Human DNA topoisomerase I poisoning causes R loop-mediated genome instability attenuated by transcription factor IIS.

DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5'-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G1 or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.

Sleep disturbance in clinical and preclinical scrapie-infected sheep measured by polysomnography.

Neurodegenerative diseases are characterised by neuronal loss and abnormal deposition of pathological proteins in the nervous system. Among the most common neurodegenerative diseases are Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease and transmissible spongiform encephalopathies (TSEs). Sleep and circadian rhythm disturbances are one of the most common symptoms in patients with neurodegenerative diseases. Currently, one of the main objectives in the study of TSEs is to try to establish an early diagnosis, as clinical signs do not appear until the damage to the central nervous system is very advanced, which prevents any therapeutic approach. In this paper, we provide the first description of sleep disturbance caused by classical scrapie in clinical and preclinical sheep using polysomnography compared to healthy controls. Fifteen sheep classified into three groups, clinical, preclinical and negative control, were analysed. The results show a decrease in total sleep time as the disease progresses, with significant changes between control, clinical and pre-clinical animals. The results also show an increase in sleep fragmentation in clinical animals compared to preclinical and control animals. In addition, sheep with clinical scrapie show a total loss of Rapid Eye Movement sleep (REM) and alterations in Non Rapid Eyes Movement sleep (NREM) compared to control sheep, demonstrating more shallow sleep. Although further research is needed, these results suggest that prion diseases also produce sleep disturbances in animals and that polysomnography could be a diagnostic tool of interest in clinical and preclinical cases of prion diseases.

What is in the fish? Collaborative trial in suspect and non-target screening of organic micropollutants using LC- and GC-HRMS.

A collaborative trial involving 16 participants from nine European countries was conducted within the NORMAN network in efforts to harmonise suspect and non-target screening of environmental contaminants in whole fish samples of bream (Abramis brama). Participants were provided with freeze-dried, homogenised fish samples from a contaminated and a reference site, extracts (spiked and non-spiked) and reference sample preparation protocols for liquid chromatography (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS). Participants extracted fish samples using their in-house sample preparation method and/or the protocol provided. Participants correctly identified 9-69 % of spiked compounds using LC-HRMS and 20-60 % of spiked compounds using GC-HRMS. From the contaminated site, suspect screening with participants' own suspect lists led to putative identification of on average ∼145 and ∼20 unique features per participant using LC-HRMS and GC-HRMS, respectively, while non-target screening identified on average ∼42 and ∼56 unique features per participant using LC-HRMS and GC-HRMS, respectively. Within the same sub-group of sample preparation method, only a few features were identified by at least two participants in suspect screening (16 features using LC-HRMS, 0 features using GC-HRMS) and non-target screening (0 features using LC-HRMS, 2 features using GC-HRMS). The compounds identified had log octanol/water partition coefficient (KOW) values from -9.9 to 16 and mass-to-charge ratios (m/z) of 68 to 761 (LC-HRMS and GC-HRMS). A significant linear trend was found between log KOW and m/z for the GC-HRMS data. Overall, these findings indicate that differences in screening results are mainly due to the data analysis workflows used by different participants. Further work is needed to harmonise the results obtained when applying suspect and non-target screening approaches to environmental biota samples.

Break-induced RNA-DNA hybrids (BIRDHs) in homologous recombination: friend or foe?

Double-strand breaks (DSBs) are the most harmful DNA lesions, with a strong impact on cell proliferation and genome integrity. Depending on cell cycle stage, DSBs are preferentially repaired by non-homologous end joining or homologous recombination (HR). In recent years, numerous reports have revealed that DSBs enhance DNA-RNA hybrid formation around the break site. We call these hybrids "break-induced RNA-DNA hybrids" (BIRDHs) to differentiate them from sporadic R-loops consisting of DNA-RNA hybrids and a displaced single-strand DNA occurring co-transcriptionally in intact DNA. Here, we review and discuss the most relevant data about BIRDHs, with a focus on two main questions raised: (i) whether BIRDHs form by de novo transcription after a DSB or by a pre-existing nascent RNA in DNA regions undergoing transcription and (ii) whether they have a positive role in HR or are just obstacles to HR accidentally generated as an intrinsic risk of transcription. We aim to provide a comprehensive view of the exciting and yet unresolved questions about the source and impact of BIRDHs in the cell.

DICER ribonuclease removes harmful R-loops.

R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.

Localized Colonic Small-Cell Carcinoma with Pathological Complete Response after Neoadjuvant Cisplatin and Etoposide: A Case Report.

Extrapulmonary small-cell carcinoma (SCC) is a rare neoplasm that shares certain features with its pulmonary counterpart and occurs predominantly in the gastrointestinal tract (GIT). It is a high-grade and poorly differentiated neuroendocrine tumor, usually diagnosed in advanced stages, with a poor prognosis and few therapeutic options in that setting. This is a case report of a 77-year-old Spanish male patient with localized SCC of the colon, who presented a pathological complete response in the surgical specimen after neoadjuvant chemotherapy with cisplatin and etoposide. To date, 5 years after surgery, the patient remains without evidence of tumor recurrence. As clinical guidelines for the management of this entity are lacking, and therefore its management has not been standardized, an attempt to summarize the current evidence in the literature was made.

Binary Graft of Poly(acrylic acid) and Poly(vinyl pyrrolidone) onto PDMS Films for Load and Release of Ciprofloxacin.

Polymers are versatile compounds which physical and chemical properties can be taken advantage of in wide applications. Particularly, in the biomedical field, polydimethylsiloxane (PDMS) is one of the most used for its high biocompatibility, easy manipulation, thermal, and chemical stability. Nonetheless, its hydrophobic nature makes it susceptible to bacterial pollution, which represents a disadvantage in this field. A potential solution to this is through the graft of stimuli-sensitive polymers that, besides providing hydrophilicity, allow the creation of a drug delivery system. In this research, PDMS was grafted with acrylic acid (AAc) and vinyl pyrrolidone (VP) in two steps using gamma radiation. The resulting material was analyzed by several characterization techniques such as infrared spectroscopy (FTIR), swelling, contact angle, critical pH, and thermogravimetric analysis (TGA), demonstrating the presence of both polymers onto PDMS films and showing hydrophilic and pH-response properties. Among the performed methods to graft, the loading and release of ciprofloxacin were successful in those samples obtained by direct irradiation method. Furthermore, the antimicrobial assays showed zones of inhibition for microorganisms such as Staphylococcus aureus and Escherichia coli.

Non-dipping blood pressure pattern in pediatricians during on-duty.

INTRODUCTION: People with a reduced nighttime dip in blood pressure have an increased cardiovascular risk. Our objective was to describe the different patterns in blood pressure (BP) among pediatricians who work in long on-duty shifts in relation with sex, medical rank and sleeping time. METHODS: Descriptive, cross-sectional, two-center study. On duty pediatric Resident physicians and pediatric Consultants were recruited between January 2018 and December 2021. RESULTS: Fifty-one physicians were included in the study (78.4% female, 66.7% Resident physicians). Resident physicians had a higher night/day ratio (0.91 vs 0.85; p<0.001) and a shorter nighttime period (3.87 vs 5.41, p<0.001) than Consultants. Physicians sleeping less than 5h had a higher night/day ratio (0.91 vs 0.87, p=0.014). Being a Resident showed a ∼4.5-fold increased risk of having a non-dipping BP pattern compared to Consultants. CONCLUSION: We found a potential link between both being a Resident and, probably, having shorter sleeping time, and the non-dipping BP pattern in physicians during prolonged shifts.

