RHO Variants and Autosomal Dominant Retinitis Pigmentosa: Insights from the Italian Genetic Landscape.

Autosomal dominant retinitis pigmentosa (AD-RP) is caused by several genes, among which RHO is one of the most investigated. This article will be focused on RHO and its role in explaining AD-RP cases in the Italian population, taking advantage of the experience of the Genomic Medicine Laboratory UILDM at the Santa Lucia Foundation IRCCS. The retrospective evaluation of the distribution of RHO variants in the Italian patients with a clinical suspicion of RP pointed out eight variants. Of them, four variants (c.632A>T, c.1040C>T, c.1030C>T, c.383_392del) were pathogenic and made it possible to confirm the diagnosis of AD-RP in nine affected patients, highlighting a lower frequency (17%) of RHO variants compared to previous studies (30-40%). In addition, this study identified four variants classified as Variants of Uncertain Significance (VUS). In conclusion, the experience of the Genomic Medicine Laboratory provides an overview of the distribution of RHO variants in the Italian population, highlighting a slightly lower frequency of these variants in our cases series compared to previous reports. However, further studies on RHO variants are essential to characterize peculiar RP phenotypes and extend the spectrum of disease associated with this gene.

SARS-CoV-2 Molecular Evolution: A Focus on Omicron Variants in Umbria, Italy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 6 million deaths worldwide, and the spread of new variants over time increased the ability of this virus to cause infection. The Omicron variant was detected for the first time in Umbria, a region of central Italy, in November 2021 and it induced an unprecedented increase in the number of infection cases. Here, we analysed 3300 SARS-CoV-2 positive samples collected in Umbria between April 2022 and December 2023. We traced the molecular evolution of SARS-CoV-2 variants over time through the Next-Generation Sequencing (NGS) approach. We assessed correlation between SARS-CoV-2 infection and patients' health status. In total, 17.3% of our samples came from patients hospitalised as a consequence of COVID-19 infection even though 81.4% of them received at least three vaccine doses. We identified only Omicron variants, and the BA.5 lineage was detected in the majority of our samples (49.2%). Omicron variants outcompeted each other through the acquisition of mutations especially in Spike glycoprotein that are fingerprints of each variant. Viral antigenic evolution confers higher immunological escape and makes a continuous improvement of vaccine formulation necessary. The continuous update of international genomic databases with sequencing results obtained by emergent pathogens is essential to manage a possible future pandemic.

Summary of ChIP-Seq Methods and Description of an Optimized ChIP-Seq Protocol.

Chromatin immunoprecipitation (ChIP) is an invaluable method to characterize interactions between proteins and genomic DNA, such as the genomic localization of transcription factors and post-translational modification of histones. DNA and proteins are reversibly and covalently crosslinked using formaldehyde. Then the cells are lysed to release the chromatin. The chromatin is fragmented into smaller sizes either by micrococcal nuclease (MN) or sonication and then purified from other cellular components. The protein-DNA complexes are enriched by immunoprecipitation (IP) with antibodies that target the epitope of interest. The DNA is released from the proteins by heat and protease treatment, followed by degradation of contaminating RNAs with RNase. The resulting DNA is analyzed using various methods, including polymerase chain reaction (PCR), quantitative PCR (qPCR), or sequencing. This protocol outlines each of these steps for both yeast and human cells. This chapter includes a contextual discussion of the combination of ChIP with DNA analysis methods such as ChIP-on-Chip, ChIP-qPCR, and ChIP-Seq, recent updates on ChIP-Seq data analysis pipelines, complementary methods for identification of binding sites of DNA binding proteins, and additional protocol information about ChIP-qPCR and ChIP-Seq.

Quadruple primary tumors in a lynch syndrome patient surviving more than 26 years with genetic analysis: a case report and literature review.

