The TRESLIN-MTBP complex couples completion of DNA replication with S/G2 transition.

It has been proposed that ATR kinase senses the completion of DNA replication to initiate the S/G2 transition. In contrast to this model, we show here that the TRESLIN-MTBP complex prevents a premature entry into G2 from early S-phase independently of ATR/CHK1 kinases. TRESLIN-MTBP acts transiently at pre-replication complexes (preRCs) to initiate origin firing and is released after the subsequent recruitment of CDC45. This dynamic behavior of TRESLIN-MTBP implements a monitoring system that checks the activation of replication forks and senses the rate of origin firing to prevent the entry into G2. This system detects the decline in the number of origins of replication that naturally occurs in very late S, which is the signature that cells use to determine the completion of DNA replication and permit the S/G2 transition. Our work introduces TRESLIN-MTBP as a key player in cell-cycle control independent of canonical checkpoints.

ATR prohibits replication catastrophe by preventing global exhaustion of RPA.

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors.

TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.

Replication Catastrophe: When a Checkpoint Fails because of Exhaustion.

Proliferating cells rely on the so-called DNA replication checkpoint to ensure orderly completion of genome duplication, and its malfunction may lead to catastrophic genome disruption, including unscheduled firing of replication origins, stalling and collapse of replication forks, massive DNA breakage, and, ultimately, cell death. Despite many years of intensive research into the molecular underpinnings of the eukaryotic replication checkpoint, the mechanisms underlying the dismal consequences of its failure remain enigmatic. A recent development offers a unifying model in which the replication checkpoint guards against global exhaustion of rate-limiting replication regulators. Here we discuss how such a mechanism can prevent catastrophic genome disruption and suggest how to harness this knowledge to advance therapeutic strategies to eliminate cancer cells that inherently proliferate under increased DNA replication stress.

Skin microbiome disturbance linked to drought-associated amphibian disease.

The onset of global climate change has led to abnormal rainfall patterns, disrupting associations between wildlife and their symbiotic microorganisms. We monitored a population of pumpkin toadlets and their skin bacteria in the Brazilian Atlantic Forest during a drought. Given the recognized ability of some amphibian skin bacteria to inhibit the widespread fungal pathogen Batrachochytrium dendrobatidis (Bd), we investigated links between skin microbiome health, susceptibility to Bd and host mortality during a die-off event. We found that rainfall deficit was an indirect predictor of Bd loads through microbiome disruption, while its direct effect on Bd was weak. The microbiome was characterized by fewer putative Bd-inhibitory bacteria following the drought, which points to a one-month lagged effect of drought on the microbiome that may have increased toadlet susceptibility to Bd. Our study underscores the capacity of rainfall variability to disturb complex host-microbiome interactions and alter wildlife disease dynamics.

The cardiorespiratory system of miniature frogs.

Anurans of the genus Brachycephalus are among the smallest vertebrates in the world, due to an extreme process of miniaturization. As an example of this process, Brachycephalus species show loss of fingers, loss of the eardrum and middle ear, bone fusions, and the presence of paravertebral plates and parotic plaque. However, no studies addressing the consequences of miniaturization on internal organs, such as the lungs and heart, are currently available. Thus, this study aimed to investigate if overall small body size has affected the cardiorespiratory system. We investigated, via dissections, individuals of four Brachycephaloidea species: Brachycephalus rotenbergae, B. pitanga, Eleutherodactylus johnstonei, and Ischnocnema parva. We observed that B. rotenbergae and B. pitanga present a reduction of the atrial septum and absence of the carotid body. On the other hand, despite being a member of the sister genus to Brachycephalus (both genera belong to the Brachycephalidae), individuals of Ischnocnema present a heart with a complete septum and carotid body; this is also observed in E. johnstonei (Eleutherodactylidae). We observed that B. rotenbergae and B. pitanga have thin skin with a one to two cell thick germ layer, and their lungs likely exhibit lower blood supply when compared to individuals of the E. johnstonei and I. parva species. Based on the observed structures, we suggest that in species of Brachycephalus, respiration is performed mainly through the skin, and their lungs may have a reduced respiratory function.

Novices' learning curve in single-port surgery using three surgical training programs.

