Editor’s Choice Articles

Background/Objectives: Temozolomide is an important drug used for the treatment of glioblastoma multiforme. Covalent conjugation of temozolomide to triplex-forming oligonucleotides could facilitate better sequence discrimination when targeted to DNA to lessen off-target effects and potentially reduce side-effects associated with conventional chemotherapy. The base sensitivity of temozolomide precludes use of basic deprotection conditions that typify the solid-supported synthesis of oligonucleotides. Methods: A novel di-iso-propylsilylene-linked solid support was developed and used in solid-supported synthesis of oligonucleotide conjugates. Results: Conditions were established whereby fully deprotected, solid-supported oligonucleotides could be prepared for derivatisation. Cleavage of the di-iso-propylsilylene linker was possible using mild, acidic conditions. Conclusions: The di-iso-propylsilylene-linked solid support was developed and shown to be compatible with base-sensitive oligonucleotide conjugate formation. The DNA triplex formation exhibited by a temozolomide oligonucleotide conjugate was equal in stability to the unconjugated control, opening new possibilities for sequence selective delivery of temozolomide to targeted DNA. Full article

Background: Medullary thyroid cancer (MTC), a neuroendocrine tumor originating from thyroid parafollicular C-cells, presents therapeutic challenges, particularly in advanced stages. While RET proto-oncogene mutations are known drivers, a comprehensive understanding of the broader somatic mutation landscape is needed to identify novel therapeutic targets and improve prognostication. This study leveraged the extensive AACR Project GENIE dataset to characterize MTC genomics. Methods: A retrospective analysis of MTC samples from GENIE examined recurrent somatic mutations, demographic/survival correlations, and copy number variations using targeted sequencing data (significance: p < 0.05). Results: Among 341 samples, RET mutations predominated (75.7%, mostly M918T), followed by HRAS (10.0%) and KRAS (5.6%), with mutual exclusivity between RET and RAS alterations. Recurrent mutations included KMT2D (5.3%), CDH11 (5.3%), ATM (5.0%), and TP53 (4.1%). NOTCH1 mutations were enriched in metastatic cases (p = 0.023). Preliminary associations included sex-linked mutations (BRAF/BRCA1/KIT in females, p = 0.028), and survival (ATM associated with longer survival, p = 0.016; BARD1/BLM/UBR5/MYH11 with shorter survival, p < 0.05), though limited subgroup sizes warrant caution. Conclusions: This large-scale genomic analysis confirms the centrality of RET and RAS pathway alterations in MTC and their mutual exclusivity. The association of NOTCH1 mutations with metastasis suggests a potential role in disease progression. While findings regarding demographic and survival correlations are preliminary, they generate hypotheses for future validation. This study enhances the genomic foundation for understanding MTC and underscores the need for integrated clinico-genomic datasets to refine therapeutic approaches. Full article

Background/Objectives: The potential of DNA as an information-dense storage medium has inspired a broad spectrum of creative systems. In particular, hybrid biomolecular systems that integrate new materials and chemistries with DNA could drive novel functions. In this work, we explore the potential for proteins to serve as molecular file addresses. We stored DNA-encoded data in yeast and leveraged yeast surface display to readily produce the protein addresses and make them easy to access on the cell surface. Methods: We generated yeast populations that each displayed a distinct protein on their cell surfaces. These proteins included binding partners for cognate antibodies as well as chromatin-associated proteins that bind post-translationally modified histone peptides. For each specific yeast population, we transformed a library of hundreds of DNA sequences collectively encoding a specific image file. Results: We first demonstrated that the yeast retained file-encoded DNA through multiple cell divisions without a noticeable skew in their distribution or a loss in file integrity. Second, we showed that the physical act of sorting yeast displaying a specific file address was able to recover the desired data without a loss in file fidelity. Finally, we showed that analog addresses can be achieved by using addresses that have overlapping binding specificities for target peptides. Conclusions: These results motivate further exploration into the advantages proteins may confer in molecular information storage. Full article

