Stress-triggered hematopoietic stem cell proliferation relies on PrimPol-mediated repriming.

Stem cell division is linked to tumorigenesis by yet-elusive mechanisms. The hematopoietic system reacts to stress by triggering hematopoietic stem and progenitor cell (HSPC) proliferation, which can be accompanied by chromosomal breakage in activated hematopoietic stem cells (HSCs). However, whether these lesions persist in their downstream progeny and induce a canonical DNA damage response (DDR) remains unclear. Inducing HSPC proliferation by simulated viral infection, we report that the associated DNA damage is restricted to HSCs and that proliferating HSCs rewire their DDR upon endogenous and clastogen-induced damage. Combining transcriptomics, single-cell and single-molecule assays on murine bone marrow cells, we found accelerated fork progression in stimulated HSPCs, reflecting engagement of PrimPol-dependent repriming, at the expense of replication fork reversal. Ultimately, competitive bone marrow transplantation revealed the requirement of PrimPol for efficient HSC amplification and bone marrow reconstitution. Hence, fine-tuning replication fork plasticity is essential to support stem cell functionality upon proliferation stimuli.

RAD51 restricts DNA over-replication from re-activated origins.

Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and genomic instability. Most of these mechanisms limit the activity of origin licensing proteins to prevent the reactivation of origins that have already been used. Here, we have investigated whether additional controls restrict the extension of re-replicated DNA in the event of origin re-activation. In a genetic screening in cells forced to re-activate origins, we found that re-replication is limited by RAD51 and enhanced by FBH1, a RAD51 antagonist. In the presence of chromatin-bound RAD51, forks stemming from re-fired origins are slowed down, leading to frequent events of fork reversal. Eventual re-initiation of DNA synthesis mediated by PRIMPOL creates ssDNA gaps that facilitate the partial elimination of re-duplicated DNA by MRE11 exonuclease. In the absence of RAD51, these controls are abrogated and re-replication forks progress much longer than in normal conditions. Our study uncovers a safeguard mechanism to protect genome stability in the event of origin reactivation.

Machine learning-aided engineering of hydrolases for PET depolymerization.

Plastic waste poses an ecological challenge1-3 and enzymatic degradation offers one, potentially green and scalable, route for polyesters waste recycling4. Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste5, and a circular carbon economy for PET is theoretically attainable through rapid enzymatic depolymerization followed by repolymerization or conversion/valorization into other products6-10. Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temperature ranges, slow reaction rates and inability to directly use untreated postconsumer plastics11. Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives12 between 30 and 50 °C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week. FAST-PETase can also depolymerize untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymatic plastic recycling at the industrial scale.

PRIMPOL-Mediated Adaptive Response Suppresses Replication Fork Reversal in BRCA-Deficient Cells.

Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.

DNA replication and replication stress response in the context of nuclear architecture.

The DNA replication process needs to be coordinated with other DNA metabolism transactions and must eventually extend to the full genome, regardless of chromatin status, gene expression, secondary structures and DNA lesions. Completeness and accuracy of DNA replication are crucial to maintain genome integrity, limiting transformation in normal cells and offering targeting opportunities for proliferating cancer cells. DNA replication is thus tightly coordinated with chromatin dynamics and 3D genome architecture, and we are only beginning to understand the underlying molecular mechanisms. While much has recently been discovered on how DNA replication initiation is organised and modulated in different genomic regions and nuclear territories-the so-called "DNA replication program"-we know much less on how the elongation of ongoing replication forks and particularly the response to replication obstacles is affected by the local nuclear organisation. Also, it is still elusive how specific components of nuclear architecture participate in the replication stress response. Here, we review known mechanisms and factors orchestrating replication initiation, and replication fork progression upon stress, focusing on recent evidence linking genome organisation and nuclear architecture with the cellular responses to replication interference, and highlighting open questions and future challenges to explore this exciting new avenue of research.

PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.

Improving Odometric Model Performance Based on LSTM Networks.

This paper presents a localization system for an autonomous wheelchair that includes several sensors, such as odometers, LIDARs, and an IMU. It focuses on improving the odometric localization accuracy using an LSTM neural network. Improved odometry will improve the result of the localization algorithm, obtaining a more accurate pose. The localization system is composed by a neural network designed to estimate the current pose using the odometric encoder information as input. The training is carried out by analyzing multiple random paths and defining the velodyne sensor data as training ground truth. During wheelchair navigation, the localization system retrains the network in real time to adjust any change or systematic error that occurs with respect to the initial conditions. Furthermore, another network manages to avoid certain random errors by using the relationship between the power consumed by the motors and the actual wheel speeds. The experimental results show several examples that demonstrate the ability to self-correct against variations over time, and to detect non-systematic errors in different situations using this relation. The final robot localization is improved with the designed odometric model compared to the classic robot localization based on sensor fusion using a static covariance.

