The Crystal Structure of a Biological Insulated Transmembrane Molecular Wire.

A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand ß-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes.

Cross-talk between Lysine-Modifying Enzymes Controls Site-Specific DNA Amplifications.

Acquired chromosomal DNA amplifications are features of many tumors. Although overexpression and stabilization of the histone H3 lysine 9/36 (H3K9/36) tri-demethylase KDM4A generates transient site-specific copy number gains (TSSGs), additional mechanisms directly controlling site-specific DNA copy gains are not well defined. In this study, we uncover a collection of H3K4-modifying chromatin regulators that function with H3K9 and H3K36 regulators to orchestrate TSSGs. Specifically, the H3K4 tri-demethylase KDM5A and specific COMPASS/KMT2 H3K4 methyltransferases modulate different TSSG loci through H3K4 methylation states and KDM4A recruitment. Furthermore, a distinct chromatin modifier network, MLL1-KDM4B-KDM5B, controls copy number regulation at a specific genomic locus in a KDM4A-independent manner. These pathways comprise an epigenetic addressing system for defining site-specific DNA rereplication and amplifications.

Resveratrol: Friend or Foe?

In this issue of Molecular Cell, Benslimane et al. (2020) perform a CRISPR-Cas9 chemogenomic screen, identifying a network of DNA replication and genome integrity genes with the nutraceutical compound Resveratrol and its analog Pterostilbene, linking these compounds to the induction of DNA replication stress in mammalian cells.

How does the microbiota control systemic innate immunity?

The intestinal microbiota has a pervasive influence on mammalian innate immunity fortifying defenses to infection in tissues throughout the host. How intestinal microbes control innate defenses in systemic tissues is, however, poorly defined. In our opinion, there are three core challenges that need addressing to advance our understanding of how the intestinal microbiota controls innate immunity systemically: first, deciphering how signals from intestinal microbes are transmitted to distal tissues; second, unraveling how intestinal microbes prime systemic innate immunity without inducing widespread immunopathology; and third, identifying which intestinal microbes control systemic immunity. Here, we propose answers to these problems which provide a framework for understanding how microbes in the intestine can regulate innate immunity systemically.

Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination.

Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR. Mechanistically, we demonstrate that PRMT5-directed methylation of RUVBL1 is critically required for the acetyltransferase activity of TIP60, promoting histone H4K16 acetylation, which facilities 53BP1 displacement from DSBs. Interestingly, RUVBL1 methylation did not affect the ability of TIP60 to facilitate ATM activation. Taken together, our findings reveal the importance of PRMT5-mediated arginine methylation during DSB repair pathway choice through its ability to regulate acetylation-dependent control of 53BP1 localization.

Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme.

Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 µm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-µs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.

Escherichia coli ST131 Associated with Increased Mortality in Bloodstream Infections from Urinary Tract Source.

Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug-resistant clone, although its clinical impact on patients with bloodstream infection (BSI) is incompletely understood. This study aims to further define the risk factors, clinical outcomes, and bacterial genetics associated with ST131 BSI. A prospectively enrolled cohort study of adult inpatients with E. coli BSI was conducted from 2002 to 2015. Whole-genome sequencing was performed with the E. coli isolates. Of the 227 patients with E. coli BSI in this study, 88 (39%) were infected with ST131. Patients with E. coli ST131 BSI and those with non-ST131 BSI did not differ with respect to in-hospital mortality (17/82 [20%] versus 26/145 [18%]; P = 0.73). However, in patients with BSI from a urinary tract source, ST131 was associated with a numerically higher in-hospital mortality than patients with non-ST131 BSI (8/42 [19%] versus 4/63 [6%]; P = 0.06) and increased mortality in an adjusted analysis (odds ratio of 5.85; 95% confidence interval of 1.44 to 29.49; P = 0.02). Genomic analyses showed that ST131 isolates primarily had an H4:O25 serotype, had a higher number of prophages, and were associated with 11 flexible genomic islands as well as virulence genes involved in adhesion (papA, kpsM, yfcV, and iha), iron acquisition (iucC and iutA), and toxin production (usp and sat). In patients with E. coli BSI from a urinary tract source, ST131 was associated with increased mortality in an adjusted analysis and contained a distinct repertoire of genes influencing pathogenesis. These genes could contribute to the higher mortality observed in patients with ST131 BSI.

