Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

Discovery of prolactin-like in lamprey: Role in osmoregulation and new insight into the evolution of the growth hormone/prolactin family.

We used a representative of one of the oldest extant vertebrate lineages (jawless fish or agnathans) to investigate the early evolution and function of the growth hormone (GH)/prolactin (PRL) family. We identified a second member of the GH/PRL family in an agnathan, the sea lamprey (Petromyzon marinus). Structural, phylogenetic, and synteny analyses supported the identification of this hormone as prolactin-like (PRL-L), which has led to added insight into the evolution of the GH/PRL family. At least two ancestral genes were present in early vertebrates, which gave rise to distinct GH and PRL-L genes in lamprey. A series of gene duplications, gene losses, and chromosomal rearrangements account for the diversity of GH/PRL-family members in jawed vertebrates. Lamprey PRL-L is produced in the proximal pars distalis of the pituitary and is preferentially bound by the lamprey PRL receptor, whereas lamprey GH is preferentially bound by the lamprey GH receptor. Pituitary PRL-L messenger RNA (mRNA) levels were low in larvae, then increased significantly in mid-metamorphic transformers (stage 3); thereafter, levels subsided in final-stage transformers and metamorphosed juveniles. The abundance of PRL-L mRNA and immunoreactive protein increased in the pituitary of juveniles under hypoosmotic conditions, and treatment with PRL-L blocked seawater-associated inhibition of freshwater ion transporters. These findings clarify the origin and divergence of GH/PRL family genes in early vertebrates and reveal a function of PRL-L in osmoregulation of sea lamprey, comparable to a role of PRLs that is conserved in jawed vertebrates.

Toxic and non-toxic cyanobacterial biomass as a resource for sustainable agriculture: A lettuce cultivation experiment.

Cyanobacteria represent a promising resource for sustainable agriculture, as they have demonstrated the ability to restore soil fertility even after death and decay. However, several cyanobacteria can also release secondary metabolites, such as cyanotoxins, which may compromise the quality of agricultural products and pose a potential risk to human health. Depending on the concentration of exposure, few studies reported deleterious effects on plant species when irrigated with cylindrospermopsin (CYN) contaminated water, impairing plant growth and leading to food product contamination, while other studies show promoting effects on plant yield. To evaluate the potential of cyanobacterial biomass (cyanotoxin-containing or not) as a sustainable resource for soil amendment, biostimulants or fertilizers for lettuce cultivation, a study was carried out that consisted of the culture of lettuce plants under controlled conditions, in soil: (1) with no extra nutrient addition (control) and supplemented with 0.6 g of freeze-dried Raphidiopsis raciborskii biomass of (2) a non-CYN-producing strain, (3) a CYN-producing strain, and (4) the same CYN-producing strain pasteurized. Results showed no significant differences in photosystem II efficiency with the amendment addition. On the contrary, shoot fresh weight significantly increased in lettuce plants grown with the cyanobacterial biomass addition, especially in condition (3). In addition, there were significant differences in mineral concentrations in lettuce leaves after the cyanobacterial biomass addition, such as K, Na, Ca, P, Mg, Mn, Zn, Cu, Mo, and Co. CYN accumulation was detected under conditions (3) and (4), with concentrations observed in descending order from roots > soil > shoot. Nevertheless, the CYN concentration in edible tissues did not exceed the WHO-proposed tolerable daily intake of 0.03 µg/kg/day. These findings suggest that incorporating cyanobacterial biomass as a soil amendment, biostimulant or fertilizer for lettuce cultivation, even with trace amounts of CYN (1-40 µg/g), may enhance plant yield without leading to cyanotoxin accumulation in edible tissues above the WHO-recommended tolerable daily intake.

Clostridioides difficile Sporulation.

Some members of the Firmicutes phylum, including many members of the human gut microbiota, are able to differentiate a dormant and highly resistant cell type, the endospore (hereinafter spore for simplicity). Spore-formers can colonize virtually any habitat and, because of their resistance to a wide variety of physical and chemical insults, spores can remain viable in the environment for long periods of time. In the anaerobic enteric pathogen Clostridioides difficile the aetiologic agent is the oxygen-resistant spore, while the toxins produced by actively growing cells are the main cause of the disease symptoms. Here, we review the regulatory circuits that govern entry into sporulation. We also cover the role of spores in the infectious cycle of C. difficile in relation to spore structure and function and the main control points along spore morphogenesis.

Assessing Cross-Border Telemedicine Data Exchange in the European Union: A Call to Action.

Background: Telemedicine offers potential benefits for health care delivery. However, evidence of cross-border telemedicine data exchange within the European Union (EU) remains limited. The objective of this communication provides a brief outline of the regulatory framework, initiatives, and challenges associated with cross-border telemedicine data exchange in the EU, setting the stage for a comprehensive evidence assessment. Methods: We explore the current regulatory landscape (European Health Data Space), existing initiatives (the European Electronic Health Record Exchange Format), and interoperability challenges (e.g., legal, technical, semantic) facing EU cross-border telemedicine data exchange. Results: There is a need for thorough evidence assessment of cross-border telemedicine and related data movements. Conclusion: Understanding the current landscape of cross-border telemedicine is crucial. This article highlights the need for evidence assessment through a formal review to inform future research and policy initiatives in this domain.

Reactive Docking: A Computational Method for High-Throughput Virtual Screenings of Reactive Species.

We describe the formalization of the reactive docking protocol, a method developed to model and predict reactions between small molecules and biological macromolecules. The method has been successfully used in a number of applications already, including recapitulating large proteomics data sets, performing structure-reactivity target optimizations, and prospective virtual screenings. By modeling a near-attack conformation-like state, no QM calculations are required to model the ligand and receptor geometries. Here, we present its generalization using a large data set containing more than 400 ligand-target complexes, 8 nucleophilic modifiable residue types, and more than 30 warheads. The method correctly predicts the modified residue in ∼85% of complexes and shows enrichments comparable to standard focused virtual screenings in ranking ligands. This performance supports this approach for the docking and screening of reactive ligands in virtual chemoproteomics and drug design campaigns.

Ringtail: A Python Tool for Efficient Management and Storage of Virtual Screening Results.

Virtual screening using molecular docking is now routinely used for the rapid evaluation of very large ligand libraries in early stage drug discovery. As the size of compound libraries which can feasibly be screened grows, so do the challenges in result management and storage. Here we introduce Ringtail, a new Python tool in the AutoDock Suite for efficient storage and analysis of virtual screening data based on portable SQLite databases. Ringtail is designed to work with AutoDock-GPU and AutoDock Vina out-of-the-box. Its modular design also allows for easy extension to support input file types from other docking software, different storage solutions, and incorporation into other applications. Ringtail's SQLite database output can dramatically reduce the required disk storage (36-46 fold) by selecting individual poses to store and by taking advantage of the relational database format. Filtering times are also dramatically reduced, requiring minutes to filter millions of ligands. Thus, Ringtail is a tool that can immediately integrate into existing virtual screening pipelines using AutoDock-GPU and Vina, and is scriptable and modifiable to fit specific user needs.

Temporal and spatial regulation of protein cross-linking by the pre-assembled substrates of a Bacillus subtilis spore coat transglutaminase.

