Cytotoxicity, genotoxicity and mutagenicity of mixed ternary mononuclear Mg complex based on valproic acid with 1,10-phenanthroline in Saccharomyces cerevisiae and V79 cells.

Valproic acid (VA) is a widely used drug for the treatment of diseases affecting the central nervous system. Due to its epigenetic modulatory potential, it has been studied for possible therapeutic application in anticancer therapies. However, the VA exhibits different side effects in its application. Thus, synthetic coordination complexes with valproate can generate promising candidates for new active drugs with reduced toxicity. In this sense, we investigated the genotoxic and mutagenic potential of the sodium valproate and of the mixed ternary mononuclear Mg complex based on VA with 1,10-phenanthroline (Phen) ligand - [Mg (Valp)2Phen], in Saccharomyces cerevisiae and V79 cells. The MTT and clonal survival assays in V79 cells indicated that the Mg complex has higher cytotoxicity than sodium valproate. A similar cytotoxicity profile is observed in yeast. This fact is possibly due to the intercalation capacity of [Mg(Valp)2Phen], inducing DNA strand breaks, as observed in the comet assay and micronucleus test. In this sense, members of the NER, HR, NHEJ and TLS repair pathways are required for the repair of DNA lesions induced by [Mg(Valp)2Phen]. Interestingly, BER proteins apparently increase the cytotoxic potential of the drug. Furthermore, the [Mg(Valp)2Phen] showed higher cytotoxicity in V79 cells and yeast when compared to sodium valproate indicating applicability as a cytotoxic agent.

RNA or DNA? Revisiting the Chemical Nature of the Cenancestral Genome.

One of the central issues in the understanding of early cellular evolution is the characterisation of the cenancestor. This includes the description of the chemical nature of its genome. The disagreements on this question comprise several proposals, including the possibility that AlkB-mediated methylation repair of alkylated RNA molecules may be interpreted as evidence of a cenancestral RNA genome. We present here an evolutionary analysis of the cupin-like protein superfamily based on tertiary structure-based phylogenies that includes the oxygen-dependent AlkB and its homologs. Our results suggest that the repair of methylated RNA molecules is the outcome of the enzyme substrate ambiguity, and doesn´t necessarily indicates that the last common ancestor was endowed with an RNA genome.

Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li-Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.

An important step towards the comprehensive sun protection: Blue-light exposure inhibits DNA repair in reconstituted human skin and a broadband sunscreen avoids this inhibition.

The field of sun protection is quickly changing and the research article by Douki et al., published in the current issue of Photochemistry and Photobiology, reported key experimental data that will certainly help the development of better sun care products. Mutagenic photoproducts (CPDs, cyclobutane pyrimidine dimers and 6-4PPs, pyrimidine-6-4-pyrimidone photoproducts) were formed in the reconstructed human epidermis (RHE) by UVB (312 nm) irradiation, and their concentrations were detected by HPLC-MS/MS as a function of time after the UVB treatment. RHE had been previously exposed or not (control) to blue light (427 nm). Both CPDs and 6-4PPs were shown to last longer in blue-light irradiated RHE, proving the inhibition of the DNA repair by blue light exposure. This is a highly relevant information because sunscreens allow people to enjoy longer periods under the sun and consequently, to endure very high doses of blue light. The work also reported results obtained with RHEs previously treated with a sunscreen formulation containing a broadband filter that offers blue-light protection. Interestingly, authors observed that the DNA repair was not significantly inhibited in RHE previously treated with the sunscreen offering broadband protection. Readers will find a scientifically sound proof of the importance of blue-light protection in sun care products.

FGF18 impairs blastocyst viability, DNA double-strand breaks and maternal recognition of pregnancy genes.