BACKGROUND: The objective of this study was to evaluate the novices' learning curves and proficiency level reached in laparoendoscopic single-site (LESS) surgery using three surgical training programs. MATERIAL AND METHODS: Participants were randomly divided into three groups, who trained in a specific practice regimen for 12 days using a laparoscopic box simulator and three tasks. Group A trained in three stages using conventional laparoscopic surgery (CLS) with straight instruments, and LESS with straight and articulating instruments for four days each. Group B trained in two stages in LESS with straight and articulating instruments for six days each. Group C trained only in LESS with articulating instruments exclusively for all 12 days. Performance was registered daily during the 12 days to evaluate the participants' progress. RESULTS: Pre- and post-training analysis of the three groups showed significant differences in performance, denoting the significant improvement in their LESS skills, with no difference between the groups. Group C reached a high level of technical competence with their specific training program in LESS, obtaining a lower asymptote and slow learning rate. CONCLUSION: Specific training programs in LESS settings using articulated instruments showed a slower learning rate than the other programs but better proficiency in the technique with the best surgical performance.

ASO Author Reflections: PSMA PET in the Prostate Cancer Primary Staging Scenario-A Reliable Method with a Remarkable Decision-Making Impact.

Prostate cancer (PCa) is the second most common malignant tumor in men in Brazil and worldwide. Although positron emission tomography (PET) prostate-specific membrane antigen (PSMA) has been used for over 10 years and several studies have been published regarding its superior performance in PCa primary staging and other scenarios, the management decision is often still based on conventional imaging information. A retrospective analysis was performed in 35 patients with PCa in the primary staging scenario with conventional exams as well as with PET PSMA. The findings of our study showed changes on the staging classification and significant impact on the therapeutic choice. PET PSMA is a dependable imaging exam and has already proved its worth and superiority when compared with conventional methods to evaluate PCa patients in primary staging and biochemical relapse, in addition to other potential roles. Prospective studies are needed to assess the outcome of patients whose management was guided by PSMA.

Impact of Prostate-Specific Membrane Antigen Positron Emission Tomography/Computed Tomography on the Therapeutic Decision of Prostate Carcinoma Primary Staging: A Retrospective Analysis at the Brazilian National Public Health System.

BACKGROUND: Prostate cancer (PCa) is the most common malignant tumor in males and conventional imaging does not provide accurate primary staging. Prostate-specific membrane antigen (PSMA) positron emission tomography (PET) presents superior performance and strongly affects therapeutic choice. OBJECTIVE: The aim of this study was to evaluate the impact of PSMA PET, compared with conventional imaging methods, on the therapeutic approach in primary staging scenarios in patients with PCa treated at the Brazilian National Public Health System. METHODS: Overall, 35 patients diagnosed with PCa were evaluated using PSMA after conventional staging imaging with multiparametric magnetic resonance (MMR) and/or total abdominal computed tomography (CT) scan and bone scintigraphy (BS). The PCa extension identified by PET was compared with conventional imaging; staging changes and the management impact were then determined. PET comparison with conventional imaging, staging, and decision-making changes was analyzed using descriptive statistics. RESULTS: PET revealed local disease (LD) in 15 (42.9%) patients, seminal vesicle invasion (SVI) in 5 (14.3%) patients, pelvic nodal impairment (PNI) in 7 (20%) patients, pelvic and distant nodes in 3 (8.6%) patients, pelvic nodes and bone metastasis in 4 (11.4%) patients, and pelvic and distant nodes and bone metastasis in 1 (2.8%) patient. Staging changes were observed in 60% of patients, with downstaging predominance (76.2%). Volume increase was identified in 11 (31.4%) patients (only 4 related to upstaging, 36.4%). The board changed management decisions for 60% of the patients. The main limitations of this study were the sample size and its retrospective nature. CONCLUSIONS: PSMA findings changed the management decisions in more than half of the patients, which made the majority eligible for locoregional treatment and avoided unnecessary procedures in the systemic disease scenario.

Impact of COVID-19 on surgical procedures for stress urinary incontinence in the Brazilian public health system.

INTRODUCTION AND HYPOTHESIS: The objective was to characterize the impact of the COVID-19 pandemic on the surgical treatment of female stress urinary incontinence (FSUI) in Brazil. METHODS: This study was conducted with population-based data from the Brazilian public health system database. We obtained data on the number of surgical procedures for FSUI in 2019 (before the coronavirus disease [COVID-19] pandemic), 2020, and 2021 (during the pandemic) in each of the 27 Brazilian states. We included official Brazilian Institute of Geography and Statistics (IBGE) data on the population, Human Development Index (HDI), and annual per capita income of each state. RESULTS: A total of 6,718 surgical procedures for FSUI were performed in the Brazilian public health system in 2019. The number of procedures was reduced by 56.2% in 2020, and an additional reduction of 7.2% was seen in 2021. The distribution of procedures by state showed important differences, ranging from 4.4 procedures/1,000,000 inhabitants in Paraíba and Sergipe to 67.6 procedures/1,000,000 inhabitants in Paraná (p<0.01) in 2019. The number of surgical procedures was higher in states with a higher HDI (p=0.0001) and per capita income (p=0.042). The decrease in the number of surgical procedures affected the whole country and its rate did not correlate with HDI (p=0.289) or per capita income (p=0.598). CONCLUSION: The impact of the COVID-19 pandemic on the surgical treatment of FSUI in Brazil was significant in 2020 and persisted in 2021. Access to surgical treatment of FSUI varied according to geographic region, HDI, and per capita income, even before COVID-19.