In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular ε-(γ-glutamyl)lysil cross-links catalyzed by transglutaminases (TGases). Because of the potential for unwanted cross-linking reactions, the activities of many TGases have been shown to be tightly controlled. Bacterial endospores are highly resilient cells in part because they are surrounded by a complex protein coat. Proteins in the coat that surrounds Bacillus subtilis endospores are crosslinked by a TGase (Tgl). Unlike other TGases, however, Tgl is produced in an active form, and efficiently catalyzes amine incorporation and protein cross-linking in vitro with no known additional requirements. The absence of regulatory factors raises questions as to how the activity of Tgl is controlled during spore coat assembly. Here, we show that substrates assembled onto the spore coat prior to Tgl production govern the localization of Tgl to the surface of the developing spore. We also show that Tgl residues important for substrate recognition are crucial for its localization. We identified the glutamyl (Q) and lysil (K) substrate docking sites and we show that residues on the Q side of Tgl are more important for the assembly of Tgl than those on the K side. Thus, the first step in the reaction cycle, the interaction with Q-substrates and formation of an acyl-enzyme intermediate, is also the determinant step in the localization of Tgl. Consistent with the idea that Tg exerts a "spotwelding" activity, cross-linking pre-formed assemblies, we show that C30 is an oblong hexamer in solution that is cross-linked in vitro into high molecular weight forms. Moreover, during the reaction, Tgl becomes part of the cross-linked products. We suggest that the dependency of Tgl on its substrates is used to accurately control the time, location and extent of the enzyme´s activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore.

SuFEx-enabled, agnostic discovery of covalent inhibitors of human neutrophil elastase.

Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC50 0.24 µM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.

Benchmarking the Performance of Irregular Computations in AutoDock-GPU Molecular Docking.

Irregular applications can be found in different scientific fields. In computer-aided drug design, molecular docking simulations play an important role in finding promising drug candidates. AutoDock is a software application widely used for predicting molecular interactions at close distances. It is characterized by irregular computations and long execution runtimes. In recent years, a hardware-accelerated version of AutoDock, called AutoDock-GPU, has been under active development. This work benchmarks the recent code and algorithmic enhancements incorporated into AutoDock-GPU. Particularly, we analyze the impact on execution runtime of techniques based on early termination. These enable AutoDock-GPU to explore the molecular space as necessary, while safely avoiding redundant computations. Our results indicate that it is possible to achieve average runtime reductions of 50% by using these techniques. Furthermore, a comprehensive literature review is also provided, where our work is compared to relevant approaches leveraging hardware acceleration for molecular docking.

AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings.

AutoDock Vina is arguably one of the fastest and most widely used open-source programs for molecular docking. However, compared to other programs in the AutoDock Suite, it lacks support for modeling specific features such as macrocycles or explicit water molecules. Here, we describe the implementation of this functionality in AutoDock Vina 1.2.0. Additionally, AutoDock Vina 1.2.0 supports the AutoDock4.2 scoring function, simultaneous docking of multiple ligands, and a batch mode for docking a large number of ligands. Furthermore, we implemented Python bindings to facilitate scripting and the development of docking workflows. This work is an effort toward the unification of the features of the AutoDock4 and AutoDock Vina programs. The source code is available at https://github.com/ccsb-scripps/AutoDock-Vina.

Improving Decision-Making for Population Health in Nonhealth Sectors in Urban Environments: the Example of the Transportation Sector in Three Megacities-the 3-D Commission.

Noncommunicable diseases (NCDs) represent a significant global public health burden. As more countries experience both epidemiologic transition and increasing urbanization, it is clear that we need approaches to mitigate the growing burden of NCDs. Large and growing urban environments play an important role in shaping risk factors that influence NCDs, pointing to the ineluctable need to engage sectors beyond the health sector in these settings if we are to improve health. By way of one example, the transportation sector plays a critical role in building and sustaining health outcomes in urban environments in general and in megacities in particular. We conducted a qualitative comparative case study design. We compared Bus Rapid Transit (BRT) policies in 3 megacities-Lagos (Africa), Bogotá (South America), and Beijing (Asia). We examined the extent to which data on the social determinants of health, equity considerations, and multisectoral approaches were incorporated into local politics and the decision-making processes surrounding BRT. We found that all three megacities paid inadequate attention to health in their agenda-setting, despite having considerable healthy transportation policies in principle. BRT system policies have the opportunity to improve lifestyle choices for NCDs through a focus on safe, affordable, and effective forms of transportation. There are opportunities to improve decision-making for health by involving more available data for health, building on existing infrastructures, building stronger political leadership and commitments, and establishing formal frameworks to improve multisectoral collaborations within megacities. Future research will benefit from addressing the political and bureaucratic processes of using health data when designing public transportation services, the political and social obstacles involved, and the cross-national lessons that can be learned from other megacities.