Embryonic mortality in cattle is high, reaching 10-40 % in vivo and 60-70 % in vitro. Death of embryos involves reduced expression of genes related to embryonic viability, inhibition of DNA repair and increased DNA damage. In follicular granulosa cells, FGF18 from the theca layer increases apoptosis and DNA damage, so we hypothesized that FGF18 may also affect the oocyte and contribute to early embryonic death. The aims of this study were to identify the effects of FGF18 on cumulus expansion, oocyte maturation and embryo development from cleavage to blastocyst stage using a conventional bovine in vitro embryo production system using ovaries of abattoir origin. Addition of FGF18 during in-vitro maturation did not affect FSH-induced cumulus expansion or rates of nuclear maturation. When FGF18 was present in the culture system, rates of cleavage were not affected however, blastocyst and expanded blastocyst development was substantially inhibited (P < 0.05), indicating a delay of blastulation. The number of phosphorylated histone H2AFX foci per nucleus, a marker of DNA damage, was higher in cleavage-stage embryos cultured with FGF18 than in those from control group (P < 0.05). Furthermore, FGF18 decreased accumulation of PTGS2 and IFNT2 mRNA in blastocysts. In conclusion, these novel findings suggest that FGF18 plays a role in the regulation of embryonic death during the early stages of development by impairing DNA double-strand break repair and expression of genes associated with embryo viability and maternal recognition of pregnancy during the progression from oocyte to expanded blastocysts.

Prevalence of DNA-Repair Gene mutations in Mexican men with prostate cancer.

INTRODUCTION AND OBJECTIVE: Mexico reported 26,742 new cases of prostate cancer in 2020. Different risk factors have been identified in the pathogenesis of prostate cancer. Among them, genetic factors and alterations or mutations in specific genes have been described in different ethnic groups worldwide. The aim of our study is to report the prevalence of germline DNA-repair gene mutations in Mexican patients with prostate cancer. MATERIAL AND METHOD: We performed germline genetic testing in 50 patients with localized prostate cancer and 50 patients with metastatic prostate cancer. Demographic, clinical, and histopathological data were collected. RESULTS: Thirty-seven germline mutations were identified in 32 patients. The most commonly affected genes were ATM in 6%, followed by FANCA (5%), and ATR (4%). BRCA2 mutations were identified in 3%. The frequency of mutations was higher in the metastatic group. DISCUSSION AND CONCLUSION: The results of our study show different mutations from those reported in different populations or regions. The use of PARP inhibitors is indicated in patients with germline mutations, specifically BRCA2, showing improvement in overall survival and progression free survival. To our knowledge, this is the first study reporting the prevalence of mutations in DNA-repair genes in Mexican patients with prostate cancer.

PARP1 in the intersection of different DNA repair pathways, memory formation, and sleep pressure in neurons.

Poly(ADP-ribose) polymerase-1 (PARP1) is a bottleneck that connects different DNA pathways during a DNA damage response. Interestingly, PARP1 has a dualist role in neurons, acting as a neuroprotector and inducer of cell death in distinct neurological diseases. Recent studies significantly expanded our knowledge of how PARP1 regulates repair pathways in neurons and uncovered new roles for PARP1 in promoting sleep to enhance DNA repair. Likewise, PARP1 is deeply associated with memory consolidation, implying that it has multiple layers of regulation in the neural tissue. In this review, we critically discuss PARP1 recent advances in neurons, focusing on its interplay with different DNA repair mechanisms, memory, and sleep. Provocative questions about how oxidative damage is accessed, and different hypotheses about the molecular mechanisms influenced by PARP1 in neurons are presented to expand the debate of future studies.

Bacillus subtilis stress-associated mutagenesis and developmental DNA repair.

