Therapeutic Ligands Antagonize Estrogen Receptor Function by Impairing Its Mobility.

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.

ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.

Coordination between Two Branches of the Unfolded Protein Response Determines Apoptotic Cell Fate.

The kinases PERK and IRE1 alleviate endoplasmic reticulum (ER) stress by orchestrating the unfolded protein response (UPR). If stress mitigation fails, PERK promotes cell death by activating pro-apoptotic genes, including death receptor 5 (DR5). Conversely, IRE1-which harbors both kinase and endoribonuclease (RNase) modules-blocks apoptosis through regulated IRE1-dependent decay (RIDD) of DR5 mRNA. Under irresolvable ER stress, PERK activity persists, whereas IRE1 paradoxically attenuates, by mechanisms that remain obscure. Here, we report that PERK governs IRE1's attenuation through a phosphatase known as RPAP2 (RNA polymerase II-associated protein 2). RPAP2 reverses IRE1 phosphorylation, oligomerization, and RNase activation. This inhibits IRE1-mediated adaptive events, including activation of the cytoprotective transcription factor XBP1s, and ER-associated degradation of unfolded proteins. Furthermore, RIDD termination by RPAP2 unleashes DR5-mediated caspase activation and drives cell death. Thus, PERK attenuates IRE1 via RPAP2 to abort failed ER-stress adaptation and trigger apoptosis.

Prevalence and Predictors of Barrett's Esophagus After Negative Initial Endoscopy: Analysis From Two National Databases.

BACKGROUND & AIMS: Guidelines suggest a single screening esophagogastroduodenoscopy (EGD) in patients with multiple risk factors for Barrett's esophagus (BE). We aimed to determine BE prevalence and predictors on repeat EGD after a negative initial EGD, using 2 large national databases (GI Quality Improvement Consortium [GIQuIC] and TriNetX). METHODS: Patients who underwent at least 2 EGDs were included and those with BE or esophageal adenocarcinoma detected at initial EGD were excluded. Patient demographics and prevalence of BE on repeat EGD were collected. Multivariate logistic regression was performed to assess for independent risk factors for BE detected on the repeat EGD. RESULTS: In 214,318 and 153,445 patients undergoing at least 2 EGDs over a median follow-up of 28-35 months, the prevalence of BE on repeat EGD was 1.7% in GIQuIC and 3.4% in TriNetX, respectively (26%-45% of baseline BE prevalence). Most (89%) patients had nondysplastic BE. The prevalence of BE remained stable over time (from 1 to >5 years from negative initial EGD) but increased with increasing number of risk factors. BE prevalence in a high-risk population (gastroesophageal reflux disease plus ≥1 risk factor for BE) was 3%-4%. CONCLUSIONS: In this study of >350,000 patients, rates of BE on repeat EGD ranged from 1.7%-3.4%, and were higher in those with multiple risk factors. Most were likely missed at initial evaluation, underscoring the importance of a high-quality initial endoscopic examination. Although routine repeat endoscopic BE screening after a negative initial examination is not recommended, repeat screening may be considered in carefully selected patients with gastroesophageal reflux disease and ≥2 risk factors for BE, potentially using nonendoscopic tools.

The Role of Intramolecular Reactions and Chemical Degradation in the Apparent Biotransformation Pathways of a Series of SYK Inhibitors.

