A Genetic Map of the Response to DNA Damage in Human Cells.

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

Genome-scale mapping of DNA damage suppressors through phenotypic CRISPR-Cas9 screens.

To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.

CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition.

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Endogenous DNA 3' Blocks Are Vulnerabilities for BRCA1 and BRCA2 Deficiency and Are Reversed by the APE2 Nuclease.

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

Fish endoparasite metacommunity in environments with different degrees of conservation in the western Brazilian Amazon.

Parasite communities correspond to the definition of metacommunity, as species interact and disperse within hosts. The present study evaluated parasite metacommunities in a tropical floodplain. The study was conducted in the Western Amazon around the municipalities of Cruzeiro do Sul, state of Acre, and Guajará, state of Amazonas, Brazil. Six sampling sites were selected and grouped into conserved and degraded environments. Fish were caught between periods of drought and flood, using passive and active sampling methods; in the laboratory, they were measured weighed, and necropsied. Parasites found were fixed, evaluated, and identified. Physical and chemical variables and environmental conservation characteristics were measured in all sites. Diversity index, ANOVA, Tukey, local contribution to beta diversity (LCBD), species contribution to beta diversity by individual species (SCBD), and variance partitioning were summarized. The α species diversity increased in conserved environments and varied between seasonal periods, mainly in detritivorous and omnivorous hosts. Local contributions to beta diversity showed significantly higher values in conserved environments for the endoparasite fauna of piscivorous and omnivorous hosts, indicating that these environments presented unique parasite infracommunities and revealing the conservation status of these environments. Variations in infracommunities were explained mainly by niche-based processes, including environmental conditions, degree of conservation, and host characteristics. Thus, these data will serve as a tool to understand the way parasite communities are structured, which is important information for the management and conservation of aquatic environments.

Anatomic and Surgical Aspects of Total Pancreatectomy in Swine.

BACKGROUND: The swine is a valuable model for preclinical research and surgical technique training. Induction of Type I diabetes is achieved by total pancreatectomy, therefore these animals may be used in several research studies, including islet transplantation field. Given the lack of information in the literature, the purpose of this work is to describe anatomic aspects of swine pancreas, the total pancreatectomy surgical technique, intra- and postoperative complications and the autopsy results. MATERIAL AND METHODS: Five hybrid male pigs, 20-35 kg, submitted to total pancreatectomy with duodenum, bile duct, and spleen preservation. Postoperatively, daily clinical assessment and capillary blood glucose collection were performed. At the end of the 30-day period or in the occurrence of serious clinical complications, euthanasia and autopsy were performed. RESULTS: The average duration of surgery was 128 minutes, without intraoperative deaths or anesthesia induction failures. The median survival was 6.6 days. Postoperative complications were weight loss (3), emesis (2), constipation (2), abdominal distension (2), diarrhea (1), and loss of appetite (1). All animals were euthanized due to serious complications. Two animals presented surgical complications (duodenal necrosis with gastroparesis and internal hernia with intestinal necrosis). The other 3 animals presented serious clinical complications related to exocrine pancreatic insufficiency due to deficiency of pancreatic enzymes. Glycemic values above 200 mg/dL were found on the first postoperative day and above 300 mg/dL on the seventh day in all animals. CONCLUSION: A model of total pancreatectomy with duodenum, spleen, and bile duct preservation in pigs was established. All animals became diabetic, however, animals without postoperative complications were euthanized due to serious complications related to pancreas exocrine insufficiency.

Enhancing soil vapor extraction with EKSF for the removal of HCHs.

This paper evaluates the combination of electrokinetic soil flushing (EKSF) with soil vapor extraction (SVE) for the removal of four hexachlorocyclohexane (HCH) isomers contained in a real matrix. Results demonstrate that the combination of EKSF and SVE can be positive, but it is required the application of high electric fields (3 V cm-1) in order to promote a higher temperature in the system, which improves the volatilization of the HCH contained in the system. Electrokinetic transport is also enhanced with the application of higher electric gradients, but these transport processes are slower than the volatilization processes, which are the primary in this system. Hence collection of species in the electrolyte wells is negligible as compared to the compound dragged with air by the SVE but the temperature increase demonstrates a good performance. Combination of EKSF with SVE can efficiently exhaust the four HCH isomers reaching a removal of more than 90% after 15 days of treatment (20% more than values attained by SVE) but it is required the application of high electric fields to promote a higher temperature in the system (to improve the volatilization) and EK transport (to improve the dragging). 1-D transport model can be easily used to estimate the average pore water velocity and the effective diffusion of each compound under the different experimental conditions tested.

Impact of Brazil Nut (Bertholletia excelsa, H.B.K.) Supplementation on Body Composition, Blood Pressure, and the Vascular Reactivity of Wistar Rats When Submitted to a Hypersodium Diet.