Telemedicina en el control de la hipertensión arterial

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Introduction: Arterial hypertension is the great challenge of health policies in the world. Its high prevalence, the lack of control (linked to poor adherence to antihypertensive treatment) and its high "burden" (as a non-communicable disease) are responsible for the increase in cardiovascular morbidity and mortality. Objetive: To evaluate, as a pilot test, the benefit of real-time telemonitoring applied to blood pressure (BP) control. Metodology: Inclusion criteria: Diagnosed hypertensives, under pharmacological treatment, assisted in the Maciel Hospital polyclinic in the period June-October 2019. Exclusion criteria: pregnancy and upper arm circumference less than 22 cm or greater than 42 cm. Home blood pressure monitoring was implemented, using a validated electronic blood pressure monitor and a smart cell phone connected to a private web platform in real time. Analytical, experimental, prospective study. Qualitative variables were expressed in absolute frequency and percentage relative frequency. Quantitative variables were expressed as mean and standard deviation. The normality of the distribution was verified using the Kolmogorov Smirnov test. Results: Of a total of 57 patients, the mean age was 59 ± 12.8 years, female sex 64.9%. Presenting 22.8% poor knowledge of the disease. Regarding the control of BP figures, 31.6% reported not checking regularly. Adherence to pharmacological treatment at the beginning of the monitoring was 33.3%. Figures lower than those described by RENATA-2, where half of the treated hypertensives complied with the treatment. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were analyzed at the beginning and end of the study. Pre-monitoring measurements were mean SBP: 138.6 ± 17.8 mmHg and mean DBP: 85.4 ± 14.8 mmHg. The records at the end of the immediate monitoring were 131.5 ± 19.9 mmHg and 81.5 ± 14.2 mmHg for PAS and PAD, respectively. Finding a significant difference between the means of the PAS (p 0.019), but not between the means of the DBP (p 0.06). Discussion: Although the percentage of patients achieving good BP control was significant, these results may be closely related to a subtype of observed performance bias, rather than the outcome of the intervention. The mean days of telemonitoring were 8, 85, number of shots was 34.12, and number of interventions performed by the observer (medication change) was 0.30. User satisfaction with the method used was 96.4%, and 100% considered it Easy or very easy to use. 87.5% would use it for long periods of time, including older adults. Conclusions: The studied method shows potential benefits for the control of BP figures, with a wide acceptance by users, facilitating access to the health system, and quality medical care.

Introdução: A hipertensão arterial é o grande desafio das políticas de saúde no mundo. Sua alta prevalência, o descontrole (ligado à baixa adesão ao tratamento anti-hipertensivo) e sua alta "carga" (como doença não transmissível) são responsáveis ​​pelo aumento da morbimortalidade cardiovascular. Objetivo: foi avaliar como teste piloto, o benefício do telemonitoramento em tempo real, aplicado ao controle da pressão arterial (PA). Metodologia: Critérios de inclusão: Hipertensos diagnosticados, em tratamento farmacológico, atendidos na policlínica do Hospital Maciel no período de junho a outubro de 2019. Critérios de exclusão: gravidez e circunferência do braço menor que 22 cm ou maior que 42 cm. Foi implementado o monitoramento domiciliar da pressão arterial, utilizando um monitor eletrônico de pressão arterial validado e um telefone celular inteligente conectado a uma plataforma web privada em tempo real. Estudo analítico, experimental, prospectivo.As variáveis ​​qualitativas foram expressas em frequência absoluta e frequência relativa percentual. As variáveis ​​quantitativas foram expressas como média e desvio padrão. A normalidade da distribuição foi verificada por meio do teste de Kolmogorov Smirnov. Resultados: De um total de 57 pacientes, a média de idade foi de 59 ± 12,8 anos, sexo feminino 64,9%. Apresentando 22,8% conhecimento ruim sobre a doença. Em relação ao controle dos valores da PA, 31,6% relataram não verificar regularmente. A adesão ao tratamento farmacológico no início do acompanhamento foi de 33,3%, valores inferiores aos descritos pelo RENATA-2, onde metade dos hipertensos tratados aderiu ao tratamento. A pressão arterial sistólica (PAS) e a pressão arterial diastólica (PAD) foram analisadas no início e no final do estudo. As medições de pré-monitoramento foram PAS média: 138,6 ± 17,8 mmHg e PAD média: 85,4 ± 14,8 mmHg. Os registros ao final do monitoramento imediato foram de 131,5 ± 19,9 mmHg e 81,5 ± 14,2 mmHg para PAS e PAD, respectivamente. Encontrando diferença significativa entre as médias do PAS (p 0,019), mas não entre as médias do DBP (p 0,06). Discussão: Embora a porcentagem de pacientes que alcançaram um bom controle da PA tenha sido significativa, esses resultados podem estar intimamente relacionados a um subtipo de viés de desempenho observado, e não ao resultado da intervenção. A média de dias de telemonitoramento foi 8, 85, o número de disparos foi 34,12, e o número de intervenções realizadas pelo observador (mudança de medicação) foi de 0,30. A satisfação dos usuários com o método utilizado foi de 96,4%, sendo que 100% consideraram fácil ou muito fácil de usar. 87,5% usariam por longos períodos de tempo, inclusive idosos adultos. Conclusões: O método estudado apresenta potenciais benefícios para o controle dos valores da PA, com ampla aceitação pelos usuários, facilitando o acesso ao sistema de saúde e assistência médica de qualidade.

