ß-barrel membrane proteins fold via hybrid-barrel intermediate states.

Gram-negative bacteria and eukaryotic organelles of bacterial origin contain outer membrane proteins that possess a transmembrane "ß-barrel" domain. The conserved ß-barrel assembly machine (BAM) and the sorting and assembly machine (SAM) are required for the folding and membrane insertion of ß-barrels in Gram-negative bacteria and mitochondria, respectively. Although the mechanisms by which ß-barrels are folded are incompletely understood, advances in cryo-electron microscopy (cryo-EM) have recently yielded unprecedented insights into their folding process. Here we highlight recent studies that show that both bacterial and mitochondrial ß-barrels fold via the formation of remarkable "hybrid-barrel" intermediate states during their interaction with the folding machinery. We discuss how these results align with a general model of ß-barrel folding.

Management of Talus Fractures.

Fractures of the talus are life-changing events. The talus is of vital importance to normal gait. Given its importance, great care is needed in diagnosing and treating these injuries. The threshold for operative treatment and accurate anatomic reduction should be low. Surgical tenets include the avoidance of extensive subperiosteal dissection to minimize vascular disruption. The complications with injuries to the talus are extensive and include avascular necrosis (AVN). Although AVN can prove to be a devastating sequela from this injury, it occurs less frequently than posttraumatic arthritis.

The patatin-like protein PlpD forms structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded ß-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal ß-barrel domain. Challenging the current dogma, we find that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring ß-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.

Molecular Machines that Facilitate Bacterial Outer Membrane Protein Biogenesis.

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a ß-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the ß-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.

In Vivo Disulfide-Bond Crosslinking to Study ß-Barrel Membrane Protein Interactions, Dynamicity, and Folding Intermediates.

Membrane-embedded ß-barrels are the major building blocks of the Gram-negative outer membrane and are involved in antibiotic resistance, virulence, and the maintenance of bacterial cell physiology. The increased frequency of multidrug resistant Gram-negative infections warrants the sharing of accessible methods for the study of ß-barrels. One such method is "in vivo disulfide-bond crosslinking" which is a highly informative and cost-effective approach to study the structure, topology, dynamicity, and function of ß-barrels in situ. The approach can also be used to identify and finely map both stable or transient interactions between ß-barrels and other interacting proteins. In this chapter, I describe the conceptual basis of in vivo disulfide-bond crosslinking and the potential pitfalls in experimental design. I also provide a general protocol for high-efficiency in vivo disulfide-bond crosslinking and modified protocols as examples for how the method can be adapted to different scenarios.

Surgical ablation of atrial fibrillation during mitral valve surgery: a systematic review and meta-analysis.

Background: Atrial fibrillation (AF) is a common tachyarrhythmia, affecting approximately 33 million people worldwide, and is frequently associated with mitral valve disease. Surgical ablation during mitral valve surgery provides an opportune circumstance for arrhythmia correction. The results of recent randomized trial data are promising, demonstrating both safety and efficacy. The aim of this systematic review is to report the efficacy and morbidity of concomitant surgical ablation for AF during mitral valve surgery. Methods: Five electronic databases were searched from inception to March 2023. All studies reporting the primary outcome, freedom from AF (FFAF), for patients with a history of AF undergoing concomitant mitral valve surgery were identified. Studies with patient cohorts less than 100 were excluded. Relevant data were extracted and a meta-analysis of proportions was conducted using a random-effects model. Survival data were pooled from original Kaplan-Meier curves and reconstructed, reporting aggregate FFAF and survival. Results: Thirty-six studies with a total of 8,340 patients were included in the systematic review. All 36 papers reported postoperative FFAF with a pooled result of 76.9% [95% confidence interval (CI): 73.8-79.9%] at a weighted mean follow-up of 40.2 months, however this result was associated with significant heterogeneity (I2=89%). A total of 31 studies reported postoperative short-term mortality, with a pooled result of 1.68% (95% CI: 1.15-2.29%). Aggregate survival at 1 to 5 years was 93.7%, 92.5%, 91.3%, 89.4%, and 87%, respectively, and aggregate FFAF for 1 to 5 years was 90.2%, 83.5%, 79.5%, 76.4% and 73.2%, respectively. Conclusions: Evaluation of the evidence suggests that concomitant ablation for AF during mitral valve surgery is both safe and efficacious. The results were associated with significant heterogeneity, reflective of variable institutional protocols, patient characteristics, and lesion sets. Randomized data with longer term follow-up would help validate these results.

The patatin-like protein PlpD forms novel structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded ß-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal ß-barrel domain. Challenging the current dogma, we found that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring ß-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.

Evaluation of an advanced oral hygiene regimen on maternity outcomes in a randomized multicenter clinical trial (Oral Hygiene and Maternity Outcomes Multicenter Study).

BACKGROUND: During pregnancy, dental plaque-induced gingivitis prevalence and subsequent risk of periodontal disease increases substantially, as a consequence of both inadequate oral hygiene and changes in sex steroid hormone levels. Although maternal periodontal disease has been associated with adverse pregnancy outcomes, including spontaneous preterm birth, previous clinical trials involving mechanical debridement (eg, scaling and root planing) to remove plaque biofilm have not yielded decreased rates of preterm birth. OBJECTIVE: We hypothesized that an advanced oral hygiene program, including an antibacterial regimen coupled with a power toothbrush and hygiene instruction, may improve the periodontal milieu and reduce the rate of spontaneous preterm birth. Therefore, we conducted a clinical trial of obstetrical patients with moderate-to-severe gingivitis who were randomized to an advanced oral hygiene program vs usual oral hygiene care, and the primary obstetrical outcome compared between the treatment groups was gestational age at delivery. STUDY DESIGN: Obstetrical patients with singleton pregnancies between 8 and 24 weeks' gestation were screened by trained dentists at 2 clinical sites for moderate-to-severe gingivitis (>30 intraoral sites with gingival bleeding on probing). Eligible participants were randomized to an advanced oral hygiene regimen, including antibacterial mouth wash, dental floss, antibacterial toothpaste, a power toothbrush, and intensive oral hygiene education vs standard oral home care instructions and regimen. The primary obstetrical outcome assessed was gestational age at delivery. The overall primary (dental) outcomes were gingival index, bleeding sites, and pocket depth (reported elsewhere). Secondary obstetrical outcomes included birthweight and incidence of preterm birth at <37 weeks' gestation. Regression models were utilized and included factors such as treatment group, gestational age at enrollment, preterm birth history, and potential markers of health disparities (ie, education, employment status). RESULTS: A total of 817 obstetrical patients underwent dental screening at the 2 sites, 789 were identified with moderate-to-severe gingivitis, 746 were randomized, and birth outcomes were available for 613 of the study participants. The gestational age at delivery was 2 days earlier in the control group (P=.040), but the clinical significance of this finding was limited (39.13 vs 38.85 weeks). Overall rates of spontaneous preterm birth <37 weeks and <34 weeks were not statistically different between the 2 treatment groups (7.0% advanced oral hygiene regimen vs 10.3% controls, OR=1.63 (0.90-3.00), P=.11; and 2.3% vs 3.9%, OR=1.88 (0.71-4.98), P=.20, respectively). However, we observed lower rates of spontaneous preterm birth among unemployed participants with less favorable socioeconomic status (4.7% advanced oral hygiene regimen vs 16.0% controls, OR=4.04 (1.34-15.12), P=.01). CONCLUSION: An advanced oral hygiene program may reduce the risk of spontaneous preterm birth among pregnant patients with moderate-to-severe gingivitis and health disparities. This is the same group that may be at highest risk of poor dental hygiene and preterm birth.

A Randomized Controlled Clinical Trial of Prenatal Oral Hygiene Education in Pregnancy-Associated Gingivitis.

INTRODUCTION: Research shows there is a significant increase in gingival inflammation during pregnancy. This study was conducted to determine if an oral health intervention (OHI), including oral hygiene education delivered by nurse-led staff and an advanced over-the-counter (OTC) oral home care regimen, improved gingival inflammation in pregnant women with moderate-to-severe gingivitis compared with a standard oral hygiene control group. METHODS: This was a multicenter, randomized, controlled, single-masked, parallel group clinical trial conducted in obstetrics clinics of 2 medical centers. A total of 750 pregnant women between 8 and 24 weeks of pregnancy with at least 20 natural teeth and moderate-to-severe gingivitis (>30 intraoral bleeding sites) were enrolled. Participants were randomized to either the OHI group, which included oral hygiene instructions supplemented with an educational video and advanced OTC antibacterial/mechanical oral hygiene products, or the control group receiving oral hygiene instructions and standard products. Both groups received oral hygiene instructions from nurse-led staff. Experienced, masked examiners measured whole mouth gingival index (GI) and periodontal probing depths (PDs) at baseline and months 1, 2, and 3. RESULTS: Participants enrolled in this study presented with moderate-to-severe gingivitis at baseline. Both the OHI and control groups exhibited significant reductions in GI (P < .001) and PD (P < .03) from baseline that persisted throughout the study period. The OHI group exhibited modest, yet statistically greater, reductions in GI (P ≤ .044) compared with the control at all time points. The reduction in PD directionally favored the OHI group, but between-group differences were small (<0.03 mm) and not statistically significant (P > .18). DISCUSSION: Significant gingivitis was prevalent among participants in this study and identifies an opportunity to improve gingival health during pregnancy by providing oral health education during the course of prenatal care when coupled with an advanced OTC oral hygiene regimen.

A Comparison of Vessel Patch Materials in Tetralogy of Fallot Patients Using Virtual Surgery Techniques.

Tetralogy of Fallot (ToF) is characterized by stenosis causing partial obstruction of the right ventricular outflow tract, typically alleviated through the surgical application of a vessel patch made from a biocompatible material. In this study, we use computational simulations to compare the mechanical performance of four patch materials for various stenosis locations. Nine idealized pre-operative ToF geometries were created by imposing symmetrical stenoses on each of three anatomical sub-regions of the pulmonary arteries of three patients with previously repaired ToF. A virtual surgery methodology was implemented to replicate the steps of vessel de-pressurization, surgical patching, and subsequent vessel expansion after reperfusion. Significant differences in patch average stress (p < 0.001) were found between patch materials. Biological patch materials (porcine xenopericardium, human pericardium) exhibited higher patch stresses in comparison to synthetic patch materials (Dacron and PTFE). Observed differences were consistent across the various stenosis locations and were insensitive to patient anatomy.

Radiographic Comparison of Heterotopic Ossification After Primary Total Ankle Arthroplasty in Fourth-Generation Implants.

LEVEL OF EVIDENCE: Level III: Retrospective comparative.

Outcomes and Complications of the INFINITY Total Ankle: A Systematic Review.

LEVEL OF EVIDENCE: IV.

A modeling framework for computational simulations of thoracic endovascular aortic repair.

Thoracic endovascular aortic repair (TEVAR) is a minimally invasive treatment for thoracic aortic conditions including aneurysms and is associated with a number of postoperative stent graft related complications. Computational simulations of TEVAR have the potential to predict surgical outcomes and complications preoperatively. When using simulations for stent graft design and prediction of complications in a population, it is difficult to generalize patient-specific TEVAR computational models due to patient variability. This study proposes a novel modeling framework for creating realistic population-based computational models of TEVAR focused on aneurysms that allow for developing various clinically relevant geometric configurations and scenarios that are not easily attainable with limited patient data. The framework includes a methodology for developing population-based thoracic aortic geometries and defining age-dependent aortic tissue material models, as well as detailed steps and boundary conditions for finite element modeling of stent graft deployment during TEVAR. The simulation framework is illustrated for predicting the formation of a bird-beak configuration, a wedge-shaped gap at the proximal end of the deployed stent graft in TEVAR that leads to incomplete seal. A baseline TEVAR simulation model was developed along with three simulations in which the value of aortic curvature, aortic arch angle, or aortic tissue properties varied from the baseline model. Analyzing the length and angle of the bird-beak configuration in each case shows that the bird-beak size is sensitive to different values of the aortic geometry highlighting the importance of using realistic parameter values.

Listeria monocytogenes Illness and Deaths Associated With Ongoing Contamination of a Multiregional Brand of Ice Cream Products, United States, 2010-2015.

BACKGROUND: Frozen foods have rarely been linked to Listeria monocytogenes illness. We describe an outbreak investigation prompted by both hospital clustering of illnesses and product testing. METHODS: We identified outbreak-associated listeriosis cases using whole-genome sequencing (WGS), product testing results, and epidemiologic linkage to cases in the same Kansas hospital. We reviewed hospital medical and dietary records, product invoices, and molecular subtyping results. Federal and state officials tested product and environmental samples for L. monocytogenes. RESULTS: Kansas officials were investigating 5 cases of listeriosis at a single hospital when, simultaneously, unrelated sampling for a study in South Carolina identified L. monocytogenes in Company A ice cream products made in Texas. Isolates from 4 patients and Company A products were closely related by WGS, and the 4 patients with known exposures had consumed milkshakes made with Company A ice cream while hospitalized. Further testing identified L. monocytogenes in ice cream produced in a second Company A production facility in Oklahoma; these isolates were closely related by WGS to those from 5 patients in 3 other states. These 10 illnesses, involving 3 deaths, occurred from 2010 through 2015. Company A ultimately recalled all products. CONCLUSIONS: In this US outbreak of listeriosis linked to a widely distributed brand of ice cream, WGS and product sampling helped link cases spanning 5 years to 2 production facilities, indicating longstanding contamination. Comprehensive sanitation controls and environmental and product testing for L. monocytogenes with regulatory oversight should be implemented for ice cream production.

Posterior Ankle Impingement Syndrome.

Posterior ankle impingement is typically seen in athletes, primarily dancers and soccer players, secondary to dynamic and repetitive push-off maneuvers and forced hyperplantarflexion. Posterior ankle impingement results from chronic, repetitive trauma to the posterior ankle capsule, flexor hallucis longus tendon, and/or os trigonum. It is important to perform a thorough workup by isolating and testing the posterior compartment muscles and obtaining proper imaging with radiographs to identify any osseous abnormalities and MRI to evaluate the soft tissue structures. Nonsurgical treatment includes activity modification, physical therapy, and steroid injections.

Acute Syndesmosis Injuries.

Ankle syndesmosis injuries include isolated ligamentous rupture, as well as fractures with ligamentous injury. These injuries can significantly affect athletes in all sports, and lead to prolonged recovery and return to sport. Adequate evaluation and diagnosis of these injuries are imperative for treatment and return to play. Many can be treated nonoperatively, but operative treatment is indicated in fractures with syndesmosis disruption and ligamentous injuries with instability. Anatomic reduction and fixation of these injuries will allow functional rehab and return to sport.

Small Molecule Antibiotics Inhibit Distinct Stages of Bacterial Outer Membrane Protein Assembly.

Several antibacterial compounds have recently been discovered that potentially inhibit the activity of BamA, an essential subunit of a heterooligomer (the barrel assembly machinery or BAM) that assembles outer membrane proteins (OMPs) in Gram-negative bacteria, but their mode of action is unclear. To address this issue, we examined the effect of three inhibitors on the biogenesis of a model E. coli OMP (EspP) in vivo. We found that darobactin potently inhibited the interaction of a conserved C-terminal sequence motif (the "ß signal") with BamA, but had no effect on assembly if added at a postbinding stage. In contrast, Polyphor peptide 7 and MRL-494 inhibited both binding and at least one later step of assembly. Taken together with previous studies that analyzed the binding of darobactin and Polyphor peptide 7 to BamA in vitro, our results strongly suggest that the two compounds inhibit BAM function by distinct competitive and allosteric mechanisms. In addition to providing insights into the properties of the antibacterial compounds, our results also provide direct experimental evidence that supports a model in which the binding of the ß signal to BamA initiates the membrane insertion of OMPs. IMPORTANCE There is a clear need to develop novel broad-spectrum antibiotics to address the global problem of antimicrobial resistance. Multiple compounds that have strong antibacterial activity have recently been described that appear to inhibit the activity of the barrel assembly machinery (BAM), an essential complex that catalyzes the assembly (i.e., folding and membrane insertion) of outer membrane proteins (OMPs) in all Gram-negative bacteria. We analyzed the effects of three of these compounds on OMP biogenesis in vivo and found that they inhibited different stages of the assembly process. Because these compounds have distinct modes of action, they can be used in combination to reduce the emergence of resistant strains. As a corollary, we obtained evidence that these compounds will be valuable tools in future studies on BAM function.

Function of the Omp85 Superfamily of Outer Membrane Protein Assembly Factors and Polypeptide Transporters.

The Omp85 protein superfamily is found in the outer membrane (OM) of all gram-negative bacteria and eukaryotic organelles of bacterial origin. Members of the family catalyze both the membrane insertion of ß-barrel proteins and the translocation of proteins across the OM. Although the mechanism(s) by which these proteins function is unclear, striking new insights have emerged from recent biochemical and structural studies. In this review we discuss the entire Omp85 superfamily but focus on the function of the best-studied member, BamA, which is an essential and highly conserved component of the bacterial barrel assembly machinery (BAM). Because BamA has multiple functions that overlap with those of other Omp85 proteins, it is likely the prototypical member of the Omp85 superfamily. Furthermore, BamA has become a protein of great interest because of the recent discovery of small-molecule inhibitors that potentially represent an important new class of antibiotics.

Multistate outbreak of Salmonella Mbandaka infections linked to sweetened puffed wheat cereal - United States, 2018.

In May of 2018, PulseNet, the national molecular subtyping network for enteric pathogens, detected a multistate cluster of illnesses caused by an uncommon molecular subtype of Salmonella serovar Mbandaka. A case was defined as an illness in a person infected with the outbreak strain of Salmonella Mbandaka with illness onset on or after 3 March 2018 and before 1 September 2018. One-hundred thirty-six cases from 36 states were identified; 35 hospitalisations and no deaths were reported. Ill people ranged in age from <1 year to 95 years (median: 57 years). When standardised questionnaires did not generate a strong hypothesis, opened-ended interviews were performed. Sixty-three of 84 (75%) ultimately reported consuming or possibly consuming a specific sweetened puffed wheat cereal in the week before illness onset. Environmental sampling performed at the cereal manufacturing facility yielded the outbreak strain. The outbreak strain was also isolated from open cereal samples from ill people's homes and from a sealed retail sample. Due to these findings, the brand owner of the product issued a voluntary recall of the cereal on 14 June 2018. Additional investigation of the manufacturing facility identified persistent environmental contamination with Salmonella Mbandaka that was closely genetically related to other isolates in the outbreak. This investigation highlights the ability of Salmonella to survive in low-moisture environments, and the potential for prolonged outbreaks linked to products with long shelf lives and large distribution areas.