Introdution: Endothelium integrity is a key that maintains vascular homeostasis but it can suffer irreversible damage by blood pressure changes, reflecting an imbalance in the maintenance of vascular homeostasis.Objective: The aim of this study was to investigate the impact of Brazil nut (Bertholletia excelsa, H.B.K.) (BN) supplementation (10% in chow, wt/wt) on the vascular reactivity of Wistar rats during chronic exposure to a sodium overload (1% in water).Methods: First, male Wistar rats were allocated into two groups: Control Group (CG) and the Hypersodic Group (HG) for 4 weeks. Afterward, the CG was divided into the Brazil Nut Group (BNG) and the HG Group into the Hypersodic Brazil Nut Group (HBNG) for a further 8 weeks, totaling 4 groups. Blood pressure was measured during the protocol. At the end of the protocol, the vascular reactivity procedure was performed. Glucose, lipid profile, lipid peroxidation, and platelet aggregation were analyzed in the serum. Body composition was determined by the carcass technique.Results: The groups that were supplemented with the BN chow presented less body mass gain and body fat mass, together with lower serum glucose levels. The HG Group presented an increase in blood pressure and a higher platelet aggregation, while the BN supplementation was able to blunt this effect. The HG Group also showed an increase in contractile response that was phenylephrine-induced and a decrease in maximum relaxation that was acetylcholine-induced when compared to the other groups.Conclusion: The BN supplementation was able to prevent an impaired vascular function in the early stages of arterial hypertension, while also improving body composition, serum glucose, and platelet aggregation.

The CIP2A-TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer.

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.

The influence of a diet based on flaxseed, an omega-3 source, during different developmental periods, on the blood pressure of rats submitted to stress.

OBJECTIVE: This study aimed to investigate a flaxseed diet during different developmental periods, and its effect on the blood pressure of rats submitted to stress. METHODS: Fifty-six male rats (F1), born from 14 rats (F0), were divided into seven groups (n = 8): flaxseed group (FG); flaxseed group gestation and lactation (FG-GL); flaxseed group weaning (FG-W); flaxseed group weaning and stress (FG-WS); flaxseed group stress (FG-S); flaxseed group gestation lactation and weaning (FG-GLW), and control Group (CG). Stress protocol was undertaken for 1 month. Blood pressure was analysed before and after the stress protocol. The left adrenal glands and serum corticosterone levels were analysed. RESULTS: Systolic blood pressure before stress was lower in all groups with flaxseed diet compared with the CG (p = .00001). After stress, CG showed higher blood pressure compared with FG, FG-GL, and FG-GLW (p = .004). The levels of corticosterone were lower in the FG between all groups (p < .000001) and the CG showed higher compared with FG-W, FG-WS, FG-GL, and FG-GLW (p < .0001). The adrenal gland did not show differences. CONCLUSIONS: Results suggest a possible factor from a flaxseed diet against the effects of stress on a blood pressure in all periods of life but especially in the gestation and lactation periods.

Development of an electronic nose to characterize odours emitted from different stages in a wastewater treatment plant.

Wastewater treatment plants have widely been described as a significant source of odour nuisance, which has led to an increase of neighbourhood complaints. Therefore, to mitigate the negative impact of odours, the detection and analysis of these emissions are required. This paper presents a measurement system based on an electronic nose for quantitative and qualitative odour analysis of samples collected from six different stages on a wastewater plant. Hence, two features vectors were performed in order to represent quantitative trends of the gaseous mixture sampled on the facility. In addition, odour fingerprints and a PCA were computed to discriminate odours from its sources and to detect relationships among the samples. This approach also comprises a dynamic dilution olfactometer. A PLS regression model was performed to predict the odour concentration by the electronic nose in term of odour units per cubic meter. The results show that the developed electronic nose is a promising and feasible instrument to characterize odours from wastewater plants.

Assessments of body composition and bone parameters of lactating rats treated with diet containing flaxseed meal (Linum usitatissinum) during post-weaning period.

INTRODUCTION: There are few studies on body composition and the effects of diet on weight postpartum women. The aim was to evaluate the body composition and bone parameters in lactating rats treated with diet containing flaxseed flour during postweaning period. METHODS: After weaning, the lactating rat were divided in control (n = 6) and experimental (F, n = 6) group, treated with 25% flaxseed flour diet. After 30 days, body composition by dual-energy X-ray absorptiometry, serum analysis, organs and intra-abdominal fat mass, femur and lumbar vertebra parameters were determined. RESULTS: The groups showed similar food intake, body mass and bone parameters. While F group showed the following: lower body (-5%), gonadal (-17%), mesenteric (-23%) and intra-abdominal (-6%) fat mass. Increase of HDL-cholesterol (+10%) and lower glucose (-15%), triglycerides (P < 0.05, -37%) and cholesterol (P < 0.05, -21%). CONCLUSIONS: The findings highlight the effects of flaxseed for control of adiposity and to maintain a healthy biochemical profile during the postnatal period.

Introducción: Hay pocos estudios sobre la composición corporal y los efectos de la dieta en mujeres en el periodo postparto. El objetivo consistió en evaluar la composición corporal y los parámetros óseos en ratas lactantes tratadas con dietas a base de linaza durante el periodo de destete. Métodos: Después del destete, las ratas lactantes fueron divididas en un grupo de control (n = 6) y un grupo experimental (F, n = 6), tratadas con una dieta a base de harina de lino al 25%. Al cabo de 30 días, se midieron los parámetros corporales mediante absorciometría de rayos X de doble energía, se realizó un análisis sérico, y se evaluó órganos y masa grasa intra-abdominal así como los parámetros en fémur y vértebras lumbares. Resultados: El grupo mostró una ingesta alimenticia similar, así como parámetros óseos y de masa corporal. Mientras que el grupo F mostró los porcentajes siguientes en masa grasa: parte inferior del cuerpo (-5%), gonadal (-17%), mesentérica (-23%) e intra-abdominal (-6%). Aumento de HDL-colesterol (+10%) y disminución de glucosa (-15%), triglicéridos (P < 0,05, -37%) y colesterol (P < 0,05, -21%). Conclusiones: Los resultados destacan los efectos del lino para el control de la adiposidad y para mantener un perfil bioquímico sano durante el periodo postnatal.