SUMMARYThe metabolic conditions that prevail during bacterial growth have evolved with the faithful operation of repair systems that recognize and eliminate DNA lesions caused by intracellular and exogenous agents. This idea is supported by the low rate of spontaneous mutations (10-9) that occur in replicating cells, maintaining genome integrity. In contrast, when growth and/or replication cease, bacteria frequently process DNA lesions in an error-prone manner. DNA repairs provide cells with the tools needed for maintaining homeostasis during stressful conditions and depend on the developmental context in which repair events occur. Thus, different physiological scenarios can be anticipated. In nutritionally stressed bacteria, different components of the base excision repair pathway may process damaged DNA in an error-prone approach, promoting genetic variability. Interestingly, suppressing the mismatch repair machinery and activating specific DNA glycosylases promote stationary-phase mutations. Current evidence also suggests that in resting cells, coupling repair processes to actively transcribed genes may promote multiple genetic transactions that are advantageous for stressed cells. DNA repair during sporulation is of interest as a model to understand how transcriptional processes influence the formation of mutations in conditions where replication is halted. Current reports indicate that transcriptional coupling repair-dependent and -independent processes operate in differentiating cells to process spontaneous and induced DNA damage and that error-prone synthesis of DNA is involved in these events. These and other noncanonical ways of DNA repair that contribute to mutagenesis, survival, and evolution are reviewed in this manuscript.

Prevalent role of homologous recombination in the repair of specific double-strand breaks in Rhizobium etli.

Double-strand breaks (DSBs) are the most dangerous injuries for a genome. When unrepaired, death quickly ensues. In most bacterial systems, DSBs are repaired through homologous recombination. Nearly one-quarter of bacterial species harbor a second system, allowing direct ligation of broken ends, known as Non-Homologous End Joining (NHEJ). The relative role of both systems in DSBs repair in bacteria has been explored only in a few cases. To evaluate this in the bacterium Rhizobium etli, we used a modified version of the symbiotic plasmid (264 kb), containing a single copy of the nifH gene. In this plasmid, we inserted an integrative plasmid harboring a modified nifH gene fragment containing an I-SceI site. DSBs were easily inflicted in vivo by conjugating a small, replicative plasmid that expresses the I-SceI nuclease into the appropriate strains. Repair of a DSB may be achieved through homologous recombination (either between adjacent or distant repeats) or NHEJ. Characterization of the derivatives that repaired DSB in different configurations, revealed that in most cases (74%), homologous recombination was the prevalent mechanism responsible for repair, with a relatively minor contribution of NHEJ (23%). Inactivation of the I-SceI gene was detected in 3% of the cases. Sequence analysis of repaired derivatives showed the operation of NHEJ. To enhance the number of derivatives repaired through NHEJ, we repeated these experiments in a recA mutant background. Derivatives showing NHEJ were readily obtained when the DSB occurred on a small, artificial plasmid in a recA mutant. However, attempts to deliver a DSB on the symbiotic plasmid in a recA background failed, due to the accumulation of mutations that inactivated the I-SceI gene. This result, coupled with the absence of derivatives that lost the nonessential symbiotic plasmid, may be due to an unusual stability of the symbiotic plasmid, possibly caused by the presence of multiple toxin-antitoxin modules.

mRNA Expression and Methylation of the RAD51, ATM, ATR, BRCA1, and BRCA2 Genes in Gastric Adenocarcinoma.

Background: Immunohistochemical prognostic significance of the homologous recombination-related proteins RAD51, ATM, BRCA1, and BRCA2 is known in gastric adenocarcinoma, one of the deadliest cancers. Objective and design: This retrospective cohort study aimed to evaluate mRNA expression and promoter methylation of some homologous recombination-related genes in this neoplasm. Methods: We evaluated mRNA expression and methylation of RAD51, ATM, ATR, BRCA1, and BRCA2 in tumor and non-tumor frozen samples from gastrectomy specimens by RT-qPCR and MS-HRM, correlating our results with previous immunohistochemistry data and prognostic features. Results: RAD51, ATR, BRCA1, BRCA2, and ATM mRNA expression was detected in 93.75% (45/48), 93.75% (45/48), 91.67% (44/48), 83.33% (40/48), and 89.58% (43/48) of the tumors; partial or complete methylation, in 94.87% (37/39), 0 (0/42), 97.56% (40/41), 100% (41/41), and 0 (0/40), respectively. Most gene pairs showed significant weak to moderate positive correlations of tumoral mRNA expression with each other: RAD51 with ATR (P = .027), BRCA1 (P < .001), and BRCA2 (P < .001); ATR with BRCA1 (P = .007), and ATM (P = .001); BRCA1 with BRCA2 (P = 0.001). BRCA1 mRNA was reduced in tumors compared with non-neoplastic mucosa (0.345 vs 1.272, P = .015) and, excluding neoadjuvant therapy cases, in T3 to T4 tumors compared with T2 (0.414 vs 0.954, P = .035). Greater tumoral RAD51 mRNA levels correlated with perineural invasion (1.822 vs 0.725, P = .010) and death (1.664 vs 0.929, P = .036), but not with survival time. There was an inverse association between nuclear immunohistochemical positivity for ATR and its mRNA levels (0.487 vs 0.907, P = .032), and no significant correlation for the other markers. Conclusions: Our results suggest RAD51, BRCA1, and BRCA2 methylation as a frequent epigenetic mechanism in gastric cancer, support the hypothesis that reduced BRCA1 expression participates in disease progression, and show an association between RAD51 mRNA and perineural invasion and mortality that may be considered unexpected, considering the former immunohistochemical studies. The lack of correlation between immunohistochemistry and mRNA, and even the inverse association, for ATR, can be seen as indicative of action of post-transcriptional or post-translational regulatory mechanisms, to be better investigated.