In vitro metabolism studies of the spleen tyrosine kinase inhibitors AZ-A and AZ-B identified four unusual metabolites. M1 (mass-to-charge ratio 411) was formed by both molecules and was common to several analogs (AZ-C to AZ-H) sharing the same core structure, appearing to derive from the complete loss of a pendent 3,4-diaminotetrahydropyran ring and pyrazole ring cleavage resulting in a nonobvious metabolite. M2-M4 were formed by AZ-A and a subset of the other compounds only and apparently resulted from a sequential loss of H2 from parent. Initial attempts to isolate M3 for identification were unsuccessful due to sample degradation, and it was subsequently found that M2 and M3 underwent sequential chemical degradation steps to M4. M4 was successfully isolated and shown by mass spectrometry and NMR spectroscopy to be a tricyclic species incorporating the pyrazole and the 3,4-diaminotetrahydropyran groups. We propose that this arises from an intramolecular reaction between the primary amine on the tetrahydropyran and a putative epoxide intermediate on the adjacent pyrazole ring, evidence for which was generated in a ß-mercaptoethanol-trapping experiment. The loss of the tetrahydropyran moiety observed in M1 was found to be enhanced in an analog that was unable to undergo the intramolecular reaction step, leading us to propose two possible reaction pathways originating from the reactive intermediate. Ultimately, we conclude that the apparently complex and unusual metabolism of this series of compounds likely resulted from a single metabolic activation step forming an epoxide intermediate, which subsequently underwent intramolecular rearrangement and/or chemical degradation to form the final observed products. SIGNIFICANCE STATEMENT: The current work provides an unusual biotransformation example showing the potential for intramolecular reactions and chemical degradation to give the appearance of complex metabolism arising from a single primary route of metabolism.

Risk Factors for Chronic Atrial Fibrillation Following Lung Lobectomy.

INTRODUCTION: Postoperative atrial fibrillation (POAF) is a common complication following lung lobectomy and is associated with increased risk of stroke, mortality, and prolonged hospital length of stay. The purpose of this study was to define the risk factors for POAF after lobectomy, hypothesizing that operative approach would be associated with risk of chronic POAF. METHODS: The TriNetX database was used to identify adult patients with no history of arrythmia receiving elective lung lobectomy for cancer from 7/6/2003-7/6/2023. Patients were categorized by approach: video-assisted thoracoscopic surgery (VATS) or open. The outcome of interest was the presence of POAF occurring at 1-3 months ("early") and 12-24 months postop ("chronic"). Propensity matching was performed to reduce bias between cohorts. RESULTS: We identified 22,998 patients: 8472 (36.8%) who received open and 14,526 (63.2%) VATS lobectomy. The rate of early POAF was 3.7% of VATS and 5.3% of open patients. The rate of chronic POAF was 5.5 % of VATS patients and 6.2% of open lobectomy patients. Propensity matching decreased bias between the approach groups, creating 7942 pairs for analysis. After matching, the risk of early POAF was greater in the open approach (5.5% open vs 3.4% VATS, risk ratio 1.607 (95% confidence interval 1.385-1.865), P < 0.001). Chronic POAF was (also) higher in the open approach (6.3% open vs 5.2% VATS, Risk Ratio 1.211 (95%CI 1.067-1.374), P = 0.003). CONCLUSIONS: Postoperative atrial fibrillation (POAF) occurs more commonly after open lobectomy, both acutely and chronically. Providers should counsel patients about the risk of chronic arrythmia after lung resection.

PharmacoDB 2.0: improving scalability and transparency of in vitro pharmacogenomics analysis.

Cancer pharmacogenomics studies provide valuable insights into disease progression and associations between genomic features and drug response. PharmacoDB integrates multiple cancer pharmacogenomics datasets profiling approved and investigational drugs across cell lines from diverse tissue types. The web-application enables users to efficiently navigate across datasets, view and compare drug dose-response data for a specific drug-cell line pair. In the new version of PharmacoDB (version 2.0, https://pharmacodb.ca/), we present (i) new datasets such as NCI-60, the Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) dataset, as well as updated data from the Genomics of Drug Sensitivity in Cancer (GDSC) and the Genentech Cell Line Screening Initiative (gCSI); (ii) implementation of FAIR data pipelines using ORCESTRA and PharmacoDI; (iii) enhancements to drug-response analysis such as tissue distribution of dose-response metrics and biomarker analysis; and (iv) improved connectivity to drug and cell line databases in the community. The web interface has been rewritten using a modern technology stack to ensure scalability and standardization to accommodate growing pharmacogenomics datasets. PharmacoDB 2.0 is a valuable tool for mining pharmacogenomics datasets, comparing and assessing drug-response phenotypes of cancer models.

The Disconnect in Intrinsic Clearance Determined in Human Hepatocytes and Liver Microsomes Results from Divergent Cytochrome P450 Activities.

