DNA Sequence Research Articles

Nomenclatural Updates in Anomospermum (Anomospermeae: Menispermaceae)

Two new combinations, Anomospermum prancei (Barneby & Krukoff) R. Ortiz and A. verruculosum (Krukoff & Barneby) R. Ortiz, are made in the Neotropical genus Anomospermum Miers (Anomospermeae–Menispermaceae) to conform with the newly revised classification based on morphology and DNA sequence data.

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 Spatio-Temporal Analysis of Hepatitis D Virus Infection in Western Brazilian Amazon

The Brazilian Amazon region is a highly endemic area for hepatitis Delta and has areas that are difficult to access. Understanding the dynamics of HDV transmission in these remote locations is important for elucidating the routes of infection. To investigate this, a molecular analysis of HDV was conducted to assess the spatio-temporal dynamics of HDV cases. Between 2010 and 2023, 35 patients were recruited from the Viral Hepatitis Outpatient Clinic in Rondônia, Brazil. Conventional PCR was used to amplify the complete HDV genome followed by nucleotide sequencing via the Sanger method. The HDV genotype was determined using maximum likelihood phylogenetic reconstruction. A Skygrid coalescent approach with a Relaxed Random Walk phylogeographic model was used for the spatio-temporal analysis. Most individuals were males (21/35), with a median age of 39 years. HDV-3 was identified in all samples (35/35; 100%). The tMRCA was estimated to be 1824, with a substitution rate of 8.2 × 10−4 substitutions/site/year. The results suggest that HDV likely entered Brazil around 1820, in the state of Amazonas, subsequently spreading to Acre and Rondônia. Notable migration events were observed starting from 2010. This study suggests that HDV-3 has a complex evolutionary history spanning over two centuries, with intricate transmission routes in different locations in Brazil.

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.

Comparative satellite DNA mapping in species of the genus Prochilodus (Teleostei, Characiformes) and its evolutionary implications.

Satellite DNA (satDNA) sequences are dynamic components of the eukaryotic genome, that can play significant roles in species diversification. The Prochilodontidae family, which includes 21 Neotropical fish species, is characterized by a conserved karyotype of 2n = 54 biarmed chromosomes, with variation in some species and populations regarding the presence or absence of B chromosomes. This study aimed to investigate whether the chromosomal distribution of specific satDNA sequences is conserved among three Prochilodus species (P. lineatus, P. costatus, and P. argenteus) regarding organization and number of loci, and to compare their genomes using comparative genomic hybridization (CGH). Our results demonstrated that most satDNA sequences share a similar distribution pattern across the three species, and CGH analysis corroborated that their karyotypes are very similar in terms of repetitive DNA distribution. We also identified a potential CENP-B box sequence within PliSat01, a satDNA located in the pericentromeric region of all analyzed species. In contrast, PliSat04 and PliSat14 displayed differential locations and variations in the number of loci per genome, underscoring the dynamic nature of repetitive sequences even in species with otherwise highly conserved genomes. These findings represent the first evidence of karyotype diversification in Prochilodus, highlighting the evolutionary dynamism of satDNA sequences.

In Silico Design of Novel EpCAM-Binding Aptamers for Targeted Delivery of RNA Therapeutics.

Aptamers are short DNA or RNA sequences that adopt 3D structures and can bind to protein targets with high binding affinity and specificity. Aptamers exhibit excellent tissue penetration, are inexpensive to produce, and can be internalized by cells. Therefore, aptamers are attractive targeting ligands to direct the delivery of theranostic agents to the desired cells. Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is aberrantly overexpressed on many epithelial-derived cancers, including on cholangiocarcinoma (CCA) cells. Its expression on treatment-resistant cancer stem cells, along with its abundance in the CCA tumor microenvironment, highlights the need to develop EpCAM-targeted therapies for CCA. Herein, an in silico approach was used to design and screen DNA aptamers capable of binding to the EpCAM monomer and homodimer. Two aptamers, PLD01 and PLD02, met the selection criteria and were validated in vitro. Both aptamers exhibited high affinity for EpCAM+ CCA cells, with negligible binding to EpCAM- leukemia cells. Modified versions of PLD01 and PLD02 were successfully incorporated into the membranes of milk-derived nanovesicles. PLD01-functionalized nanovesicles enabled EpCAM-targeted delivery of the therapeutic cargo to CCA cells. In summary, these EpCAM-targeting aptamers can be utilized to direct the delivery of theranostic agents to EpCAM-expressing cells.

Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq) to Study DNA Methylation Using Low Amounts of DNA

Background/Objectives: DNA methylation is a key epigenetic mark involved in regulating gene expression. Aberrant DNA methylation contributes to various human diseases, including cancer, autoimmune disorders, atherosclerosis, and cardiovascular diseases. While whole-genome bisulfite sequencing and methylated DNA immunoprecipitation (MeDIP) are standard techniques for studying DNA methylation, they are typically limited to a few samples per run, making them expensive and low-throughput. Therefore, an automation-friendly method is needed to increase throughput and reduce costs without compromising data quality. Methods and Results: We developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed a 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis. Conclusions: Mx-MeDIP-Seq is a cost-effective and efficient method for high-throughput DNA methylation analysis, offering faster processing and reduced sample requirements. This technique makes DNA methylome analysis more accessible for large-scale studies.