X-linked congenital adrenal hypoplasia: Report of long clinical follow-up and description of a new complex variant in the NR0B1 gene.

Adrenal hypoplasia congenita, attributed to NR0B1 pathogenic variants, accounts for more than 50% of the incidence of primary adrenal insufficiency in children. Although more than 250 different deleterious variations have been described, no genotype-phenotype correlation has been defined to date. We report a case of an adopted boy who reported the onset of an adrenal crisis at 2 weeks of age, requiring replacement therapy with mineralocorticoids and glucocorticoids for 4 months. For 3 years, he did well without treatment. At almost 4 years of age, the disorder was restarted. A long follow-up showed the evolution of hypogonadotropic hypogonadism. Molecular studies on NR0B1 revealed a novel and deleterious deletion-insertion-inversion-deletion complex rearrangement sorted in the 5'-3' direction, which is described as follows: (1) deletion of the intergenic region (between TASL and NR0B1 genes) and 5' region, (2) insertion of a sequence containing 37 bp at the junction of the intergenic region of the TASL gene and a part of exon 1 of the NR0B1 gene, (3) inversion of a part of exon 1, (4) deletion of the final portion of exon 1 and exon 2 and beginning of the 3'UTR region, (5) maintenance of part of the intergenic sequence (between genes MAGEB1 and NR0B1, telomeric sense), (6) large posterior deletion, in the same sense. The path to molecular diagnosis was challenging and involved several molecular biology techniques. Evaluating the breakpoints in our patient, we assumed that it was a nonrecurrent rearrangement that had not yet been described. It may involve a repair mechanism known as nonhomologous end-joining (NHEJ), which joins two ends of DNA in an imprecise manner, generating an "information scar," represented herein by the 37 bp insertion. In addition, the local Xp21 chromosome architecture with sequences capable of modifying the DNA structure could impact the formation of complex rearrangements.

Enzymes for dermatological use.

Skin is the ultimate barrier between body and environment and prevents water loss and penetration of pathogens and toxins. Internal and external stressors, such as ultraviolet radiation (UVR), can damage skin integrity and lead to disorders. Therefore, skin health and skin ageing are important concerns and increased research from cosmetic and pharmaceutical sectors aims to improve skin conditions and provide new anti-ageing treatments. Biomolecules, compared to low molecular weight drugs and cosmetic ingredients, can offer high levels of specificity. Topically applied enzymes have been investigated to treat the adverse effects of sunlight, pollution and other external agents. Enzymes, with a diverse range of targets, present potential for dermatological use such as antioxidant enzymes, proteases and repairing enzymes. In this review, we discuss enzymes for dermatological applications and the challenges associated in this growing field.

Decreased DNA repair capacity caused by exposure to metal mixtures is modulated by the PARP1 rs1136410 variant in newborns from a polluted metropolitan area.

