the diversity in rice-fish symbiosis was significantly higher than in pond culture (p<0.05). Meanwhile, the abundance of Brevundimonas, Pseudomonas, and Ralstonia in rice-fish symbiosis was significantly higher than those in pond culture (p<0.05), but Pirellulaceae unclassified and Cyanobium_PCC-6307 were opposite. The number of 8 predictive functional tilapia genes in the rice-fish symbiosis was higher than in the pond culture. Correlation analysis showed that the abundance of Ralstonia was positively correlated with the contents of Valine, Phenylalanine, and Docosahexaenoic acid, respectively. Our study shows that the rice-fish symbiosis could improve muscle nutrient profile and intestinal flora diversity in tilapia. This study will help to improve the rice-fish symbiosis model and provide a scientific basis for ecological aquaculture and healthy fish production. [ABSTRACT FROM AUTHOR]

The T7 gene 3 product, T7 endonuclease I, acts on various substrates with DNA structures, including Holliday junctions, heteroduplex DNAs and single-mismatch DNAs. Genetic analyses have suggested the occurrence of DNA recombination, replication and repair in Escherichia coli. In this study, T7 endonuclease I digested UV-irradiated covalently closed circular plasmid DNA into linear and nicked plasmid DNA, suggesting that the enzyme generates single- and double-strand breaks (SSB and DSB). To further investigate the biochemical functions of T7 endonuclease I, we have analysed endonuclease activity in UV-induced DNA substrates containing a single lesion, cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts (6–4PP). Interestingly, the leading cleavage site for CPD by T7 endonuclease I is at the second and fifth phosphodiester bonds that are 5′ to the lesion of CPD on the lesion strand. However, in the case of 6–4PP, the cleavage pattern on the lesion strand resembled that of CPD, and T7 endonuclease I could also cleave the second phosphodiester bond that is 5′ to the adenine–adenine residues opposite the lesion, indicating that the enzyme produces DSB in DNA containing 6–4PP. These findings suggest that T7endonuclease I accomplished successful UV damage repair by SSB in CPD and DSB in 6–4PP. [ABSTRACT FROM AUTHOR]

Intracellular parasites of the genus Glugea Thélohan, 1891 (Microsporidia) comprise about 34 putative species capable of causing high morbidity and mortality in freshwater and marine teleost fishes. In this study, we report on the first mass mortality event associated with Glugea sp. infecting free‐ranging round sardinella Sardinella aurita in the southern Tyrrhenian Sea (Italy). Here, we describe the ultrastructure of mature spores of this microsporidian and characterize it molecularly, as well as report its phylogenetic position. Most of the affected fish showed an irregular swelling of its abdomen. At necropsy, a variable number of xenomas, spherical to ellipsoidal in shape, were found in the peritoneal cavity strongly attached to the viscera of all fish. Histological analysis revealed varying severity of chronic inflammation along with occasional necrosis in visceral organs associated with multiple xenoma proliferation. These pathological findings were considered the main cause of this mass mortality event. Morphologically, the present material was closely related to G. sardinellesis and G. thunni. The phylogenetically closest taxa to the newly SSU rDNA sequence were G. thunni and an erroneusly identified  G. plecoglossi, which were very closely related to each other, also suggesting that all these sequences might belong to the same species. [ABSTRACT FROM AUTHOR]

Recombinant protein production technology is widely applied to the manufacture of biologics used as drug substances and industrial proteins such as recombinant enzymes and bioactive proteins. Various heterologous protein production systems have been developed using prokaryotic and eukaryotic hosts. Especially methylotrophic yeast in eukaryotic hosts is suggested to be particularly valuable because such systems have the following advantages: protein secretion into culture broth, eukaryotic quality control systems, a post-translational modification system, rapid growth, and established recombinant DNA tools and technologies such as strong promoters, effective selection markers, and gene knock-in and -out systems. Many methylotrophic yeasts such as the genera Candida, Ogataea, and Komagataella have been studied since methylotrophic yeast was first isolated in 1969. The methanol-consumption-related genes in methylotrophic yeast are strongly and strictly regulated under methanol-containing conditions. The well-regulated gene expression systems under the methanol-inducible gene promoter lead to the potential application of heterologous protein production in methylotrophic yeast. In this review, we describe the recent progress of heterologous protein production technology in methylotrophic yeast and introduce Ogataea minuta as an alternative production host as a substitute for K. phaffii and O. polymorpha. [ABSTRACT FROM AUTHOR]