Newborn screening for propionic, methylmalonic acidemia and vitamin B12 deficiency. Analysis of 588,793 newborns.

OBJECTIVES: We present the results of our experience in the diagnosis and follow up of the positive cases for propionic, methylmalonic acidemias and cobalamin deficiencies (PA/MMA/MMAHC) since the Expanded Newborn Screening was implemented in Madrid Region. METHODS: Dried blood samples were collected 48 h after birth. Amino acids and acylcarnitines were quantitated by MS/MS. Newborns with alterations were referred to the clinical centers for follow-up. Biochemical and molecular genetic studies for confirmation of a disease were performed. RESULTS: In the period 2011-2020, 588,793 children were screened, being 953 of them were referred to clinical units for abnormal result (192 for elevated C3 levels). Among them, 88 were false positive cases, 85 maternal vitamin B12 deficiencies and 19 were confirmed to suffer an IEM (8 PA, 4 MMA, 7 MMAHC). Ten out 19 cases displayed symptoms before the NBS results (6 PA, 1 MMA, 3 MMAHC). C3, C16:1OH+C17 levels and C3/C2 and C3/Met ratios were higher in newborns with PA/MMA/MMAHC. Cases diagnosed with B12 deficiency had mean B12 levels of 187.6 ± 76.9 pg/mL and their mothers 213.7 ± 95.0; 5% of the mothers were vegetarian or had poor eating while 15% were diagnosed of pernicious anemia. Newborns and their mothers received treatment with B12 with different posology, normalizing their levels and the secondary alterations disappeared. CONCLUSIONS: Elevated C3 are a frequent cause for abnormal result in newborn screening with a high rate of false positive cases. Presymptomatic diagnosis of most of PA and some MMA/MMAHC is difficult. Vitamin B12 deficiency secondary to maternal deprivation is frequent with an heterogenous clinical and biochemical spectrum.

A role for the Saccharomyces cerevisiae Rtt109 histone acetyltransferase in R-loop homeostasis and associated genome instability.

The stability of the genome is occasionally challenged by the formation of DNA-RNA hybrids and R-loops, which can be influenced by the chromatin context. This is mainly due to the fact that DNA-RNA hybrids hamper the progression of replication forks, leading to fork stalling and, ultimately, DNA breaks. Through a specific screening of chromatin modifiers performed in the yeast Saccharomyces cerevisiae, we have found that the Rtt109 histone acetyltransferase is involved in several steps of R-loop-metabolism and their associated genetic instability. On the one hand, Rtt109 prevents DNA-RNA hybridization by the acetylation of histone H3 lysines 14 and 23 and, on the other hand, it is involved in the repair of replication-born DNA breaks, such as those that can be caused by R-loops, by acetylating lysines 14 and 56. In addition, Rtt109 loss renders cells highly sensitive to replication stress in combination with R-loop-accumulating THO-complex mutants. Our data evidence that the chromatin context simultaneously influences the occurrence of DNA-RNA hybrid-associated DNA damage and its repair, adding complexity to the source of R-loop-associated genetic instability.

Activation of the integrated stress response is a vulnerability for multidrug-resistant FBXW7-deficient cells.

