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Abstract The integrated stress response (ISR) enables cells to cope with a variety of insults, but its specific contribution to downstream cellular outputs remains unclear. Using a synthetic tool, we selectively activate the ISR without co-activation of parallel pathways and define the resulting cellular state with multi-omics profiling. We identify time- and dose-dependent gene expression modules, with ATF4 driving only a small but sensitive subgroup that includes amino acid metabolic enzymes. This ATF4 response affects cellular bioenergetics, rerouting carbon utilization towards amino acid production and away from the tricarboxylic acid cycle and fatty acid synthesis. We also find an ATF4-independent reorganization of the lipidome that promotes DGAT-dependent triglyceride synthesis and accumulation of lipid droplets. While DGAT1 is the main driver of lipid droplet biogenesis, DGAT2 plays an essential role in buffering stress and maintaining cell survival. Together, we demonstrate the sufficiency of the ISR in promoting a previously unappreciated metabolic state.

Hip arthroscopy is commonly performed to treat femoroacetabular impingement syndrome. A post-assisted arthroscopic hip preservation surgery approach provides joint distraction for central-compartment access. Owing to the location, compression of the post in the perineum may cause injuries to the pudendal nerve, perineal soft tissue, or genitourinary system. A postless technique significantly reduces the risk of these complications. Postless arthroscopy uses friction between the patient’s torso and the table surface to permit distraction without the post. An air arthrogram, general anesthesia with muscle paralysis, and variable degrees of Trendelenburg positioning reduce the force needed for joint distraction. Early postless literature suggested Trendelenburg angles of approximately 15°, which may be disorienting to surgeons and compromise the precision and accuracy of the surgical procedure. With the described technique, hip arthroscopy can be effectively performed with a Trendelenburg angle of less than 5° in nearly every case. Understanding the physics of postless hip arthroscopy using free-body diagrams of inclined planes with friction permits surgeons to understand the required Trendelenburg angle of the bed, the force of traction for any patient given his or her body habitus, and the coefficient of static friction of the table surface to achieve a minimum amount of joint distraction.

Purpose: To evaluate online, self-reported pudendal nerve or perineal injuries related to the use of a perineal post during hip arthroscopy. Methods: Public posts on Reddit and the Health Organization for Pudendal Education were searched to identify anonymous individuals reporting symptoms of pudendal nerve or perineal injury following hip arthroscopy. Included posts were by any individual with a self-reported history of hip arthroscopy who developed symptoms of pudendal nerve injury or damage to the perineal soft tissues. Demographic information and details about a person’s symptoms and concerns were collected from each post. Descriptive statistics were used to analyze the data. Results: Twenty-three online posts reported on a perineal post-related complication following hip arthroscopy. Sex information was available in 16 (70%) posts (8 male, 8 female). Twenty-two posts reported a sensory injury, and 4 posts reported a motor injury with sexual consequences (sexual dysfunction, dyspareunia, impotence). Symptom duration was available in 15 (65%) posts (8 temporary, 7 permanent). Permanent symptoms included paresthesia of the perineum or genitals (7) and sexual complaints (5). Two posts stated they were counseled preoperatively about the possibility of this injury. Zero patients reported that a postless hip arthroscopy alternative was an option made available to them before surgery. Conclusions: A high incidence of permanent pudendal nerve, perineal skin, and genitourinary/sexual complications are self-reported and discussed online by patients who have undergone post-assisted hip arthroscopy. These patients report being uninformed and undereducated about the possibility of sustaining a post-related complication. No patient reported being informed of postless hip arthroscopy preoperatively. Clinical Relevance: Identifying and evaluating self-reported patient information in online medical forums can provide important information about patient experiences and outcomes.

Background: Robotic-assisted total knee arthroplasty (rTKA) has been shown to reduce the number of alignment outliers and to improve component positioning compared to manual TKA (mTKA). The primary purpose of this investigation was to compare the frequency of achieving target postoperative limb alignment and component positioning for rTKA vs mTKA. Methods: A retrospective comparative study was performed on 250 patients undergoing primary TKA by 2 fellowship-trained arthroplasty surgeons. Surgeon A performed predominantly rTKA (103 cases) with the ROSA system (Zimmer Biomet, Warsaw, IN) and less frequently mTKA (44 cases) with conventional instrumentation. Surgeon B performed only mTKA (103 cases). Target limb alignment for surgeon A was 0° for all cases and for surgeon B was 2° varus for varus knees and 0° for valgus knees. Radiographic measurements were determined by 2 reviewers. Target zone was set at ± 2 degrees from the predefined target. Results: When comparing rTKA to mTKA performed by different surgeons, there were no differences in the percentage within the target zone (57.28% vs 53.40%, P = .575), but rTKA did result in a greater percentage for cases with preoperative valgus (71.42% vs 44.12%, P = .031). Patient-reported Outcomes Measurement Information System Global-10 physical scores were statistically higher at both 3 (P = .016) and 6 months (P = .001) postoperatively for rTKA compared to mTKA performed by different surgeons. Conclusions: Although experienced surgeons can achieve target limb alignment correction with similar frequency when comparing rTKA to mTKA for all cases, rTKA may achieve target limb alignment with more accuracy for preoperative valgus deformity. Level of Evidence: Retrospective Cohort Study, Level III.

