DNA Patterns Research Articles

RNA Captures More Cations than DNA: Insights from Molecular Dynamics Simulations.

The distribution of cations around nucleic acids is essential for a broad variety of processes ranging from DNA condensation and RNA folding to the detection of biomolecules in biosensors. Predicting the exact distribution of ions remains challenging since the distribution and, hence, a broad variety of nucleic acid properties depend on the salt concentration, the valency of the ions, and the ion type. Despite the importance, a general theory to quantify ion-specific effects for highly charged biomolecules is still lacking. Moreover, recent experiments reveal that despite their similar building blocks, DNA and RNA duplexes can react differently to the same ionic conditions. The aim of our current work is to provide a comprehensive set of molecular dynamics simulations using more than 180 μs of simulation time. For the mono- and divalent cations Li+, Na+, K+, Cs+, Ca2+, Sr2+, and Ba2+, the simulations allow us to reveal the ion-specific distributions and binding patterns for DNA and RNA duplexes. The microscopic insights from the simulations display the origin of ion-specificity and shed light on the question of why DNA and RNA show opposing behavior in the same ionic conditions. Finally, the detailed binding patterns from the simulations reveal why RNA can capture more cations than DNA.

Molecular Identification of Host Blood Meals and Detection of Blood Parasites in Culicoides Latreille (Diptera: Ceratopogonidae) Collected from Phatthalung Province, Southern Thailand.

Simple SummaryBiting midges, which feed on a variety of hosts, transmit a wide range of harmful human and animal viruses and parasites. According to recent studies in Thailand, biting midges may act as potential vectors for leishmaniasis and trypanosomiasis. The purpose of this study is to look for Leishmania and Trypanosoma DNA in biting midges obtained in the field in a leishmaniasis-endemic area in Phatthalung province, Southern Thailand. In addition, we analysed engorged midges for host blood DNA and screened the collected midges for avian haemosporidian parasites. According to our findings, biting midges have a diversified feeding habit and may be able to transmit various pathogens, including trypanosomatid and avian haemosporidian parasites.Five hundred and fifty-nine female biting midges were collected, and seventeen species in six subgenera (Avaritia, Haemophoructus, Hoffmania, Meijerehelea, Remmia, and Trithecoides) and two groups (Clavipalpis and Shortti) were identified. The dominant Culicoides species was C. peregrinus (30.94%), followed by C. subgenus Trithecoides. From blood meal analysis of engorged biting midges, they were found to feed on cows, dogs, pigs, and avians. The majority of blood preferences of biting midges (68%; 49/72) displayed a mixed pattern of host blood DNA (cow and avian). The overall non-engorged biting midge field infectivity rate was 1.44 % (7/487). We detected Leucocytozoon sp. in three Culicoides specimens, one from each species: C. fulvus, C. oxystoma, and C. subgenus Trithecoides. Crithidia sp. was found in two C. peregrinus specimens, and Trypanosoma sp. and P. juxtanucleare were separately found in two C. guttifer. More consideration should be paid to the capacity of biting midges to transmit pathogens such as avian haemosporidian and trypanosomatid parasites. To demonstrate that these biting midges are natural vectors of trypanosomatid parasites, additional research must be conducted with a greater number of biting midges in other endemic regions.

Genistein anticancer efficacy during induced oral squamous cell carcinoma: an experimental study

BackgroundAbout 7 million people die from various types of cancer every year representing nearly 12.5% of deaths worldwide. This fact raises the demand to develop new, effective anticancer, onco-suppressive, and chemoprotective agents for the future fighting of cancers. Genistein exhibits pleiotropic functions in cancer, metabolism, and inflammation. It functions as an antineoplastic agent through its effect on the cell cycle, apoptotic processes, angiogenesis, invasion, and metastasis.Aim of the studyThe current study aimed to study the genistein onco-suppressive effects during 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters’ buccal pouch utilizing flow cytometry analysis (FMA), as a fast-diagnosing tool, in addition to the histopathology.Material and methodsThe buccal mucosa of adult male Syrian hamsters was painted with paraffin oil only (group 1), DMBA mixed in mineral oil (group 2), or orally administrated genistein along with painting DMBA (group 2B). The buccal mucosa was utilized for flow cytometric analysis and histopathological examination.ResultsGrossly, DMBA-induced carcinogenesis started at the 9th week. Progressive signs appeared in the following weeks reaching to large ulcerative oral masses and exophytic nodules at the 21st week. Histologically, invasive well-differentiated oral squamous cell carcinoma (OSCC) appeared in the underlying tissues from the 12th week, showing malignant criteria. Genistein had delayed clinicopathological change, which started 6 weeks later, than the DMBA-painted hamsters, as mild epithelial dysplastic changes. This became moderate during the last 6 weeks, without dysplastic changes. Flow cytometry revealed that DMBA led to considerable variation in DNA proliferation activity, aneuploid DNA pattern, in 47.22% of hamsters and significantly raised the S-phase fragment (SPF) values, which drastically reduced after genistein treatment.ConclusionTaken together, genistein could be employed as an onco-suppressive agent for carcinogenesis. Moreover, FMA could be used as an aiding fast tool for diagnosis of cancer.