Immunological design of commensal communities to treat intestinal infection and inflammation.

The immunological impact of individual commensal species within the microbiota is poorly understood limiting the use of commensals to treat disease. Here, we systematically profile the immunological fingerprint of commensals from the major phyla in the human intestine (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) to reveal taxonomic patterns in immune activation and use this information to rationally design commensal communities to enhance antibacterial defenses and combat intestinal inflammation. We reveal that Bacteroidetes and Firmicutes have distinct effects on intestinal immunity by differentially inducing primary and secondary response genes. Within these phyla, the immunostimulatory capacity of commensals from the Bacteroidia class (Bacteroidetes phyla) reflects their robustness of TLR4 activation and Bacteroidia communities rely solely on this receptor for their effects on intestinal immunity. By contrast, within the Clostridia class (Firmicutes phyla) it reflects the degree of TLR2 and TLR4 activation, and communities of Clostridia signal via both of these receptors to exert their effects on intestinal immunity. By analyzing the receptors, intracellular signaling components and transcription factors that are engaged by different commensal species, we identify canonical NF-κB signaling as a critical rheostat which grades the degree of immune stimulation commensals elicit. Guided by this immunological analysis, we constructed a cross-phylum consortium of commensals (Bacteroides uniformis, Bacteroides ovatus, Peptostreptococcus anaerobius and Clostridium histolyticum) which enhances innate TLR, IL6 and macrophages-dependent defenses against intestinal colonization by vancomycin resistant Enterococci, and fortifies mucosal barrier function during pathological intestinal inflammation through the same pathway. Critically, the setpoint of intestinal immunity established by this consortium is calibrated by canonical NF-κB signaling. Thus, by profiling the immunological impact of major human commensal species our work paves the way for rational microbiota reengineering to protect against antibiotic resistant infections and to treat intestinal inflammation.

Secondary Degeneration of Oligodendrocyte Precursor Cells Occurs as Early as 24 h after Optic Nerve Injury in Rats.

Optic nerve injury causes secondary degeneration, a sequela that spreads damage from the primary injury to adjacent tissue, through mechanisms such as oxidative stress, apoptosis, and blood-brain barrier (BBB) dysfunction. Oligodendrocyte precursor cells (OPCs), a key component of the BBB and oligodendrogenesis, are vulnerable to oxidative deoxyribonucleic acid (DNA) damage by 3 days post-injury. However, it is unclear whether oxidative damage in OPCs occurs earlier at 1 day post-injury, or whether a critical 'window-of-opportunity' exists for therapeutic intervention. Here, a partial optic nerve transection rat model of secondary degeneration was used with immunohistochemistry to assess BBB dysfunction, oxidative stress, and proliferation in OPCs vulnerable to secondary degeneration. At 1 day post-injury, BBB breach and oxidative DNA damage were observed, alongside increased density of DNA-damaged proliferating cells. DNA-damaged cells underwent apoptosis (cleaved caspase3+), and apoptosis was associated with BBB breach. OPCs experienced DNA damage and apoptosis and were the major proliferating cell type with DNA damage. However, the majority of caspase3+ cells were not OPCs. These results provide novel insights into acute secondary degeneration mechanisms in the optic nerve, highlighting the need to consider early oxidative damage to OPCs in therapeutic efforts to limit degeneration following optic nerve injury.

Imipenem/Relebactam Resistance in Clinical Isolates of Extensively Drug Resistant Pseudomonas aeruginosa: Inhibitor-Resistant ß-Lactamases and Their Increasing Importance.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections are a major clinical challenge. Many isolates are carbapenem resistant, which severely limits treatment options; thus, novel therapeutic combinations, such as imipenem-relebactam (IMI/REL), ceftazidime-avibactam (CAZ/AVI), ceftolozane-tazobactam (TOL/TAZO), and meropenem-vaborbactam (MEM/VAB) were developed. Here, we studied two extensively drug-resistant (XDR) P. aeruginosa isolates, collected in the United States and Mexico, that demonstrated resistance to IMI/REL. Whole-genome sequencing (WGS) showed that both isolates contained acquired GES ß-lactamases, intrinsic PDC and OXA ß-lactamases, and disruptions in the genes encoding the OprD porin, thereby inhibiting uptake of carbapenems. In one isolate (ST17), the entire C terminus of OprD deviated from the expected amino acid sequence after amino acid G388. In the other (ST309), the entire oprD gene was interrupted by an ISPa1328 insertion element after amino acid D43, rendering this porin nonfunctional. The poor inhibition by REL of the GES ß-lactamases (GES-2, -19, and -20; apparent Ki of 19 ± 2 µM, 23 ± 2 µM, and 21 ± 2 µM, respectively) within the isolates also contributed to the observed IMI/REL-resistant phenotype. Modeling of REL binding to the active site of GES-20 suggested that the acylated REL is positioned in an unstable conformation as a result of a constrained Ω-loop.