FBXW7 is one of the most frequently mutated tumor suppressors, deficiency of which has been associated with resistance to some anticancer therapies. Through bioinformatics and genome-wide CRISPR screens, we here reveal that FBXW7 deficiency leads to multidrug resistance (MDR). Proteomic analyses found an upregulation of mitochondrial factors as a hallmark of FBXW7 deficiency, which has been previously linked to chemotherapy resistance. Despite this increased expression of mitochondrial factors, functional analyses revealed that mitochondria are under stress, and genetic or chemical targeting of mitochondria is preferentially toxic for FBXW7-deficient cells. Mechanistically, the toxicity of therapies targeting mitochondrial translation such as the antibiotic tigecycline relates to the activation of the integrated stress response (ISR) in a GCN2 kinase-dependent manner. Furthermore, the discovery of additional drugs that are toxic for FBXW7-deficient cells showed that all of them unexpectedly activate a GCN2-dependent ISR regardless of their accepted mechanism of action. Our study reveals that while one of the most frequent mutations in cancer reduces the sensitivity to the vast majority of available therapies, it renders cells vulnerable to ISR-activating drugs.

Detection of R-Loops by In Vivo and In Vitro Cytosine Deamination in Saccharomyces cerevisiae.

R-loops are transcriptional by-products formed by a hybrid of the nascent RNA molecule with its DNA template and the displaced nontemplate DNA strand. The single stranded nature of the displaced nontemplate strand makes it vulnerable to attack. This property is used in nature to cause directed mutagenesis and breaks by the action of the activation-induced cytosine deaminase (AID) enzyme and can thus be exploited to detect the presence of R-loops even when they form at low frequencies by overexpressing this enzyme in vivo or by in vitro treatment with the bisulfite anion, which further allows nucleotide resolution. This is of particular relevance given the fact that R-loops have the potential to hamper DNA replication and repair, threatening genome integrity. Here, we describe the protocols used in the yeast Saccharomyces cerevisiae to infer the presence of R-loops through increased AID-induced DNA damage, measured as increased recombination or Rad52 foci formation as well as to detect single R-loop molecules and determine their length at particular genomic sites via bisulfite treatment and amplification.

Expression of immune checkpoint molecules programmed death protein 1, programmed death-ligand 1 and inducible T-cell co-stimulator in mycosis fungoides and Sézary syndrome: association with disease stage and clinical outcome.

BACKGROUND: The relationship between immune checkpoint status and disease outcome is a major focus of research in cutaneous T-cell lymphoma (CTCL), a disfiguring neoplastic dermatological disorder. Mycosis fungoides (MF) and Sézary syndrome (SS) are the two most common types of CTCL. OBJECTIVES: The aim was to evaluate the immune checkpoint markers programmed death protein 1 (PD1), inducible T-cell co-stimulator (ICOS) and programmed death-ligand 1 (PD-L1) in skin biopsies from patients with CTCL relative to disease stage and overall survival. METHODS: This consecutive case series enrolled 47 patients: 57% had stage IA-IIA disease and 43% had stage IIB-IVA2 disease (including seven with SS). RESULTS: PD1, PD-L1 and ICOS expression was seen in all biopsies. Notably, PD-L1 was predominantly expressed on histiocytes/macrophages, but focal expression on CTCL cells was seen. High expression of either ICOS or PD-L1 was associated with advanced-stage disease (P = 0·007 for both) and with the appearance of large-cell transformation (LCT), a histopathological feature associated with a poor prognosis (ICOS: P = 0·02; PD-L1: P = 0·002). PD1 expression was not significantly associated with disease stage (P = 0·12) or LCT (P = 0·49), but expression was high in SS biopsies. A high combined checkpoint marker score (PD1, PD-L1 and ICOS) was associated with advanced-stage disease (P = 0·001), LCT (P = 0·021) and lower overall survival (P = 0·014). CONCLUSIONS: These findings demonstrate the existence of a complex immunoregulatory microenvironment in CTCL and support the development of immunotherapies targeting ICOS and PD-L1 in advanced disease.