Two populations of Aporcelaimellus obtusicaudatus were isolated from the soils of sugarcane and Paddy fields of district Dehradun (30° 19' N, 78°03' E and 960 meter above sea level), Uttarakhand, India. Based on morphological, morphometrical and molecular studies, the nematode was identified as Aporcelaimellus obtusicaudatus. Both isolates characterized by having body size 1.6-2.9 mm (SF5) and 1.8-3.1 mm (SF8) long, lip region offset by deep constriction, lips angular and 15.5-19.8 μm (SF5) and 14.5-18.8 μm (SF8) broad, odontostyle dorylemoides with wide lumen 19-22 μm long in (SF5) and 20-23 μm long in (SF8), odontostyle aperture half of its length, neck length 416-631 μm long (SF5) and 430-594 μm long (SF8), pharyngeal expansion 220.3-367 μm and 234-327.5 μm long or occupying 52-58% and 54-55% of the total neck length in both isolates, female genital system amphidelphic with transverse vulva and opaque pars refringens vaginae, tail short and rounded to conical with inner cuticle. Molecular analyses utilizing segments of the 28S rDNA and 18S rDNA placed Aporcelaimellus obtusicaudatus in a clade with other A. obtusicaudatus reported from different geography Ireland, South Africa, China and UK. A comparative study of species of genus Aporcelaimellus is also provided. [ABSTRACT FROM AUTHOR]

An integrated and projected‐based laboratory course was described, integrating interconnected knowledge points and biochemistry and molecular biology techniques on a research project‐based system. The program, which served as an essential extension of theoretical courses to practice, was conducted with a sophomore of basic medical science who had completed the course in medical biochemistry and molecular biology. This course engaged students in learning "genetic manipulation" and "recombinant DNA technology" to understand the target gene's role in disease mechanics, thus altering evaluation and treatment for clinical disease. Students could master applied and advanced techniques, such as cell culture, transfection, inducing exogenous fusion protein expression, purifying protein and its concentration assay, quantitative polymerase chain reaction, and western bot analysis. This laboratory exercise links laboratory practices with the methods of current basic research. Students need to complete the experimental design report and laboratory report, which could be advantageous for improving their ability to write lab summaries and scientific papers in the future. The reliability and validity analyses were conducted on the questionnaire, and we examined students' satisfaction with the course and their gains from the course. The student feedback was generally positive, indicating that the exercise helped consolidate theoretical knowledge, increase scientific research enthusiasm, and provide a powerful tool to be a better person and make informed decisions. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies are widely used in all fields of biology and medicine. The emergence and development of the technology for their production revolutionized immunology and allowed the creation of not only new diagnostic methods, but also many effective drugs. The purpose of our review is to analyze and summarize relevant data concerning the technology of obtaining monoclonal antibodies. We have analyzed information from 70 modern literary sources devoted to various methods of obtaining them. In this review, we tried to cover the entire range of methods used to obtain monoclonal antibodies today.