Cryptic lineages and standing genetic variation across independent cane toad introductions.

Widespread introduced species can be leveraged to investigate the genetic, ecological and adaptive processes underlying rapid evolution and range expansion, particularly the contributions of genetic diversity to adaptation. Rhinella marina, the cane toad, has been a focus of invasion biology for decades in Australia. However, their introduction history in North America is less clear. Here, we investigated the roles of introduction history and genetic diversity in establishment success of cane toads across their introduced range. We used reduced representation sequencing (ddRAD) to obtain 34,000 SNPs from 247 toads in native (French Guiana, Guyana, Ecuador, Panama, Texas) and introduced (Bermuda, southern Florida, northern Florida, Hawai'i, Puerto Rico) populations. Unlike all other cane toad introductions, we found that Florida populations were more closely related to native Central American lineages (R. horribilis), than to native Southern American lineages (R. marina). Furthermore, we found high levels of diversity and population structure in the native range, corroborating suggestions that R. marina is a species complex. We also found that introduced populations exhibit only slightly lower genetic diversity than native populations. Together with demographic analyses, this indicates founding populations of toads in Florida were larger than previously reported. Lastly, within R. marina, only one of 245 putatively adaptive SNPs showed fixed differences between native and introduced ranges, suggesting that putative selection in these introduced populations is based upon existing genetic variation. Our findings highlight the importance of genetic sequencing in understanding biological introductions and hint at the role of standing genetic variation in range expansion.

Functional urology: Practice patterns and training aspirations among urologists in Brazil.

PURPOSE: Urology has rapidly evolved during the last decades, incorporating new technologies faster than most specialties. The challenge to maintain a competent workforce that is prepared to deliver proper contemporary treatment has become increasingly difficult and newly graduated urologists throughout the world typically lack the skills to practice many urological subspecialties. We performed a survey to evaluate the practice patterns and educational aspirations in functional urology (FU) among Brazilian urologists. METHODS: A web-based survey was sent to board-certified Brazilian urologists to collect data on clinical practice and training aspirations in four subareas of FU: female urology, urodynamics, postprostatectomy incontinence (PPI), and neurourology. We evaluated urologists' clinical and surgical workload in each subarea and investigated educational training aspirations to identify areas and training formats of interest. RESULTS: A total of 366 urologists (mean age 47.7 + 10.7 years) completed the survey. Mean time since completion of residency was 17.9 + 11.9 years. Of the respondents, 176 (53%) perform urodynamics, 285 (83.1%) SUI surgeries, 159 (47.6%) PPI surgeries, 194 (58.1%) third line OAB procedures, 168 (48.9%) pelvic organ prolapse (POP), and 88 (26.3%) bladder augmentation. Mid-urethral sling is the most performed SUI surgery and transobturator is the preferred route (64.0%). For those performing POP surgery, 40.5% use mesh in at least 50% of their cases, and the vaginal route is used in most cases (75.4%) for apical prolapse. For PPI, 64.6% use artificial sphincter in most surgeries and only 8.1% perform at least 5 surgeries/year. Being fellowship-trained and working in an academic hospital are associated with a higher chance of being active in FU. Most urologists are interested in receiving training in PPI, female SUI, and POP and a hands-on course is the preferred educational method (81%). CONCLUSIONS: Most urologists in Brazil are involved in the evaluation and treatment of FU patients, but few have a large volume of patient visits and surgical procedures. Completing a fellowship program and working in an academic practice are associated with a higher chance of being a FU practitioner. There is a high interest in training for PPI, female SUI, and POP.

Presence of invasive American bullfrogs may reduce infectious disease in a native frog species.

Amphibians breeding in aquatic environments may encounter a myriad of threats during their life cycle. One species known to prey on native amphibians in aquatic habitats is the invasive North American bullfrog Lithobates catesbeianus, which, besides being a voracious predator and competitor, often acts as a pathogen carrier and disease superspreader because it tolerates high infection loads of the frog-killing fungus Batrachochytrium dendrobatidis (Bd). Here, we hypothesized that the presence of the bullfrogs in microcosms should either (1) decrease Bd disease severity in native frog species by discouraging them from using the aquatic environment, or (2) increase the mortality of the native species. We tested these 2 mutually exclusive hypotheses by co-housing the snouted treefrog Scinax x-signatus (native to our study area) with L. catesbeianus in the laboratory, exposing them to Bd, and using qPCR analysis to quantify the resulting Bd infection loads in the native frogs. Our experiment had the following replicated treatments: (1) native-only treatment (3 individuals of S. x-signatus), (2) native-predominant treatment (2 S. x-signatus + 1 L. catesbeianus), and (3) exotic-predominant treatment (1 S. x-signatus + 2 L. catesbeianus). We found that Bd infection loads in the native S. x-signatus were highest in the native-only treatment, and lowest in the exotic-predominant treatment, indicating that bullfrogs may discourage native frogs from occupying the aquatic habitat, thus reducing encounter rates between native frogs and the waterborne pathogen. This effect could be driven by the bullfrogs' predatory behavior and their high philopatry to aquatic habitats. Our results highlight that predation risk adds to the complexity of host-species interactions in Bd epidemiology.

Chytrid fungus in amphibians from the lowland Brazilian Amazon.

Infectious diseases are one of the main threats to biodiversity. The fungus Batrachochytrium dendrobatidis (Bd) is associated with several amphibian losses around the globe, and environmental conditions may dictate the success of pathogen spread. The Brazilian Amazon has been considered climatically unsuitable for chytrid fungus, but additional information on Bd dynamics in this ecoregion is still lacking. We sampled 462 amphibians (449 anurans, 4 caudatans and 9 caecilians), representing 57 species from the Brazilian Amazon, and quantified Bd infections using qPCR. We tested whether abiotic variables predicted the risk of Bd infections, and tested for relationships between biotic variables and Bd. Finally, we experimentally tested the effects of Bd strains CLFT 156 and CLFT 102 (from the southern and northern Atlantic Forest, respectively) on Atelopus manauensis. We detected higher Bd prevalence than those previously reported for the Brazilian Amazon, and positive individuals in all 3 orders of amphibians sampled. Both biotic and abiotic predictors were related to prevalence, and no variable explained infection load. Moreover, we detected higher Bd prevalence in forested than open areas, while the host's reproductive biology was not a factor. We detected higher mortality in the experimental group infected with CLFT 156, probably because this strain was isolated from a region characterized by discrepant climatic conditions (latitudinally more distant) when compared with the host's sampling site in Amazon. The lowland Brazilian Amazon is still underexplored and future studies targeting all amphibian orders are essential to better understand Bd infection dynamics in this region.

Steerable Surgical Instrument for Conventional and Single-Site Minimally Invasive Surgery.

Background. This article aims to present an innovative design of a steerable surgical instrument for conventional and single-site minimally invasive surgery (MIS), which improves the dexterity and maneuverability of the surgeon while offering a solution to the limitations of current tools. Methods. The steerable MIS instrument consists of a deflection structure with a curved sliding joints design that articulates the distal tip in two additional degrees of freedom (DoFs), relative to the instrument shaft, using transmission by cables. A passive ball-joint mechanism articulates the handle relative to the instrument shaft, improves wrist posture, and prevents collision of instrument handles during single-site MIS procedures. The two additional DoFs of the articulating tip are activated by a thumb-controlled device, using a joystick design mounted on the handle. This steerable MIS instrument was developed by additive manufacturing in a 3D printer using PLA polymer. Results. Prototype testing showed a maximum tip deflection of 60° in the left and right directions, with a total deflection of 120°. With the passive ball-joint fully offset, the steerable tip achieved a deflection of 90° for the right and 40° for the left direction, with a total deflection of 130°. Furthermore, the passive ball-joint mechanism in the handle obtained a maximum range of motion of 60°. Conclusions. This steerable MIS instrument concept offers an alternative to enhance the application fields of conventional and single-site MIS, increasing manual dexterity of the surgeon and the ability to reach narrow anatomies from other directions.

The chromatin scaffold protein SAFB1 renders chromatin permissive for DNA damage signaling.

Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling.

Acute hydroxyurea-induced replication blockade results in replisome components disengagement from nascent DNA without causing fork collapse.

During S phase, replication forks can encounter several obstacles that lead to fork stalling, which if persistent might result in fork collapse. To avoid this collapse and to preserve the competence to restart, cells have developed mechanisms that maintain fork stability upon replication stress. In this study, we aimed to understand the mechanisms involved in fork stability maintenance in non-transformed human cells by performing an isolation of proteins on nascent DNA-mass spectrometry analysis in hTERT-RPE cells under different replication stress conditions. Our results show that acute hydroxyurea-induced replication blockade causes the accumulation of large amounts of single-stranded DNA at the fork. Remarkably, this results in the disengagement of replisome components from nascent DNA without compromising fork restart. Notably, Cdc45-MCM-GINS helicase maintains its integrity and replisome components remain associated with chromatin upon acute hydroxyurea treatment, whereas replisome stability is lost upon a sustained replication stress that compromises the competence to restart.

Amphibian chytrid infections along the highest elevational gradient of the Brazilian Atlantic Forest.

Environmental variation along elevational gradients shapes conditions for pathogen development, which influences disease outcomes. Chytridiomycosis is a non-vectored disease caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd) and is responsible for massive declines of amphibian populations all over the world. Several biotic and abiotic factors are known to influence Bd infection dynamics in amphibians, including temperature and host species richness. Here, we quantified Bd prevalence and load along an elevational gradient in the Caparaó National Park (CNP), Brazil, and tested for associations of Bd infections with elevation, temperature, and species richness. We hypothesized that Bd infections would increase as local species richness decreased with elevation. We detected Bd along the entire elevational gradient and found a negative association between infection load and elevation. We did not detect significant associations between infection prevalence and elevation. Our findings are consistent with other wide elevational gradient studies, but are contrary to 2 other studies performed in the Atlantic Forest. We did not find the minimum elevational range that should be sampled to detect the influence of elevation on Bd variation. Our study represents the widest elevational gradient that has been sampled in Brazil and contributes to a better understanding of Bd distribution and dynamics in natural systems.

Seasonal variation of Batrachochytrium dendrobatidis in a threatened anuran species from Uruguay.

Chytridiomycosis, an emergent infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd), is considered one of the drivers of the current amphibian biodiversity loss. To inform endangered species conservation efforts, it is essential to improve our knowledge about the abiotic and biotic factors that influence Bd infection dynamics in the wild. Here, we analyzed variation of Bd infection in the redbelly toad Melanophryniscus montevidensis, a threatened bufonid from Uruguay. We tested the influence of temperature, precipitation, season, and host population size on Bd prevalence and intensity. Additionally, considering the sub-lethal effects of Bd, we tested if these variables, potentially through their effect on Bd, also explain the variation in host body condition. We determined a high Bd prevalence of 41% (100/241), and that population size influenced both Bd prevalence and infection intensity. We identified an effect of precipitation and season on Bd infection intensity and an effect of season on toad body condition. In addition, we found a negative effect of infection intensity on body condition; moreover, while some toads cleared the infection, their body condition did not improve, suggesting a long-term cost. This is the first report on host population size as an important factor in Bd infection dynamics in a threatened anuran species, and seasonal demographic changes appear to play an important role in the dynamics. Finally, we highlight the need for monitoring Bd in this and other endangered amphibian populations, especially those within the genus Melanophryniscus, which includes several Endangered and Data Deficient species in South America.

Forest cover influences chytrid infections in populations of Boana curupi, a threatened treefrog of south Brazil.

Complex interactions among hosts, pathogens, and the environment affect the vulnerability of amphibians to the emergence of infectious diseases such as chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Boana curupi is a forest-dwelling amphibian endemic to the southern Atlantic Forest of South America, a severely fragmented region. Here, we evaluated whether abiotic factors (including air and water temperature, relative air humidity, and landscape) are correlated with chytrid infection intensity and prevalence in B. curupi. We found individuals infected with Bd in all populations sampled. Prevalence ranged from 25-86%, and the infection burden ranged from 1 to over 130000 zoospore genomic equivalents (g.e.) (mean ± SD: 4913 ± 18081 g.e.). The infection load differed among populations and was influenced by forest cover at scales of 100, 500, and 1000 m, with the highest infection rates recorded in areas with a higher proportion of forest cover. Our results suggest that the fungus is widely distributed in the populations of B. curupi in southern Brazil. Population and disease monitoring are necessary to better understand the relationships between host, pathogen, and environment, especially when, as in the case of B. curupi, threatened species are involved.