Membraneless channels sieve cations in ammonia-oxidizing marine archaea.

Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle1,2. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.

LipIDens: simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities.

Effectiveness of out-patient based acute heart failure care: a pilot randomised controlled trial.

OBJECTIVES: Acute heart failure (AHF) hospitalisation is associated with 10% mortality. Outpatient based management (OPM) of AHF appeared effective in observational studies. We conducted a pilot randomised controlled trial (RCT) comparing OPM with standard inpatient care (IPM). METHODS: We randomised patients with AHF, considered to need IV diuretic treatment for ≥2 days, to IPM or OPM. We recorded all-cause mortality, and the number of days alive and out-of-hospital (DAOH). Quality of life, mental well-being and Hope scores were assessed. Mean NHS cost savings and 95% central range (CR) were calculated from bootstrap analysis. Follow-up: 60 days. RESULTS: Eleven patients were randomised to IPM and 13 to OPM. There was no statistically significant difference in all-cause mortality during the index episode (1/11 vs 0/13) and up to 60 days follow-up (2/11 vs 2/13) [p = .86]. The OPM group accrued more DAOH {47 [36,51] vs 59 [41,60], p = .13}. Two patients randomised to IPM (vs 6 OPM) were readmitted [p = .31]. Hope scores increased more with OPM within 30 days but dropped to lower levels than IPM by 60 days. More out-patients had increased total well-being scores by 60 days (p = .04). OPM was associated with mean cost savings of £2658 (95% CR 460-4857) per patient. CONCLUSIONS: Patients with acute HF randomised to OPM accrued more days alive out of hospital (albeit not statistically significantly in this small pilot study). OPM is favoured by patients and carers and is associated with improved mental well-being and cost savings.

Structure of the native chemotaxis core signaling unit from phage E-protein lysed E. coli cells.

IMPORTANCE: Bacterial chemotaxis is a ubiquitous behavior that enables cell movement toward or away from specific chemicals. It serves as an important model for understanding cell sensory signal transduction and motility. Characterization of the molecular mechanisms underlying chemotaxis is of fundamental interest and requires a high-resolution structural picture of the sensing machinery, the chemosensory array. In this study, we combine cryo-electron tomography and molecular simulation to present the complete structure of the core signaling unit, the basic building block of chemosensory arrays, from Escherichia coli. Our results provide new insight into previously poorly-resolved regions of the complex and offer a structural basis for designing new experiments to test mechanistic hypotheses.

A new class of protein sensor links spirochete pleomorphism, persistence, and chemotaxis.

IMPORTANCE: A new class of bacterial protein sensors monitors intracellular levels of S-adenosylmethionine to modulate cell morphology, chemotaxis, and biofilm formation. Simultaneous regulation of these behaviors enables bacterial pathogens to survive within their niche. This sensor, exemplified by Treponema denticola CheWS, is anchored to the chemotaxis array and its sensor domain is located below the chemotaxis rings. This position may allow the sensor to directly interact with the chemotaxis histidine kinase CheA. Collectively, these data establish a critical role of CheWS in pathogenesis and further illustrate the impact of studying non-canonical chemotaxis proteins.

Rampant asexual reproduction and limited dispersal in a mangrove population of the coral Porites divaricata.

Corals are critical to marine biodiversity. Reproduction and dispersal are key to their resilience, but rarely quantified in nature. Exploiting a unique system-a fully censused, longitudinally characterized, semi-isolated population inhabiting mangroves-we used 2bRAD sequencing to demonstrate that rampant asexual reproduction most likely via parthenogenesis and limited dispersal enable the persistence of a natural population of thin-finger coral (Porites divaricata). Unlike previous studies on coral dispersal, knowledge of colony age and location enabled us to identify plausible parent-offspring relationships within multiple clonal lineages and develop tightly constrained estimates of larval dispersal; the best-fitting model indicates dispersal is largely limited to a few metres from parent colonies. Our results explain why this species is adept at colonizing mangroves but suggest limited genetic diversity in mangrove populations and limited connectivity between mangroves and nearby reefs. As P. divaricata is gonochoristic, and parthenogenesis would be restricted to females (whereas fragmentation, which is presumably common in reef and seagrass habitats, is not), mangrove populations likely exhibit skewed sex ratios. These findings suggest that coral reproductive diversity can lead to distinctly different demographic outcomes in different habitats. Thus, coral conservation will require the protection of the entire coral habitat mosaic, and not just reefs.

Concurrent validity of machine learning-classified functional upper extremity use from accelerometry in chronic stroke.

Objective: This study aims to investigate the validity of machine learning-derived amount of real-world functional upper extremity (UE) use in individuals with stroke. We hypothesized that machine learning classification of wrist-worn accelerometry will be as accurate as frame-by-frame video labeling (ground truth). A second objective was to validate the machine learning classification against measures of impairment, function, dexterity, and self-reported UE use. Design: Cross-sectional and convenience sampling. Setting: Outpatient rehabilitation. Participants: Individuals (>18 years) with neuroimaging-confirmed ischemic or hemorrhagic stroke >6-months prior (n = 31) with persistent impairment of the hemiparetic arm and upper extremity Fugl-Meyer (UEFM) score = 12-57. Methods: Participants wore an accelerometer on each arm and were video recorded while completing an "activity script" comprising activities and instrumental activities of daily living in a simulated apartment in outpatient rehabilitation. The video was annotated to determine the ground-truth amount of functional UE use. Main outcome measures: The amount of real-world UE use was estimated using a random forest classifier trained on the accelerometry data. UE motor function was measured with the Action Research Arm Test (ARAT), UEFM, and nine-hole peg test (9HPT). The amount of real-world UE use was measured using the Motor Activity Log (MAL). Results: The machine learning estimated use ratio was significantly correlated with the use ratio derived from video annotation, ARAT, UEFM, 9HPT, and to a lesser extent, MAL. Bland-Altman plots showed excellent agreement between use ratios calculated from video-annotated and machine-learning classification. Factor analysis showed that machine learning use ratios capture the same construct as ARAT, UEFM, 9HPT, and MAL and explain 83% of the variance in UE motor performance. Conclusion: Our machine learning approach provides a valid measure of functional UE use. The accuracy, validity, and small footprint of this machine learning approach makes it feasible for measurement of UE recovery in stroke rehabilitation trials.

Domestic violence assault during the first year of the COVID-19 pandemic: a longitudinal community study.

BACKGROUND: The consequences of the COVID-19 pandemic have been far-reaching, disproportionately impacting vulnerable populations. Of particular concern is the impact on individuals experiencing domestic violence (DV), an urgent public health issue. There have been numerous reports of pandemic-related surges in DV, and it has been speculated that prolonged periods of state-mandated isolation may be the source of these surges. The current study utilized publicly available records to examine fluctuations in DV coinciding with COVID-19 lockdown restrictions in a diverse metropolitan county. METHODS: Data were extracted from local police blotters and mapping engines in Orange County, California (United States), documenting police-reported DV assault. All incidents were coded for time to examine the time course of DV among other types of assault, allowing for a longitudinal view of incidents over a 66-week window. Changepoint analyses were used to determine whether and when DV assaults changed when mapped with coinciding tightening or loosening of restrictions county-wide. Piecewise regression analyses evaluated whether any detected fluctuations were statistically meaningful. RESULTS: In Santa Ana, rates saw a small but significant spike in the week following the first major lockdown in March 2020 (b = .04, SE = .02, t = 2.37, p = .01), remaining stable at this higher level thereafter (b = -.003, SE = .003, t = -1.29, p = .20). In Anaheim, no meaningful change in DV assault rates was observed at any time interval. CONCLUSION: Results suggest that surges in DV vary between communities and that systemic issues may set the stage for the surge of an already endemic problem.

Borrelia miyamotoi: A Comprehensive Review.

Borrelia miyamotoi is an emerging tick-borne pathogen in the Northern Hemisphere and is the causative agent of Borrelia miyamotoi disease (BMD). Borrelia miyamotoi is vectored by the same hard-bodied ticks as Lyme disease Borrelia, yet phylogenetically groups with relapsing fever Borrelia, and thus, has been uniquely labeled a hard tick-borne relapsing fever Borrelia. Burgeoning research has uncovered new aspects of B. miyamotoi in human patients, nature, and the lab. Of particular interest are novel findings on disease pathology, prevalence, diagnostic methods, ecological maintenance, transmission, and genetic characteristics. Herein, we review recent literature on B. miyamotoi, discuss how findings adapt to current Borrelia doctrines, and briefly consider what remains unknown about B. miyamotoi.

Structural basis for SMAC-mediated antagonism of caspase inhibition by the giant ubiquitin ligase BIRC6.

Certain inhibitor of apoptosis (IAP) family members are sentinel proteins that prevent untimely cell death by inhibiting caspases. Antagonists, including second mitochondria-derived activator of caspases (SMAC), regulate IAPs and drive cell death. Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP with dual E2 and E3 ubiquitin ligase activity, regulates programmed cell death through unknown mechanisms. We show that BIRC6 directly restricts executioner caspase-3 and -7 and ubiquitinates caspase-3, -7, and -9, working exclusively with noncanonical E1, UBA6. Notably, we show that SMAC suppresses both mechanisms. Cryo-electron microscopy structures of BIRC6 alone and in complex with SMAC reveal that BIRC6 is an antiparallel dimer juxtaposing the substrate-binding module against the catalytic domain. Furthermore, we discover that SMAC multisite binding to BIRC6 results in a subnanomolar affinity interaction, enabling SMAC to competitively displace caspases, thus antagonizing BIRC6 anticaspase function.

