High-throughput Sequencing Studies Research Articles

Reproductive Tract Microbiota of Mares.

The female reproductive tract microbiota is a complex community of microorganisms that might be crucial in maintaining a healthy reproductive environment. Imbalances in the bacterial community (dysbiosis) and the reduction of beneficial organisms and pathogen proliferation are associated with disease. Endometritis is a common cause of fertility problems in mares, and it is still challenging to diagnose and treat based on routine culture results of certain microorganisms. Although high-throughput sequencing studies provide helpful information regarding the composition of the reproductive tract microbiota in mares, there are still challenges in defining a "normal" microbiota. The primary objective of this literature review is to summarize the current knowledge regarding the microbiota present in the reproductive tract of mares, including the vagina, cervix, and uterus. The second objective is to describe the relevant factors that can impact the reproductive microbiota of mares, including the estrous cycle stage, the type of species (genera) investigated, season, and geographic location. The rationality of identifying the normal microbiota in the reproductive tract of a mare will likely aid in understanding the impact of the microbiota on the host's reproductive health and contribute to the treatment and prevention of equine sub and infertility issues.

Widespread and active piezotolerant microorganisms mediate phenolic compound degradation under high hydrostatic pressure in hadal trenches

Phenolic compounds, as well as other aromatic compounds, have been reported to be abundant in hadal trenches. Although high-throughput sequencing studies have hinted at the potential of hadal microbes to degrade these compounds, direct microbiological, genetic and biochemical evidence under in situ pressures remain absent. Here, a microbial consortium and a pure culture of Pseudomonas, newly isolated from Mariana Trench sediments, efficiently degraded phenol under pressures up to 70 and 60 MPa, respectively, with concomitant increase in biomass. By analyzing a high-pressure (70 MPa) culture metatranscriptome, not only was the entire range of metabolic processes under high pressure generated, but also genes encoding complete phenol degradation via ortho- and meta-cleavage pathways were revealed. The isolate of Pseudomonas also contained genes encoding the complete degradation pathway. Six transcribed genes (dmpKLMNOPsed) were functionally identified to encode a multicomponent hydroxylase catalyzing the hydroxylation of phenol and its methylated derivatives by heterogeneous expression. In addition, key catabolic genes identified in the metatranscriptome of the high-pressure cultures and genomes of bacterial isolates were found to be all widely distributed in 22 published hadal microbial metagenomes. At microbiological, genetic, bioinformatics, and biochemical levels, this study found that microorganisms widely found in hadal trenches were able to effectively drive phenolic compound degradation under high hydrostatic pressures. This information will bridge a knowledge gap concerning the microbial aromatics degradation within hadal trenches.

Human embryonic genetic mosaicism and its effects on development and disease.

Nearly every mammalian cell division is accompanied by a mutational event that becomes fixed in a daughter cell. When carried forward to additional cell progeny, a clone of variant cells can emerge. As a result, mammals are complex mosaics of clones that are genetically distinct from one another. Recent high-throughput sequencing studies have revealed that mosaicism is common, clone sizes often increase with age and specific variants can affect tissue function and disease development. Variants that are acquired during early embryogenesis are shared by multiple cell types and can affect numerous tissues. Within tissues, variant clones compete, which can result in their expansion or elimination. Embryonic mosaicism has clinical implications for genetic disease severity and transmission but is likely an under-recognized phenomenon. To better understand its implications for mosaic individuals, it is essential to leverage research tools that can elucidate the mechanisms by which expanded embryonic variants influence development and disease.

SeqKit2: A Swiss army knife for sequence and alignment processing.

In the era of ubiquitous high-throughput sequencing studies, there is a growing need for analysis tools that are not just performant but also comprehensive and user-friendly enough to cater to both novice and advanced users. This article introduces SeqKit2, the next iteration of the widely used sequence analysis tool SeqKit, featuring expanded functionality, performance optimizations, and support for additional compression methods. Retaining a pragmatic subcommand architecture, SeqKit2 represents substantial enhancement through the inclusion of 19 additional subcommands, expanding its overall repertoire to a total of 38 in eight categories. The new subcommands add functionality such as amplicon processing and robust, error-tolerant parsing of sequence records. In addition, three subcommands designed for real-time analysis are added for periodic monitoring of properties of FASTQ and Binary Alignment/Map alignment records and real-time streaming from multiple sequence files. The performance of SeqKit2 is benchmarked against the old version of SeqKit, Bioawk, Seqtk, and SeqFu tools. SeqKit2 consistently outperforms its predecessor, albeit with marginally higher memory usage, while maintaining competitive runtimes against other tools. With its broad functionality, proven usability, and ongoing development driven by user feedback, we hope that bioinformaticians will find SeqKit2 useful as a "Swiss army knife" of sequence and alignment processing-equally adept at facilitating ad hoc analyses and seamlessly integrating into larger pipelines.