Microbial contamination is a critical challenge for the pharmaceutical industry, especially in thermosensitive sterile products, and can compromise their quality and safety. The accurate identification of microorganisms is essential to trace sources of contamination and adopt corrective measures. Although MALDI-TOF MS technology has revolutionized this process, its database limitations necessitate the use of complementary methods, such as sequencing 16S rRNA genes, housekeeping genes, and, in some cases, the entire genome. Advances in sequencing have expanded genomic taxonomy, increasing the accuracy of bacterial identification. The integration of these approaches significantly improves the reliability of identification, overcoming the limitations of isolated methods. Full article

In B-cell chronic lymphocytic leukemia (B-CLL), hypodiploidy is a rare but aggressive subtype of the disease with a very bad prognosis. Hypodiploidy, in contrast to normal B-CLL chromosomal aberrations, is marked by widespread genomic instability, which promotes treatment resistance and quick illness development. Its persistence after treatment implies that chromosomal loss gives cancerous clones a selection edge, which is made worse by telomere malfunction and epigenetic changes. Since thorough genetic profiling has a major impact on patient outcomes, advanced diagnostic methods are crucial for early detection. Treatment approaches must advance beyond accepted practices because of its resistance to traditional medicines. Hematopoietic stem cell transplantation (HSCT) and chimeric antigen receptor (CAR) T-cell therapy are two potential new therapeutic modalities. Relapse and treatment-related morbidity continue to be limiting concerns, despite the noteworthy improvements in outcomes in high-risk CLL patients receiving HSCT. Although more research is required, CAR T-cell treatment is effective in treating recurrent B-ALL and may also be used to treat B-CLL with hypodiploidy. Novel approaches are essential for enhancing patient outcomes and redefining therapeutic success when hypodiploidy challenges established treatment paradigms. Hypodiploidy is an uncommon yet aggressive form of B-CLL that has a very bad prognosis. Hypodiploidy represents significant chromosomal loss and structural imbalance, which contributes to a disordered genomic environment, in contrast to more prevalent cytogenetic changes. This instability promotes resistance to certain new drugs as well as chemoimmunotherapy and speeds up clonal evolution. Its persistence after treatment implies that hypodiploid clones have benefits in survival, which are probably strengthened by chromosomal segregation issues and damaged DNA repair pathways. Malignant progression and treatment failure are further exacerbated by telomere erosion and epigenetic dysregulation. The need for more sensitive molecular diagnostics is highlighted by the fact that standard karyotyping frequently overlooks hypodiploid clones, particularly those concealed by endoreduplication, despite the fact that these complications make early and correct diagnosis crucial. Hypodiploidy requires a move toward individualized treatment because of their link to high-risk genetic traits and resistance to conventional regimens. Although treatments like hematopoietic stem cell transplantation and CAR T-cells show promise, long-term management is still elusive. To improve long-term results and avoid early relapse, addressing this cytogenetic population necessitates combining high-resolution genomic technologies with changing therapy approaches. Full article

Background/Objectives: Genetic abnormalities play a pivotal role in patient risk stratification, therapeutic decision-making, and elucidating the disease pathogenesis in hematological malignancies. In multiple myeloma (MM) and acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS), numerous recurring genetic aberrations are well documented. Fluorescence in situ hybridization (FISH) is a cornerstone of clinical diagnostics for detecting these abnormalities. Conventionally, FISH assesses up to two biomarkers, with one or two circles per slide, but this approach faces challenges when cancer cell yields are limited, particularly in post-treatment follow-up specimens. Methods: To overcome this limitation, we developed a multi-well method, enabling the simultaneous testing of multiple biomarkers on a single microscopic slide. This study included 53 MM and 129 AML/MDS cases. Results: With a cohort of 182 patients, 1016 FISH assays performed on multi-well slides accurately detected diagnostic genetic aberrations previously identified by karyotyping and/or FISH, achieving a sensitivity and specificity of 100%. The use of multi-well slides achieved up to a 2.5-fold increase in the number of wells per slide while achieving more than a 3-fold reduction in the reagent volume compared to traditional methods. This advancement leverages distinct FISH signal patterns to strategically combine biomarkers within multiple wells, suitable for specimens from diagnosis, follow-ups, and relapses, regardless of the cancer cell quantity. Conclusions: The multi-well approach enhances the accessibility to comprehensive biomarker analysis, reducing both the processing time and costs. Beyond MM and AML/MDS, this technique holds promise for use with other hematological malignancies with limited sample volumes, offering an efficient, cost-effective solution for precision diagnostics. Full article