Photocatalytic Removal of the Greenhouse Gas Nitrous Oxide by Liposomal Microreactors.

Nitrous oxide (N2 O) is a potent greenhouse and ozone-reactive gas for which emissions are growing rapidly due to increasingly intensive agriculture. Synthetic catalysts for N2 O decomposition typically contain precious metals and/or operate at elevated temperatures driving a desire for more sustainable alternatives. Here we demonstrate self-assembly of liposomal microreactors enabling catalytic reduction of N2 O to the climate neutral product N2 . Photoexcitation of graphitic N-doped carbon dots delivers electrons to encapsulated N2 O Reductase enzymes via a lipid-soluble biomolecular wire provided by the MtrCAB protein complex. Within the microreactor, electron transfer from MtrCAB to N2 O Reductase is facilitated by the general redox mediator methyl viologen. The liposomal microreactors use only earth-abundant elements to catalyze N2 O removal in ambient, aqueous conditions.

Microbiota-mediated protection against antibiotic-resistant pathogens.

Colonization by the microbiota provides one of our most effective barriers against infection by pathogenic microbes. The microbiota protects against infection by priming immune defenses, by metabolic exclusion of pathogens from their preferred niches, and through direct antimicrobial antagonism. Disruption of the microbiota, especially by antibiotics, is a major risk factor for bacterial pathogen colonization. Restoration of the microbiota through microbiota transplantation has been shown to be an effective way to reduce pathogen burden in the intestine but comes with a number of drawbacks, including the possibility of transferring other pathogens into the host, lack of standardization, and potential disruption to host metabolism. More refined methods to exploit the power of the microbiota would allow us to utilize its protective power without the drawbacks of fecal microbiota transplantation. To achieve this requires detailed understanding of which members of the microbiota protect against specific pathogens and the mechanistic basis for their effects. In this review, we will discuss the clinical and experimental evidence that has begun to reveal which members of the microbiota protect against some of the most troublesome antibiotic-resistant pathogens: Klebsiella pneumoniae, vancomycin-resistant enterococci, and Clostridioides difficile.

Shigella sonnei infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence.

Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.

The power of national acoustic tracking networks to assess the impacts of human activity on marine organisms during the COVID-19 pandemic.

COVID-19 restrictions have led to an unprecedented global hiatus in anthropogenic activities, providing a unique opportunity to assess human impact on biological systems. Here, we describe how a national network of acoustic tracking receivers can be leveraged to assess the effects of human activity on animal movement and space use during such global disruptions. We outline variation in restrictions on human activity across Australian states and describe four mechanisms affecting human interactions with the marine environment: 1) reduction in economy and trade changing shipping traffic; 2) changes in export markets affecting commercial fisheries; 3) alterations in recreational activities; and 4) decline in tourism. We develop a roadmap for the analysis of acoustic tracking data across various scales using Australia's national Integrated Marine Observing System (IMOS) Animal Tracking Facility as a case study. We illustrate the benefit of sustained observing systems and monitoring programs by assessing how a 51-day break in white shark (Carcharodon carcharias) cage-diving tourism due to COVID-19 restrictions affected the behaviour and space use of two resident species. This cessation of tourism activities represents the longest break since cage-diving vessels started day trips in this area in 2007. Long-term monitoring of the local environment reveals that the activity space of yellowtail kingfish (Seriola lalandi) was reduced when cage-diving boats were absent compared to periods following standard tourism operations. However, white shark residency and movements were not affected. Our roadmap is globally applicable and will assist researchers in designing studies to assess how anthropogenic activities can impact animal movement and distributions during regional, short-term through to major, unexpected disruptions like the COVID-19 pandemic.

