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Purpose The purpose of this study is (1) to determine if, when optimizing modern techniques, medial opening-wedge osteotomies can effectively maintain tibial slope and (2) to determine how different magnitude coronal plane corrections affect tibial slope. Methods Proximal tibial osteotomies (PTOs) were performed on 10 fresh frozen cadaveric knees leaving a consistent lateral hinge, using either a 5mm or a 10mm trapezoidal wedged osteotomy plate. Techniques including posterior plate placement, a trapezoidal, sloped plate, and knee hyperextension were used during plate fixation to help close the anterior osteotomy gap. Medial coronal proximal tibia angle and posterior tibial slope were measured pre-osteotomy, after a 5mm implant, and after a 10mm implant using true anteroposterior and lateral fluoroscopic images. Three independent observers performed all radiographic measurements and intraclass correlation coefficients were calculated. Results The 5mm and 10mm osteotomy plates increased the coronal medial proximal tibia angle by a mean of 3.4° (range, 3.0° to 3.7°) and 7.3° (range, 6.7° to 7.7°), respectively. The 5mm and 10mm trapezoidal wedged osteotomy plates decreased the posterior tibial slope by a mean of 0.9° (range, 0.5° to 1.2°) and 0.3° (range, 0° to 0.6°), respectively. Intraobserver reliability was found to be high for both the coronal medial proximal tibia angle (ICC = 0.897, [0.821-0.946]) and the tibial slope measurements (ICC = 0.761, [0.672-0.826]). Conclusions When optimizing the medial opening-wedge proximal tibial osteotomy, including utilization of a sagittally-oriented hinge, placement of a trapezoidal plate posteriorly, and fixation with knee hyperextension, posterior tibial slope can be maintained regardless of the degree of coronal correction. Clinical Relevance Tibial slope has a significant effect on cruciate ligament stress and a better understanding of coronal plane correction and its effect on tibial slope is critical when performing proximal tibia osteotomies.

Protease-activated receptor 1 (PAR1) is widely expressed in humans and mice, and is activated by a variety of proteases, including thrombin. Recently, we showed that PAR1 contributes to the innate immune response to viral infection. Mice with a global deficiency of PAR1 expressed lower levels of CXCL10 and had increased Coxsackievirus B3 (CVB3)-induced myocarditis compared with control mice. In this study, we determined the effect of cell type-specific deletion of PAR1 in cardiac myocytes (CMs) and cardiac fibroblasts (CFs) on CVB3-induced myocarditis. Mice lacking PAR1 in either CMs or CFs exhibited increased CVB3 genomes, inflammatory infiltrates, macrophages and inflammatory mediators in the heart and increased CVB3-induced myocarditis compared with wild-type controls. Interestingly, PAR1 enhanced poly I:C induction of CXCL10 in rat CFs but not in rat neonatal CMs. Importantly, activation of PAR1 reduced CVB3 replication in murine embryonic fibroblasts and murine embryonic cardiac myocytes. In addition, we showed that PAR1 reduced autophagy in murine embryonic fibroblasts and rat H9c2 cells, which may explain how PAR1 reduces CVB3 replication. These data suggest that PAR1 on CFs protects against CVB3-induced myocarditis by enhancing the anti-viral response whereas PAR1 on both CMs and fibroblasts inhibits viral replication.

Background The percutaneous knotless repair technique for Achilles tendon ruptures utilizes a Percutaneous Achilles Repair System (PARS) device for suturing the proximal tendon and 2 suture anchors for fixing the sutures into the calcaneus. Determining the best position of the suture anchors may optimize the strength of this repair. Methods Twelve pairs of human ankle cadaveric specimens were randomly assigned to receive suture anchors placed at 45°, 90°, or 135° from the sagittal plane. The anchors were tensioned according to a protocol representing progressive, postoperative rehabilitation. Load, number of loading cycles, displacement, and mode of failure were recorded. Results With the anchors placed at 45°, 90°, and 135°, the ultimate failure loads were mean 265 ± 64 N, 264 ± 75 N, and 279 ± 40 N, and the total number of loading cycles were mean 459 ± 166, 466 ± 158, and 469 ± 110, respectively. The effect of anchor angle on failure load, number of loading cycles, and displacement was not statistically significant. Visually, the anchors at 45° and 90° demonstrated sutures cutting through the bone. Conclusion We found no statistically significant difference in pullout strength between the 3 different anchor angles. Sutures cutting through the bone may be a concern with acute anchor angles. This suggests that a 135° anchor angle may result in a lower risk of tendon elongation with the percutaneous knotless repair technique. Levels of Evidence Cadaveric laboratory study

Background: Acute ankle sprains are common injuries. The anterior talofibular (ATFL) and calcaneofibular ligaments (CFL) are the most injured lateral structures. However, controversy exists on the optimal surgical treatment when the injury is both acute and severe or becomes chronic and unstable. Studies have evaluated the biomechanics of these ligaments, but no studies have robotically evaluated injury effects and surgical treatment of ATFL or ATFL and CFL injuries. Purpose: To quantitatively evaluate biomechanical effects of ATFL and CFL lesions, ATFL repair, ATFL and CFL repair, and augmentation of ATFL on ankle stability. Study Design: Controlled laboratory study. Methods: Ten nonpaired cadaveric ankles were tested using a 6 degrees of freedom robot. Each ankle underwent testing in the following states sequentially: (1) intact, (2) ATFL cut, (3) CFL cut, (4) ATFL repair + CFL cut, (5) ATFL repair + CFL repair, and (6) ATFL repair with augmentation with suture tape + CFL repair. Testing included 88 N anterior drawer and 5 N·m varus talar tilt tests at 0° and 30° of plantarflexion, and 88 N Cotton test at 0° of plantarflexion. Results: After all surgical treatments ankles still had increased laxity compared with intact state testing, except after augmented ATFL repair + CFL repair in anterior drawer testing at 30° of plantarflexion ( P = .393). Sectioning the CFL caused a significant increase in talar tilt compared with the ATFL cut state at 0° ( P < .001) and 30° of plantarflexion ( P < .001), but no increase in anterior drawer or Cotton tests. Conclusion: Complete native stability may not be attainable at time zero repair with the tested treatments. The option that best returned stability in anterior translation was augmented ATFL repair with nonaugmented CFL repair. The importance of the CFL as a primary ligamentous stabilizer for talar tilt was confirmed. Clinical Relevance: Evaluating lateral ankle stability and treatment with a 6 degrees of freedom robot should help delineate optimal treatment options. Findings in this study show that none of the repair methods at time zero restored kinematics to the intact state. Of the tested states, the augmented ATFL repair with CFL repair was the best option for controlling anterior translation at time zero. The importance of addressing the CFL to correct talar tilt instability was suggested as was the importance of a period of immobilization before beginning protected rehabilitation. The benefit of ATFL repair augmentation with suture tape is in limiting the postoperative motion in an anterior drawer motion to just 0.5 to 1 mm, but there was no significant improvement to talar tilt even with CFL repair, suggesting that further consideration should be given to CFL augmentation in future studies.

Context The pattern and quantity of insulin required for high-protein high-fat (HPHF) meals is not well understood. Objective This study aimed to determine the amount and delivery pattern of insulin required to maintain euglycemia for 5 hours after consuming a HPHF meal compared with a low-protein low-fat (LPLF) meal. Methods This randomized crossover clinical trial, conducted at 2 Australian pediatric diabetes centers, included 10 patients (12-21 years of age) with type 1 diabetes for ≥ 1 year. Participants were randomized to HPHF meal (60 g protein, 40 g fat) or LPLF meal (5 g protein, 5 g fat) with identical carbohydrate content (30 g). A modified insulin clamp technique was used to determine insulin requirements to maintain postprandial euglycemia for 5 hours. Total mean insulin requirements over 5 hours were measured. Results The total mean insulin requirements for the HPHF meal were significantly greater than for the LPLF meal (11.0 [CI 9.2, 12.8] units vs 5.7 [CI 3.8, 7.5] units; P = 0.001). Extra intravenous insulin was required for HPHF: 0 to 2 hours (extra 1.2 [CI 0.6, 1.6] units/h), 2 to 4 hours (extra 1.1 [CI 0.6, 1.6] units/h), and 4 to 5 hours (extra 0.6 [CI 0.1, 1.1] units/h) after the meal. There were marked inter-individual differences in the quantity of additional insulin (0.3 to 5 times more for HPHF) and the pattern of insulin delivery (0%-85% of additional insulin required in the first 2 hours). Conclusion The addition of protein and fat to a standardized carbohydrate meal almost doubled the mean insulin requirement, with most participants requiring half of the additional insulin in the first 2 hours.

In individuals with type 1 diabetes, chronic hyperglycaemia impairs aerobic fitness. However, the effect of acute marked hyperglycaemia on aerobic fitness is unclear, and the impact of insulin level has not been examined. In this study, we explored if acute hyperglycaemia with higher or low insulin levels affects $$ \dot{V}{\mathrm{O}}_{2\mathrm{peak}} $$ and other exercise performance indicators in individuals with type 1 diabetes. Eligible participants were aged 14 to 30 years, with complication-free, type 1 diabetes and HbA1c ≤ 75 mmol/mol (≤9%). Participants exercised in a clinical laboratory under three clamp (constant insulin, variable glucose infusion) conditions: euglycaemia (5 mmol/l) with 20 mU [m2 BSA]−1 min−1 insulin (where BSA is body surface area) (Eu20); hyperglycaemia (17 mmol/l) with 20 mU [m2 BSA]−1 min−1 insulin (Hyper20); and hyperglycaemia (17 mmol/l) with 5 mU [m2 BSA]−1 min−1 insulin (Hyper5) on separate days. Participants and the single testing assessor were blinded to condition, with participants allocated to randomised testing condition sequences as they were consecutively recruited. Standardised testing (in order) conducted on each of the three study days included: triplicate 6 second sprint cycling, grip strength, single leg static balance, vertical jump and modified Star Excursion Balance Test, ten simple and choice reaction times and one cycle ergometer $$ \dot{V}{\mathrm{O}}_{2\mathrm{peak}} $$ test. The difference between conditions in the aforementioned testing measures was analysed, with the primary outcome being the difference in $$ \dot{V}{\mathrm{O}}_{2\mathrm{peak}} $$ . Twelve recreationally active individuals with type 1 diabetes (8 male, mean ± SD 17.9 ± 3.9 years, HbA1c 61 ± 11 mmol/mol [7.7 ± 1.0%], 7 ± 3 h exercise/week) were analysed. Compared with Eu20, $$ \dot{V}{\mathrm{O}}_{2\mathrm{peak}} $$ was lower in Hyper20 (difference 0.17 l/min [95% CI 0.31, 0.04; p = 0.02] 6.6% of mean Eu20 level), but Hyper5 was not different (p = 0.39). Compared with Eu20, sprint cycling peak power was not different in Hyper20 (p = 0.20), but was higher in Hyper5 (64 W [95% CI 13, 115; p = 0.02] 13.1%). Hyper20 reaction times were not different (simple: p = 0.12) but Hyper5 reaction times were slower (simple: 11 milliseconds [95% CI 1, 22; p = 0.04] 4.7%) than Eu20. No differences between Eu20 and either hyperglycaemic condition were observed for the other testing measures (p > 0.05). Acute marked hyperglycaemia in the higher but not low insulin state impaired $$ \dot{V}{\mathrm{O}}_{2\mathrm{peak}} $$ but to a small extent. Acute hyperglycaemia had an insulin-dependent effect on sprint cycling absolute power output and reaction time but with differing directionality (positive for sprint cycling and negative for reaction time) and no effect on the other indicators of exercise performance examined. We find that acute hyperglycaemia is not consistently adverse and does not impair overall exercise performance to an extent clinically relevant for recreationally active individuals with type 1 diabetes. This research was funded by Diabetes Research Western Australia and Australasian Paediatric Endocrine Group grants.

Background Protease‐activated receptor 1 (PAR1) is expressed in various immune cells and in the lung. We showed that PAR1 plays a role in Coxsackievirus B3 infection by enhancing toll‐like receptor 3‐dependent interferon‐β expression in cardiac fibroblasts. Objectives We investigated the role of PAR1 in a mouse model of influenza A virus (IAV) infection. Methods We used mice with either a global deficiency of PAR1, cell type‐specific deficiencies of PAR1, or mutation of PAR1 at the R41 or R46 cleavage sites. Results PAR1‐deficient mice had increased CXCL1 expression in the lung, increased neutrophil recruitment, increased protein levels in the bronchoalveolar lavage fluid, and increased mortality after IAV infection compared with control mice infected with IAV. Results from mice with cell type‐specific deletion of PAR1 indicated that PAR1 expression by hematopoietic cells suppressed CXCL1 expression, whereas PAR1 expression by endothelial cells enhanced CXCL1 expression in response to IAV infection. PAR1 activation also enhanced polyinosinic:polycytodylic acid induction of interleukin‐8 in a human endothelial cell line. Mutation of the R46 cleavage site of PAR1 was associated with increased CXCL1 expression in the lung in response to IAV infection, which suggested that R46 signaling suppresses CXCL1 expression. Conclusions These results indicate that PAR1 expression by different cell types and activation by different proteases modulates the immune response during IAV infection.

Purpose The purpose of this meta-analysis and systematic review was to critically evaluate the biomechanical outcomes of different fixation constructs for a variety of biceps tenodesis techniques in cadaveric models based on both type of fixation and location. Methods A PROSPERO-registered systematic review (CCRD42018109243) of the current literature was conducted with the terms “long head of biceps” AND “tenodesis” AND “biomechanics” and numerous variations thereof in the PubMed, Embase, and Cochrane databases, yielding 1,460 abstracts. After screening by eligibility criteria, 18 full-text articles were included. The individual biomechanical factors evaluated included ultimate load to failure (in newtons), stiffness (in newtons per millimeter), and cyclic displacement (in millimeters). After reviewing the included literature, we performed a quality analysis of the studies (Quality Appraisal for Cadaveric Studies scale score) and a meta-analysis comparing raw mean differences in data between the suprapectoral and subpectoral fixation location groups, as well as between the fixation construct groups. Results Among the 18 included studies, 347 cadaveric specimens were evaluated for ultimate load to failure, stiffness, and cyclic displacement when comparing both location (suprapectoral vs subpectoral) and tenodesis fixation type (interference screw vs cortical button, suture anchor, or all–soft-tissue techniques). Interference screw fixation showed significantly greater mean stiffness by 8.0 N/mm (P = .013) compared with the other grouped techniques but did not show significant differences when evaluated for ultimate load to failure and cyclic displacement (P = .28 and P = .18, respectively). Additionally, no difference in construct strength was seen when comparing the fixation strength of suprapectoral versus subpectoral techniques for stiffness, ultimate load to failure, and cyclic loading (P = .47, P = .053, and P = .13, respectively). Conclusions In this meta-analysis, no significant biomechanical differences were found when the results were stratified by specific surgical technique (interference screw vs other tenodesis techniques) and location (suprapectoral vs subpectoral biceps tenodesis). Clinical Relevance As a result of this study, when biomechanically evaluating specific tenodesis constructs, the individual clinician has the liberty of choosing the fixation technique based on his or her preference and knowledge of shortcomings of each type of fixation construct.

Introduction In neonates and infants, epidural analgesia has gained popularity as a means of providing postoperative analgesia, limiting opioid-related adverse effects and improving the postoperative course. In addition to a local anesthetic agent, adjunctive agents may be added to further augment analgesia. Clonidine is an α2-adrenergic agonist that is frequently added to single-shot caudal analgesia, but there are limited data regarding its use in a continuous epidural infusion, especially in patients ≤12 months of age. Methods We retrospectively reviewed the hospital records of neonates and infants who received postoperative epidural infusions with 2-chloroprocaine, and clonidine was identified over a 4-year period. Results The study cohort included 52 neonates and infants ranging in age from 0 to 12 months and in weight from 2.1 to 10.1 kilograms. The catheters were dosed with either 1.5% 2-chloroprocaine (n=47) or 3% 2-chloroprocaine (n=5) with clonidine (median concentration 0.2 µg/mL) infused at a median rate of 0.72 mL/kg/hour. Pain scores were uniformly ≤3 at all evaluation points during the first 72 postoperative hours with a limited need for supplemental systemic opioids. No serious adverse effects were noted. Conclusion With the recognized limitations of a retrospective study, these preliminary data demonstrate the safety of adding clonidine to an epidural infusion of 2-chloroprocaine in neonates and infants less than 12 months of age. Future studies are needed to determine its analgesic efficacy compared to 2-chloroprocaine alone and the optimal clonidine concentration for postoperative epidural infusions.

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation., In Vibrio cholerae, a type IVa pilus (T4aP) binds to exogenous DNA, and threads this DNA through the outer membrane secretin, PilQ. Here authors present the cryoEM structure of PilQ from native V. cholerae cells and design a series of mutants to reversibly regulate VcPilQ gate dynamics.

Purpose To investigate the isolated and combined effects of medial patellofemoral ligament (MPFL) and medial patellotibial ligament (MPTL) deficiency and reconstruction on patellofemoral kinematics. Methods Sixteen matched-paired female cadaveric knee specimens with a mean age of 53.5 years (range, 26-65) were tested in 5 conditions: (1) intact, (2) MPFL or MPTL cut, (3) MPFL and MPTL combined cut, (4) MPFL or MPTL reconstruction, and (5) MPFL and MPTL combined reconstruction. Dynamic testing allowed continuous analysis of kinematics from 0° to 90° of knee flexion. Knees were also tested statically using a lateral load of 45 N at 0°, 30°, 60°, and 90° of flexion. In both dynamic and static loading tests, a motion capture system detected patellar position for each testing state to distinguish changes in patellar kinematics. Random-intercepts linear mixed-effects models were used to compare patellar kinematics. Results The MPFL is the primary restraint to lateral translation of the patella at all knee flexion angles. MPTL deficiency alone did not create significant patella instability, but further increased instability when the MPFL was deficient. Isolated MPFL and combined reconstruction provided improved stability. Through full range of motion native patella tracking was best recreated with combined ligament reconstruction. Conclusions The MPFL plays the greatest role in medial patellar stability, but the MPTL appears to have an influence on patella tracking. This study provides further understanding to the impact of the MPFL and MPTL on patellofemoral motion with implications for reconstruction to improve stability and optimize patellofemoral tracking. Clinical Relevance This study provides further understanding of the role of the MPFL and MPTL on patellofemoral motion with implications for reconstruction to improve stability and optimize patellofemoral tracking.