This study determined the effects of administering a glycoprotein with equine chorionic gonadotropin (eCG)-like activity (eCG-like) on corpus luteum (CL) area, serum progesterone concentrations, incidence of multiple ovulations (MOV), estrus expression rate (EER), and pregnancy to timed AI (P/TAI) in Angus cattle synchronized with a 5-d Co-Synch protocol. On Day −8, cattle were body condition scored (BCS), and received a 1.0 g progesterone intravaginal device (IVD) and 100 μg GnRH. On Day −3, the IVDs were removed and 500 μg cloprostenol was administered intramuscularly (i.m.). Cattle were randomly assigned into one of two groups: eCG-like (heifers, n = 232, primiparous, n = 148, and multiparous cows = 485; 300 IU (heifers) and 400 IU (cows) eCG-like i.m. on Day −3), or Control (heifers, n = 240, primiparous, n = 151, and multiparous cows, n = 478; no eCG-like). On Day −2, cattle received a second dose of 500 μg cloprostenol, and on Day 0, 100 μg GnRH was given concurrently with TAI. Estrus expression rate was assessed by observing the tail paint rubbed off in a subset of heifers (n = 372) and all cows on Day 0. Transrectal ultrasonography was used to evaluate the presence of CL on Day −8 and to diagnose P/TAI on Day 30–35. In a subset of cattle (heifers = 194 and multiparous cows = 87), CL area, serum progesterone concentrations, and incidence of MOV were evaluated on Day 7. Heifers, primiparous, and multiparous cows were analyzed separately. Treatment with eCG-like did not affect (P > 0.1) EER in heifers. Estrus expression rate was increased (P ≤ 0.03) in primiparous (68.9 % vs 45.0 %) and multiparous (75.5 % vs. 68.8 %) cows treated with eCG-like compared with Controls. Pregnancy/TAI was increased (P < 0.01) in heifers (65.2 % vs 48.3 %) and primiparous cows (48.3 % vs. 35.1 %) treated with eCG-like than Controls. In multiparous cows with a BCS ≤4 P/TAI was increased (P = 0.03) in the eCG-like group (47.7 %) than the Control group (34.8 %) but was similar (P > 0.1) between treatment groups in multiparous cows with a BCS ≥4.5. The eCG-like treatment increased (P < 0.05) CL area in heifers and multiparous cows and tended (P = 0.10) to elevate serum progesterone concentrations only in heifers. However, it did not affect (P > 0.1) the incidence of MOV in heifers and multiparous cows. Glycoprotein eCG-like administration increased fertility in heifers and primiparous cows, but in multiparous the effect of eCG-like on fertility was associated with BCS. eCG-like treatment in a 5-day Co-Synch synchronization protocol: • Increases estrus expression rate in primiparous and multiparous cows. • Increases P/TAI in heifers, primiparous cows, and multiparous cows with a BCS ≤ 4. • Increases CL area in heifers and multiparous cows. • Does not affect the incidence of multiple ovulations in heifers and multiparous cows. [ABSTRACT FROM AUTHOR]

Recombinant protein expression is a fundamental aspect of both synthetic biology and biotechnology as well as a field unto itself. Microalgae, with their eukaryotic cellular machinery, high lipid content, cost-effective cultivation conditions, safety profile for human consumption, and environmentally friendly attributes, are a promising system for protein expression or metabolic engineering for sustainable chemical production. Amongst the incredible diversity of microalgae species, Chlorella spp. are heavily studied due to their high growth efficiency, potential for low-cost cultivation, and well-characterized scale-up process for large-scale cultivation. This review aims to comprehensively examine the ongoing advancements in the bioengineering of Chlorella spp. for recombinant protein production and its biotechnological applications. This includes genetic elements such as promoters, terminators, reporters and markers, enhancers, and tags successfully used in Chlorella spp.

Saccharomyces cerevisiae always faces various environmental stresses during industrial-scale fermentation, including oxidation, high temperature, acid, ethanol, and hyperosmosis. Therefore, screening and breeding of strains with high tolerance to these stresses is of great significance for industrial applications. It is well known that DNA recombination enzyme, nucleic acid repair enzyme, and other proteins involved in nucleic acid repair pathway play important roles in tolerance to abiotic stresses. Based on that, the effects of endogenous nucleic acid excision and repair genes (RAD16, RAD7, RAD23 and RAD4) and exogenous genes (UVRA) on the tolerance of S. cerevisiae to different stresses were investigated by gene editing. The results showed that overexpression of RAD16, RAD7, RAD23 and RAD4 could improve the tolerance against osmotic stress in S. cerevisiae. Furthermore, overexpression of UVRA gene from bacterium Acetobacter pasteurii could also improve the tolerance against osmotic stress, suggesting that the function of UVRA was conserved between eukaryotic and procaryotic cells. This results were beneficial to improvement of the tolerance of S. cerevisiae strains, especially under high osmotic stress, and also provide a novel strategy to reveal the mechanism of tolerance in yeast. [ABSTRACT FROM AUTHOR]

Background: Non-conventional yeasts hold significant potential as biorefinery cell factories for microbial bioproduction. Currently, gene editing systems used for these yeasts rely on antibiotic and auxotrophic selection mechanisms. However, the drawbacks of antibiotics, including high costs, environmental concerns, and the dissemination of resistance genes, make them unsuitable for large-scale industrial fermentation. For auxotrophic selection system, the engineered strains harboring auxotrophic marker genes are typically supplemented with complex nutrient-rich components instead of precisely defined synthetic media in large-scale industrial fermentations, thus lack selection pressure to ensure the stability of heterologous metabolic pathways. Therefore, it is a critical to explore alternative selection systems that can be adapted for large-scale industrial fermentation. Results: Here, a novel glucose-dependent selection system was developed in a high pullulan-producing non-conventional strain A. melanogenum P16. The system comprised a glucose-deficient chassis cell Δpfk obtained through the knockout of the phosphofructokinase gene (PFK) and a series of chromosomal integration plasmids carrying a selection marker PFK controlled by different strength promoters. Utilizing the green fluorescent protein gene (GFP) as a reporter gene, this system achieved a 100% positive rate of transformation, and the chromosomal integration numbers of GFP showed an inverse relationship with promoter strength, with a customizable copy number ranging from 2 to 54. More importantly, the chromosomal integration numbers of target genes remained stable during successive inoculation and fermentation process, facilitated simply by using glucose as a cost-effective and environmental-friendly selectable molecule to maintain a constant and rigorous screening pressure. Moreover, this glucose-dependent selection system exhibited no significant effect on cell growth and product synthesis, and the glucose-deficient related selectable marker PFK has universal application potential in non-conventional yeasts. Conclusion: Here, we have developed a novel glucose-dependent selection system to achieve customizable and stable multilocus chromosomal integration of target genes. Therefore, this study presents a promising new tool for genetic manipulation and strain enhancement in non-conventional yeasts, particularly tailored for industrial fermentation applications. [ABSTRACT FROM AUTHOR]

Holliday junction resolution is a crucial process in homologous recombination and DNA double-strand break repair. Complete Holliday junction resolution requires two stepwise incisions across the center of the junction, but the precise mechanism of metal ion-catalyzed Holliday junction cleavage remains elusive. Here, we perform a metal ion-triggered catalysis in crystals to investigate the mechanism of Holliday junction cleavage by MOC1. We capture the structures of MOC1 in complex with a nicked Holliday junction at various catalytic states, including the ground state, the one-metal ion binding state, and the two-metal ion binding state. Moreover, we also identify a third metal ion that may aid in the nucleophilic attack on the scissile phosphate. Further structural and biochemical analyses reveal a metal ion-mediated allosteric regulation between the two active sites, contributing to the enhancement of the second strand cleavage following the first strand cleavage, as well as the precise symmetric cleavage across the Holliday junction. Our work provides insights into the mechanism of metal ion-catalyzed Holliday junction resolution by MOC1, with implications for understanding how cells preserve genome integrity during the Holliday junction resolution phase. The precise mechanism of metal-ion catalysis in Holliday junction resolution remains elusive. Here, the authors describe a metal ion-mediated nick and counter-nick mechanism of Holliday junction cleavage by MOC1. [ABSTRACT FROM AUTHOR]

SUMMARY: Deficiency in chromatin assembly factor‐1 (CAF‐1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication‐dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild‐type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single‐strand annealing pathway of homology‐dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF‐1 dysfunction. Significance Statement: We explore the contribution of KU70 to the specific genome instability of plants dysfunctional in chromatin assembly factor‐1 (fas1 plants) expressed as a progressive loss of telomeres and rDNA repeats. While KU dysfunction does not markedly affect rDNA instability in fas1 plants, it fully suppresses the progressive loss of telomeres in fas1 plants through activation of alternative telomere lengthening. [ABSTRACT FROM AUTHOR]

Cultivated potato (Solanum tuberosum) is a major crop worldwide. It occupies the second place after cereals (corn, rice, and wheat). This important crop is threatened by the Oomycete Phytophthora infestans, the agent of late blight disease. This pathogen was first encountered during the Irish famine during the 1840s and is a reemerging threat to potatoes. It is mainly controlled chemically by using fungicides, but due to health and environmental concerns, the best alternative is resistance. When there is no disease, no treatment is required. In this study, we present a summary of the ongoing efforts concerning resistance breeding of potato against this devastating pathogen, P. infestans. This work begins with the search for and selection of resistance genes, whether they are from within or from outside the species. The genetic methods developed to date for gene mining, such as effectoromics and GWAS, provide researchers with the ability to identify genes of interest more efficiently. Once identified, these genes are cloned using molecular markers (MAS or QRL) and can then be introduced into different cultivars using somatic hybridization or recombinant DNA technology. More innovative technologies have been developed lately, such as gene editing using the CRISPR system or gene silencing, by exploiting iRNA strategies that have emerged as promising tools for managing Phytophthora infestans, which can be employed. Also, gene pyramiding or gene stacking, which involves the accumulation of two or more R genes on the same individual plant, is an innovative method that has yielded many promising results. All these advances related to the development of molecular techniques for obtaining new potato cultivars resistant to P. infestans can contribute not only to reducing losses in agriculture but especially to ensuring food security and safety. [ABSTRACT FROM AUTHOR]

Graphene-based warm uterus acupoint paste (GWUAP) is an emerging non-drug alternative therapy for the treatment of primary dysmenorrhea (PD), but the underlying mechanism is still unclear. SD female rats were randomly divided into control group, model group and treatment group to explore the mechanism of GWUAP in the treatment of PD. Combined with 16S rDNA and fecal metabolomics, the diversity of microbiota and metabolites in each group was comprehensively evaluated. In this study, GWUAP reduced the torsion score of PD model rats, improved the pathological morphology of uterine tissue, reduced the pathological damage score of uterine tissue, and reversed the expression levels of inflammatory factors, pain factors and sex hormones. The 16 S rDNA sequencing of fecal samples showed that the abundance of Lactobacillus in the intestinal flora of the model group decreased and the abundance of Romboutsia increased, while the abundance of Lactobacillus in the intestinal flora of the treatment group increased and the abundance of Romboutsia decreased, which improved the imbalance of flora diversity in PD rats. In addition, 32 metabolites related to therapeutic effects were identified by metabolomics of fecal samples. Moreover, there is a close correlation between fecal microbiota and metabolites. Therefore, the mechanism of GWUAP in the treatment of PD remains to be further studied. [ABSTRACT FROM AUTHOR]

Physella acuta is a freshwater snail native to North America. Understanding the phylogeography and genetic structure of P. acuta will help elucidate its evolution. In this study, we used mitochondrial (COI and 16S rDNA) and nuclear (ITS1) markers to identify the species and examine its genetic diversity, population structure, and demographic history of P. acuta in Thailand. Phylogenetic and network analyses of P. acuta in Thailand pertained to clade A, which exhibits a global distribution. Analysis of the genetic structure of the population revealed that the majority of pairwise comparisons showed no genetic dissimilarity. An isolation-by-distance test indicates no significant correlation between genetic and geographical distances among P. acuta populations, suggesting that gene flow is not restricted by distance. Demographic history and haplotype network analyses suggest a population expansion of P. acuta, as evidenced by the star-like structure detected in the median-joining network. Based on these results, we concluded that P. acuta in Thailand showed gene flow and recent population expansion. Our findings provide fundamental insights into the genetic variation of P. acuta in Thailand. [ABSTRACT FROM AUTHOR]

Introduction: Currently, there are 38 valid species of freshwater stingrays, and these belong to the subfamily Potamotrygoninae. However, cytogenetic information about this group is limited, with studies mainly using classical techniques, Giemsa, and C-banding. Methods: In this study, we used classical and molecular cytogenetic techniques – mapping of 18S and 5S rDNA and simple sequence repeats (SSRs) – in order to investigate the karyotypic composition of Potamotrygon schroederi and reveal the karyoevolutionary trends of this group. Results: The species presented 2n = 66 chromosomes with 18m + 12sm + 16st + 20a, heterochromatic blocks distributed in the centromeric regions of all the chromosomes, and terminal blocks in the q arm of pairs 2 and 3. Mapping of 18S rDNA regions revealed multiple clusters on pairs 2 and 7 and a homolog of pair 24. The 5S rDNA region was found in the pericentromeric portion of the subtelocentric pair 16. Furthermore, dinucleotide SSRs sequences were found in the centromeric and terminal regions of different chromosomal pairs, with preferential accumulation in pair 17. In addition, we identified conspicuous blocks of (GATA)n and (GACA)n sequences colocalized with the 5S rDNA (pair 16). Conclusion: In general, this study corroborates the general trend of a reduction in 2n in the species of Potamotrygoninae subfamily. Moreover, we found that the location of rDNA regions is very similar among Potamotrygon species, and the SSRs accumulation in the second subtelocentric pair (17) seems to be a common trait in this genus. [ABSTRACT FROM AUTHOR]