BACKGROUND: DNA damage caused by exposure to metal mixtures and the potential modulating role of genes involved in DNA repair and the antioxidant response have not been evaluated in newborns. AIM: The aim was to evaluate the association between prenatal exposure to metal mixtures and DNA repair capacity (DRC) in newborns from the Metropolitan Area of Mexico City (MAMC), a heavily polluted area, and the impact of variants in genes involved in DNA repair and the antioxidant response on this association. METHODS: We analyzed cord blood samples obtained at delivery from 125 healthy newborns from the MAMC. Twenty-four elements were determined by inductively coupled plasma mass spectrometry (ICP‒MS), but only 12 (Cu, I, Se, Zn, As, Ba, Cs, Mn, Sb, Sr, Pb, and Ti) were quantified in most samples. DRC was assessed by the challenge-comet assay, and OGG1, PARP1, and NFE2L2 genotyping was performed with TaqMan probes. Metal mixtures were identified and analyzed using principal component analysis (PCA) and weighted quantile sum (WQS) regression. Independent adjusted linear regression models were used to evaluate the associations. RESULTS: A null DRC was observed in 46% of newborns. The metals with the highest concentrations were Mn, Sr, Ti, and Pb. Essential elements showed normal levels. Only the mixture characterized by increased As, Cs, Cu, Se, and Zn levels was inversely associated with DRC. As was the principal contributor (37.8%) in the negative direction in the DRC followed by Ba and Sb, according to the WQS regression. Newborns carrying of the derived (G) allele of the PARP1 rs1136410 variant showed decreased DRC by exposure to some potentially toxic metals (PTMs) (As, Cs, and Ba). CONCLUSION: Prenatal exposure to metal mixtures negatively affected DRC in newborns, and the PARP1 rs1136410 variant had a modulating role in this association.

Evaluation of Meibomian gland dysfunction using meibography in patients with xeroderma pigmentosum

ABSTRACT Purpose: To assess Meibomian gland dysfunction using meibography in patients with xeroderma pigmentosum and correlate with ocular surface changes. Methods: This cross-sectional study evaluated patients with xeroderma pigmentosum. All patients underwent a comprehensive and standardized interview. The best-corrected visual acuity of each eye was determined. Detailed ophthalmic examination was conducted, including biomicroscopy examination of the ocular surface, Schirmer test type I, and meibography, and fundus examination was also performed when possible. Meibomian gland dysfunction was assessed by non-contact meibography using Oculus Keratograph® 5M (OCULUS Inc., Arlington, WA, USA). Saliva samples were collected using the Oragene DNA Self-collection kit (DNA Genotek Inc., Ottawa, Canada), and DNA was extracted as recommended by the manufacturer. Factors associated with abnormal meiboscores were assessed using generalized estimating equation models. Results: A total of 42 participants were enrolled, and 27 patients underwent meibography. The meiboscore was abnormal in the upper eyelid in 8 (29.6%) patients and in the lower eyelid in 17 (62.9%). The likelihood of having abnormal meiboscores in the lower eyelid was 16.3 times greater than that in the upper eyelid. In the final multivariate model, age (p=0.001), mutation profile (p=0.006), and presence of ocular surface malignant tumor (OSMT) (p=0.014) remained significant for abnormal meiboscores. For a 1-year increase in age, the likelihood of abnormal meiboscores increased by 12%. Eyes with OSMT were 58.8 times more likely to have abnormal meiboscores than eyes without ocular surface malignant tumor. Conclusion: In the final model, age, xeroderma pigmentosum profile, previous cancer, and clinical alterations on the eyelid correlated with a meiboscore of ≥2. Meibomian gland dysfunction was common in patients with xeroderma pigmentosum, mainly in the lower eyelid. The severity of Meibomian gland dysfunction increases with age and is associated with severe eyelid changes.

The dark side of SIRT7.

