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Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its cat

The U.S. Department of Health and Human Services obtains information about U.S. registered nurses through the periodic National Sample Survey of Registered Nurses (NSSRN). Occupational health nurses comprise less than 1% of the U.S. nursing population and published NSSRN reports usually include only estimates of the total occupational health nurse population and minimal information about occupational health nurses' characteristics. The objectives of this study were to develop a knowledge base of occupational health nurses' characteristics; examine characteristics that may influence entry and retention in occupational health nursing practice; and explore indications of demand for occupational health nurses. Descriptive and inferential statistics were used in a secondary analysis of data from recent (1992 to 2004) NSSRN. The findings are reported in two parts. This article, Part I, provides descriptive data about occupational health nurses based on responses to the 1992 through 2004 NSSRN questionnaires. Part II will provide findings from analysis of 2004 responses indicative of occupational health nurses' entry, retention, and demand characteristics. [ABSTRACT FROM AUTHOR]

Occupational health nurses are challenged by the complexity of the laws and regulations governing their practice. Establishing a practice-based regulatory framework and applying an efficient review process simplifies that challenge. For those interested in expanding this framework beyond laws and regulations, a number of agencies and organizations that establish standards and recommendations relevant to the occupational health setting exist, such as the Centers for Disease Control and Prevention (www.cdc.gov), the National Institute for Occupational Safety and Health (www.cdc.gov/niosh), and the American Conference of Governmental Industrial Hygienists (www.acgih.org). Materials published by these agencies (e.g., the CDC 'Adult Immunization Schedule;' NIOSH 'Recommendations for Protecting Outdoor Workers from West Nile;' American Conference of Governmental Industrial Hygienists 'Guide to Occupational Exposure Values,' 'TLVs,' and 'BEIs') are often of value to occupational health nurses and worth their attention. [ABSTRACT FROM AUTHOR]

The objective of the study was to identify factors that affected the implementation of an inpatient case management program in rural hospitals. The hospitals studied were from the Western New York Rural Health Care Cooperative. Five of the hospitals implemented the program in 1992. A qualitative evaluation was conducted by analyzing tape-recorded interviews with nurses and chief executive officers to identify obstacles to and facilitators of program implementation. Many obstacles to implementation could be traced to workload and time constraints, physician autonomy concerns, and limited nursing staff and physician participation. Implementation was facilitated foremost by the effort and supportive attitudes of nursing leaders and hospital chief executive officers. This study concluded that it should be possible to successfully implement conceptually sound managed care and case management programs in rural hospitals, but it will require a relatively long period of support, especially from hospital administration and nursing leaders. [ABSTRACT FROM AUTHOR]

Bow hunter's syndrome (BHS) is a rare condition resulting from vertebrobasilar insufficiency secondary to mechanical occlusion or stenosis of the vertebral artery (VA) due to head rotation. Traditionally, surgical intervention with C1-C2 fusion or VA decompression was the mainstay of therapy. Endovascular intervention was rarely performed to treat BHS. We reviewed the neurointerventional database from July 2005 to October 2010 to identify all cases of BHS treated with VA stenting. Here we report clinical, technical, and outcome data for 4 patients with BHS who were treated with VA stenting. In all 4 of these patients, stenting was performed in the V2 segment (C2-C6) of the VA without significant technical difficulties. All patients reported symptomatic relief, and only minor or no residual stenosis was detected by dynamic digital subtraction angiography. Our findings indicate that VA stenting for the treatment of BHS is feasible, safe, and clinically effective. Endovascular techniques might offer an alternative, minimally invasive therapy for the treatment of BHS. [ABSTRACT FROM AUTHOR]

Bruton tyrosine kinase inhibitors (BTKi) have transformed the treatment of B-cell malignancies, but intolerance has often led to their discontinuation. The phase 1/2 BRUIN study evaluated pirtobrutinib, a highly selective non-covalent (reversible) BTKi, in patients with R/R B-cell malignancies (NCT03740529). Pirtobrutinib was investigated in 127 patients with intolerance to at least one prior BTKi therapy in the absence of progressive disease. The most common adverse event (AE) leading to BTKi discontinuation was cardiac disorders (n=40, 31.5%), specifically atrial fibrillation (n=30, 23.6%). The median follow-up was 17.4 months and the median time on pirtobrutinib was 15.3 months. The most common reasons for pirtobrutinib discontinuation were progressive disease (26.8%), AE (10.2%), or death (5.5%). The most frequent treatment-emergent AEs were fatigue (39.4%) and neutropenia (37.0%). Among patients who discontinued a prior BTKi for a cardiac issue, 75% had no recurrence of their cardiac AE. No patient discontinued pirtobrutinib for the same AE that led to discontinuation of the prior BTKi. In 78 CLL/SLL and 21 MCL patients intolerant to prior BTKi, ORR to pirtobrutinib was 76.9% and 81.0%, respectively. Median PFS for CLL/SLL was 28.4 months and was not estimable for MCL. These results suggest that pirtobrutinib was safe, well-tolerated, and an efficacious option in patients with prior BTKi-intolerance.

Introduction: Patient self-scheduling of medical appointments is becoming more common in many medical institutions. However, the complexity of scheduling multiple specialties, following scheduling guidelines, and managing appointment access requires a variety of processes for a diverse inventory of self-schedulable appointment types., Methods: From 7 unique patient self-scheduling methods, we captured counts of successfully self-scheduled and completed appointments. A process map was created to show the paths of 5 different primary self-scheduling processes (new appointment self-scheduling) and 2 secondary self-scheduling processes (existing appointment self-rescheduling)., Results: There were 7 unique processes that led to 733,651 successfully self-scheduled completed visits from January 1 to December 31, 2023 at a multisite, multispecialty clinic. The self-scheduling processes consisted of the following: (1) Ticket offer (appointment "ticket" offers for specific visits generated by a provider order or system rules), the software "ticket" sent to the patient permits "admission" to self-schedule calendar templates (341,591 uses, 46.6%); (2) direct self-scheduled visit for prequalified visit types (203,593 uses, 27.6%); (3) self-reschedule option (patient option to reschedule existing appointment, 79,706 uses, 10.9%); (4) new patient self-scheduled visit via clinic website (does not require portal access, 54,367 uses, 7.4%). (5) automated waitlist self-rescheduled visit (38,649 uses, 5.3%); (6) automated waitlist self-scheduled visit of previously unscheduled visit (10,939 uses, 1.5%); and (7) self-triage self-scheduled visit (4806 uses, 0.7%)., Conclusion: The processes for self-scheduling are expanding. Our multispecialty clinic has implemented 7 different processes to help patients successfully self-schedule medical appointments. Some of the processes occur before initial scheduling (such as self-triage), and some are implemented after successful scheduling has already occurred (self-rescheduling option and self-rescheduling aided by an automated waitlist). Continued research is needed to look for measures of success beyond the ability to complete a self-scheduled visit, including the accuracy of the booking (right provider, location, and length of visit)., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

Venetoclax is a BCL2 inhibitor used in chronic lymphocytic leukemia (CLL) which can cause tumor lysis syndrome (TLS). We aimed to determine the incidence of and risk factors for TLS among patients with CLL/small lymphocytic lymphoma (SLL) who received treatment with venetoclax at our institution from 1/1/2016 to 12/31/2020. We included 616 venetoclax escalations among 136 pts with CLL. 74 pts (54%) underwent escalation exclusively outpatient, 35 (26%) had at least one planned hospitalization and 27 (20%) were escalated exclusively inpatient. During venetoclax initiation, 86% of pts received allopurinol, 71% intravenous hydration, 18% phosphate binders, and 10% prophylactic rasburicase. Among the entire cohort, 7 pts (5.1%) developed laboratory TLS by modified Cairo Bishop criteria and none developed clinical TLS. Incidence of laboratory TLS was 15% for those escalated exclusively inpatient, 2.9% for those with any prophylactic hospitalization and 2.7% for those escalated exclusively outpatient. Those who developed TLS were more likely to have higher TLS risk, and no additional risk factors were identified. In this single institution retrospective cohort study, laboratory TLS was observed, though clinical TLS was not. Prophylactic measures, including use of IV hydration, may have contributed to low rates of observed TLS in the outpatient setting., (Copyright © 2024 American Society of Hematology.)

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation., Competing Interests: RD, AE, GC, PA, IY, BF, MT, IS No competing interests declared, SV, RL S.J.V.D. and R.M.L. are listed as inventors on a patent United States application: 17/505,561 for the use of chitinases to treat fibrotic lung disease. S.J.V.D., R.M.L., and J.S.F. are listed as inventors on a patent for mutant chitinases with enhanced expression and activity, JF S.J.V.D. and R.M.L. are listed as inventors on a patent for the use of chitinases to treat fibrotic lung disease. United States application: 17/505,561 S.J.V.D., R.M.L., and J.S.F. are listed as inventors on a patent for mutant chitinases with enhanced expression and activity, (© 2023, Díaz et al.)

