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The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM -mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM -mutant backgrounds are different to those in BRCA1 -mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation. Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. Here, the authors provide evidence that these hypersensitivities reflect a crucial role for ATM at damaged replication forks being to prevent toxic DNA end-joining leading to chromosome fusions and cell death.

Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities, and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). Here, we identify the deubiquitylating enzyme USP48 as synthetic viable for FA-gene deficiencies by performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2). Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA. Fanconi anemia is a rare disease caused by defective DNA interstrand crosslink repair. Here the authors observe that USP48 deficiencies reduce chromosomal instability in FA-defective cells, suggesting it might be a potential therapeutic target.

Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be monomeric, which rules out a role of the TPR domain in self-association.

Background It is well known that concurrent increases in both maximal strength and aerobic capacity are associated with improvements in sports performance as well as overall health. One of the most popular training methods used for achieving these objectives is resistance circuit-based training. Objective The objective of the present systematic review with a meta-analysis was to evaluate published studies that have investigated the effects of resistance circuit-based training on maximum oxygen uptake and one-repetition maximum of the upper-body strength (bench press exercise) in healthy adults. Methods The following electronic databases were searched from January to June 2016: PubMed, Web of Science and Cochrane. Studies were included if they met the following criteria: (1) examined healthy adults aged between 18 and 65 years; (2) met the characteristics of resistance circuit-based training; and (3) analysed the outcome variables of maximum oxygen uptake using a gas analyser and/or one-repetition maximum bench press. Results Of the 100 articles found from the database search and after all duplicates were removed, eight articles were analysed for maximum oxygen uptake. Of 118 healthy adults who performed resistance circuit-based training, maximum oxygen uptake was evaluated before and after the training programme. Additionally, from the 308 articles found for one-repetition maximum, eight articles were analysed. The bench press one-repetition maximum load, of 237 healthy adults who performed resistance circuit-based training, was evaluated before and after the training programme. Significant increases in maximum oxygen uptake and one-repetition maximum bench press were observed following resistance circuit-based training. Additionally, significant differences in maximum oxygen uptake and one-repetition maximum bench press were found between the resistance circuit-based training and control groups. Conclusions The meta-analysis showed that resistance circuit-based training, independent of the protocol used in the studies, is effective in increasing maximum oxygen uptake and one-repetition maximum bench press in healthy adults. However, its effect appears to be larger depending on the population and training characteristics. For large effects in maximum oxygen uptake, the programme should include ~14–30 sessions for ~6–12 weeks, with each session lasting at least ~20–30 min, at intensities between ~60 and 90% one-repetition maximum. For large effects in one-repetition maximum bench press, the data indicate that intensity should be ~30–60% one-repetition maximum, with sessions lasting at least ~22.5–60 min. However, the lower participant’s baseline fitness level may explain the lighter optimal loads used in the circuit training studies where greater strength gains were reported.

Mutations in the ATM tumor suppressor confer hypersensitivity to DNA-damaging agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase poison topotecan. Thus, we establish that loss of terminal components of the non-homologous end-joining (NHEJ) machinery or the BRCA1-A complex specifically confers topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib is due to delayed homologous recombination repair at DNA-replication-fork-associated double-strand breaks (DSBs), resulting in toxic NHEJ-mediated chromosome fusions. Accordingly, restoring legitimate repair in ATM-deficient cells, either by preventing NHEJ DNA ligation or by enhancing DSB-resection by BRCA1-A complex inactivation, markedly suppresses this toxicity. Our work suggests opportunities for patient stratification in ATM-deficient cancers and when using ATM inhibitors in the clinic, and identifies additional therapeutic vulnerabilities that might be exploited when such cancers evolve drug resistance.

ObjectiveWe aimed to analyze the association between inflammatory cytokine levels (IFN-a2, IFN-b, IFN-g, IL10 and BLyS) and disease activity for a 12 months of follow-up in SLE patients.MethodsA longitudinal, observational prospective study with evaluations at baseline and follow-up visits every 3 months in SLE patients (SLICC 2012 criteria) and 65 healthy controls was performed. In SLE patients complete laboratory test, clinical evaluation and SLEDAI score was carried out. We analyzed inflammatory cytokines serum levels by colorimetric methods in all cases.Results45 SLE patients (86.7% female) participated in the study, with a mean age at diagnosis of 32.8 (16.2) years and a mean time of disease evolution of 17.9 (11.4) years. The 28.9% of patients showed SLEDAI>6 at the basal visit. The 66.7% were under glucocorticoid treatment, 44.4% under immunosupressants (methotrexate, azatioprine, belimumab or mycophenolate) and 66.7% under antimalarials. SLEDAI and inflammatory cytokine levels during follow-up is shown in table 1.Abstract P9 Table 1 CONTROLS N=65 Mean (SD) SLE V0 N=45 Mean (SD) SLE V3 N=45 Mean (SD) SLE V6 N=45 Mean (SD) SLE V9 N=45 Mean (SD) SLE V12 N=45 Mean (SD) SLEDAI score --- 6.09 (5.38) 3.53 (3.41) 5.09 (4.06) 3.71 (2.87) 3.64 (2.61) IFN-alpha2 (pg/mL) 85.53 (104.72) 202.59 (608.11) 115.24 (219.33) 170.6 (634.44) 103.21 (194.39) 157.22 (523.12) IFN-beta (pg/mL) 38.49 (35.43) 74.61 (91.24) 72.65 (114.59) 76.2 (101.37) 77.23 (114.21) 74.55 (97.51) IFN-gamma (pg/mL) 150.95 (133.14) 257.18 (413.65) 293.62 (395.65) 334.26 (485.59) 289.19 (354.71) 370.53 (794.94) IL-10 (pg/mL) 5.66 (5.45) 14.35 (21.07) 15.48 (16.92) 11.79 (15.23) 11.02 (11.14) 11.86 (11.76) BLyS (pg/mL) 2576.86 (882.64) 3073.4 (2198.6) 6232.42 (4838.81) 4111.58 (3593.74) 4812.91 (3786.77) 5432.31 (4747.82) Statistical analysis showed significant association between SLEDAI score and IL-10 (P=0.014) and IFNa2 (0.009), as well as a tendency with IFN-beta (P=0.057), independently of the time of follow-up. Regarding to clinical activity biomarkers, we observed an association between high levels of antidsDNA and elevated IFN-beta (P=0.005) and IFN-gamma (P=0.038), and low levels of C3 and an increment in IL-10 (P=0.006).Patients under antimalarials treatment during follow-up exhibit low levels of IL-10 (P=0.012) and those under belimumab treatment showed high levels of BLyS (P<0.001). No influence of age at diagnosis, time of evolution, vitamin D levels, corticoids and tobacco use in cytokine levels was observed.SLE patients were categorized by normal or high level of the five cytokines, based on the cytokine level above 2 SD of the mean in healthy controls. Despite the fact that no specific cytokine profile associated with clinical activity was observed, those patients with high SLEDAI score had increased levels of IL10.ConclusionWe observed an association between IL-10, IFN-alpha2, IFN-beta and IFN-gamma levels with clinical activity, independently of the time of follow-up. IL-10 levels may be influenced by antimalarial treatment and BLyS levels by belimumab treatment.

ObjectiveSeveral drugs have been implicated in the development of de novo systemic lupus erythematosus (SLE), unmasking of quiescent SLE or causing an exacerbation of previously diagnosed SLE. Our aim is to describe the causative drugs, clinical and serological features of patients diagnosed of Drug-Induced lupus (DIL) in our Rheumatology Department.MethodsA total of 445 patients diagnosed with SLE treated in our Rheumatology Department from 2012 to 2023 were retrospectively screened for the fulfilment of DIL criteria through the search of medical electronic records. Demographic, clinical and laboratory data were summarised using descriptive statistics.ResultsWe identified 18 patients diagnosed with DIL, representing a prevalence of 4% among all SLE patients in our Department. There was a female preponderance and a young age at disease onset. Patients’ clinical and serological characteristics are shown in table 1. Only three drugs (infliximab, adalimumab and sulfasalazine) were identified as causative agents of DIL, anti-TNF being the most common. Most patients were treated for a condition different from a rheumatic disease, mainly inflammatory bowel disease (IBD). Median time to symptom onset after drug initiation ranged from 3 to 194 weeks (median 50.4). Peripheral arthritis and skin rash were the most frequent symptoms, with 4 patients (22%) presenting both at onset. Serologically, only 2 patients were ANA negative, but tested positive for anti-dsDNA. After drug withdrawal, ANA titre showed a slow decreasing trend over time, as well as anti-dsDNA antibodies. However, only 2 patients lost ANA-positivity through follow-up. Remarkably, more than half of the patients tested positive for antiphospholipid antibodies.ConclusionDIL showed a prevalence of 4% in our Rheumatology Department. Anti-TNF agents were the most common drugs causing DIL. ANA tend to decrease over time, but only become undetectable in a few patients. Antiphospholipid antibodies are common in our DIL patients. Age at onset is earlier than previously reported, probably because causative drugs are being used in younger populations.Abstract P28 Table 1Patients’ clinical and serological characteristics Age at onset (median years; IQR) 37.6 ± 19.6 Gender (n,%) Female 14 (78%) Male 4 (22%) Baseline diagnosis Inflammatory Bowel Disease 8 (44%) Inflammatory arthritis 4 (22%) Hidrosadenitis 3 (17%) Psoriasis 2 (11%) Nonspecific Orbital Inflammation 1 (6%) Drug Infliximab 12 (66%) Adalimumab 5 (28%) Sulfasalazine 1 (6%) Time to onset after drug initiation (median weeks, range) 50.4 (3 – 194) Symptoms at onset Peripheral arthritis 9 (50%) Skin rash 4 (22%) Peripheral arthritis AND skin rash 4 (22%) Inflammatory arthralgia 1 (6%) Autoantibodies profile ANA positivity 16 (89%) Median ANA titre 1/320 Low C3 and/or C4 5 (28%) Anti-Ro 1 (6%) Antihistone positivity 0 Anti-La 0 Anti-Sm 0 Antiphospholipid antibodies (AAF) 10 (55.5%) 1 AAF 5 (28%) 2 AAF 4 (22%) 3 AAF 1 (6%)

ObjectiveThe objective is to analyze the association between therapeutic adherence and disease activity in SLE patients.MethodsAn observational, prospective study in SLE patients (SLICC/ACR criteria), treated whit subcutaneous Belimumab (200 mg/week) was made. Disease activity was measured by SLEDAI in three consecutive visits, and it was considered clinical worsening an increase of SLEDAI-score of ≥4 point. Persistence and adherence of Belimumab data during the follow-up were collected and were calculated based on the number of drug dispensing. Poor therapeutic adherence was established under the 95%.ResultsThirty-one prescriptions of Belimumab were registered (83.9% women) with a mean age of 48.1 (14.9) years. Time since the diagnosis was 12.5 (6.29) years and treatment period were 2.2 (1.4) years. Fifteen patients were considered as non-adherent (48.4%).Persistence and disease activity data in each group were showed in the table 1.Abstract P149 Table 1Poor adherence was secondary to clinical improvement (66.67%), recurrent infections (13.33%), surgery (6.67%), pregnancy (6.67%) and inability to drug collect due to COVID-19 pandemic (6.67%). Non-adherent group showed worse SLEDAI-score than adherent group in V0, despite of a similar SLEDAI-score at V2 in both groups was observed. There was an association between poor therapeutic adherence and high delta_SLEDAI (p=0.046).ConclusionsWe observed an association between poor therapeutic adherence and delta_SLEDAI. The high SLEDAI-score at the beginning of the study in non-adherent group would be due to clinical manifestations, despite of the similar serological activity in both groups.

This dissertation explores how the nexus between the government and the private sector can generate misallocation of resources in the economy, and how government tools used to reduce this problem can impact the private sector. In the first chapter joint with Felipe González we notice that political transitions are associated with significant economic changes, but little is known about how firms fare across regimes. We study Chile's transition to democracy and show that firms in the dictator's network make critical investments in physical capital before the transition takes place. These investments are made possible by government banks during the dictatorship and allow them to improve their market position in the new regime. Our results show how market distortions can be transferred across regimes and suggest limited changes in the distribution of economic power after a democratization. The second chapter joint with Emanuele Colonnelli investigates the impact of anti-corruption audits on the private sector. In particular, we study whether firms are affected by the disclosure of information about corporate misconduct. We focus on a natural experiment given by a large Brazilian anti-corruption audit program, and we estimate both a difference-in-difference and an event study model that rely on the random timing of the audits for identification. Exploiting a special feature of the auditors' instruction manual, we are able to manually construct a dataset on more than 20,000 corruption cases. This dataset is then linked to matched employer-employee data, to the universe of federal procurement contracts and government loans, as well as to in-court prosecutions. We first show that government anti-corruption audits have significant real effects on the local economy. We then find that corrupt firms suffer a drop in employment and total wages after the information about their misconduct is revealed to the public. Concurrently, the probability that the corrupt firm fires the managers or CEO increases, leading to a restructuring at the top of the organization. We conclude by discussing the channels and mechanisms at play, and the potential policy implications. Finally in the third chapter joint with Emanuele Colonnelli and Edoardo Teso, we ask how the political career of politicians affects their labor market returns. We collect data on the labor market history of Brazilian local candidates, leveraging close elections to obtain exogenous variation in political power. We find no evidence of a return to office in the labor market outside of politics. On the contrary, we show that losers obtain substantial gains following their unsuccessful race, which come in the form of better employment opportunities in the public sector. These benefits exist only for unelected candidates aligned with the mayor in power, are increasing in a candidate's electoral performance, and are strictly tied to the fortunes of a candidate's party. These findings are consistent with an insurance mechanism in which parties and coalitions reward candidates for their electoral efforts in the negative state in which they fail to be elected. We provide evidence suggesting that this system creates incentives to increase the size of public sector employment and leads to misallocation of skills in the public sector.