Sirtuin 7 (SIRT7) is a member of the sirtuin family and has emerged as a key player in numerous cellular processes. It exhibits various enzymatic activities and is predominantly localized in the nucleolus, playing a role in ribosomal RNA expression, DNA damage repair, stress response and chromatin compaction. Recent studies have revealed its involvement in diseases such as cancer, cardiovascular and bone diseases, and obesity. In cancer, SIRT7 has been found to be overexpressed in multiple types of cancer, including breast cancer, clear cell renal cell carcinoma, lung adenocarcinoma, prostate adenocarcinoma, hepatocellular carcinoma, and gastric cancer, among others. In general, cancer cells exploit SIRT7 to enhance cell growth and metabolism through ribosome biogenesis, adapt to stress conditions and exert epigenetic control over cancer-related genes. The aim of this review is to provide an in-depth understanding of the role of SIRT7 in cancer carcinogenesis, evolution and progression by elucidating the underlying molecular mechanisms. Emphasis is placed on unveiling the intricate molecular pathways through which SIRT7 exerts its effects on cancer cells. In addition, this review discusses the feasibility and challenges associated with the development of drugs that can modulate SIRT7 activity.

ALT-EJ genera rearreglos cromosómicos en respuesta a Etopósido en células humanas con los principales sistemas de reparación de rupturas de doble cadena comprometidos / ALT-EJ originates chromosomal rearrangements in response to Etoposide in human cells with the main DNA double-strand break repair systems compromised

RESUMEN La droga antitumoral Etopósido (ETO) induce rupturas de doble cadena en el ADN (RDC) y promueve el desarrollo de neoplasias secundarias en los pacientes tratados. Dos mecanismos principales, recombinación homóloga (HR) y reunión de extremos no-homólogos clásica (c-NHEJ) reparan las RDC. Cuando HR y c-NHEJ son defectuosas, la vía alternativa de reunión de extremos (alt-EJ) dependiente de PARP-1 está implicada. Se examinó la participación de alt-EJ en la progresión de las RDC inducidas por ETO en la fase G2 de células humanas. Se establecieron células HeLa deficientes en HR (inhibición de cohesina RAD21, HeLa RAD21kd) y su control no-silenciada (HeLa NS). Las células se trataron con ETO en presencia del inhibidor químico de DNA-PKcs (DNA-PKi, c-NHEJ). En ambas líneas celulares, la inducción de RDC (γH2AX+) por ETO en la fase G2 aumentó respecto a sus controles. La reparación incorrecta en células deficientes en DNA-PKcs y RAD21 originó un incremento sinérgico de intercambio de cromátidas y de cromosomas dicéntricos en la primera y segunda metafase, respectivamente. En cambio, la frecuencia de cromosomas dicéntricos se redujo en células deficientes en PARP-1 (HeLa PARP- 1kd) luego del tratamiento con ETO. En células binucleadas HeLa RAD21kd, DNA-PKi/ETO acrecentó el porcentaje de células con ≥20 focos γH2AX en la fase G1-posmitótica y de micronúcleos a las 96 h. Una mayor acumulación en G2/M se observó en HeLa NS tratadas con DNA-PKi/ETO en relación a HeLa RAD21kd a las 8 h. El ciclo celular se reanudó en HeLa NS a las 16 h, sin embargo, la acumulación se mantuvo en HeLa RAD21kd. Los rearreglos cromosómicos obtenidos con DNA-PKcs y RAD21 disfuncionales y su disminución en células HeLa PARP-1kd, sugieren que alt-EJ contribuye a su formación.

