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CHO cells are major hosts for the industrial production of therapeutic proteins and their production stability is of considerable economic significance. It is widely known that CHO cells can rapidly acquire genetic alterations, which affects their genetic homogeneity over time. However, the role of non-genetic mechanisms, including epigenetic mechanisms such as DNA methylation, remains poorly understood. We have now used whole-genome bisulfite sequencing to establish single-base methylation maps of eight independent CHO cell lines. Our results identify CpG islands and low-methylated regions as conserved elements with dynamic DNA methylation. Interestingly, methylation patterns were found to cluster clearly along the three main branches of CHO evolution, with no directional changes over short culture periods. Furthermore, multi-ome single-cell sequencing of 9833 nuclei from three independent cultures revealed dynamic subpopulation structures characterized by robust expression differences in pathways related to protein production. Our findings thus provide novel insights into the epigenetic landscape and heterogeneity of CHO cells and support the development of epigenetic biomarkers that trace the emergence of subpopulations in CHO cultures. • We generated the most diverse dataset of CHO methylomes known to date. • We generated the first multi-ome single-cell sequencing dataset of CHO cells. • We identified low-methylated regions as novel dynamically methylated elements. • We identified a novel association between DNA methylation and CHO cell evolution. • We show that epigenetic subpopulations are related to protein production. [ABSTRACT FROM AUTHOR]

Secondary structures are non-canonical arrangements of nucleic acids due to intra-strand interactions, including base pairing, stacking, or other higher-order features that deviate from the standard double-helical conformation. While these structures are extensively studied in RNA, they can also form when DNA becomes single stranded, creating topological roadblocks that can impact essential DNA-based processes such as replication, transcription, and repair, ultimately affecting genome stability. The availability of a complete linear sequence of human genomes, including repetitive loci, enables the prediction of DNA secondary structures comparing across various regions. Here, we evaluate the intrinsic properties of linear single-stranded DNA sequences derived from sampling specialized human loci such as centromeres, pericentromeres, ribosomal DNA (rDNA), and coding regions from the CHM13 genome. Our comparative analysis of predicted secondary structures across human chromosomes revealed the heightened presence, complexity, and instability of secondary structures within the centromere, which gradually decreased toward the pericentromere onto chromosomes' arms, on average lowest in coding regions. Notably, centromeric repeats exhibited the highest level of topological complexity within both the active and divergent domains, even when compared to other repetitive tandem satellites, such as rDNA in acrocentric chromosomes. Our findings provide evidence of the intrinsic self-hybridizing properties of centromere repeats, which are capable of generating complex topological structures that may functionally correlate with chromosome missegregation, especially when centromeric chromatin is disrupted. Processes such as long non-coding RNA transcription, recombination, and other mechanisms that dechromatinize and unwind stretches of linear DNA in these regions create in vivo opportunities for the DNA acrobatics hereby predicted. [Display omitted] DNA-based transactions can unwind the double helix, promoting the formation of secondary structures through intra-strand interactions and non-canonical folding. Our study predicts that human centromere sequences harbor more of these complex and unstable DNA structures than other genomic regions, potentially driving chromosome segregation errors and genomic instability. [ABSTRACT FROM AUTHOR]

Background: Antithrombin (AT) is a serine protease inhibitor which exerts its anticoagulant effect through binding to serine residues in the active centers of procoagulant serine proteases. Its deficiency is associated with increased risk of venous thrombosis. We aim to investigate the pathogenic mechanism of two natural mutants (W221C and M284R) in inherited AT deficiency. Methods: We analyzed 9 unrelated patients with inherited AT deficiency by extracting peripheral blood DNA and sequencing the SERPINC1 gene after amplification by polymerase chain reaction. Enzyme-linked immunosorbent assay and heparin affinity chromatography were used to assess AT secretion and purification efficiency. The mutant AT models were evaluated via computational simulations. Results: Among the 9 patients with inherited AT deficiency, 8 patients had type I AT deficiency, and one patient had type II AT deficiency with subtype of reactive site mutation. Seven of them experienced venous thrombotic events and all patients were found genetic mutations including missense (n = 6), deletion (n = 2) and insertion (n = 1). Two point mutations, W221C and M284R, were identified and were hypothesized to affect AT by destabilizing the central β-sheet. Based on immunoassays and heparin purification, the W221C mutant may impair AT secretion, whereas M284R mutant decreased the total AT production (696.8 ± 151.6 ng/ml versus 3833.72 ± 315.4 ng/ml, p = 0.029). Both mutants delayed the peak of AT release in heparin affinity chromatography. Conclusions: Our study demonstrates that two mutations in SERPINC1 gene altered the production and structure of AT by in vitro protein expression and functional studies, including protein secretion and production. These findings enhance our understanding of the genetic basis of AT deficiency and its possible clinical implications. [ABSTRACT FROM AUTHOR]

Biological mechanisms related to cancer development can leave distinct molecular fingerprints in tumours. By leveraging multi-omics and epidemiological information, we can unveil relationships between carcinogenesis processes that would otherwise remain hidden. Our integrative analysis of DNA methylome, transcriptome, and somatic mutation profiles of kidney tumours linked ageing, epithelial–mesenchymal transition (EMT), and xenobiotic metabolism to kidney carcinogenesis. Ageing process was represented by associations with cellular mitotic clocks such as epiTOC2, SBS1, telomere length, and PBRM1 and SETD2 mutations, which ticked faster as tumours progressed. We identified a relationship between BAP1 driver mutations and the epigenetic upregulation of EMT genes (IL20RB and WT1), correlating with increased tumour immune infiltration, advanced stage, and poorer patient survival. We also observed an interaction between epigenetic silencing of the xenobiotic metabolism gene GSTP1 and tobacco use, suggesting a link to genotoxic effects and impaired xenobiotic metabolism. Our pan-cancer analysis showed these relationships in other tumour types. Our study enhances the understanding of kidney carcinogenesis and its relation to risk factors and progression, with implications for other tumour types. Synopsis: Integrative analysis of multi-omics and epidemiological data implicated ageing, epithelial-mesenchymal transition (EMT), and xenobiotic metabolism as biological mechanisms driving clear cell renal cell carcinoma (ccRCC). Cellular mitotic ageing is a major source of variance between ccRCC tumours, with faster ticking mitotic clocks (epiTOC2, SBS1, and telomere length), genomic instability and PBRM1 and SETD2 mutations related to tumour progression. There is a relationship between BAP1 driver mutations, the epigenetic activation of EMT related genes (IL20RB and WT1), tumour immune infiltration, and worse survival outcomes. Epigenetic silencing of GSTP1, especially in smokers, points to impaired xenobiotic metabolism and increased genotoxic risk in ccRCC tumours. These biological mechanisms were also observed across other cancer types, highlighting broader implications for tumour progression. Integrative analysis of multi-omics and epidemiological data implicated ageing, epithelial-mesenchymal transition (EMT), and xenobiotic metabolism as biological mechanisms driving clear cell renal cell carcinoma (ccRCC). [ABSTRACT FROM AUTHOR]

The BRAF V600E substitution, predominantly found in melanoma and metastatic colorectal cancer (mCRC), is associated with aggressive traits and poor prognosis. Molecular testing of the BRAF mutational status is crucial for guiding therapeutic decisions, driving the necessity for new rapid diagnostic technologies. Here, a novel LAMP‐based assay is presented for fast and selective amplification of mutated DNA, coupled with simple electrochemical detection on electrode biochips. The assay demonstrated high selectivity across cell lines and in 51 cancer patients, detecting mutations in both tumor tissue DNA and circulating tumor DNA (ctDNA) in serum. Clear discrimination between the mutated and wild‐type sequences is achieved, in perfect agreement with either NGS or ddPCR. The bioassay offers a simple and efficient alternative to traditional PCR methods for point‐of‐care diagnostics, particularly in settings with limited equipment and resources. [ABSTRACT FROM AUTHOR]

ABSTRACT Background and Aim Methods Results Conclusions The T cell receptor (TCR) can recognize a vast number of antigens and is closely associated with the pathogenesis of various diseases including autoimmune diseases and malignancies. However, the clinical significance of the TCR repertoire and its post‐treatment changes remain unclear in liver diseases.We performed next‐generation sequencing (NGS)‐based TCR analysis using DNA obtained from peripheral blood mononuclear cells (PBMCs) of healthy donors (HD, n = 5), primary biliary cholangitis (PBC, n = 5), autoimmune hepatitis (AIH, n = 5), and hepatocellular carcinoma (HCC, n = 5) and evaluated the changes after treatment.Baseline TCR repertoire analysis demonstrated that TCR clonotype usage is restricted and diversity is low in all three disease groups (PBC, AIH, and HCC), particularly in PBC and AIH compared to HD (p < 0.05). Following treatment, clonotype usage and diversity did not change significantly, except in AIH, where diversity decreased further (p < 0.05 for clone Shannon diversity and clone evenness). Disease‐specific usage of TCR beta genes and specific changes after therapy were observed in all groups. Analysis of clonotypes shared with other individuals (public clonotypes) revealed that nine public clonotypes in PBC, eight in AIH, and eight in HCC disappeared after treatment. Motif analysis identified one characteristic motif (NQPQH) in PBC.The diversity of the TCR repertoire, TCR beta chain usage, clonotypes, and motifs and their post‐treatment changes are disease‐specific in each liver disease, indicating that further TCR repertoire studies are needed to accelerate the understanding of liver disease pathogenesis from an immunological perspective. [ABSTRACT FROM AUTHOR]

• Essential oil from whole plant of Ocimum viride was studied for cytotoxicity against HL-60 cells. • Cytotoxicity was mediated by inducing apoptosis in the cells as observed by various microscopic and flow cytometry techniques. • Apoptotic pathway involved here was caspase −9 dependent intrinsic pathway. Initiator caspases −9 and −8 directly stimulated caspase-3 and −6, that ultimately leads to apoptotic activities in HL-60 cells. The present study discusses the apoptotic potential of essential oil from whole plant of Ocimum viride (EOV) against human promyelocytic leukemia HL-60 cells. IC 50 value of EOV on HL-60 cells was 0.087, 0.0503, and 0.034 µl/ml respectively at 6, 12, and 24 h. Several parameters were used to access the role of EOV in inducing apoptosis in human HL-60 cancer cells, including nuclear binding fluoroprobes, electron micrographs, DNA laddering, and annexin V-FITC/PI affinity assay. Quantification of BrdU incorporation was done to determine the degree of DNA damage. It was further determined that EOV has an effect on the DNA cell cycle and mitochondria, which was observed in the form of a prominent subdivision of G 0 peaks in the analysis of the DNA cell cycle, as well as a reduction in the mitochondrial membrane potential. A higher level of cytosolic cytochrome-c was associated with these events. Bcl-2 depletion and increase in ROS were also analysed. Both initiator caspases namely caspase-9 and -8 activated caspase-3 which eventually leads to apoptosis. These effects revealed that EOV has apoptosis inducing potential towards HL-60 cells and can be a promising candidate for further anticancer studies. [ABSTRACT FROM AUTHOR]

Specimen-based taxonomic research is essential for understanding diversity and setting management frameworks for threatened mammal faunas, and ancient DNA techniques are increasingly used to extract information from taxonomically relevant historical specimens. The largest survivors of the depleted Caribbean mammal fauna are hutias in the genus Capromys , which is usually interpreted as containing a single species, C. pilorides. Previous studies have demonstrated genetic differentiation of Capromys populations across Cuba, but infrageneric species diversity and nomenclature remain unclear. We conducted ancient DNA analysis of historical Capromys samples using cytochrome b and complete mitogenome data sets, and including the 19th-century holotypes of 2 species now considered synonyms, C. fournieri and C. geayi. Our analyses identify distinct western and central/eastern Capromys clades that diverged 1.75 Mya based upon mitogenome data. These clades are separated by the Havana–Matanzas Channel, which represented a barrier to dispersal throughout the Neogene–Quaternary. Divergence date comparisons with other hutia species provide support for interpreting divergence between Capromys populations as species-level differentiation. Although we were unable to yield amplifiable DNA from the C. fournieri holotype, our analyses confidently assign the C. geayi holotype to the western Capromys clade. We therefore recognize 2 extant Capromys species: C. geayi (western Cuba) and C. pilorides (central/eastern Cuba and Cayman Islands). [ABSTRACT FROM AUTHOR]

PURPOSE: The highly aggressive undifferentiated sarcomatoid carcinoma (USC) subtype of pancreatic ductal adenocarcinoma (PDAC) remains poorly characterized because of its rarity. Previous case reports suggest that immune checkpoint inhibitors could be a promising treatment strategy, but the prevalence of established predictive biomarkers of response is largely unknown. The objective of this study was to leverage comprehensive genomic profiling of USC PDAC tumors to determine the prevalence of biomarkers associated with potential response to targeted therapies. METHODS: USC tumors (n = 20) underwent central pathology review by a board-certified gastrointestinal pathologist to confirm the diagnosis. These samples were compared with non-USC PDAC tumors (N = 5,562). Retrospective analysis of DNA and RNA next-generation sequencing data was performed. RESULTS: USC PDACs were more frequently PD-L1+ by immunohistochemistry than non-USC PDAC (63% v 16%, respectively, P <.001). Furthermore, USC PDAC had an increase in neutrophils (8.99% v 5.55%, P =.005) and dendritic cells (1.08% v 0.00%, q = 0.022) and an increased expression of PDCD1LG2 (4.6% v 1.3%, q = 0.001), PDCD1 (2.0% v 0.8%, q = 0.060), and HAVCR2 (45.9% v 21.7%, q = 0.107) than non-USC PDAC. Similar to non-USC PDAC, KRAS was the most commonly mutated gene (86% v 90%, respectively, P = 1). CONCLUSION: To our knowledge, this work represents the largest molecular analysis of USC tumors to date and showed an increased expression of immune checkpoint genes in USC tumors. These findings provide evidence for further investigation into immune checkpoint inhibitors in USC tumors. @fabermdphd @cancerassassin1 et al report comprehensive analysis of sarcomatoid pancreatic carcinomas. [ABSTRACT FROM AUTHOR]

The article discusses a case series of three children with ATM-mutated medulloblastoma, a common malignant brain tumor in childhood. It explores the genetic and clinical features of medulloblastoma, emphasizing the importance of molecular characterization for prognosis and treatment management. The study highlights the impact of germline ATM variants on cancer risk and radiation-induced toxicity, with one patient experiencing severe radiation-induced brainstem necrosis due to a germline ATM variant. The article underscores the potential of precision oncology in guiding personalized treatment decisions based on genetic predispositions to radiosensitivity. [Extracted from the article]

PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) is typically treated with agents directly or indirectly targeting the androgen receptor (AR) pathway. However, such treatment is limited by resistance mechanisms, including the development of activating mutations in the AR ligand-binding domain (AR -LBD). METHODS: This study evaluated a database of over 15,000 patients with advanced prostate cancer (PC) undergoing comprehensive circulating-tumor DNA analysis (Guardant360, Redwood City, CA) between 2014 and 2021, with associated clinical information from administrative claims (GuardantINFORM database). RESULTS: Of 15,705 patients with PC included, 54% had mCRPC at the time of their blood draw. Of those, 49% had previous treatment with an AR pathway inhibitor (ARPi). AR -LBD mutation prevalence was 15% in patients with mCRPC who were untreated with a next-generation ARPi, 22% in those after one line of ARPi therapy, and 24% in those after two lines of ARPi treatment. Next-generation ARPi treatment yielded an increase in AR L702H and T878A/S mutations after abiraterone, and an increase in AR L702H and F877L mutations after enzalutamide. AR -LBD+ patients demonstrated unique biology, including increased concurrent mutations in the cell-cycle, wingless-related integration site, homologous recombination repair, and phospho-inositide 3-kinase pathways (all P <.0005), and greater low-level (copy number <10) AR amplifications (P =.0041). AR -LBD+ patients exhibited worse overall survival (OS) relative to a matched cohort of AR -LBD– patients (50.1 v 60.7 months, unadjusted log-rank P =.013). CONCLUSION: This large database analysis demonstrates that AR -LBD mutation prevalence increases after next-generation ARPi use. AR -LBD+ tumors demonstrate unique biology (more oncogenic pathway mutations and low-level AR amplification) and reduced OS. These findings inform the development of novel therapies designed to circumvent AR-mediated therapeutic resistance. [ABSTRACT FROM AUTHOR]

This paper reports the first observation of a giant kōkopu (Galaxias argenteus) spawning site in a tidal waterway and illustrates the usefulness of DNA barcoding for opportunistic discoveries that in this case involved the collection of a single egg. We describe key characteristics of the spawning site and compare them with the only other recorded giant kōkopu spawning location which was found in a much different environmental context far inland. This is also a novel observation of giant kōkopu and īnanga (G. maculatus) spawning on the same section of riverbank. Eggs from the two species occupied the same vertical elevation band, which together the consideration of tidal heights and the egg development stage suggest that the spawning event was triggered by a tidal cue. We discuss the implications of more widespread giant kōkopu spawning in tidal waterways, including historical perspectives contributing to their decline and the potential to reverse these trends. [ABSTRACT FROM AUTHOR]

BACKGROUND: Crustaceans of the superfamily Penaeoidea (e.g., shrimps and prawns) are among the most commercially available aquatic products worldwide. However, there are few studies regarding not only the presence but also the characteristics of mislabelling in these food products. Such information would be helpful for consumers in order to avoid the typical problems associated with mislabelling (e.g., health and economic issues). For this reason, this work considers Penaeoidea mislabelling by comparing different products (frozen, fresh, boiled), and sources (hypermarkets, supermarkets and fishmongers) from Spain (Europe). RESULTS: A total of 94 samples from 55 different products were collected, representing 19 different species from 13 genera. Mitochondrial DNA (COI gene) was amplified, revealing mislabelling in almost 30% of supermarket products and almost exclusively found in frozen samples (95% of the total) regardless of its price. In addition, products from the Pacific Ocean seem to be particularly susceptible to mislabelling. CONCLUSIONS: All in all, recommendations for the consumer in order to avoid mislabelling of prawns include purchasing them fresh from fishmongers; aquaculture products must not be avoided. This study represents, to our knowledge, the first attempt to provide recommendations to consumers based on DNA analyses in order to avoid mislabelling in food products. Further research is therefore required to provide such recommendations in different food products, particularly those that are processed, packaged and/or frozen. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Passports are the number one form of individual identification when crossing borders resulting in counterfeit or modified passports being used to allow illegal travel. Here we report on the use of Diamond Dye to detect cells deposited by touch onto parts of the photograph page of a passport. The passport page was divided into a grid of 11 rectangles of approximately equal dimensions. Thumbprints were deposited at 10 of the 11 squares, with the 11th left blank as a negative control. The location and number of deposited cells were determined and recorded. Cellular material was removed from each square separately, using a moistened swab or a tapelift. Tapelifting was better suited for the substrate resulting in an increase in DNA recovered. The highest total quantity of DNA isolated from the 10 samples post extraction was 0.162 ng. Short tandem repeat data were obtained from all 10 samples using the GlobalFilerTM PCR Amplification Kit. The DNA profiles were single source or two-person mixtures and the donor was always the major contributor. These data show the potential to visualize cellular material deposited by touch and then generate STR data from an item such as a passport after only brief physical contact. [ABSTRACT FROM AUTHOR]

DNA samples used in forensic DNA analysis, especially casework evidence samples, are usually collected in diverse and harsh environments and often contain PCR inhibitors. DNA extracted from casework evidence samples is likely to have a low concentration. Additionally, the PCR clean-up process to remove PCR inhibitors can decrease DNA concentration. Therefore, through this study, we aimed to compare the inhibitor tolerance of short tandem repeat (STR) kits and identify their strengths and weaknesses in order to effectively apply and utilize the kits in forensic science. Inhibitor tolerance analysis of STR kits was conducted by measuring the peak height, limit of detection (LOD), and peak balance. The effect of the inhibitor on the peak height variation was determined using the effective concentration 50 (EC50) value. Inhibitor tolerance indicators, such as the LOD and peak balance of each kit, were measured in the presence of four inhibitors, including humic acid, haematin, calcium phosphate, and indigo carmine. No significant difference was observed for the inhibitors for each of the STR kits tested, with the exception of the Investigator® 24plex QS kit which had a significantly higher tolerance for calcium phosphate. [ABSTRACT FROM AUTHOR]

An effective, practical approach for incorporating molecular biology tools to biology and biochemistry students is described. For 6 weeks, students are exposed to bacterial transformation, plasmid DNA purification and analysis, parasite transfection, ectopic expression of fluorescent proteins, fluorescence microscopy, and flow cytometry analysis. This research-oriented laboratory course delves into theoretical and practical concepts of basic techniques in molecular biology to emphasize the logical connections between the obtained results and the resolution of problems that arise daily in a research laboratory, training the students in planning, conducting, discussing, and presenting their own experiments and results. [ABSTRACT FROM AUTHOR]

Homologous blood transfusion (HBT) is used for doping in endurance sports since the 1960s. The blood comes from a compatible donor, that is, someone with a compatible ABO and rhesus blood group. Despite been prohibited by the IOC in 1985, no detection method was available until 2003. Then came the idea to use red blood cells (RBC) minor blood groups antigens that constitute an "identity" card of someone's RBC to detect the presence of a second RBC population. The method validated for doping control samples uses flow cytometry after incubation of isolated RBC with eight to 12 primary antibodies against specific minor blood groups antigens. The presence of double populations of RBC is revealed by a major and a minor peak in a fluorescence histogram. The sensitivity was estimated sufficient to detect HBT for a few weeks. Despite the complexity and cost of the method, right after its application in 2004, several cases of HBT were identified but the number of cases dropped rapidly over the years. In the 2010s, other ways to detect HBT were developed and evaluated: indirect detection using the Athlete Biological Passport approach, and a few years later forensic DNA analysis to establish the presence of two different DNA in a blood sample after HBT. Despite the high specificity of the latter, the sensitivity was recently questioned in vivo. Nowadays, the flow cytometry method remains the method of choice for HBT detection and recent investigations helped to simplify the method and increase its specificity and sensitivity. [ABSTRACT FROM AUTHOR]

The Atlantic sturgeon Acipenser oxyrinchus oxyrinchus is an anadromous species with a wide range along the Atlantic coast of North America. Because of overharvest and a variety of other anthropogenic stressors, the numbers of estuaries that currently host successful natural reproduction and the abundances of extant populations are depressed compared to historic numbers, resulting in its listing under the US Endangered Species Act as five Distinct Population Segments (DPS). Atlantic sturgeon are vulnerable to stressors not only within their natal estuaries but also at distant locales because of the highly migratory behavior of their subadult and adult life stages. In this study, we used our previously derived microsatellite DNA catalogue of 13 reference spawning populations and Individual-Based Assignment testing to determine the origin of 202 adult Atlantic sturgeon that were collected from the lower Altamaha River during spring, several months prior to their fall upriver spawning. We found that approximately one third (37%) of these adults assigned to populations other than the Altamaha, almost all (96%) to other populations within the South Atlantic DPS, a finding consistent with our earlier acoustic telemetry results. These results have management implications, including the likelihood of recolonization of depleted populations in the South Atlantic DPS and the compilation of reference population genetic profiles used in population delineation and mixed-stock analyses. [ABSTRACT FROM AUTHOR]

Diverse, traditional landraces of indica rice with varying pigmentations are grown at the foothills of Eastern Himalayas. Interaction between two rice genes Rc and Rd is the major determining factor for diverse pigmentations. Rd regulates the synthesis of anthocyanidin, whereas Rc encodes a transcription factor for the expression of proanthocyanidins in rice. This study involved 24 genotypes of rice with varying pigmentation, collected from the Himalayan region and Eastern India for appraisal of genetic variability, character association, diversity regarding yield and grain-quality traits, and detection of sequence variation concerning Rc and Rd loci. Results revealed substantial genetic variability with significant genotype × year interaction for most of the traits. The tested genotypes were grouped into nine clusters based on Euclidean distance. Analyses of coding DNA and amino acid sequences revealed non-functional Rc gene with 14-bp deletion without or with a single nucleotide addition in the sixth exon, which led to premature stop codon formation or frameshift mutation in most of the black and white rice on the contrary to red rice genotypes. Furthermore, only a single white rice genotype was detected with a premature stop codon formation in the analyzed region of Rd. That mutation might be the probable cause for obstruction in anthocyanin and proanthocyanidin biosynthesis. Further studies are needed to identify the point mutations and premature stop codon (if any) in the Rd locus of various indica rice genotypes from these regions to unveil the evolutionary relevance of the Rd allele for aberrated dihydroflavonol-4-reductase protein formation. [ABSTRACT FROM AUTHOR]

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to identify risk factors for NSCLP by analyzing polymorphisms in IRF6 rs2013162 and MTHFR A1298C rs1801131 in the Deutero Malay Population in Indonesia. Setting: DNA isolation from venous blood samples was done followed by PCR and PCR-RFLPs method. Patients/Participants: 115 NSCLP subjects and 120 healthy control subjects. Main Outcome Measure(s): The odds ratio (OR) determined to evaluate the risk factors is the main outcome measure. Material and Methods: The study is a case-control design using samples from the venous blood of 115 NSCLP subjects and 120 healthy control subjects. After DNA was extracted, the PCR-RFLPs method was performed using the DdeI restriction enzyme on 100 blood samples of the IRF6 rs2013162 group and Mboll restriction enzyme on 135 blood samples of the MTHFR A1298C rs1801131 group. The Chi-Square test was used with the Exact Fisher alternatives, depending on the expected count value. Results: The results showed that the T mutant allele (OR = 4.125, P <.05) and GT genotype (OR = 21.00, P <.05) of IRF6 rs2013162 and the C mutant allele (OR = 3.781, P <.05), AC genotype (OR = 5, P <.05) and CC genotype (OR = 9,681, P <.05) of the MTHFR A1298C is associated to a greater risk of NSCLP. Conclusions: IRF6 rs2013162 and MTHFR A1298C rs1801131 gene polymorphisms are strongly associated with NSCLP among the Deutero Malay population in the Indonesian population. [ABSTRACT FROM AUTHOR]

Background: Several studies suggest that child maltreatment (CM) is a risk factor for a wide range of adverse biological and psychological outcomes, such as cocaine use disorder (CUD). CM and CUD are independently associated with altered telomere length (TL), but the mechanisms that underpin this relationship remain unknown. The aim of this study was to examine TL changes in cocaine-addicted individuals with and without CM. Methods: This study comprised 29 cocaine-addicted individuals with a DSM-5 diagnosis of CUD and 29 healthy controls. All subjects completed questionnaires to evaluate the presence of CM (CTQ-SF) and their perceived parental care quality (PBI). Based on the responses, the CUD sample was divided into subgroups, while the control group was selected based on the absence of CM. Blood was collected from all participants, DNA was extracted, and TL was measured using quantitative real-time PCR. Results: CUD patients with a history of CM had the shortest TL among subgroups. Further, we observed a linear relationship between "paternal care" levels experienced in childhood and TL. Conclusion: These results support the association between telomere shortening with CM and drug addiction and suggest that greater parental care is a protective factor against stressors that affect TL. [ABSTRACT FROM AUTHOR]

Biotechnological analysis of DNA microarray genes provides valuable insights into the discovery and treatment of diseases such as cancer. It may also be crucial for the prevention and treatment of other genetic diseases. However, due to the large number of features and dimensions in a DNA microarray, the “curse of dimensions” problem is very common. Many machine learning methods require an effective subset of input genes to achieve high accuracy. Unfortunately, extracting features (genes) is an inherently NP-hard problem. Recently, the use of metaheuristics to overcome the NP-hardness of the feature extraction problem has attracted the attention of many researchers. In this paper, we use the combination of fuzzy entropy and Giza Pyramid Construction (GPC) for feature selection. First, redundant features in the microarray dataset are removed using the fuzzy entropy approach. GPC is then used to reduce the execution time. This results in the selection of a near-optimal subset of genes for cancer detection. Dimensionality reduction with GPC followed by classification with Convolutional Neural Network (CNN) creates a synergy to increase efficiency. The proposed method is tested on five well-known cancer patient datasets: leukemia, lymphoma, MLL, ovarian, and SRBCT. The performance of CNN was also measured with four well-known classifiers, including K-nearest neighbor, naïve Bayesian, decision tree, and logistic regression. Our results show that, on average, CNN has the highest accuracy, recall, precision, and F-measure in all datasets. [ABSTRACT FROM AUTHOR]

Liquid biopsies, in particular, analysis of cell-free DNA, are expected to revolutionize the current landscape of cancer diagnostics and treatment. However, the existing methods for cfDNA-based liquid biopsies for cancer have certain limitations, such as fragment interruption and GC bias, which are likely to be resolved by the emerging Oxford Nanopore Technologies (ONT), characterized by long read-length, fast read-times, high throughput, and polymerase chain reaction-free. In this review, we summarized the current literatures regarding the feasibility and applications of cfDNA-based liquid biopsies using ONT for cancer management, a possible game-changer that we believe is promising in detecting multimodal biomarkers and can be applied in a wide range of oncology utilities including early screening, diagnosis, and treatment monitoring. [ABSTRACT FROM AUTHOR]

Background: With the increasing incidence of breast lesions, the differential diagnosis between benign lesions and breast cancer (BCa) has become a big challenge. Host peripheral blood mononuclear cells (PBMCs) could undergo changes in DNA methylation upon disease progression. However, the clinical value of DNA methylation of PBMCs in differentiating benign lesions and BCa is still unclear. Methods: DNA of PBMCs was isolated and the methylation status of PBMCs in patients with BCa and benign breast nodules was detected by using Illumina Infinium methylation EPIC array. The specific methylation targets were validated by pyrosequencing, Targeted Bisulfite Sequencing Assay, and Multiplex Methylation PCR Assay(MMPA). Results: cg26977936, cg23351954, and cg27209741 were validated as differentially methylated and showed the potential diagnostic values (sensitivity and specificity were 90.0%/53.3%, 43.3%/90.0%, 90.0%/43.3%, respectively). Moreover, a diagnostic model was established using these 3 CGs through logistic regression analysis, and the AUC reached 0.837. Next, this diagnostic model was validated in another, independent cohort with Targeted Bisulfite Sequencing Assay, and the clinical value in distinguishing benign and malignant breast disease was also confirmed (AUC = 0.827, P < 0.05). Finally, to better meet the need for the clinical test, we further validated the differential diagnostic efficacy of the 2 hypermethylated DMPs by establishing a Multiplex Methylation PCR Assay by coupling the 5'-flap endonuclease activity of Taq DNA polymerase and molecular beacon reporters. Conclusions: DNA methylation changes in PBMCs showed great potential in discriminating BCa patients from breast benign lesion patients and may serve as a novel predictor in clinical applications. [ABSTRACT FROM AUTHOR]

In the era of transcriptomics, the role of epigenetics in the study of cancers in females has gained increasing recognition. This article explores the impact of epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNA, on cancers in females, including breast, cervical, and ovarian cancers (1). Our findings suggest that these epigenetic markers not only influence tumor onset, progression, and metastasis but also present novel targets for therapeutic intervention. Detailed analyses of DNA methylation patterns have revealed aberrant events in cancer cells, particularly promoter region hypermethylation, which may lead to silencing of tumor suppressor genes. Furthermore, we examined the complex roles of histone modifications and long non-coding RNAs in regulating the expression of cancer-related genes, thereby providing a scientific basis for developing targeted epigenetic therapies. Our research emphasizes the importance of understanding the functions and mechanisms of epigenetics in cancers in females to develop effective treatment strategies. Future therapeutic approaches may include drugs targeting specific epigenetic markers, which could not only improve therapeutic outcomes but also enhance patient survival and quality of life. Through these efforts, we aim to offer new perspectives and hope for the prevention, diagnosis, and treatment of cancers in females. [ABSTRACT FROM AUTHOR]

Background: The underlying pathogenesis of pemphigus vulgaris, an autoimmune skin disorder, remains incompletely understood. An integrative analysis comprising DNA methylation, mRNA expression, and proteomic data in patients with pemphigus vulgaris was conducted to identify potential pathogenic contributors and explore the molecular mechanisms involved in its pathogenesis. Results: The analysis revealed differentially methylated regions (DMRs) in the promoter, exon, intron, and downstream regions in the peripheral blood DNA of patients with pemphigus vulgaris. Associations between methylation levels and both transcriptomic and proteomic profiles revealed that differentially expressed genes between patients with pemphigus vulgaris and healthy controls were primarily linked to biological functions such as platelet activation and coagulation, cellular adhesion, and immunoglobulin binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis highlighted notable pathway abnormalities, including those related to platelet activation, focal adhesions, tight junctions, and infectious inflammatory responses. Notably, genes such as FGA (fibrinogen alpha chain), VWF (von Willebrand factor), and ACTG1 (actin gamma 1) were dysregulated, with a prominent role in platelet activation. Conclusion: The dysregulation of genes such as FGA, VWF, and ACTG1 suggests that alterations in their transcription and expression may contribute to the pathogenesis of pemphigus vulgaris. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Methods Results Conclusions Odontogenic keratocyst (OKC) is a partial manifestation of Gorlin syndrome (GS), resulting from the abnormal activation of the hedgehog signaling pathway. OKC predominantly occurs in young adults and is mostly asymptomatic at the time of initial diagnosis. As OKC is asymptomatic, GS can be challenging to diagnose in certain instances. In this study, we attempted to identify asymptomatic GS from sporadic OKC cases using a previously developed gene panel for GS.Genomic DNA was extracted from patient samples. These DNA samples were analyzed using the AmpliSeq Custom DNA Panel (Illumina), which was specifically designed to target four previously established genes (PTCH1, PTCH2, SMO, and SUFU). Mutations from patients were predicted using tools, such as MutationTaster, CADD, and Polyphen‐2.Thirty‐one patients with OKC were included: 22 sporadic, 9 syndromic, 14 cases with dentigerous cysts, and 3 patients with orthokeratinized odontogenic cysts. One patient with sporadic OKC carried 50% genetic mutation in the cyst and blood, indicative of GS. PTCH1 mutations were found in one of the 14 patients with dentigerous cysts, 3 of the 17 first‐time sporadic cases, and all four recurrent cases. Resected OKC tissues revealed a PTCH1 mutation.We found one patient with GS from those diagnosed with sporadic OKC. Our findings suggest that PTCH1 mutations are associated with postoperative recurrence of OKC, implying that hedgehog‐related gene variations may contribute to jaw cyst development and improve the prognosis of OKC. [ABSTRACT FROM AUTHOR]

Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis, and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)–based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100 pM. The equation describing the standard curve was y = 24.10x + 1594, with R2 = 0.9914. The limit of detection was as low as 4.42 pM for genomic DNA, and a plasmid containing an M. tuberculosis–specific sequence was detected at 5 copy/μL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50 min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology. [ABSTRACT FROM AUTHOR]

Circulating tumor DNA (ctDNA) has shown potential as a non-invasive tumor biomarker in neuroblastoma. Previous studies used generic assays for detection of selected predefined oncogenic variants as markers of ctDNA, which limits the sensitivity and excludes a subset of patients from analysis. Here we assessed patient-specific ctDNA analysis for treatment evaluation and detection of relapse in neuroblastoma. We generated personalized sequencing panels targeting 10 tumor-specific single nucleotide variants (SNVs) for each patient and performed ctDNA analysis of 136 plasma samples collected longitudinally in 13 children with neuroblastoma. ctDNA was detected using ultra-deep next generation sequencing with unique molecular identifiers to eliminate polymerase-induced errors. We found that the levels of ctDNA at diagnosis correlated with risk group and decreased gradually during effective treatment. All samples collected during follow-up in patients without disease relapse were ctDNA-negative. All four relapses were associated with elevated ctDNA levels, and a majority of the analyzed SNVs were detected at time of relapse. In one case, ctDNA became positive 78 days before the relapse was diagnosed with routine assessment and increased by over a thousandfold before the start of additional treatment. Overall, ctDNA was more uniformly elevated at diagnosis, showed less putative false positive results, and was more sensitive for detection of relapse compared to five serum or urine tumor markers used in clinical routine. In conclusion, personalized ctDNA analysis is suitable for clinical monitoring of tumor burden and may be used in all neuroblastoma patients regardless of risk group or tumor genetics. [ABSTRACT FROM AUTHOR]

Background: Sudden unexplained death (SUD) is a devastating event in the young. Despite efforts to identify causal genetic variants, many cases remain unexplained after genetic screening. This study aimed to investigate an alternative potential contributor to SUD by studying the human methylome using the MethylationEPIC v2.0 BeadChip kit in heart tissue from SUD cases. The genome-wide methylation results of the SUD cases were compared to the results of a control cohort. The SUD cases were divided into three main groups based on their autopsy reports, heart morphology and histopathology (primaryN: macroscopically and histologically normal heart; primaryCM: macroscopically or histologically abnormal heart, suspected cardiomyopathies; and secondary: myocardial damage due to other underlying conditions). The main focus of this study was to identify differentially methylated regions (DMRs) between the case groups and the control cohort. Results: We identified DMRs for both the primaryN and primaryCM groups, whereas the secondary group yielded no such results. In the primaryN cases, the corresponding genes for each DMR led to the identification of genes with common biological pathways. Some were associated with heart morphology (e.g. heart outflow tract morphogenesis or trabecular morphogenesis), but the majority belonged to more general cellular regulatory pathways (e.g. transcription coactivator activity, long non-coding RNAs, etc.). Although no common pathways were found for the primaryCM group, some common regulatory molecular functions were identified, such as p53 binding and transcription coactivator activity. Conclusions: Our study is the first to investigate the whole human methylome in heart tissue of SUD cases. We propose that there are observable differences in the methylation patterns of the case groups that may have contributed to SUD. Still, further studies are required to improve our understanding of the impact of methylation levels on SUD risk and to pinpoint methylation-based screening opportunities for SUD relatives. [ABSTRACT FROM AUTHOR]

Background: Cancer-associated fibroblasts (CAFs) are essential components of the tumor microenvironment and play a critical role in cancer progression. Numerous studies have identified significant molecular differences between CAFs and normal tissue-associated fibroblasts (NAFs). In this study, we isolated CAFs and NAFs from liver tumors and conducted a comprehensive analysis of their DNA methylation profiles, integrating our finding with data from studies on other cancer types. Results: Our analysis revealed that several CAF samples exhibited aberrant DNA methylation patterns, which corresponded with altered gene expression levels. Notably, DNA methylation at liver CAF-specific CpG sites was linked to survival outcomes in liver cancer datasets. An integrative analysis using publicly available datasets from various cancer types, including lung, prostate, esophageal, and gastric cancers, uncovered common epigenetic abnormalities across these cancers. Among the consistently altered CpGs were cg09809672 (EDARADD), cg07134930 (HDAC4), and cg05935904 (intergenic). These methylation changes were associated with prognosis across multiple cancer types. Conclusion: The activation of CAFs by the tumor microenvironment seems to be associated with distinct epigenetic modifications. Remarkably, similar genomic regions tend to undergo hypomethylation in CAFs across different studies and cancer types. Our findings suggest that CAF-associated DNA methylation changes hold potential as prognostic biomarkers. However, further research and validation are necessary to develop and apply such signatures in a clinical setting. [ABSTRACT FROM AUTHOR]

Background: Forensic analysis heavily relies on DNA analysis techniques, notably autosomal Single Nucleotide Polymorphisms (SNPs), to expedite the identification of unknown suspects through genomic database searches. However, the uniqueness of an individual's genome sequence designates it as Personal Identifiable Information (PII), subjecting it to stringent privacy regulations that can impede data access and analysis, as well as restrict the parties allowed to handle the data. Homomorphic Encryption (HE) emerges as a promising solution, enabling the execution of complex functions on encrypted data without the need for decryption. HE not only permits the processing of PII as soon as it is collected and encrypted, such as at a crime scene, but also expands the potential for data processing by multiple entities and artificial intelligence services. Methods: This study introduces HE-based privacy-preserving methods for SNP DNA analysis, offering a means to compute kinship scores for a set of genome queries while meticulously preserving data privacy. We present three distinct approaches, including one unsupervised and two supervised methods, all of which demonstrated exceptional performance in the iDASH 2023 Track 1 competition. Results: Our HE-based methods can rapidly predict 400 kinship scores from an encrypted database containing 2000 entries within seconds, capitalizing on advanced technologies like Intel AVX vector extensions, Intel HEXL, and Microsoft SEAL HE libraries. Crucially, all three methods achieve remarkable accuracy levels (ranging from 96% to 100%), as evaluated by the auROC score metric, while maintaining robust 128-bit security. These findings underscore the transformative potential of HE in both safeguarding genomic data privacy and streamlining precise DNA analysis. Conclusions: Results demonstrate that HE-based solutions can be computationally practical to protect genomic privacy during screening of candidate matches for further genealogy analysis in Forensic Genetic Genealogy (FGG). [ABSTRACT FROM AUTHOR]

Summary Tomato is a major global crop. However, its production is limited by Botrytis cinerea. Due to the toxicity of postharvest pesticide application, alternative control methods such as priming are being investigated. Plants were treated with β‐aminobutyric acid (BABA) at two developmental stages and resistance against B. cinerea was tested in fruit tissue and in progenies. DNA methylation and RNA sequencing were conducted to characterise the (epi)genetic changes associated with long‐lasting resistance. Grafting experiments were done to assess the systemic nature of this signal, which was further characterised by small RNA (sRNA) sequencing of scions. Only BABA‐treated seedlings displayed induced resistance (IR). DNA methylation analysis revealed seedling‐specific changes, which occurred in the context of lower basal methylation. BABA‐IR was found to be transmissible from primed rootstock to grafted unprimed scions. In these scions, we identified a subset of mobile 24 nt sRNAs associated with genes showing primed expression during infection in fruit. Our results demonstrate the functional association of a systemic signal with long‐lasting IR and priming. Through integrated omics approaches, we have identified markers of long‐lasting priming in tomato fruit which could also serve as targets for durable resistance in other crops. [ABSTRACT FROM AUTHOR]

AbstractThe potential of neurotransmitters and neural hormones as possible G-quadruplex DNA binders was analyzed using fluorescence spectroscopy, surface-enhanced Raman spectroscopy (SERS), DNA melting analysis, and molecular docking. G-quadruplex sequences, (GGC)3 and G4C2, with roles in Fragile X syndrome and amyotrophic lateral sclerosis (ALS), respectively, were selected, and their interactions with melatonin, serotonin, and gamma-aminobutyric acid (GABA), were studied. Both melatonin and serotonin demonstrated strong interactions with the DNA sequences with hydrogen bonding being the primary mode of interaction, with some non-intercalative interactions involving the π systems. GABA demonstrated much weaker interactions and may not be a suitable candidate as a probe for low concentrations of G-quadruplex DNA. [ABSTRACT FROM AUTHOR]

Ecological surveys of living things based on DNAs from environmental samples are attractive. However, despite simplicity of water sampling from the target environment, it is still necessary to transport the samples to the laboratory for DNA analysis based on skillful next-generation sequencers. To perform DNA-oriented surveys based on a simple protocol without any special training, we demonstrated, in this study, the detection of genes from cell-containing environmental waters using gene sensor arrays that require no DNA labeling and no external indicators. Cell-suspended PBS or river water were used as models of environmental waters containing living things, and DNA samples were prepared by PCR amplification. Ferrocene-terminated probes were synthesized and immobilized on an electrode array to develop a sensor array. The sensor array showed a large response to a target DNA complementary to the probe and no response to a mismatched DNA, indicating sequence-specific detection. For DNA samples prepared from the cells in PBS, they showed good responses similar to those for the target DNA. They also significantly detected DNA samples from the cells in river water at a general environmental concentration (38 cells mL−1) with 28-fold larger responses than those for 0 cells mL−1. [ABSTRACT FROM AUTHOR]

Simple Summary: Genetic methods can currently be used to identify the species of an animal on the basis of the animal itself, its fragments or tissues, or products obtained from it. This study presents typical molecular markers used in species identification. It presents a procedure comparing mtDNA primers used to determine the species of animal from which an item secured at a border crossing (a keychain) was made. Analysis of mitochondrial DNA, specifically the cytochrome b gene (cyt b), has become an essential tool for species identification. In the case of degraded samples, in which DNA is fractionated, universal primers, which are highly effective at amplifying the target region, are necessary. The material analysed in this study was a keychain made of bone, which was secured at a border crossing due to the suspicion that it was made of ivory. Due to processing of the bone and the likelihood of DNA degradation, five pairs of universal primers with different product lengths (from 148 to 990 base pairs) were used for species identification. Fragments of mtDNA from the cyt b and the 12S rRNA and 16S rRNA subunits were analysed. The analysis showed that only one pair of primers (L15601/H15748) enabled identification of the species, which is very common in samples with highly degraded DNA. The material was bone tissue belonging to the species Bos taurus (cattle). Species identification by molecular methods is extremely important in analysis of material when the species cannot be identified on the basis of morphological characteristics. [ABSTRACT FROM AUTHOR]

The cosmopolitan genus Hedysarum L. (Fabaceae) is divided into sections Hedysarum, Stracheya, and Multicaulia. This genus includes many valuable medicinal, melliferous, and forage species. The species taxonomy and genome relationships within the sections are still unclear. We examined intra- and interspecific diversity in the section (sect.) Hedysarum based on repeatome analyses using NGS data, bioinformatic technologies, and chromosome FISH mapping of 35S rDNA, 5S rDNA, and the identified satellite DNA families (satDNAs). A comparison of repeatomes of H. alpinum, H. theinum, and H. flavescens revealed differences in their composition. However, similarity in sequences of most satDNAs indicated a close relationship between genomes within sect. Hedysarum. New effective satDNA chromosomal markers were detected, which is important for karyotype analyses within Hedysarum. Intra- and interspecific variability in the chromosomal distribution patterns of the studied markers were revealed, and species karyograms were constructed. These results provided new insight into the karyotype structures and genomic diversity within sect. Hedysarum, clarified the systematic position of H. sachalinense and H. arcticum, and confirmed the distant genomic relationships between species from sections Hedysarum and Multicaulia. Our findings are important for further comparative genome studies within the genus Hedysarum. [ABSTRACT FROM AUTHOR]

Direct in situ imaging of nucleic acids on biological samples is advantageous for rapid analysis without DNA extraction. However, traditional nucleic acid amplification in aqueous solutions tends to lose spatial information because of the high mobility of molecules. Similar to a cellular matrix, hydrogels with biomimetic 3D nanoconfined spaces can limit the free diffusion of nucleic acids, thereby allowing for ultrafast in situ enzymatic reactions. In this study, hydrogel‐based in situ space‐confined interfacial amplification (iSCIA) is developed for direct imaging of single nucleic acid and single pathogen on biological samples without formaldehyde fixation. With a polyethylene glycol hydrogel coating, nucleic acids on the sample are nanoconfined with restricted movement, while in situ amplification can be successfully performed. As a result, the nucleic acids are lighted‐up on the large‐scale surface in 20 min, with a detection limit as low as 1 copy/10 cm2. Multiplex imaging with a deep learning model is also established to automatically analyze multiple targets. Furthermore, the iSCIA imaging of pathogens on plant leaves and food is successfully used to monitor plant health and food safety. The proposed technique, a rapid and flexible system for in situ imaging, has great potential for food, environmental, and clinical applications. [ABSTRACT FROM AUTHOR]

Diaporthe species cause Diaporthe dieback (DD) in many grape‐growing countries causing general vine decline, shoot dieback and dead spurs, with perennial cankers and internal trunk necrosis. These pathogens are also the causal agents of Phomopsis cane and leaf spot, a common foliar disease affecting grapevine around the world. In this study, we specifically investigated DD affecting nursery vines and commercial vineyards in Uruguay. A collection of 59 Diaporthe isolates obtained from symptomatic and asymptomatic wood samples of commercial grapevines (nine cultivars) and nursery vines (including cuttings of four scion cultivars and two rootstocks, and six scion–rootstock combinations of grafted plants), was characterized based on DNA phylogenetic analysis, phenotypical characteristics and pathogenicity tests. Diaporthe ampelina was the most frequent species (86%), followed by D. foeniculina (9%), D. eres (3%) and D. terebinthifolii (2%). All species were isolated from nursery material, but D. ampelina was the only one found in field vines. The pathogenicity of all species was confirmed, with D. ampelina being the most virulent, causing the longest necrotic lesions on both green shoots and lignified canes. Further studies are needed to comprehend the epidemiology of DD and to better understand its relationship with Phomopsis cane and leaf spot in order to design a management programme to prevent the disease development. [ABSTRACT FROM AUTHOR]

Background: DNA methylation forms 5-methylcytosine and its regulation in the hippocampus is critical for learning and memory. Indeed, dysregulation of DNA methylation is associated with neurological diseases. Alzheimer's disease (AD) is the predominant of dementia and a neurodegenerative disorder. Methods: We examined the learning and memory function in 3- and 9-month-old wild-type and 5xfamiliar Alzheimer's disease (5xFAD) transgenic mice by performing the object recognition memory and Y-maze tests, and identified the hippocampal amyloid beta burden. To investigate the epigenetically regulated genes involved in the development or neuropathology of AD, we performed genome-wide DNA methylation sequencing and RNA sequencing analyses in the hippocampus of 9-month-old wild-type and 5xFAD tg mice. To validate the genes inversely regulated by epigenetics, we confirmed their methylation status and mRNA levels. Results: At 9 months of age, 5xFAD tg mice showed significant cognitive impairment and amyloid-beta plaques in the hippocampus. DNA methylation sequencing identified a total of 13,777 differentially methylated regions, including 4484 of hyper- and 9293 of hypomethylated regions, that are associated with several gene ontology (GO) terms including 'nervous system development' and 'axon guidance'. In RNA sequencing analysis, we confirmed a total of 101 differentially expressed genes, including 52 up- and 49 downregulated genes, associated with GO functions such as 'positive regulation of synaptic transmission, glutamatergic' and 'actin filament organization'. Through further integrated analysis of DNA methylation and RNA sequencing, three epigenetically regulated genes were selected: thymus cell antigen 1, theta (Thy1), myosin VI (Myo6), and filamin A-interacting protein 1-like (Filip1l). The methylation level of Thy1 decreased and its mRNA levels increased, whereas that of Myo6 and Filip1l increased and their mRNA levels decreased. The common functions of these three genes may be associated with the neural cytoskeleton and synaptic plasticity. Conclusions: We suggest that the candidate genes epigenetically play a role in AD-associated neuropathology (i.e., amyloid-beta plaques) and memory deficit by influencing neural structure and synaptic plasticity. Furthermore, counteracting dysregulated epigenetic changes may delay or ameliorate AD onset or symptoms. [ABSTRACT FROM AUTHOR]

The genus Ficus of the family Moraceae, is one of the largest genera of angiosperms, with diverse pharmaceutical applications and biological activities. The traditional approaches based on the morphological traits have been frequently implemented for taxonomical identification of the different taxa of Ficus, however, encompassing these features are quite laborious, due to the dependence of these phenotypic traits on the environmental conditions. So, authenticating the taxonomical identity of the Ficus taxa with molecular barcoding and metabolic profiling, as relatively stable traits, could be a relevant approach for confirming the traditional phenotypic traits of this genus. Nine species of the genus Ficus namely F. amplissima Sm., F. benjamina L. F. binnendijkii, F. drupacea var. pubescens, F. elastica Roxb., F. microcarpa L., F. religiosa L., F. tinctoria subsp. gibbosa and F. virens var. sublancelata in Egypt, were selected for this study. From the anatomical features, three species of subsection Urostigma, F. religiosa, F. virens var. sublanceolata have cystoliths on the abaxial layer, whereas in F. amplissima it was on the adaxial layer. The UPGMA dendrogram of the studied Ficus taxa has been generated from the 21 anatomical characters, categorized the studied taxa into two clusters (I and II) of average distance ~ 3.5, each cluster has been further divided into subclusters I and II. The sub-cluster I includes F. religiosa, F. virens var. sublanceolata and F. tinctoria subsp. gibbosa were grouped together to subsection Urostigma, while the sub-cluster II of the cluster I includes F. benjamina and F. amplissima. From the DNA barcoding analysis, three clusters I, II and III were emerged, the cluster I includes F. benjamina, F. binnendjikee, and F. amplissima. The cluster II, F. virens var. sublanceolata and F. religiosa that belong to subsection Urostigma, while, the cluster III includes F. elastica and F. drupacea var. pubescens, F. microcarpa that belongs to subsection Conosycea. From the metabolic profiling of Ficus species, the major compounds; H-cycloprop-azulen-7-ol, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, 2-(9-octadecenyloxy), pentadecanoic acid, phytol, sitosterol and 9,12-octadecadienoic acid were the common among the taxa, with an obvious fluctuation, that could be a chemotaxonomic markers for these species of Ficus. Based on the metabolic profiling, two distinct clusters I and II were evolved, the cluster I involve F. elastica, F. benjamina, F. drupacea var. pubescens, F. amplissima, while, the cluster II had F. tinctoria subsp. gibbosa and F. religiosa. The fluctuation on the metabolites of the tested Ficus species could be a metabolic fingerprint for each species. So, the delamination of the tested plants based on their anatomical traits was typically matched to the separation based on the ITS sequence analysis. [ABSTRACT FROM AUTHOR]

Viruses play an important role in microbial community structure and biodiversity by lysing host cells, and can also affect host metabolic pathways by expressing auxiliary metabolic genes (AMGs). As a unique low-latitude, high-altitude seasonal plateau wetland in China, Napahai has high research value. However, studies on the genetic diversity of AMGs and viruses associated with DNA biosynthesis have not been reported. Based on metagenomics, with the phylogenetic tree, PCoA, and α diversity analysis, we found that three DNA biosynthesis-related viral AMGs (cobS, mazG, and purM) in the Napahai plateau wetland were rich in genetic diversity, uniqueness, and differences compared with other habitats and host sources. Through the KEGG metabolic pathway and metabolic flow analysis of Pseudomonas mandelii (SW-3) and phage (VSW-3), the AMGs (cobS, mazG, and purM) genes of the three related viruses involved in DNA biosynthesis were upregulated and their expression increased significantly. In general, we systematically described the genetic diversity of AMGs associated with DNA biosynthesis in plateau wetland ecosystems and clarified the contribution of viral AMGs in the Napahai plateau wetland to DNA biosynthesis, as well as the changes of metabolites and genes. It further expands the understanding of phage-host interactions, which is of great significance for further revealing the role of viral AMGs in the biological evolution and biogeochemical cycle of wetland ecosystems. [ABSTRACT FROM AUTHOR]

Introduction: This study investigated the impact of creep-feeding supplementation on the genome methylation of the Longissimus thoracis (LT) muscle in crossbred beef cattle (Bos taurus × Bos indicus). Methods: The experiment involved 48 uncastrated F1 Angus-Nellore males (half-siblings), which were divided into two groups: NCF – no creep-feeding (n = 24) and CF – creep-feeding (n = 24). After weaning at 210 days, all animals were feedlot finished for 180 days under identical conditions. LT muscle biopsies were collected at weaning for genomic DNA methylation analysis by reduced representation bisulfite sequencing (RRBS). Results and discussion: The groups differed significantly (CF > NCF: p < 0.05) to weaning weight (243.57±5.70 vs. 228.92±5.07kg), backfat thickness (12.96±0.86 vs. 10.61±0.42mm), LT muscle marbling score (366.11±12.39 vs. 321.50±13.65), and LT intramuscular fat content (5.80±0.23 vs. 4.95±0.20%). The weights at the beginning of the experiment and at slaughter (390 days) did not differ significantly. Mean methylation levels were higher in CF with 0.18% more CpG, 0.04% CHG, and 0.03% CHH. We identified 974 regions with differential methylation (DMRs: > 25% and q < 0.05), which overlapped with 241 differentially methylated genes (DMGs). Among these genes, 108 were hypermethylated and 133 were hypomethylated in CF group. Notably, 39 of these DMGs were previously identified as differentially expressed genes (DEGs: log2 fold change [0.5]) in the same animal groups. Over-representation analysis highlighted epigenetic regulations related to muscle growth, PPAR signaling, adipogenesis, insulin response, and lipid metabolism. Key DMGs/DEGs included: ACAA1, SORBS1, SMAD3, TRIM63, PRKCA, DNMT3A, RUNX1, NRG3 , and SLC2A8. These epigenetic changes improved the performance of supplemented animals up to weaning and enhanced meat quality traits, particularly higher intramuscular fat. The results provided insights into the intricate interplay between nutrition, epigenetics, gene expression and phenotypes in beef cattle production. [ABSTRACT FROM AUTHOR]

Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate‐limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate <8% at 95% confidence) by manipulating the interplay between the DNA replication biochemistry (via the amplicon GC content) and the microscale circulatory flow inside a PCR tube is reported. Surprisingly, optimal performance depends on selecting primer sequences that replicate high GC content amplicons, contradicting established PCR primer design rules. This innovative thermocycling approach accelerates PCR to speeds rivaling ultra‐fast instruments, enabling rapid, repeatable isothermal DNA analysis across a range of targets relevant to diagnostics and pathogen detection. [ABSTRACT FROM AUTHOR]

Forensic case samples collected in sexual assaults typically contain DNA from multiple sources, which complicates short‐tandem repeat (STR) profiling. These samples are typically sent to a laboratory to separate the DNA from sperm and non‐sperm sources prior to analysis. Here, the automation and miniaturization of these steps using digital microfluidics (DMF) is reported, which may eventually enable processing sexual assault samples outside of the laboratory, at the point of need. When applied to vaginal swab samples collected up to 12 h post‐coitus (PC), the new method identifies single‐source (male) STR profiles. When applied to samples collected 24–72 h PC, the method identifies mixed STR profiles, suggesting room for improvement and/or potential for data deconvolution. In sum, an automated, miniaturized sample pre‐processing method for separating the DNA contained in sexual assault samples is demonstrated. This type of automated processing using DMF, especially when combined with Rapid DNA Analysis, has the potential to be used for processing of sexual assault samples in hospitals, police offices, and other locations outside of the laboratory. [ABSTRACT FROM AUTHOR]

Globally, gastric cancer (GC) ranks among the most prevalent forms of malignancy, posing a significant health burden. Epigenetic modifications, predominantly characterized by alterations in DNA methylation patterns, have been linked to a diverse array of neoplastic processes. Here, we undertake a comprehensive analysis of the DNA methylation signature in GC, with the aim to discover the potential diagnostic epigenetic biomarkers. Utilizing the Illumina 935 K BeadChip, we conducted a genome-wide exploration of DNA methylation patterns in four paired samples of GC tissues and adjacent non-cancerous counterparts. The bisulfite-pyrosequencing (n = 7) was employed to the quantification for methylated gene. The pubic databases including GWAS Catalog, TCGA and GEO were used. The immunohistochemistry and qRT-PCR analysis were performed. In contrast to adjacent tissues, GC tissues manifested pronounced hypermethylation patterns specifically within the promoter cytosine-phosphate-guanine (CpG) islands, indicating localized epigenetic alterations. DNA methylome analysis further revealed 4432 differentially-methylated probes (DMPs), with the gene PRKCB exhibited the most prominent average DNA methylation disparity (mean Δβ = 0.353). Pyrosequencing validation confirmed three DMPs within the PRKCB promoter (cg08406370, cg00735962, and cg18526361). Notably, the mean methylation levels of PRKCB were inversely correlated with mRNA expression levels in the GWAS Catalog. Furthermore, both mRNA and protein expression levels of PRKCB were significantly reduced in GCs when compared to their adjacent non-cancerous counterparts, verified by TCGA and GEO database. Our study reveals significant DNA methylation alterations in GC and emphasizes the pivotal role of PRKCB gene hypermethylation in conferring GC risk, which offers fresh perspectives for advancing diagnostic approaches and therapeutic strategies for GC. [ABSTRACT FROM AUTHOR]

With recent advances, nuclear genome data for phylogenomic analyses can now be sequenced from minuscule quantities of DNA 1 and from specimens that are more than a million years old. 2 DNA analysis from hair is a well-established approach 3 widely used in forensic science 4 and wildlife conservation. 5 Hair samples can be effectively decontaminated 6 and can be used to identify the mammalian species from which the hair was shed. 7,8 We aimed to use advances optimized for degraded DNA to systematically identify dietary prey species from hair compacted in the teeth of two Tsavo lions that lived during the 1890s in Kenya (see description of samples in the STAR Methods and Patterson 9 and Kerbis Peterhans and Gnoske 10 for background on the Tsavo "man-eaters"). Analysis of hair DNA identified giraffe, human, oryx, waterbuck, wildebeest, and zebra as prey and also identified hair that originated from lion. DNA preservation allowed for analyses of complete mitogenome profiles of zebra, giraffe, and lion. Giraffe mitogenomes are phylogeographically partitioned, and we found that the lions ate at least two individuals that belong to a subspecies of Masai giraffe (Giraffa tippelskirchi tippelskirchi) typically found in southeast Kenya. The lion mitogenome from a hair sample was identical to the Tsavo lion endogenous mitogenome and most closely matched other East African lions from Kenya and Tanzania. Our approach enables a better understanding of the hunting behaviors, diets, and ecology of historical individuals, populations, and species and holds promise for extinct populations and species. [Display omitted] • Prey hair in the Tsavo lions' tooth cavities contains complete mitogenomes • Cavities contained hair from giraffe, human, oryx, waterbuck, wildebeest, and zebra • A comparison of hair DNA to endogenous lion DNA indicates self- or allogrooming • At least two giraffes from a subspecies typical of Tsavo were eaten de Flamingh et al. use ancient DNA and bioinformatic methodologies to systematically identify dietary prey species from hair compacted in the teeth of two Tsavo "man-eating" lions that lived during the 1890s in Kenya. Analysis of hair DNA identified giraffe, human, oryx, waterbuck, wildebeest, and zebra as prey and also identified hair from lion. [ABSTRACT FROM AUTHOR]

Background: The heterogeneous and complex genetic landscape of NSCLC impacts the clinical outcomes of patients who will eventually develop resistance to osimertinib. Liquid biopsy (LB) analysis as a minimally invasive approach is a key step to efficiently identify resistance mechanisms and adjust to proper subsequent treatments. Materials and methods: In the present study, we combined plasma-cfDNA and CTC analysis from 30 NSCLC patients in samples collected before treatment and at the progression of disease (PD). We detected molecular alterations at the DNA mutation (EGFR, PIK3CA, KRAS G12C, BRAF V600E), DNA methylation (RASSF1A, BRMS1, FOXA1, SLFN1, SHISA3, RARβ,, WIF-1, RASSF10 and APC), gene expression (CK-19, CK-18, CK-8, AXL, TWIST-1, PD-L1, PIM-1, Vimentin, ALDH-1 , and B2M) and chromosomal level (HER2 and MET amplification) as possible resistance mechanisms and druggable targets. We also studied the expression of PD-L1 in single CTCs using immunofluorescence. Results: In some cases, T790M resistance EGFR mutation was detected at baseline in CTCs but not in the corresponding plasma cfDNA. PIK3CA mutations were detected only in plasma-cfDNA but not in corresponding CTCs. KRAS G12C and BRAF V600E mutations were not detected in the samples analyzed. MET amplification was detected in the CTCs of two patients before treatment whereas HER2 amplification was detected in the CTCs of three patients at baseline and in one patient at PD. DNA methylation analysis revealed low concordance between CTCs and cfDNA, indicating the complementary information obtained through parallel LB analysis. Results from gene expression analysis indicated high rates of vimentin-positive CTCs detected at all time points during osimertinib. Moreover, there was an increased number of NSCLC patients at PD harboring CTCs positive in PD-L1. AXL and PIM-1 expression detected in CTCs during treatment suggesting new possible therapeutic strategies. Discussion: Our results reveal that comprehensive liquid biopsy analysis can efficiently represent the heterogeneous molecular landscape and provide prominent information on subsequent treatments for NSCLC patients at PD since druggable molecular alterations were detected in CTCs. [ABSTRACT FROM AUTHOR]

Objective: High‐risk human papillomaviruses (HPV) are an established cause of oropharyngeal cancer. Their relationship with oral cancer remains unclear with detection ranging from 0% to 100%. HPV DNA detection or evidence of exposure alone is insufficient to conclude causality. This systematic review assesses the extent of bias in studies of HPV detection in cancers of the oral cavity. Methods: PubMed, Ovid MEDLINE, EMBASE, and PsycInfo databases were searched for observational studies reporting the effect of HPV in oral cavity specific cancers. Results: All 15 included studies presented HPV DNA detection or serum HPV‐antibodies, none included mRNA E6/E7 analysis. Cases with oral cancer had 5.36 times (95% CI 3.29–8.72) higher odds of having HPV detected compared to controls. The odds of HPV detection were higher in cell‐based (OR 6.93; 95% CI 0.82–58.55) and tissue samples (OR 5.28; 95% CI 3.41–8.18) than blood‐based samples (OR 3.36; 95% CI 1.53–7.40). Conclusion: When cancer site is clearly differentiated between oropharynx and oral cavity, 12 studies showed strong association between HPV and oral cancer, but the available estimates lack internal validity due to inconsistent measurements, high confounding, and lack of gold standard testing. There is not high‐quality evidence to conclude a causal relationship of HPV with oral cancer. [ABSTRACT FROM AUTHOR]