Single cell data provides cellular resolution on gene expression, but is rarely mined for isoforms. Analysis of 3' isoforms across ~250 Drosophila cell types reveals the cellular bases for numerous tissue-specific 3' programs, identifies new 3' programs, and nominates candidate trans-acting factors

Summary: Nonsense-mediated RNA decay (NMD) degrades transcripts carrying premature termination codons. NMD is thought to prevent the synthesis of toxic truncated proteins. However, whether loss of NMD results in widespread production of truncated proteins is unclear. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD), features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Using a cell-based model of FSHD, we show production of truncated proteins from physiological NMD targets and find that RNA-binding proteins are enriched for aberrant truncations. The NMD isoform of one RNA-binding protein, SRSF3, is translated to produce a stable truncated protein, which is detected in FSHD patient-derived myotubes. Ectopic expression of truncated SRSF3 confers toxicity, and its downregulation is cytoprotective. Our results delineate the genome-scale impact of NMD loss. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

Yisha Li,1 Ran Dai,2 Yeongjin Gwon,2 Stephen I Rennard,3 Barry J Make,4 Dinah Foer,5 Matthew J Strand,4 Erin Austin,6 Kendra A Young,1 John E Hokanson,1 Katherine A Pratte,7 Rebecca Conway,1 Gregory L Kinney1 On behalf of COPDGene investigators1Department of Epidemiology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 2Department of Biostatistics, School of Public Health, University of Nebraska Medical Center, Omaha, NE, USA; 3Division of Pulmonary, Critical Care and Sleep Medicine, University of Nebraska Medical Center, Omaha, NE, USA; 4Department of Medicine, National Jewish Health, Denver, CO, USA; 5Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 6Mathematical and Statistical Sciences, University of Colorado Denver, Denver, CO, USA; 7Department of Biostatistics, National Jewish Health, Denver, CO, USACorrespondence: Gregory L Kinney, Department of Epidemiology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA, Tel +1 303-724-4437, Email GREG.KINNEY@CUANSCHUTZ.EDU

Abstract Obesity creates a localized inflammatory reaction in the adipose, altering secretion of adipocyte-derived factors that contribute to pathologies including cancer. We have previously shown that adiponectin inhibits pancreatic cancer by antagonizing leptin-induced STAT3 activation. Yet, the effects of adiponectin on pancreatic cancer cell metabolism have not been addressed. In these studies, we have uncovered a novel metabolic function for the synthetic adiponectin-receptor agonist, AdipoRon. Treatment of PDAC cells with AdipoRon led to mitochondrial uncoupling and loss of ATP production. Concomitantly, AdipoRon-treated cells increased glucose uptake and utilization. This metabolic switch further correlated with AMPK mediated inhibition of the prolipogenic factor acetyl coenzyme A carboxylase 1 (ACC1), which is known to initiate fatty acid catabolism. Yet, measurements of fatty acid oxidation failed to detect any alteration in response to AdipoRon treatment, suggesting a deficiency for compensation. Additional disruption of glycolytic dependence, using either a glycolysis inhibitor or low-glucose conditions, demonstrated an impairment of growth and survival of all pancreatic cancer cell lines tested. Collectively, these studies provide evidence that pancreatic cancer cells utilize metabolic plasticity to upregulate glycolysis in order to adapt to suppression of oxidative phosphorylation in the presence of AdipoRon.

Total knee arthroplasty is one of the most widely performed surgical procedures today. Its widespread popularity has helped drive innovation and improvement in the field. Different schools of thought have developed regarding the best way to perform this operation. Specifically, there are controversaries regarding the best alignment philosophy for the femoral and tibial components to optimize implant stability and longevity. Traditionally, neutral mechanical alignment has been the preferred alignment target. More recently, some surgeons advocate for alignment matching the patient's pre-arthritic anatomic alignment (“physiologic” varus or valgus), which has been described as kinematic alignment. Functional alignment is a hybrid technique that focuses on the coronal plane minimizing soft tissue releases. To date, there is no evidence demonstrating superiority of one method over another. There is growing popularity of robotic surgical techniques to improve accuracy of implant position and alignment. The choice of alignment philosophy is an important aspect of robotic assisted TKA surgery and has the potential to clarify the optimal alignment technique.

Ma, Hernandez et al develop a 3D culture-transplant system to enable studies of cancer metastasis using patient-derived xenograft (PDX) tumor cells. Using this system, they find that OXPHOS inhibition attenuates the lung metastatic capacity of breast cancer cells and that overexpression of the metabolic enzyme NME1 increases lung metastasis.

Synthetic mimics of glycoproteins and proteoglycans have wide applications in biomedicine, yet preparation has been challenged by their high grafting and glycosylation densities. Here the authors show one-pot dual-catalysis polymerization of glycan-bearing α-amino acid N-carboxyanhydrides to form glycopolypeptide brushes.