Study design A bibliometric review of current literature. Objective The purpose of this study was to identify and analyze the 100 most cited articles in spinal disc arthroplasty (SDA) research. Summary of background data In the last several decades, SDA has been widely performed all over the world, with increasing popularity of cervical disc arthroplasty (CDA). While there is a large number of articles on this topic, to our knowledge, there is no bibliometric analysis yet. Methods All databases from the Web of Science were searched in a three-step approach. The information of the 100 most cited studies was collected, including title, first and last author, year of publication, journal, total citations, geographic origin, subspecialty, and types of artificial intervertebral disc for further analysis. Results The 100 most-cited articles were published from 1966 to 2015 in 9 different journals and were cited from 66 to 346 times. A total of 11 countries contributed to the 100 articles and the United States topped the list, with 54 articles, followed by Germany and France, with 10 and nine articles, respectively. There were more studies in CDA (n = 53) than lumbar disc arthroplasty (n = 35). Most of the studies reported clinical and radiographic outcomes (n = 33). The most productive periods were from 2006 to 2010. The majority of publications were in Spine, which published 43 articles. In total, 12 authors published more than two articles on the list. Conclusion Of the top 100 most cited articles on SDA, cervical papers outnumbered lumbar articles and the United States had 55 articles, with no other countries having more than 10. Our paper can help readers determine which of the thousands of articles on this topic are the most impactful and important ones to be familiar with. Level of evidence 3.

Background:The acetabular labrum contains free nerve endings, and an unstable labrum can result in increased femoral head movement during hip motion. This can be caused by chondrolabral junction (CLJ) separation, especially in association with pincer-type femoroacetabular impingement, and may contribute to hip pain.Hypothesis:Rim resection alone has no effect on suction seal biomechanics. Further, separation of the CLJ changes hip suction seal biomechanics when compared with those of the native state, whereas repair and refixation with suture anchors restore these biomechanical parameters.Study Design:Controlled laboratory study.Methods:A total of 12 fresh-frozen human cadaveric hips were used in this study. Hips were mounted in a saline bath on a dynamic tensile testing machine and were distracted at a rate of 0.5 mm/s from neutral position. A total of 3 parameters (force, displacement, and intra-articular pressure) were measured throughout testing. Before testing, hips were randomly allocated to 1 of 2 groups: 1 that included the CLJ separation (CLJ Cut group) and 1 that did not (CLJ Intact group). Hips were tested in the following states: (1) native, (2) rim trimming, (3) separated CLJ (CLJ Cut group only), and (4) labral repair/refixation. For each group a linear mixed-effects model was used to compare biomechanical parameters between states.Results:Rim trimming did not affect any suction seal parameters relative to those of the native state. In the CLJ Cut group, no significant difference in distance to break the suction seal was observed for any states compared with that of the native state. In the CLJ Intact group, the distance to break the suction seal was significantly shorter in the labral refixation state (1.8 mm) than the native state (5.6 mm; P = .002). The maximum distraction force (62.1 ± 54.1 N) and the peak negative pressure (−36.6 ± 24.2 kPa) of the labral repair/refixation state were significantly lower than those of the native state in both groups (93.4 ± 41.7 N, P = .01; –60.7 ± 20.4 kPa, P = .02).Conclusion:Rim trimming did not change the biomechanical properties of the labral suction seal. Labral refixation resulted in a shorter distance to break the labral suction seal. This indicates that labral mobility is reduced by the labral refixation procedure, which could be beneficial in postoperative pain relief and labral healing.Clinical Relevance:The labral refixation reduced labral mobility, which could be beneficial for both pain relief and labral healing to the acetabulum after pincer-type femoroacetabular impingement resection.

The endonuclease Artemis is responsible for opening DNA hairpins during V(D)J recombination and for processing a subset of pathological DNA double-strand breaks. Artemis is an attractive target for the development of therapeutics to manage various B cell and T cell tumors, because failure to open DNA hairpins and accumulation of chromosomal breaks may reduce the proliferation and viability of pre-T and pre-B cell derivatives. However, structure-based drug discovery of specific Artemis inhibitors has been hampered by a lack of crystal structures. Here, we report the structure of the catalytic domain of recombinant human Artemis. The catalytic domain displayed a polypeptide fold similar overall to those of other members in the DNA cross-link repair gene SNM1 family and in mRNA 3'-end-processing endonuclease CPSF-73, containing metallo-β-lactamase and β-CASP domains and a cluster of conserved histidine and aspartate residues capable of binding two metal atoms in the catalytic site. As in SNM1A, only one zinc ion was located in the Artemis active site. However, Artemis displayed several unique features. Unlike in other members of this enzyme class, a second zinc ion was present in the β-CASP domain that leads to structural reorientation of the putative DNA-binding surface and extends the substrate-binding pocket to a new pocket, pocket III. Moreover, the substrate-binding surface exhibited a dominant and extensive positive charge distribution compared with that in the structures of SNM1A and SNM1B, presumably because of the structurally distinct DNA substrate of Artemis. The structural features identified here may provide opportunities for designing selective Artemis inhibitors.

To report the clinical and structural outcomes for non-pseudoparalytic irreparable posterosuperior rotator cuff tears treated with superior capsule reconstruction (SCR) using dermal allograft (DA).Patients who underwent SCR using DA with a mean thickness of 3 mm for irreparable posterosuperior rotator cuff tears and underwent surgery at least 2 years earlier were included. Outcomes were assessed prospectively by the American Shoulder and Elbow Surgeons (ASES), Single Assessment Numeric Evaluation, and Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH) scores; patient satisfaction; and visual analog scale for pain. Structurally, acromiohumeral distances (AHDs) were assessed both preoperatively and postoperatively (standard radiographs). Graft integrity was assessed by magnetic resonance imaging. Clinical failures were reported.We included 22 patients with a mean age of 56 years (range, 41-65 years) and a mean follow-up period of 2.1 years (range, 2-3 years). The ASES score improved from 54.0 to 83.9 (P.001); the Single Assessment Numeric Evaluation score improved from 44.9 to 71.4 (P.001); and Quick Disabilities of the Arm, Shoulder and Hand score (QuickDASH) improved from 37.6 to 16.2 (P = .001). Of the patients, 85% achieved an improvement in the ASES score that exceeded the minimal clinically important difference (11.1 points). The median patient satisfaction rating was 8.5 (range, 1-10). The median preoperative visual analog scale score decreased from 4 to 0 (range, 0-3) postoperatively (P.001). Complete radiographs of 19 of 22 patients (86%) were obtained at a mean of 5.2 months (range, 1.4-10 months) postoperatively and showed a significant increase in the mean AHD from 7.0 mm preoperatively to 8.3 mm postoperatively (P = .029). Postoperative magnetic resonance imaging scans were obtained in 95% of the patients (21 of 22) at a mean of 2.5 months (range, 0.3-10.2 months) postoperatively and showed graft integrity rates of 100% (21 of 21) on the tuberosity side, 76% (16 of 21) at the midsubstance, and 81% (17 of 21) on the glenoid side. No significant differences in clinical outcome scores (P0.930) were found in patients with intact grafts versus those with torn grafts. The number of previous shoulder surgical procedures was a negative predictor of clinical outcome. There was 1 clinical failure.SCR using DA for irreparable tears improves outcomes with high satisfaction and high graft integrity at short-term follow-up. Graft integrity, although correlated with an increased AHD, had no correlation with clinical outcomes at final follow-up.Level IV, case series.

Ponds that form on sea ice can cause it to thin or break-up, which can promote calving from an adjacent ice shelf. Studies of sea ice ponds have predominantly focused on Arctic ponds formed by in situ melting/ponding. Our study documents another mechanism for the formation of sea ice ponds. Using Landsat 8 and Sentinel-2 images from the 2015–16 to 2018–19 austral summers, we analyze the evolution of sea ice ponds that form adjacent to the McMurdo Ice Shelf, Antarctica. We find that each summer, meltwater flows from the ice shelf onto the sea ice and forms large (up to 9 km2) ponds. These ponds decrease the sea ice's albedo, thinning it. We suggest the added mass of runoff causes the ice to flex, potentially promoting sea-ice instability by the ice-shelf front. As surface melting on ice shelves increases, we suggest that ice-shelf surface hydrology will have a greater effect on sea-ice stability.

ObjectiveTo evaluate whether vascular health in young children was associated with exposure to a 6-week episode of coal mine fire smoke or environmental tobacco smoke (ETS) in a retrospective cohort study.MethodsThree years after a coal mine fire in Victoria, Australia, we investigated the vascular health of children either in utero (n=75) or aged 2.5) exposures were estimated based on their daily locations throughout the fire period. Multivariable linear regression models were used to test for associations between the fire-related PM2.5 and outcomes adjusted for relevant covariates including ETS.ResultsIn the postnatal-exposure group, each 10 µg/m³ increase in mean PM2.5 level was independently positively associated with PWV (β=0.116, p=0.028). When these two groups were combined, there was an association between mean PM2.5 and increased PWV in those children who had ETS exposure (β=0.148, p=0.033) or whose mothers smoked (β=0.151, p=0.011), but not in those not exposed to ETS or maternal smoking.ConclusionsThree years after a coal mine fire, infants aged up to 2 years at the time of exposure have increases in vascular stiffness. Although no adverse effects were observed in the in uterus exposure group, further follow-up study is needed to elucidate the long-term effects of coal mine fire smoke exposure.

Background: Increased posterior tibial slope and posterior medial meniscus root tears increase the force experienced by the anterior cruciate ligament (ACL) and predispose patients to higher rates of primary ACL injury or ACL graft failure after an ACL reconstruction (ACLR). However, the interplay among sagittal plane tibial slope, medial meniscus root tears, and ACLR graft force remains inadequately defined. Purpose/Hypothesis: The purpose was to quantify the effect of sagittal plane tibial slope on ACLR graft force at varying knee flexion angles with an intact medial meniscus, a posterior medial meniscus root tear, and a medial meniscus root repair. Our null hypothesis was that changes in slope and meniscal state would have no effect on the forces experienced by the ACLR graft. Study Design: Controlled laboratory study. Methods: Ten male fresh-frozen cadaveric human knees underwent a posteriorly based high tibial osteotomy. A spanning external fixator and wedges of varying sizes were used to stabilize the osteotomy and allow for accurate slope adjustment. After ACLR, specimens were compressed with a 1000-N axial load at flexion angles of 0° and 30° for each of the 3 meniscal states and at tibial slopes of 0° to 15° at 3° increments. Graft loads were recorded through a force transducer clamped to the graft. Results: Increasing tibial slope led to a linear increase in ACLR graft force at 0° and 30° of knee flexion. Posterior medial meniscus root tear led to significant increases in ACLR graft forces over the intact state, while root repair restored the function of the medial meniscus as a secondary stabilizer. At 30° of knee flexion, the tibial slope effect on ACLR graft force was potentiated in the root tear state as compared with the intact and root repair states—test of interaction effect: t(139) = 2.67 ( P = .009). Conclusion: Increases in tibial slope lead to a linear increase in ACLR graft forces, and this effect is magnified in the setting of a posterior medial meniscus root tear. At slopes >12°, a slope-changing osteotomy could be considered in the setting of a revision ACLR with a concomitant medial meniscus root tear. Clinical Relevance: Defining the relationship between tibial slope and varying states of meniscal insufficiency can help determine when it may be necessary to perform a slope-decreasing proximal tibial osteotomy before ACLR and meniscal repair.

Inflammasomes are a group of multimolecular intracellular complexes assembled around several innate immune proteins. Recognition of a diverse range of microbial, stress and damage signals by inflammasomes results in direct activation of caspase‐1, which subsequently induces the only known form of secretion of active interleukin‐1β and interleukin‐18. Although the importance of interleukin‐1β in the periodontium is not questioned, the impact of inflammasomes in periodontal disease and its potential for therapeutics in periodontology is still in its very early stages. Increasing evidence in preclinical models and human data strongly implicate the involvement of inflammasomes in a number of inflammatory, autoinflammatory and autoimmune disorders. Here we review: (a) the currently known inflammasome functions, (b) clinical/preclinical data supporting inflammasome involvement in the context of periodontal and comorbid diseases and (c) potential therapies targeting inflammasomes. To clarify further the inflammasome involvement in periodontitis, we present analyses of data from a large clinical study (n = 5809) that measured the gingival crevicular fluid‐interleukin‐1β and grouped the participants based on current periodontal disease classifications. We review data on 4910 European‐Americans that correlate 16 polymorphisms in the interleukin‐1B region with high gingival crevicular fluid‐interleukin‐1β levels. We show that inflammasome components are increased in diseased periodontal tissues and that the caspase‐1 inhibitor, VX‐765, inhibits ~50% of alveolar bone loss in experimental periodontitis. The literature review further supports that although patients clinically present with the same phenotype, the disease that develops probably has different underlying biological pathways. The current data indicate that inflammasomes have a role in periodontal disease pathogenesis. Understanding the contribution of different inflammasomes to disease development and distinct patient susceptibility will probably translate into improved, personalized therapies.

Pollen is a well-established trigger of asthma and allergic rhinitis, yet concentration-response relationships, lagged effects, and interactions with other environmental factors remain poorly understood. Smartphone technology offers an opportunity to address these challenges using large, multi-year datasets that capture individual symptoms and exposures in real time. We aimed to characterise associations between six pollen types and respiratory symptoms logged by users of the AirRater smartphone app in Tasmania, Australia. We analyzed 44,820 symptom reports logged by 2272 AirRater app users in Tasmania over four years (2015-2019). With these data we evaluated associations between daily respiratory symptoms and atmospheric pollen concentrations. We implemented Poisson regression models, using the case time series approach designed for app-sourced data. We assessed potentially non-linear and lagged associations with (a) total pollen and (b) six individual pollen taxa. We adjusted for seasonality and meteorology and tested for interactions with particulate air pollution (PM2.5). We found evidence of non-linear associations between total pollen and respiratory symptoms for up to three days following exposure. For total pollen, the same-day relative risk (RR) increased to 1.31 (95% CI: 1.26-1.37) at a concentration of 50 grains/m3 before plateauing. Associations with individual pollen taxa were also non-linear with some diversity in shapes. For all pollen taxa the same-day RR was highest. The interaction between total pollen and PM2.5 was positive, with risks associated with pollen significantly higher in the presence of high concentrations of PM2.5. Our results support a non-linear response between airborne pollen and respiratory symptoms. The association was strongest on the day of exposure and synergistic with particulate air pollution. The associations found with Dodonaea and Myrtaceae highlight the need to further investigate the role of Australian native pollen types in allergic respiratory disease.

The bacterial flagellar motor is a complex macromolecular machine whose function and self-assembly present a fascinating puzzle for structural biologists. Here, we report that in diverse bacterial species, cell lysis leads to loss of the cytoplasmic switch complex and associated ATPase before other components of the motor. This loss may be prevented by the formation of a cytoplasmic vesicle around the complex. These observations suggest a relatively loose association of the switch complex with the rest of the flagellar machinery. IMPORTANCE We show in eight different bacterial species (belonging to different phyla) that the flagellar motor loses its cytoplasmic switch complex upon cell lysis, while the rest of the flagellum remains attached to the cell body. This suggests an evolutionary conserved weak interaction between the switch complex and the rest of the flagellum which is important to understand how the motor evolved. In addition, this information is crucial for mimicking such nanomachines in the laboratory.

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

Background: Superior capsule reconstruction (SCR) has been shown to improve shoulder function and reduce pain in patients with isolated irreparable supraspinatus tendon tears. However, the effects of SCR on biomechanics in a shoulder with an extensive posterosuperior rotator cuff tear pattern remain unknown. Purpose/Hypothesis: The purpose was to (1) establish a dynamic robotic shoulder model, (2) assess the influence of rotator cuff tear patterns, and (3) assess the effects of SCR on superior humeral head translation after a posterosuperior rotator cuff tear. It was hypothesized that a posterosuperior rotator cuff tear would increase superior humeral head translation when compared with the intact and supraspinatus tendon–deficient state and that SCR would reduce superior humeral head translation in shoulders with massive rotator cuff tears involving the supraspinatus and infraspinatus tendons. Study Design: Controlled laboratory study. Methods: Twelve fresh-frozen cadaveric shoulders were tested using a robotic arm. Kinematic testing was performed in 4 conditions: (1) intact, (2) simulated irreparable supraspinatus tendon tear, (3) simulated irreparable supra- and infraspinatus tendon tear, and (4) SCR using a 3 mm–thick dermal allograft (DA). Kinematic testing consisted of static 40-N superior force tests at 0°, 30°, 60°, and 90° of abduction and dynamic flexion, abduction, and scaption motions. In each test, the superior translation of the humeral head was reported. Results: In static testing, SCR significantly reduced humeral superior translation compared with rotator cuff tear at all abduction angles. SCR restored the superior stability back to native at 60° and 90° of abduction, but the humeral head remained significantly and superiorly translated at neutral position and at 30° of abduction. The results of dynamic testing showed a significantly increased superior translation in the injured state at lower elevation angles, which diminished at higher elevation, becoming nonsignificant at elevation >75°. SCR reduced the magnitude of superior translation across all elevation angles, but translation remained significantly different from the intact state up to 60° of elevation. Conclusion: Massive posterosuperior rotator cuff tears increased superior glenohumeral translation when compared with the intact and supraspinatus tendon–insufficient rotator cuff states. SCR using a 3-mm DA partially restored the superior stability of the glenohumeral joint even in the presence of a simulated massive posterosuperior rotator cuff tear in a static and dynamic robotic shoulder model. Clinical Relevance: The biomechanical performance concerning glenohumeral stability after SCR in shoulders with large posterosuperior rotator cuff tears is unclear and may affect clinical outcomes in daily practice.

Maternal ingestion of naphthalene-containing mothballs is an uncommon cause of perinatal toxicity. Naphthalene toxicity is associated with methemoglobinemia, hypotension, hemolytic anemia, and hyperbilirubinemia, as well as other hepatic, renal, and respiratory complications. Naphthalene exposure is a common cause of toxicity in older children, but is rarely described in neonates. The neonatal cases described in the literature focus primarily on maternal inhalation as opposed to ingestion. We present a case of perinatal toxicity due to repeated maternal ingestion of naphthalene-containing mothballs during pregnancy. The patient presented with methemoglobinemia, hypotension, hemolytic anemia, and hyperbilirubinemia. Sepsis or pulmonary hypertension were the initial working diagnoses, as the mother did not provide the history of ingestion until after the patient’s clinical status worsened. This case highlights the importance of obtaining a thorough maternal history and considering maternal ingestion when the etiology of symptoms is not clear.

Aim: Understanding past distributions of people across the landscape is key to understanding how people used, affected and related to the natural environment. Here, we use habitat suitability modelling to represent the landscape distribution of Tasmanian Aboriginal archaeological sites and assess the implications for patterns of past human activity. Location: Tasmania, Australia. Methods: We developed a RandomForest ‘habitat suitability' model of site records in the Tasmanian Aboriginal Heritage Register. We applied a best‐effort bias correction, considered 31 predictor variables relating to climate, topography and resource proximity, and used a variable selection procedure to optimize the final model. Model uncertainty was assessed via bootstrapping and we ran an analogous MaxEnt model as a cross‐validation exercise. Results: The results from the RandomForest and MaxEnt models are highly congruent. The strongest environmental predictors of site occurrence include distance to coast, elevation, soil clay content, topographic roughness and distance to inland water. The highest habitat suitability scores are distributed across a wide range of environments in central, northern and eastern Tasmania, including coastal areas, inland water body margins and forests and savannas in the drier parts of Tasmania. With the exception of coastal areas much of western Tasmania has low habitat suitability scores, consistent with theories of low‐density Holocene Tasmanian Aboriginal settlement in this region. Main conclusions: Our modelling suggests Tasmanian Aboriginal people occupied a heterogeneity of habitats but targeted coastal areas around the whole island, and drier, less steep and/or open forest and savanna environments in the central lowlands. The western interior was identified as being rarely used by Aboriginal people in the Holocene, with the exception of isolated pockets of habitat; yet whether this is a true reflection of Aboriginal‐resourceuse demands increased archaeological surveys, particularly in the Tasmanian Wilderness World Heritage Area.

Background: We aimed to explore the impact of externally worn diabetes technologies on sexual behavior and activity, body image, and anxiety in adopters and nonadopters of these devices. Methods: People with type 1 diabetes aged 16-60 years living in Western Australia were invited to complete an online survey. Results: Of the 289 respondents (mean age 34.3 years), 45% used continuous subcutaneous insulin infusion (CSII) and 35% used continuous glucose monitoring (CGM). Approximately half of CSII users stated that the pump interferes with sex. Of these, 75% disconnect their pump during sexual activity to avoid this issue. Comfort during sex influenced the location of the CSII insertion site in 22% of respondents, with the abdomen being preferred. One in four non-CSII users cited sex-related concerns as a factor for not adopting the technology. CGM interfered with sexual activity in 20% of users, but did not commonly affect CGM placement (only 18%). Sexual activity was reported as a factor for not adopting the technology in 10% of non-CGM users. No differences in body dissatisfaction ( P = .514) or anxiety ( P = .304) between CSII and non-CSII users were observed. No differences in sexual activity and behavior between technology users and nontechnology users were observed. Conclusion: Wearable technologies impact upon sexual activity and this influences the decision to adopt the technology. Despite this, technology users are similar in terms of sexual behavior, anxiety, and body image compared to nontechnology users. Where appropriate, these data can be used to identify potential concerns, address strategies to mitigate them, and inform people with diabetes when considering adopting external technologies.

Background: Limited biomechanical data exist for dual small plate fixation of midshaft clavicle fractures, and no prior study has concurrently compared dual small plating to larger superior or anteroinferior single plate and screw constructs. Purpose: To biomechanically compare dual small orthogonal plating, superior plating, and anteroinferior plating of midshaft clavicle fractures by use of a cadaveric model. Study Design: Descriptive laboratory study. Methods: The study used 18 cadaveric clavicle specimens (9 pairs total), and 3 plating techniques were studied: anteroinferior, superior, and dual. The dual plating technique used smaller diameter plates and screws (1.6-mm thickness) than the other, single plate techniques (3.3-mm thickness). Each of the 9 clavicle pairs was randomly assigned a combination of 2 plating techniques, and randomization was used to determine which techniques were used for the right and left specimens. Clavicles were plated and then osteotomized to create an inferior butterfly fracture model, which was then fixed with a single interfragmentary screw. Clavicle specimens were then potted for mechanical testing. Initial bending, axial, and torsional stiffness of each construct was determined through use of a randomized nondestructive cyclic testing protocol followed by load to failure. Results: No significant differences were found in cyclical axial ( P = .667) or torsional ( P = .526) stiffness between plating groups. Anteroinferior plating demonstrated significantly higher cyclical bending stiffness than superior plating ( P = .005). No significant difference was found in bending stiffness between dual plating and either anteroinferior ( P = .129) or superior plating ( P = .067). No significant difference was noted in load to failure among plating methods ( P = .353). Conclusion: Dual plating with a smaller plate-screw construct is biomechanically similar to superior and anteroinferior single plate fixation that uses larger plate-screw constructs. No significant differences were found between dual plating and either superior or anteroinferior single plating in axial, bending, or torsional stiffness or in bending load to failure. Dual small plating is a viable option for fixing midshaft clavicle fractures and may be a useful low-profile technique that avoids a larger and more prominent plate-screw construct. Clinical Relevance: Plate prominence and hardware irritation are commonly reported complaints and reasons for revision surgery after plate fixation of midshaft clavicle fractures. Dual small plate fixation has been used to improve cosmetic acceptability, minimize hardware irritation, and decrease reoperation rate. Biomechanically, dual small plate fixation performed similarly to larger single plate fixation in this cadaveric model of butterfly fracture.

Background: Although posterior medial meniscal root (PMMR) repairs are often successful, postoperative meniscal extrusion after a root repair has been identified as a potential clinical problem. Purpose/Hypothesis: The purpose was to quantitatively evaluate the tibiofemoral contact mechanics and extent of meniscal extrusion after a PMMR repair. It was hypothesized that the addition of a centralization suture (into the posterior medial tibial plateau) would help restore normal joint load-bearing characteristics and restore the native amount of meniscal extrusion after a root tear. Furthermore, we hypothesized that the amount of meniscal extrusion would be greatest in loaded and flexed knees when measured at the posterior border of the medial collateral ligament (MCL). Study Design: Controlled laboratory study. Methods: Meniscal extrusion and tibiofemoral contact mechanics were measured using 3-dimensional digitization and pressure sensors in 10 nonpaired, human cadaveric knees. The PMMR of each knee was tested under 6 states: (1) intact; (2) type 2A PMMR tear; (3) anatomic transtibial pull-out root repair; (4) anatomic transtibial pull-out repair with centralization; (5) nonanatomic transtibial pull-out repair; and (6) nonanatomic transtibial pull-out repair with centralization, with randomization of the order of conditions 3 and 4, and 5 and 6. The testing protocol loaded knees with a 1000-N axial compressive force at 4 flexion angles (0°, 30°, 60°, 90°) in each state. Meniscal extrusion was measured with a 3-dimensional coordinate digitizer at 0° and 90° in both the loaded and unloaded states and calculated from the difference from the articular margin of the tibia to the periphery of the meniscus. Peak contact pressure, contact area, and total contact pressure were also recorded for all states at all flexion angles. Statistical analysis investigated the independent effects of flexion, state, and loading using 3 distinct 2-factor models. Results: Differences in the contact mechanics between repair techniques were most notable at higher flexion angles, demonstrating significantly higher average and peak contact pressures for nonanatomic repair states when compared with anatomic repairs with and without centralization (all P < .05). In unloaded knees at full extension, the magnitude of medial meniscal extrusion was significantly higher at the posterior border of the MCL compared with the posterior medial tibia ( P < .001) and adjacent to the root attachment on the tibia locations ( P < .001). Both anatomic repair states had no significant difference in the degree of extrusion when compared with the intact state. Conclusion: The anatomic transtibial pull-out root repair and the anatomic transtibial pull-out root repair with centralization techniques best restored contact mechanics of the knee and meniscal extrusion when compared with root tear and nonanatomic repair states at time zero. There were no significant differences in contact pressure or magnitude of extrusion between the anatomic repair state and the anatomic repair with centralization state. We found that extrusion is best measured in the coronal plane at the posterior border of the MCL for unloaded knees. However, the degree of extrusion increased as the knee was loaded and flexed to 90°. Clinical Relevance: When there are concerns about meniscal extrusion with a medial meniscal root repair, the addition of a centralization suture may be beneficial for patients in reducing pathologic meniscal extrusion and restoring joint contact mechanics.

Background:Platelet-rich plasma (PRP) and bone marrow concentrate (BMC) are orthobiologic therapies with numerous growth factors and other bioactive molecules. Before the clinical utility of PRP and BMC is optimized as a combined therapy or monotherapy, an improved understanding of the components and respective concentrations is necessary.Purpose:To prospectively measure and compare anabolic, anti-inflammatory, and proinflammatory growth factors, cytokines, and chemokines in bone marrow aspirate (BMA), BMC, whole blood, leukocyte-poor PRP (LP-PRP), and leukocyte-rich PRP (LR-PRP) from samples collected and processed concurrently on the same day from patients presenting for elective knee surgery.Study Design:Cross-sectional study; Level of evidence, 3.Methods:Patients presenting for elective knee surgery were prospectively enrolled over a 3-week period. Whole blood from peripheral venous draw and BMA from the posterior iliac crest were immediately processed via centrifugation and manual extraction methods to prepare LR-PRP, LP-PRP, and BMC samples, respectively. BMA, BMC, whole blood, LR-PRP, and LP-PRP samples were immediately assayed and analyzed to measure protein concentrations.Results:BMC had a significantly higher interleukin 1 receptor antagonist (IL-1Ra) concentration than all other preparations (all P < .0009). LR-PRP also had a significantly higher IL-1Ra concentration than LP-PRP ( P = .0006). There were no significant differences in IL-1Ra concentration based on age, sex, body mass index, or chronicity of injury in all preparations. LR-PRP had significantly higher concentrations of platelet-derived growth factor AA (PDGF-AA) and PDGF-AB/BB than all other preparations (all P < .0006). LR-PRP also had significantly higher concentrations of matrix metalloproteinase 1 (MMP-1) and soluble CD40 ligand than all other preparations (all P < .004). LP-PRP had significantly higher concentrations of MMPs, namely MMP-2, MMP-3, and MMP-12, than all other preparations (all P < .007).Conclusion:BMC is a clinically relevant source of anti-inflammatory biologic therapy that may be more effective in treating osteoarthritis and for use as an intra-articular biologic source for augmented healing in the postsurgical inflammatory and healing phases, owing to its significantly higher concentration of IL-1Ra as compared with LR-PRP and LP-PRP. Additionally, LR-PRP had a significantly higher concentration of IL-1Ra than LP-PRP. In cases where increased vascularity and healing are desired for pathological or injured tissues, including muscle and tendon, LR-PRP may be optimal given its higher overall concentrations of PDGF, TGF-β, EGF, VEGF, and soluble CD40 ligand.

Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography and immunofluorescence microscopy to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. Electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that, interestingly, are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Together, these results revealed that the Dot/Icm complex assembles in an ‘axial-to-peripheral’ pattern.

Aims To determine whether pre-exercise ingestion of carbohydrates to maintain stable glycaemia during moderate-intensity exercise results in excessive hyperglycaemia if combined with repeated sprints in individuals with Type 1 diabetes. Methods Eight overnight-fasted people with Type 1 diabetes completed the following four 40-min exercise sessions on separate days in a randomized counterbalanced order under basal insulinaemic conditions: continuous moderate-intensity exercise at 50% V ˙ O 2 peak; intermittent high-intensity exercise (moderate-intensity exercise interspersed with 4-s sprints every 2 min and a final 10-s sprint); continuous moderate-intensity exercise with prior carbohydrate intake (~10 g per person); and intermittent high-intensity exercise with prior carbohydrate intake. Venous blood was sampled during and 2 h after exercise to measure glucose and lactate levels. Results The difference in marginal mean time-averaged area under the blood glucose curve between continuous moderate-intensity exercise + prior carbohydrate and intermittent high-intensity exercise + prior carbohydrate during exercise and recovery was not significant [0.2 mmol/l (95% CI -0.7, 1.1); P = 0.635], nor was the difference in peak blood glucose level after adjusting for baseline level [0.2 mmol/l (95% CI -0.7, 1.1); P = 0.695]. The difference in marginal mean time-averaged area under the blood glucose curve between continuous moderate-intensity and intermittent high-intensity exercise during exercise and recovery was also not significant [-0.2 mmol/l (95% CI -1.2, 0.8); P = 0.651]. Conclusions When carbohydrates are ingested prior to moderate-intensity exercise, adding repeated sprints is not significantly detrimental to glycaemic management in overnight fasted people with Type 1 diabetes under basal insulin conditions.

Global sea-level rise is caused, in part, by more rapid ice discharge from Antarctica, following the removal of the restraining forces of floating ice-shelves after their break-up. A trigger of ice-shelf break-up is thought to be stress variations associated with surface meltwater ponding and drainage, causing flexure and fracture. But until now, there have been no direct measurements of these processes. Here, we present field data from the McMurdo Ice Shelf, Antarctica, showing that the filling, to ~2 m depth, and subsequent draining, by overflow and channel incision, of four surface lakes causes pronounced and immediate ice-shelf flexure over multiple-week timescales. The magnitude of the vertical ice-shelf deflection reaches maxima of ~1 m at the lake centres, declining to zero at distances of, Meltwater ponding on top of ice shelves is thought to play a role in ice-shelf flexure and fracture, however in-situ evidence of these mechanisms is lacking. Here, the authors provide field-based evidence showing the impact of the filling and draining of four surface lakes on ice-shelf flexure in Antarctica.

Background:Historically, radial meniscal tears were treated with partial or near-total meniscectomy, which usually resulted in poor outcomes. Radial meniscal tears function similar to a total meniscectomy and are challenging to treat. Repair of radial meniscal tears should be performed to prevent joint deterioration and the need for salvage procedures in the future.Purpose/Hypothesis:The purpose was to compare 3 repair techniques for radial tears of the medial meniscus: the 2-tunnel, hybrid, and hybrid tunnel techniques. It was hypothesized that there would be no differences among the 3 groups in regard to gapping and ultimate failure strength.Study Design:Controlled laboratory study.Methods:Thirty human male cadaver knees (10 matched pairs, n = 20; 10 unpaired, n = 10) were used to compare the 2-tunnel, hybrid, and hybrid tunnel repairs. A complete radial tear was made at the midbody of the medial meniscus. Repairs were performed according to the described techniques. Specimens were potted and mounted on a universal material testing machine where each specimen was cyclically loaded for 1000 cycles before experiencing a pull to failure. Gap distances at the tear site, ultimate failure load, and failure location were measured and recorded.Results:After 1000 cycles of cyclic loading, there were no significant differences in displacement among the 2-tunnel repair (3.0 ± 1.7 mm), hybrid repair (3.0 ± 0.9 mm), and hybrid tunnel repair (2.3 ± 1.0 mm; P = .4042). On pull-to-failure testing, there were also no significant differences in ultimate failure strength among the 2-tunnel repair (259 ± 103 N), hybrid repair (349 ± 149 N), and hybrid tunnel repair (365 ± 146 N; P = .26). However, the addition of vertical mattress sutures to act as a “rip stop” significantly reduced the likelihood of the sutures pulling through the meniscus during pull-to-failure testing for the hybrid and hybrid tunnel repairs (4 of 16, 25%) as compared with the 2-tunnel repair (7 of 9, 78%; P = .017).Conclusion:The results showed equivalent biomechanical testing with regard to gap distance and pull-to-failure strength among the 3 repairs. The addition of the vertical mattress sutures to act as a rip stop was effective in preventing meniscal cutout through the meniscus.Clinical Relevance:Effective healing of radial meniscal tears after repair is paramount to prevent joint deterioration and symptom development. Each tested repair showed a biomechanically equivalent and stable construct to use to repair radial meniscal tears. The authors recommend that rip stop vertical mattress sutures be used, especially in poor-quality meniscal tissue, to prevent suture cutout.

PURPOSE To quantify and compare normative catabolic and anabolic factor concentrations in leukocyte-rich platelet-rich plasma (LR-PRP) at various time points, including baseline, 1 week after initiating naproxen use, and after a 1-week washout period. METHODS Asymptomatic healthy donors aged between 18 and 70 years were recruited (average age, 36.6 years; range, 25-64 years). Subjects were excluded from the study if they were actively taking any prescribed medications or nonsteroidal anti-inflammatory drugs (NSAIDs) or if they had any of the following at present or previously: blood or immunosuppression disorders, cancer, osteonecrosis, rheumatoid arthritis, avascular necrosis, NSAID intolerance, gastrointestinal or peptic ulcer disease, or kidney dysfunction. The anabolic factors vascular endothelial growth factor, fibroblast growth factor 2, platelet-derived growth factor AB (PDGF-AB), and platelet-derived growth factor AA (PDGF-AA) and the catabolic factors interleukin (IL) 1β, IL-6, IL-8, and tumor necrosis factor α in LR-PRP were measured. Peripheral blood was drawn at 3 time points: baseline, after 1 week of naproxen use, and after a 1-week washout period. RESULTS The angiogenic factors PDGF-AA (44% decrease in median) and PDGF-AB (47% decrease) significantly declined from baseline (P < .05) after 1 week of naproxen use. There was a significant recovery (P < .05) of PDGF-AA (94% increase) and PDGF-AB (153% increase) levels after the 1-week washout period, with a return to baseline levels. The catabolic factor IL-6 also had a significant decline from baseline (77% decrease in median, P < .05) after 1 week of naproxen use. After a 1-week washout period, the IL-6 level was similar to the baseline level (130% increase, P < .05). CONCLUSIONS Naproxen use diminished several biological factors in LR-PRP; however, a 1-week washout period was sufficient for the recovery of PDGF-AA, PDGF-AB, and IL-6 to return to baseline levels. Tumor necrosis factor α, IL-1β, IL-8, vascular endothelial growth factor, and fibroblast growth factor 2 did not show differences between the 3 time points of data collection. Discontinuing NSAIDs for a minimum of 1 week before LR-PRP treatment may improve certain biological factor levels. LEVEL OF EVIDENCE Level II, prospective comparative study.

Supplemental Digital Content is available in the text., Background: Ambulance data provide a useful source of population-based and spatiotemporally resolved information for assessing health impacts of air pollution in nonhospital settings. We used the clinical records of paramedics to quantify associations between particulate matter (PM2.5) and diabetic, cardiovascular, and respiratory conditions commonly managed by those responding to calls for emergency ambulance services. Methods: We evaluated 394,217 paramedic assessments from three states in Southeastern Australia (population 13.2 million) and daily PM2.5 concentrations modeled at 5 km resolution from 2009 to 2014. We used a time-stratified, case-crossover analysis adjusted for daily meteorology to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for each clinical outcome per 10 µg/m3 increase in daily PM2.5 at lags from 0 to 2 days. Results: Increased PM2.5 was associated with increased odds of paramedic assessments of hypoglycemia (OR = 1.07; 95% CI = 1.02, 1.12, lag 0), arrhythmia (OR = 1.05; 95% CI = 1.02, 1.09, lag 0), heart failure (OR = 1.07; 95% CI = 1.02, 1.12, lag 1), faint (OR = 1.09; 95% CI = 1.04–1.13, lag 0), asthma (OR = 1.06; 95% CI = 1.01, 1.11, lag 1), chronic obstructive pulmonary disease (OR = 1.07; 95% CI = 1.01, 1.13, lag 1), and croup (OR = 1.09; 95% CI = 1.02, 1.17). We did not identify associations with cerebrovascular outcomes. Conclusions: Ambulance data enable the evaluation of important clinical syndromes that are often initially managed in nonhospital settings. Daily PM2.5 was associated with hypoglycemia, faint, and croup in addition to the respiratory and cardiovascular outcomes that are better established.

A data-processing pipeline to solve structures from tomograms of protein nanocrystals is presented and validated using simulated crystals. The robustness of this workflow to radiation damage is assessed to investigate optimal data-collection strategies., Structure-determination methods are needed to resolve the atomic details that underlie protein function. X-ray crystallography has provided most of our knowledge of protein structure, but is constrained by the need for large, well ordered crystals and the loss of phase information. The rapidly developing methods of serial femtosecond crystallography, micro-electron diffraction and single-particle reconstruction circumvent the first of these limitations by enabling data collection from nanocrystals or purified proteins. However, the first two methods also suffer from the phase problem, while many proteins fall below the molecular-weight threshold required for single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure-determination methods. Here, a data-processing scheme is presented that combines routines from X-ray crystallography and new algorithms that have been developed to solve structures from tomograms of nanocrystals. This pipeline handles image-processing challenges specific to tomographic sampling of periodic specimens and is validated using simulated crystals. The tolerance of this workflow to the effects of radiation damage is also assessed. The simulations indicate a trade-off between a wider tilt range to facilitate merging data from multiple tomograms and a smaller tilt increment to improve phase accuracy. Since phase errors, but not merging errors, can be overcome with additional data sets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.

Gas vesicles (GVs) are cylindrical or spindle‐shaped protein nanostructures filled with air and used for flotation by various cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells, as well as their properties in imaging applications. Despite its importance, reported diameters for the same types of GV differ depending on the method used for its assessment. Here, we provide an explanation for these discrepancies and utilize electron microscopy (EM) techniques to accurately estimate the diameter of the most commonly studied types of GVs. We show that during air drying on the EM grid, GVs flatten, leading to a ~1.5‐fold increase in their apparent diameter. We demonstrate that GVs' diameter can be accurately determined by direct measurements from cryo‐EM samples or alternatively indirectly derived from widths of flat collapsed and negatively stained GVs. Our findings help explain the inconsistency in previously reported data and provide accurate methods to measure GVs dimensions.

The bacterial flagellum consists of a long extracellular filament that is rotated by a motor embedded in the cell envelope. While flagellar assembly has been extensively studied, the disassembly process remains less well understood. In addition to the programmed flagellar ejection that occurs during the life cycle of Caulobacter crescentus, we and others have recently shown that many bacterial species lose their flagella under starvation conditions, leaving relic structures in the outer membrane. However, it remains unknown whether the programmed flagellar ejection of C. crescentus leaves similar relics or not. Here, we imaged the various stages of the C. crescentus life cycle using electron cryo-tomography (cryo-ET) and found that flagellar relic subcomplexes, akin to those produced in the starvation-induced process, remain as a result of flagellar ejection during cell development. This similarity suggests that the programmed flagellar ejection of C. crescentus might share a common evolutionary path with the more general, and likely more ancient (Kaplan et al., 2020), starvation-related flagellar loss.

Cryo-ET of photodamaged plasma membrane reveals shedding from protrusions that depends on F-actin dynamics, Myo1a, and Vps4B., Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.

Significance The complement system is integral to innate immunity and host defense. However, inappropriate activation causes host tissue damage and disease. In health, this is prevented by a complex protein network that includes the factor H proteins. Understanding control of complement is critical to treat complement-mediated disease. We demonstrate that a gain-of-function mutant factor H–related 5 protein (FHR5) results in glomerular damage. The mutant interfered with complement regulation within the kidney, resulting in complement accumulation within glomeruli and kidney damage. Administration of a complement regulator with enhanced surface regulatory activity reduced mutant-associated glomerular complement. FHR5 can disrupt the homeostatic regulation of complement within the kidney, and targeting FHR5 represents a way to treat some types of complement-mediated kidney injury., Genetic variation within the factor H–related (FHR) genes is associated with the complement-mediated kidney disease, C3 glomerulopathy (C3G). There is no definitive treatment for C3G, and a significant proportion of patients develop end-stage renal disease. The prototypical example is CFHR5 nephropathy, through which an internal duplication within a single CFHR5 gene generates a mutant FHR5 protein (FHR5mut) that leads to accumulation of complement C3 within glomeruli. To elucidate how abnormal FHR proteins cause C3G, we modeled CFHR5 nephropathy in mice. Animals lacking the murine factor H (FH) and FHR proteins, but coexpressing human FH and FHR5mut (hFH-FHR5mut), developed glomerular C3 deposition, whereas mice coexpressing human FH with the normal FHR5 protein (hFH-FHR5) did not. Like in patients, the FHR5mut had a dominant gain-of-function effect, and when administered in hFH-FHR5 mice, it triggered C3 deposition. Importantly, adeno-associated virus vector-delivered homodimeric mini-FH, a molecule with superior surface C3 binding compared to FH, reduced glomerular C3 deposition in the presence of the FHR5mut. Our data demonstrate that FHR5mut causes C3G by disrupting the homeostatic regulation of complement within the kidney and is directly pathogenic in C3G. These results support the use of FH-derived molecules with enhanced C3 binding for treating C3G associated with abnormal FHR proteins. They also suggest that targeting FHR5 represents a way to treat complement-mediated kidney injury.

AirRater is a free smartphone app developed in 2015, supporting individuals to protect their health from environmental hazards. It does this by providing (i) location-specific and near real-time air quality, pollen and temperature information and (ii) personal symptom tracking functionality. This research sought to evaluate user perceptions of AirRater’s usability and effectiveness. We collected demographic data and completed semi-structured interviews with 42 AirRater users, identified emergent themes, and used two frameworks designed to understand and support behavior change—the Behavior Change Wheel (BCW) and the Protective Action Decision Model (PADM)—to interpret results. Of the 42 participants, almost half indicated that experiencing symptoms acted as a prompt for app use. Information provided by the app supported a majority of the 42 participants to make decisions and implement behaviors to protect their health irrespective of their location or context. The majority of participants also indicated that they shared information provided by the app with family, friends and/or colleagues. The evaluation also identified opportunities to improve the app. Several study limitations were identified, which impacts the generalizability of results beyond the populations studied. Despite these limitations, findings facilitated new insights into motivations for behavior change, and contribute to the existing literature investigating the potential for smartphone apps to support health protection from environmental hazards in a changing climate.

Commotio cordis is the second leading cause of sudden cardiac death in young athletes. Currently available chest protectors on the market are ineffective in preventing cases of commotio cordis in young athletes who play baseball. This study focused on using contour maps to identify specific baseball impact locations to the chest that may result in instances of commotio cordis to children during baseball games. By identifying these vulnerable locations, we may design and develop chest protectors that can provide maximum protection to prevent commotio cordis in young athletes. Simulation cases were run using the validated CHARM-10 chest model, a detailed finite element model representing an average 10-year-old child's chest. A baseball model was developed in company with the chest model, and then used to impact the chest at different locations. A 7 × 8 impact location matrix was designed with 56 unique baseball impact simulations. Left ventricle strain and pressure, reaction force between the baseball and chest, and rib deformations were analyzed. Left ventricle strain was highest from baseball impacts directly over the left ventricle (0.34) as well as impacts slightly lateral and superior to the cardiac silhouette (0.34). Left ventricle pressure was highest with impacts directly over the left ventricle (82.94 kPa). We have identified the most dangerous impact locations resulting in high left ventricle strain and pressure. This novel study provided evidence of where to emphasize protective materials for establishing effective chest protectors that will minimize instances of commotio cordis in young athletes.

Short-term interaction between Acanthamoeba and M. avium has been demonstrated to increase infectivity in human and murine models of infection, establishing the paradigm that amoebae “train” M. avium in the environment by selecting for phenotypes capable of enduring in human cells. We investigated this phenomenon further by determining the consequence of long-term amoebae adaptation on M. avium subsp. hominissuis persistence in host cells., Free-living amoebae are ubiquitous in aquatic environments and act as environmental reservoirs for nontuberculous mycobacteria. Mycobacterium avium subsp. hominissuis recovered from Acanthamoeba has been demonstrated to be more virulent in both human and murine models. Here, we investigate the persistence of M. avium subsp. hominissuis after short-term (2 weeks) and long-term (42 weeks) coculture in Acanthamoeba lenticulata. We hypothesize that A. lenticulata-adapted M. avium subsp. hominissuis demonstrates phenotypic and genomic changes facilitating intracellular persistence in naive Acanthamoeba and human macrophages. M. avium subsp. hominissuis CFU in coculture with A. lenticulata were recorded every 2 weeks for up to 60 weeks. While A. lenticulata-associated M. avium subsp. hominissuis CFU did not significantly change across 60 weeks of coculture, longer adaptation time in amoebae reduced the colony size. Isolates recovered after 2 or 42 weeks of amoeba coculture were referred to as “early-adapted” and “late-adapted” M. avium subsp. hominissuis, respectively. Whole-genome sequencing was performed on amoeba-adapted isolates with pan-genome comparisons to the original M. avium subsp. hominissuis isolate. Next, amoeba-adapted isolates were assessed for their persistence in A. lenticulata, Acanthamoeba castellanii, and human THP-1 macrophages. Multiplex cytokine/chemokine analyses were conducted on THP-1 culture supernatants. Compared to the original isolate, counts of late-adapted M. avium subsp. hominissuis were reduced in Acanthamoeba and, contrary to expectations, lower counts were also observed in THP-1 macrophages, with concomitant decreases in tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and macrophage inflammatory protein 1 beta (MIP-1β), suggesting that host adaptation may influence the inflammatory properties of M. avium. IMPORTANCE Short-term interaction between Acanthamoeba and M. avium has been demonstrated to increase infectivity in human and murine models of infection, establishing the paradigm that amoebae “train” M. avium in the environment by selecting for phenotypes capable of enduring in human cells. We investigated this phenomenon further by determining the consequence of long-term amoeba adaptation on M. avium subsp. hominissuis persistence in host cells. We monitored genomic changes across long-term Acanthamoeba coculture and report significant changes to the M. avium subsp. hominissuis genome in response to amoeba adaptation and reduced colony size. Furthermore, we examined isolates cocultured with A. lenticulata for 2 or 42 weeks and provide biological evidence that long-term coculture in amoebae reduces M. avium persistence in human macrophages.

FtsZ is thought to generate constrictive force to divide the cell, possibly via one of two predominant models in the field. In one, FtsZ filaments overlap to form complete rings which constrict as filaments slide past each other to maximize lateral contact., To divide, bacteria must constrict their membranes against significant force from turgor pressure. A tubulin homolog, FtsZ, is thought to drive constriction, but how FtsZ filaments might generate constrictive force in the absence of motor proteins is not well understood. There are two predominant models in the field. In one, FtsZ filaments overlap to form complete rings around the circumference of the cell, and attractive forces cause filaments to slide past each other to maximize lateral contact. In the other, filaments exert force on the membrane by a GTP-hydrolysis-induced switch in conformation from straight to bent. Here, we developed software, ZCONSTRICT, for quantitative three-dimensional (3D) simulations of Gram-negative bacterial cell division to test these two models and identify critical conditions required for them to work. We find that the avidity of any kind of lateral interactions quickly halts the sliding of filaments, so a mechanism such as depolymerization or treadmilling is required to sustain constriction by filament sliding. For filament bending, we find that a mechanism such as the presence of a rigid linker is required to constrain bending to within the division plane and maintain the distance observed in vivo between the filaments and the membrane. Of these two models, only the filament bending model is consistent with our lab’s recent observation of constriction associated with a single, short FtsZ filament. IMPORTANCE FtsZ is thought to generate constrictive force to divide the cell, possibly via one of two predominant models in the field. In one, FtsZ filaments overlap to form complete rings which constrict as filaments slide past each other to maximize lateral contact. In the other, filaments exert force on the membrane by switching conformation from straight to bent. Here, we developed software, ZCONSTRICT, for three-dimensional (3D) simulations to test these two models. We find that a mechanism such as depolymerization or treadmilling are required to sustain constriction by filament sliding. For filament bending, we find that a mechanism that constrains bending to within the division plane is required to maintain the distance observed in vivo between the filaments and the membrane.

Introduction While telemedicine usage has increased due to the COVID-19 pandemic, there remains little consensus about how spine surgeons perceive virtual care. The purpose of this study was to explore international perspectives of spine providers on the challenges and benefits of telemedicine. Methods Responses from 485 members of AO Spine were analyzed, covering provider perceptions of the challenges and benefits of telemedicine. All questions were optional, and blank responses were excluded from analysis. Results The leading challenges reported by surgeons were decreased ability to perform physical examinations (38.6%), possible increased medicolegal exposure (19.3%), and lack of reimbursement parity compared to traditional visits (15.5%). Fewer than 9.0% of respondents experienced technological issues. On average, respondents agreed that telemedicine increases access to care for rural/long-distance patients, provides societal cost savings, and increases patient convenience. Responses were mixed about whether telemedicine leads to greater patient satisfaction. North Americans experienced the most challenges, but also thought telemedicine carried the most benefits, whereas Africans reported the fewest challenges and benefits. Age did not affect responses. Conclusion Spine surgeons are supportive of the benefits of telemedicine, and only a small minority experienced technical issues. The decreased ability to perform the physical examination was the top challenge and remains a major obstacle to virtual care for spine surgeons around the world, although interestingly, 61.4% of providers did not acknowledge this to be a major challenge. Significant groundwork in optimizing remote physical examination maneuvers and achieving legal and reimbursement clarity is necessary for widespread implementation.

Student engagement can encompass many different levels of interaction. Whether it be between student and instructor, among students themselves, with the actual subject matter, or with the various resources and departments on campus, getting and keeping students engaged is a challenging proposition. Frequently, students say they fear speaking in public, being called upon in class, going to the whiteboard, and being singled out by an instructor. Collectively, these fears could be conceptualized as a wish or need for anonymity. Faculty have cited student actions such as participation, question asking, volunteering, office hour visits, and favorable body language as preferred behaviors. These preferences could be conceptualized as a wish or need for engagement.

During extreme air pollution events, such as bushfires, public health agencies often recommend that vulnerable individuals visit a nearby public building with central air conditioning to reduce their exposure to smoke. However, there is limited evidence that these “cleaner indoor air shelters” reduce exposure or health risks. We quantified the impact of a “cleaner indoor air shelter” in a public library in Port Macquarie, NSW, Australia when concentrations of fine particulate matter (PM2.5) were elevated during a local peat fire and nearby bushfires. Specifically, we evaluated the air quality improvements with central air conditioning only and with the use of portable high efficiency particulate air (HEPA) filter air cleaners. We measured PM2.5 from August 2019 until February 2020 by deploying pairs of low-cost PM2.5 sensors (i) inside the main library, (ii) in a smaller media room inside the library, (iii) outside the library, and (iv) co-located with regulatory monitors located in the town. We operated two HEPA cleaners in the media room from August until October 2019. We quantified the infiltration efficiency of outdoor PM2.5 concentrations, defined as the fraction of the outdoor PM2.5 concentration that penetrates indoors and remains suspended, as well as the additional effect of HEPA cleaners on PM2.5 concentrations. The infiltration efficiency of outdoor PM2.5 into the air-conditioned main library was 30%, meaning that compared to the PM2.5 concentration outdoors, the concentrations of outdoor-generated PM2.5 indoors were reduced by 70%. In the media room, when the HEPA cleaners were operating, PM2.5 concentrations were reduced further with a PM2.5 infiltration efficiency of 17%. A carefully selected air-conditioned public building could be used as a cleaner indoor air shelter during episodes of elevated smoke emissions. Further improvements in indoor air quality within the building can be achieved by operating appropriately sized HEPA cleaners.

Nontuberculous mycobacteria (NTM) are environmental organisms that can cause opportunistic pulmonary disease with species diversity showing significant regional variation. In the United States, Hawai&rsquo, i shows the highest rate of NTM pulmonary disease. The need for improved understanding of NTM reservoirs led us to identify NTM from patient respiratory specimens and compare NTM diversity between outdoor and indoor locations in Hawai&rsquo, i. A total of 545 water biofilm samples were collected from 357 unique locations across Kaua&rsquo, i (n = 51), O&rsquo, ahu (n = 202), Maui (n = 159), and Hawai&rsquo, i Island (n = 133) and divided into outdoor (n = 179) or indoor (n = 366) categories. rpoB sequence analysis was used to determine NTM species and predictive modeling applied to develop NTM risk maps based on geographic characteristics between environments. M. chimaera was frequently identified from respiratory and environmental samples followed by M. chelonae and M. abscessus, yet significantly less NTM were consistently recovered from outdoor compared to indoor biofilms, as exemplified by showerhead biofilm samples. While the frequency of M. chimaera recovery was comparable between outdoor and indoor showerhead biofilms, phylogenetic analyses demonstrate similar rpoB gene sequences between all showerhead and respiratory M. chimaera isolates, supporting outdoor and indoor environments as possible sources for pulmonary M. chimaera infections.

Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our understanding of how large biomolecular complexes look like, how they assemble, disassemble, function, and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques, e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection, and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light and electron microscopy (cryo-CLEM).