Introduction: Pirtobrutinib, a highly selective, noncovalent (reversible) Bruton tyrosine kinase inhibitor, has demonstrated promising efficacy in B-cell malignancies and is associated with low rates of discontinuation and dose reduction. Pirtobrutinib is administered until disease progression or toxicity, necessitating an understanding of the safety profile in patients with extended treatment., Methods: Here we report the safety of pirtobrutinib in patients with relapsed/refractory B-cell malignancies with extended (≥12 months) drug exposure from the BRUIN trial. Assessments included median time-to-first-occurrence of adverse events (AEs), dose reductions, and discontinuations due to treatment-emergent AEs (TEAEs) and select AEs of interest (AESIs)., Results: Of 773 patients enrolled, 326 (42%) received treatment for ≥12 months. In the extended exposure cohort, the median time-on-treatment was 19 months. The most common all-cause TEAEs were fatigue (32%) and diarrhea (31%). TEAEs leading to dose reduction occurred in 23 (7%) and discontinuations in 11 (3%) extended exposure patients. One patient had a fatal treatment-related AE (COVID-19 pneumonia). Infections (73.0%) were the most common AESI with a median time-to-first-occurrence of 7.4 months. Majority of TEAEs and AESIs occurred during the first year of therapy., Conclusions: Pirtobrutinib therapy continues to demonstrate an excellent safety profile amenable to long-term administration without evidence of new or worsening toxicity signals., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Systematic analysis of molecular recognition is critical for understanding the biological function of macromolecules. For the immunomodulatory protein D-dopachrome tautomerase (D-DT), the mechanism of protein-ligand interactions is poorly understood. Here, 17 carefully designed protein variants and wild type (WT) D-DT were interrogated with an array of complementary techniques to elucidate the structural basis of ligand recognition. Utilization of a substrate and two selective inhibitors with distinct binding profiles offered previously unseen mechanistic insights into D-DT-ligand interactions. Our results demonstrate that the C-terminal region serves a key role in molecular recognition via regulation of the active site opening, protein-ligand interactions, and conformational flexibility of the pocket's environment. While our study is the first comprehensive analysis of molecular recognition for D-DT, the findings reported herein promote the understanding of protein functionality and enable the design of new structure-based drug discovery projects.

Background: Self-scheduling of medical visits is becoming more common but the complexity of applying multiple requirements for self-scheduling has hampered implementation. Mayo Clinic implemented self-scheduling in 2019 and has been increasing its portfolio of self-schedulable visits since then. Our aim was to show measures quantifying the complexity associated with medical visit scheduling and to describe how opportunities and challenges of scheduling complexity apply in self-scheduling., Methods: We examined scheduled visits from January 1, 2022, through August 24, 2023. For seven visit categories, we counted all unique visit types that were scheduled, for both staff-scheduled and self-scheduled. We examined counts of self-scheduled visit types to identify those with highest uptake during the study period., Results: There were 9555 unique visit types associated with 20.8 M (million) completed visits. Self-scheduled visit types accounted for 4.0% (838,592/20,769,699) of the completed total visits. Of seven visit categories, self-scheduled established patient visits, testing visits, and procedure visits accounted for 93.5% (784,375/838,592) of all self-scheduled visits. Established patient visits in primary care (10 visit types) accounted for 273,007 (32.6%) of all self-scheduled visits. Testing visits (blood and urine testing, 2 visit types) accounted for 183,870 (21.9%) of all self-scheduled visits. Procedure visits for screening mammograms, bone mineral density, and immunizations (8 visit types) accounted for 147,358 (17.6%) of all self-scheduled visits., Conclusion: Large numbers of unique visit types comprise a major challenge for self-scheduling. Some visit types are more suitable for self-scheduling. Guideline-based procedure visits such as screening mammograms, bone mineral density exams, and immunizations are examples of visits that have high volumes and can be standardized for self-scheduling. Established patient visits and laboratory testing visits also can be standardized for self-scheduling. Despite the successes, there remain thousands of specific visit types that may need some staff-scheduler intervention to properly schedule., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

Background: Self-scheduling of medical visits is becoming available at many medical institutions. We aimed to examine the self-scheduled visit counts and rate of growth of self-scheduled visits in a multispecialty practice., Methods: For 85 weeks extending from January 1, 2022 through August 24, 2023, we examined self-scheduled visit counts for over 1500 self-scheduled visit types. We compared completed self-scheduled visit counts to all scheduled completed visit counts for the same visit types. We collected counts of the most frequently self-scheduled visit types for each week and examined the change over time. We also determined the proportion that each visit type was self-scheduled., Results: There were 20,769 699 completed visits during the course of the study that met the criteria for inclusion. Self-scheduled visits accounted for 4.0% of all completed visits (838 592/20,769 699). Over the 85-week span, self-scheduled visits rose from 3.0% to 5.3% of the total. There were 1887 unique visit types that were associated with completed visits. There were just 6 appointment visit types of the total 1887 self-scheduled visit types that accounted for 50.7% of the total 838 592 self-scheduled visits. Those 6 visit types were a lab blood test visit (19.5%, 163 K visits), two Family Medicine office visit types (13.0%, 109 K visits), a screening mammogram visit type (6.6%, 55 K visits), a scheduled express care visit type (6%, 50 K visits) and a COVID immunization visit type (5.7%, 48 K visits). Twenty-one visit types that were self-scheduled accounted for 75% of the total self-scheduled visits. Four seasonal visits, accounting for 10.6% of the total self-scheduled visits, were responsible for almost all the non-linear change in self-scheduling., Conclusion: Self-scheduling accounted for a small but growing percent of all outpatient scheduled visits in a multispecialty, multisite practice. A wide range of visit types can be successfully self-scheduled., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

Introduction: Online symptom checkers are a way to address patient concerns and potentially offload a burdened healthcare system. However, safety outcomes of self-triage are unknown, so we reviewed triage recommendations and outcomes of our institution's depression symptom checker., Methods: We examined endpoint recommendations and follow-up encounters seven days afterward during 2 December 2021 to 13 December 2022. Patients with an emergency department visit or hospitalization within seven days of self-triaging had a manual review of the electronic health record to determine if the visit was related to depression, suicidal ideation, or suicide attempt. Charts were reviewed for deaths within seven days of self-triage., Results: There were 287 unique encounters from 263 unique patients. In 86.1% (247/287), the endpoint was an instruction to call nurse triage; in 3.1% of encounters (9/287), instruction was to seek emergency care. Only 20.2% (58/287) followed the recommendations given. Of the 229 patients that did not follow the endpoint recommendations, 121 (52.8%) had some type of follow-up within seven days. Nearly 11% (31/287) were triaged to endpoints not requiring urgent contact and 9.1% (26/287) to an endpoint that would not need any healthcare team input. No patients died in the study period., Conclusions: Most patients did not follow the recommendations for follow-up care although ultimately most patients did receive care within seven days. Self-triage appears to appropriately sort patients with depressed mood to emergency care. On-line self-triaging tools for depression have the potential to safely offload some work from clinic personnel., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

The ability to create efficient artificial enzymes for any chemical reaction is of great interest. Here, we describe a computational design method for increasing the catalytic efficiency of de novo enzymes by several orders of magnitude without relying on directed evolution and high-throughput screening. Using structural ensembles generated from dynamics-based refinement against X-ray diffraction data collected from crystals of Kemp eliminases HG3 ( k cat / K M 125 M -1 s -1 ) and KE70 ( k cat / K M 57 M -1 s -1 ), we design from each enzyme ≤10 sequences predicted to catalyze this reaction more efficiently. The most active designs display k cat / K M values improved by 100-250-fold, comparable to mutants obtained after screening thousands of variants in multiple rounds of directed evolution. Crystal structures show excellent agreement with computational models, with catalytic contacts present as designed and transition-state root-mean-square deviations of ≤0.65 Å. Our work shows how ensemble-based design can generate efficient artificial enzymes by exploiting the true conformational ensemble to design improved active sites.

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp 17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Chronic lymphocytic leukaemia (CLL) is a clonal B-cell malignancy and remains a chronic disease despite improvements in clinical outcomes since the use of targeted therapies. Both clinical and biological parameters are important for determining prognosis. Unlike other mature B-cell lymphomas, translocations involving the immunoglobulin heavy chain (IGH) locus are uncommon in CLL. There have been few case reports of CLL harbouring t(14;18)/IGH::BCL2 and t(14;19)/IGH::BCL3. Here we describe the first two cases of patients with CLL with documented t(14;18)(q32;q21)/IGH::MALT1. Both cases in this report were associated with lower-risk biological parameters. Thus, FISH testing for MALT1 in cases with unknown IGH translocation partners in the setting of CLL should be implemented in clinical practice to better define such cases., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics., (© 2023. The Author(s).)

Background: Left atrial myopathy has been implicated in atrial fibrillation (AF)-related stroke and embolic stroke of undetermined source (ESUS)., Objective: To use advanced cardiac magnetic resonance (CMR) imaging techniques, including left atrial (LA) strain and 4D flow CMR, to identify atrial myopathy in patients with ESUS., Methods: 20 patients with ESUS and no AF or other cause for stroke, and 20 age and sex-matched controls underwent CMR with 4D flow analysis. Markers of LA myopathy were assessed including LA size, volume, ejection fraction, and strain. 4D flow CMR was performed to measure novel markers of LA stasis such as LA velocities and the LA residence time distribution time constant (RTDtc). These markers of LA myopathy were compared between the two groups., Results: There was no significant difference in: CMR-calculated LA velocities or LA total, passive or active ejection fractions between the groups. There was no significant difference in CMR-derived reservoir, conduit or contractile average longitudinal strain between the ESUS and control groups (22.9 vs 22.6%, p=0.379, 11.2 ± 3.5 vs 12.4 ± 2.6% p=0.224, 10.8 ± 3.2 vs 10.4 ± 2.3%, p=0.625 respectively). Similarly, RTDtc was not significantly longer in ESUS patients compared to controls (1.3 ± 0.2 vs 1.2 ± 0.2, p=0.1)., Conclusions: There were no significant differences in any CMR marker of atrial myopathy in ESUS patients compared to healthy controls, likely reflecting the multiple possible aetiologies of ESUS suggesting that the role LA myopathy plays in ESUS is smaller than previously thought., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

Structural plasticity of enzymes dictates their function. Yet, our ability to rationally remodel enzyme conformational landscapes to tailor catalytic properties remains limited. Here, we report a computational procedure for tuning conformational landscapes that is based on multistate design of hinge-mediated domain motions. Using this method, we redesign the conformational landscape of a natural aminotransferase to preferentially stabilize a less populated but reactive conformation and thereby increase catalytic efficiency with a non-native substrate, resulting in altered substrate selectivity. Steady-state kinetics of designed variants reveals activity increases with the non-native substrate of approximately 100-fold and selectivity switches of up to 1900-fold. Structural analyses by room-temperature X-ray crystallography and multitemperature nuclear magnetic resonance spectroscopy confirm that conformational equilibria favor the target conformation. Our computational approach opens the door to targeted alterations of conformational states and equilibria, which should facilitate the design of biocatalysts with customized activity and selectivity., (© 2023. Springer Nature Limited.)

Background: Although online self-triage is easily accessible, little is known about the patients who use self-triage or their subsequent diagnoses. We compared ear/hearing self-triage subsequent diagnoses to ear/hearing visit diagnoses in emergency departments (ED) and ambulatory clinics across the United States., Methods: We compared International Classification of Diseases version 10 (ICD10) coded diagnoses following online self-triage for ear/hearing concerns with those from national ED and ambulatory clinic samples. We used data from the Centers for Disease Control (CDC) National Hospital Ambulatory Medical Care Survey (NHAMCS) and National Ambulatory Medical Care Survey (NAMCS) for comparison. Using matched ear/hearing diagnostic categories for those aged 1 and over, we compared self-triage diagnosis frequencies with national ED and ambulatory diagnosis frequencies., Results: Following ear/hearing self-triage, there were 1092 subsequent office visits with a primary diagnosis code. For five frequently diagnosed ear/hearing conditions (i.e., suppurative and nonsuppurative otitis media [OM], otalgia, otitis externa, and cerumen impaction), there was a strong correlation between diagnosis counts made following self-triage and estimated counts of national ED visit diagnoses (r = 0.94; CI 95% [0.37 to 0.99]; p  = .016, adjusted r 2  = 0.85). Seven diagnoses were available to compare with the national ambulatory sample; correlation was r = 0.79; CI 95% [0.08 to 0.97]; p  = .037, adjusted r 2  = 0.54. For ages 1 and over, estimated hospital admissions from the national ED visits for ear/hearing were 0.76%, CI 95% [0.28-2.1%]; estimated total national ear/hearing ED visits were 7.5 million (for 4 years, 2016 through 2019)., Conclusion: The strong correlation of ear-related self-triage diagnoses with national ED diagnoses and the low hospitalization risk for these diagnoses suggests that there is an opportunity for self-triage of ear/hearing concerns to decrease ED visits for these symptoms., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

Covalent Bruton's tyrosine kinase inhibitors (cBTKi) have led to a paradigm shift in the treatment of chronic lymphocytic leukemia (CLL). These targeted oral therapies are administered as standard treatments in both the front-line and relapsed and/or refractory settings. Given their administration as a continuous therapy with a "treat-to-progression" strategy, limitations of their use include discontinuation due to toxicity or from progression of the disease. Non-covalent Bruton's tyrosine kinase inhibitors (ncBTKi) distinguish themselves by binding reversibly to the BTK target, which may address the limitations of toxicity and acquired resistance seen with cBTKi. Several ncBTKis have been studied preclinically and in clinical trials, including pirtobrutinib and nemtabrutinib. Pirtobrutinib, which is now FDA approved for relapsed and/or refractory mantle cell lymphoma (MCL), has shown outstanding safety and preliminary efficacy in CLL in phase 1 and 2 clinical trials, with phase 3 trials underway. This agent may fill an unmet medical need for CLL patients requiring treatment after a cBTKi. Pirtobrutinib is particularly promising for the treatment of "double exposed" CLL, defined as CLL requiring treatment after both a cBTKi and venetoclax. Some patients have now developedacquired resistance to pirtobrutinib, and resistance mechanisms (including novel acquired mutations in BTK outside of the C481 position) have been recently described. Further study regarding the mechanisms of resistance to pirtobrutinib in patients without prior cBTKi exposure, as well as the potential for cross-resistance between cBTKi and ncBTKis, may be important to help inform where ncBTKis will ultimately fit in the treatment sequencing paradigm for CLL. An emerging clinical challenge is the treatment of CLL after ncBTKi discontinuation. Novel therapeutic strategies are being investigated to address the treatment of patients following disease progression on ncBTKis. Such strategies include novel agents (BTK degraders, bispecific antibody therapy, CAR T-cell therapy, PKC-beta inhibitors) as well as combination approaches incorporating a ncBTKi (e.g., pirtobrutinib and venetoclax) that may help overcome this acquired resistance.

National and global efforts have led to significant improvements in breast health and diagnosis, globally (Lukong, 2017). These achievements, however, are not even. Focusing on the case of breast cancer in the UK, we argue that enduring forms of medical racism leave Black women more vulnerable to advanced forms of the disease, explaining higher mortality rates and later-stage diagnosis. In particular, we show how a lack of dedicated policy, inadequate data collection, and a lack of representation conspire to place Black women at additional and unnecessary risk of worse breast cancer outcomes. We thus propose key recommendations to address the ethnic disparities in and make steps to decolonise breast cancer care. These are early screening for at-risk groups, community-led interventions, and more and better representation of Black women and their risks in breast cancer resources., Competing Interests: Conflict of interest There are no conflicts of interest by any authors to report, (Copyright © 2023. Published by Elsevier Ltd.)

An evolutionary debate contrasts the importance of genetic convergence versus genetic redundancy. In genetic convergence, the same adaptive trait evolves because of similar genetic changes. In genetic redundancy, the adaptive trait evolves using different genetic combinations, and populations might not share the same genetic changes. Here we address this debate by examining single nucleotide polymorphisms (SNPs) associated with the rapid evolution of character displacement in Anolis carolinensis populations inhabiting replicate islands with and without a competitor species (1Spp and 2Spp islands, respectively). We identify 215-outliers SNPs that have improbably large FST values, low nucleotide variation, greater linkage than expected and that are enriched for genes underlying animal movement. The pattern of SNP divergence between 1Spp and 2Spp populations supports both genetic convergence and genetic redundancy for character displacement. In support of genetic convergence: all 215-outliers SNPs are shared among at least three of the five 2Spp island populations, and 23% of outlier SNPS are shared among all five 2Spp island populations. In contrast, in support of genetic redundancy: many outlier SNPs only have meaningful allele frequency differences between 1Spp and 2Spp islands on a few 2Spp islands. That is, on at least one of the 2Spp islands, 77% of outlier SNPs have allele frequencies more similar to those on 1Spp islands than to those on 2Spp islands. Focusing on genetic convergence is scientifically rigorous because it relies on replication. Yet, this focus distracts from the possibility that there are multiple, redundant genetic solutions that enhance the rate and stability of adaptive change., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE).)

Background: Self-triage is becoming more widespread, but little is known about the people who are using online self-triage tools and their outcomes. For self-triage researchers, there are significant barriers to capturing subsequent healthcare outcomes. Our integrated healthcare system was able to capture subsequent healthcare utilization of individuals who used self-triage integrated with self-scheduling of provider visits., Methods: We retrospectively examined healthcare utilization and diagnoses after patients had used self-triage and self-scheduling for ear or hearing symptoms. Outcomes and counts of office visits, telemedicine interactions, emergency department visits, and hospitalizations were captured. Diagnosis codes associated with subsequent provider visits were dichotomously categorized as being associated with ear or hearing concerns or not. Nonvisit care encounters of patient-initiated messages, nurse triage calls, and clinical communications were also captured., Results: For 2168 self-triage uses, we were able to capture subsequent healthcare encounters within 7 days of the self-triage for 80.5% (1745/2168). In subsequent 1092 office visits with diagnoses, 83.1% (891/1092) of the uses were associated with relevant ear, nose and throat diagnoses. Only 0.24% (4/1662) of patients with captured outcomes were associated with a hospitalization within 7 days. Self-triage resulted in a self-scheduled office visit in 7.2% (126/1745). Office visits resulting from a self-scheduled visit had significantly fewer combined non-visit care encounters per office visit (fewer combined nurse triage calls, patient messages, and clinical communication messages) than office visits that were not self-scheduled (-0.51; 95% CI, -0.72 to -0.29; P  < .0001)., Conclusion: In an appropriate healthcare setting, self-triage outcomes can be captured in a high percentage of uses to examine for safety, patient adherence to recommendations, and efficiency of self-triage. With the ear or hearing self-triage, most uses had subsequent visit diagnoses relevant to ear or hearing, so most patients appeared to be selecting the appropriate self-triage pathway for their symptoms., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

Temperature is an important state variable that governs the behavior of microscopic systems, yet crystallographers rarely exploit temperature changes to study the structure and dynamics of biological macromolecules. In fact, approximately 90% of crystal structures in the Protein Data Bank were determined under cryogenic conditions, because sample cryocooling makes crystals robust to X-ray radiation damage and facilitates data collection. On the other hand, cryocooling can introduce artifacts into macromolecular structures, and can suppress conformational dynamics that are critical for function. Fortunately, recent advances in X-ray detector technology, X-ray sources, and computational data processing algorithms make non-cryogenic X-ray crystallography easier and more broadly applicable than ever before. Without the reliance on cryocooling, high-resolution crystallography can be combined with various temperature perturbations to gain deep insight into the conformational landscapes of macromolecules. This Chapter reviews the historical reasons for the prevalence of cryocooling in macromolecular crystallography, and discusses its potential drawbacks. Next, the Chapter summarizes technological developments and methodologies that facilitate non-cryogenic crystallography experiments. Finally, the chapter discusses the theoretical underpinnings and practical aspects of multi-temperature and temperature-jump crystallography experiments, which are powerful tools for understanding the relationship between the structure, dynamics, and function of proteins and other biological macromolecules., (Copyright © 2023. Published by Elsevier Inc.)

As patients continue to live longer with chronic lymphocytic leukemia, it has become evident that there is an unmet treatment need for patients who have progressed on multiple lines of therapy. In this article, we attempt to define the "double refractory" patient as resistant to both Bruton's tyrosine kinase inhibitors (BTKi) and venetoclax for which prognosis is poor and there remains no standard of care. We further examine the mechanism of resistance to these targeted agents and discuss the current landscape for managing this patient population. Finally, we explore data supporting promising new agents, including non-covalent BTKi, chimeric antigen receptor T cells, and additional classes of agents currently in development., (© 2022 Wiley Periodicals LLC.)

Purpose: Ibrutinib has transformed the management of chronic lymphocytic leukemia (CLL), though its use is limited by toxicity and resistance. In this study, we utilized an "add on" approach for patients who had been treated with ibrutinib in the front-line or relapsed/refractory settings with detectable MRD. Umbralisib and ublituximab (U2) were added on to ibrutinib, patients were treated until achieving undetectable-MRD (U-MRD), and then they entered a period of treatment-free observation (TFO)., Patients and Methods: Patients were eligible if they received ibrutinib in any line of therapy for at least 6 months and had detectable MRD (flow cytometry, <1 cell in 10-4 cutoff for U-MRD). U2 was added to ibrutinib, and patients were monitored serially for MRD. Once U-MRD was achieved or a total of 24 cycles were administered, patients entered a period of TFO. The primary study objective was rate of U-MRD. Secondary endpoints included safety and durability of clinical benefit after treatment discontinuation., Results: Twenty-eight patients were enrolled of whom 27 were evaluable for efficacy. Patients received ibrutinib for a median of 21 months (range 7-67) prior to study enrollment. Fourteen patients (52%) have achieved U-MRD per protocol whereas 78% had at least one U-MRD evaluation. Seventeen patients (63%) have entered TFO after a median of 6.4 months on triplet therapy. Progression-free survival at 12 months was estimated at 95%. Grade ≥3 adverse events were hypertension 7%, diarrhea 4%, and increased ALT/AST 4%., Conclusions: This triplet approach utilizes the addition of U2 to ibrutinib as an MRD-driven time-limited therapy. This therapy was well tolerated and effective. TFO following this therapy appears durable in ongoing follow-up., (©2022 American Association for Cancer Research.)

Introduction: The COVID 19 pandemic increased the need for rapid and accurate diagnostic testing for COVID. When testing became available, a systems response was needed to efficiently accommodate the high-volume flow of patients who needed testing. Self-scheduling of COVID testing was developed to help patients safely and efficiently schedule their COVID testing online or with a mobile app., Methods: We captured the counts of COVID test appointments, time patients spent in scheduling COVID test appointments, appointment lead times, and no-shows for COVID test appointments. For 17 months of self-scheduling, we retrospectively compared self-scheduling with the concurrent staff scheduling of COVID tests., Results: From November 2020 through March 2022 there were 619 104 scheduled appointments for COVID testing with 22% (136 252) being self-scheduled. For asymptomatic self-scheduled COVID tests, accounting for 10.3% (63 605/619 104) of total COVID tests scheduled, median time to self-schedule was 3.1 min, interquartile range (IQR) [2.4,4.7]. For symptomatic self-schedulers accounting for 11.7% (72 647/619 104) of total COVID tests scheduled, the median time to self-triage and self-schedule was 5.8 min, IQR[4.3,8.9]. Self-scheduled COVID appointments increased to 44% (42 387/97 086) of the total COVID appointments during the peak month of January 2022. Median appointment lead time for symptomatic self-scheduled COVID test appointments was 6.6 h compared to 2.9 h ( P  < .0001) for symptomatic staff scheduled appointments. However, adjusting for the 24% (32 194/135 252) that self-scheduled during hours when testing was unavailable, the median appointment lead time for symptomatic self-scheduled patients dropped to 3.6 h. No-shows were 2.5% for self-scheduled appointments compared to 3.0% no-shows that were staff scheduled (odds ratio 0.83, P  < .0001)., Conclusion: COVID testing was self-scheduled for a large percent of scheduled COVID tests, taking patients only a few minutes to complete. Self-scheduling use increased over time, associated with a decreasing use of staff scheduled appointments and lower no-shows., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Understanding the cortical representations of movements and their stability can shed light on improved brain-machine interface (BMI) approaches to decode these representations without frequent recalibration. Here, we characterize the spatial organization (somatotopy) and stability of the bilateral sensorimotor map of forearm muscles in an incomplete-high spinal-cord injury study participant implanted bilaterally in the primary motor and sensory cortices with Utah microelectrode arrays (MEAs). We built representation maps by recording bilateral multiunit activity (MUA) and surface electromyography (EMG) as the participant executed voluntary contractions of the extensor carpi radialis (ECR), and attempted motions in the flexor carpi radialis (FCR), which was paralytic. To assess stability, we repeatedly mapped and compared left- and right-wrist-extensor-related activity throughout several sessions, comparing somatotopy of active electrodes, as well as neural signals both at the within-electrode (multiunit) and cross-electrode (network) levels. Wrist motions showed significant activation in motor and sensory cortical electrodes. Within electrodes, firing strength stability diminished as the time increased between consecutive measurements (hours within a session, or days across sessions), with higher stability observed in sensory cortex than in motor, and in the contralateral hemisphere than in the ipsilateral. However, we observed no differences at network level, and no evidence of decoding instabilities for wrist EMG, either across timespans of hours or days, or across recording area. While map stability differs between brain area and hemisphere at multiunit/electrode level, these differences are nullified at ensemble level., (© 2022. The Author(s).)

Background: The wide variety of patient-reported outcome measures used to assess outcomes following total joint arthroplasty can present a substantial methodological obstacle when attempting to compare information across studies or between institutions. A simple solution is to create crosswalks that reliably convert scores between patient-reported outcome measures. Our goal is to create and validate crosswalks between the commonly used Western Ontario & McMaster Universities Osteoarthritis Index (WOMAC) and short-form versions of the Hip Disability and Osteoarthritis Outcome Score Joint Replacement (HOOS JR)/Knee Injury and Osteoarthritis Outcome Score (KOOS JR.)., Methods: Patients in our joint replacement registry were included if they underwent primary total hip arthroplasty (n = 4649) or total knee arthroplasty (n = 3750) for osteoarthritis between May 2007 and February 2012. We used their preoperative and 2-year postoperative HOOS scores (n = 6351) or KOOS scores (n = 4688) to generate the patients' WOMAC and HOOS JR/KOOS JR scores. The equipercentile equating method was applied to create 10 crosswalks: HOOS JR/KOOS JR to WOMAC Total (WOMAC-T), and WOMAC-T, WOMAC Pain (WOMAC-P), WOMAC Stiffness (WOMAC-S), and WOMAC Function (WOMAC-F) to HOOS JR/KOOS JR. Crosswalk validity was assessed by comparing actual and derived scores using Spearman's rank correlation coefficients in a bootstrapped cohort., Results: All 10 crosswalks showed strong positive correlations ranging from 0.846 (WOMAC-S to KOOS JR) to 0.981 (HOOS JR to WOMAC-T)., Conclusion: We created and validated 10 crosswalks between WOMAC and HOOS JR/KOOS JR. We recommend using the crosswalks between WOMAC-T and HOOS JR/KOOS JR when possible, as they demonstrated the highest correlation. WOMAC-F or WOMAC-P should be used in favor of WOMAC-S if only subscores are available. The HOOS JR/KOOS JR should only be converted to a WOMAC-T., Level of Evidence: Level III., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Mac1) removes adenosine diphosphate (ADP) ribosylation posttranslational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the coronavirus disease 2019 pandemic. Here, we determined neutron and x-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase the potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a reevaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

Mantle cell lymphoma is a rare B-cell non-Hodgkin lymphoma that is clinically and biologically heterogeneous. Risk stratification at the time of diagnosis is critical. One of the most powerful prognostic indices is the Mantle Cell Lymphoma International Prognostic Index-Combined, which integrates an estimate of proliferation (Ki67 index) with the standard Mantle Cell Lymphoma International Prognostic Index clinical factors. In addition, the presence of TP53 mutation is associated with suboptimal response to intensive chemoimmunotherapy and particularly dismal survival outcomes. Given their excellent activity in the relapsed/refractory setting, increasingly, biologically targeted therapeutics-such as covalent Bruton tyrosine kinase inhibitors, lenalidomide, and venetoclax-are being incorporated into "chemotherapy-free" regimens and in combination with established chemoimmunotherapy backbones for treatment-naïve mantle cell lymphoma. In addition, risk-adapted treatment programs are increasingly being studied. These programs tailor treatment according to baseline prognostic factors (e.g., presence of TP53 mutation) and may incorporate biomarkers of response such as minimal residual disease assessment. Although still investigational, these studies present an opportunity to move beyond the biology-agnostic, historical fitness-based treatment selection paradigm and toward a more personalized, tailored treatment approach in mantle cell lymphoma. After Bruton tyrosine kinase inhibitor failure, many promising standard or investigational therapies exist, including CAR T-cell therapy (including brexucabtagene autoleucel and lisocabtagene maraleucel), bispecific antibody therapy targeting CD20-CD3, zilovertamab vedotin (an antibody-drug conjugate that targets ROR1), and the noncovalent Bruton tyrosine kinase inhibitor pirtobrutinib. These new therapies show promising efficacy, even among high-risk patients, and will likely translate to improvements in survival outcomes for patients with progressive mantle cell lymphoma following treatment with a Bruton tyrosine kinase inhibitor.

SignificanceComputational protein design promises to advance applications in medicine and biotechnology by creating proteins with many new and useful functions. However, new functions require the design of specific and often irregular atom-level geometries, which remains a major challenge. Here, we develop computational methods that design and predict local protein geometries with greater accuracy than existing methods. Then, as a proof of concept, we leverage these methods to design new protein conformations in the enzyme ketosteroid isomerase that change the protein's preference for a key functional residue. Our computational methods are openly accessible and can be applied to the design of other intricate geometries customized for new user-defined protein functions.

Background: Covalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood., Methods: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors., Results: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors., Conclusions: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.)., (Copyright © 2022 Massachusetts Medical Society.)