Photographic grading for trachoma diagnosis within trachoma impact surveys in Amhara region, Ethiopia.

BACKGROUND: As countries reach the trachoma elimination threshold and cases of trachomatous inflammation follicular (TF) become rare, it becomes difficult to train survey graders to recognize clinical signs. We assess the use of photography as a grading tool, the efficiency of an in-country grading center and the comparability of field and photographic grading. METHODS: During January-February 2017 surveys in Amhara, Ethiopia, field graders assessed TF, trachomatous inflammation intense (TI) and trachomatous scarring (TS). Photographs were taken from each conjunctiva and later graded at the Gondar Grading Center (GGC) at the University of Gondar in Amhara. Two trained ophthalmology residents graded each set of photographs and a third grader provided an adjudicating grade when needed. RESULTS: A total of 4953 photographs of 2477 conjunctivae from 1241 participants in 10 communities were graded over 5 d at the GGC. Six examined participants were not photographed. Agreement between field and photographic grades were for TF: percent agreement (PA) 96.7%, κ=0.70 (95% confidence interval [CI] 0.64 to 0.77; for TI: PA 94.7%, κ=0.32 (95% CI 0.20 to 0.43); and for TS: PA 83.5%, κ=0.22 (95% CI 0.15 to 0.29). CONCLUSIONS: Conjunctival photography may be a solution for programs near the elimination threshold where there are few available community cases for training field graders.

High-resolution reconstruction of a Jumbo-bacteriophage infecting capsulated bacteria using hyperbranched tail fibers.

The Klebsiella jumbo myophage ϕKp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combine cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, microbiological and machine learning approaches to explore the capsid, tail, and tail fibers of ϕKp24. We determine the structure of the capsid and tail at 4.1 Šand 3.0 Šresolution. We observe the tail fibers are branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide ϕKp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Our study provides structural and functional insight into how ϕKp24 adapts to the variable surfaces of capsulated bacterial pathogens, which is useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.

Mechanisms of E. coli chemotaxis signaling pathways visualized using cryoET and computational approaches.

Chemotaxis signaling pathways enable bacteria to sense and respond to their chemical environment and, in some species, are critical for lifestyle processes such as biofilm formation and pathogenesis. The signal transduction underlying chemotaxis behavior is mediated by large, highly ordered protein complexes known as chemosensory arrays. For nearly two decades, cryo-electron tomography (cryoET) has been used to image chemosensory arrays, providing an increasingly detailed understanding of their structure and function. In this mini-review, we provide an overview of the use of cryoET to study chemosensory arrays, including imaging strategies, key results, and outstanding questions. We further discuss the application of molecular modeling and simulation techniques to complement structure determination efforts and provide insight into signaling mechanisms. We close the review with a brief outlook, highlighting promising future directions for the field.

Structure and activity of particulate methane monooxygenase arrays in methanotrophs.

Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.

Machine Learning Improves Functional Upper Extremity Use Capture in Distal Radius Fracture Patients.

Current outcome measures, including strength/range of motion testing, patient-reported outcomes (PROs), and motor skill testing, may provide inadequate granularity in reflecting functional upper extremity (UE) use after distal radius fracture (DRF) repair. Accelerometry analysis also has shortcomings, namely, an inability to differentiate functional versus nonfunctional movements. The objective of this study was to evaluate the accuracy of machine learning (ML) analyses in capturing UE functional movements based on accelerometry data for patients after DRF repair. In this prospective study, six patients were enrolled 2-6 weeks after DRF open reduction and internal fixation (ORIF). They all performed standardized activities while wearing a wrist accelerometer, and the data were analyzed by an ML algorithm. These activities were also videotaped and evaluated by visual inspection. Our novel ML algorithm was able to predict from accelerometry data whether the limb was performing a movement rated as functional, with accuracy of 90.4% ± 3.6% for within-subject modeling and 79.8% ± 8.9% accuracy for between-subject modeling. The application of ML algorithms to accelerometry data allowed for capture of functional UE activity in patients after DRF open reduction and internal fixation and accurately predicts functional UE use. Such analyses could improve our understanding of recovery and enhance routine postoperative rehabilitation in DRF patients.

Medial septum activation improves strategy switching once strategies are well-learned via bidirectional regulation of dopamine neuron population activity.

Strategy switching is a form of cognitive flexibility that requires inhibiting a previously successful strategy and switching to a new strategy of a different categorical modality. It is dependent on dopamine (DA) receptor activation and release in ventral striatum and prefrontal cortex, two primary targets of ventral tegmental area (VTA) DA projections. Although the circuitry that underlies strategy switching early in learning has been studied, few studies have examined it after extended discrimination training. This may be important as DA activity and release patterns change across learning, with several studies demonstrating a critical role for substantia nigra pars compacta (SNc) DA activity and release once behaviors are well-learned. We have demonstrated that medial septum (MS) activation simultaneously increased VTA and decreased SNc DA population activity, as well as improved reversal learning via these actions on DA activity. We hypothesized that MS activation would improve strategy switching both early in learning and after extended training through its ability to increase VTA DA population activity and decrease SNc DA population activity, respectively. We chemogenetically activated the MS of male and female rats and measured their performance on an operant-based strategy switching task following 1, 10, or 15 days of discrimination training. Contrary to our hypothesis, MS activation did not affect strategy switching after 1 day of discrimination training. MS activation improved strategy switching after 10 days of training, but only in females. MS activation improved strategy switching in both sexes after 15 days of training. Infusion of bicuculline into the ventral subiculum (vSub) inhibited the MS-mediated decrease in SNc DA population activity and attenuated the improvement in strategy switching. Intra-vSub infusion of scopolamine inhibited the MS-mediated increase in VTA DA population activity but did not affect the improvement in strategy switching. Intra-vSub infusion of both bicuculline and scopolamine inhibited the MS-mediated effects on DA population activity in both the SNc and VTA and completely prevented the improvement in strategy switching. These data indicate that MS activation improves strategy switching once the original strategy has been sufficiently well-learned, and that this may occur via the MS's regulation of DA neuron responsivity.

Routine Lower Extremity Screening Ultrasound Protocols in Trauma Patients Are Not Cost Effective.

BACKGROUND: Despite prophylaxis, deep vein thrombosis (DVT) and pulmonary embolism remain dreaded complications following traumatic injury and are associated with significant morbidity and mortality. Screening ultrasound (US) protocols have been employed in trauma centers for early detection of lower extremity (LE) deep venous thrombosis. We hypothesized that screening lower extremity venous duplex US would not prove cost effective in our trauma population who receives early pharmacologic prophylaxis. METHODS: Data was collected for one year on all adult trauma patients admitted to the trauma service from December 2019 to 2020. DVT screening US was obtained at 3 days after admission for patients with long bone or pelvic fracture, spinal cord injury, immobility, and/or spinal fracture requiring surgery. Screening US was obtained at 7 days for all others and repeated weekly until discharge. Data was retrospectively collected and analyzed. RESULTS: Exactly 1365 patients met inclusion criteria with median ISS 12 (IQR, 9-17), median age 56 (IQR, 36-73 years), and with majority blunt injuries (90.7%). A total of 1369 screening US were performed finding 27 DVTs (2%). The total cost of screening for the year analyzed amounted to over $270,000 with 50.7 screening US needed to detect 1 DVT. This resulted in an average screening cost of over $10,000 for the detection of a single DVT. DISCUSSION: In trauma patients receiving early pharmacologic prophylaxis, routine LE screening US protocols to detect LE DVT are not cost effective.

Isolation-by-distance and genetic parentage analysis provide similar larval dispersal estimates.

Larval exchange among marine populations is a vital driver of population dynamics and has the potential to inform conservation actions, but accurately measuring dispersal remains challenging. Consequently, empirical dispersal kernels have been measured for only a few marine species. Here, we obtained indirect dispersal estimates using an isolation-by-distance (IBD) model in the coral reef fish Elacatinus lori and assessed the accuracy of these estimates by comparing them to direct measurements of dispersal from genetic parentage analysis. Specifically, drawing on the IBD slope and effective population density, we indirectly estimated sigma (σ), the spread of a dispersal distribution. While the spread of the directly measured distribution was σ = 3.93 km (95% CI: 3.29-4.71 km), the spread of the IBD distribution was σ = 4.10 km (95% CI: 3.23-5.03) and σ = 2.90 km (95% CI: 2.26-3.59), assuming a random or monogamous mating system, respectively. Parameterizing Laplace dispersal kernels with these values of σ yielded patterns that were remarkably similar to a kernel fit to the direct parentage data. We also found that, like many marine fishes, E. lori has a large effective population size. However, uncertainty in effective size did not ultimately have a strong effect on the IBD-based dispersal estimates. Taken together, these findings illustrate that accurate dispersal estimates can be produced by indirect IBD methods and suggest that this more feasible approach to estimating dispersal may be broadly applicable to the study of marine larval dispersal.