A National Cost Analysis of Community Interventions to Prevent Underage Drinking and Prescription Drug Misuse.

This prospective cost analysis addresses a gap in the prevention literature by providing estimates of the typical real-world costs to implement community interventions focused on preventing underage drinking and prescription drug misuse. The study uses cost data reported by more than 400 community subrecipients participating in a national cross-site evaluation of the Substance Abuse and Mental Health Services Administration's Strategic Prevention Framework Partnerships for Success grant program during 2013-2017. Community subrecipient organizations completed an annual Web-based survey to report their intervention costs. The analysis compares the relative startup and annual ongoing implementation costs of different prevention strategies and services. Partnerships for Success communities implemented a wide variety of interventions. Annual ongoing implementation was typically more costly than intervention startup. Costs were generally similar for population-level interventions, such as information dissemination and environmental strategies, and individual-level interventions, such as prevention education and positive alternative activities. However, population-level interventions reached considerably more people and consequently had much lower costs per person. Personnel contributed the most to intervention costs, followed by intervention supplies and overhead. Startup costs for initial training and costs for incentives, ongoing training, and in-kind contributions (nonlabor) during ongoing implementation were not typically reported. This study informs prevention planning by providing detailed information about the costs of classes of interventions used in communities, outside of research settings.

Defining a Safe Zone for Percutaneous Screw Fixation of Posterior Malleolar Fractures.

Posterior malleolar fractures require fixation to confer stability to the ankle. Although some have suggested that fractures involving less than 25% of the articular surface require no intervention, estimation of malleolar size on plain imaging is inaccurate. Some posterior malleolar fractures may be particularly suitable for posterior-to-anterior percutaneous screw fixation of the posterior malleolus via a posterolateral approach. We hypothesized that there may be a safe zone in the posterolateral ankle, identifiable with reliable anatomic landmarks, that might allow safe percutaneous screw placement for fracture fixation. The study protocol involved Step 1, in which multiple Kirschner wires were used in a single cadaveric specimen to attempt to identify a safe zone entry point in the posterior ankle, and Step 2, in which a single wire was used in each of six additional cadaveric specimens to test the ability to safely replicate the use of that entry point. In Step 1, a safe zone entry point was identified, located immediately lateral to the Achilles tendon and 1 cm above the level of the tip of the medial malleolus, when visualizing the posterior ankle. In Step 2, using these landmarks and an image intensifier, single wires were then successfully placed in the other six specimens without injury to any significant structure. If confirmed in clinical studies, the safe zone entry point that we have identified could potentially be used to facilitate posterior-to-anterior percutaneous fixation in patients with posterior malleolar fractures for whom open reduction may not be required or may be contraindicated.

A Latent Class Analysis of Prevention Approaches Used to Reduce Community-Level Prescription Drug Misuse in Adolescents and Young Adults.

The Substance Abuse and Mental Health Services Administration's Strategic Prevention Framework Partnerships for Success (PFS) program supports community-based organizations (CBOs) across the United States in implementing evidence-based prevention interventions to reduce substance use in adolescents and young adults. Little attention has been paid to how CBOs combine interventions to create comprehensive community-specific prevention approaches, or whether different approaches achieve similar community-level effects on prescription drug misuse (PDM). We used PFS evaluation data to address these gaps. Over 200 CBOs reported their prevention intervention characteristics, including strategy type (e.g., prevention education, environmental strategies) and number of unique interventions. Evaluation staff coded whether each intervention was an evidence-based program, practice, or policy (EBPPP). Latent Class Analysis of seven characteristics (use of each of five strategy types, use of one or more EBPPP, and number of interventions implemented) identified six prevention approach profiles: High Implementation EBPPP, Media Campaigns, Environmental EBPPP, High Implementation Non-EBPPP, Prevention Education, and Other Information Dissemination. All approaches except Media Campaigns and Other Information Dissemination were associated with significant reductions in community-level PDM. These approaches may need to be paired with other, more direct, prevention activities to effectively reduce PDM at the community level. However, similar rates of change in PDM across all 6 prevention approaches suggests only weak evidence favoring use of the other four approaches. Community-based evaluations that account for variability in implemented prevention approaches may provide a more nuanced understanding of community-level effects. Additional work is needed to help CBOs identify the most appropriate approach to use based on their target communities' characteristics and resources.