908P Evaluation of anxiety, distress and satisfaction with a multi-cancer early detection test

A multi-cancer early detection (MCED) test using methylation patterns in plasma cell-free DNA to detect a cancer signal and predict site of origin was evaluated in the prospective PATHFINDER study. Because MCED testing has the potential to cause anxiety and distress, participant-reported outcomes were assessed.

Enhancing anti-tumour innate immunity by targeting the DNA damage response and pattern recognition receptors in combination with radiotherapy.

Radiotherapy is one of the most effective and frequently used treatments for a wide range of cancers. In addition to its direct anti-cancer cytotoxic effects, ionising radiation can augment the anti-tumour immune response by triggering pro-inflammatory signals, DNA damage-induced immunogenic cell death and innate immune activation. Anti-tumour innate immunity can result from recruitment and stimulation of dendritic cells (DCs) which leads to tumour-specific adaptive T-cell priming and immunostimulatory cell infiltration. Conversely, radiotherapy can also induce immunosuppressive and anti-inflammatory mediators that can confer radioresistance. Targeting the DNA damage response (DDR) concomitantly with radiotherapy is an attractive strategy for overcoming radioresistance, both by enhancing the radiosensitivity of tumour relative to normal tissues, and tipping the scales in favour of an immunostimulatory tumour microenvironment. This two-pronged approach exploits genomic instability to circumvent immune evasion, targeting both hallmarks of cancer. In this review, we describe targetable DDR proteins (PARP (poly[ADP-ribose] polymerase); ATM/ATR (ataxia–telangiectasia mutated and Rad3-related), DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) and Wee1 (Wee1-like protein kinase) and their potential intersections with druggable immunomodulatory signalling pathways, including nucleic acid-sensing mechanisms (Toll-like receptors (TLR); cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) and retinoic acid-inducible gene-I (RIG-I)-like receptors), and how these might be exploited to enhance radiation therapy. We summarise current preclinical advances, recent and ongoing clinical trials and the challenges of therapeutic combinations with existing treatments such as immune checkpoint inhibitors.

Methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA methyltransferases

DNA methylation is one important epigenetic modification that involves in regulating multiple cellular functions. DNA methyltransferases (MTases) maintain methylation patterns in genomic DNA, and their aberrant activities may lead to multiple diseases including cancers. However, the efficient sensing of multiple DNA MTases is limited by the diversity of catalytic substrates and the rareness of methylation-dependent restriction endonucleases. Herein, inspired by the intrinsic superiorities of light-up RNA aptamers and unique properties of T7-based transcription amplification, we construct a novel methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA MTases. Taking advantage of ultrahigh specificity of MDREs toward methylated DNAs, high efficiency of bicyclic cascade amplifications and high signal-to-noise ratio of light-up RNA aptamer-fluorophore pairs, this nanosensor exhibits good specificity and high sensitivity with a detection limit of 3.77 × 10−3 U/mL for M.SssI and 7.21 × 10−5 U/mL for Dam. Furthermore, it can screen potential inhibitors and quantify M.SssI and Dam MTases activities in E. coli cells and human serum samples. Notably, this assay can be completed homogeneously within 2 h in a single tube in a label-free manner, providing a powerful platform for sensing multiple DNA MTases in biomedical research and clinical therapeutics.

DNA-POINT: DNA Patterning of Optical Imprint for Nanomaterials Topography.

Engineering well-defined scale-spanning structures through transfer of diverse biomolecules and materials to a surface is of tremendous interest in life sciences research yet remains profoundly challenging. Here, we report a novel method, termed as DNA patterning of optical imprint for nanomaterials topography (DNA-POINT), for rapid photopatterning of large area, geometrically complex surfaces via light-responsive DNA. Our method employs top-down multiphoton-driven patterning of azobenzene-modified DNA strands, offering precise position control of molecules and nanoparticles along the axial plane and a template for bottom-up self-assembly of multiple layers of different chemical composition along the vertical plane. We demonstrate the surface patterning of plasmonic gold nanoparticles, fluorophore-labeled oligonucleotides, and multiple layers consisting of molecule-nanoparticle hybrid patterns into preconceived shapes without compromising on the functionality of the biomolecules. Furthermore, we exhibit scanning mode operation of DNA-POINT, thereby paving the way for maskless and cleanroom-free fast fabrication of biochips for high-throughput diagnostics and biosensing applications.

AhrC Negatively Regulates Streptococcus mutans Arginine Biosynthesis.

ABSTRACTStreptococcus mutans is a primary cariogenic pathogen in humans. Arginine metabolism is required for bacterial growth. In S. mutans, however, the involvement of transcription factors in regulating arginine metabolism is unclear. The purpose of this study was to investigate the function and mechanism of ArgR family transcription factors in S. mutans. Here, we identified an ArgR (arginine repressor) family transcription factor named AhrC, which negatively regulates arginine biosynthesis and biofilm formation in S. mutans. The ahrC in-frame deletion strain exhibited slow growth and significantly increased intracellular arginine content. The strain overexpressing ahrC showed reduced intracellular arginine content, decreased biofilm biomass, reduced production of water-insoluble exopolysaccharides (EPS), and different biofilm structures. Furthermore, global gene expression profiles revealed differential expression levels of 233 genes in the ahrC-deficient strain, among which genes related to arginine biosynthesis (argJ, argB, argC, argD, argF, argG, argH) were significantly upregulated. In the ahrC overexpression strain, there are 89 differentially expressed genes, mostly related to arginine biosynthesis. The conserved DNA patterns bound by AhrC were identified by electrophoretic mobility shift assay (EMSA) and DNase I footprinting. In addition, the analysis of β-galactosidase activity showed that AhrC acted as a negative regulator. Taken together, our findings suggest that AhrC is an important transcription factor that regulates arginine biosynthesis gene expression and biofilm formation in S. mutans. These findings add new aspects to the complexity of regulating the expression of genes involved in arginine biosynthesis and biofilm formation in S. mutans.IMPORTANCE Arginine metabolism is essential for bacterial growth. The regulation of intracellular arginine metabolism in Streptococcus mutans, one of the major pathogens of dental caries, is unclear. In this study, we found that the transcription factor AhrC can directly and negatively regulate the expression of N-acetyl-gamma-glutamyl-phosphate reductase (argC), thus regulating arginine biosynthesis in S. mutans. In addition, the ahrC overexpression strain exhibited a significant decrease in biofilm and water-insoluble extracellular polysaccharides (EPS). This study adds new support to our understanding of the regulation of intracellular arginine metabolism in S. mutans.

Association between Escherichia coli with NotI-restriction resistance and urinary tract infections.

Escherichia coli is the most common cause of urinary tract infections (UTIs). It is widely accepted that uropathogenic E. coli (UPEC) mainly emerge from the distal gut microbiota. Identification of bacterial characteristics that are able to differentiate UPEC from fecal commensal strains will facilitate the development of novel strategies to detect and monitor the spread of UPEC. Fifty fecal commensal, 83 UTI-associated and 40 biliary tract infection (BTI)-associated E. coli isolates were analyzed. The NotI restriction patterns of chromosomal DNA in the isolates were determined by pulse-field gel electrophoresis. The phylogenetic types and the presence of 9 known virulence genes of each isolate were determined by PCR analyses. Additionally, the susceptibilities of the isolates to antibiotics were revealed. Then the associations of NotI resistance with UTI-associated isolates, phylotypes, and antibiotic resistance were assessed. NotI resistance was correlated with UTI-associated isolates, compared to the fecal isolates. Consistently, NotI-resistant isolates harbored a greater number of virulence factors and mainly belonged to phylotype B2. Additionally NotI resistance was correlated with chloramphenicol resistance among the bacteria. Among the fecal, UTI-associated and BTI-associated groups, the distribution of NotI-resistant group B2 isolates was correlated with UTI-associated bacteria. NotI resistance alone is a potential marker for distinguishing fecal strains and UPEC, while the combination of NotI resistance and B2 phylogeny is a candidate marker to differentiate UPEC from fecal and other extraintestinal pathogenic E. coli. Additionally, NotI resistance may be valuable for assessing the potential of chloramphenicol resistance of E. coli.

The diagnostic potential of plaque-specific methylation patterns in cell-free dna

Background and Aims : There is an urgent and unmet need for early, non-invasive diagnosis of atherosclerosis that can identify high-risk patients with potentially symptomatic plaques. Recent breakthroughs in the use of circulating fragments of cell-free DNA (cfDNA), have provided major advances in early cancer detection. Furthermore, it has been shown that, using tissue-specific methylation patterns, the contribution of different tissue origins to cfDNA can be quantified. Here we aim to identify plaque-specific methylation patterns and unravel the biological processes behind this epigenetic signature. We hypothesize that an increased contribution of plaque-derived cfDNA, as a proxy for the accumulation of atherosclerotic plaque, can be measured in cardiovascular disease patients.Methods: DNA methylation patterns were obtained for plaque samples from 50 female and 50 male patients that underwent carotid endarterectomy (CEA) from the Athero-Express Biobank using the Infinium HumanMethylation450 Beadchip Array. cfDNA methylation from 18 patients, spanning three cardiovascular clinical cohorts, was measured using the NEBNext Enzymatic Methyl-seq kit.Results: We were able to identify a plaque-specific methylation signature encompassing 200 CpG sides, of which a portion was found to be involved in atherosclerotic processes. Furthermore, we built a comprehensive human methylation atlas using methylation data on 18 different tissue and cell types, including plaque, which is used for the deconvolution of cfDNA.Conclusions: Our plaque-centric methylation atlas together with our well-established biobanks, offer the unique opportunity to investigate cfDNA composition in patients spanning various types of cardiovascular disease. We believe this comprehensive methylation framework would help unravel the potential of cfDNA as an innovative non-invasive biomarker for cardiovascular disease. Background and Aims : There is an urgent and unmet need for early, non-invasive diagnosis of atherosclerosis that can identify high-risk patients with potentially symptomatic plaques. Recent breakthroughs in the use of circulating fragments of cell-free DNA (cfDNA), have provided major advances in early cancer detection. Furthermore, it has been shown that, using tissue-specific methylation patterns, the contribution of different tissue origins to cfDNA can be quantified. Here we aim to identify plaque-specific methylation patterns and unravel the biological processes behind this epigenetic signature. We hypothesize that an increased contribution of plaque-derived cfDNA, as a proxy for the accumulation of atherosclerotic plaque, can be measured in cardiovascular disease patients. Methods: DNA methylation patterns were obtained for plaque samples from 50 female and 50 male patients that underwent carotid endarterectomy (CEA) from the Athero-Express Biobank using the Infinium HumanMethylation450 Beadchip Array. cfDNA methylation from 18 patients, spanning three cardiovascular clinical cohorts, was measured using the NEBNext Enzymatic Methyl-seq kit. Results: We were able to identify a plaque-specific methylation signature encompassing 200 CpG sides, of which a portion was found to be involved in atherosclerotic processes. Furthermore, we built a comprehensive human methylation atlas using methylation data on 18 different tissue and cell types, including plaque, which is used for the deconvolution of cfDNA. Conclusions: Our plaque-centric methylation atlas together with our well-established biobanks, offer the unique opportunity to investigate cfDNA composition in patients spanning various types of cardiovascular disease. We believe this comprehensive methylation framework would help unravel the potential of cfDNA as an innovative non-invasive biomarker for cardiovascular disease.

DNA-RNA hybrid G-quadruplex tends to form near the 3′ end of telomere overhang

Telomeric repeat-containing RNA (TERRA) has been suggested to participate in telomere maintenance. TERRA consisting of UUAGGG repeats is capable of forming an intermolecular G-quadruplex (GQ) with single-stranded TTAGGG-repeat DNA in the telomere 3′ overhang. To explore the structural features and potential functions of this DNA-RNA hybrid GQ (HGQ), we used single-molecule FRET to study the folding patterns of DNA with four to seven telomeric tandem repeats annealed with a short RNA consisting of two or five telomeric repeats. Our data highlight that RNA prefers to form DNA-RNA HGQ near the 3′ end of telomeric DNA. Furthermore, the unfolding of secondary structures by a complementary C-rich sequence was observed for DNA GQ but not for DNA-RNA HGQ, which demonstrated the enhanced stability of the telomere 3′ end via hybridization with RNA. These conformational and physical properties of telomeric DNA-RNA HGQ suggest that TERRA might limit access to the 3′ end of the telomeric DNA overhang, which is known to be critical for the interaction with telomerase and other telomere-associated proteins.