The estimation of seismic reservoir properties faces difficulties such as delineating deceptive fluid contacts, over-estimating reserves and drilling dry holes. To overcome these problems, sequence stratigraphic and rock physics analyses that were constrained by the compaction trend were implemented in the Sigma Field. Sigma field is located in the Coastal Swamp depobelt, in the distal part. Seismic data from the field and a total of three wells, Sigma-30, 26, and 25 were provided for the analyses. Biostratigraphic data was also given for the field and dated to shale marker Cassidulina-7. The stratigraphic sequence analysis of the wells in field indicate they were deposited in a regressive, transgressive and aggradational episodes, whereas the entire depositional history shows predominantly regressive episode. Three field reservoirs, SM-14 (LST), SM-04 (TST), and SM-02 (HST) were delineated. Depositional analysis of the landscape from sequence stratigraphic analyses of the field indicates that Sigma field reservoirs were deposited in a front region of the delta. Trend to compaction has been generated for the well. Sigma wells showed a normal trend toward compaction. Rock physics analysis of the reservoirs of interest reveals that LST and TST reservoirs have been difficult to distinguish against HST reservoirs which are easy to classify regardless of their depth for fluids in a Vp/Vs crossplot versus acoustic impedance. In the analysis, a pattern is suggested for TST reservoirs. Therefore, this research work shows that fluid discrimination becomes simpler as we go basin ward as the sorting of the reservoir rocks increases with finer grain sand sizes.

Our aim was to identify proteins that reflect an acute systemic response to prolonged hyperbaric stress and discover potential biomarker pathways for pulmonary O2 toxicity. The study was a double-blind, randomized, crossover design in trained male Navy diver subjects. Each subject completed two dry resting hyperbaric chamber dives separated by a minimum of one week. One dive exposed the subject to 6.5 h of 100% oxygen (O2) at 2ATA. The alternate dive exposed the subjects to an enhanced air nitrox mixture (EAN) containing 30.6% O2 at the same depth for the same duration. Venous blood samples collected before (PRE) and after (POST) each dive were prepared and submitted to LC-MS/MS analysis (2 h runs). A total of 346 total proteins were detected and analyzed. A total of 12 proteins were significantly increased at EANPOST (vs. EANPRE), including proteins in hemostasis and immune signaling and activation. Significantly increased proteins at O2PRE (vs. O2POST) included neural cell adhesion molecule 1, glycoprotein Ib, catalase, hemoglobin subunit beta, fibulin-like proteins, and complement proteins. EANPOST and O2POST differed in biomarkers related to coagulation, immune signaling and activation, and metabolism. Of particular interest is (EANPOST vs. O2POST), which is protective against oxidative stress.

Abstract Background The sequence content of the 3′ UTRs of many mRNA transcripts is regulated through alternative polyadenylation (APA). The study of this process using RNAseq data, though, has been historically challenging. Results To combat this problem, we developed LABRAT, an APA isoform quantification method. LABRAT takes advantage of newly developed transcriptome quantification techniques to accurately determine relative APA site usage and how it varies across conditions. Using LABRAT, we found consistent relationships between gene-distal APA and subcellular RNA localization in multiple cell types. We also observed connections between transcription speed and APA site choice as well as tumor-specific transcriptome-wide shifts in APA isoform abundance in hundreds of patient-derived tumor samples that were associated with patient prognosis. We investigated the effects of APA on transcript expression and found a weak overall relationship, although many individual genes showed strong correlations between relative APA isoform abundance and overall gene expression. We interrogated the roles of 191 RNA-binding proteins in the regulation of APA isoforms, finding that dozens promote broad, directional shifts in relative APA isoform abundance both in vitro and in patient-derived samples. Finally, we find that APA site shifts in the two classes of APA, tandem UTRs and alternative last exons, are strongly correlated across many contexts, suggesting that they are coregulated. Conclusions We conclude that LABRAT has the ability to accurately quantify APA isoform ratios from RNAseq data across a variety of sample types. Further, LABRAT is able to derive biologically meaningful insights that connect APA isoform regulation to cellular and molecular phenotypes.

Abstract Background People who inject drugs (PWID) suffer high morbidity and mortality from injection related infections (IRI). The inpatient setting is an ideal opportunity to treat underlying substance use disorder (SUD), but it is unclear how often this occurs. Objectives To quantify the utilization of behavioral health services for PWID during inpatient admissions for IRI. Methods Data for all hospital admissions in Florida in FY2017 were obtained from the Agency for Healthcare Administration. Hospitalization for IRI were obtained using a validated ICD-10 algorithm and treatment for substance use disorder was quantified using ICD-10-Procedure Coding System (ICD-10-PCS) codes. Result Among the 20,001 IRI admissions, there were 230 patients who received behavioral health services as defined by ICD-10-PCS SAT codes for treatment for SUD. Conclusions In a state with a large number of IRI, only a very small portion of admissions received behavioral health services. Increased efforts should be directed to studying referral patterns among physicians and other providers caring for this population and increasing utilization of behavioral health services.