The incidence of multiple primary tumors(MPTs) is on the rise in recent years, but patients having four or more primary tumors is still rare. Lynch syndrome (LS) patients have a high risk of developing MPTs. NGS sequencing could identify the genetic alterations in different tumors to make a definite diagnosis of uncommon cases in clinical practice. Here, we report the case of a 66-year-old female patient who develops four MPTS between the ages of 41 and 66, that is sigmoid colon cancer, acute non-lymphocytic leukemia, urothelial carcinoma and ascending colon cancer. She has survived for more than 26 years since the first discovery of tumor. Targeted sequencing indicates that she has a pathogenic germline mutation in the exon 13 of MSH2, and her 2020 ureteral cancer sample and 2023 colon cancer sample have completely different mutation profiles. To the best of our knowledge, this is the first case of multiple primary tumors with an acute non-lymphocytic leukemia in LS patients.

Comparing full variation profile analysis with the conventional consensus method in SARS-CoV-2 phylogeny.

This study proposes a novel approach to studying severe acute respiratory syndrome coronavirus 2 virus mutations through sequencing data comparison. Traditional consensus-based methods, which focus on the most common nucleotide at each position, might overlook or obscure the presence of low-frequency variants. Our method, in contrast, retains all sequenced nucleotides at each position, forming a genomic matrix. Utilizing simulated short reads from genomes with specified mutations, we contrasted our genomic matrix approach with the consensus sequence method. Our matrix methodology, across multiple simulated datasets, accurately reflected the known mutations with an average accuracy improvement of 20% over the consensus method. In real-world tests using data from GISAID and NCBI-SRA, our approach demonstrated an increase in reliability by reducing the error margin by approximately 15%. The genomic matrix approach offers a more accurate representation of the viral genomic diversity, thereby providing superior insights into virus evolution and epidemiology.

Meat-Borne-Parasite: A Nanopore-Based Meta-Barcoding Work-Flow for Parasitic Microbiodiversity Assessment in the Wild Fauna of French Guiana.

French Guiana, located in the Guiana Shield, is a natural reservoir for many zoonotic pathogens that are of considerable medical or veterinary importance. Until now, there has been limited data available on the description of parasites circulating in this area, especially on protozoan belonging to the phylum Apicomplexa; conversely, the neighbouring countries describe a high parasitic prevalence in animals and humans. Epidemiological surveillance is necessary, as new potentially virulent strains may emerge from these forest ecosystems, such as Amazonian toxoplasmosis. However, there is no standard tool for detecting protozoa in wildlife. In this study, we developed Meat-Borne-Parasite, a high-throughput meta-barcoding workflow for detecting Apicomplexa based on the Oxford Nanopore Technologies sequencing platform using the 18S gene of 14 Apicomplexa positive samples collected in French Guiana. Sequencing reads were then analysed with MetONTIIME pipeline. Thanks to a scoring rule, we were able to classify 10 samples out of 14 as Apicomplexa positive and reveal the presence of co-carriages. The same samples were also sequenced with the Illumina platform for validation purposes. For samples identified as Apicomplexa positive by both platforms, a strong positive correlation at up to the genus level was reported. Overall, the presented workflow represents a reliable method for Apicomplexa detection, which may pave the way for more comprehensive biomonitoring of zoonotic pathogens.

Composition of the sputum bacterial microbiome of patients with different pathomorphological forms of non-small-cell lung cancer.