Candidate drugs may exhibit higher unbound intrinsic clearances (CLint,u) in human liver microsomes (HLMs) relative to human hepatocytes (HHs), posing a challenge as to which value is more predictive of in vivo clearance (CL). This work was aimed at better understanding the mechanism(s) underlying this 'HLM:HH disconnect' via examination of previous explanations, including passive permeability limited CL or cofactor exhaustion in hepatocytes. A series of structurally related, passively permeable (Papps > 5 × 10-6 cm/s), 5-azaquinazolines were studied in different liver fractions, and metabolic rates and routes were determined. A subset of these compounds demonstrated a significant HLM:HH (CLint,u ratio 2-26) disconnect. Compounds were metabolized via combinations of liver cytosol aldehyde oxidase (AO), microsomal cytochrome P450 (CYP) and flavin monooxygenase (FMO). For this series, the lack of concordance between CLint,u determined in HLM and HH contrasted with an excellent correlation of AO dependent CLint,u determined in human liver cytosol[Formula: see text], r2 = 0.95, P < 0.0001). The HLM:HH disconnect for both 5-azaquinazolines and midazolam was as a result of significantly higher CYP activity in HLM and lysed HH fortified with exogenous NADPH relative to intact HH. Moreover, for the 5-azaquinazolines, the maintenance of cytosolic AO and NADPH-dependent FMO activity in HH, relative to CYP, supports the conclusion that neither substrate permeability nor intracellular NADPH for hepatocytes were limiting CLint,u Further studies are required to identify the underlying cause of the lower CYP activities in HH relative to HLM and lysed hepatocytes in the presence of exogenous NADPH. SIGNIFICANCE STATEMENT: Candidate drugs may exhibit higher intrinsic clearance in human liver microsomes relative to human hepatocytes, posing a challenge as to which value is predictive of in vivo clearance. This work demonstrates that the difference in activity determined in liver fractions results from divergent cytochrome P450 but not aldehyde oxidase or flavin monooxygenase activity. This is inconsistent with explanations including substrate permeability limitations or cofactor exhaustion and should inform the focus of further studies to understand this cytochrome P450 specific disconnect phenomenon.

Targeting MCL-1 and BCL-2 with polatuzumab vedotin and venetoclax overcomes treatment resistance in R/R non-Hodgkin lymphoma: Results from preclinical models and a Phase Ib study.

The treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge. Here, we identify the pro-survival BCL-2 protein family member MCL-1 as a resistance factor for the BCL-2 inhibitor venetoclax in non-Hodgkin lymphoma (NHL) cell lines and primary NHL samples. Mechanistically, we show that the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system. This targeted MCL-1 antagonism, when combined with venetoclax and the anti-CD20 antibodies obinutuzumab or rituximab, results in tumor regressions in preclinical NHL models, which are sustained even off-treatment. In a Phase Ib clinical trial (NCT02611323) of heavily pre-treated patients with relapsed or refractory NHL, 25/33 (76%) patients with follicular lymphoma and 5/17 (29%) patients with diffuse large B-cell lymphoma achieved complete or partial responses with an acceptable safety profile when treated with the recommended Phase II dose of polatuzumab vedotin in combination with venetoclax and an anti-CD20 antibody.

Inferring population connectivity in eastern massasauga rattlesnakes (Sistrurus catenatus) using landscape genetics.

Assessing the environmental factors that influence the ability of a threatened species to move through a landscape can be used to identify conservation actions that connect isolated populations. However, direct observations of species' movement are often limited, making the development of alternate approaches necessary. Here we use landscape genetic analyses to assess the impact of landscape features on the movement of individuals between local populations of a threatened snake, the eastern massasauga rattlesnake (Sistrurus catenatus). We linked connectivity data with habitat information from two landscapes of similar size: a large region of unfragmented habitat and a previously studied fragmented landscape consisting of isolated patches of habitat. We used this analysis to identify features of the landscape where modification or acquisition would enhance population connectivity in the fragmented region. We found evidence that current connectivity was impacted by both contemporary land-cover features, especially roads, and inherent landscape features such as elevation. Next, we derived estimates of expected movement ability using a recently developed pedigree-based approach and least-cost paths through the unfragmented landscape. We then used our pedigree and resistance map to estimate resistance polygons of the potential extent for S. catenatus movement in the fragmented landscape. These polygons identify possible sites for future corridors connecting currently isolated populations in this landscape by linking the impact of future habitat modification or land acquisition to dispersal ability in this species. Overall, our study shows how modeling landscape resistance across differently fragmented landscapes can identify habitat features that affect contemporary movement in threatened species in fragmented landscapes and how this information can be used to guide mitigation actions whose goal is to connect isolated populations.

USP7 small-molecule inhibitors interfere with ubiquitin binding.

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Preoperative Paralabral Cysts Predict More Significant Chondral Damage but Not 2-Year Functional Outcomes Following Arthroscopic Labral Repair.

PURPOSE: To investigate whether paralabral cysts identified incidentally on preoperative magnetic resonance imaging (MRI/MRA) predict 2-year functional outcomes after arthroscopic acetabular labral repair. METHODS: Prospectively collected data for patients undergoing primary hip arthroscopy by a single surgeon from 2014 to 2020 were retrospectively reviewed. Included patients were ≥18 years and completed baseline patient-reported outcome measures (PROMs) with additional follow-up at 3, 6, 12, and 24 months. Exclusion criteria were labral debridement, hip dysplasia, advanced hip osteoarthritis (Tönnis >1), or previous ipsilateral hip surgery. Patients were stratified based on the presence of paralabral cysts identified on MRI/MRA. Primary outcomes were International Hip Outcome Tool (iHOT-33) and modified Harris Hip Score (mHHS). Secondary outcomes included other PROMs and the visual analog pain scale. Outcomes were compared between cohorts using linear mixed-effects models and Fisher's exact tests. Sensitivity analyses accounted for preoperative PROMs, nonlinear improvement trajectories, and relevant baseline characteristics. RESULTS: Of the 182 included hips (47.8% female; mean ± standard deviation age, 36.9 ± 11.4), 30 (16.4%) had paralabral cysts. During the 2-year study period, there were no significant differences between patients with and without paralabral cysts in terms of iHOT-33 scores (weighted difference = 1.60; 95% confidence interval [CI], -5.09, 8.28; P = .64), mHHS scores (weighted difference = 0.56; 95% CI, -4.16, 5.28; P = .82), or any secondary outcomes (except for HOS-Sports Subscale at 3 months [mean difference = -11.85; 95% CI, -22.85, -0.84; P = .035]). Furthermore, there were no significant differences in clinically meaningful outcomes (P > .05 for all), revision rates (P = 1.00), or conversion to total hip arthroplasty between cohorts (P = 1.00). These results held across all sensitivity analyses. CONCLUSIONS: Although preoperative paralabral cysts were associated with worse cam impingement and more severe chondral damage observed intraoperatively, they did not predict 2-year functional outcomes or clinically meaningful improvements, suggesting that incidentally discovered paralabral cysts are not a contraindication for arthroscopic labral repair. LEVEL OF EVIDENCE: Level III, retrospective cohort study.

Operative management of rotator cuff tears: identifying disparities in access on a national level.

BACKGROUND: The purpose of this study was to identify nationwide disparities in the rates of operative management of rotator cuff tears based on race, ethnicity, insurance type, and socioeconomic status. METHODS: Patients diagnosed with a full or partial rotator cuff tear from 2006 to 2014 were identified in the Healthcare Cost and Utilization Project's National Inpatient Sample database using International Classification of Diseases, Ninth Revision diagnosis codes. Bivariate analysis using chi-square tests and adjusted, multivariable logistic regression models were used to evaluate differences in the rates of operative vs. nonoperative management for rotator cuff tears. RESULTS: This study included 46,167 patients. When compared with white patients, adjusted analysis showed that minority race and ethnicity were associated with lower rates of operative management for Black (adjusted odds ratio [AOR]: 0.31, 95% confidence interval [CI]: 0.29-0.33; P < .001), Hispanic (AOR: 0.49, 95% CI: 0.45-0.52; P < .001), Asian or Pacific Islander (AOR: 0.72, 95% CI: 0.61-0.84; P < .001), and Native American patients (AOR: 0.65, 95% CI: 0.50-0.86; P = .002). In comparison to privately insured patients, our analysis also found that self-payers (AOR: 0.08, 95% CI: 0.07-0.10; P < .001), Medicare beneficiaries (AOR: 0.76, 95% CI: 0.72-0.81; P < .001), and Medicaid beneficiaries (AOR: 0.33, 95% CI: 0.30-0.36; P < .001) had lower odds of receiving surgical intervention. Additionally, relative to those in the bottom income quartile, patients in all other quartiles experienced nominally higher rates of operative repair; these differences were statistically significant for the second quartile (AOR: 1.09, 95% CI: 1.03-1.16; P = .004). CONCLUSION: There are significant nationwide disparities in the likelihood of receiving operative management for rotator cuff tear patients of differing race/ethnicity, payer status, and socioeconomic status. Further investigation is needed to fully understand and address causes of these discrepancies to optimize care pathways.