Three-Dimensional Genomics: A New Perspective and Therapeutic Strategies in Prostate Cancer Research

Prostate cancer (PC) poses significant health risks to men globally. Enhancing our understanding of prostate cancer biology is crucial for facilitating early diagnosis and effective treatment strategies. A multitude of high-throughput sequencing studies, encompassing whole genome resequencing, transcriptome sequencing, and genome-wide association studies, have unearthed vital point mutations, structural variations, and epigenomic alterations linked to prostate cancer. These findings have significantly enriched our knowledge of the genomic framework for prostate cancer. Still, these investigations have predominantly centered on the one- or two-dimensional landscape of the genome. Research in three-dimensional genomics underscores the critical role of the genome's three-dimensional spatial structure in maintaining normal cellular functions. Additionally, it demonstrates that dysregulation of key genes in numerous cancers relates to the chromatin's spatial organization across various levels. This article explores the intricate three-dimensional architecture of chromosomes. It outlines the progressive development of techniques used in three-dimensional genomic research and synthesizes the application of these techniques in the study of prostate cancer biology. Furthermore, it proposes potential therapeutic strategies for prostate cancer.

Current trends, limitations and future research in the fungi?

The field of mycology has grown from an underappreciated subset of botany, to a valuable, modern scientific discipline. As this field of study has grown, there have been significant contributions to science, technology, and industry, highlighting the value of fungi in the modern era. This paper looks at the current research, along with the existing limitations, and suggests future areas where scientists can focus their efforts, in the field mycology. We show how fungi have become important emerging diseases in medical mycology. We discuss current trends and the potential of fungi in drug and novel compound discovery. We explore the current trends in phylogenomics, its potential, and outcomes and address the question of how phylogenomics can be applied in fungal ecology. In addition, the trends in functional genomics studies of fungi are discussed with their importance in unravelling the intricate mechanisms underlying fungal behaviour, interactions, and adaptations, paving the way for a comprehensive understanding of fungal biology. We look at the current research in building materials, how they can be used as carbon sinks, and how fungi can be used in biocircular economies. The numbers of fungi have always been of great interest and have often been written about and estimates have varied greatly. Thus, we discuss current trends and future research needs in order to obtain more reliable estimates. We address the aspects of machine learning (AI) and how it can be used in mycological research. Plant pathogens are affecting food production systems on a global scale, and as such, we look at the current trends and future research needed in this area, particularly in disease detection. We look at the latest data from High Throughput Sequencing studies and question if we are still gaining new knowledge at the same rate as before. A review of current trends in nanotechnology is provided and its future potential is addressed. The importance of Arbuscular Mycorrhizal Fungi is addressed and future trends are acknowledged. Fungal databases are becoming more and more important, and we therefore provide a review of the current major databases. Edible and medicinal fungi have a huge potential as food and medicines, especially in Asia and their prospects are discussed. Lifestyle changes in fungi (e.g., from endophytes, to pathogens, and/or saprobes) are also extremely important and a current research trend and are therefore addressed in this special issue of Fungal Diversity.

Host DNA depletion methods and genome-centric metagenomics of bovine hindmilk microbiome.