Improving Mobile Robot Maneuver Performance Using Fractional-Order Controller.

In this paper, the low-level velocity controller of an autonomous vehicle is studied. The performance of the traditional controller used in this kind of system, a PID, is analyzed. This kind of controller cannot follow ramp references without error, so when the reference implies a change in the speed, the vehicle cannot follow the proposed reference, and there is a significant difference between the actual and desired vehicle behaviors. A fractional controller is proposed which changes the ordinary dynamics allowing faster responses for small times, at the cost of slower responses for large times. The idea is to take advantage of this fact to follow fast setpoint changes with a smaller error than that obtained with a classic non-fractional PI controller. Using this controller, the vehicle can follow variable speed references with zero stationary error, significantly reducing the difference between reference and actual vehicle behavior. The paper presents the fractional controller, studies its stability in function of the fractional parameters, designs the controller, and tests its stability. The designed controller is tested on a real prototype, and its behavior is compared to a standard PID controller. The designed fractional PID controller overcomes the results of the standard PID controller.

Low Cost Magnetic Field Control for Disabled People.

Our research presents a cost-effective navigation system for electric wheelchairs that utilizes the tongue as a human-machine interface (HMI) for disabled individuals. The user controls the movement of the wheelchair by wearing a small neodymium magnet on their tongue, which is held in place by a suction pad. The system uses low-cost electronics and sensors, including two electronic compasses, to detect the position of the magnet in the mouth. One compass estimates the magnet's position while the other is used as a reference to compensate for static magnetic fields. A microcontroller processes the data using a computational algorithm that takes the mathematical formulations of the magnetic fields as input in real time. The system has been tested using real data to control an electric wheelchair, and it has been shown that a trained user can effectively use tongue movements as an interface for the wheelchair or a computer.

Psychosocial health of school-aged children during the initial COVID-19 safer-at-home school mandates in Florida: a cross-sectional study.

BACKGROUND: Given the emerging literature regarding the impacts of lockdown measures on mental health, this study aims to describe the psychosocial health of school-aged children and adolescents during the COVID-19 Safer-at-Home School mandates. METHODS: A cross-sectional study was conducted in April 2020 (n = 280) among K-12 students at a research school in North Central Florida. Bivariate analysis and logistic and multinomial logistic regression models were used to examine socio-demographic and knowledge, attitude, and practice (KAP) predictors of indicators of anxiety-related, depressive, and obsessive-compulsive disorder(OCD)-related symptoms. Outcomes (anxiety, OCD, and depressive related symptoms) were measured by indices generated based on reported symptoms associated with each psychosocial outcome. RESULTS: Loss of household income was associated with increased risk for all three index-based outcomes: depressive symptoms [aOR = 3.130, 95% CI = (1.41-6.97)], anxiety-related symptoms [aOR = 2.531, 95%CI = (1.154-5.551)], and OCD-related symptoms [aOR = 2.90, 95%CI = (1.32-6.36)]. Being female was associated with being at higher risk for depressive symptoms [aOR = 1.72, 95% CI = (1.02-2.93)], anxiety-related symptoms [aOR = 1.75, 95% CI = (1.04-2.97)], and OCD-related symptoms [aOR = 1.764, 95%CI = (1.027-3.028)]. Parental practices protective against COVID-19 were associated with children being at higher risk of depressive symptoms [aOR = 1.55, 95% CI = (1.04-2.31)]. Lower school level was associated with children being at higher risk of anxiety-related and OCD-related symptoms. CONCLUSIONS: As the COVID-19 pandemic continues, schools should prioritize mental health interventions that target younger, female students, and children of families with income loss. Limiting the spread of COVID-19 through school closure may exacerbate negative psychosocial health outcomes in children, thus school administrators should move quickly to target those at greatest risk.

Functional interplay between c-Myc and Max in B lymphocyte differentiation.

The Myc family of oncogenic transcription factors regulates myriad cellular functions. Myc proteins contain a basic region/helix-loop-helix/leucine zipper domain that mediates DNA binding and heterodimerization with its partner Max. Among the Myc proteins, c-Myc is the most widely expressed and relevant in primary B lymphocytes. There is evidence suggesting that c-Myc can perform some of its functions in the absence of Max in different cellular contexts. However, the functional in vivo interplay between c-Myc and Max during B lymphocyte differentiation is not well understood. Using in vivo and ex vivo models, we show that while c-Myc requires Max in primary B lymphocytes, several key biological processes, such as cell differentiation and DNA replication, can initially progress without the formation of c-Myc/Max heterodimers. We also describe that B lymphocytes lacking Myc, Max, or both show upregulation of signaling pathways associated with the B-cell receptor. These data suggest that c-Myc/Max heterodimers are not essential for the initiation of a subset of important biological processes in B lymphocytes, but are required for fine-tuning the initial response after activation.

A new targeted treatment for patients with a germline BRCA mutation: olaparib in pancreatic cancer.

Pancreatic cancer has a poor prognosis. Focused efforts in the development of novel treatments of this disease have led to the approval of new combinations. Improvements in knowledge of the biology of these tumors have been made, and it is now widely accepted that a proportion of patients have potentially targetable altered genes. One such gene is BRCA, which confers sensibility to PARP inhibitors. Olaparib, an oral PARP inhibitor, initially demonstrated activity in Phase II clinical trials including germline BRCA-mutated patients. This was confirmed in a Phase III clinical trial in pancreatic cancer patients with a germline BRCA mutation. After the results of this study, new scenarios have been evoked. We review the development of olaparib in pancreatic cancer.

MODELING INTER-KINGDOM REGULATION OF INFLAMMATORY SIGNALING IN HUMAN INTESTINAL EPITHELIAL CELLS.

The human gastrointestinal (GI) tract is colonized by a highly diverse and complex microbial community (i.e., microbiota). The microbiota plays an important role in the development of the immune system, specifically mediating inflammatory responses, however the exact mechanisms are poorly understood. We have developed a mathematical model describing the effect of indole on host inflammatory signaling in HCT-8 human intestinal epithelial cells. In this model, indole modulates transcription factor nuclear factor κ B (NF-κB) and produces the chemokine interleukin-8 (IL-8) through the activation of the aryl hydrocarbon receptor (AhR). Phosphorylated NF-κB exhibits dose and time-dependent responses to indole concentrations and IL-8 production shows a significant down-regulation for 0.1 ng/mL TNF-α stimulation. The model shows agreeable simulation results with the experimental data for IL-8 secretion and normalized NF-κB values. Our results suggest that microbial metabolites such as indole can modulate inflammatory signaling in HTC-8 cells through receptor-mediated processes.

Improving Odometric Accuracy for an Autonomous Electric Cart.

In this paper, a study of the odometric system for the autonomous cart Verdino, which is an electric vehicle based on a golf cart, is presented. A mathematical model of the odometric system is derived from cart movement equations, and is used to compute the vehicle position and orientation. The inputs of the system are the odometry encoders, and the model uses the wheels diameter and distance between wheels as parameters. With this model, a least square minimization is made in order to get the nominal best parameters. This model is updated, including a real time wheel diameter measurement improving the accuracy of the results. A neural network model is used in order to learn the odometric model from data. Tests are made using this neural network in several configurations and the results are compared to the mathematical model, showing that the neural network can outperform the first proposed model.

Recent Advances by In Silico and In Vitro Studies of Amyloid-ß 1-42 Fibril Depicted a S-Shape Conformation.

The amyloid-ß 1-42 (Aß1-42) peptide is produced by proteolytic cleavage of the amyloid precursor protein (APP) by sequential reactions that are catalyzed by γ and ß secretases. Aß1-42, together with the Tau protein are two principal hallmarks of Alzheimer's disease (AD) that are related to disease genesis and progression. Aß1-42 possesses a higher aggregation propensity, and it is able to form fibrils via nucleated fibril formation. To date, there are compounds available that prevent Aß1-42 aggregation, but none have been successful in clinical trials, possibly because the Aß1-42 structure and aggregation mechanisms are not thoroughly understood. New molecules have been designed, employing knowledge of the Aß1-42 structure and are based on preventing or breaking the ionic interactions that have been proposed for formation of the Aß1-42 fibril U-shaped structure. Recently, a new Aß1-42 fibril S-shaped structure was reported that, together with its aggregation and catalytic properties, could be helpful in the design of new inhibitor molecules. Therefore, in silico and in vitro methods have been employed to analyze the Aß1-42 fibril S-shaped structure and its aggregation to obtain more accurate Aß1-42 oligomerization data for the design and evaluation of new molecules that can prevent the fibrillation process.