Reduction of Doppler and Range Ambiguity Using AES-192 Encryption-Based Pulse Coding.

This research investigates the use of a Binary Phase Shift Key (BPSK) sequence derived from the 192-bit key Advanced Encryption Standard (AES-192) algorithm for radar signal modulation to mitigate Doppler and range ambiguities. The AES-192 BPSK sequence has a non-periodic nature resulting in a single large and narrow main lobe in the matched filter response but also produces undesired periodic side lobes that can be mitigated through the use of a CLEAN algorithm. The performance of the AES-192 BPSK sequence is compared to an Ipatov-Barker Hybrid BPSK code, which effectively extends the maximum unambiguous range but has some limitations in terms of signal processing requirements. The AES-192 based BPSK sequence has the advantage of having no maximum unambiguous range limit, and when the pulse location within the Pulse Repetition Interval (PRI) is randomized, the upper limit on the maximum unambiguous Doppler frequency shift is greatly extended.

Drift-Free Joint Angle Calculation Using Inertial Measurement Units without Magnetometers: An Exploration of Sensor Fusion Methods for the Elbow and Wrist.

Joint angles of the lower extremities have been calculated using gyroscope and accelerometer measurements from inertial measurement units (IMUs) without sensor drift by leveraging kinematic constraints. However, it is unknown whether these methods are generalizable to the upper extremity due to differences in motion dynamics. Furthermore, the extent that post-processed sensor fusion algorithms can improve measurement accuracy relative to more commonly used Kalman filter-based methods remains unknown. This study calculated the elbow and wrist joint angles of 13 participants performing a simple ≥30 min material transfer task at three rates (slow, medium, fast) using IMUs and kinematic constraints. The best-performing sensor fusion algorithm produced total root mean square errors (i.e., encompassing all three motion planes) of 6.6°, 3.6°, and 2.0° for the slow, medium, and fast transfer rates for the elbow and 2.2°, 1.7°, and 1.5° for the wrist, respectively.

Evaluation of statistical approaches for association testing in noisy drug screening data.

BACKGROUND: Identifying associations among biological variables is a major challenge in modern quantitative biological research, particularly given the systemic and statistical noise endemic to biological systems. Drug sensitivity data has proven to be a particularly challenging field for identifying associations to inform patient treatment. RESULTS: To address this, we introduce two semi-parametric variations on the commonly used concordance index: the robust concordance index and the kernelized concordance index (rCI, kCI), which incorporate measurements about the noise distribution from the data. We demonstrate that common statistical tests applied to the concordance index and its variations fail to control for false positives, and introduce efficient implementations to compute p-values using adaptive permutation testing. We then evaluate the statistical power of these coefficients under simulation and compare with Pearson and Spearman correlation coefficients. Finally, we evaluate the various statistics in matching drugs across pharmacogenomic datasets. CONCLUSIONS: We observe that the rCI and kCI are better powered than the concordance index in simulation and show some improvement on real data. Surprisingly, we observe that the Pearson correlation was the most robust to measurement noise among the different metrics.

Reproducible pharmacogenomic profiling of cancer cell line panels.

The use of large-scale genomic and drug response screening of cancer cell lines depends crucially on the reproducibility of results. Here we consider two previously published screens, plus a later critique of these studies. Using independent data, we show that consistency is achievable, and provide a systematic description of the best laboratory and analysis practices for future studies.