Bovine mastitis is a multi-etiological and complex disease, resulting in serious economic consequences for dairy farmers and industry. In recent years, the microbiological evaluation of raw milk has been investigated in-depth using next-generation sequencing approaches such as metataxonomic analysis. Despite this, host DNA is a major concern in the shotgun metagenomic sequencing of microbial communities in milk samples, and it represents a big challenge. In this study, we aimed to evaluate different methods for host DNA depletion and/or microbial DNA enrichment and assess the use of PCR-based whole genome amplification in milk samples with high somatic cell count (SCC) by using short- and long-read sequencing technologies. Our results evidenced that DNA extraction performed differently in terms of host DNA removal, impacting metagenome composition and functional profiles.. Moreover, the ratio of SCC/bacteria ultimately impacts microbial DNA yield, and samples with low SCC (SCC below 100,000 cells/mL) are the most problematic. When milk samples with high SCC (SCC above 200,000 cells/mL) underwent multiple-displacement amplification (MDA), we successfully recovered high-quality metagenome-assembled genomes (MAGs), and long-read sequencing was feasible even for samples with low DNA concentration. By associating MDA and short-read sequencing, we recovered two times more MAGs than in untreated samples, and an ongoing co-infection not reported by traditional methods was detected for mastitis pathogen. Overall, this new approach will improve the detection of mastitis-associated microorganisms and make it possible to examine host-microbiome interactions in bovine mastitis.IMPORTANCENext-generation sequencing technologies have been widely used to gain new insights into the diversity of the microbial community of milk samples and dairy products for different purposes such as microbial safety, profiling of starter cultures, and host-microbiome interactions. Milk is a complex food matrix, and additionally, the presence of host nucleic acid sequences is considered a contaminant in untargeted high-throughput sequencing studies. Therefore, genomic-centric metagenomic studies of milk samples focusing on the health-disease status in dairy cattle are still scarce, which makes it difficult to evaluate the microbial ecophysiology of bovine hindmilk. This study provides an alternative method for genome-centric metagenome studies applied to hindmilk samples with high somatic cell content, which is indispensable to examining host-microbiome interactions in bovine mastitis.

Hepatoviruses promote very-long-chain fatty acid and sphingolipid synthesis for viral RNA replication and quasi-enveloped virus release.

Virus-induced changes in host lipid metabolism are an important but poorly understood aspect of viral pathogenesis. By combining nontargeted lipidomics analyses of infected cells and purified extracellular quasi-enveloped virions with high-throughput RNA sequencing and genetic depletion studies, we show that hepatitis A virus, an hepatotropic picornavirus, broadly manipulates the host cell lipid environment, enhancing synthesis of ceramides and other sphingolipids and transcriptionally activating acyl-coenzyme A synthetases and fatty acid elongases to import and activate long-chain fatty acids for entry into the fatty acid elongation cycle. Phospholipids with very-long-chain acyl tails (>C22) are essential for genome replication, whereas increases in sphingolipids support assembly and release of quasi-enveloped virions wrapped in membranes highly enriched for sphingomyelin and very-long-chain ceramides. Our data provide insight into how a pathogenic virus alters lipid flux in infected hepatocytes and demonstrate a distinction between lipid species required for viral RNA synthesis versus nonlytic quasi-enveloped virus release.

Population Genomics of Pooled Samples: Unveiling Symbiont Infrapopulation Diversity and Host-Symbiont Coevolution.

Microscopic symbionts represent crucial links in biological communities. However, they present technical challenges in high-throughput sequencing (HTS) studies due to their small size and minimal high-quality DNA yields, hindering our understanding of host-symbiont coevolution at microevolutionary and macroevolutionary scales. One approach to overcome those barriers is to pool multiple individuals from the same infrapopulation (i.e., individual host) and sequence them together (Pool-Seq), but individual-level information is then compromised. To simultaneously address both issues (i.e., minimal DNA yields and loss of individual-level information), we implemented a strategic Pool-Seq approach to assess variation in sequencing performance and categorize genetic diversity (single nucleotide polymorphisms (SNPs)) at both the individual-level and infrapopulation-level for microscopic feather mites. To do so, we collected feathers harboring mites (Proctophyllodidae: Amerodectes protonotaria) from four individual Prothonotary Warblers (Parulidae: Protonotaria citrea). From each of the four hosts (i.e., four mite infrapopulations), we conducted whole-genome sequencing on three extraction pools consisting of different numbers of mites (1 mite, 5 mites, and 20 mites). We found that samples containing pools of multiple mites had more sequencing reads map to the feather mite reference genome than did the samples containing only a single mite. Mite infrapopulations were primarily genetically structured by their associated individual hosts (not pool size) and the majority of SNPs were shared by all pools within an infrapopulation. Together, these results suggest that the patterns observed are driven by evolutionary processes occurring at the infrapopulation level and are not technical signals due to pool size. In total, despite the challenges presented by microscopic symbionts in HTS studies, this work highlights the value of both individual-level and infrapopulation-level sequencing toward our understanding of host-symbiont coevolution at multiple evolutionary scales.

