A Dynamic Search Process Underlies MicroRNA Targeting.

Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target messenger RNAs for repression. Here, we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2-4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2-8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes the target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.

Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute.

Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

Factor structure of posttraumatic stress disorder (PTSD) in persons with serious mental illness.

BACKGROUND: Trauma and posttraumatic stress disorder (PTSD) are common among individuals with serious mental illness (SMI; e.g., schizophrenia, schizoaffective disorder, bipolar disorder, treatment refractory major depressive disorder), with resultant functional impairment. Previous studies have not evaluated the factor structure of the PTSD Checklist (PCL) among persons with SMI. AIMS: This study evaluated the factor structure of the PCL in two large SMI samples from public mental health treatment sectors screened for PTSD using the PCL. METHODS: Four different models of PTSD were tested using confirmatory factor analyses. RESULTS: Results indicated that the DSM-5 4-factor model (intrusion, avoidance, numbing, and hyperarousal) had the best fit. Further, the DSM-5 4-factor model demonstrated measurement invariance. CONCLUSIONS: Results supported the suitability of the DSM-5 4-factor model of PTSD among people with SMI.

A novel role for PGE2-EP4 in the developmental programming of the mouse ductus arteriosus: consequences for vessel maturation and function.

The ductus arteriosus (DA) is a vascular shunt that allows oxygenated blood to bypass the developing lungs in utero. Fetal DA patency requires vasodilatory signaling via the prostaglandin E2 (PGE2) receptor EP4. However, in humans and mice, disrupted PGE2-EP4 signaling in utero causes unexpected patency of the DA (PDA) after birth, suggesting another role for EP4 during development. We used EP4-knockout (KO) mice and acute versus chronic pharmacological approaches to investigate EP4 signaling in DA development and function. Expression analyses identified EP4 as the primary EP receptor in the DA from midgestation to term; inhibitor studies verified EP4 as the primary dilator during this period. Chronic antagonism recapitulated the EP4 KO phenotype and revealed a narrow developmental window when EP4 stimulation is required for postnatal DA closure. Myography studies indicate that despite reduced contractile properties, the EP4 KO DA maintains an intact oxygen response. In newborns, hyperoxia constricted the EP4 KO DA but survival was not improved, and permanent remodeling was disrupted. Vasomotion and increased nitric oxide (NO) sensitivity in the EP4 KO DA suggest incomplete DA development. Analysis of DA maturity markers confirmed a partially immature EP4 KO DA phenotype. Together, our data suggest that EP4 signaling in late gestation plays a key developmental role in establishing a functional term DA. When disrupted in EP4 KO mice, the postnatal DA exhibits signaling and contractile properties characteristic of an immature DA, including impairments in the first, muscular phase of DA closure, in addition to known abnormalities in the second permanent remodeling phase.NEW & NOTEWORTHY EP4 is the primary EP receptor in the ductus arteriosus (DA) and is critical during late gestation for its development and eventual closure. The "paradoxical" patent DA (PDA) phenotype of EP4-knockout mice arises from a combination of impaired contractile potential, altered signaling properties, and a failure to remodel associated with an underdeveloped immature vessel. These findings provide new mechanistic insights into women who receive NSAIDs to treat preterm labor, whose infants have unexplained PDA.

A fast temperature-programmed second-dimension column for comprehensive two-dimensional gas chromatography.

The short analysis time and constant temperature environment in the second dimension of two-dimensional comprehensive chromatography frequently causes wraparound problems, especially for complex high boilers. This problem can be solved by temperature programming on the second column, but since this requires heating and cooling the column in a matter of seconds, it is difficult to implement. In this study, we describe a method of accomplishing rapid heating and cooling with a resistively heated column cooled by compressed air. Critical to this method is minimizing the lag time between the actual temperature and the reported temperature by using the column heating element as the temperature sensor, virtually eliminating the danger of overshooting the temperature setpoint. This technique facilitates a ramp rate of up to 100 °C/s with minimal overshooting-well beyond the requirements of gas chromatography. A single-layer column bundle design allows a compressed-air cooling device to cool the column from 200 to 50 °C at an average rate of -21 °C/s. The secondary dimension temperature programming is facilitated by the longer secondary dimension time made possible by the direct flow modulation method. We evaluated the performance of the single-layer column bundle and demonstrated this method by applying it to a gasoline sample. We also compared this method with the traditional isothermal approach and found that use of the secondary temperature program reduced the naphthalene retention time from 12.1 to 6.3 s and its peak width at half height from 846 to 126 ms.

Moving past neonicotinoids and honeybees: A systematic review of existing research on other insecticides and bees.

Synthetic pesticides (e.g. herbicides, fungicides and insecticides) are used widely in agriculture to protect crops from pests, weeds and disease. However, their use also comes with a range of environmental concerns. One key concern is the effect of insecticides on non-target organisms such as bees, who provide pollination services for crops and wild plants. This systematic literature review quantifies the existing research on bees and insecticides broadly, and then focuses more specifically on non-neonicotinoid insecticides and non-honeybees. We find that articles on honeybees (Apis sp.) and insecticides account for 80% of all research, with all other bees combined making up 20%. Neonicotinoids were studied in 34% of articles across all bees and were the most widely studied insecticide class for non-honeybees overall, with almost three times as many studies than the second most studied class. Of non-neonicotinoid insecticide classes and non-honeybees, the most studied were pyrethroids and organophosphates followed by carbamates, and the most widely represented bee taxa were bumblebees (Bombus), followed by leaf-cutter bees (Megachile) and mason bees (Osmia). Research has taken place across several countries, with the highest numbers of articles from Brazil and the US, and with notable gaps from countries in Asia, Africa and Oceania. Mortality was the most studied effect type, while sub-lethal effects such as on behaviour were less studied. Few studies tested how the effect of insecticides were influenced by multiple pressures, such as climate change and co-occurring pesticides (cocktail effects). As anthropogenic pressures do not occur in isolation, we suggest that future research also addresses these knowledge gaps. Given the changing global patterns in insecticide use, and the increasing inclusion of both non-honeybees and sub-lethal effects in pesticide risk assessment, there is a need for expanding research beyond its current state to ensure a strong scientific evidence base for the development of risk assessment and associated policy.

Reliability of velocity pulsatility in small vessels on 3Tesla MRI in the basal ganglia: a test-retest study.

OBJECTIVE: Recent work showed the feasibility of measuring velocity pulsatility in the perforating arteries at the level of the BG using 3T MRI. However, test-retest measurements have not been performed, yet. This study assessed the test-retest reliability of 3T MRI blood flow velocity measurements in perforating arteries in the BG. MATERIALS AND METHODS: Two-dimensional phase-contrast cardiac gated (2D-PC) images were acquired for 35 healthy controls and repeated with and without repositioning. 2D-PC images were processed and analyzed, to assess the number of detected perforating arteries (Ndetected), mean blood flow velocity (Vmean), and velocity pulsatility index (vPI). Paired t-tests and Bland-Altman plots were used to compare variance in outcome parameters with and without repositioning, and limits of agreement (LoA) were calculated. RESULTS: The LoA was smallest for Vmean (35%) and highest for vPI (79%). Test-retest reliability was similar with and without repositioning of the subject. DISCUSSION: We found similar LoA with and without repositioning indicating that the measurement uncertainty is dominated by scanner and physiological noise, rather than by planning. This enables to study hemodynamic parameters in perforating arteries at clinically available scanners, provided sufficiently large sample sizes are used to mitigate the contribution of scanner- and physiological noise.

Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition.

Argonaute proteins use microRNAs (miRNAs) to identify mRNAs targeted for post-transcriptional repression. Biochemical assays have demonstrated that Argonaute functions by modulating the binding properties of its miRNA guide so that pairing to the seed region is exquisitely fast and accurate. However, the mechanisms used by Argonaute to reshape the binding properties of its small RNA guide remain poorly understood. Here, we identify a structural element, α-helix-7, in human Argonaute2 (Ago2) that is required for speed and fidelity in binding target RNAs. Biochemical, structural, and single-molecule data indicate that helix-7 acts as a molecular wedge that pivots to enforce rapid making and breaking of miRNA:target base pairs in the 3' half of the seed region. These activities allow Ago2 to rapidly dismiss off-targets and dynamically search for seed-matched sites at a rate approaching the limit of diffusion.

The octopamine receptor, OA2B2, modulates stress resistance and reproduction in Nilaparvata lugens Stål (Hemiptera: Delphacidae).

The brown planthopper (BPH), Nilaparvata lugens (Stål) is a resurgent pest of rice crops throughout Asia. We recently discovered that octopamine (OA) and OA2B2 operate in the BPH mating system, where it mediates a wide range of molecular, physiological and behavioural changes. Here, we report on outcomes of experiments designed to test the hypothesis that OA/OA2B2 signalling mediates responses to three abiotic stressors, starvation, high temperature (37 °C), and induced oxidative stress. We found per os RNAi-mediated OA2B2 silencing led to significantly decreased survival, measured in days, following exposure to each of these stressors. We selected a biologically costly process, reproductive biology, as a biotic stressor. Silencing of OA2B2 led to decreased total protein content in ovaries and fat bodies, downregulated expression of vitellogenin (Vg) and Vg receptor (VgR), inhibited fat body Vg protein synthesis, shortened the oviposition period, prolonged the preoviposition period, reduced the number of laid eggs, body weight and female longevity. In addition, the silencing treatments also led to inhibited ovarian development, and ovarian Vg uptake, reduced numbers of egg masses and offspring and lower hatching rates and population growth index. These data support our hypothesis that OA2B2 acts in mediating BPH resistance to biotic and abiotic stressors.

Novel anterior coracoglenoid line utilizing magnetic resonance imaging (MRI) corresponds with critical glenoid bone loss.

OBJECTIVE: Glenoid bone loss is estimated using a best-fit circle method and requires software tools that may not be available. Our hypothesis is that a vertical reference line drawn parallel to the long axis of the glenoid and passing through the inflection point of the coracoid and glenoid will represent a demarcation line of approximately 20% of the glenoid. Our aim is to establish a more efficient method to estimate a surgical threshold for glenoid insufficiency. METHODS: Fifty patients with normal glenoid anatomy were randomly chosen from an orthopedic surgeon's database. Two orthopedic surgeons utilized T1-weighted sagittal MRIs and the coracoglenoid line technique to determine the percentage of bony glenoid anterior to vertical line. Two musculoskeletal radiologists measured the same 50 glenoids using the circle technique. Differences were determined using dependent t test. Reliability was compared using interclass correlation coefficient and Kappa. Validity was compared using Pearson correlation coefficient. RESULTS: Mean surface area of the glenoid anterior to the vertical line was on average 21.69% ± 3.12%. Surface area of the glenoid using the circle method was on average 20.86% ± 2.29%. Inter-rater reliability of the circle method was 0.553 (fair). Inter-rater reliability of the vertical line technique was 0.83 (excellent). There was a linear relationship between circle and vertical line measurements, r = 0.704 (moderate to high). CONCLUSION: The coracoglenoid line appears to represent a line of demarcation of approximately 21% of glenoid bone anterior to the coracoglenoid line. Our technique was found to be reliable, valid, and accurate.

Outcome Prediction of Postanoxic Coma: A Comparison of Automated Electroencephalography Analysis Methods.

BACKGROUND: To compare three computer-assisted quantitative electroencephalography (EEG) prediction models for the outcome prediction of comatose patients after cardiac arrest regarding predictive performance and robustness to artifacts. METHODS: A total of 871 continuous EEGs recorded up to 3 days after cardiac arrest in intensive care units of five teaching hospitals in the Netherlands were retrospectively analyzed. Outcome at 6 months was dichotomized as "good" (Cerebral Performance Category 1-2) or "poor" (Cerebral Performance Category 3-5). Three prediction models were implemented: a logistic regression model using two quantitative features, a random forest model with nine features, and a deep learning model based on a convolutional neural network. Data from two centers were used for training and fivefold cross-validation (n = 663), and data from three other centers were used for external validation (n = 208). Model output was the probability of good outcome. Predictive performances were evaluated by using receiver operating characteristic analysis and the calculation of predictive values. Robustness to artifacts was evaluated by using an artifact rejection algorithm, manually added noise, and randomly flattened channels in the EEG. RESULTS: The deep learning network showed the best overall predictive performance. On the external test set, poor outcome could be predicted by the deep learning network at 24 h with a sensitivity of 54% (95% confidence interval [CI] 44-64%) at a false positive rate (FPR) of 0% (95% CI 0-2%), significantly higher than the logistic regression (sensitivity 33%, FPR 0%) and random forest models (sensitivity 13%, FPR, 0%) (p < 0.05). Good outcome at 12 h could be predicted by the deep learning network with a sensitivity of 78% (95% CI 52-100%) at a FPR of 12% (95% CI 0-24%) and by the logistic regression model with a sensitivity of 83% (95% CI 83-83%) at a FPR of 3% (95% CI 3-3%), both significantly higher than the random forest model (sensitivity 1%, FPR 0%) (p < 0.05). The results of the deep learning network were the least affected by the presence of artifacts, added white noise, and flat EEG channels. CONCLUSIONS: A deep learning model outperformed logistic regression and random forest models for reliable, robust, EEG-based outcome prediction of comatose patients after cardiac arrest.

Comparison of Antimicrobial-Resistant Escherichia coli Isolates from Urban Raccoons and Domestic Dogs.

Wildlife can be exposed to antimicrobial-resistant bacteria (ARB) via multiple pathways. Spatial overlap with domestic animals is a prominent exposure pathway. However, most studies of wildlife-domestic animal interfaces have focused on livestock and little is known about the wildlife-companion animal interface. Here, we investigated the prevalence and phylogenetic relatedness of extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli from raccoons (Procyon lotor) and domestic dogs (Canis lupus familiaris) in the metropolitan area of Chicago, IL, USA. To assess the potential importance of spatial overlap with dogs, we explored whether raccoons sampled at public parks (i.e., parks where people and dogs could enter) differed in prevalence and phylogenetic relatedness of ESC-R E. coli to raccoons sampled at private parks (i.e., parks where people and dogs could not enter). Raccoons had a significantly higher prevalence of ESC-R E. coli (56.9%) than dogs (16.5%). However, the richness of ESC-R E. coli did not vary by host species. Further, core single-nucleotide polymorphism (SNP)-based phylogenetic analyses revealed that isolates did not cluster by host species, and in some cases displayed a high degree of similarity (i.e., differed by less than 20 core SNPs). Spatial overlap analyses revealed that ESC-R E. coli were more likely to be isolated from raccoons at public parks than raccoons at private parks, but only for parks located in suburban areas of Chicago, not urban areas. That said, ESC-R E. coli isolated from raccoons did not genetically cluster by park of origin. Our findings suggest that domestic dogs and urban/suburban raccoons can have a diverse range of ARB, some of which display a high degree of genetic relatedness (i.e., differ by less than 20 core SNPs). Given the differences in prevalence, domestic dogs are unlikely to be an important source of exposure for mesocarnivores in urbanized areas. IMPORTANCE Antimicrobial-resistant bacteria (ARB) have been detected in numerous wildlife species across the globe, which may have important implications for human and animal health. Wildlife can be exposed to ARB via numerous pathways, including via spatial overlap with domestic animals. However, the interface with domestic animals has mostly been explored for livestock and little is known about the interface between wild animals and companion animals. Our work suggests that urban and suburban wildlife can have similar ARB to local domestic dogs, but local dogs are unlikely to be a direct source of exposure for urban-adapted wildlife. This finding is important because it underscores the need to incorporate wildlife into antimicrobial resistance surveillance efforts, and to investigate whether certain urban wildlife species could act as additional epidemiological pathways of exposure for companion animals, and indirectly for humans.

Operative temperature analysis of the honey bee Apis mellifera.

A key challenge for linking experiments of organisms performed in a laboratory environment to their performance in more complex environments is to determine thermal differences between a laboratory and the energetically complex terrestrial ecosystem. Studies performed in the laboratory do not account for many factors that contribute to the realized temperature of an organism in its natural environment. This can lead to modelling approaches that use experimentally derived data to erroneously link the air temperature in a laboratory to air temperatures in energetically heterogenous ecosystems. Traditional solutions to this classic problem assume that animals in an isotropic, isothermal chamber behave either as pure heterothermic ectotherms (body temperature=chamber temperature) or homeothermic endotherms (body temperature is entirely independent of chamber temperature). This approach may not be appropriate for endothermic insects which exist as an intermediate between strongly thermoregulating endotherms and purely thermoconforming species. Here, we use a heat budget modelling approach for the honey bee Apis mellifera to demonstrate that the unique physiology of endothermic insects may challenge many assumptions of traditional biophysical modelling approaches. We then demonstrate under modelled field-realistic scenarios that an experiment performed in a laboratory has the potential to both overestimate and underestimate the temperature of foraging bees when only air temperature is considered.

DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute.

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

Seasonal and daily shifts in behavior and resource selection: how a carnivore navigates costly landscapes.

The dynamic environmental conditions in highly seasonal systems likely have a strong influence on how species use the landscape. Animals must balance seasonal and daily changes to landscape risk with the underlying resources provided by that landscape. One way to balance the seasonal and daily changes in the costs and benefits of a landscape is through behaviorally-explicit resource selection and temporal partitioning. Here, we test whether resource selection of coyotes (Canis latrans) in Cape Breton Highlands National Park, Nova Scotia, Canada is behaviorally-explicit and responsive to the daily and seasonal variation to presumed costs and benefits of moving on the landscape. We used GPS data and local convex hulls to estimate space use and Hidden Markov Models to estimate three types of movement behavior: encamped, foraging, and traveling. We then used integrated step-selection analysis to investigate behaviorally explicit resource selection across times of day (diurnal, crepuscular, and nocturnal) and season (snow-free and snow). We found that throughout the day and seasonally coyotes shifted foraging behavior and altered behavior and resource choices to avoid moving across what we could be a challenging landscape. These changes in behavior suggest that coyotes have a complex response to land cover, terrain, and linear corridors that are not only scale dependent but also vary by behavior, diel period, and season. By examining the resource selection across three axes (behavior, time of day, and season), we have a more nuanced understanding of how a predator balances the cost and benefits of a stochastic environment.

Why Argonaute is needed to make microRNA target search fast and reliable.

MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the search process, Argonaute (Ago) proteins come together with miRNA, forming a dynamic search complex. In this review we use the language of free-energy landscapes to discuss recent single-molecule and high-resolution structural data in the light of theoretical work appropriated from the study of transcription-factor search. We suggest that experimentally observed internal states of the Ago-miRNA search complex may have the explicit biological function of speeding up search while maintaining specificity.

Secretion of secretory phospholipase A2 into Spodoptera exigua larval midgut lumen and its role in lipid digestion.

In insects, lipid digestion is controversial because insects have no bile salts to solubilize dietary lipids. One hypothesis is that a secretory type of phospholipase A2 (sPLA2 ) provides lysophospholipid (LPL) from dietary phospholipids (PLs). We identified a sPLA2 , Se-sPLA2 , in beet armyworm, Spodoptera exigua, that hydrolyses PLs at sn-2. Our goal was to investigate its role in lipid digestion. Se-sPLA2 was expressed in the entire alimentary canal. Incubating the isolated midgut in a cell culture medium led to secretion of Se-sPLA2 and other proteins. Ex vivo RNA interference (RNAi) of Se-sPLA2 expression in isolated midgut culture led to significantly decreased Se-sPLA2 secretion into the medium. Feeding double-stranded RNA specific to Se-sPLA2 to larvae suppressed sPLA2 activity in gut contents. A recombinant Se-sPLA2 was susceptible to benzylideneacetone (BZA), a specific PLA2 inhibitor. After feeding BZA to larvae, we recorded significant decreases in gut content sPLA2 activity, body growth and total haemolymph lipid contents. RNAi against Se-sPLA2 resulted in reduced digestibility. Addition of a specific LPL, 1-palmitoyl-sn-glycero-3-phosphocholine, to BZA-treated larvae rescued digestibility and larval growth. These results strongly bolster our hypothesis that Se-sPLA2 secreted from the midgut acts in lipid digestion by providing necessary LPL to solubilize dietary neutral lipids.

Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry.

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3ß3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3ß3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.

Histopathology of Bordetella pertussis in the Baboon Model.

Pertussis is a severe respiratory disease caused by Bordetella pertussis The classic symptoms of pertussis include paroxysmal coughing with an inspiratory whoop, posttussive vomiting, cyanosis, and persistent coryzal symptoms. Infants under 2 months of age experience more severe disease, with most deaths occurring in this age group. Most of what is known about the pathology of pertussis in humans is from the evaluation of fatal human infant cases. The baboon model of pertussis provides the opportunity to evaluate the histopathology of severe but nonfatal pertussis. The baboon model recapitulates the characteristic clinical signs of pertussis observed in humans, including leukocytosis, paroxysmal coughing, mucus production, heavy colonization of the airway, and transmission of the bacteria between hosts. As in humans, baboons demonstrate age-related differences in clinical presentation, with younger animals experiencing more severe disease. We examined the histopathology of 5- to 6-week-old baboons, with the findings being similar to those reported for fatal human infant cases. In juvenile baboons, we found that the disease is highly inflammatory and concentrated to the lungs with signs of disease that would typically be diagnosed as acute respiratory distress syndrome (ARDS) and bronchopneumonia. In contrast, no significant pathology was observed in the trachea. Histopathological changes in the trachea were limited to cellular infiltrates and mucus production. Immunohistostaining revealed that the bacteria were localized to the surface of the ciliated epithelium in the conducting airways. Our observations provide important insights into the pathology of pertussis in typical, severe but nonfatal pertussis cases in a very relevant animal model.

Evidence for a Partially Stalled γ Rotor in F1-ATPase from Hydrogen-Deuterium Exchange Experiments and Molecular Dynamics Simulations.

F1-ATPase uses ATP hydrolysis to drive rotation of the γ subunit. The γ C-terminal helix constitutes the rotor tip that is seated in an apical bearing formed by α3ß3. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we interrogated E. coli F1-ATPase by hydrogen-deuterium exchange (HDX) mass spectrometry. Rotation of γ caused greatly enhanced deuteration in the γ C-terminal helix. The HDX kinetics implied that most F1 complexes operate with an intact rotor at any given time, but that the rotor tip is prone to occasional unfolding. A molecular dynamics (MD) strategy was developed to model the off-axis forces acting on γ. MD runs showed stalling of the rotor tip and unfolding of the γ C-terminal helix. MD-predicted H-bond opening events coincided with experimental HDX patterns. Our data suggest that in vitro operation of F1-ATPase is associated with significant rotational resistance in the apical bearing. These conditions cause the γ C-terminal helix to get "stuck" (and unfold) sporadically while the remainder of γ continues to rotate. This scenario contrasts the traditional "greasy bearing" model that envisions smooth rotation of the γ C-terminal helix. The fragility of the apical rotor tip in F1-ATPase is attributed to the absence of a c10 ring that stabilizes the rotation axis in intact FoF1. Overall, the MD/HDX strategy introduced here appears well suited for interrogating the inner workings of molecular motors.