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 (>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.

Coccidiosis 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. A 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. Our 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. This 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.

Development of Simple Sequence Repeat of Monochamus alternatus (Coleoptera: Cerambycidae) Based on Restriction Site-Associated DNA Sequencing

Monochamus alternatus, a pest posing a serious threat to coniferous species, such as Pinus massoniana, has had devastating effects on pine forests due to its association with Bursaphelenchus xylophilus. The creation of unique simple sequence repeat (SSR) primers for M. alternatus is crucial, as there has been little study of the species’ phylogeography. The aim of this study was to identify and create polymorphic SSR primers by sequencing samples of M. alternatus obtained from three different sampling points using the restriction site-associated DNA sequencing (Red-seq) approach. Subsequently, supplementary samples were integrated, and genetic typing was performed utilizing the identified polymorphic primers. Through comprehensive analysis, a total of 95,612 SSR loci were identified. Among these, mononucleotide repeats (51.43%), dinucleotide repeats (28.79%), and trinucleotide repeats (16.74%) predominated among the SSR motif types. Ultimately, 18 pairs of SSR primers were screened out, demonstrating stable amplification and high polymorphism. Genetic typing revealed that the mean number of alleles (Na) for these primer pairs ranged from 3 to 8, observed heterozygosity (Ho) ranged from 0.133 to 0.733, polymorphic information content (PIC) ranged from 0.294 and 0.783, and Shannon’s index (I) ranged from 0.590 to 1.802. This study effectively produced 16 pairs of SSR primers that can be applied to different populations of M. alternatus. As a result, important tools for furthering studies on the phylogeography of pine wood nematodes, creating genetic maps, gene mapping, and carrying out in-depth investigations into gene function have been made available.

The Complete Mitochondrial Genome of Nephropsis grandis: Insights into the Phylogeny of Nephropidae Mitochondrial Genome.

The systematic phylogeny of Pleocyemata species, particularly within the family Nephropidae, remains incomplete. In order to enhance the taxonomy and systematics of Nephropidae within the evolutionary context of Pleocyemata, we embarked upon a comprehensive study aiming to elucidate the phylogenetic position of Nephropsis grandis. Consequently, we determined the complete mitochondrial DNA sequence for N. grandis. The circular genome spans a length of 15,344bp and exhibits a gene composition analogous to that observed in other metazoans, encompassing a comprehensive set of 37 genes. Additionally, the genome features an AT-rich region. The rRNAs exhibited the highest AT content among the 37 genes (70.41%), followed by tRNAs (67.42%) and protein-coding genes (PCGs) (62.76%). The absence of a dihydrouracil arm in trnS1 prevented the formation of the canonical cloverleaf secondary structure. Selective pressure analysis indicated that the PCGs underwent purifying selection. The Ka/Ks ratios for cox1, cox2, cox3, and cob were considerably lower compared to other PCGs, implying strong purifying selection acting upon these particular genes. The mitochondrial gene order in N. grandis was consistent with the reported order in ancestral Pleocyemata. Phylogenetic revealed that N. grandis forms a cluster with the genus Metanephrops, and this cluster further groups with Homarus and the genus Nephrops within the Nephropidae family. These findings provide robust support for N. grandis as an ancestral member of the Nephropidae family. This study highlights the significance of employing complete mitochondrial genomes in phylogenetic analysis and deepens our understanding of the evolution of the Nephropidae family.

Population structure of Desmophyllum pertusum found along the United States eastern continental margin

ObjectiveThe connectivity and genetic structuring of populations throughout a region influence a species’ resilience and probability of recovery from anthropogenic impacts. By gaining a comprehensive understanding of population connectivity, more effective management can be prioritized. To assess the connectivity and population genetic structure of a common cold-water coral species, Desmophyllum pertusum (Lophelia pertusa), we performed Restriction-site Associated DNA Sequencing (RADseq) on individuals from nine sites ranging from submarine canyons off New England to the southeastern coast of the United States (SEUS) and the Gulf of Mexico (GOM). Fifty-seven individuals and 3,180 single-nucleotide polymorphisms (SNPs) were used to assess genetic differentiation.ResultsHigh connectivity exists among populations along the SEUS, yet these populations were differentiated from those to the north off New England and in Norfolk Canyon along the North Atlantic coast of the United States, as well as those in the GOM. Interestingly, Norfolk Canyon, located just north of North Carolina, and GOM populations exhibited low levels of genetic differentiation, corroborating previous microsatellite analyses and signifying gene flow between these populations. Increasing sample sizes from existing populations and including additional sampling sites over a larger geographic range would help define potential source populations and reveal fine-scale connectivity patterns among D. pertusum populations.

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 >97% reduction in liver PCSK9 mRNA, and >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.