Developing diagnostic tools for canine periodontitis: combining molecular techniques and machine learning models

BackgroundDental plaque microbes play a key role in the development of periodontal disease. Numerous high-throughput sequencing studies have generated understanding of the bacterial species associated with both canine periodontal health and disease. Opportunities therefore exist to utilise these bacterial biomarkers to improve disease diagnosis in conscious-based veterinary oral health checks. Here, we demonstrate that molecular techniques, specifically quantitative polymerase chain reaction (qPCR) can be utilised for the detection of microbial biomarkers associated with canine periodontal health and disease.ResultsOver 40 qPCR assays targeting single microbial species associated with canine periodontal health, gingivitis and early periodontitis were developed and validated. These were used to quantify levels of the respective taxa in canine subgingival plaque samples collected across periodontal health (PD0), gingivitis (PD1) and early periodontitis (PD2). When qPCR outputs were compared to the corresponding high-throughput sequencing data there were strong correlations, including a periodontal health associated taxa, Capnocytophaga sp. COT-339 (rs =0.805), and two periodontal disease associated taxa, Peptostreptococcaceae XI [G-4] sp. COT-019 (rs=0.902) and Clostridiales sp. COT-028 (rs=0.802). The best performing models, from five machine learning approaches applied to the qPCR data for these taxa, estimated 85.7% sensitivity and 27.5% specificity for Capnocytophaga sp. COT-339, 74.3% sensitivity and 67.5% specificity for Peptostreptococcaceae XI [G-4] sp. COT-019, and 60.0% sensitivity and 80.0% specificity for Clostridiales sp. COT-028.ConclusionsA qPCR-based approach is an accurate, sensitive, and cost-effective method for detection of microbial biomarkers associated with periodontal health and disease. Taken together, the correlation between qPCR and high-throughput sequencing outputs, and early accuracy insights, indicate the strategy offers a prospective route to the development of diagnostic tools for canine periodontal disease.

CRISPR-based knockout and base editing confirm the role of MYRF in heart development and congenital heart disease.

High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.

The emerging role of epitranscriptome in shaping stress responses in plants.

RNA modifications and editing changes constitute 'epitranscriptome' and are crucial in regulating the development and stress response in plants. Exploration of the epitranscriptome and associated machinery would facilitate the engineering of stress tolerance in crops. RNA editing and modifications post-transcriptionally decorate almost all classes of cellular RNAs, including tRNAs, rRNAs, snRNAs, lncRNAs and mRNAs, with more than 170 known modifications, among which m6A, Ψ, m5C, 8-OHG and C-to-U editing are the most abundant. Together, these modifications constitute the "epitranscriptome", and contribute to changes in several RNA attributes, thus providing an additional structural and functional diversification to the "cellular messages" and adding another layer of gene regulation in organisms, including plants. Numerous evidences suggest that RNA modifications have a widespread impact on plant development as well as in regulating the response of plants to abiotic and biotic stresses. High-throughput sequencing studies demonstrate that the landscapes of m6A, m5C, Am, Cm, C-to-U, U-to-G, and A-to-I editing are remarkably dynamic during stress conditions in plants. GO analysis of transcripts enriched in Ψ, m6A and m5C modifications have identified bonafide components of stress regulatory pathways. Furthermore, significant alterations in the expression pattern of genes encoding writers, readers, and erasers of certain modifications have been documented when plants are grown in challenging environments. Notably, manipulating the expression levels of a few components of RNA editing machinery markedly influenced the stress tolerance in plants. We provide updated information on the current understanding on the contribution of RNA modifications in shaping the stress responses in plants. Unraveling of the epitranscriptome has opened new avenues for designing crops with enhanced productivity and stress resilience in view of global climate change.

The endosymbiont and the second bacterial circle of entomopathogenic nematodes.