Water-Soluble Naked Gold Nanoclusters Are Not Luminescent.

Here, the synthesis of water-dispersible naked gold nanoclusters (AuNCnaked ) is reported by a simple reduction of HAuCl4 with NaOH at room temperature, and it is shown that they are non-luminescent. They are then easily passivated with different thiols and adenosine monophosphate, leading to luminescent NCs. This is an important finding because the photoluminescence of the passivated NCs can now be clearly attributed to the ligand-AuNC surface interaction. These results are also highly relevant from the point of view of the preparation of luminescent NCs from the same NC batch. This strategy can be valuable for the preparation of a broad range of nano(bio)composites.

The importance of incorporating systems thinking and One Health in global health classrooms: findings from a One Health simulation activity.

There are several challenges and opportunities in health education in global health. Given the field's rapid expansion, demand for including systems thinking and One Health (a unifying approach that considers human, animal, and environmental health) in global health courses has recently increased. Simulation activities provide an avenue to attain and assess learning objectives that foster critical and systems thinking. This study carried out a One Health simulation activity in an undergraduate global health course, conducted a focus group discussion, and obtained responses from written questionnaires from students who participated in the activity. Data were analyzed using thematic analysis. Results show that the One Health simulation was instrumental for students to understand the complex interactions between different actors and stakeholders in global health systems. The One Health simulation also improved class dynamics, peer-to-peer interactions, and collaborations in the remaining part of the course. The activity helped assess two of the critical thinking learning objectives of the course, and there was some evidence that student agency and confidence may have been improved. Evidence shows that the activity helped students understand the principles of systems thinking and apply them in complex scenarios. Findings support including interactive simulation activities in global health courses to include elements of system science and One Health into classroom activities innovatively and engagingly.

Climate change and health in the Sahel: a systematic review.

The Sahel region is projected to be highly impacted by the more frequent hazards associated with climate change, including increased temperature, drought and flooding. This systematic review examined the evidence for climate change-related health consequences in the Sahel. The databases used were Medline (PubMed), Embase (Ovid), Web of Science (Clarivate) and CABI Global Health. Hand searches were also conducted, which included directly engaging Sahelian researchers and hand-searching in the African Journals Online database. Of the 4153 studies found, 893 were identified as duplicates and the remaining 3260 studies were screened (title and abstract only) and then assessed for eligibility. A total of 81 studies were included in the systematic review. Most studies focused on vector-borne diseases, food security, nutrition and heat-related stress. Findings suggest that mosquito distribution will shift under different climate scenarios, but this relationship will not be linear with temperature, as there are other variables to consider. Food insecurity, stunting (chronic malnutrition) and heat-related mortality are likely to increase if no action is taken owing to the projected impact of climate change on environmental factors and agriculture. Seventy-one per cent of manuscripts (n = 58) had first authors from institutions in North America or Europe, of which 39.7% (n = 23) included co-authors from African institutions.

A microfluidic co-culture model for investigating colonocytes-microbiota interactions in colorectal cancer.

Changes in the abundance of certain bacterial species within the colorectal microbiota correlate with colorectal cancer (CRC) development. While carcinogenic mechanisms of single pathogenic bacteria have been characterized in vitro, limited tools are available to investigate interactions between pathogenic bacteria and both commensal microbiota and colonocytes in a physiologically relevant tumor microenvironment. To address this, we developed a microfluidic device that can be used to co-culture colonocyte spheroids and colorectal microbiota. The device was used to explore the effect of Fusobacterium nucleatum, an opportunistic pathogen associated with colorectal cancer development in humans, on colonocyte gene expression and microbiota composition. F. nucleatum altered the transcription of genes involved in cytokine production, epithelial-to-mesenchymal transition, and proliferation in colonocytes in a contact-independent manner; however, most of these effects were significantly diminished by the presence of commensal microbiota. Interestingly, F. nucleatum significantly altered the abundance of multiple bacterial clades associated with mucosal immune responses and cancer development in the colon. Our results highlight the importance of evaluating the potential carcinogenic activity of pathogens in the context of a commensal microbiota, and the potential to discover novel inter-species microbial interactions in the CRC microenvironment.

Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50 = 20 µM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.