ABSTRACT The antitumor drug Etoposide (ETO) induces DNA double-strand breaks (DSB) and is associated with the development of secondary neoplasms in treated patients. DSB are repaired by two main mechanisms, homologous recombination (HR) and classical non-homologous end joining (c-NHEJ). When HR and c-NHEJ are defective, DSB are repaired by the PARP-1-dependent alternative end-joining (alt-EJ) pathway. The involvement of alt-EJ in the progression of DSB induced by ETO in the G2 phase of human cells was analyzed. HeLa cells deficient in HR (cohesin RAD21 inhibition, HeLa RAD21kd) and their nonsilencing control (HeLa NS) were established. Cells were treated with ETO in the presence of a chemical inhibitor of DNA-PKcs (DNA-PKi, c-NHEJ). In both cell lines, ETO-induced DSB (γH2AX+) in G2 phase were increased compared to their controls. The incorrect repair of DSB in DNA-PKcs- and RAD21-deficient cells caused a synergistic augment in chromatid exchanges and dicentric chromosomes in the first and second metaphase, respectively. In contrast, the frequency of dicentric chromosomes was reduced in PARP-1-deficient cells (HeLa PARP-1kd) following ETO treatment. In HeLa RAD21kd binucleated cells, DNA-PKi/ETO increased the percentage of cells with ≥20 γH2AX foci in the G1-postmitotic phase and of micronuclei at 96 h. A greater accumulation in G2/M was observed in HeLa NS treated with DNA-PKi/ ETO compared with HeLa RAD21kd at 8 h. The cell cycle restarted in HeLa NS at 16 h; however, the G2/M accumulation was maintained in HeLa RAD21kd. Chromosomal rearrangements obtained when DNAPKcs and RAD21 were absent and their decrease in HeLa PARP-1kd cells suggest that alt-EJ contributes to their formation.

Could violet-blue lights increase the bacteria resistance against ultraviolet radiation mediated by photolyases?

Studies have demonstrated bacterial inactivation by radiations at wavelengths between 400 and 500 nm emitted by low-power light sources. The phototoxic activity of these radiations could occur by oxidative damage in DNA and membrane proteins/lipids. However, some cellular mechanisms can reverse these damages in DNA, allowing the maintenance of genetic stability. Photoreactivation is among such mechanisms able to repair DNA damages induced by ultraviolet radiation, ranging from ultraviolet A to blue radiations. In this review, studies on the effects of violet and blue lights emitted by low-power LEDs on bacteria were accessed by PubMed, and discussed the repair of ultraviolet-induced DNA damage by photoreactivation mechanisms. Data from such studies suggested bacterial inactivation after exposure to violet (405 nm) and blue (425-460 nm) radiations emitted from LEDs. However, other studies showed bacterial photoreactivation induced by radiations at 348-440 nm. This process occurs by photolyase enzymes, which absorb photons at wavelengths and repair DNA damage. Although authors have reported bacterial inactivation after exposure to violet and blue radiations emitted from LEDs, pre-exposure to such radiations at low fluences could activate the photolyases, increasing resistance to DNA damage induced by ultraviolet radiation.

Toxicity assessment and DNA repair kinetics in HEK293 cells exposed to environmentally relevant concentrations of Glyphosate (Roundup® Control Max).

Glyphosate is a systemic, non-selective, pre and post-emergence wide range herbicide. In 2015, IARC classified Glyphosate as "a probable carcinogenic agent for humans". The aim of this study was to evaluate the cytotoxicity and genotoxicity of the commercial formulation of glyphosate (Roundup® Control Max) at environmentally relevant concentrations and measure the potential effect of this herbicide over the cell capacity to repair DNA damage. HEK293 cells were exposed to 5 concentrations of Roundup® Control Max equivalent to 0.7; 7; 70; 700 and 3,500 µg/L glyphosate acid, for 1, 4 and 24 h. Cytotoxicity was quantified by the Trypan Blue staining method and by the MTT assay, while genotoxicity and evaluation of DNA damage repair kinetics were analyzed through the alkaline comet assay. In all treatments, cell viability was higher than 80%. The three highest glyphosate concentrations-70 µg/L, 700 µg/L, and 3,500 µg/L-increased levels of DNA damage compared to the control at the three exposure times tested. Finally, concerning the kinetics of DNA damage repair, cells initially exposed to 3,500 µg/L of glyphosate for 24 h were unable to repair the breaks in DNA strands even after 4 h of incubation in culture medium. The present study demonstrated for the first time that Roundup® Control Max may induce genetic damage and cause alterations in the DNA repair system in human embryonic kidney cells even at concentrations found in blood and breast milk of people exposed through residues of the herbicide in food, which values have been poorly assessed or not studied yet according to the existent literature.