Single host-symbiont interactions should be reconsidered from the perspective of the pathobiome. We revisit here the interactions between entomopathogenic nematodes (EPNs) and their microbiota. We first describe the discovery of these EPNs and their bacterial endosymbionts. We also consider EPN-like nematodes and their putative symbionts. Recent high-throughput sequencing studies have shown that EPNs and EPN-like nematodes are also associated with other bacterial communities, referred to here as the second bacterial circle of EPNs. Current findings suggest that some members of this second bacterial circle contribute to the pathogenic success of nematodes. We suggest that the endosymbiont and the second bacterial circle delimit an EPN pathobiome.

Evaluation of circulating tumor DNA by electropherogram analysis and methylome profiling in high-risk neuroblastomas.

Liquid biopsy has emerged as a promising, non-invasive diagnostic approach in oncology because the analysis of circulating tumor DNA (ctDNA) reflects the precise status of the disease at diagnosis, progression, and response to treatment. DNA methylation profiling is also a potential solution for sensitive and specific detection of many cancers. The combination of both approaches, DNA methylation analysis from ctDNA, provides an extremely useful and minimally invasive tool with high relevance in patients with childhood cancer. Neuroblastoma is an extracranial solid tumor most common in children and responsible for up to 15% of cancer-related deaths. This high death rate has prompted the scientific community to search for new therapeutic targets. DNA methylation also offers a new source for identifying these molecules. However, the limited blood sample size which can be obtained from children with cancer and the fact that ctDNA content may occasionally be diluted by non-tumor cell-free DNA (cfDNA) complicate optimal quantities of material for high-throughput sequencing studies. In this article, we present an improved method for ctDNA methylome studies of blood-derived plasma from high-risk neuroblastoma patients. We assessed the electropherogram profiles of ctDNA-containing samples suitable for methylome studies, using 10 ng of plasma-derived ctDNA from 126 samples of 86 high-risk neuroblastoma patients, and evaluated several bioinformatic approaches to analyze DNA methylation sequencing data. We demonstrated that enzymatic methyl-sequencing (EM-seq) outperformed bisulfite conversion-based method, based on the lower proportion of PCR duplicates and the higher percentage of unique mapping reads, mean coverage, and genome coverage. The analysis of the electropherogram profiles revealed the presence of nucleosomal multimers, and occasionally high molecular weight DNA. We established that 10% content of the mono-nucleosomal peak is sufficient ctDNA for successful detection of copy number variations and methylation profiles. Quantification of mono-nucleosomal peak also showed that samples at diagnosis contained a higher amount of ctDNA than relapse samples. Our results refine the use of electropherogram profiles to optimize sample selection for subsequent high-throughput analysis and support the use of liquid biopsy followed by enzymatic conversion of unmethylated cysteines to assess the methylomes of neuroblastoma patients.

Abstract 3745: GRAS1 long non-coding RNA binds and stabilizes NF-kB activating protein to protect lung cancer cells from DNA damage

Abstract Long non-coding RNAs (lncRNAs) control numerous biological processes, including epigenetic regulation, gene expression, proliferation and metabolism. However, the detailed biological mechanisms by which lncRNAs regulate cellular processes are still poorly understood. We identified a set of growth regulator non-coding RNAs based on data mining of CRISPR/Cas9 screens and high-throughput sequencing studies from patient normal and tumor cells. We hypothesized that growth regulator lncRNAs play a role in cancer development by recruiting effector proteins to regulate gene expression. In our screen, the Growth Regulator Antisense 1 RNA (GRAS1) was identified as a key factor controlling growth in non-small cell lung cancer cells. Loss of GRAS1 resulted in altered expression of genes involved in DNA repair, cell cycle progression, and p53 and NF-ΚB signaling pathways. Extensive DNA damage occurred within 48 hours after GRAS1 knockdown, with a significant increase in the number of γH2AX loci visualized by immunofluorescence assay. We used RNA antisense purification combined with mass spectrometry to identify the NF-ΚB activating protein (NKAP) as the top directly interacting partner of GRAS1 lncRNA. NKAP stability decreased after GRAS1 knockdown, and this phenotype could be reversed with proteasome inhibition. Further, RelA levels were decreased in GRAS1 knockdown cells. We found that GRAS1 lncRNA binds and stabilizes NKAP to enable survival and protect against DNA damage in lung cancer cells. Citation Format: Tong Su, Bobby Kong, Jonathan Zhu, Colleen A. McHugh. GRAS1 long non-coding RNA binds and stabilizes NF-kB activating protein to protect lung cancer cells from DNA damage. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3745.