Backround/Objectives: Mobile genetic elements (MGEs), in general, and transposable elements (TEs), in particular, constitute a major part of almost every eukaryotic genome, and several types of such elements have been classified based on size, genetic structure and transposition intermediate. Methods: The fast-growing availability of whole genome sequences of species across the living world provides almost unlimited possibilities for in-depth molecular analyses of all kinds, including the search for TEs. The aim of the present study was to perform the first molecular description and characterization of selected MGEs in leeches, namely, short interspersed nuclear element (SINE), long interspersed nuclear element (LINE) and long terminal repeat (LTR) retrotransposons. Results: Several representatives of all three groups of TEs could be identified, and some of the newly described elements display unique structural features compared to the archetype elements of the respective groups. Conclusions: Non-model organisms like leeches are an excellent source for new information on long-term studied objects like TEs and may provide new insights into the diversity and the putative biological impact of these MGEs. Full article

Background: The success of forensic genetics has led to considerable numbers of DNA samples that must be stored. For example, the genetic casework unit of the forensic institute of the French gendarmerie analyzes more than 70,000 casework samples per year mainly from swabs that are fully consumed during DNA extraction. The only way to process further analyses is to preserve DNA. Currently, the most common technique used for the long-term preservation of DNA is to freeze the extracted DNA at −20 °C or −80 °C. However, this preservation method involves significant constraints (large equipment), risks (equipment failure), and is not ecologically sustainable due to its high energy consumption. Many solutions for DNA preservation at room temperature exist based either on fibrous supports or on anhydrobiosis. However, few studies have examined the efficiency of these systems in preserving very-low DNA amounts, such as those in forensic samples (≤1 ng), while ensuring full recovery and the ability to retest the samples many years later. Methods: We choose to evaluate the ability of the anhydrobiosis technology from GenTegra^® LLC to preserve DNA extracts from one month to one accelerated year from different DNA quantities (from 1 ng to 0.2 ng) and sources (NIST, mocked samples, and true casework mixtures). We studied the quantity, integrity of DNA, and also the quality of the STR genetic profiles obtained. Results and Conclusions: Our results prove the high potential of this technology to preserve and to allow an effective recovery of the DNA extracts for forensic purposes. Full article

Background/Objectives: DNA methylation profiles have emerged as robust biomarkers of ageing, leading to the development of “epigenetic clocks” that estimate biological age. Most established clocks (e.g., Horvath’s 353-CpG pan-tissue clock and Hannum’s 71-CpG blood clock) require dozens to hundreds of CpG sites. This study presents a novel saliva-specific epigenetic clock built on 10 sites identified from Illumina MethylationEPIC (850 k) array data. Methods: Saliva DNA methylation was analysed from 3408 individuals (age range 15–89 years, 68% male, 32% female, no diagnosed disease) from the Muhdo Health Ltd. dataset (2022–2024), and 10 CpG sites were selected where methylation levels showed the strongest positive correlations with chronological age (Pearson r = 0.48–0.66, p < 1 × 10−20). These CpGs map to genes involved in developmental and metabolic pathways (including ELOVL2, CHGA, OTUD7A, PRLHR, ZYG11A, and GPR158). A linear combination of the 10 methylation sites was used to calculate a “DNA methylation age”. Results: The 10-CpG clock’s predictions were highly correlated with chronological age (r = 0.80, R2 = 0.64), with a mean absolute error of ~5.5 years. Its performance, while slightly less precise than Horvath’s or Hannum’s multi-CpG clocks, is notable given the minimal marker set. It was observed that all 10 clock CpGs undergo age-related hypermethylation. The biological significance of these loci is discussed, along with the potential health and forensic applications of a saliva-based epigenetic age predictor. Conclusions: This study demonstrates that a saliva-specific epigenetic clock using only 10 CpG sites can capture a substantial portion of age-related DNA methylation changes, providing a cost-effective tool for age estimation. Full article