A Benchmark of In-House Homologous Recombination Repair Deficiency Testing Solutions for High-Grade Serous Ovarian Cancer Diagnosis.

Homologous recombination deficiency (HRD) has become an important prognostic and predictive biomarker for patients with high-grade serous ovarian cancer who may benefit from poly-ADP ribose polymerase inhibitors (PARPi) and platinum-based therapies. HRD testing provides relevant information to personalize patients' treatment options and has been progressively incorporated into diagnostic laboratories. Here, we assessed the performance of an in-house HRD testing system deployable in a diagnostic clinical setting, comparing results from two commercially available next-generation sequencing (NGS)-based tumor tests (SOPHiA DDMTM HRD Solution and AmoyDx® (HRD Focus Panel)) with the reference assay from Myriad MyChoice® (CDx). A total of 85 ovarian cancer samples were subject to HRD testing. An overall strong correlation was observed across the three assays evaluated, regardless of the different underlying methods employed to assess genomic instability, with the highest pairwise correlation between Myriad and SOPHiA (R = 0.87, p-value = 3.39 × 10-19). The comparison of the assigned HRD status to the reference Myriad's test revealed a positive predictive value (PPV) and negative predictive value (NPV) of 90.9% and 96.3% for SOPHiA's test, while AmoyDx's test achieved 75% PPV and 100% NPV. This is the largest HRD testing evaluation using different methodologies and provides a clear picture of the robustness of NGS-based tests currently offered in the market. Our data shows that the implementation of in-house HRD testing in diagnostic laboratories is technically feasible and can be reliably performed with commercial assays. Also, the turnaround time is compatible with clinical needs, making it an ideal alternative to offer to a broader number of patients while maintaining high-quality standards at more accessible price tiers.

ROCK inhibition reduces the sensitivity of mutant p53 glioblastoma to genotoxic stress through a Rac1-driven ROS production.

Resistance to radio and chemotherapy in Glioblastoma (GBM) is correlated with its malignancy, invasiveness, and aggressiveness. The Rho GTPase pathway plays important roles in these processes, but its involvement in the GBM response to genotoxic treatments remains unsolved. Inhibition of this signaling pathway has emerged as a promising approach for the treatment of CNS injuries and diseases, proving to be a strong candidate for therapeutic approaches. To this end, Rho-associated kinases (ROCK), classic downstream effectors of small Rho GTPases, were targeted for pharmacological inhibition using Y-27632 in GBM cells, expressing the wild-type or mutated p53 gene, and exposed to genotoxic stress by gamma ionizing radiation (IR) or cisplatin (PT). The use of the ROCK inhibitor (ROCKi) had opposite effects in these cells: in cells expressing wild-type p53, ROCKi reduced survival and DNA repair capacity (reduction of γH2AX foci and accumulation of strand breaks) after stress promoted by IR or PT; in cells expressing the mutant p53 protein, both treatments promoted longer survival and more efficient DNA repair, responses further enhanced by ROCKi. The target DNA repair mechanisms of ROCK inhibition were, respectively, an attenuation of NHEJ and NER pathways in wild-type p53 cells, and a stimulation of HR and NER pathways in mutant p53 cells. These effects were accompanied by the formation of reactive oxygen species (ROS) induced by genotoxic stress only in mutant p53 cells but potentiated by ROCKi and reversed by p53 knockdown. N-acetyl-L-cysteine (NAC) treatment or Rac1 knockdown completely eliminated ROCKi's p53-dependent actions, since ROCK inhibition specifically elevated Rac-GTP levels only in mutant p53 cells. Combining IR or PT and ROCKi treatments broadens our understanding of the sensitivity and resistance of, respectively, GBM expressing wild-type or mutant p53 to genotoxic agents. Our proposal may be a determining factor in improving the efficiency and assertiveness of CNS antitumor therapies based on ROCK inhibitors. SIGNIFICANCE: The use of ROCK inhibitors in association with radio or chemotherapy modulates GBM resistance and sensitivity depending on the p53 activity, suggesting the potential value of this protein as therapeutic target for tumor pre-sensitization strategies.