DNA, Volume 4, Issue 4 (December 2024) – 16 articles

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

Background: Major depressive disorder (MDD) is a disease that has been increasingly affecting more people worldwide. The dopamine D2 receptor (DRD2), encoded by the DRD2 gene, plays critical roles in the brain, one of which is related to reward processes. Aims: The following systematic review aims to analyze the DRD2/ANKK1 TaqIA (rs1800497) polymorphism’s A1 genotype frequency fluctuation in MDD patients and determine its influence on MDD. Methods: Four databases were searched, and the consequent articles were analyzed following the inclusion criteria per the PECOS strategy, resulting in five selected articles. Results: Interestingly, although two articles showed that the A1 allele presence significantly increases the risk of MDD manifestation, most articles did not find a significant association between this DRD2 gene variant and MDD. Conclusions: Most of the included studies were dated, indicating the need for more studies to address the results’ non-conformity with different populations. Full article

The relatively rare proximal 17q12 microdeletion, including the deletion of the HNF1B gene, is associated with renal cysts and diabetes syndrome (RCAD). This genomic rearrangement results in a wide range of phenotypes, including renal cysts and diabetes, which are consistent with maturity-onset diabetes of the young type 5 (MODY5), Mullerian aplasia/dysgenesis, autism spectrum disorder and schizophrenia, speech delay, learning difficulties, transient neonatal hypercalcemia, and neonatal cholestasis. We describe a girl with a 17q12 microdeletion identified using CGH array analysis (about 1.4 Mb, including HNF1B and LHX1 genes). The same deletion was identified in her mother. The proband had shown cystic and hypodysplastic bilateral kidneys since birth and hypertension, while her mother had bilateral renal cysts and diabetes. Despite suggestive findings in the girl and in the mother, no clinical suspicion arose, and genetic testing was carried out only after referral to a pediatric nephrologist. In children, the identification of 17q12 microdeletion may have a significant impact on the diagnosis, prognosis, and management of renal disease and early-onset type II diabetes. This family with a 17q12 microdeletion confirms intrafamilial phenotypic variability and highlights the importance of including it early on in the analysis of the diagnostic workup of children with renal cystic diseases. Full article