Recent studies have shown that the bacterial microbiome of the respiratory tract influences the development of lung cancer. Changes in the composition of the microbiome are observed in patients with chronic inflammatory processes. Such microbiome changes may include the occurrence of bacteria that cause oxidative stress and that are capable of causing genome damage in the cells of the host organism directly and indirectly. To date, the composition of the respiratory microbiome in patients with various histological variants of lung cancer has not been studied. In the present study, we determined the taxonomic composition of the sputum microbiome of 52 patients with squamous cell carcinoma of the lung, 52 patients with lung adenocarcinoma and 52 healthy control donors, using next-generation sequencing (NGS) on the V3-V4 region of the bacterial gene encoding 16S rRNA. The sputum microbiomes of patients with different histological types of lung cancer and controls did not show significant differences in terms of the species richness index (Shannon); however, the patients differed from the controls in terms of evenness index (Pielou). The structures of bacterial communities (beta diversity) in the adenocarcinoma and squamous cell carcinoma groups were also similar; however, when analyzed according to the matrix constructed by the Bray-Curtis method, there were differences between patients with squamous cell carcinoma and healthy subjects, but not between those with adenocarcinoma and controls. Using the LEFse method it was possible to identify an increase in the content of Bacillota (Streptococcus and Bacillus) and Actinomycetota (Rothia) in the sputum of patients with squamous cell carcinoma when compared with samples from patients with adenocarcinoma. There were no differences in the content of bacteria between the samples of patients with adenocarcinoma and the control ones. The content of representatives of the genera Streptococcus, Bacillus, Peptostreptococcus (phylum Bacillota), Prevotella, Macellibacteroides (phylum Bacteroidota), Rothia (phylum Actinomycetota) and Actinobacillus (phylum Pseudomonadota) was increased in the microbiome of sputum samples from patients with squamous cell carcinoma, compared with the control. Thus, the sputum bacterial microbiome of patients with different histological types of non-small-cell lung cancer has significant differences. Further research should be devoted to the search for microbiome biomarkers of lung cancer at the level of bacterial species using whole-genome sequencing.

Accelerating targeted mosquito control efforts through mobile West Nile virus detection.

BACKGROUND: Different mosquito control strategies have been implemented to mitigate or prevent mosquito-related public health situations. Modern mosquito control largely relies on multiple approaches, including targeted, specific treatments. Given this, it is becoming increasingly important to supplement these activities with rapid and mobile diagnostic capacities for mosquito-borne diseases. We aimed to create and test the applicability of a rapid diagnostic system for West Nile virus that can be used under field conditions. METHODS: In this pilot study, various types of adult mosquito traps were applied within the regular mosquito monitoring activity framework for mosquito control. Then, the captured specimens were used for the detection of West Nile virus RNA under field conditions with a portable qRT-PCR approach within 3-4 h. Then, positive samples were subjected to confirmatory RT-PCR or NGS sequencing in the laboratory to obtain genome information of the virus. We implemented phylogenetic analysis to characterize circulating strains. RESULTS: A total of 356 mosquito individuals representing 7 species were processed in 54 pools, each containing up to 20 individuals. These pools were tested for the presence of West Nile virus, and two pools tested positive, containing specimens from the Culex pipiens and Anopheles atroparvus mosquito species. As a result of subsequent sequencing, we present the complete genome of West Nile virus and Bagaza virus. CONCLUSIONS: The rapid identification of infected mosquitoes is the most important component of quick response adulticide or larvicide treatments to prevent human cases. The conceptual framework of real-time surveillance can be optimized for other pathogens and situations not only in relation to West Nile virus. We present an early warning system for mosquito-borne diseases and demonstrate its application to aid rapid-response mosquito control actions.

Evaluation of the Microbiome Identification of Forensically Relevant Biological Fluids: A Pilot Study.

In forensic sciences, body fluids, or biological traces, are a major source of information, and their identification can play a decisive role in criminal investigations. Currently, the nature of biological fluids is assessed using immunological, physico-chemical, mRNA and epigenetic methods, but these have limits in terms of sensitivity and specificity. The emergence of next-generation sequencing technologies offers new opportunities to identify the nature of body fluids by determining bacterial communities. The aim of this pilot study was to assess whether analysis of the bacterial communities in isolated and mixed biological fluids could reflect the situation observed in real forensics labs. Several samples commonly encountered in forensic sciences were tested from healthy volunteers: saliva, vaginal fluid, blood, semen and skin swabs. These samples were analyzed alone or in combination in a ratio of 1:1. Sequencing was performed on the Ion Gene StudioTM S5 automated sequencer. Fluids tested alone revealed a typical bacterial signature with specific bacterial orders, enabling formal identification of the fluid of interest, despite inter-individual variations. However, in biological fluid mixtures, the predominance of some bacterial microbiomes inhibited interpretation. Oral and vaginal microbiomes were clearly preponderant, and the relative abundance of their bacterial communities and/or the presence of common species between samples made it impossible to detect bacterial orders or genera from other fluids, although they were distinguishable from one another. However, using the beta diversity, salivary fluids were identified and could be distinguished from fluids in combination. While this method of fluid identification is promising, further analyses are required to consolidate the protocol and ensure reliability.

Idiopathic erythrocytosis: a germline disease?

Polycythemia Vera (PV) is typically caused by V617F or exon 12 JAK2 mutations. Little is known about Polycythemia cases where no JAK2 variants can be detected, and no other causes identified. This condition is defined as idiopathic erythrocytosis (IE). We evaluated clinical-laboratory parameters of a cohort of 56 IE patients and we determined their molecular profile at diagnosis with paired blood/buccal-DNA exome-sequencing coupled with a high-depth targeted OncoPanel to identify a possible underling germline or somatic cause. We demonstrated that most of our cohort (40/56: 71.4%) showed no evidence of clonal hematopoiesis, suggesting that IE is, in large part, a germline disorder. We identified 20 low mutation burden somatic variants (Variant allelic fraction, VAF, < 10%) in only 14 (25%) patients, principally involving DNMT3A and TET2. Only 2 patients presented high mutation burden somatic variants, involving DNMT3A, TET2, ASXL1 and WT1. We identified recurrent germline variants in 42 (75%) patients occurring mainly in JAK/STAT, Hypoxia and Iron metabolism pathways, among them: JAK3-V722I and HIF1A-P582S; a high fraction of patients (48.2%) resulted also mutated in homeostatic iron regulatory gene HFE-H63D or C282Y. By generating cellular models, we showed that JAK3-V722I causes activation of the JAK-STAT5 axis and upregulation of EPAS1/HIF2A, while HIF1A-P582S causes suppression of hepcidin mRNA synthesis, suggesting a major role for these variants in the onset of IE.

Construction of heat stress regulation networks based on Illumina and SMRT sequencing data in potato.

Potato (Solanum tuberosum L.) is one of the most important tuber food crops in the world; however, the cultivated potatoes are susceptible to high temperature, by which potato production is adversely affected. Understanding the coping mechanism of potato to heat stress is essential to secure yield and expand adaptability under environmental conditions with rising temperature. However, the lack of heat-related information has significantly limited the identification and application of core genes. To gain deeper insights into heat tolerance genes, next-generation sequencing and single-molecule real-time sequencing were used to learn the transcriptional response of potato to heat stress and 13,159 differentially expressed genes (DEGs) were identified in this study. All DEGs were grouped into 12 clusters using the K-means clustering algorithm. Gene Ontology enrichment analysis revealed that they were involved in temperature signaling, phytohormone, and protein modification. Among them, there were 950 differentially expressed transcription factors (DETFs). According to the network analysis of DETFs at the sixth hour under heat stress, we found some genes that were previously reported to be associated with photoperiodic tuberization, StCO (CONSTANS), tuber formation, StBEL11 (BEL1-LIKE 11), and earliness in potato, StCDF1 (CYCLING DOF FACTOR 1) responding to temperature. Furthermore, we verified the relative expression levels using quantitative real-time polymerase chain reaction, and the results were consistent with the inferences from transcriptomes. In addition, there were 22,125 alternative splicing events and 2,048 long non-coding RNAs. The database and network established in this study will extend our understanding of potato response to heat stress. It ultimately provided valuable resources for molecular analysis of heat stress response in potato and cultivation of potato varieties with heat tolerance.

Genetic Diversity of Type A Influenza Viruses Found in Swine Herds in Northwestern Poland from 2017 to 2019: The One Health Perspective.

Influenza A viruses (IAV) are still a cause of concern for public health and veterinary services worldwide. With (-) RNA-segmented genome architecture, influenza viruses are prone to reassortment and can generate a great variety of strains, some capable of crossing interspecies barriers. Seasonal IAV strains continuously spread from humans to pigs, leading to multiple reassortation events with strains endemic to swine. Due to its high adaptability to humans, a reassortant strain based on "human-like" genes could potentially be a carrier of avian origin segments responsible for high virulence, and hence become the next pandemic strain with unseen pathogenicity. The rapid evolution of sequencing methods has provided a fast and cost-efficient way to assess the genetic diversity of IAV. In this study, we investigated the genetic diversity of swine influenza viruses (swIAVs) collected from Polish farms. A total of 376 samples were collected from 11 farms. The infection was confirmed in 112 cases. The isolates were subjected to next-generation sequencing (NGS), resulting in 93 full genome sequences. Phylogenetic analysis classified 59 isolates as genotype T (H1avN2g) and 34 isolates as genotype P (H1pdmN1pdm), all of which had an internal gene cassette (IGC) derived from the H1N1pdm09-like strain. These data are consistent with evolutionary trends in European swIAVs. The applied methodology proved to be useful in monitoring the genetic diversity of IAV at the human-animal interface.

Diagnosis of PTEN mosaicism: the relevance of additional tumor DNA sequencing. A case report and review of the literature.

BACKGROUND: PTEN hamartoma syndrome (PHTS) is an autosomal dominant disorder characterized by pathogenic variants in the tumor suppressor gene phosphatase and tensin homolog (PTEN). It is associated with an increased risk of muco-cutaneous features, hamartomatous tumors, and cancers. Mosaicism has been found in a few cases of patients with de novo PHTS, identified from blood samples. We report a PHTS patient with no variant identified from blood sample. Constitutional PTEN mosaicism was detected through sequencing of DNA from different tumoral and non-tumoral samples. CASE PRESENTATION: Our patient presented clinical Cowden syndrome at 56 years of age, with three major criteria (macrocephaly, Lhermitte Duclos disease, oral papillomatosis), and two minor criteria (structural thyroid lesions, esophageal glycogenic acanthosis). Deep sequencing of PTEN of blood leukocytes did not reveal any pathogenic variants. Exploration of tumoral (colonic ganglioneuroma, esophageal papilloma, diapneusia fibroids) and non-tumoral stomach tissues found the same PTEN pathogenic variant (NM_000314.4 c.389G > A; p.(Arg130Gln)), with an allelic frequency of 12 to 59%, confirming genomic mosaicism for Cowden syndrome. CONCLUSIONS: This case report, and review of the literature, suggests that systematic tumor analysis is essential for patients presenting PTEN hamartoma syndrome in the absence of any causal variant identified in blood leukocytes, despite deep sequencing. In 65 to 70% of cases of clinical Cowden syndrome, no pathogenic variant in the PTEN is observed in blood samples: mosaicism may explain a significant number of these patients. Tumor analysis would improve our knowledge of the frequency of de novo variations in this syndrome. Finally, patients with mosaicism for PTEN may not have a mild phenotype; medical care identical to that of heterozygous carriers should be offered.

Metagenomic Study of Fungal Microbial Communities in Two PDO Somontano Vineyards (Huesca, Spain): Effects of Age, Plant Genotype, and Initial Phytosanitary Status on the Priming and Selection of their Associated Microorganisms.

The study of microbial communities associated with different plants of agronomic interest has allowed, in recent years, to answer a number of questions related to the role and influence of certain microbes in key aspects of their autoecology, such as improving the adaptability of the plant host to different abiotic or biotic stresses. In this study, we present the results of the characterization, through both high-throughput sequencing and classical microbiological methods, of the fungal microbial communities associated with grapevine plants in two vineyards of different ages and plant genotypes located in the same biogeographical unit. The study is configured as an approximation to the empirical demonstration of the concept of "microbial priming" by analyzing the alpha- and beta-diversity present in plants from two plots subjected to the same bioclimatic regime to detect differences in the structure and taxonomic composition of the populations. The results were compared with the inventories of fungal diversity obtained by culture-dependent methods to establish, where appropriate, correlations between both microbial communities. Metagenomic data showed a differential enrichment of the microbial communities in the two vineyards studied, including the populations of plant pathogens. This is tentatively explained due to factors such as the different time of exposure to microbial infection, different plant genotype, and different starting phytosanitary situation. Thus, results suggest that each plant genotype recruits differential fungal communities and presents different profiles of associated potential microbial antagonists or communities of pathogenic species.

Combined Drug with Antibacterial Effect Supports the Normal Intestinal Microflora.

Widespread use of antibiotics leads to an imbalance of normal intestinal microflora and to the development of multidrug resistance. The problem can be solved by administration of the antibiotics in combination with the drugs that have an immunotropic effect. We studied the effect of the drug containing technologically processed affinity purified antibodies to IFNγ, CD4 receptor, ß2-microglobulin of MHC class I, and ß2-domain of MHC II combined with antibiotics on the composition of intestinal microflora of pigs and the total number of microbiome resistance genes. Using the methods of NGS sequencing and quantitative PCR, we found that the drug contributes to the maintenance of normal microflora and, consequently, to the symbiotic relationship of the host with microflora, and prevents the reproduction of pathogenic bacterial species. Analysis for the presence of the resistance genes of gastrointestinal microorganisms showed that the drug does not affect the qualitative and quantitative composition of these genes of the intestinal microbiome.

Unique features of the TCR repertoire of reactivated memory T cells in the experimental mouse tumor model.

T cell engineering with T cell receptors (TCR) specific to tumor antigens has become a breakthrough towards personalized cancer adoptive cell immunotherapy. However, the search for therapeutic TCRs is often challenging, and effective strategies are strongly required for the identification and enrichment of tumor-specific T cells that express TCRs with superior functional characteristics. Using an experimental mouse tumor model, we studied sequential changes in TCR repertoire features of T cells involved in the primary and secondary immune responses to allogeneic tumor antigens. In-depth bioinformatics analysis of TCR repertoires showed differences in reactivated memory T cells compared to primarily activated effectors. After cognate antigen re-encounter, memory cells were enriched with clonotypes that express α-chain TCR with high potential cross-reactivity and enhanced strength of interaction with both MHC and docked peptides. Our findings suggest that functionally true memory T cells could be a better source of therapeutic TCRs for adoptive cell therapy. No marked changes were observed in the physicochemical characteristics of TCRß in reactivated memory clonotypes, indicative of the dominant role of TCRα in the secondary allogeneic immune response. The results of this study could further contribute to the development of TCR-modified T cell products based on the phenomenon of TCR chain centricity.

Impact of Intestinal Microbiota on Growth Performance of Suckling and Weaned Piglets.

Small-scale studies investigating the relationship between pigs' intestinal microbiota and growth performance have generated inconsistent results. We hypothesized that on farms under favorable environmental conditions (e.g., promoting sow nest-building behavior, high colostrum production, low incidence of diseases and minimal use of antimicrobials), the piglet gut microbiota may develop toward a population that promotes growth and reduces pathogenic bacteria. Using 16S rRNA gene amplicon sequencing, we sampled and profiled the fecal microbiota from 170 individual piglets throughout suckling and postweaning periods (in total 670 samples) to track gut microbiota development and its potential association with growth. During the suckling period, the dominant genera were Lactobacillus and Bacteroides, the latter being gradually replaced by Clostridium sensu scricto 1 as piglets aged. The gut microbiota during the nursery stage, not the suckling period, predicted the average daily growth (ADG) of piglets. The relative abundances of SCFA-producing genera, in particular Faecalibacterium, Megasphaera, Mitsuokella, and Subdoligranulum, significantly correlated with high ADG of weaned piglets. In addition, the succession of the gut microbiota in high-ADG piglets occurred faster and stabilized sooner upon weaning, whereas the gut microbiota of low-ADG piglets continued to mature after weaning. Overall, our findings suggest that weaning is the major driver of gut microbiota variation in piglets with different levels of overall growth performance. This calls for further research to verify if promotion of specific gut microbiota, identified here at weaning transition, is beneficial for piglet growth. IMPORTANCE The relationship between pigs' intestinal microbiota and growth performance is of great importance for improving piglets' health and reducing antimicrobial use. We found that gut microbiota variation is significantly associated with growth during weaning and the early nursery period. Importantly, transitions toward a mature gut microbiota enriched with fiber-degrading bacteria mostly complete upon weaning in piglets with better growth. Postponing the weaning age may therefore favor the development of fiber degrading gut bacteria, conferring the necessary capacity to digest and harvest solid postweaning feed. The bacterial taxa associated with piglet growth identified herein hold potential to improve piglet growth and health.

Mitochondrial DNA Isolation from Plants.

For most eukaryotes, sequencing and assembly of the mitochondrial DNA (mtDNA) is possible by starting the analysis from total cellular DNA, but the exploration of the mtDNA of plants is more challenging because of the low copy number, limited sequence conservation, and complex structure of the mtDNA. The very large size of the nuclear genome of many plant species and the very high ploidy of the plastidial genome further complicate the analysis, sequencing, and assembly of plant mitochondrial genomes. An enrichment of mtDNA is therefore necessary. For this, plant mitochondria are purified prior to mtDNA extraction and purification. The relative enrichment in mtDNA can be assessed by qPCR, while the absolute enrichment can be deduced from the proportion of NGS reads mapping to each of the three genomes of the plant cell. Here we present methods for mitochondrial purification and mtDNA extraction applied to different plant species and tissues, and compare the mtDNA enrichment obtained with the different procedures.

Locus-Specific Bisulfate NGS Sequencing of GSTP1, RNF219, and KIAA1539 Genes in the Total Pool of Cell-Free and Cell-Surface-Bound DNA in Prostate Cancer: A Novel Approach for Prostate Cancer Diagnostics.

The locus-specific methylation of three genes (GSTP1, RNF219, and KIAA1539, also known as FAM214B) in the total pool of blood cell-free DNA, including cell-free DNA from plasma and cell-surface-bound DNA, of patients with prostate cancer and healthy donors was studied on the MiSeq platform. Our study found a higher methylation index of loci for total cell-free DNA compared with cell-free DNA. For total cell-free DNA, the methylation of GSTP1 in each of the 11 positions provided a complete separation of cancer patients from healthy donors, whereas for cell-free DNA, there were no positions in the three genes allowing for such separation. Among the prostate cancer patients, the minimum proportion of GSTP1 genes methylated in any of the 17 positions was 12.1% of the total circulated DNA fragments, and the minimum proportion of GSTP1 genes methylated in any of the 11 diagnostically specific positions was 8.4%. Total cell-free DNA was shown to be more convenient and informative as a source of methylated DNA molecules circulating in the blood than cell-free DNA.

Discovery of Aptamers Against Cell Surface Markers Using Ligand-Guided Selection.

Oligonucleotide ligands (DNA, RNA, or XNA), also known as aptamers, are selected against various target molecules using an iterative, evolutionary process called systematic evolution of ligands by exponential enrichment (SELEX). To select aptamers against complex cell surface proteins in their native state, a variant of SELEX termed ligand-guided selection (LIGS) was recently introduced. The significance of LIGS is rooted in its strategy of exploiting the selection step in SELEX to identify highly specific aptamers against known cell surface markers. Thus, in LIGS, a higher-affinity secondary ligand, such as a monoclonal antibody (mAb) to a whole-cell bound to an evolved SELEX library, is introduced to outcompete sequences against the mAb targeting cell surface protein or induce a conformational switch to destabilize the aptamer-surface cell surface protein resulting in elution of the sequences. Here, we describe the detailed method of LIGS utilized in identifying aptamers against T-cell receptor cluster of differentiation three complex (TCR-CD3) expressed in human T-cells and T-cell leukemia.