Tardigrades, also known as “water bears”, are microscopic invertebrates capable of surviving extreme conditions, including extreme temperatures, intense radiation, and the vacuum of space. Recent studies have unveiled a novel nucleosome-binding protein in the tardigrade Ramazzottius varieornatus, known as the damage suppressor protein (Dsup). This protein has proven essential for enabling tardigrades to thrive in the most challenging environmental conditions, highlighting its pivotal role in their remarkable survival capabilities. Dsup is a highly disordered protein with DNA-binding abilities that reduces DNA damage and enhances cell survival and viability caused by several stresses such as oxidative stress, UV exposure, and X-ray and ionizing radiation. In this review, we summarized articles describing the protective role of Dsup upon different stressors across diverse organisms, including bacteria, yeast, plants, and animals (cell lines and organisms). The multifaceted properties of Dsup open avenues for biotechnological applications, such as developing stress-resistant crops and innovative biomaterials for DNA manipulation. Furthermore, investigations into its potential in space exploration, particularly in protecting organisms from space radiation, underscore its relevance in extreme environments. Full article

Background/Objectives: Ancient DNA (aDNA) research workflows heavily depend on efficient aDNA extraction and NGS library preparation. In this study, we compared some of the commonly used laboratory protocols and compared the source of the bone material for sufficient and reliable results. Methods: We executed a three-phase study. First, we analyzed about 2000 previously processed archaic bone samples and conducted a comparative analysis. The second phase involved a controlled experiment of five ancient individuals, with internal control, to further investigate the efficiency of some of the methods. In the third phase, we made a comparison between the efficiency of two enzymes used for library preparation. Results: Samples made from Pars petrosa resulted in the highest yield of endogenous DNA and longer fragment sizes compared to tooth or skeletal samples. DNA extraction made by MinElute columns preserved slightly longer fragments than the handmade silica suspension. NGS libraries indexed using AccuPrime Pfx produced slightly more consistent insert sizes compared to GoTaq G2. Samples prepared with GoTaq G2 contained slightly more unique molecules. The duplication rates showed no significant impact from enzyme choice. Conclusions: Pars petrosa remains the most reliable source of aDNA, with the extraction method using MinElute columns. While AccuPrime Pfx ensures precise NGS library preparation, a more economical choice of the GoTaq G2 enzyme is a viable alternative for degraded archaic samples. Full article

Background/Objective: Oligodeoxynucleotides (ODNs) containing base-labile modifications such as N4-acetyldeoxycytidine (4acC), N6-acetyladenosine (6acA), N2-acetylguanosine (2acG), and N4-methyoxycarbonyldeoxycytidine (4mcC) are highly challenging to synthesize because standard ODN synthesis methods require deprotection and cleavage under strongly basic and nucleophilic conditions, and there is a lack of ideal alternative methods to solve the problem. The objective of this work is to explore the capability of the recently developed 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) method for the incorporation of multiple 4acC modifications into a single ODN molecule and the feasibility of using the method for the incorporation of the 6acA, 2acG and 4mcC modifications into ODNs. Methods: The sensitive ODNs were synthesized on an automated solid phase synthesizer using the Dmoc group as the linker and the methyl Dmoc (meDmoc) group for the protection of the exo-amino groups of nucleobases. Deprotection and cleavage were achieved under non-nucleophilic and weakly basic conditions. Results: The 4acC, 6acA, 2acG, and 4mcC were all found to be stable under the mild ODN deprotection and cleavage conditions. Up to four 4acC modifications were able to be incorporated into a single 19-mer ODN molecule. ODNs containing the 6acA, 2acG, and 4mcC modifications were also successfully synthesized. The ODNs were characterized using RP HPLC, capillary electrophoresis, gel electrophoresis and MALDI MS. Conclusions: Among the modified nucleotides, 4acC has been found in nature and proven beneficial to DNA duplex stability. A method for the synthesis of ODNs containing multiple 4acC modifications is expected to find applications in biological studies involving 4acC. Although 6acA, 2acG, and 4mcC have not been found in nature, a synthetic route to ODNs containing them is expected to facilitate projects aimed at studying their biophysical properties as well as their potential for antisense, RNAi, CRISPR, and mRNA therapeutic applications. Full article

The ability of a nucleic acid molecule to self-replicate is the driving force behind the evolution of cellular life and the transition from RNA to DNA as the genetic material. Thus, the physicochemical properties of genome replication, such as the requirement for a terminal hydroxyl group for de novo DNA synthesis, are conserved in all three domains of life: eukaryotes, bacteria, and archaea. Canonical DNA replication is initiated from specific chromosomal sequences termed origins. Early bacterial models of DNA replication proposed origins as regulatory points for spatiotemporal control, with replication factors acting on a single origin on the chromosome. In eukaryotes and archaea, however, replication initiation usually involves multiple origins, with complex spatiotemporal regulation in the former. An alternative replication initiation mechanism, recombination-dependent replication, is observed in every cellular domain (and viruses); DNA synthesis is initiated instead from the 3′ end of a recombination intermediate. In the domain archaea, species including Haloferax volcanii are not only capable of initiating DNA replication without origins but grow faster without them. This raises questions about the necessity and nature of origins. Why have archaea retained such an alternative DNA replication initiation mechanism? Might recombination-dependent replication be the ancestral mode of DNA synthesis that was used during evolution from the primordial RNA world? This review provides a historical overview of major advancements in the study of DNA replication, followed by a comparative analysis of replication initiation systems in the three domains of life. Our current knowledge of origin-dependent and recombination-dependent DNA replication in archaea is summarised. Full article

Background/Objectives: The agriculture sector faces significant challenges due to global climate change, environmental stressors, and rapid population growth, compounded by unsustainable farming practices. This study investigates the potential of the endophytic bacterial strain B.B.Sf.2, isolated from the bark of Salvia fruticosa and identified as Bacillus velezensis through phylogenomic analyses. Methods: To address these issues, eco-friendly techniques, such as the application of plant-associated microbes, are gaining attention. Genome mining revealed numerous secondary metabolite biosynthetic gene clusters associated with plant growth promotion, biocontrol, colonization, and defense elicitation. Results: The strain exhibited strong antagonistic activity against phytopathogens, mediated by diffusible and volatile compound production, along with plant-growth-promoting traits and environmental adaptability. Genome mining revealed numerous secondary metabolite biosynthetic gene clusters associated with plant growth promotion, biocontrol, colonization, and defense elicitation. B.B.Sf.2 effectively inhibited Colletotrichum species causing olive anthracnose and suppressed Botrytis cinerea, the gray mold pathogen, in post-harvest studies on infected fruits. Bioautography of ethyl acetate extracts demonstrated bioactivity against B. cinerea, attributed to iturin-like metabolites. The extracts maintained bioactive properties regardless of fungal interaction. Furthermore, the strain significantly promoted the growth of Arabidopsis thaliana via diffusible and volatile compounds. Conclusions: Our results highlight the multifunctional potential of B.B.Sf.2 as a biocontrol and growth-promoting agent, warranting further evaluation in field applications to enhance sustainable agriculture. Full article

Our genome has evolved a complex network of information designed to precisely regulate gene transcription. Commonly known as cis-regulatory elements, they represent those parts of DNA that are highly sensitive to environmental changes in the form of associated multi-protein complexes. Oxygen levels are an important environmental factor influencing a range of cellular activities, including cell survival. To respond to changes in oxygen levels, cells have developed an efficient and precise system for regulating gene expression. Cis-regulatory elements are the key hubs of this response and control the activation of the transcriptional response to hypoxia. In this review, we will discuss the complex genomic and epigenomic structures that are modulated by oxygen and control the activity of cis-regulatory elements and the adaptations to variations in O₂ availability. Full article

Pathogenic variants in the KAT6A gene cause KAT6A syndrome, a neurodevelopmental disorder characterised by intellectual disability (ID), developmental delay, speech and language challenges, feeding difficulties, and skeletal abnormalities. This scoping review synthesises current knowledge on KAT6A syndrome, identifies key research themes, and supports the mission of advocacy groups like the KAT6 Foundation. A systematic search of five databases (Ovid MEDLINE, Ovid EMBASE, PubMed, Web of Science, and Scopus) was conducted from 1990 to 2024, including peer-reviewed articles, preprints, and conference abstracts published from 2022 onward. Of 771 citations retrieved, 111 full-text articles were reviewed, with 62 meeting the inclusion criteria. Data were synthesised into six themes: (1) the genotype and phenotype map, revealing a broad phenotypic spectrum with common features like ID, absent speech, and craniofacial dysmorphism, as well as rare features such as severe aplastic anaemia and pancraniosynostosis; (2) the neurodevelopmental profile, detailing communication deficits, sleep disturbances, and impaired adaptive functioning; (3) the epigenetic and developmental roles of KAT6A, highlighting its critical function in histone acetylation, chromatin remodelling, and gene regulation; (4) molecular biomarkers, identifying distinct DNA methylation episignatures and dysregulated cellular pathways; (5) drug discovery, with preliminary studies suggesting that pantothenate and L-carnitine may mitigate mitochondrial dysfunction and histone acetylation deficits, while RSPO2 overexpression reverses cognitive impairment in animal models; (6) phenotypic overlap with Rett syndrome and KAT6B-related disorders. This review underscores the complexity and variability of KAT6A syndrome, highlighting the need for multidisciplinary approaches to improving diagnosis, management, and development of therapies. Future research should focus on longitudinal studies, underrepresented phenotypes, biomarker identification, and robust therapeutic trials to enhance outcomes for affected individuals and their families. Full article

Background/Objectives: Branch dieback and canker diseases caused by Cytospora species adversely impact the health of woody plants worldwide. Results: During this survey, 59 Cytospora isolates were obtained from symptomatic trees and shrubs growing in southwest Ontario and Saskatchewan, Canada. A DNA barcoding approach combined with morphological characterization identified 15 known species of Cytospora associated with these diseases: C. chrysosperma, C. curvata, C. euonymina, C. hoffmannii, C. kantschavelii, C. leucosperma, C. leucostoma, C. nitschkeana, C. piceae, C. populina, C. pruinopsis, C. pruinosa, C. ribis, C. schulzeri, and C. sorbina. The most common species isolated from multiple hosts were C. sorbina (10), C. chrysosperma (8), C. nitschkeana (6), and C. pruinosa (6). A wide range of host associations, including non-conifer species, was observed for C. piceae. Conclusions: The obtained results contribute to the study of diversity, host affiliation, geographical distribution, and pathogenicity of Cytospora species occurring on woody plants in both natural habitats and agricultural systems. The findings support the effectiveness of using DNA barcodes in fungal taxonomy and plant pathology studies. Full article

DNA damage and repair have been central themes in cellular biology research. Broadly, DNA damage is understood as modifications to canonical nucleotides that disrupt their function during transcription and replication. A deeper biochemical understanding of DNA damage is essential, as the genome governs all cellular processes. We can classify DNA damage according to whether the modifications to the nucleic acid scaffold are chemically or enzymatically initiated. This distinction is important because chemical modifications are often irreversible, sometimes sparse, and difficult to detect or control spatially and replicate systematically. This can result in genomic damage or modifications to nucleotides in the nucleotide pool, which is less commonly studied. In contrast, enzymatic modifications are typically induced by the cell for specific purposes and are under strong regulatory control. Enzymatic DNA modifications also present a degree of sequence specificity and are often reversible. However, both types of DNA modifications contribute to cellular aging when poorly repaired and, as a result, remain incompletely understood. This review hopes to gather less studied mechanisms in nucleotide modifications and show research gaps in our current understanding of nucleotide biology. By examining the implications of these mechanisms on DNA modifications, in the nucleotide pool and genome, we may gain insights into innovative strategies for mitigating the effects of cellular aging. Full article

Background/Objectives: Thyroid hormones are key regulators in hepatic metabolic pathways. Although they regulate various hepatic genes, only a few are known to be under direct transcriptional regulation through thyroid hormone receptors. To better understand the roles of thyroid hormones in the liver, it is critical to identify thyroid hormone-responsive genes at the cellular level. Methods: A cDNA microarray analysis was applied to primary cultures of rat hepatic cells treated with triiodothyronine (T3) at 10⁻⁹ M for 24 h to identify the differentially expressed genes. The identified gene expressions were further examined in vivo using F344 rats. The reporter gene assay was performed to investigate the transcriptional activity of the upstream region of the gene. Results: A limited number of genes were listed, and only three of them, pyridoxal kinase (Pdxk), phosphoenolpyruvate carboxykinase 1 (Pck1), and solute carrier family 17 member 2 (Slc17a2), were confirmed to be upregulated by quantitative RT-PCR. The mRNA expression of these genes increased in the livers of F344 rats after T3 injection, suggesting the physiological relevance in vivo. There are two partially conserved thyroid hormone-responsive elements (TREs) in the upstream region of the rat Pdxk gene. The reporter gene assay indicated that an imperfect TRE (5′-gGGTCAxxxxAGGaCt-3′) located at −2146 was sufficient for the thyroid hormone-induced transcription of the gene. Conclusions: The present study identified novel T3-responsive genes, pdxk and Slc17a2. Promoter analyses showed that a single TRE in the pdxk gene accounts for the transcriptional regulation by T3. Full article

Cancers that arise from germline mutations of breast cancer associated gene 1 (BRCA1), which is a crucial player in homologous recombination (HR) DNA repair, are vulnerable to DNA-damaging agents such as platinum and PARP inhibitors (PARPis). Increasing evidence suggests that BRCA1 is an essential driver of all phases of the cell cycle, thereby maintaining orderly steps during cell cycle progression. Specifically, loss of BRCA1 activity causes the S-phase, G₂/M, spindle checkpoints, and centrosome duplication to be dysregulated, thereby blocking cell proliferation and inducing apoptosis. In vertebrates, loss of HR genes such as BRCA1 and/or BRCA2 is lethal, since HR is a prerequisite for genome integrity. Thus, cancer cells utilize alternative DNA repair pathways such as non-homologous end joining (NHEJ) to cope with the loss of BRCA1 function. In this review, we attempt to update and discuss how these novel components are crucial for regulating DNA damage repair (DDR) in BRCA1-deficient cancers. Full article

Background: Triple-negative breast cancer (TNBC) is a highly aggressive subtype with limited effective treatments available, including targeted therapies, often leading to poor prognosis. Mitotic checkpoint kinase BUB1 is frequently overexpressed in TNBC and correlates with poor survival outcomes suggesting its potential as a therapeutic target. This study explores the cytotoxicity of TNBC cells to BUB1 inhibition, alone or in combination with radiation and demonstrates that ferroptosis, an iron-dependent form of programmed cell death, has a role. Methods: TNBC cell lines (SUM159, MDA-MB-231, and BT-549) were treated with a BUB1 inhibitor BAY1816032 (BUB1i) alone or in combination with the ferroptosis activator RSL3 with or without 4 Gy irradiation. Cell viability assays were conducted to assess treatment effects, qPCR analyses measured expression of key ferroptosis markers including ACSL4, GPX4, PTGS2, SLC7A11, NCOA4, IREB2, NFS1, and TFRC expression, and TBARS assay measured the lipid peroxidation levels. Ferroptosis specificity was confirmed through co-treatment with the ferroptosis inhibitor Ferrostatin-1 (F-1). Results: In all TNBC cell lines studied, BUB1 inhibition significantly induced ferroptosis, marked by increased expression of ACSL4 and PTGS2, decreased expression of GPX4 and SLC7A11, and increased lipid peroxidation levels. The combination of BUB1i with RSL3 further amplified these ferroptotic markers, suggesting at least an additive effect, which was not present with the combination of BUB1i and radiation. Co-treatment with Ferrostatin-1 reversed the expression of ferroptosis markers, suggesting that BUB1i-mediated cell death may involve ferroptotic signaling in TNBC cell lines. Conclusions: This study demonstrates that BUB1 inhibition may independently induce ferroptosis in TNBC cell lines, which is enhanced when combined with a ferroptosis activator. Further research is warranted to delineate the molecular mechanism of BUB1-mediated ferroptosis in TNBC. Full article

Background and Methods: We assessed the prokaryotic and eukaryotic diversity present in non-vegetated and vegetated soils on King George Island, Maritime Antarctic, in combination with measurements of carbon dioxide fluxes. Results: For prokaryotes, 381 amplicon sequence variants (ASVs) were assigned, dominated by the phyla Actinobacteriota, Acidobacteriota, Pseudomonadota, Chloroflexota, and Verrucomicrobiota. A total of 432 eukaryotic ASVs were assigned, including representatives from seven kingdoms and 21 phyla. Fungi dominated the eukaryotic communities, followed by Viridiplantae. Non-vegetated soils had higher diversity indices compared with vegetated soils. The dominant prokaryotic ASV in non-vegetated soils was Pyrinomonadaceae sp., while Pseudarthrobacter sp. dominated vegetated soils. Mortierella antarctica (Fungi) and Meyerella sp. (Viridiplantae) were dominant eukaryotic taxa in the non-vegetated soils, while Lachnum sp. (Fungi) and Polytrichaceae sp. (Viridiplantae) were dominant in the vegetated soils. Measured CO₂ fluxes indicated that the net ecosystem exchange values measured in vegetated soils were lower than ecosystem respiration in non-vegetated soils. However, the total flux values indicated that the region displayed positive ecosystem respiration values, suggesting that the soils may represent a source of CO₂ in the atmosphere. Conclusions: Our study revealed the presence of rich and complex communities of prokaryotic and eukaryotic organisms in both soil types. Although non-vegetated soils demonstrated the highest levels of diversity, they had lower CO₂ fluxes than vegetated soils, likely reflecting the significant biomass of photosynthetically active plants (mainly dense moss carpets) and their resident organisms. The greater diversity detected in exposed soils may influence future changes in CO₂ flux in the studied region, for which comparisons of non-vegetated and vegetated soils with different microbial diversities are needed. This reinforces the necessity for studies to monitor the impact of resident biota on CO₂ flux in different areas of Maritime Antarctica, a region strongly impacted by climatic changes. 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/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: 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: 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