Abstract 4764: The therapeutic potential of lnc-HLX-2-7 in group 3 medulloblastomas in children

Abstract Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the non-coding RNA genome, in particular long non-coding RNAs (lncRNAs), contributes to MB formation and tumor progression. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 MBs. In this study, we report that lnc-HLX-2-7 RNA specifically accumulates in the HLX (host gene of lnc-HLX-2-7) promoter region and activates HLX expression by recruiting multiple factors including enhancer elements. RNA sequencing and chromatin immunoprecipitation revealed that HLX directly binds to the promoters of several tumor-promoting genes, including MYC, and activates their expression. Furthermore, intravenous treatment with antisense oligonucleotides targeting lnc-HLX-2-7 coated with cerium-oxide nanoparticle (CNP-lnc-HLX-2-7) reduced tumor growth (40-50%) in intracranial MB xenograft mouse model (n=10, p<0.01, t-test). We found that the combinatorial therapy of CNP-lnc-HLX-2-7 and cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared to CNP-lnc-HLX-2-7 monotherapy (n=10, p<0.01, t-test) only. We report here the importance of the lnc-HLX-2-7-HLX-MYC axis in regulating group 3 MB progression and provide a strong rationale for using lnc-HLX-2-7 as a specific and potent therapeutic target for the group 3 MBs in children. Citation Format: Keisuke Katsushima, Bongyong Lee, Menglang Yuan, Stacie Stapleton, George Jallo, Sudipta Seal, Charles G. Eberhart, Ranjan J. Perera. The therapeutic potential of lnc-HLX-2-7 in group 3 medulloblastomas in children. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4764.

Gastrointestinal microbiome and cholelithiasis: Current status and perspectives

Cholelithiasis is a common digestive disease affecting 10% to 15% of adults. It imposes significant global health and financial burdens. However, the pathogenesis of cholelithiasis involves several factors and is incompletely elucidated. In addition to genetic predisposition and hepatic hypersecretion, the pathogenesis of cholelithiasis might involve the gastrointestinal (GI) microbiome, consisting of microorganisms and their metabolites. High-throughput sequencing studies have elucidated the role of bile, gallstones, and the fecal microbiome in cholelithiasis, associating microbiota dysbiosis with gallstone formation. The GI microbiome may drive cholelithogenesis by regulating bile acid metabolism and related signaling pathways. This review examines the literature implicating the GI microbiome in cholelithiasis, specifically gallbladder stones, choledocholithiasis, and asymptomatic gallstones. We also discuss alterations of the GI microbiome and its influence on cholelithogenesis.

Morphological Continua Make Poor Species: Genus-Wide Morphometric Survey of the European Bee Orchids (Ophrys L.).