Supraspinatus pathology on MRI is associated with degree of weakness on dynamic clinical strength testing.

OBJECTIVE: To analyze shoulder strength and function in patients presenting with possible supraspinatus pathology and to ascertain if these clinical findings are associated with severity of supraspinatus pathology on MRI. MATERIALS AND METHODS: In total, 171 patients with presumptive rotator cuff pathology and with preserved strength on standard rotator cuff examination were prospectively recruited. Patients were subjected to bilateral shoulder strength testing employing dynamometry; this included isometric strength testing at 90° of abduction, followed by eccentric assessment of isotonic strength from full abduction through the full range of motion until the arm rested at the patient's side. We calculated absolute strength and symptomatic-to-asymptomatic arm (S/A) strength ratios. On subsequent shoulder MRI, supraspinatus pathology was designated into one of seven categories. The association between strength measurements and MRI findings was analyzed. RESULTS: Increasing lesion severity on MRI was associated with both decreasing absolute strength (no tear [59.9 N] to full-thickness tear [44.2 N]; P = 0.036) and decreasing S/A strength ratios during isotonic testing (no tear [91.9%] to full-thickness tear [65.3%]; P = 0.022). In contrast, there were no significant relationships between imaging severity and absolute strength or S/A strength ratios on isometric testing. CONCLUSION: Severity of supraspinatus pathology on MRI was associated with dynamic clinical function. These results validate the clinical correlation between MRI designations of supraspinatus pathology and function and suggest the need for future work to investigate utility of dynamic (versus isometric) rotator cuff physical examination maneuvers.

Accelerated versus standard physical therapy in patients with transtendinous rotator cuff repair: a propensity-matched cohort study.

BACKGROUND: Partial-thickness rotator cuff tears that remain symptomatic despite conservative management can be repaired operatively through a transtendinous approach. Although transtendinous repairs have been linked to superior long-term biomechanical outcomes compared with other surgical techniques, they are classically associated with early postoperative stiffness and a slower rate of recovery. PURPOSE: To examine the impact of expediting the physical therapy (PT) regimen after transtendinous repair on postoperative range of motion and complications. METHODS: The first 61 patients to receive accelerated PT after transtendinous repair were compared with a historical cohort of 61 patients who underwent standard postoperative management. The patients were propensity matched on age, sex, smoking status, and biceps procedure performed at the time of rotator cuff repair. Primary outcome measures included active range of motion (AROM) in forward flexion, abduction, external rotation, and internal rotation at 2 weeks, 6 weeks, 3 months, and 6 months postoperatively. Secondary outcome measures included development of severe stiffness or symptomatic rotator cuff retear at 1-year follow-up. Patients with full-thickness tears and those undergoing revision surgery or tear-completion and repair were excluded. RESULTS: The accelerated PT cohort showed significantly increased AROM at 6 weeks and 3 months postoperatively. At 6 weeks, AROM in forward flexion (137.0° vs. 114.9°; P < .001), abduction (126.1° vs. 105.3°; P = .009), and external rotation (51.7° vs. 36.5°; P = .005) were all significantly higher in the accelerated PT cohort. A similar increase was seen at 3 months, with superior forward flexion (147.5° vs. 134.0°; P = .01) and external rotation (57.7° vs. 44.0°; P = .008) in patients who received accelerated PT. Severe postoperative stiffness was significantly less common in the accelerated PT cohort (3.3% vs. 18.0%; P = .02), and there were no symptomatic retears (0.00%) in the accelerated PT cohort as compared with 1 symptomatic retear (1.64%) in the standard PT cohort (P = 1.00). CONCLUSION: Accelerated PT after transtendinous rotator cuff repair is associated with significant improvement in AROM at 6 weeks and 3 months postoperatively. Further, the early motion may help obviate the development of severe postoperative stiffness without any evidence of higher rotator cuff retear rates. LEVEL OF EVIDENCE: Level III; Retrospective Cohort Comparison; Treatment Study.

The alpha-1 subunit of the Na+,K+-ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells.

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug.