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DNA, Volume 5, Issue 1 (March 2025) – 9 articles

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18 pages, 1239 KiB  
Review
Contribution of Androgen Receptor CAG Repeat Polymorphism to Human Reproduction
by Alessandro Ciarloni, Nicola delli Muti, Nicola Ambo, Michele Perrone, Silvia Rossi, Sara Sacco, Gianmaria Salvio and Giancarlo Balercia
DNA 2025, 5(1), 9; https://doi.org/10.3390/dna5010009 (registering DOI) - 8 Feb 2025
Abstract
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 [...] Read more.
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
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24 pages, 3436 KiB  
Article
Transcription Factor Inhibition as a Potential Additional Mechanism of Action of Pyrrolobenzodiazepine (PBD) Dimers
by Julia Mantaj, Paul J. M. Jackson, Richard B. Parsons, Tam T. T. Bui, David E. Thurston and Khondaker Miraz Rahman
DNA 2025, 5(1), 8; https://doi.org/10.3390/dna5010008 - 5 Feb 2025
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Abstract
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 [...] Read more.
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
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10 pages, 2422 KiB  
Article
Partial Proliferating Cell Nuclear Antigen Functional Knockout Impairs Cisplatin Resistance and Clonogenic Potential in Lung Adenocarcinoma Cells
by Ana Paula Morelli, Nathalia Quintero-Ruiz, Mariana Camargo Silva Mancini, Isadora Carolina Betim Pavan, Isabelle Lima Flores, Luiz Guilherme Salvino Silva, Matheus Brandemarte Severino, Rosangela Maria Neves Bezerra and Fernando Moreira Simabuco
DNA 2025, 5(1), 7; https://doi.org/10.3390/dna5010007 - 2 Feb 2025
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Abstract
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 [...] Read more.
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
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8 pages, 640 KiB  
Perspective
The Evolutionary Reasons of Epigenetics
by Giorgio Camilloni
DNA 2025, 5(1), 6; https://doi.org/10.3390/dna5010006 - 30 Jan 2025
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Abstract
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 [...] Read more.
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
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18 pages, 3544 KiB  
Article
MafB Transcription Factor Involved in IRD-Induced AKI (Acute Kidney Injury) Phenotype Attenuation and Inflammation Resolution
by Dhouha Daassi
DNA 2025, 5(1), 5; https://doi.org/10.3390/dna5010005 - 17 Jan 2025
Viewed by 589
Abstract
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, [...] Read more.
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 MafBf/f::Lys-Cre mice kidneys compared to their littermates, MafBf/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
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13 pages, 1991 KiB  
Article
Outcomes of Broader Genomic Profiling in Metastatic Colorectal Cancer: A Portuguese Cohort Study
by Ricardo Roque, Rita Santos, Luís Guilherme Santos, Rita Coelho, Isabel Fernandes, Gonçalo Cunha, Marta Gonçalves, Teresa Fraga, Judy Paulo and Nuno Bonito
DNA 2025, 5(1), 4; https://doi.org/10.3390/dna5010004 - 14 Jan 2025
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Abstract
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 [...] Read more.
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
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15 pages, 5138 KiB  
Review
Targeting Thyroid Hormone Receptor Interacting Protein (TRIP13) for Cancer Therapy: A Promising Approach
by Surya P. Singh, Krishnendu Goswami, Gopal Pathuri, Chinthalapally V. Rao and Venkateshwar Madka
DNA 2025, 5(1), 3; https://doi.org/10.3390/dna5010003 - 6 Jan 2025
Viewed by 609
Abstract
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 [...] Read more.
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
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14 pages, 754 KiB  
Article
Preliminary Results of Reduced Polymerase Chain Reaction (PCR) Volumes When Analysing Low Template DNA Samples with Globalfiler™ and Yfiler™ Plus Kits
by Jesús Martínez-Gómez, Sheila Laso-Izquierdo, Araceli Vera-Yánez, José Juan Fernández-Serrano and Cláudia Gomes
DNA 2025, 5(1), 2; https://doi.org/10.3390/dna5010002 - 1 Jan 2025
Viewed by 1107
Abstract
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 [...] Read more.
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
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15 pages, 2887 KiB  
Communication
Generation of Cas9 Knock-In Culex quinquefasciatus Mosquito Cells
by Elizabeth Walsh, Tran Zen B. Torres, Brian C. Prince and Claudia Rückert
DNA 2025, 5(1), 1; https://doi.org/10.3390/dna5010001 - 1 Jan 2025
Viewed by 832
Abstract
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 [...] Read more.
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
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