Despite (or perhaps because of) intensive multidisciplinary research, opinions on the optimal number of species recognised within the Eurasian orchid genus Ophrys range from nine to at least 400. The lower figure of nine macrospecies is based primarily on seeking small but reliable discontinuities in DNA 'barcode' regions, an approach subsequently reinforced and finessed via high-throughput sequencing studies. The upper figure of ca. 400 microspecies reflects the morphological authoritarianism of traditional taxonomy combined with belief in extreme pollinator specificity caused by reliance on pollination through pseudo-copulation, enacted by bees and wasps. Groupings of microspecies that are less inclusive than macrospecies are termed mesospecies. Herein, we present multivariate morphometric analyses based on 51 characters scored for 457 individual plants that together span the full morphological and molecular diversity within the genus Ophrys, encompassing 113 named microspecies that collectively represent all 29 mesospecies and all nine macrospecies. We critique our preferred morphometric approach of accumulating heterogeneous data and analysing them primarily using principal coordinates, noting that our conclusions would have been strengthened by even greater sampling and the inclusion of data describing pseudo-pheromone cocktails. Morphological variation within Ophrys proved to be exceptionally multidimensional, lacking strong directional trends. Multivariate clustering of plants according to prior taxonomy was typically weak, irrespective of whether it was assessed at the level of macrospecies, mesospecies or microspecies; considerable morphological overlap was evident even between subsets of the molecularly differentiable macrospecies. Characters supporting genuine taxonomic distinctions were often sufficiently subtle that they were masked by greater and more positively correlated variation that reflected strong contrasts in flower size, tepal colour or, less often, plant size. Individual macrospecies appear to represent morphological continua, within which taxonomic divisions are likely to prove arbitrary if based exclusively on morphological criteria and adequately sampled across their geographic range. It remains unclear how much of the mosaic of subtle character variation among the microspecies reflects genetic versus epigenetic or non-genetic influences and what proportion of any contrasts observed in gene frequencies can be attributed to the adaptive microevolution that is widely considered to dictate speciation in the genus. Moreover, supplementing weak morphological criteria with extrinsic criteria, typically by imposing constraints on geographic location and/or supposed pollinator preference, assumes rather than demonstrates the presence of even the weakest of species boundaries. Overall, it is clear that entities in Ophrys below the level of macrospecies have insufficiently structured variation, either phenotypic or genotypic, to be resolved into discrete, self-circumscribing ("natural") entities that can legitimately be equated with species as delimited within other less specialised plant genera. Our search for a non-arbitrary (meso)species concept competent to circumscribe an intermediate number of species has so far proven unsuccessful.

A Discussion of the Contemporary Prediction Models for Atrial Fibrillation.

Atrial Fibrillation is a complex disease state with many environmental and genetic risk factors. While there are environmental factors that have been shown to increase an individual's risk of atrial fibrillation, it has become clear that atrial fibrillation has a genetic component that influences why some patients are at a higher risk of developing atrial fibrillation compared to others. This review will first discuss the clinical diagnosis of atrial fibrillation and the corresponding rhythm atrial flutter. We will then discuss how a patients' risk of stroke can be assessed by using other clinical co-morbidities. We will then review the clinical risk factors that can be used to help predict an individual patient's risk of atrial fibrillation. Many of the clinical risk factors have been used to create several different risk scoring methods that will be reviewed. We will then discuss how genetics can be used to identify individuals who are at higher risk for developing atrial fibrillation. We will discuss genome-wide association studies and other sequencing high-throughput sequencing studies. Finally, we will touch on how genetic variants derived from a genome-wide association studies can be used to calculate an individual's polygenic risk score for atrial fibrillation. An atrial fibrillation polygenic risk score can be used to identify patients at higher risk of developing atrial fibrillation and may allow for a reduction in some of the complications associated with atrial fibrillation such as cerebrovascular accidents and the development of heart failure. Finally, there is a brief discussion of how artificial intelligence models can be used to predict which patients will develop atrial fibrillation.

DDDR-31. THE THERAPEUTIC POTENTIAL OF THE LONG NON-CODING RNA LNC-HLX-2-7 IN GROUP 3 MEDULLOBLASTOMAS IN CHILDREN

Abstract Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the non-coding RNA genome, in particular long non-coding RNAs (lncRNAs), contributes to MB formation and tumor progression. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 MBs. In this study, we report that lnc-HLX-2-7 RNA specifically accumulates in the HLX (host gene of lnc-HLX-2-7) promoter region and activates HLX expression by recruiting multiple factors including enhancer elements. RNA sequencing and chromatin immunoprecipitation revealed that HLX directly binds to the promoters of several tumor-promoting genes, including MYC, and activates their expression. Furthermore, intravenous treatment with antisense oligonucleotides targeting lnc-HLX-2-7 coated with cerium-oxide nanoparticle (CNP-lnc-HLX-2-7) reduced tumor growth (40-50%) in intracranial MB xenograft mouse model (n = 10, p < 0.01, t-test). We found that the combinatorial therapy of CNP-lnc-HLX-2-7 and cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared to CNP-lnc-HLX-2-7 monotherapy (n = 10, p < 0.01, t-test) only. We report here the importance of the lnc-HLX-2-7-HLX-MYC axis in regulating group 3 MB progression and provide a strong rationale for using lnc-HLX-2-7 as a specific and potent therapeutic target for the group 3 MBs in children.