Abstract Background Transthyretin and immunoglobulin light-chain amyloidoses cause amyloid deposition throughout various organ systems. Recent evidence suggests that soft tissue amyloid deposits may lead to orthopedic conditions before cardiac manifestations occur. Pharmacologic treatments reduce further amyloid deposits in these patients. Thus, early diagnosis improves long term survival. Questions/purposes The primary purpose of this systematic review was to characterize the association between amyloid deposition and musculoskeletal pathology in patients with common orthopedic conditions. A secondary purpose was to determine the relationship between amyloid positive biopsy in musculoskeletal tissue and the eventual diagnosis of systemic amyloidosis. Methods We performed a systematic review using PRISMA guidelines. Inclusion criteria were level I-IV evidence articles that analyzed light-chain or transthyretin amyloid deposits in common orthopedic surgeries. Study methodological quality, risk of bias, and recommendation strength were assessed using MINORS, ROBINS-I, and SORT. Results This systematic review included 24 studies for final analysis (3606 subjects). Amyloid deposition was reported in five musculoskeletal pathologies, including carpal tunnel syndrome (transverse carpal ligament and flexor tenosynovium), hip and knee osteoarthritis (synovium and articular cartilage), lumbar spinal stenosis (ligamentum flavum), and rotator cuff tears (tendon). A majority of studies reported a mean age greater than 70 for patients with TTR or AL positive amyloid. Conclusions This systematic review has shown the presence of amyloid deposition detected at the time of common orthopedic surgeries, especially in patients ≥70 years old. Subtyping of the amyloid has been shown to enable diagnosis of systemic light-chain or transthyretin amyloidosis prior to cardiac manifestations. Level of evidence Level IV.

Although venous aneurysms are rare, typically asymptomatic, and most commonly found incidentally on imaging studies, patients with this pathology can develop pulmonary emboli owing to these aneurysms acting as a nidus for thrombus formation. There is no clear consensus regarding conservative management with anticoagulation vs operative intervention as the best treatment of deep venous aneurysms. We report the clinical course and surgical treatment of a patient presenting with both bilateral common femoral vein and bilateral popliteal venous aneurysms who had a known history of prior symptomatic pulmonary emboli.

Background: Medial column procedures are commonly used to treat progressive collapsing foot deformity (PCFD) reconstruction. The aim of this research is to present the clinical results of plantar plating for medial naviculocuneiform (NC) arthrodesis when NC joint pathology contributes to medial arch collapse. The authors hypothesized that lag screws with a plantar neutralization plate would result in a satisfactory NC joint fusion rate. Methods: A single-surgeon, retrospective case series was performed on patients with flexible PCFD who underwent NC arthrodesis using lag screws and a contoured neutralization plate applied plantarly across the medial NC joint as part of PCFD reconstruction. Thirteen patients (11 females, 2 males; mean age 53.1 [34-62] years) between 2016 and 2019 were identified for inclusion. Mean follow-up was 25.2 ± 12.7 months. Preoperative and postoperative anteroposterior talo–first metatarsal angle, lateral talo–first metatarsal angle, talonavicular coverage angle, and calcaneal pitch were measured. Union was evaluated radiologically. AOFAS midfoot scores were recorded at final follow-up. Results: All parameters demonstrated a significant improvement. Fusion was confirmed in 11 of 13 patients (85%) at a mean 5.7 ± 2.1 months. One patient required a revision of their NC fusion because of symptomatic nonunion. There were no cases of symptomatic plantar hardware. Conclusion: The results of this small cohort series suggest that lag screw with plantar plate NC arthrodesis yielded generally improved short-term radiographic and clinical outcomes in PCFD patients with medial arch collapse through the NC joint. Level of Evidence : Level IV, retrospective case series.

Yisha Li,1 Margaret Ragland,1 Erin Austin,2 Kendra Young,1 Katherine Pratte,3 John E Hokanson,1 Terri H Beaty,4 Elizabeth A Regan,3 Stephen I Rennard,5 Christina Wern,6 Michael R Jacobs,7 Ruth Tal-Singer,8 Barry J Make,3 Gregory L Kinney1 On Behalf of theCOPDGene investigators1Department of Epidemiology, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 2Mathematical and Statistical Sciences, University of Colorado Denver, Denver, CO, USA; 3National Jewish Health, Denver, CO, USA; 4Bloomberg School of Public Health, University of John Hopkins, Baltimore, MD, USA; 5Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NB, USA; 6Department of Clinical Pharmacy, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA; 7School of Pharmacy, Temple University, PA, Pennsylvania, USA; 8COPD Foundation, Washington, D.C., USACorrespondence: Gregory L KinneyDepartment of Epidemiology, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO, USATel +1 303-724-4437Email GREG.KINNEY@CUANSCHUTZ.EDUPurpose: Medication patterns include all medications in an individual’s clinical profile. We aimed to identify chronic co-morbidity treatment patterns through medication use among COPDGene participants and determine whether these patterns were associated with mortality, acute exacerbations of chronic obstructive pulmonary disease (AECOPD) and quality of life.Materials and Methods: Participants analyzed here completed Phase 1 (P1) and/or Phase 2 (P2) of COPDGene. Latent class analysis (LCA) was used to identify medication patterns and assign individuals into unobserved LCA classes. Mortality, AECOPD, and the St. George’s Respiratory Questionnaire (SGRQ) health status were compared in different LCA classes through survival analysis, logistic regression, and Kruskal–Wallis test, respectively.Results: LCA identified 8 medication patterns from 32 classes of chronic comorbid medications. A total of 8110 out of 10,127 participants with complete covariate information were included. Survival analysis adjusted for covariates showed, compared to a low medication use class, mortality was highest in participants with hypertension+diabetes+statin+antiplatelet medication group. Participants in hypertension+SSRI+statin medication group had the highest odds of AECOPD and the highest SGRQ score at both P1 and P2.Conclusion: Medication pattern can serve as a good indicator of an individual’s comorbidities profile and improves models predicting clinical outcomes.Keywords: COPDGene, co-morbidities, latent class analysis, medication patterns, mortality