Integrating Social Determinants in Decision-Making Processes for Health: Insights from Conceptual Frameworks-the 3-D Commission.

The inclusion of social determinants of health offers a more comprehensive lens to fully appreciate and effectively address health. However, decision-makers across sectors still struggle to appropriately recognise and act upon these determinants, as illustrated by the ongoing COVID-19 pandemic. Consequently, improving the health of populations remains challenging. This paper seeks to draw insights from the literature to better understand decision-making processes affecting health and the potential to integrate data on social determinants. We summarised commonly cited conceptual approaches across all stages of the policy process, from agenda-setting to evaluation. Nine conceptual approaches were identified, including two frameworks, two models and five theories. From across the selected literature, it became clear that the context, the actors and the type of the health issue are critical variables in decision-making for health, a process that by nature is a dynamic and adaptable one. The majority of these conceptual approaches implicitly suggest a possible role for data on social determinants of health in decision-making. We suggest two main avenues to make the link more explicit: the use of data in giving health problems the appropriate visibility and credibility they require and the use of social determinants of health as a broader framing to more effectively attract the attention of a diverse group of decision-makers with the power to allocate resources. Social determinants of health present opportunities for decision-making, which can target modifiable factors influencing health-i.e. interventions to improve or reduce risks to population health. Future work is needed to build on this review and propose an improved, people-centred and evidence-informed decision-making tool that strongly and explicitly integrates data on social determinants of health.

Guidelines of the Brazilian Society of Oncologic Surgery for pelvic exenteration in the treatment of cervical cancer.

BACKGROUND AND OBJECTIVES: The primary treatment for locally advanced cases of cervical cancer is chemoradiation followed by high-dose brachytherapy. When this treatment fails, pelvic exenteration (PE) is an option in some cases. This study aimed to develop recommendations for the best management of patients with cervical cancer undergoing salvage PE. METHODS: A questionnaire was administered to all members of the Brazilian Society of Surgical Oncology. Of them, 68 surgeons participated in the study and were divided into 10 working groups. A literature review of studies retrieved from the National Library of Medicine database was carried out on topics chosen by the participants. These topics were indications for curative and palliative PE, preoperative and intraoperative evaluation of tumor resectability, access routes and surgical techniques, PE classification, urinary, vaginal, intestinal, and pelvic floor reconstructions, and postoperative follow-up. To define the level of evidence and strength of each recommendation, an adapted version of the Infectious Diseases Society of America Health Service rating system was used. RESULTS: Most conducts and management strategies reviewed were strongly recommended by the participants. CONCLUSIONS: Guidelines outlining strategies for PE in the treatment of persistent or relapsed cervical cancer were developed and are based on the best evidence available in the literature.

Structural basis for the stabilization of amyloidogenic immunoglobulin light chains by hydantoins.

Misfolding and aggregation of immunoglobulin light chains (LCs) leads to the degeneration of post-mitotic tissue in the disease immunoglobulin LC amyloidosis (AL). We previously reported the discovery of small molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which slow or stop the LC aggregation cascade at the outset. A predominant structural category of kinetic stabilizers emerging from the high-throughput screen are coumarins substituted at the 7-position, which bind at the interface between the two variable domains of the light chain dimer. Here, we report the binding mode of another, more polar, LC kinetic stabilizer chemotype, 3,5-substituted hydantoins. Computational docking, solution nuclear magnetic resonance experiments, and x-ray crystallography show that the aromatic substructure emerging from the hydantoin 3-position occupies the same LC binding site as the coumarin ring. Notably, the hydantoin ring extends beyond the binding site mapped out by the coumarin hits. The hydantoin ring makes hydrogen bonds with both LC monomers simultaneously. The alkyl substructure at the hydantoin 5-position partially occupies a novel binding pocket proximal to the pocket occupied by the coumarin substructure. Overall, the hydantoin structural data suggest that a larger area of the LC variable-domain-variable-domain dimer interface is amenable to small molecule binding than previously demonstrated, which should facilitate development of more potent full-length LC kinetic stabilizers.

Initial Analysis of the Arylomycin D Antibiotics.

The arylomycins are a class of natural product antibiotics that inhibit bacterial type I signal peptidase and are under development as therapeutics. Four classes of arylomycins are known, arylomycins A-D. Previously, we reported the synthesis and analysis of representatives of the A, B, and C classes and showed that their spectrum of activity has the potential to be much broader than originally assumed. Along with a comparison of the mechanism of acquired and innate resistance, this led us to suggest that the arylomycins are latent antibiotics, antibiotics that once possessed broad-spectrum activity, but which upon examination today, have only narrow spectrum activity due to prior selection for resistance in the course of the competition with other microorganisms that drove their evolution in the first place. Interestingly, actinocarbasin, the only identified member of the arylomycin D class, has been reported to have activity against MRSA. To confirm and understand this activity, several actinocarbasin derivatives were synthesized. We demonstrate that the previously reported structure of actinocarbasin is incorrect, identify what is likely the correct scaffold, confirm that scaffold has activity against MRSA, and determine the origin of this activity.

D3R Grand Challenge 4: prospective pose prediction of BACE1 ligands with AutoDock-GPU.

In this paper we describe our approaches to predict the binding mode of twenty BACE1 ligands as part of Grand Challenge 4 (GC4), organized by the Drug Design Data Resource. Calculations for all submissions (except for one, which used AutoDock4.2) were performed using AutoDock-GPU, the new GPU-accelerated version of AutoDock4 implemented in OpenCL, which features a gradient-based local search. The pose prediction challenge was organized in two stages. In Stage 1a, the protein conformations associated with each of the ligands were undisclosed, so we docked each ligand to a set of eleven receptor conformations, chosen to maximize the diversity of binding pocket topography. Protein conformations were made available in Stage 1b, making it a re-docking task. For all calculations, macrocyclic conformations were sampled on the fly during docking, taking the target structure into account. To leverage information from existing structures containing BACE1 bound to ligands available in the PDB, we tested biased docking and pose filter protocols to facilitate poses resembling those experimentally determined. Both pose filters and biased docking resulted in more accurate docked poses, enabling us to predict for both Stages 1a and 1b ligand poses within 2 Å RMSD from the crystallographic pose. Nevertheless, many of the ligands could be correctly docked without using existing structural information, demonstrating the usefulness of physics-based scoring functions, such as the one used in AutoDock4, for structure based drug design.

Comparison of affinity ranking using AutoDock-GPU and MM-GBSA scores for BACE-1 inhibitors in the D3R Grand Challenge 4.

Molecular docking has been successfully used in computer-aided molecular design projects for the identification of ligand poses within protein binding sites. However, relying on docking scores to rank different ligands with respect to their experimental affinities might not be sufficient. It is believed that the binding scores calculated using molecular mechanics combined with the Poisson-Boltzman surface area (MM-PBSA) or generalized Born surface area (MM-GBSA) can predict binding affinities more accurately. In this perspective, we decided to take part in Stage 2 of the Drug Design Data Resource (D3R) Grand Challenge 4 (GC4) to compare the performance of a quick scoring function, AutoDock4, to that of MM-GBSA in predicting the binding affinities of a set of [Formula: see text]-Amyloid Cleaving Enzyme 1 (BACE-1) ligands. Our results show that re-scoring docking poses using MM-GBSA did not improve the correlation with experimental affinities. We further did a retrospective analysis of the results and found that our MM-GBSA protocol is sensitive to details in the protein-ligand system: (i) neutral ligands are more adapted to MM-GBSA calculations than charged ligands, (ii) predicted binding affinities depend on the initial conformation of the BACE-1 receptor, (iii) protonating the aspartyl dyad of BACE-1 correctly results in more accurate binding affinity predictions.