P464: COMPARED TO CLASSICAL CYTOGENETICS OPTICAL GENOME MAPPING (OGM) DETECTS MULTIPLE ADDITIONAL STRUCTURAL CHROMOSOMAL ABERRATIONS IN PEDIATRIC ACUTE MYELOID LEUKEMIA (AML)

Background: AML is the 2nd most frequently diagnosed blood cancer in children affecting approximately 130 patients annually in Germany. Pediatric AML is a heterogeneous malignancy that has multiple genetic aberrations. This is relevant for classification, prognosis and selection of optimal therapy. To detect these alterations established procedures (karyotyping, FISH, RNA-based techniques) are used with well-known limitations (resolution of karyotyping and FISH, respectively; cell cultivation is necessary to generate metaphases; cost-intensiveness; necessity of experienced and intensively trained staff). OGM is an emerging technique addressing these limitations. Based on extraction of ultra-high weight DNA followed by fluorescent labelling at the sequence CTTAAG a barcode-like pattern of the genomic DNA is created. The DNA strands labelled in this way are stretched and drawn as single ultra-long fragments through nanochannels. The results are translated into molecule maps which are compared to the reference genome (resolution down to 500 bp) allowing the identification of genetic alterations. This method combines high resolution with picturing almost whole chromosomes without cell cultivation. Aims: The aim of this study was to test to what extend OGM might supplement classical cytogenetics (CCG) to detect risk defining genetic aberrations at initial diagnosis in pediatric patients with AML. Methods: We analyzed 24 specimen of pediatric AML, MPAL and bilineage leukemia patients by OGM starting from stored frozen material (bone marrow, peripheral blood) obtained at initial diagnosis. Results of OGM were compared with karyotyping and FISH. Primer walking and breakpoint spanning PCR were used to validate newly detected aberrations by OGM in selected cases. Applying breakpoint-spanning PCR, validated aberrations were used as markers for assessment of minimal residual disease (MRD) in selected cases during AML treatment. Results: Overall, we detected discrepant results between CCG and OGM in 17/24 (70%) cases including 9 cases in which karyotyping detected aberrations which were not found by OGM. However, these aberrations were not relevant for risk stratification. In general, focusing on genetic aberrations important for risk stratification the results between CCG and OGM were concordant in 23/24 (96%) cases. In a single case OGM was able to detect a high-risk aberration which was missed by CCG. In total, 33 previously unknown genetic alterations were identified by OGM. Out of these four newly detected variants (2 deletions, 2 translocations) were validated by PCR. The translocations t(2;12) and t(8,12) both affected gene ETV6 on chr. 12 with different fusion partners (chr. 2: AC064875.1; chr. 8: NSMCE2). In both translocations sequences at the breakpoints were determined. Two deletions affecting genes RNF157 on chr. 17 and MEF2B on chr. 19 were validated. These aberrations had not been described before in pediatric AML. In 2 cases - both without a previously identified MRD marker – the newly detected translocations by OGM were used for MRD monitoring. Summary/Conclusion: OGM considerably expands the range of techniques to optimize the diagnosis of pediatric AML and has much potential to omit limitations of classical cytogenetics. Furthermore, the results will contribute to a better understanding of the leukemogenesis of pediatric AML. In addition, OGM offers the possibility to identify new aberrations which can serve as patient specific MRD markers especially in cases without a previously known MRD marker.

Abstract 3738: Monitoring chemotherapy response status of colorectal cancer patients using liquid biopsy samples

Abstract Background and aims: The early detection of colorectal cancer (CRC) recurrence and the monitoring of therapeutic response are crucial steps in the determination and modification of treatment strategies. The currently used gold standard tumor markers such as CEA and CA 19-9 and the different imaging techniques have several limitations in many cases. Therefore, we aimed to establish a liquid biopsy-based approach for tracking tumor dynamics in post-operative non-metastatic (n=32) and metastatic (n=23) CRC patients. Methods: Blood samples were collected from each patient before and during chemotherapy, and finally, patients were classified according to disease outcome. Longitudinal investigations of the total amount, global and local (SFRP2 and SDC2 genes) DNA methylation pattern of cell-free DNA (cfDNA) fraction were performed. The plasma concentration of homocysteine was also determined, as it is one of the main components of the DNA methylation process, and its level defines the methylation potential. We examined how the parameters mentioned above were affected depending on the different therapy responses. Results: The average cfDNA amount was significantly higher (p<0.05) in patients with recurrent cancer (30.4±17.6ng) and progressive disease (PD) (44.3±34.5ng) than individuals who achieved remission (REM) (13.2±10.0ng). More than 10% elevation of cfDNA from first to last sample collection was detected in 92% of PD patients, while reduced cfDNA concentration was observed in 67% of the non-metastatic CRC patients with REM. An effective differentiation was detected between patients achieving remission and showing tumor progression based on cfDNA level with 94% sensitivity and 81% specificity. The average global cfDNA methylation was determined by bisulfite pyrosequencing analysis of long interspersed nuclear element-1 (LINE-1), and it was significantly lower (p<0.05) in the PD group compared to people with remission (71.0±6.7% vs. 78.9±2.0%). Methylation level changes between the study beginning and end indicated a decline (75.5±3.4% vs. 68.2±8.4%) in PD; in contrast, we found a reverse change in remission. The mean relative change of homocysteine concentration revealed an opposite trend with the global DNA methylation suggesting a linkage between these parameters, as it showed an increase (+12.5%) in the case of PD and a decrease (-15.8%) in patients with REM. Regarding local DNA methylation, SFRP2 and SDC2 genes revealed higher promoter hypermethylation in the PD set compared to patients with remission. Conclusion: Our study offers the possibility to monitor the therapeutic response during chemotherapy with a minimally-invasive blood-based method that combines the analysis of cfDNA, its global and local DNA methylation pattern, and homocysteine level. Citation Format: Barbara Kinga Bartak, Tamás Fodor, Alexandra Kalmár, Zsófia Brigitta Nagy, Sára Zsigrai, Krisztina Andrea Szigeti, William Kothalawala, Gábor Valcz, Péter Igaz, István Takács, Magdolna Dank, Béla Molnár. Monitoring chemotherapy response status of colorectal cancer patients using liquid biopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3738.

Abstract 3385: Pre-diagnosis plasma cell-free DNA methylation profiling reveals signatures of cancers up to7 years prior to clinical detection

Abstract Cancer survival rates are significantly improved when detected at early stages, particularly when the tumour is still localised to the tissue of origin. However, effective screening tools for early cancer detection is currently limited to a subset of cancer types. Profiling cell-free DNA (cfDNA) patterns has emerged as a prominent non-invasive biomarker for detection and subtyping of cancers. However, owing to difficulties in observing the early development of human malignancies as cancers are often detected once they become symptomatic, most cancer biomarker and evolution studies to date have primarily examined the genomics from solid tumour or liquid biopsies following a diagnosis. Utilizing cfDNA as a screening tool for early cancer detection requires profiling of blood plasma samples collected from asymptomatic individuals prior to the diagnosis of cancers to enable assessment of the earliest detectability and predictive performance of potential biomarkers. Here, we leverage the Canadian Partnership for Tomorrow’s Health Project (CanPaTH), to profile blood plasma collected prior to the clinical detection of underlying cancers. Specifically, we use cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq), a highly sensitive assay for profiling cfDNA methylomes, to profile over 300 blood plasma samples collected up to seven years prior to the detection of a breast, prostate or pancreatic cancer, in addition to matched controls with no history of cancer free through follow-up. We identified differentially methylated signatures in pre-diagnosis cfDNA that discriminated cancer-free controls from pre-diagnosis cancer cases up to seven years before diagnosis, and demonstrated that these markers were reflective of differentially methylated in cancer tissue relative to normal tissue and peripheral blood leukocytes. Further, predictive modelling reveals that cfDNA methylation markers in blood are predictive of pre-diagnosis breast cancer cases, achieving an average test AUROC of 0.75 (95% CI: 0.70 - 0.80). Predictive models trained solely with pre-diagnosis cfDNA methylation samples were also predictive of prostate and pancreatic cancer samples collected following diagnosis, achieving an average test AUROCs of 0.95 (95% CI: 0.93-0.96) and 0.96 (95% CI: 0.94-0.97) respectively. In our current studies, we focus specifically on breast, prostate and pancreatic cancer cases, and are extending this to further pan-cancer applications in subsequent investigations. Citation Format: Nicholas Cheng, David Soave, Kimberly Skead, Tom Ouellette, Scott Bratman, Daniel De Carvalho, Philip Awadalla. Pre-diagnosis plasma cell-free DNA methylation profiling reveals signatures of cancers up to7 years prior to clinical detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3385.

DNA patterning utilizing mixed monolayer template by phase separation between hydrocarbons and fluorocarbons and evaluation of second-order structural maintenance ability

Adsorption of DNA molecules on mixed phase-separated monolayers composed of a hydrocarbon-containing comb copolymer with an s-triazine ring and a fluorinated s-triazine derivative and their fluorescence emission behavior were investigated. DNA molecules derived from salmon testes were selectively adsorbed only on the diamino-s-triazine ring of the hydrogenated copolymer in the monolayer from the subphase. A mixed monolayer of a hydrocarbon-based comb copolymer and an s-triazine derivative with fluorocarbons exhibited phase separation behavior on water surfaces and indicated expansion behavior, reflecting interactions on the subphase containing DNA molecules. The phase-separated morphology changed depending on the mixing ratio of the hydrogenated copolymer and fluorinated s-triazine derivative. After DNA adsorption, the height of the domain formed by the hydrocarbon chain was selectively increased. In addition, because the DNA molecules adsorbed on the phase-separated template film maintain their fluorescence emission ability, the second-order structure, which is the origin of the emission ability, was evaluated using circular dichroism spectroscopy. The phase-separated, template-adsorbed DNA molecules maintained the same positive Cotton effect as in solution and were predicted to retain exquisite and delicate steric structure, leading to functional occurrence.

Interrelationship amongst varieties of edible mushroom through Molecular marker Study

The information regarding the DNA sequences of plant is very limited. When knowledge of the DNA sequence of the targeted genome is unavailable several PCR based techniques (RAPD, ITS, AFLP, SSR markers, SNP markers) are utilized to accumulate information about the genetic adaptability. Random Amplification of Polymorphic DNA is a technique by which the random segments of DNA are amplified. Short nucleotide primers (8–12 nucleotides) are used to proceed with the PCR using genomic DNA, for fragments to amplify. The primers bind somewhere in the sequence. By comparing the banding matrix the DNA patterns can be ascertained. RAPD has been utilized to characterize, trace the phylogeny of diverse plant species. Internal transcribed spacer (ITS) is to a non-functional RNA situated between structural ribosomal RNAs (rRNA) on a transcript. The sequence of rRNA precursor transcript is the 5' external transcribed sequence (5' ETS), 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA and the 3'ETS read from 5' to 3' direction. The complete sequence repeats themselves in tandem array for thousands of copies separated by regions of non-transcribed DNA termed non-transcribed spacer (NTS) or intergenic spacer (IGS). There is the presence of two ITS regions in eukaryotes. The first one located between 18S and 5.8S rRNA genes is called ITS1, while the next in the sequence is ITS2 that lies between 5.8S and 26S rRNA genes in plants. Each eukaryotic ribosomal cluster follows the sequence of the 5' external transcribed sequence (5' ETS), trailed by the 18S rRNA gene, the ITS1 and the 5.8S rRNA gene, the ITS2, the 28S rRNA gene, and finally the 3' ETS. ETS and ITS pieces are excised and rapidly degraded during rRNA maturation. In our study four different gilled mushrooms were analyzed for RAPD and ITS study.

Heterogeneous hydration patterns of G-quadruplex DNA

G-quadruplexes (GQs) are guanine-rich, non-canonical nucleic acid structures that play fundamental roles in biological processes. Their structure and function are strongly influenced by their hydration shells. Although extensively studied through various experimental and computational methods, hydration patterns near DNA remain under debate due to the chemically and topologically heterogeneous nature of the exposed surface. In this work, we employed all-atom molecular dynamics (MD) simulation to study the hydration patterns of GQ DNA. The Drude oscillator model was used in MD simulation as a computationally efficient method for modeling electronic polarization in DNA ion solutions. Hydration structure was analyzed in terms of radial distribution functions and high-density three-dimensional hydration sites. Analysis of hydration dynamics focused on self-diffusion rates and orientation time correlation at different structural regions of GQ DNA. The results show highly heterogeneous hydration patterns in both structure and dynamics; for example, there are several insular high-density sites in the inner channel, and ‘spine of water’ in the groove. For water inside the loop, anomalous diffusion is present over a long time scale, but for water around the phosphate group and groove, diffusion becomes normal after ∼ 30 ps. These essentially correspond to deeply buried structural water and strong interaction with DNA, respectively.

Association of dynamic changes in serum levels of HBV DNA and risk of hepatocellular carcinoma

Background and aimsWe aimed to examine the risk of HCC associated with the long-term change patterns in HBV DNA levels.MethodsWe conducted a longitudinal study of 6,301 participants with chronic HBV infection (CHB) from October 2012 to June 2019 and measured serum levels of HBV DNA at enrollment and during follow-up. The dynamic change patterns of HBV DNA were identified by group-based trajectory models. The associations between change patterns of HBV DNA and HCC were estimated using Cox regression models.ResultsDuring 35,112 person-years of follow-up, 182 participants developed HCC (518.34 per 105 person-years). Five trajectory groups of repeated measurement of HBV DNA were identified. The risk of HCC was significantly higher for the “high, fast-declined” group whose HBV DNA spontaneously decreased from > 2000 IU/mL at baseline compared with those with persistent undetected HBV DNA (reference group; 963.96 per 105 person-years, HR = 2.62, 95% CI, 1.82 to 3.77, P < 0.001). In addition, the “rebound” group whose HBV DNA level increased from undetectable level to > 20,000 IU/mL from baseline to the end of follow-up also showed an obviously higher cumulative HCC incidence rate (1193.29 per 105 person-years, HR = 4.17, 95% CI, 1.87 to 9.31, P < 0.001). The positive association remained stable after taking the potential effect of time-dependent antiviral treatment into account.ConclusionsSignificant variability in serum levels of HBV DNA presented during long-term follow-up. Regular monitoring of serum levels of HBV DNA and antiviral treatment are required for the clinical management of CHB patients, as well as those with undetected HBV DNA.

Nucleosome footprinting in plasma cell-free DNA for the pre-surgical diagnosis of ovarian cancer

Fragmentation patterns of plasma cell-free DNA (cfDNA) are known to reflect nucleosome positions of cell types contributing to cfDNA. Based on cfDNA fragmentation patterns, the deviation in nucleosome footprints was quantified between diagnosed ovarian cancer patients and healthy individuals. Multinomial modeling was subsequently applied to capture these deviations in a per sample nucleosome footprint score. Validation was performed in 271 cfDNAs pre-surgically collected from women with an adnexal mass. We confirmed that nucleosome scores were elevated in invasive carcinoma patients, but not in patients with benign or borderline disease. Combining nucleosome scores with chromosomal instability scores assessed in the same cfDNA improved prediction of malignancy. Nucleosome scores were, however, more reliable to predict non-high-grade serous ovarian tumors, which are characterized by low chromosomal instability. These data highlight that compared to chromosomal instability, nucleosome footprinting provides a complementary and more generic read-out for pre-surgical diagnosis of invasive disease in women with adnexal masses.

The Microbiome in Osteoarthritis: a Narrative Review of Recent Human and Animal Model Literature.

The microbiome has recently emerged as a powerful contributor to health and illness in chronic, systemic disorders. Furthermore, new microbiome niches beyond traditional gut locations are frequently being described. Over the past 5years, numerous pivotal studies have demonstrated associations between changes in various microbiome niches and the development of osteoarthritis (OA). Herein, we review the most impactful recent literature, including microbiome associations with disease and the potential therapeutic value of microbiome manipulation. The gut microbiome of human OA patients is enriched in specific bacterial clades, most notably Streptococcus, which correlates with OA pain, Firmicutes, and others. Most studies have focused on knee OA, although one publication demonstrated positive associations with 3 gut microbiome clades in hand OA. OA can be easily distinguished from RA by evaluating differences in oral microbiome composition. Most studies have also demonstrated a reduction in richness of the gut microbiome (alpha diversity) associated with OA. Several studies have identified bacterial signatures within human knee and hip cartilage, synovial fluid, and synovial tissue and have described changes in these patterns occurring with the development of OA. In animal models of OA, high-fat diet-induced obesity has been the most well-studied OA risk factor associated with changes in the microbiome, with numerous bacterial clades changed within the gut microbiome and associated with OA. Also in animal models, various oral supplementations, including dietary fiber, probiotics including Lactobacillus species, and cecal microbiome transplantation have all shown improvements in OA histopathology or cartilage healing. Microbiome changes are strongly associated with the OA disease process and with individual OA risk factors related to both the gut microbiome and the microbial DNA patterns in the joint. Microbiome-directed interventions have the potential to prevent or reduce the progression of OA. Future studies should investigate the mechanistic underpinnings of these microbiome associations and further define the therapeutic potential of microbiome augmentation.