Summary: The Mixed Lineage Leukemia (MLL1, KMT2A) gene is critical for development and maintenance of hematopoietic stem cells (HSCs), however, whether this protein is limiting for HSC development is unknown due to lack of physiologic model systems. Here, we develop an MLL1-inducible embryonic stem cell (ESC) system and show that induction of wild-type MLL1 during ESC differentiation selectively increases hematopoietic potential from a transitional c-Kit+/Cd41+ population in the embryoid body and also at sites of hematopoiesis in embryos. Single-cell sequencing analysis illustrates inherent heterogeneity of the c-Kit+/Cd41+ population and demonstrates that MLL1 induction shifts its composition toward multilineage hematopoietic identities. Surprisingly, this does not occur through increasing Hox or other canonical MLL1 targets but through an enhanced Rac/Rho/integrin signaling state, which increases responsiveness to Vla4 ligands and enhances hematopoietic commitment. Together, our data implicate a Rac/Rho/integrin signaling axis in the endothelial to hematopoietic transition and demonstrate that MLL1 actives this axis. : In this article, Ernst and colleagues show that overexpression of wild-type, non-fusion MLL1/KMT2A increases the hematopoietic potential of ESC-derived and embryonic progenitors. MLL1 expression selectively enhances hematopoietic commitment from hemogenic endothelium through an unexpected Rac/Rho/integrin signaling, adhesion-mediated mechanism. Keywords: KMT2A, progenitor heterogeneity, single-cell RNA sequencing, hemogenic endothelium, EMP, embryonic hematopoiesis

Macrophages play a dual role in tumor initiation and progression, with both tumor-promoting and tumor-suppressive effects; hence, it is essential to understand the distinct responses of macrophages to tumor progression and therapy. Mild hyperthermia has gained importance as a therapeutic regimen against cancer due to its immunogenic nature, efficacy, and potential synergy with other therapies, yet the response of macrophages to molecular signals from hyperthermic cancer cells has not yet been clearly defined. Due to limited response rate of breast cancer to conventional therapeutics the development, and understanding of alternative therapies like hyperthermia is pertinent. In order to determine conditions corresponding to mild thermal dose, cytotoxicity of different hyperthermic temperatures and treatment durations were tested in normal murine macrophages and breast cancer cell lines. Examination of exosome release in hyperthermia-treated cancer cells revealed enhanced efflux and a larger size of exosomes released under hyperthermic stress. Exposure of naïve murine macrophages to exosomes released from 4T1 and EMT-6 cells posthyperthermia treatment, led to an increased expression of specific macrophage activation markers. Further, exosomes released by hyperthermia-treated cancer cells had increased content of heat shock protein 70 (Hsp70). Together, these results suggest a potential immunogenic role for exosomes released from cancer cells treated with mild hyperthermia.

Category: Midfoot/Forefoot Introduction/Purpose: Medial column procedures are a common adjunct to progressive collapsing foot deformity (PCFD) correction. In particular, naviculocuneiform (NC) arthrodesis could be performed when degeneration or instability at the NC joint is contributing to arch collapse. Plantar plate fusion resists gapping along the tension side of the joint, potentially reducing risk of non-union and recurrent deformity. Additionally, by placing the hardware in a plantar location there is an increased soft tissue envelope to function in a protective matter and eliminate the possibility of hardware prominence and tendon irritation. The purpose of this study was to analyze short-term clinical and radiographic outcomes of NC fusion using a lag screw with plantar plating technique. Methods: A single-surgeon retrospective case series was performed on patients with PCFD and medial arch collapse treated with NC arthrodesis using a lag screw plus plantar plating technique between January 2016 and December 2019, with a minimum six month clinical and radiographic follow up. Patients undergoing revision NC joint arthrodesis, patients with incomplete imaging, or an arthrodesis technique that did not utilize NC joint plantar plating were excluded. Demographic data, perioperative data, complications, and reoperations were recorded from electronic medical records. Pre-operative AOFAS midfoot scores were calculated at the initial clinic visit and then again at the patients' most recent follow-up. In the case of most recent follow up occurring prior to six months post-operatively, a telemedicine visit was performed. Anteroposterior (AP) and lateral talo-first metatarsal angles, talonavicular coverage angle, and calcaneal pitch were measured independently by three authors. Fusion was confirmed radiographically and computed topography when union was in question. Results: Nineteen patients (15 female, 4 male; 11 right, 8 left) with a mean age of 56.6 years and a mean follow-up of 21.0 +- 13.4 months were reviewed. A mean 3.4 concomitant procedures were performed at the time of NC fusion. AP talo-first metatarsal angle (18.9° to 5.2°), lateral talo-first metatarsal angel (-18.7° to -4.4°), talonavicular coverage angle (28.1° to 7.4°), and calcaneal pitch (14.6° to 20.1°) all improved significantly (p

