DNA Sequence Research Articles

Ferulic Acid Protects Human Lens Epithelial Cells Against UVA-Induced Oxidative Damage by Downregulating the DNA Demethylation of the Keap1 Promoter.

Ultraviolet (UV) radiation-triggered production of reactive oxygen species (ROS) is a primary contributor to apoptosis in human lens epithelial cells (HLECs), which can ultimately result in cataract formation. The nuclear factor erythroid-2-related factor 2 (Nrf2)-Kelch ECH associating protein 1 (Keap1) pathway, a fundamental oxidative stress regulation mechanism, plays a crucial role in the development of cataracts. Ferulic acid (FA), recognized for its potent antioxidant properties can activate the Nrf2 signaling pathway to mitigate oxidative damage and cell apoptosis. In this study, we have demonstrated the protective effects of FA in reducing UVA-induced oxidative damage and apoptosis in HLECs through the modulation of the Keap1/Nrf2 pathway, as evidenced by both cellular and animal experiments. HLECs and Lens were exposed to 10 J/cm2 UVA radiation with or without prior treatment with FA. We found that UVA radiation increased oxidative damage and cell apoptosis in HLECs, ultimately leading to opacification of rat lenses, while FA was able to attenuate both oxidative damage and cell apoptosis in HLECs and reduce the degree of lens opacification. FA upregulated the expression of antioxidant response factors of the Keap1/Nrf2 pathway and downregulated the expression of apoptosis-related genes in HLECs, as demonstrated by Western blot and RT-qPCR analyses. We also found that UVA radiation increased the degree of demethylation of the Keap1 promoter in HLECs, whereas FA reduced the level of Keap1 promoter demethylation as determined by DNA sequencing. Additionally, UVA upregulated the expression of DNA active demethylase of the Keap1 promoter in HLECs, Dnmt1, Dnmt3a, and Dnmt3b, as shown by immunofluorescence, Western blot, and RT-qPCR, however, FA attenuated the activity of the passive demethylase TET1 in addition to the active demethylases. These results demonstrated that UVA radiation can cause oxidative damage, cell apoptosis, and rat lens opacification by increasing the demethylation of the Keap1 promoter in lens epithelial cells. Conversely, FA was shown to reduce oxidative damage, inhibit cell apoptosis, and decrease rat lens opacification by increasing the methylation of the Keap1 promoter. These findings suggest that FA could be therapeutically beneficial in preventing and mitigating cataracts induced by UVA radiation.

Somatic reversion in Wiskott-Aldrich syndrome: Case reports and mechanistic insights.

This report describes two brothers from India and a Chinese patient with somatic reversion of an inherited deleterious mutation in the WAS gene. Both the Indian siblings had inherited a single nucleotide deletion causing a frameshift mutation (c.1190del, p.Pro397Argfs*48) (variant 1: marked in blue) from the mother. Another variant (variant 2: marked in red), a 12-nucleotide deletion at position 1188-1199 (c.1188_1199del, p.P401_P404del) was also found, which resulted in restoration of the frame and subsequent rescue of the protein sequence. DNA sequencing from buccal mucosal cells revealed only the inherited variant (variant 1), while no reversion mutation was identified in the mucosal cells. Similarly, the Chinese patient was found to have a novel germline 14-base duplication (ACGAAAATGCTTGG) c.120_132 + 1dup (variant 1). This resulted in abolishment of the original splice junction coupled with the creation of a new junction 14 bases 3' and a frameshift mutation with predicted protein truncation p. Thr45Aspfs*. DNA from the patient's PBMC showed co-existence of wild-type and mutated sequences, but only the mutant was present in the buccal cells. Genomic and mRNA analysis of the isolated CD3+ T lymphocytes, CD3- mononuclear cells, and EBV-transformed B lymphocytes indicated that the reverant variant (germline variant was restored to wild-type sequence) were selectively found in CD3+ T lymphocytes.

All-in-one bimodal DNA and RNA next-generation sequencing panel for integrative diagnosis of glioma

Previously, we constructed a DNA-based next-generation sequencing (NGS) panel for an integrated diagnosis of gliomas according to the 2021 World Health Organization classification system. The aim of the current study was to evaluate the feasibility of a modified panel to include fusion gene detection via RNA-based analysis. Using this bimodal DNA/RNA panel, we analyzed 210 cases of gliomas and others to identify fusion genes in addition to gene alterations, including TERT promoter (TERTp) mutation and 1p/19q co-deletion, in formalin-fixed paraffin-embedded tissues. Of the 210 patients, fusion genes were detected in tumors of 35 patients. Eighteen of 112 glioblastomas (GBs) harbored fusion genes, including EGFR and FGFR3 fusions. In IDH-mutant astrocytoma, 6 of 30 cases showed fusion genes such as MET and NTRK2 fusions. Eleven molecular GBs and 20 not-elsewhere-classified cases harbored no gene fusions. Other 11 tumors including ependymoma, pilocytic astrocytoma, diffuse hemispheric glioma, infant-type hemispheric glioma, and solitary fibrous tumors exhibited diagnostic fusion genes. Overall, our results suggest that the all-in-one bimodal DNA/RNA panel is reliable for detecting diagnostic gene alterations in accordance with the latest WHO classification. The integrative pathological and molecular strategy could be valuable in confirmation of diagnosis and selection of treatment options for brain tumors.

Differential modulation of polycomb-associated histone marks by cBAF, pBAF, and gBAF complexes.

Chromatin regulators alter the physical properties of chromatin to make it more or less permissive to transcription by modulating another protein's access to a specific DNA sequence through changes in nucleosome occupancy or histone modifications at a particular locus. Mammalian SWI/SNF complexes are a group of ATPase-dependent chromatin remodelers. In mouse embryonic stem cells, there are three primary forms of mSWI/SNF: canonical BAF (cBAF), polybromo-associated BAF (pBAF), and GLTSCR-associated BAF (gBAF). Nkx2-9 is bivalent, meaning nucleosomes at the locus have active and repressive modifications. In this study, we used unique BAF subunits to recruit each of the three complexes to Nkx2-9 using dCas9-mediated inducible recruitment (FIRE-Cas9). We show that recruitment of cBAF complexes leads to a significant loss of the polycomb repressive-2 H3K27me3 histone mark and polycomb repressive-1 and repressive-2 complex proteins, whereas gBAF and pBAF do not. Moreover, nucleosome occupancy alone cannot explain the loss of these marks. Our results demonstrate that cBAF has a unique role in the direct opposition of polycomb-associated histone modifications that gBAF and pBAF do not share.

Using metabolomics to explore the effects of epigenetic-modification strategies on the metabolites of Acanthus ilicifolius L. endophytic fungi against ovarian cancer

Ovarian cancer is a serious threat to women's health and safety. So far, people have discovered more than 130 small molecule compounds of natural origin for anti-tumor, of which approximately 50% are of microbial origin. The Acanthus ilicifolius L. species is primarily distributed in the Guangdong, Hainan, and Guangxi regions of China and grows in tidally accessible coastal areas. Recent studies have revealed that Acanthus ilicifolius L. extracts are endowed with a range of pharmacological properties, including anti-inflammatory, hepatoprotective, antioxidant, and antitumor activities. Endophytic fungi are commonly found in the healthy tissue and organs of medicinal plants. These fungi and the plants they inhabit form mutually beneficial symbiotic relationships. Endophytic fungi produce a series of secondary metabolites, with active substances having shown great economic value and applications prospects in drug research and development as well as for the biological control of plant diseases. Secondary metabolites production by endophytic fungi is regulated by specific gene clusters, and several techniques have been used to stimulate the secondary metabolic processes of fungi, including epigenetic-modification and OSMAC (one strain many compounds) strategies, co-culturing, and gene modification. Among these, epigenetic modification has been shown to be effective; this strategy involves the addition of small-molecule epigenetic modifiers to the culture medium, thereby activating silenced biosynthetic gene clusters without altering the DNA sequences of the fungi. This approach facilitates the expression of silenced genes in endophytic fungi, thereby increasing the number and diversity of secondary metabolites. Furthermore, it assists in overcoming the inhibition of microbial secondary-metabolite synthesis under laboratory conditions, and enhances silenced-gene expressions. The advent of novel analytical techniques and bioinformatics has provided a comprehensive, multifaceted, and holistic understanding of fungal metabolism through the development of metabolomics as a research platform. However, few studies have combined anti-ovarian cancer-activity screening with metabolomic approaches in the search for activity-differentiating metabolites from endophytic fungi under the intervention of epigenetic modifiers. Herein, we investigated the impact of epigenetic modifiers on the secondary metabolites of the endophytic Diaporthe goulteri fungus from Acanthus ilicifolius L. to determine their potential anti-ovarian cancer activities. Crude extracts were obtained by controlling three variables: the number of fermentation days, the type of epigenetic modifier, and its concentration, with activities screened using the CCK-8 (cell counting kit-8) method. Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was subsequently employed for non-targeted metabolomic analysis. A multivariate statistical analysis model was constructed using principal component analysis and orthogonal partial least squares-discriminant analysis, which combines model and variable importance projection, with qualitative screening performed and significant changes (variable importance in the projection (VIP)≥1; P<0. 05) determined. Fifteen differential metabolites were identified in the fungal and epigenetic modification group, primarily comprising polyketides, amino acids, derivatives, alkaloids, and organic acids, including prenderol, glycine, valine, 2-ethylcaproic acid, rubratoxin B, finasteride, 6-silaspiro[5.5]undecane, 1-(2-nitrophenoxy)octane, heptadecene, 1-pentadecene, 11-ketoetiocholanolone, 3-(1-ethyl-1,3,3-trimethyl-2,3-dihydro-1H-inden-5-yl)butanal, N2-benzoylarginine, tabutrex, (3aR,6S,6aS)-6-(4-hydroxy-2-methoxy-2-butanyl)-4,4-dimethylhexahydro-1(2H)-pentalenone, and 8-aminoquinoline. The expressions of prenderol, 1-(2-nitrophenoxy)octane, 3-(1-ethyl-1,3,3-trimethyl-2,3-dihydro-1H-inden-5-yl)butanal, N2-benzoylarginine, and 8-aminoquinoline were downregulated, whereas the expressions of the remaining 10 substances were upregulated. Polyketides were the main components that exhibited higher expressions. This study showed that latent active differential metabolites can be searched by combining anti-ovarian cancer-activity screening with metabolomics analysis, thereby providing a reference for the further development of Acanthus ilicifolius L. resources and the subsequent targeted isolation of active compounds.

Sequencing and characterization of complete mitogenome DNA of worldwide turkey (Meleagris gallopavo) populations

The history of turkey (Meleagris gallopavo) domestication can be traced back to the period between 700 and 200 BC in Mexico. This process involved multiple contributors and resulted in the development of modern local turkey breeds. This research investigates the complete mitochondrial diversity across a diverse range of local turkeys. Seventy-three turkeys were sampled from various populations, including autochthonous Italian breeds, an American breed (Narragansett), as well as wild turkeys from the USA and Mexico. The mitochondrial DNA (mtDNA) was employed as a powerful tool for biodiversity and breed phylogeny investigation. An analysis of the entire mtDNA was conducted to identify breed-specific unique traits, mitochondrial-specific characteristics, and the phylogenetic relationship among turkey populations. A total of 44 polymorphic sites were identified. Brianzolo and Narragansett birds were characterized as genetically homogeneous populations. Thirty-two different haplotypes were identified when our samples were compared with mtDNA D-loop of 96 online available turkeys from various geographical countries. H1 and H2, differing for one mutation, were the most abundant, comprising 132 of the 185 sequences. H1 included samples coming from every region, while H2 was predominantly characterized by Italian samples. USA and Mexican samples appear to be more variable in their mtDNA than the other populations.

Crispr and plant pathology: Revolutionizing disease resistance in crops

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has revolutionized plant pathology by providing a precise and efficient tool for enhancing disease resistance in crops. This gene-editing technique enables scientists to modify specific DNA sequences in plants, allowing for the targeted improvement of traits related to pathogen defense. By altering genes responsible for susceptibility to diseases, CRISPR facilitates the development of crops that are more resistant to bacterial, fungal, and viral pathogens, thereby reducing the reliance on chemical pesticides. Additionally, CRISPR can be used to boost plant immunity and improve resilience against emerging plant diseases that threaten global food security. The technology's accuracy and speed have accelerated breeding programs, enabling researchers to respond rapidly to agricultural challenges posed by climate change and the evolution of new pathogens. This article explores the breakthroughs achieved through CRISPR in plant pathology, highlighting case studies of its application in major crops such as rice, wheat, and tomatoes. Furthermore, it discusses the ethical, regulatory, and environmental considerations of using CRISPR for crop improvement, alongside its potential to revolutionize sustainable agriculture by reducing disease-induced yield losses and promoting food security.

A redescription and new generic placement of the New Zealand marine-associated jumping spider Marpissa marina (Goyen, 1892) (Araneae: Salticidae: Euophryini)

ABSTRACT The jumping spider Marpissa marina (Goyen, 1892) is endemic to Aotearoa New Zealand. In this paper, we redescribe the species. Marpissa marina was placed in the Northern Hemisphere genus Marpissa due to its subjective similarity to spiders in that genus. The original description lacked modern morphological diagnoses and useful illustrations. The description was based largely on the female, and therefore lacked the taxonomically important characters seen in the male pedipalp. Previous literature has suggested M. marina may be related to the Australian genus, Maratus Karsch, 1878. We applied an integrative taxonomic approach based on diagnostic morphological characters and molecular analysis for a more detailed species description and assignment into an appropriate genus. Morphological features indicate that M. marina belongs to Maratus. This is supported by phylogenetic analyses using DNA sequences from the nuclear gene Actin 5C and the mitochondrial gene 16S ribosomal RNA. We transfer Marpissa marina into the genus Maratus, as Maratus marinus (Goyen, 1892), new combination.

Genetic and Haplotype Diversity of Clarias gariepinus (Burchell 1822) Based on Cytochrome c Oxidase I (COI) Gene

The evaluation of mitochondrial DNA and genetic analysis is helpful for economically significant species. Clarias gariepinus is a critical species in aquaculture. This study investigates the genetic diversity and population differentiation of C. gariepinus from 19 countries using 164 sequences of the mitochondrial DNA’s Cytochrome c oxidase I (COI) gene. The haplotype analysis revealed a total of 17 haplotypes, with a nucleotide diversity (π) of 0.012 and a haplotype diversity (Hd) of 0.87. The results of an AMOVA and fixation index indicated significant genetic variation and structure among the populations. Additionally, neutrality tests and mismatch distribution analysis supported the hypothesis of under-purifying selection in C. gariepinus. The findings suggested that the population did not experience expansion. In conclusion, the genetic analysis highlighted substantial variation among C. gariepinus populations from different locations, providing valuable insights for the global management of this species.

First Molecular Characterization of Small Ruminant Lentiviruses in Hungarian Goat Population

In 2023, a molecular study was conducted on the Hungarian goat population to determine genotypes and subtypes of small ruminant lentiviruses (SRLV) infecting these herds. Ten goat herds seropositive for SRLV infection according to a serosurvey conducted earlier in Hungary were selected, and 135 adult goats (&gt;1 year old) were blood sampled. The two-stage nested real-time PCR (nRT-PCR) was used to detect proviral DNA of SRLV and distinguish between two main viral genotypes (A and B). PCR products were submitted for Sanger dideoxy sequencing, and phylogenetic and molecular evolutionary analyses were conducted on the 200–250 bp-long proviral DNA sequences from the end of long terminal repeat (LTR) region and beginning of gag gene using the MEGA11 software. Reference strains included strains most identical to Hungarian sequences according to the Standard Nucleotide BLAST and prototypic strains for the relevant genotypes and subtypes. Proviral DNA of SRLV was detected in goats from all ten tested herds. A single SRLV genotype was detected in 6 herds—genotype A in three herds and B also in three herds. In four herds, mixed infection with genotypes A and B was confirmed. In total, 110/135 seropositive goats tested positive in the nRT-PCR (81.5%): 49/110 goats (44.5%) for genotype A, 54/110 goats (49.1%) for genotype B, and 7/110 goats (6.4%) for both genotypes. Hungarian sequences belonged to subtypes A1/A18, A2, and subtype B1. This is the first study which shows that Hungarian goats are infected by SRLV belonging to both genotypes A and B.

Morphological and molecular identification of Eimeria rajasthani (coccidia: Eimeriidae) in the dromedary camel (Camelus dromedarius) in Riyadh, Saudi Arabia

IntroductionCoccidiosis is a serious parasitic disease in camels caused by an intestinal protozoan parasite of the genus Eimeria, which is linked to significant causes of reduced milk and meat production. In Saudi Arabia, scare literature focused on the coprological investigation of dromedary camels (Camelus dromedarius). To determine the taxonomic status of camel parasite species, we performed morphological characterization of oocysts and genetic analysis (18S rRNA and ITS-1 gene regions) of Eimeria species collected from camels in Riyadh, Saudi Arabia.MethodsA total of 150 faecal samples were obtained from camels at the old camel market. These samples were tested for the presence of Eimeria oocysts using the conventional floatation technique before being sporulated in a 2.5% potassium dichromate solution. Eimeria oocysts were morphologically and molecularly examined and identified, and the infection rate of parasitic infections was determined.ResultsOur findings revealed that the overall frequency of oocysts was 30%. The identified species was Eimeria rajasthani, which had a typical ellipsoidal oocyst shape. Oocystic polar granule, micropyle, micropylar cap, and oocyst residuum are not visible. Sporocysts are oval with stieda body. Sporocyst residuum contains many granules and sporozoites with refractile bodies and nuclei. Genetic analyses of the sequence data from the partial 18S rRNA and ITS-1 gene regions revealed that the sequences obtained from E. rajasthani oocysts are related to DNA sequences reported from E. lamae from the Alpaca from China, particularly the 18S rRNA sequences.ConclusionThis study emphasized the need to use molecular phylogenetic tools to describe camel intestinal coccidian parasites with traditional morphology-based approaches to better understand their biology. For camel husbandry and disease control, more studies should be conducted to better understand the epidemiology of these protozoan parasites.

Effect of single parenteral administration of marbofloxacin on bacterial load and selection of resistant Enterobacteriaceae in the fecal microbiota of healthy pigs

BackgroundAntimicrobial resistance (AMR) is a global concern impacting both humans, animals and their environment. The use of oral antimicrobials in livestock, particularly in pigs, has been identified as a driver in the selection of AMR bacteria. The aim of the present study was to evaluate the effects of a single intramuscular (IM) dose of marbofloxacin (8 mg/kg) on Enterobacteriaceae and E. coli populations, as well as on fluoroquinolone resistance within the fecal microbiota of pigs. Twenty healthy pigs, 60-days old, were divided into two groups: a treated group (n = 13) and a control group (n = 7) and were monitored over a 28-day experimental period. Fecal samples were collected from all animals for the isolation of E. coli and Salmonella strains. The minimum inhibitory concentration (MIC) of marbofloxacin for the isolates recovered on MacConkey agar supplemented with 1 or 4 µg/mL of marbofloxacin and for some generic E. coli isolates (recovered from MacConkey agar not supplemented with marbofloxacin) was determined using the broth microdilution method. Genomic DNA was extracted from the confirmed bacterial strains and sequenced using the Sanger method to identify mutations in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes.ResultsThe single IM administration of marbofloxacin resulted in a significant decrease in Enterobacteriaceae and E. coli fecal populations from days 1 to 3 post- treatment. No Salmonella isolates were detected in either group, and no marbofloxacin-resistant E. coli isolates were identified. The MIC of the selected generic E. coli strains (n = 100) showed an increase to up to 0.5 µg/mL between days 1 and 3 post-treatment but remained below the clinical breakpoint of marbofloxacin resistance (4 µg/mL). Sequencing of these isolates revealed no mutations in gyrA and parC genes.ConclusionsThe present study showed that this dosing regimen of marbofloxacin significantly decreases the fecal shedding of Enterobacteriaceae and E. coli populations in pigs, while limiting the selection of marbofloxacin-resistant E. coli isolates. These findings warrant validation in sick pigs to support the selective use of this antibiotic solely in cases of clinical disease, thereby minimizing the reliance on conventional (metaphylactic) group treatments in pigs.

Vishniacozyma floricola sp. nov., a flower-related tremellomycetous yeast species from Europe.

During the course of two independent studies conducted in Hungary and Spain, four conspecific yeast strains were isolated from flowers of different plant species. DNA sequences of two barcoding regions, the D1/D2 domain of the LSU rRNA gene and the internal transcribed spacer (ITS) region (ITS1-5.8S rRNA gene-ITS2), revealed that the four strains represent an undescribed Vishniacozyma (family Bulleribasidiaceae, Basidiomycota) species. In terms of pairwise sequence similarities and according to our phylogenetic analyses of the concatenated DNA sequences of the ITS region and the D1/D2 domain of the LSU rRNA gene, the undescribed species is most closely related to Vishniacozyma melezitolytica, a yeast species of phylloplane origin. The novel species differs from the type strain of V. melezitolytica by 8 substitutions and 3 insertion/deletion (indels) and 11 substitutions and 5 indels along the D1/D2 domain of the LSU rRNA gene and the ITS region, respectively. In addition to the DNA sequence divergences, the two species differ in some physiological characters as well. We propose the species Vishniacozyma floricola sp. nov. to accommodate the above-noted strains (holotype, NCAIM Y.02320; isotype, CBS 18939; MycoBank number, 856028).

DNA barcode identification of a tropical liver fluke (Fasciola gigantica) in cattle from Oyo, southwestern Nigeria

BackgroundFascioliasis is a major parasite illness that affects ruminants, both domesticated and free, and has an impact on public health and animals’ productivity. The genetic diversity of Fasciola species in cattle from Oyo, Oyo State, Nigeria is yet not well understood. In this study, the genetic diversity of Fasciola gigantica in slaughtered cattle in Oyo was examined using a molecular-based approach targeting the mitochondrial cytochrome C oxidase subunit I (COI) gene region.ResultsOne hundred flukes were gathered from slaughtered cattle, and their COI gene sequences were analyzed using maximum likelihood methods. The results of phylogenetic analysis showed genetic similarities between Nigerian F. gigantica and isolates from Algeria, Egypt, Iran, Sudan, China, Japan and Nigeria. The predominant DNA substitution was the A to T transversion, while the least common substitution was the G to A transition.ConclusionOur results show how useful the COI gene region is for examining intraspecific differences between F. gigantica isolates. The genetic similarity observed among the sampled F. gigantica populations suggests the value of mitochondrial DNA sequences as a marker for the accurate identification and characterization of Fasciola species across different ruminants.

DNMT3A CHIP-driver mutations ignite the bone marrow

Abstract Background Clonal hematopoiesis of indeterminate potential (CHIP) is an acquired, genetic cardiovascular (CV) risk factor affecting about 10% of the general population at the age of 70 years. Most CHIP-driver mutations affect the epigenetic regulator gene DNMT3A. Experimental studies suggest a causal link between CHIP and inflammation-driven atherosclerosis, but the underlying mechanisms of how few mutant clones provoke adverse clinical outcomes in patients remain obscure. Methods Single monocytes and bone marrow cells from DNMT3A-mutation carriers undergoing coronary angiography and open-heart surgery underwent RNA and genomic DNA sequencing in parallel on a single cell level. This approach enabled us to compare mutant and non-mutant cells directly within carriers, and to non-carrier cells. Atherosclerotic chimeric mice reconstituted with a mixture of Dnmt3a-deficient (CD45.2) and -competent (CD45.1) bone marrow served as an experimental model to compare mutant to non-mutant macrophages in atherosclerotic lesions. Results Surprisingly, RNA/gDNA parallel sequencing revealed that gene expression profiles of mutant monocytes resembled those of non-mutant monocytes in carriers of DNMT3A-CHIP mutations (n=4). Collectively, however, monocytes of DNMT3A mutation carriers were more inflammatory than those of matched non-carrier patients. Mutant hematopoietic stem and progenitor cells, analyzed by RNA/gDNA parallel sequencing in two carriers, expressed more activation and inflammatory markers compared to non-mutant cells within the same bone marrow, and intraindividual differences declined as cells differentiated into monocytes. Macrophages were isolated from atherosclerotic aortas of mixed bone marrow chimeric mice based on CD45.1 (non-mutant) or CD45.2 (mutant or non-mutant controls) expression and subjected to single cell RNA sequencing. Inflammation markers were higher expressed in non-mutant macrophages within carrier mice than in non-mutant macrophages of non-carrier chimeras, and showed relatively small differences compared to Dnmt3a-deficient macrophages. Conclusions Our experimental findings in humans and mice support a pathomechanistic model in which few DNMT3A-CHIP mutation-carrying hematopoietic stem cells stimulate non-mutant cells within the bone marrow, thereby augmenting inflammation downstream in all circulating monocytes and their progeny in atherosclerotic lesions.

An epigenetic editor targeting human PCSK9 efficiently and durably lowers Low Density Lipoprotein Cholesterol (LDL-C) in non-human primates

Abstract Background Reducing the risk of atherosclerotic cardiovascular disease is dependent on both the magnitude and cumulative duration of LDL-C lowering. However, in clinical practice achieving treatment goals is often hampered by low adherence to standard of care. Although PCSK9 inhibitors represent an effective option for LDL-C lowering, they require chronic life-long treatment. Epigenetic editing is a new therapeutic approach designed to durably silence genes by leveraging nature’s endogenous cellular mechanism for gene regulation via CpG methylation without the inherent genotoxic risks of genome editing approaches that nick or cut the DNA. We are developing a PCSK9 epigenetic editor (PCSK9-EE) to durably silence PCSK9, with the promise of lifelong reduction in LDL-C. Methods Here, we describe the development of an epigenetic editor targeting human PCSK9. This epigenetic editor consists of a DNA targeting component fused to a transcriptional repressor domain and a DNA methyltransferase domain. PCSK9-EE was first screened and evaluated for specificity and efficacy in primary human hepatocytes (PHH). In vivo efficacy and durability were then evaluated in a transgenic mouse carrying the human PCSK9 genomic locus (hPCSK9-Tg mouse) and non-human primates (NHP). Results Our initial screen of PCSK9-EEs identified several hits that efficiently suppressed secreted PCSK9 levels in immortalized liver cells. We confirmed that our top PCSK9-EE candidates robustly decreased PCSK9 secretion in PHH and were found to be highly specific at targeting PCSK9 as assessed by RNA-seq, methylation array, and whole-genome methylation sequencing. To examine in vivo activity, we treated hPCSK9-Tg mice with a single administration of a lipid nanoparticle delivering mRNA encoding our PCSK9-EE and observed &amp;gt;97% reduction in liver PCSK9 mRNA, and &amp;gt;98% reduction in plasma PCSK9 for the duration of the study (one year). We then delivered PCSK9-EE in NHP and observed a robust reduction in plasma PCSK9 with a concomitant reduction in LDL-C. These effects were found to be durable for at least 6 months following a single administration of PCSK9-EE in NHP. To establish a mechanistic relationship between PCSK9-EE’s molecular action and durable PCSK9 silencing, we performed serial liver biopsies in each NHP approximately 2 months apart. Robust CpG methylation at the PCSK9 genomic locus was observed in the first liver biopsy and was comparable to that observed in the second liver biopsy suggesting that PCSK9-EE induced stable CpG methylation in NHP livers. Conclusions We believe PCSK9-EE may offer an effective, safe, and durable approach to suppress hepatic PCSK9 expression and achieve long-lasting reduction in LDL-C. Furthermore, epigenetic editing may hold promise for one-and-done approaches for the treatment of atherosclerotic cardiovascular disease without cutting, nicking, or altering the DNA sequence.

The Mutscore metapredictor: a new application of artificial intelligence in cardiogenetics

Abstract Introduction The rapid development of high-throughput next generation sequencing has shifted the limits of genetic knowledge from variant sequencing to variant interpretation. The current standards recommend that sequence variants are interpreted according to a comprehensive analysis including, among others, computational data. The two major types of in-silico predictive tools encompass those predicting the theoretical variant effect on splicing and algorithms predicting the potential damage of missense variants. However, overall for missense variants, prediction accuracy is often limited and results are sometimes inconsistent between software programs. Purpose We aimed at testing the predictive performance of a new predictive algorithm (Mutscore) for missense variants based on a machine learning approach, in our cohort of families referred for hereditary cardiac diseases. Methods We retrospectively reviewed DNA sequencing results from 230 families (from 01.07.2014 till 01.07.2023) addressed to our center for channelopathy or hereditary cardiomyopathy. Missense variants were identified and evaluated according to the current standards of the American College of Medical Genetics and Genomics. Variants were analysed with the commonly used in-silico tools CADD, Polyphen, Alpha-missense and Revel. We further applied the Mutscore, a new metapredictor integrating 16 existing predictive tools to data on variant topographical location. Results Among our 230 families, we detected 252 missense variants (class I-II 2.4%, class III 62.3%, class IV and V 15.9% and 19.4% respectively). We observed a significant positive correlation (r = 0.59, p=0.000) between the Mutscore and the interpretation of variants according to standards. The Mutscore had a better predictive performance than Polyphen (AUC 0.83 vs 0.67, p=0.007), Alpha-missense (AUC 0.87 vs 0.79, p=0.047) and CADD (AUC 0.87 vs 0.70, p=0.0001). Compared to Revel, the Mutscore had a comparable predictive performance (0.89 vs 0.87, p=NS), but a better sensitivity (75% vs 66%) at the maximum tolerated false positive rate of 10%. Figure 1. Focusing on variants of uncertain significance (VUS), we applied Mutscore cut-off values which were identified to reclassify variants in our previous study (1). Importantly, the Mutscore reclassified 45% of VUS into likely benign/pathogenic variants. Figure 2. Conclusions The Mutscore, through its metaprediction approach integrating data on variant topographical location, improves the accuracy of pathogenicity prediction as compared to three out of four algorithms commonly used in clinical practice, and seems to represent a valuable tool contributing to VUS disambiguation.

Genome-Wide Data Uncover Cryptic Diversity With Multiple Reticulation Events in the Balkan-Anatolian Cardamine (Brassicaceae) Species Complex.

Plant species diversity may be considerably underestimated, especially in evolutionarily complex genera and in diversity hotspots that have enabled long-term species persistence and diversification, such as the Balkan Peninsula. Here, we address the topic of underexplored plant diversity and underlying evolutionary and biogeographic processes by investigating the hygrophytic mountain species complex of Cardamine acris s.l. distributed in the Balkans (three subspecies within C. acris) and northwestern Anatolia (C. anatolica). We performed a series of phylogenetic and phylogeographic analyses based on restriction-site associated DNA sequencing (RADseq) and target enrichment (Hyb-Seq) data in combination with habitat suitability modelling. We found C. anatolica as a clade nested within the Balkan C. acris, probably resulting from a founder event, and uncovered three allopatric cryptic lineages within C. acris subsp. acris, allowing us to recognise a total of six entities in this complex. We observed the deepest genetic split within C. acris subsp. acris in the western Balkans, which was at odds with taxonomy and showed no distribution gap. We inferred vicariance as the most likely process for population divergence in the Balkans, accompanied by gene flow between the recognised entities, which was consistent with the modelled habitat suitability dynamics. Furthermore, we discovered several polyploid populations in C. acris, representing both pure intra- and inter-lineage hybrid polyploids, but detected only minor traces of hybridization with related congeners. Overall, our results illustrate that diverse evolutionary processes may influence the history of mountain plant species in the Balkan Peninsula, including vicariance, reticulation, polyploidization and cryptic diversification.

Phytoplasma DNA Enrichment from Sugarcane White Leaves for Shotgun Sequencing Improvement

Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma sacchari, poses a significant threat to sugarcane cultivation. An obligate parasite, phytoplasma is difficult to culture in laboratory conditions, making the isolation of its DNA from the massive amount of plant host DNA extremely challenging. Yet, the appropriate amount and quality of plant microbiome-derived DNA are key for high-quality DNA sequencing data. Here, a simple, cost-effective, alternative method for DNA isolation was applied using a guanidine-HCl-hydroxylated silica (GuHCl-Silica)-based method and microbiome DNA enrichment based on size-selective low-molecular-weight (LMW) DNA by PEG/NaCl precipitation. qPCR analysis revealed a significant enrichment of phytoplasma DNA in the LMW fraction. Additionally, the NEBNext Microbiome DNA enrichment kit was utilized to further enrich microbial DNA, demonstrating a remarkable increase in the relative abundance of phytoplasma DNA to host DNA. Shotgun sequencing of the isolated DNA gave high-quality data on the metagenome assembly genome (MAG) of Ca. Phytoplasma sacchari SCWL with completeness at 95.85 and 215× coverage. The results indicate that this combined approach of PEG/NaCl size selection and microbiome enrichment is effective for obtaining high-quality genomic data from phytoplasma, surpassing previous methods in efficiency and resource utilization. This low-cost method not only enhances the recovery of microbiome DNA from plant hosts but also provides a robust framework for studying plant pathogens in complex plant models.

Hop2-Mnd1 functions as a DNA sequence fidelity switch in Dmc1-mediated DNA recombination

Homologous recombination during meiosis is critical for chromosome segregation and also gives rise to genetic diversity. Genetic exchange between homologous chromosomes during meiosis is mediated by the recombinase Dmc1, which is capable of recombining DNA sequences with mismatches. The Hop2-Mnd1 complex mediates Dmc1 activity. Here, we reveal a regulatory role for Hop2-Mnd1 in restricting substrate selection. Specifically, Hop2-Mnd1 upregulates Dmc1 activity with DNA substrates that are either fully homologous or contain DNA mismatches, and it also acts against DNA strand exchange between substrates solely harboring microhomology. By isolating and examining salient Hop2-Mnd1 separation-of-function variants, we show that suppressing illegitimate DNA recombination requires the Dmc1 filament interaction attributable to Hop2-Mnd1 but not its DNA binding activity. Our study provides mechanistic insights into how Hop2-Mnd1 helps maintain meiotic recombination fidelity.