Editor’s Choice Articles

Background: Walleye (Sander vitreus), a valuable sportfish and an important ecological apex predator, exhibits genetic structuring across their range and localized structuring as a result of stocking. Methods: Walleye from 17 sampling locations across West Virginia were sequenced using a ddRAD protocol, generating various SNP datasets to assess population structuring and genomic diversity, with specific emphasis on the native Eastern Highlands strain. Different minor allele frequency filter thresholds were tested to assess impacts on genetic diversity and differentiation metrics. Results: High genetic differentiation was observed between the Eastern Highlands and Great Lakes strains, with further sub-structuring within the Eastern Highlands strain between the Ohio River populations and the other populations. Increasing MAF thresholds generally reduced the distinctiveness of clusters, but the overall inference of the number of clusters was minimally impacted. Genetic diversity metrics indicated some variability among Eastern Highlands walleye populations, with isolated populations, including the New River and Summersville Lake, showing higher inbreeding coefficients. MAF filters generally increased diversity metrics, but the trend of diversity metrics among populations remained relatively consistent. Several SNPs were found to be potentially undergoing selection, with the minor allele frequencies of these SNPs being found to be highest in Summersville Lake, highlighting potential adaptive divergence between the riverine populations and a large lentic system. Conclusions: The use of any MAF filter generated the same trends of population structuring and genomic diversity inferences regardless of the MAF threshold used. Further management of Eastern Highlands walleye in West Virginia needs to emphasize protecting the genetic integrity of the Kanawha River population and ongoing genomic screening of broodstock to conserve native genetic diversity. Full article

Background/Objectives: Estimating carbon content for cells is often necessary but difficult. In many biological, oceanographic, and marine biogeochemical studies, information on phytoplankton species composition and their biomass contribution to the community is essential. However, it is technically challenging to estimate the biomass of individual species in a natural assemblage. DNA analysis has the potential to profile species composition and estimate species-specific carbon biomass simultaneously. However, this requires an established relationship between carbon biomass and DNA content with species resolution using a measurable DNA index such as rDNA. Methods: In this study, DNA, rDNA, and carbon contents were measured for species from major phytoplankton phyla grown in different growth stages and under different nutrient and temperature conditions. Correlations between these parameters were examined. Results: Our data resulted in significant log-log regression equations: Log C = 0.8165 × Log DNA + 2.407 (R² = 0.9577, p < 0.0001), Log rDNA = 0.7472 × Log DNA − 0.0289 (R² = 0.9456, p < 0.0001), and Log C = 1.09 × Log rDNA + 2.41 (R² = 0.9199, p < 0.0001). Furthermore, similar strong regression functions were found when incorporating previously published data on a wide range of organisms including bacteria, plants, and animals. Conclusions: Carbon biomass is significantly correlated with DNA and rDNA abundances in phytoplankton and other organisms. The regression equations we developed offer a tool for estimating phytoplankton carbon biomass using DNA or rDNA and serve as a foundation for establishing similar models for other organisms. Full article

Background/Objectives: Some G-quadruplex (G4)-forming nucleic acid sequences bind a hemin cofactor to enhance its peroxidase-like activity. This has been implemented in a variety of bioanalytical assays benefiting from analyte-dependent peroxidation of a chromogenic organic substrate (e.g., ABTS) to produce a color change. Adenine and cytosine nucleotides in the vicinity of the G4 hemin-binding site promote the peroxidation reaction. In this work, the effect of G4 loop and flanking nucleotides on the colorimetric signal of split hybridization probes utilizing hemin-G4 signal reporters was tested. Methods: G4s varying by loop sequences and flanking nucleotides were tested with hemin for ABTS peroxidation (A₄₂₀), and the signal was compared with that produced by the most catalytically efficient complexes reported in the literature using one-way ANOVA and post hoc pairwise comparison with Tukey’s HSD test. The best G4s were used as signal transducers in the split peroxidase deoxyribozyme (sPDz) probes for sensing two model nucleic acid analytes, as well as in a cascade system, where the analyte-dependent assembly of an RNA-cleaving deoxyribozyme 10–23 results in G4 release. Results: Intramolecular G4s (G₃T)₃G₃TC or G₃T₃G₃ATTG₃T₃G₃ were found to be the most efficient hemin PDzs. When splitting intramolecular G4 for the purpose of sPDz probe design, the addition of a flanking d(TC) sequence at one of the G4 halves or d(ATT) in a loop connecting the second and third G-tracts helps boost analyte-dependent signal intensity. However, for the cascade system, the effect of d(TC) or d(ATT) in the released G4 was not fully consistent with the data reported for intramolecular G4-hemin complexes. Conclusions: Our findings offer guidance on the design of split hybridization probes utilizing the peroxidase-like activity of G4-hemin complexes as a signal transducer. Full article

Background: Despite increased attention to double-jointedness or joint hypermobility as seen in connective tissue dysplasias like Ehlers–Danlos syndrome, improved clinical DNA correlations are needed to reduce decadal delays in diagnosis. Methods: To this end, patterns of history (among 80) and physical (among 40) findings are compared for 121 Ehlers–Danlos syndrome patients with recurring variants in collagen type I, II, III, V, VI, VII, IX, XI, and XII genes and novel ones in type XV, XVII, XVIII, and XXVII. Results: A recognizable tissue laxity–dysautonomia profile that transcended collagen biochemical class, triple helix component, mutation type, or presence of accessory DNA variants was defined with a few exceptions. Patients with novel variations experienced severe symptoms at younger ages (6–10 versus 14–18 years) and those with collagen type III variations had more than one significant difference in finding frequencies (spinal disk issues 75% versus 49%; bloating-reflux 100% versus 69%; migraines or menorrhagia 92% versus 53%). Conclusions: These results suggest that collagen DNA variants of diverse gene and molecular type can demonstrate EDS disposition and hasten its diagnosis when distress and disease become manifest. Full article

The advancement of modern genomics has led to the large-scale industrial production of molecular data and scientific outcomes. Simultaneously, conventional DNA character alignments (sequence alignments) are utilized for DNA-based phylogenetic analyses without further recoding procedures or any a priori determination of character polarity, contrary to the requirements of foundations of phylogenetic systematics. These factors are the primary reasons why the binary perspective has not been implemented in modern molecular phylogenetics. In this study, we demonstrate how to recode conventional DNA data into various types of binary matrices, either unpolarized or with established polarity. Despite its historical foundation, our analytical approach to DNA sequence data has not been adequately explored since the inception of the molecular age. Binary representations of conventional DNA alignments allow for the analysis of molecular data from a purely comparative or static perspective. Furthermore, we show that the binarization of DNA data possesses broad mathematical and cultural connotations, making them intriguing regardless of their applications to different phylogenetic procedures. Full article

Background: Exon 1 of the gene encoding for the androgen receptor (AR) contains a polymorphic sequence of variably repeated CAG triplets ranging from 11 to 36. The number of triplets appears to inversely correlate with receptor transcriptional activity, conditioning the peripheral effects of testosterone. Methods: We conducted a narrative review to explore the current evidence regarding the relationship between the number of CAG repeats and the human reproductive system. Results: We found several articles that investigate the relationship between CAG polymorphism and the male reproductive system, suggesting a possible modulatory effect on spermatogenesis, sexual function, prostate cancer, and testicular cancer. Similarly, in women, evidence has emerged to support a possible relationship between CAG repeat number and breast cancer, polycystic ovary syndrome (PCOS), and recurrent spontaneous abortions (RSAs). Unfortunately, the data in the current literature are largely discordant, largely due to an important influence of ethnicity on the variability of the CAG polymorphism, and partly due to the quality of the available studies. Conclusions: In the current state of the art, the study of CAG polymorphism does not have a sufficient literature base to allow its use in common clinical practice. However, it represents an interesting research target and, in the future, as new evidence emerges, it could help to elucidate some pathogenetic aspects of human reproductive disorders. Full article

Background: The pyrrolobenzodiazepine (PBD) dimer SJG-136 reached Phase II clinical trials in ovarian cancer and leukaemia in the UK and USA in the 2000s. Several structural analogues of SJG-136 are currently in clinical development as payloads for Antibody-Drug Conjugates (ADCs). There is growing evidence that PBDs exert their pharmacological effects through inhibition of transcription factors (TFs) in addition to arrest at the replication fork, DNA strand breakage, and inhibition of enzymes including endonucleases and RNA polymerases. Hence, PBDs can be used to target specific DNA sequences to inhibit TFs as a novel anticancer therapy. Objective: To explore the ability of SJG-136 to bind to the cognate sequences of transcription factors using a previously described HPLC/MS method, to obtain preliminary mechanistic evidence of its ability to inhibit transcription factors (TF), and to determine its effect on TF-dependent gene expression. Methods: An HPLC/MS method was used to assess the kinetics and thermodynamics of adduct formation between the PBD dimer SJG-136 and the cognate recognition sequence of the TFs NF-κB, EGR-1, AP-1, and STAT3. CD spectroscopy, molecular dynamics simulations, and gene expression analyses were used to rationalize the findings of the HPLC/MS study. Results: Notable differences in the rate and extent of adduct formation were observed with different DNA sequences, which might explain the variations in cytotoxicity of SJG-136 observed across different tumour cell lines. The differences in adduct formation result in variable downregulation of several STAT3-dependent genes in the human colon carcinoma cell line HT-29 and the human breast cancer cell line MDA-MB-231. Conclusions: SJG-136 can disrupt transcription factor-mediated gene expression, which contributes to its exceptional cytotoxicity in addition to the DNA-strand cleavage initiated by its ability to crosslink DNA. Full article

Background/Objectives: Lung cancer ranks as the leading cause of cancer-related deaths globally and is highly associated with cisplatin resistance due to both intrinsic and extrinsic mechanisms. Proliferating Cell Nuclear Antigen (PCNA) plays a critical role in molecular processes, such as DNA replication and repair, chromatin structure maintenance, and cell cycle progression. PCNA is known as a molecular marker for proliferation and an excellent inhibition target to shut down highly proliferative cells. One of the mechanisms of cisplatin resistance is the increase in DNA repair, and studies have reported an association between PCNA, lung cancer, and cisplatin treatment. The present study aimed to characterize the absence of PCNA in A549 lung adenocarcinoma cells. Methods: Employing a CRISPR/Cas9 gene-editing approach, we generated a monoclonal cell culture, termed PKO (PCNA knockout). Results: PKO cells exhibited a residual PCNA expression, significantly decreased clonogenic potential and ubiquitylation at K164 residue. IC50 assay suggested that PKO cells could not acquire cisplatin resistance when compared to PX. After cisplatin treatment, PKO cells presented impaired ubiquitylation and did not have increased STAT3 phosphorylation (Tyr705), a previously characterized mechanism of cisplatin resistance. Conclusions: We suggest that PCNA participates in cisplatin resistance in A549, partially by DNA damage tolerance through failure on PCNA monoubiquitylation, and its inhibition may be an approach to circumvent cisplatin resistance. Full article

Epigenetic modifications affecting DNA, RNA, and proteins can alter the functional state of a gene and heavily interfere with gene expression. These processes are typically transient, and the predominant form of inheritance is mitotic, with a small fraction of transgenerational modifications. It is therefore reasonable to ask what forces drive this acquisition in living beings, where certain variations in phenotype do not correspond to changes in the DNA sequence. Full article

In this research, we induced acute kidney injury (AKI) by ischemia-reperfusion injury (IRI), one of its main causes. Then, we assessed kidney dysfunction by CRE (creatinine)/BUN (serum blood urea nitrogen) levels and histological analysis. Surprisingly, kidney macrophages, initially not expressing MafB and c-Maf, expressed both of them 48 h after bilateral ischemia renal disease (double IRD; dIRD), supporting their possible roles in the disease. We speculated that the M2 macrophages involved in AKI repair might be the source of MafB and c-Maf after injury and that these two transcription factors could have a significant role in the disease. Considering that IL-4/IL-13-induced M2a is the main contributor to AKI recovery and that MafB is upregulated under the effect of these two cytokines combined, we chose to focus on MafB analysis and aimed to examine its potential role in IRD. Previous studies have not examined the role of MafB in ischemic renal disease (IRD). In this study, we demonstrated a significant loss of brush borders, accumulation of intraluminal debris, and extensive damage to the anatomical structure of the MafB^f/f::Lys-Cre mice kidneys compared to their littermates, MafB^f/f, which are considered as a negative control in the entire paper. This was marked by the enlarged tubules, a significant decrease in mature macrophages (F4/80⁺ cells), and, therefore, worsening of the disease in the absence of MafB and delay/failure of the early signs of ischemia recovery. Importantly, these MafB cKO mice presented higher mortality, caused by the abrogation of the intraluminal debris clearance, and died after 48 h from IRD, suggesting the involvement of MafB in the signaling pathway of this pathology. Therefore, we found evidence that MafB attenuates IRD. Full article

Background: Colorectal cancer (CRC) is the third most diagnosed cancer globally and the second leading cause of cancer-related deaths. Despite advancements, metastatic CRC (mCRC) has a five-year survival rate below 20%. Next-generation sequencing (NGS) is recommended nowadays to guide mCRC treatment; however, its clinical utility when compared with traditional molecular testing in mCRC is debated due to limited survival improvement and cost-effectiveness concerns. Methods: This retrospective study included mCRC patients (≥18 years) treated at a single oncology centre who underwent NGS during treatment planning. Tumour samples were analysed using either a 52-gene Oncomine™ Focus Assay or a 500+-gene Oncomine™ Comprehensive Assay Plus. Variants were classified by clinical significance (ESMO ESCAT) and potential benefit (ESMO-MCBS and OncoKBTM). The Mann–Whitney and Chi square tests were used to compare characteristics of different groups, with significance at p < 0.05. Results: Eighty-six metastatic colorectal cancer (mCRC) patients were analysed, all being MMR proficient. Most cases (73.3%) underwent sequencing at diagnosis of metastatic disease, using primary tumour samples (74.4%) and a focused NGS assay (75.6%). A total of 206 somatic variants were detected in 86.0% of patients, 31.1% of which were classified as clinically significant, predominantly KRAS mutations (76.6%), with G12D and G12V variants as the most frequent. Among 33.7% RAS/BRAF wild-type patients, 65.5% received anti-EGFR therapies. Eleven patients (12.8%) had other actionable variants which were ESCAT level I-II, including four identified as TMB-high, four KRAS G12C, two BRAF V600E, and one HER2 amplification. Four received therapies classified as OncoKbTM level 1–2 and ESMO-MCBS score 4, leading to disease control in three cases. Conclusions: NGS enables the detection of rare variants, supports personalised treatments, and expands therapeutic options. As new drugs emerge and genomic data integration improves, NGS is poised to enhance real-world mCRC management. Full article

TRIP13 is a member of the large AAA+ ATPase protein superfamily that plays a crucial role in the precise segregation of chromosomes during mitosis. The abnormal function of TRIP13 has diverse functions, including mitotic processes, DNA repair pathways, and spindle assembly checkpoints, which may contribute to chromosomal instability (CIN). Emerging evidence suggests that the overexpression of TRIP13, observed in many cancers, plays a significant role in drug resistance, autophagy, and immune invasion. Recently, significant advances have been made in identifying TRIP13-associated signaling pathways that have been implicated in cancer progression. Several small molecules that specifically inhibit TRIP13 function and reduce cancer cell growth have been developed. Combination treatments, including TRIP13 inhibitors and other anticancer drugs, have shown promising results. While these findings are promising, TRIP13 inhibitors are awaiting clinical trials. This review discusses recent progress in understanding the oncogenic function of TRIP13 and its possible therapeutic targets, which could be exploited as an attractive option for cancer management. Full article

Background/Objectives: One of the significant challenges in forensic casework is the analysis of samples with degraded or poorly concentrated genetic material. The utilisation of the GlobalFiler™ and Yfiler Plus™ kits has unquestionably enhanced the efficacy of genetic profiling in challenging samples, facilitating the analysis of alleles that were previously undetectable with alternative kits. Therefore, the main objective of this work was to verify the efficiency of these kits in analysing forensic samples, progressively reducing the amplification volumes. To further optimise genetic profiling, it was essential not only to assess the behaviour of the alleles but also to prevent allelic loss. Methods: A series of reaction volume reduction studies were conducted, evaluating the performance of genetic profiles in both controlled samples (positive controls) and low template DNA samples (0.01 ng/µL). Results: The results demonstrate that it is effective to obtain complete genetic profiles from the amplification of optimal samples in reduced volumes of 12, 6 or 3 µL with GlobalFiler™ and Yfiler™ Plus. Conclusions: The limiting factor in obtaining complete genetic profiles is the amount of DNA available, rather than the amplification volume. Furthermore, reducing the amplification volume from DNA extracts of low template DNA samples proportionally increases the number of allelic dropouts. Full article

Background/Objectives: Culex species mosquitoes are globally distributed and transmit several pathogens that impact animal and public health, including West Nile virus, Usutu virus, and Plasmodium relictum. Despite their relevance, Culex species are less widely studied than Aedes and Anopheles mosquitoes. To expand the genetic tools used to study Culex mosquitoes, we previously developed an optimized plasmid for transient Cas9 and single-guide RNA (sgRNA) expression in Culex quinquefasciatus cells to generate gene knockouts. Here, we established a monoclonal cell line that consistently expresses Cas9 and can be used for screens to determine gene function or antiviral activity. Methods: We used this system to perform the successful gene editing of seven genes and subsequent testing for potential antiviral effects, using a simple single-guide RNA (sgRNA) transfection and subsequent virus infection. Results: We were able to show antiviral effects for the Cx. quinquefasciatus genes dicer-2, argonaute-2b, vago, piwi5, piwi6a, and cullin4a. In comparison to the RNAi-mediated gene silencing of dicer-2, argonaute-2b, and piwi5, our Cas9/sgRNA approach showed an enhanced ability to detect antiviral effects. Conclusions: We propose that this cell line offers a new tool for studying gene function in Cx. quinquefasciatus mosquitoes that avoids the use of RNAi. This short study also serves as a proof-of-concept for future gene knock-ins in these cells. Our cell line expands the molecular resources available for vector competence research and will support the design of future research strategies to reduce the transmission of mosquito-borne diseases. Full article

Background: Rice, one of the main foods in Brazil and the world, is sensitive to chilling (0–15 °C), especially in the germination and reproductive stages. Chilling causes delayed germination and affects coleoptile elongation at the S3 stage (needlepoint), causing poor plant establishment, stunted growth, and non-vigorous plants, also impacting weed management. Elucidating the mechanisms responsible for resilience under cold conditions helps the development of tolerant cultivars. Transcription factors (TFs) act in stress response signaling, making them indispensable in the tolerance mechanism. Objective: Thus, this study aimed to identify and characterize the expression profile of transcription factors in the response to chilling stress in rice at the germination stage. Methods: To determine the transcriptional profile of 2408 genes belonging to 56 TF families, RNAseq was performed on the shoot tissue of seedlings of Oro (chilling-tolerant) and Tio Taka (chilling-sensitive) genotypes grown under control conditions (25 °C) and chilling stress (13 °C) until the S3 stage. Results: Of the total genes analyzed, 22% showed significant differential expression in the analyzed cultivars. There were 117 genes that showed significant differential expression in the tolerant cultivar, 60 of which were downregulated and 57 upregulated. In the sensitive cultivar, 248 genes had a significant differential expression, of which 98 genes were downregulated and 150 genes were upregulated. A total of 170 genes encoding TFs were commonly and significantly differentially expressed in the tolerant and sensitive genotypes. Conclusions: Here, we revealed potential new targets involved in the regulation of chilling stress in rice at the S3 stage. Full article

The detection of viral DNA is considered crucial in both diagnosis and prognosis. Nowadays, molecular diagnostic approaches represent the most promising tools for the clinical detection of viral infections. This review aims to investigate the most used and promising DNA-based technologies for viral detection, focusing on herpesviruses because of their ability to undergo latent and reactivation cycles, persisting lifelong in the host in association with several diseases. Molecular technologies, such as PCR-based assays, enhance sensitivity and specificity in identifying viral DNA from clinical samples such as blood, cerebrospinal fluid and saliva, indicating PCR and its derivatives as the gold standard methods for herpesvirus detection. In conclusion, this review underscores the need for continuous innovation in diagnostic methodologies to address the complexities of herpesvirus identification in different clinical samples. Full article

Cellular senescence is a response to endogenous and exogenous stresses, including telomere dysfunction, oncogene activation, and persistent DNA damage. In particular, radiation damage induces oxidative base damage and bond breaking in the DNA double-helix structure, which are treated by dedicated enzymatic repair pathways. In this review, we discuss the correlation between senescence and the accumulation of non-repaired single-strand breaks, as can occur during radiation therapy treatments. Recent in vitro cell irradiation experiments using high-energy photons have shown that single-strand breaks may be preferentially produced at the borders of the irradiated region, inducing senescence in competition with the apoptosis end-point typically induced by double-strand breaks. Such a particular response to radiation damage has been proposed as a possible cause of radiation-induced second primary cancer, as cells with an accumulation of non-repaired single-strand breaks might evade the senescent state at much later times. In addition, we highlight the peculiarities of strand-break repair pathways in relation to the base-excision pathway that repairs several different DNA oxidation defects. Full article

Background/Objectives: This study explores the genome sequencing data from the infection of Pseudomonas fluorescens UFV 041 by the bacteriophage Pijolavirus UFJF_PfSW6, aiming to identify and characterize prophages induced in the host bacterium during the infection. Methods: Scaffolds from sequencing data were analyzed, and reads were mapped to identify potential prophages using phage-to-host coverage metrics. The putative prophage scaffold was annotated, taxonomically classified, and its integration in the host bacterium was verified by PCR amplification of two target genes. We also tested whether mitomycin treatment could induce the prophage to enter the lytic cycle. Results: The prophage UFJF_PfPro was identified with a high phage-to-host coverage ratio. Its genome is 32,700 bp in length, containing 42 genes, 3 terminators, and 11 promoters, with 98.84% completeness. PCR confirmed its integration into P. fluorescens UFV 041, but mitomycin treatment did not induce the lytic cycle. The UFJF_PfPro genome shares 38.60% similarity with the closest lytic phages in the Phitrevirus genus, below genus and species assignment thresholds. A viral proteomic tree clustered UFJF_PfPro with Phitrevirus in a clade representing the Peduoviridae family. Conclusions: The UFJF_PfPro is a prophage integrated into the P. fluorescens UFV 041 genome, but we were unable to induce it to enter the lytic cycle using mitomycin treatment. The genome of UFJF_PfPro encodes all structural proteins typical of the Caudoviricetes class and shares low genomic similarity with species of the genus Phitrevirus, suggesting that UFJF_PfPro represents a new genus and species within the Peduoviridae family. Full article

The purpose of this study is to improve the identification of Culicidae species from the Vale Ribeira region, São Paulo state, Brazil. Adults were collected in the municipalities of Cananeia and Pariquera-Açu and morphologically identified. Molecular analyses were performed on sequences of COI barcode and a fragment of the D2 expansion of the 28S ribosomal RNA gene generated from field collected mosquitoes. The analyses included species delimitation, phylogeny, and interspecific genetic distances using the Kimura 2-parameter model. Species included in the analyses were Aedes perventor, Aedes scapularis, Aedes serratus/Aedes nubilus, Aedes serratus s.s., Aedes terrens, Haemagogus capricornii, Haemagogus leucocelaenus, Haemagogus janthinomys, Kerteszia bellatrix, Kerteszia cruzii, Psorophora ferox, Psorophora forceps, Sabethes conditus, and Wyeomyia confusa. COI sequences from specimens collected at other localities were included in the analysis for comparison. Results of barcode RESL analysis showed that specimens of Ps. ferox and Hg. janthinomys split into three clusters for each species. Similarly, sequences of Ke. bellatrix and Ke. cruzii were recovered in two groups for each species. Distinct from other species included in analyses, Ps. ferox and Ps. forceps shared 100% similarity in the D2 fragment sequenced. Overall, the analysis of COI barcode sequences revealed the following key findings: (1) the presence of subclades within Hg. janthinomys, with its division into three groups suggests that this species may represent a species complex; (2) Ke. bellatrix from the Atlantic tropical rainforest shares 95.59% sequence similarity with a specimen from the type locality, indicating that specimens from Southeastern Brazil may belong to an unidentified species within the Ke. bellatrix complex; (3) Ke. cruzii also represents a species complex; and (4) D2 sequences successfully identified most species studied, apart from Ps. forceps and Ps. ferox. Full article

Background/Objectives: DAPK-1 plays a crucial role among molecules that may be affected by DNA hypermethylation. The aim of this study is to investigate the DNA methylation of DAPK-1 gene in oral potentially malignant disorders (OPMDs) and oral squamous cell carcinoma (OSCC) compared to normal oral epithelium and to evaluate the possible role of methylated DAPK-1 as an indicator of the early onset of malignant transformation of oral potentially malignant disorders. Methods: The paraffin embedded tissue samples were retrieved from the archives of the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece and St Lukas Hospital of Thessaloniki, Greece during the period of 2014–2019. The tissue samples included 83 OPMDs samples, 39 OSCC samples and 12 samples of normal oral epithelium. The PCR process followed, targeting four different DAPK-1 gene primers. Results: Regarding OSCC, it was found that all 39 OSCCs samples were methylated in DAPK-1 promoter region, whereas only 2 out of 12 normal tissues samples showed DAPK-1 promoter hypermethylation (p < 0.001 Fisher’s exact test). A total of 17 out of 83 OPMDs were DAPK-1 methylated (five erosive oral lichen planus samples, three non-dysplastic oral leukoplakias, eight mildly dysplastic oral leukoplakias and one sample belonging to the group of moderately and severely dysplastic oral leukoplakia). Conclusions: Since epigenetic changes occur early in carcinogenesis and are potentially reversible, they could be used as disease biomarkers for diagnosis, prognosis and prediction, as well as therapeutic targets. DAPK-1 methylation is mostly present in the early stages of dysplasia as well as in all cases of oral cancer. Full article

Living systems are capable on the one hand of eliciting a coordinated response to changing environments (also known as adaptation), and on the other hand, they are capable of reproducing themselves. Notably, adaptation to environmental change requires the monitoring of the surroundings, while reproduction requires monitoring oneself. These two tasks appear separate and make use of different sources of information. Yet, both the process of adaptation as well as that of reproduction are inextricably coupled to alterations in genomic DNA expression, while a cell behaves as an indivisible unity in which apparently independent processes and mechanisms are both integrated and coordinated. We argue that at the most basic level, this integration is enabled by the unique property of the DNA to act as a double coding device harboring two logically distinct types of information. We review biological systems of different complexities and infer that the inter-conversion of these two distinct types of DNA information represents a fundamental self-referential device underlying both systemic integration and coordinated adaptive responses. Full article

Separation of duplex strands of nucleic acids is a vital process in the nucleic acid metabolism and survival of all living organisms. Helicases are defined as enzymes that are intended to unwind the double-stranded nucleic acids. Helicases play a prominent role in the cold adaptation of plants and bacteria. Cold stress can increase double-strand DNA breaks, generate reactive oxygen species, cause DNA methylation, and stabilize the secondary structure of RNA molecules. In this review, we discuss how helicases play important roles in adaptive responses to cellular stress caused by low temperature conditions, particularly in bacteria and plants. We also provide a glimpse of the eminence of helicase function over nuclease when an enzyme has both helicase and nuclease functions. Full article

Genetic engineering has revolutionized our ability to modify microorganisms for various applications in agriculture, medicine, and industry. This review examines recent advances in genetic engineering techniques for bacteria, fungi, and oomycetes, with a focus on CRISPR-Cas systems. In bacteria, CRISPR-Cas9 has enabled precise genome editing, enhancing applications in antibiotic production and metabolic engineering. For fungi, despite challenges associated with their complex cell structures, CRISPR/Cas9 has advanced the production of enzymes and secondary metabolites. In oomycetes, significant plant pathogens, modified Agrobacterium-mediated transformation, and CRISPR/Cas12a have contributed to developing disease-resistant crops. This review provides a comparative analysis of genetic engineering efficiencies across these microorganisms and addresses ethical and regulatory considerations. Future research directions include refining genetic tools to improve efficiency and expand applicability in non-model organisms. This comprehensive overview highlights the transformative potential of genetic engineering in microbiology and its implications for addressing global challenges in agriculture, medicine, and biotechnology. Full article

Objective: The study aimed to optimize protocols for the joint extraction of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from 0.025 × 10⁶ CFU of Candida albicans, targeting to overcome the challenges in the extraction of these genetic materials. Materials and methods: From this, treated silicon carbide (SiC) granules were added to fungal samples from methods 1, 2, and 3 obtained from aliquots of BHI or Sabouraud medium to cause cell lysis and enable the isolation of these macromolecules by phenol and chloroform. The concentration and integrity of the extracted nucleic acids were analyzed, respectively, by spectrophotometry using the A₂₆₀/A₂₈₀ ratios and 1% agarose gel electrophoresis. Results: Therefore, method 3 is the one that most comprises samples considered pure of both DNA and RNA, simultaneously. Furthermore, the presence of intact RNAs corresponding to the base pair size such as 5.8 S rRNA and tRNA was verified during electrophoresis, considering the particularities of RNA, which makes it very unstable and easily degraded. Conclusions: Thus, it results in a faster and simpler method in addition to obtain promising results using minimal amounts of biological sample and offering a valuable alternative for small laboratories to work with molecular biology. Full article

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

Background: Freeze-tolerant animals undergo significant physiological and biochemical changes to overcome challenges associated with prolonged whole-body freezing. In wood frog Rana sylvatica (now Lithobates sylvaticus), up to 65% of total body water freezes in extracellular ice masses and, during this state of suspended animation, it is completely immobile and displays no detectable brain, heart, or respirometry activity. To survive such extensive freezing, frogs integrate various regulatory mechanisms to ensure quick and smooth transitions into or out of this hypometabolic state. One such rapid and reversible regulatory molecule capable of coordinating many aspects of biological functions is microRNA. Herein, we present a large-scale analysis of the biogenesis and regulation of microRNAs in wood frog liver over the course of a freeze–thaw cycle (control, 24 h frozen, and 8 h thawed). Methods/Results: Immunoblotting of key microRNA biogenesis factors showed an upregulation and enhancement of microRNA processing capacity during freezing and thawing. This was followed with RT-qPCR analysis of 109 microRNA species, of which 20 were significantly differentially expressed during freezing and thawing, with the majority being upregulated. Downstream bioinformatics analysis of miRNA/mRNA targeting coupled with in silico protein–protein interactions and functional clustering of biological processes suggested that these microRNAs were suppressing pro-growth functions, including DNA replication, mRNA processing and splicing, protein translation and turnover, and carbohydrate metabolism. Conclusions: Our findings suggest that this enhanced miRNA maturation capacity might be one key factor in the vital hepatic miRNA-mediated suppression of energy-expensive processes needed for long-term survival in a frozen state. Full article

Background/Objectives: Telomeres consist of repetitive nucleotide sequences and associated proteins that safeguard chromosome ends from degradation and fusion with neighboring chromosomes. As cells divide, telomeres shorten due to the end-replication problem and oxidative stress, ultimately contributing to cellular senescence. Telomeres therefore play a role in cellular health and aging. Measuring telomere length has emerged as a significant biomarker in various fields of research, including aging, cancer, and chronic diseases. Accurate measurement of telomere length is critical for interpreting research findings and clinical applications. Variability in measurement techniques can lead to inconsistent results, underscoring the need for standardized protocols. Methods and Results: The Telomere Research Network (TRN), an initiative from the National Institute of Aging and National Institute of Environmental Health Sciences, has established recommended guidelines to standardize the measurement of telomere length using qPCR to ensure accuracy and reproducibility in population-based studies. The monochrome multiplex quantitative PCR (MMqPCR) assay has emerged as a robust method endorsed by the TRN for its accuracy and reproducibility in quantifying telomere length in epidemiology ad population based studies. The absolute telomere length (aTL) qPCR assay is currently being evaluated by the TRN for its capability to utilize an oligomer standard, enabling the generation of absolute telomere lengths. The oligomer feature facilitates a more direct comparison of results across experiments and laboratories. Conclusions: This paper outlines a novel dual-labeled multiplex aTL method by incorporating dual-labeled multiplex probes to measure average absolute telomere length, providing a clear advantage over the relative telomere length assay, which quantifies the ratio of telomeric repeats to single-copy gene numbers. Full article

Melanoma is a highly aggressive type of skin cancer. Metastatic melanoma tumors have historically featured a particularly poor prognosis and have often been considered incurable. Recent advances in targeted therapeutic interventions have radically changed the landscape in metastatic melanoma management, significantly increasing the overall survival of patients. Hyperactive BRAF is the most common mutational event found in metastatic melanoma and its inhibition has proven to be a successful approach in a number of patients. Unfortunately, initial tumor retreat is followed by relapse in most cases, highlighting the elusiveness of finding a widely effective treatment. Melanoma tumors often carry a particularly high number of mutations in what is known as a high level of inter- and intra-patient tumor heterogeneity, driving resistance to treatment. The various mutations that are present in these tumors, in addition to impacting the root cause of the malignancy and the potential for therapeutic interventions, have also been known to arise during tumor clonal evolution leading to the establishment of drug resistance, a major issue in melanoma management. Full article

Diffuse large B cell lymphoma, not otherwise specified (DLBCL, NOS) is the most common type of non-Hodgkin lymphoma (NHL). Significant efforts have been focused on utilizing advanced genomic technologies to further subclassify DLBCL, NOS into clinically relevant subtypes. These efforts have led to the implementation of novel algorithms to support optimal risk-oriented therapy and improvement in the overall survival of DLBCL patients. The pathogenesis of DLBCL at the molecular level indicates copy number variation (CNV) as one of the major forms of genetic alterations in the somatic mutational landscape. Random deregulation that results in complex breaks of chromosomes and restructuring of shattered chromosomal segments is called chromothripsis. Gene expression changes influenced by chromothripsis have been reported in cancer and congenital diseases. This chaotic phenomenon results in complex CNV, gene fusions, and amplification and loss of tumor suppressor genes. We present herein a summary of the most clinically relevant genomic aberrations, with particular focus on copy number aberrations in a case that highlights DLBCL, NOS arising from relapsed Hodgkin lymphoma. The focus of our study was to understand the relationship between the clinical, morphological, and genomic abnormalities in DLBCL, NOS through multiple techniques for therapeutic considerations. Full article

Oxidative stress-mediated biomolecular damage is a characteristic feature of ionizing radiation (IR) injury, leading to genomic instability and chronic health implications. Specifically, a dose- and linear energy transfer (LET)-dependent persistent increase in oxidative DNA damage has been reported in many tissues and biofluids months after IR exposure. Contrary to low-LET photon radiation, high-LET IR exposure is known to cause significantly higher accumulations of DNA damage, even at sublethal doses, compared to low-LET IR. High-LET IR is prevalent in the deep space environment (i.e., beyond Earth’s magnetosphere), and its exposure could potentially impair astronauts’ health. Therefore, the development of biomarkers to assess and monitor the levels of oxidative DNA damage can aid in the early detection of health risks and would also allow timely intervention. Among the recognized biomarkers of oxidative DNA damage, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OxodG) has emerged as a promising candidate, indicative of chronic oxidative stress. It has been reported to exhibit differing levels following equivalent doses of low- and high-LET IR. This review discusses 8-OxodG as a potential biomarker of high-LET radiation-induced chronic stress, with special emphasis on its potential sources, formation, repair mechanisms, and detection methods. Furthermore, this review addresses the pathobiological implications of high-LET IR exposure and its association with 8-OxodG. Understanding the association between high-LET IR exposure-induced chronic oxidative stress, systemic levels of 8-OxodG, and their potential health risks can provide a framework for developing a comprehensive health monitoring biomarker system to safeguard the well-being of astronauts during space missions and optimize long-term health outcomes. Full article

Mammalian cell lines devoid of mitochondrial DNA (mtDNA) are indispensable in studies aimed at elucidating the contribution of mtDNA to various cellular processes or interactions between nuclear and mitochondrial genomes. However, the repertoire of tools for generating such cells (also known as rho-0 or ρ⁰ cells) remains limited, and approaches remain time- and labor-intensive, ultimately limiting their availability. Ethidium bromide (EtBr), which is most commonly used to induce mtDNA loss in mammalian cells, is cytostatic and mutagenic as it affects both nuclear and mitochondrial genomes. Therefore, there is growing interest in new tools for generating ρ⁰ cell lines. Here, we examined the utility of 2′,3′-dideoxycytidine (ddC, zalcitabine) alone or in combination with EtBr for generating ρ⁰ cell lines of mouse and human origin as well as inducing the ρ⁰ state in mouse/human somatic cell hybrids. We report that ddC is superior to EtBr in both immortalized mouse fibroblasts and human 143B cells. Also, unlike EtBr, ddC exhibits no cytostatic effects at the highest concentration tested (200 μM), making it more suitable for general use. We conclude that ddC is a promising new tool for generating mammalian ρ⁰ cell lines. Full article

Children born from pregnancy complications are at higher risk of chronic diseases in adulthood. Identifying which children born from a complicated pregnancy are likely to suffer from later chronic disease is important in order to intervene to prevent or delay the onset of disease. This study examined the associations between the major pregnancy complications (gestational diabetes, high blood pressure, small- and large for gestational age, and preterm birth) and child telomere length, a biomarker of chronic disease risk. This was a population-based longitudinal analysis using data from the Longitudinal Study of Australian Children. The primary outcome is telomere length, measured in 11–12-year-old children. Multivariable linear regression was used to estimate the association between pregnancy complications and child telomere length, adjusting for a range of a priori confounders. Data from 841 families were used. One in four pregnancies (27.1%) featured a pregnancy complication. In the adjusted analysis, there was no association between pregnancy complications and child telomere length (high blood pressure: mean difference (95% CI): 0.00 (−0.12, 0.12); gestational diabetes (0.05 (−0.10, 0.19)); small for gestational age (0.07 (−0.04, 0.19)); large for gestational age (−0.06 (−0.15, 0.03)); and preterm birth (−0.10 (−0.21, 0.01)). Our results do not support the notion that telomere length is shorter in children born to mothers after a pregnancy complication. Methodological considerations should be rigorous to improve the reproducibility of findings. Full article

DNA knots are deleterious for living cells if not removed. Several theoretical and simulation approaches address the question of how topoisomerases select the intermolecular passages that preferentially lead to unknotting rather than to the knotting of randomly fluctuating DNA molecules, but the formation of knots in vivo remains poorly understood. DNA knots form in vivo in non-replicating and replicating molecules, and supercoiling as well as intertwining are thought to play a crucial role in both the formation and resolution of DNA knots by topoisomerase IV. To confirm this idea, we used two-dimensional agarose gel electrophoresis run with different concentrations of chloroquine to demonstrate that non-replicating pBR322 plasmids grown in a topoisomerase I-defective E. coli strain (RS2λ) were more negatively supercoiled than in a wild-type strain (W3110) and, concurrently, showed significantly fewer knots. In this way, using wild-type and E. coli mutant strains, we confirmed that one of the biological functions of DNA supercoiling is to reduce the formation of DNA knots. Full article

Ionizing radiation induces many different types of DNA lesions. But one of its characteristics is to produce complex DNA damage, of which tandem DNA damage has received much attention, owing to its promise of distinctive biological properties. Oxidative stresses in response to inflammation in tissues and metal-catalyzed reactions that result in generation of radicals also form these DNA lesions. In this minireview, we have summarized the formation of the tandem lesions as well as the replication and repair studies carried out on them after site-specific synthesis. Many of these lesions are resistant to the traditional base excision repair, so that they can only be repaired by the nucleotide excision repair pathway. They also block DNA replication and, when lesion bypass occurs, it may be significantly error-prone. Some of these tandem DNA lesions may contribute to ageing, neurological diseases, and cancer. Full article

Chromatin is the complex of DNA and associated proteins found in the nuclei of living organisms. How it is organized is a major research field as it has implications for replication, repair, and gene expression. This review summarizes the current state of the chromatin organization field, with a special focus on chromatin motor complexes cohesin and condensin. Containing the highly conserved SMC proteins, these complexes are responsible for organizing chromatin during cell division. Additionally, research has demonstrated that condensin and cohesin also have important functions during interphase to shape the organization of chromatin and regulate expression of genes. Using the model organism C. elegans, the authors review the current knowledge of how these complexes perform such diverse roles and what open questions still exist in the field. Full article

Embryogenesis is characterized by dynamic chromatin remodeling and broad changes in chromosome architecture. These changes in chromatin organization are accompanied by transcriptional changes, which are crucial for the proper development of the embryo. Several independent mechanisms regulate this process of chromatin reorganization, including the segregation of chromatin into heterochromatin and euchromatin, deposition of active and repressive histone modifications, and the formation of 3D chromatin domains such as TADs and LADs. These changes in chromatin structure are directly linked to developmental milestones such as the loss of developmental plasticity and acquisition of terminally differentiated cell identities. In this review, we summarize these processes that underlie this chromatin reorganization and their impact on embryogenesis in the nematode C. elegans. Full article

The PowerPlex Fusion 6C PCR™ amplification kit provides a strong discriminatory power for human identification. We have validated the kit with a reduced volume (12.5 µL) and as part of the validation we compared the efficiency of the polymerase chain reaction (PCR) prepared manually and on Hamilton Microlab^® Autolys STAR Biorobot. Three years of casework data has been also included in the validation. Optimisation was carried out on different types of samples (blood, saliva, semen) and DNA was extracted robotically. Tests were conducted at two different cycle numbers (30;32), followed by analysis on both the Applied Biosystems^TM 3500 and 3500 xL Genetic Analyzer instruments (Applied Biosystems®, Foster City, CA, USA). When the PCR was prepared manually, no allele dropout was observed over 0.15 ng input DNA. Whereas when the PCR was prepared robotically, dropout already appeared at the level of 0.15 ng input DNA. In cases when increased cycle number was utilised, an increasing number of dropouts started to arise from 0.075 ng total input DNA. Despite the fact that robotically prepared PCR produced more missing alleles than the manually prepared PCR, using the optimal 0.5 ng input DNA, both methods proved to be reliable. Based on the results, our half-volume protocol is robust, and after three years of application it has proven to be effective with respect to a large number of casework samples. Full article

Motivation: Clustered DNA-lesions are predominantly induced by ionizing radiation, particularly by high-LET particles, and considered as lethal damage. Quantification of this specific type of damage as a function of radiation parameters such as LET, dose rate, dose, and particle type can be informative for the prediction of biological outcome in radiobiological studies. This study investigated the induction and complexity of clustered DNA damage for three different types of particles at an LET range of 0.5–250 keV/µm. Methods: Nanometric volumes (36.0 nm³) of 15 base-pair DNA with its hydration shell was modeled. Electron, proton, and alpha particles at various energies were simulated to irradiate the nanometric volumes. The number of ionization events, low-energy electron spectra, and chemical yields for the formation of °OH, H°, ${\mathrm{e}}_{\mathrm{a}\mathrm{q}}^{-}$, and H₂O₂ were calculated for each particle as a function of LET. Single- and double-strand breaks (SSB and DSB), base release, and clustered DNA-lesions were computed from the Monte-Carlo based quantification of the reactive species and measured yields of the species responsible for the DNA lesion formation. Results: The total amount of DNA damage depends on particle type and LET. The number of ionization events underestimates the quantity of DNA damage at LETs higher than 10 keV/µm. Minimum LETs of 9.4 and 11.5 keV/µm are required to induce clustered damage by a single track of proton and alpha particles, respectively. For a given radiation dose, an increase in LET reduces the number of particle tracks, leading to more complex clustered DNA damage, but a smaller number of separated clustered damage sites. Conclusions: The dependency of the number and the complexity of clustered DNA damage on LET and fluence suggests that the quantification of this damage can be a useful method for the estimation of the biological effectiveness of radiation. These results also suggest that medium-LET particles are more appropriate for the treatment of bulk targets, whereas high-LET particles can be more effective for small targets. Full article

Zooplankton and ichthyoplankton community assessments depend on species diagnostics, yet morphological identifications are time-consuming, require taxonomic expertise, and are hampered by a lack of diagnostic characters, particularly for larval stages. Metabarcoding can identify multiple species in communities from short DNA sequences in comparison to reference databases. To evaluate species resolution across phylogenetic groups and food webs of zooplankton and ichthyoplankton, we compare five metabarcode mitochondrial (mt)DNA markers from gene regions of (a) cytochrome c oxidase subunit I, (b) cytochrome b, (c) 16S ribosomal RNA, and (d) 12S ribosomal RNA for DNA extracted from net tows in the Northeastern Pacific Ocean’s Salish Sea across seven sites and two seasons. Species resolved by metabarcoding are compared to invertebrate morphological identifications and biomass estimates. Results indicate that species resolution for different zooplankton and ichthyoplankton taxa can markedly vary among gene regions and markers in comparison to morphological identifications. Thus, researchers seeking “universal” metabarcoding should take caution that several markers and gene regions likely will be needed; all will miss some taxa and yield incomplete overlap. Species resolution requires careful attention to taxon marker selection and coverage in reference sequence repositories. In summary, combined multi-marker metabarcoding and morphological approaches improve broadscale zooplankton diagnostics. Full article

Teratozoospermia, a complex male fertility disorder affecting sperm morphology, has been linked to AURKC, SPATA16, and SUN5 gene defects. However, the sheer volume of SNPs in these genes necessitates prioritization for comprehensive analysis. This study focuses on the often-overlooked untranslated region (UTR) variants in these genes, aiming to assess their association with teratozoospermia and prioritize them. We employed a multi-step filtering process, including functional significance assessment (RegulomeDB, 3DSNP v2.0, SNPinfo (FuncPred)), evaluation of gene expression impacts in testis tissue using GTEx, and assessment of miRNA binding site effects (PolymiRTS Database 3.0, miRNASNP v3). Additionally, we used SNPnexus to evaluate their conservation and association with diseases. In AURKC, we identified six UTR SNPs (rs11084490, rs58264281, rs35582299, rs533889458, rs2361127, rs55710619), two of which influenced gene expression in testis, while others affected the binding sites of 29 miRNAs or were located in transcription-factor binding sites. Three of these SNPs were also found to be associated with spermatogenic failure according to previous studies indicating a potential regulatory role in teratozoospermia, too. For SPATA16, two 3′ UTR variants, rs146640459 and rs148085657, were prioritized, with the latter impacting miRNA binding sites. In SUN5, three 3′ UTR variants (rs1485087675, rs762026146, rs1478197315) affected miRNA binding sites. It should be noted that none of the above variants was identified in a conserved region. Our findings shed light on the potential regulatory roles of these SNPs in teratozoospermia and lay the foundation for future research directions in this area. Full article

DNA methyltransferase 1 (DNMT1) is the enzyme primarily responsible for propagation of the methylation pattern in cells. Mutations in DNMT1 have been linked to the development of adult-onset neurodegenerative disorders; these disease-associated mutations occur in the regulatory replication foci-targeting sequence (RFTS) domain of the protein. The RFTS domain is an endogenous inhibitor of DNMT1 activity that binds to the active site and prevents DNA binding. Here, we examine the impact of the disease-associated mutation A554V on normal RFTS-mediated inhibition of DNMT1. Wild-type and mutant proteins were expressed and purified to homogeneity for biochemical characterization. The mutation increased DNA binding affinity ~8-fold. In addition, the mutant enzyme exhibited increased DNA methylation activity. Circular dichroism (CD) spectroscopy revealed that the mutation does not significantly impact the secondary structure or relative thermal stability of the isolated RFTS domain. However, the mutation resulted in changes in the CD spectrum in the context of the larger protein; a decrease in relative thermal stability was also observed. Collectively, this evidence suggests that A554V disrupts normal RFTS-mediated autoinhibition of DNMT1, resulting in a hyperactive mutant enzyme. While the disease-associated mutation does not significantly impact the isolated RFTS domain, the mutation results in a weakening of the interdomain stabilizing interactions generating a more open, active conformation of DNMT1. Hyperactive mutant DNMT1 could be responsible for the increased DNA methylation observed in affected individuals. Full article

The DNA photocleavage effect of halogenated O-carbamoyl derivatives of 4-MeO-benzamidoxime under UVB and UVA irradiation was studied in order to identify the nature, position, and number of halogens on the carbamoyl moiety that ensure photoactivity. F, Cl, and Br-phenyl carbamate esters (PCME) exhibited activity with the p-Cl-phenyl derivative to show excellent photocleavage against pBR322 plasmid DNA. m-Cl-PCME has diminished activity, whereas the presence of two halogen atoms reduced DNA photocleavage. The substitution on the benzamidoxime scaffold was irrelevant to the activity. The mechanism of action indicated function in the absence of oxygen, probably via radicals derived from the N-O bond homolysis of the carbamates and in air via hydroxyl radicals and partially singlet oxygen. The UVA-vis area of absorption of the nitro-benzamidoxime p-Cl-PCMEs allowed for the investigation of their potential efficacy as photopesticides under UVA irradiation against the whitefly Bemisia tabaci, a major pest of numerous crops. The m-nitro derivative exhibited a moderate specificity against the adult population. Nymphs were not affected. The compound was inactive in the dark. This result may allow for the development of lead compounds for the control of agricultural insect pests that can cause significant economic damage in crop production. Full article

Nucleic acid methylations are important genetic and epigenetic biomarkers. The formation and removal of these markers is related to either methylation or demethylation. In this review, we focus on the demethylation or oxidative modification that is mediated by the 2-oxoglutarate (2-OG)/Fe(II)-dependent AlkB/TET family enzymes. In the catalytic process, most enzymes oxidize 2-OG to succinate, in the meantime oxidizing methyl to hydroxymethyl, leaving formaldehyde and generating demethylated base. The AlkB enzyme from Escherichia coli has nine human homologs (ALKBH1-8 and FTO) and the TET family includes three members, TET1 to 3. Among them, some enzymes have been carefully studied, but for certain enzymes, few studies have been carried out. This review focuses on the kinetic properties of those 2-OG/Fe(II)-dependent enzymes and their alkyl substrates. We also provide some discussions on the future directions of this field. Full article

Both the number of cells and the collective genome of the gut microbiota outnumber their mammalian hosts, and the metabolic and physiological interactions of the gut microbiota with the host have not yet been fully characterized. Cancer remains one of the leading causes of death, and more research into the critical events that can lead to cancer and the importance of the gut microbiota remains to be determined. The gut microbiota can release microbial molecules that simulate host endogenous processes, such as inflammatory responses, or can alter host metabolism of ingested substances. Both of these reactions can be beneficial or deleterious to the host, and some can be genotoxic, thus contributing to cancer progression. This review focused on the molecular evidence currently available on the mechanistic understanding of how the gut microbiota are involved in human carcinogenesis. We first reviewed the key events of carcinogenesis, especially how DNA damage proceeds to tumor formulation. Then, the current knowledge on host DNA damage attributed to the gut microbiota was summarized, followed by the genotoxic endogenous processes the gut microbiota can induce. Finally, we touched base on the association between specific gut microbiota dysbiosis and different types of cancer and concluded with the up-to-date knowledge as well as future research direction for advancing our understanding of the relationship between the gut microbiota and cancer development. Full article

DNA G-quadruplexes (G4s) are non-canonical secondary structures formed in guanine-rich sequences. Within the human genome, G4s are found in regulatory regions such as gene promoters and telomeres to control replication, transcription, and telomere lengthening. In the cellular context, there are several proteins named as G4-binding proteins (G4BPs) that interact with G4s, either anchoring upon, stabilizing, and/or unwinding them. These proteins may play different key roles in the regulation of the endogenous G4 landscape and its associated functions. The present review summarizes the current literature on G4BPs in terms of their targets and functions, providing updated insights into the regulation of G4s in living organisms. Full article

Understanding the self-assembly and hybridization properties of DNA oligonucleotides in confined spaces can help to improve their applications in biotechnology and nanotechnology. This study investigates the effects of spatial confinement in the pores of hydrogels on the thermal stability of DNA oligonucleotide structures. The preparation of oligonucleotides embedded in agarose gels was simple, whereas the preparation of oligonucleotides embedded in polyacrylamide gels was required to remove unpolymerized monomers. In the latter case, a method for rehydrating a washed dry gel with a buffer solution containing oligonucleotides was developed. Fluorescence measurements of oligonucleotides bearing fluorescent probes revealed no significant influence of the internal environment of the gel pores on the stability of DNA duplex, hairpin, and G-quadruplex structures. Moreover, the effects of poly(ethylene glycol) on the stability of DNA structures in the gels were similar to those in solutions. It is likely that the oligonucleotides are not strongly constrained in the gels and may be preferentially located in a water-rich environment in the gel matrix. The gel preparation was also applied to the assessment of the stability of DNA structures under the conditions of a reduced number of water molecules. The studies using hydrogels provide insights into the ability of self-assembly and hybridization of oligonucleotides in confined environments and under low-water-content conditions. Full article

DNA glycosylases promote genomic stability by initiating base excision repair (BER) in both the nuclear and mitochondrial genomes. Several of these enzymes have overlapping substrate recognition, through which a degree of redundancy in lesion recognition is achieved. For example, OGG1 and NEIL1 both recognize and release the imidazole-ring-fragmented guanine, FapyGua as part of a common overall pathway to cleanse the genome of damaged bases. However, these glycosylases have many differences, including their differential breadth of substrate specificity, the contrasting chemistries through which base release occurs, the subsequent steps required to complete the BER pathway, and the identity of specific protein-binding partners. Beyond these differences, the complexities and differences of their in vivo biological roles have been primarily elucidated in studies of murine models harboring a knockout of Neil1 or Ogg1, with the diversity of phenotypic manifestations exceeding what might have been anticipated for a DNA glycosylase deficiency. Pathologies associated with deficiencies in nuclear DNA repair include differential cancer susceptibilities, where Ogg1-deficient mice are generally refractory to carcinogenesis, while deficiencies in Neil1-deficient mice confer cancer susceptibility. In contrast to NEIL1, OGG1 functions as a key transcription factor in regulating inflammation and other complex gene cascades. With regard to phenotypes attributed to mitochondrial repair, knockout of either of these genes results in age- and diet-induced metabolic syndrome. The adverse health consequences associated with metabolic syndrome can be largely overcome by expression of a mitochondrial-targeted human OGG1 in both wild-type and Ogg1-deficient mice. The goal of this review is to compare the roles that NEIL1 and OGG1 play in maintaining genomic integrity, with emphasis on insights gained from not only the diverse phenotypes that are manifested in knockout and transgenic mice, but also human disease susceptibility associated with polymorphic variants. Full article

DNA–Protein cross-links (DPCs) are cytotoxic DNA lesions with a protein covalently bound to the DNA. Although much has been learned about the formation, repair, and biological consequences of DPCs in the nucleus, little is known regarding mitochondrial DPCs. This is due in part to the lack of robust and specific methods to measure mitochondrial DPCs. Herein, we reported an enzyme-linked immunosorbent assay (ELISA)-based method for detecting mitochondrial DPCs formed between DNA and mitochondrial transcription factor A (TFAM) in cultured human cells. To optimize the purification and detection workflow, we prepared model TFAM-DPCs via Schiff base chemistry using recombinant human TFAM and a DNA substrate containing an abasic (AP) lesion. We optimized the isolation of TFAM-DPCs using commercial silica gel-based columns to achieve a high recovery yield for DPCs. We evaluated the microplate, DNA-coating solution, and HRP substrate for specific and sensitive detection of TFAM-DPCs. Additionally, we optimized the mtDNA isolation procedure to eliminate almost all nuclear DNA contaminants. For proof of concept, we detected the different levels of TFAM-DPCs in mtDNA from HEK293 cells under different biological conditions. The method is based on commercially available materials and can be amended to detect other types of DPCs in mitochondria. Full article

DNA damage is induced by exogenous and endogenous sources, creating a variety of lesions. However, the cellular repair machinery that addresses and corrects this damage must contend with the fact that genomic DNA is sequestered in the nucleoprotein complex of chromatin. As the minimal unit of DNA compaction, the nucleosome core particle (NCP) is a major determinant of repair and poses unique barriers to DNA accessibility. This review outlines how the base excision repair (BER) pathway is modulated by the NCP and describes the structural and dynamic factors that influence the ability of BER enzymes to find and repair damage. Structural characteristics of the NCP such as nucleobase positioning and occupancy will be explored along with factors that impact the dynamic nature of NCPs to increase mobilization of nucleosomal DNA. We will discuss how altering the dynamics of NCPs initiates a domino effect that results in the regulation of BER enzymes. Full article

Genetic instability can result from increases in DNA damage and/or alterations in DNA repair proteins and can contribute to disease development. Both exogenous and endogenous sources of DNA damage and/or alterations in DNA structure (e.g., non-B DNA) can impact genome stability. Multiple repair mechanisms exist to counteract DNA damage. One key DNA repair protein complex is ERCC1-XPF, a structure-specific endonuclease that participates in a variety of DNA repair processes. ERCC1-XPF is involved in nucleotide excision repair (NER), repair of DNA interstrand crosslinks (ICLs), and DNA double-strand break (DSB) repair via homologous recombination. In addition, ERCC1-XPF contributes to the processing of various alternative (i.e., non-B) DNA structures. This review will focus on the processing of alternative DNA structures by ERCC1-XPF. Full article