Introduction:. The COVID-19 pandemic created an unprecedented challenge for orthopaedic surgery residency applicants when away rotations were canceled and interviews were held virtually. The authors hypothesized that (1) Instagram would have more total social media accounts compared with Twitter or Facebook, (2) most social media accounts on all platforms would be created during 2020, and (3) the average number of Instagram followers would be higher among highly ranked programs. Methods:. A list of Accreditation Council for Graduate Medical Education-accredited orthopaedic surgery residency programs (n = 202) that were ranked by reputation was acquired from Doximity. Public Instagram, Facebook, and Twitter profiles of orthopaedic surgery residency programs were identified through a manual search. Variables assessed on each social media platform were all available summary statistics provided by the social media platform. Instagram accounts were categorized into 5 tiers based on numeric reputation ranking, with 40 programs in each tier (except tier 5 with 42 programs). Descriptive statistics were calculated, and continuous data were presented as mean ± standard deviation. Goodness-of-fit regression values were calculated for observed trends. Results:. One-hundred seventy-four social media accounts were identified. There was a higher percentage of Instagram accounts (66.1%) compared with Facebook (13.2%) or Twitter (20.7%). From 2010 to 2020, an exponential increase (R2 = 0.88) of social media accounts was observed during 2020. Instagram profiles (77.2%, 98/115 [95 in 2020, 3 in 2021]) were created after the cancelation of away rotations in May 2020. Instagram profiles had 1,029 ± 522.4 average followers, more than Facebook and Twitter. Tier 1 residency programs had the largest average follower count with 1,462.4 ± 584.3 followers. Conclusion:. Social media presence increased exponentially during 2020. Instagram was the social media platform with the most accounts. Many Instagram accounts were created during 2020, and most were created after away rotations were canceled during the COVID-19 pandemic. Instagram accounts averaged the most followers. Top tier orthopaedic residency programs had a greater number of accounts and average followers than lower tier programs.

ABSTRACT Prostaglandin E2 (PGE2) is an essential immunomodulatory lipid released by cells in response to infection with many bacteria, yet its function in macrophage-mediated bacterial clearance is poorly understood. Yersinia overall inhibits the inflammatory circuit, but its effect on PGE2 production is unknown. We hypothesized that one of the Yersinia effector proteins is responsible for the inhibition of PGE2 biosynthesis. We identified that yopB-deficient Y. enterocolitica and Y. pseudotuberculosis deficient in the secretion of virulence proteins via a type 3 secretion system (T3SS) failed to inhibit PGE2 biosynthesis in macrophages. Consistently, COX-2-mediated PGE2 biosynthesis is upregulated in cells treated with heat-killed or T3SS-deficient Y. pseudotuberculosis but diminished in the presence of a MAPK/ERK inhibitor. Mutants expressing catalytically inactive YopJ induce similar levels of PGE2 as heat-killed or ΔyopB Y. pseudotuberculosis, reversed by YopJ complementation. Shotgun proteomics discovered host pathways regulated in a YopJ-mediated manner, including pathways regulating PGE2 synthesis and oxidative phosphorylation. Consequently, this study identified that YopJ-mediated inhibition of MAPK signal transduction serves as a mechanism targeting PGE2, an alternative means of inflammasome inhibition by Yersinia. Finally, we showed that EP4 signaling supports macrophage function in clearing intracellular bacteria. In summary, our unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription, thereby enhancing the intracellular survival of yersiniae. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic molecule to improve the outcomes of specific bacterial infections. Since other pathogens encode YopJ homologs, this mechanism is expected to be present in other infections. IMPORTANCE PGE2 is a critical immunomodulatory lipid, but its role in bacterial infection and pathogen clearance is poorly understood. We previously demonstrated that PGE2 leads to macrophage polarization toward the M1 phenotype and stimulates inflammasome activation in infected macrophages. Finally, we also discovered that PGE2 improved the clearance of Y. enterocolitica. The fact that Y. enterocolitica hampers PGE2 secretion in a type 3 secretion system (T3SS)-dependent manner and because PGE2 appears to assist macrophage in the clearance of this bacterium indicates that targeting of the eicosanoid pathway by Yersinia might be an adaption used to counteract host defenses. Our study identified a mechanism used by Yersinia that obstructs PGE2 biosynthesis in human macrophages. We showed that Y. pseudotuberculosis interferes with PGE2 biosynthesis by using one of its T3SS effectors, YopJ. Specifically, YopJ targets the host COX-2 enzyme responsible for PGE2 biosynthesis, which happens in a MAPK/ER-dependent manner. Moreover, in a shotgun proteomics study, we also discovered other pathways that catalytically active YopJ targets in the infected macrophages. YopJ was revealed to play a role in limiting host LPS responses, including repression of EGR1 and JUN proteins, which control transcriptional activation of proinflammatory cytokine production such as interleukin-1β. Since YopJ has homologs in other bacterial species, there are likely other pathogens that target and inhibit PGE2 biosynthesis. In summary, our study’s unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic target.

Coccidiosis is one of the most economically important diseases of poultry. This study determined the preponderance of chicken Eimeria in southern Nigeria and assessed the parasite’s resistance to three anticoccidial drugs: Amprolium hydrochloride; Amprolium hydrochloride + Sulfaquinoxaline-Sodium; and Toltrazuril. Multiplex PCR amplification of the SCAR region was used to confirm Eimeria preponderance. Resistance was assessed following the inoculation of 2.32 × 105 infective oocysts into broilers. Data on weight gain, feed intake, feed conversion and fecal oocyst shed were recorded. At 7 days post inoculation 9 birds per treatment were sacrificed and assessed for macroscopic lesions in four intestinal regions. Percent optimum anticoccidial activity (POAA), Anticoccidial index (ACI) and Anticoccidial sensitivity test (AST) were used to access resistance. The preponderance of Eimeria spp. were E. tenella (77%), E. necatrix (55%), E. acervulina (44%) and E. mitis (11%), with multi-species infection occurring in 55% of samples assessed. Fecal oocyst shedding was low (P

Hibernators dramatically lower metabolism to save energy while fasting for months. Prolonged fasting challenges metabolic homeostasis, yet small-bodied hibernators emerge each spring ready to resume all aspects of active life, including immediate reproduction. The liver is the body’s metabolic hub, processing and detoxifying macromolecules to provide essential fuels to brain, muscle and other organs throughout the body. Here we quantify changes in liver gene expression across several distinct physiological states of hibernation in 13-lined ground squirrels, using RNA-seq to measure the steady-state transcriptome and GRO-seq to measure transcription for the first time in a hibernator. Our data capture key timepoints in both the seasonal and torpor-arousal cycles of hibernation. Strong positive correlation between transcription and the transcriptome indicates that transcriptional control dominates the known seasonal reprogramming of metabolic gene expression in liver for hibernation. During the torpor-arousal cycle, however, discordance develops between transcription and the steady-state transcriptome by at least two mechanisms: 1) although not transcribed during torpor, some transcripts are unusually stable across the torpor bout; and 2) unexpectedly, on some genes, our data suggest continuing, slow elongation with a failure to terminate transcription across the torpor bout. While the steady-state RNAs corresponding to these read through transcripts did not increase during torpor, they did increase shortly after rewarming despite their simultaneously low transcription. Both of these mechanisms would assure the immediate availability of functional transcripts upon rewarming. Integration of transcriptional, post-transcriptional and RNA stability control mechanisms, all demonstrated in these data, likely initiate a serial gene expression program across the short euthermic period that restores the tissue and prepares the animal for the next bout of torpor.

Salmonella Typhimurium is a causative agent of nontyphoidal salmonellosis, for which there is a lack of a clinically approved vaccine in humans. As an intracellular pathogen, Salmonella impacts many cellular pathways. However, the intercellular communication mechanism facilitated by host-derived small extracellular vesicles (EVs), such as exosomes, is an overlooked aspect of the host responses to this infection. We used a comprehensive proteome-based network analysis of exosomes derived from Salmonella-infected macrophages to identify host molecules that are trafficked via these EVs. This analysis predicted that the host-derived small EVs generated during macrophage infection stimulate macrophages and promote activation of T helper 1 (Th1) cells. We identified that exosomes generated during infection contain Salmonella proteins, including unique antigens previously shown to stimulate protective immune responses against Salmonella in murine studies. Furthermore, we showed that host EVs formed upon infection stimulate a mucosal immune response against Salmonella infection when delivered intranasally to BALB/c mice, a route of antigen administration known to initiate mucosal immunity. Specifically, the administration of these vesicles to animals stimulated the production of anti-Salmonella IgG antibodies, such as anti-OmpA antibodies. Exosomes also stimulated antigen-specific cell-mediated immunity. In particular, splenic mononuclear cells isolated from mice administered with exosomes derived from Salmonella-infected antigen-presenting cells increased CD4+ T cells secreting Th1-type cytokines in response to Salmonella antigens. These results demonstrate that small EVs, formed during infection, contribute to Th1 cell bias in the anti-Salmonella responses. Collectively, this study helps to unravel the role of host-derived small EVs as vehicles transmitting antigens to induce Th1-type immunity against Gram-negative bacteria. Understanding the EV-mediated defense mechanisms will allow the development of future approaches to combat bacterial infections.

Hibernation is a physiological and behavioral phenotype that minimizes energy expenditure. Hibernators cycle between profound depression and rapid hyperactivation of multiple physiological processes, challenging our concept of mammalian homeostasis. How the hibernator orchestrates and survives these extremes while maintaining cell to organismal viability is unknown. Here, we enhance the genome integrity and annotation of a model hibernator, the 13-lined ground squirrel. Our new assembly brings this genome to near chromosome-level contiguity and adds thousands of previously unannotated genes. These new genomic resources were used to identify 6,505 hibernation-related, differentially-expressed and processed transcripts using RNA-seq data from three brain regions in animals whose physiological status was precisely defined using body temperature telemetry. A software tool, squirrelBox, was developed to foster further data analyses and visualization. SquirrelBox includes a comprehensive toolset for rapid visualization of gene level and cluster group dynamics, sequence scanning of k-mer and domains, and interactive exploration of gene lists. Using these new tools and data, we deconvolute seasonal from temperature-dependent effects on the brain transcriptome during hibernation for the first time, highlighting the importance of carefully timed samples for studies of differential gene expression in hibernation. The identified genes include a regulatory network of RNA binding proteins that are dynamic in hibernation along with the composition of the RNA pool. In addition to passive effects of temperature, we provide evidence for regulated transcription and RNA turnover during hibernation. Significant alternative splicing, largely temperature dependent, also occurs during hibernation. These findings form a crucial first step and provide a roadmap for future work toward defining novel mechanisms of tissue protection and metabolic depression that may 1 day be applied toward improving human health.

Rheumatoid arthritis (RA)-associated lung disease is a leading cause of mortality in RA, yet the mechanisms linking lung disease and RA remain unknown. Using an established murine model of RA-associated lung disease combining collagen-induced arthritis (CIA) with organic dust extract (ODE)-induced airway inflammation, differences among lung immune cell populations were analyzed by single cell RNA-sequencing. Additionally, four lung myeloid-derived immune cell populations including macrophages, monocytes/macrophages, monocytes, and neutrophils were isolated by fluorescence cell sorting and gene expression was determined by NanoString analysis. Unsupervised clustering revealed 14 discrete clusters among Sham, CIA, ODE, and CIA+ODE treatment groups: 3 neutrophils (inflammatory, resident/transitional, autoreactive/suppressor), 5 macrophages (airspace, differentiating/recruited, recruited, resident/interstitial, and proliferative airspace), 2 T-cells (differentiating and effector), and a single cluster each of inflammatory monocytes, dendritic cells, B-cells and natural killer cells. Inflammatory monocytes, autoreactive/suppressor neutrophils, and recruited/differentiating macrophages were predominant with arthritis induction (CIA and CIA+ODE). By specific lung cell isolation, several interferon-related and autoimmune genes were disproportionately expressed among CIA and CIA+ODE (e.g. Oasl1, Oas2, Ifit3, Gbp2, Ifi44, and Zbp1), corresponding to RA and RA-associated lung disease. Monocytic myeloid-derived suppressor cells were reduced, while complement genes (e.g. C1s1 and Cfb) were uniquely increased in CIA+ODE mice across cell populations. Recruited and inflammatory macrophages/monocytes and neutrophils expressing interferon-, autoimmune-, and complement-related genes might contribute towards pro-fibrotic inflammatory lung responses following airborne biohazard exposures in setting of autoimmune arthritis and could be predictive and/or targeted to reduce disease burden.

Summary: Ependymoma (EPN) is a brain tumor commonly presenting in childhood that remains fatal in most children. Intra-tumoral cellular heterogeneity in bulk-tumor samples significantly confounds our understanding of EPN biology, impeding development of effective therapy. We, therefore, use single-cell RNA sequencing, histology, and deconvolution to catalog cellular heterogeneity of the major childhood EPN subgroups. Analysis of PFA subgroup EPN reveals evidence of an undifferentiated progenitor subpopulation that either differentiates into subpopulations with ependymal cell characteristics or transitions into a mesenchymal subpopulation. Histological analysis reveals that progenitor and mesenchymal subpopulations co-localize in peri-necrotic zones. In conflict with current classification paradigms, relative PFA subpopulation proportions are shown to determine bulk-tumor-assigned subgroups. We provide an interactive online resource that facilitates exploration of the EPN single-cell dataset. This atlas of EPN cellular heterogeneity increases understanding of EPN biology.

Objective: The aim of this research is to present a novel computer-aided decision support tool in analyzing, quantifying, and evaluating the retinal blood vessel structure from fluorescein angiogram (FA) videos.Methods: The proposed method consists of three phases: (i) image registration for large motion removal from fluorescein angiogram videos, followed by (ii) retinal vessel segmentation, and lastly, (iii) segmentation-guided video magnification. In the image registration phase, individual frames of the video are spatiotemporally aligned using a novel wavelet-based registration approach to compensate for the global camera and patient motion. In the second phase, a capsule-based neural network architecture is employed to perform the segmentation of retinal vessels for the first time in the literature. In the final phase, a segmentation-guided Eulerian video magnification is proposed for magnifying subtle changes in the retinal video produced by blood flow through the retinal vessels. The magnification is applied only to the segmented vessels, as determined by the capsule network. This minimizes the high levels of noise present in these videos and maximizes useful information, enabling ophthalmologists to more easily identify potential regions of pathology.Results: The collected fluorescein angiogram video dataset consists of 1, 402 frames from 10 normal subjects (prospective study). Experimental results for retinal vessel segmentation show that the capsule-based algorithm outperforms a state-of-the-art convolutional neural networks (U-Net), obtaining a higher dice coefficient (85.94%) and sensitivity (92.36%) while using just 5% of the network parameters. Qualitative analysis of these videos was performed after the final phase by expert ophthalmologists, supporting the claim that artificial intelligence assisted decision support tool can be helpful for providing a better analysis of blood flow dynamics.Conclusions: The authors introduce a novel computational tool, combining a wavelet-based video registration method with a deep learning capsule-based retinal vessel segmentation algorithm and a Eulerian video magnification technique to quantitatively and qualitatively analyze FA videos. To authors' best knowledge, this is the first-ever development of such a computational tool to assist ophthalmologists with analyzing blood flow in FA videos.

Assignment of cell types from single-cell RNA sequencing (scRNA-seq) data remains a time-consuming and error-prone process. Current packages for identity assignment use limited types of reference data and often have rigid data structure requirements. We developed the clustifyr R package to leverage several external data types, including gene expression profiles to assign likely cell types using data from scRNA-seq, bulk RNA-seq, microarray expression data, or signature gene lists. We benchmark various parameters of a correlation-based approach and implement gene list enrichment methods. clustifyr is a lightweight and effective cell-type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr