Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

Performance characteristics of genus or species-specific Polymerase Chain Reaction (PCR) for the microbial diagnosis of joint infections: A systematic review and meta-analysis.

Joint infections cause significant morbidity and mortality. Rapid diagnosis enables prompt initiation of appropriate antimicrobial therapy and surgical treatment. We conducted a systematic review and meta-analysis to evaluate the accuracy of genus- or species-specific polymerase chain reaction (PCR) in diagnosing joint infections. The literature databases were searched for articles from January 2010 to December 2022. The meta-analysis using the split component synthesis (SCS) method, included 20 studies with 2,457 adult participants. The pooled sensitivity, specificity, diagnostic odds ratio, and AUC of PCR were 49 % (95 % CI [37.9-60.2]), 95.7 % (95 % CI [91.6-97.8]), 21.32, and 0.82 respectively. Sensitivity was highest for sonicate fluid and lowest for periprosthetic tissue. The mean turnaround time to results was 4.7 hours (SD 1.1). PCR is a favourable option for diagnosing joint infections due to its rapid results, but it has low sensitivity. To enhance diagnostic yield, the test should be used in conjunction with other methods.

Delocalized quinolinium-macrocyclic peptides, an atypical chemotype for CNS penetration.

Macrocyclic drugs can address an increasing range of molecular targets but enabling central nervous system (CNS) access to these drugs has been viewed as an intractable problem. We designed and synthesized a series of quinolinium-modified cyclosporine derivatives targeted to the mitochondrial cyclophilin D protein. Modification of the cation to enable greater delocalization was confirmed by x-ray crystallography of the cations. Critically, greater delocalization improved brain concentrations. Assessment of the compounds in preclinical assays and for pharmacokinetics identified a molecule JP1-138 with at least 20 times the brain levels of a non-delocalized compound or those reported for cyclosporine. Levels were maintained over 24 hours together with low hERG potential. The paradigm outlined here could have widespread utility in the treatment of CNS diseases.

Engineered Heart Tissues for Standard 96-Well Tissue Culture Plates.

Engineered heart tissues (EHTs) have been shown to be a valuable platform for disease investigation and therapeutic testing by increasing human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturity and better recreating the native cardiac environment. The protocol detailed in this chapter describes the generation of miniaturized EHTs (mEHTs) incorporating hiPSC-CMs and human stromal cells in a fibrin hydrogel. This platform utilizes an array of silicone posts designed to fit in a standard 96-well tissue culture plate. Stromal cells and hiPSC-CMs are cast in a fibrin matrix suspended between two silicone posts, forming an mEHT that produces synchronous muscle contractions. The platform presented here has the potential to be used for high throughput characterization and screening of disease phenotypes and novel therapeutics through measurements of the myocardial function, including contractile force and calcium handling, and its compatibility with immunostaining.

Effect of Data-Processing Methods on Acceleration Summary Metrics of GNSS Devices in Elite Australian Football.

This study aimed to measure the differences in commonly used summary acceleration metrics during elite Australian football games under three different data processing protocols (raw, custom-processed, manufacturer-processed). Estimates of distance, speed and acceleration were collected with a 10-Hz GNSS tracking technology device from fourteen matches of 38 elite Australian football players from one team. Raw and manufacturer-processed data were exported from respective proprietary software and two common summary acceleration metrics (number of efforts and distance within medium/high-intensity zone) were calculated for the three processing methods. To estimate the effect of the three different data processing methods on the summary metrics, linear mixed models were used. The main findings demonstrated that there were substantial differences between the three processing methods; the manufacturer-processed acceleration data had the lowest reported distance (up to 184 times lower) and efforts (up to 89 times lower), followed by the custom-processed distance (up to 3.3 times lower) and efforts (up to 4.3 times lower), where raw data had the highest reported distance and efforts. The results indicated that different processing methods changed the metric output and in turn alters the quantification of the demands of a sport (volume, intensity and frequency of the metrics). Coaches, practitioners and researchers need to understand that various processing methods alter the summary metrics of acceleration data. By being informed about how these metrics are affected by processing methods, they can better interpret the data available and effectively tailor their training programs to match the demands of competition.

Circulating Tumor DNA Predicts Early Recurrence Following Locoregional Therapy for Oligometastatic Colorectal Cancer.

(1) Background: Local therapies offer a potentially curative approach for patients with oligometastatic colorectal cancer (CRC). An evidence-based consensus recommendation for systemic therapy following definitive locoregional therapy is lacking. Tumor-informed circulating tumor DNA (ctDNA) might provide information to help guide management in this setting. (2) Methods: A multi-institutional retrospective study was conducted, including patients with CRC that underwent curative-intent locoregional therapy to an isolated site of metastatic disease, followed by tumor-informed ctDNA assessment. The Kaplan-Meier method and log-rank tests were used to compare disease-free survival based on ctDNA results. ctDNA test performance was compared to carcinoembryonic antigen (CEA) test results using McNemar's test. (3) Results: Our study cohort consisted of 87 patients treated with locoregional interventions who underwent ctDNA testing. The initial ctDNA test post-intervention was positive in 28 patients and negative in 59 patients. The median follow-up time was 14.0 months. Detectable ctDNA post-intervention was significantly associated with early disease recurrence, with a median disease-free survival (DFS) of 6.63 months compared to 21.30 months in ctDNA-negative patients (p < 0.001). ctDNA detected a numerically higher proportion of recurrences than CEA (p < 0.097). Post-intervention systemic therapy was not associated with improved DFS (p = 0.745). (4) Conclusions: ctDNA results are prognostically important in oligometastatic CRC, and further prospective studies are urgently needed to define its role in guiding clinical decisions.

Selection for early reproduction leads to accelerated aging and extensive metabolic remodeling in Drosophila melanogaster populations.

Experimental evolution studies that feature selection on life-history characters are a proven approach for studying the evolution of aging and variation in rates of senescence. Recently, the incorporation of genomic and transcriptomic approaches into this framework has led to the identification of hundreds of genes associated with different aging patterns. However, our understanding of the specific molecular mechanisms underlying these aging patterns remains limited. Here, we incorporated extensive metabolomic profiling into this framework to generate mechanistic insights into aging patterns in Drosophila melanogaster . Specifically, we characterized metabolomic change over time associated with accelerated aging in populations of D. melanogaster under selection for early reproduction compared to their controls. Using this data we: i) evaluated the evolutionary repeatability across the metabolome; ii) evaluated the value of the metabolome as a predictor of "biological age" in this system; and iii) identified specific metabolic pathways associated with accelerated aging. Generally, our findings suggest that the metabolome is a reliable predictor of age and senescence in populations that share a recent evolutionary history. Metabolomic analysis revealed that generations of selection for early reproduction resulted in highly repeatable alterations to the metabolome. Specifically, changes in carbohydrate, amino acid, and TCA cycle-related metabolite abundances over time point to metabolic remodeling that favors rapid early reproduction with long-term consequences for carbohydrate and protein utilization.

Reduced Auditory Perception and Brain Response with Quiet TMS Coil.

BACKGROUND: Electromagnetic forces in transcranial magnetic stimulation (TMS) coils generate a loud clicking sound that produces confounding auditory activation and is potentially hazardous to hearing. To reduce this noise while maintaining stimulation efficiency similar to conventional TMS coils, we previously developed a quiet TMS double containment coil (qTMS-DCC). OBJECTIVE: To compare the stimulation strength, perceived loudness, and EEG response between qTMS-DCC and a commercial TMS coil. METHODS: Nine healthy volunteers participated in a within-subject study design. The resting motor thresholds (RMTs) for qTMS-DCC and MagVenture Cool-B65 were measured. Psychoacoustic titration matched the Cool-B65 loudness to qTMS-DCC pulsed at 80, 100, and 120% RMT. Event-related potentials (ERPs) were recorded for both coils. The psychoacoustic titration and ERPs were acquired with the coils both on and 6 cm off the scalp, the latter isolating the effects of airborne auditory stimulation from body sound and electromagnetic stimulation. The ERP comparisons focused on a centro-frontal region that encompassed peak responses in the global signal. RESULTS: RMT did not differ significantly between the coils, with or without the EEG cap on the head. qTMS-DCC was perceived to be substantially quieter than Cool-B65. For example, qTMS-DCC at 100% coil-specific RMT sounded like Cool-B65 at 34% RMT. The general ERP waveform and topography were similar between the two coils, as were early-latency components, indicating comparable electromagnetic brain stimulation in the on-scalp condition. qTMS-DCC had a significantly smaller P180 component in both on-scalp and off-scalp conditions, supporting reduced auditory activation. CONCLUSIONS: The stimulation efficiency of qTMS-DCC matched Cool-B65, while having substantially lower perceived loudness and auditory-evoked potentials. Highlights: qTMS coil is subjectively and objectively quieter than conventional Cool-B65 coilqTMS coil at 100% motor threshold was as loud as Cool-B65 at 34% motor thresholdAttenuated coil noise reduced auditory N100 and P180 evoked response componentsqTMS coil enables reduction of auditory activation without masking.

Substrate Trapping in Polyketide Synthase Thioesterase Domains: Structural Basis for Macrolactone Formation.

Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the termina PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TE DAP ) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TE DAP , the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 Å from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide new insights to facilitate TE engineering and optimization.

Numerical Model for Electrogenic Transport by the ATP-dependent Potassium Pump KdpFABC.

In vitro assays of ion transport are an essential tool for understanding molecular mechanisms associated with ATP-dependent pumps. Because ion transport is generally electrogenic, principles of electrophysiology are applicable, but conventional tools like patch clamp are ineffective due to relatively low turnover rates of the pumps. Instead, assays have been developed to measure either voltage or current generated by transport activity of a population of molecules either in cell-derived membrane fragments or after reconstituting purified protein into proteoliposomes. In order to understand the nuances of these assays and to characterize effects of various operational parameters, we have developed a numerical model to simulate data produced by two relevant assays: fluorescence from voltage sensitive dyes and current recorded by capacitive coupling on solid supported membranes. Parameters of the model, which has been implemented in Python, are described along with underlying principles of the computational algorithm. Experimental data from KdpFABC, a K+ pump associated with P-type ATPases, are presented and model parameters have been adjusted to mimic these data. In addition, effects of key parameters such as non-selective leak conductance and turnover rate are demonstrated. Finally, simulated data are used to illustrate the effects of capacitive coupling on measured current and to compare alternative methods for quantification of raw data.

Mapping of long interspersed element-1 (L1) insertions by TIPseq provides information about sub chromosomal genetic variation in human embryos.

PURPOSE: Retrotransposons play important roles during early development when they are transiently de-repressed during epigenetic reprogramming. Long interspersed element-1 (L1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. We applied the Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize and map L1 insertions in human embryos. METHODS: Sixteen cryopreserved, genetically tested, human blastocysts, were accessed from consenting couples undergoing IVF at NYU Langone Fertility Center. Additionally, four trios (father, mother, and embryos) were also evaluated. scTIPseq was applied to map L1 insertions in all samples, using L1 locations reported in the 1000 Genomes as controls. RESULTS: Twenty-nine unknown and unique insertions were observed in the sixteen embryos. Most were intergenic; no insertions were located in exons or immediately upstream of genes. The location or number of unknown insertions did not differ between euploid and aneuploid embryos, suggesting they are not merely markers of aneuploidy. Rather, scTIPseq provides novel information about sub-chromosomal structural variation in human embryos. Trio analyses showed a parental origin of all L1 insertions in embryos. CONCLUSION: Several studies have measured L1 expression at different stages of development in mice, but this study for the first time reports unknown insertions in human embryos that were inherited from one parent, confirming no de novo L1 insertions occurred in parental germline or during embryogenesis. Since one-third of euploid embryo transfers fail, future studies would be useful for understanding whether these sub-chromosomal genetic variants or de novo L1 insertions affect embryo developmental potential.

Prognoses Associated With Palliative Performance Scale Scores in Modern Palliative Care Practice.

Importance: The Palliative Performance Scale (PPS) is one of the most widely used prognostic tools for patients with serious illness. However, current prognostic estimates associated with PPS scores are based on data that are over a decade old. Objective: To generate updated prognostic estimates by PPS score, care setting, and illness category, and examine how well PPS predicts short- and longer-term survival. Design, Setting, and Participants: This prognostic study was conducted at a large academic medical center with robust inpatient and outpatient palliative care practices using electronic health record data linked with data from California Vital Records. Eligible participants included patients who received a palliative care consultation between January 1, 2018, and December 31, 2020. Data analysis was conducted from November 2022 to February 2024. Exposure: Palliative care consultation with a PPS score documented. Main Outcomes and Measures: The primary outcomes were predicted 1-, 6-, and 12-month mortality and median survival of patients by PPS score in the inpatient and outpatient settings, and performance of the PPS across a range of survival times. In subgroup analyses, mortality risk by PPS score was estimated in patients with cancer vs noncancer illnesses and those seen in-person vs by video telemedicine in the outpatient setting. Results: Overall, 4779 patients (mean [SD] age, 63.5 [14.8] years; 2437 female [51.0%] and 2342 male [49.0%]) had a palliative care consultation with a PPS score documented. Of these patients, 2276 were seen in the inpatient setting and 3080 were seen in the outpatient setting. In both the inpatient and outpatient settings, 1-, 6-, and 12-month mortality were higher and median survival was shorter for patients with lower PPS scores. Prognostic estimates associated with PPS scores were substantially longer (2.3- to 11.7-fold) than previous estimates commonly used by clinicians. The PPS had good ability to discriminate between patients who lived and those who died in the inpatient setting (integrated time-dependent area under the curve [iAUC], 0.74) but its discriminative ability was lower in the outpatient setting (iAUC, 0.67). The PPS better predicted 1-month survival than longer-term survival. Mortality rates were higher for patients with cancer than other serious illnesses at most PPS levels. Conclusions and Relevance: In this prognostic study, prognostic estimates associated with PPS scores were substantially longer than previous estimates commonly used by clinicians. Based on these findings, an online calculator was updated to assist clinicians in reaching prognostic estimates that are more consistent with modern palliative care practice and specific to the patient's setting and diagnosis group.

Metabolic Tumor Volume on 18-Fluorodeoxyglucose Positron Emission Tomography as a Prognostic Marker of Survival in Patients With Locally Advanced or Metastatic Neuroendocrine Neoplasms Treated With 177Lutetium-DOTA-Octreotate Peptide Receptor Radionuclide Therapy.

OBJECTIVE: We investigated metabolic tumor volume (MTV) and total lesion glycolysis (TLG) on pre-treatment FDG-PET as prognostic markers for survival in patients with metastatic neuroendocrine neoplasms (NENs) receiving peptide receptor radionuclide therapy (PRRT). METHODS: A retrospective review of patients with metastatic NENs receiving PRRT was undertaken. Pre-treatment FDG-PET images were analyzed and variables collected included MTV and TLG (dichotomized by median into high vs low). Main Outcomes were overall survival (OS) and progression-free survival (PFS) by MTV and TLG (high vs low). RESULTS: One hundred five patients were included. Median age was 64 years (50% male). Main primary NEN sites were small bowel (43.8%) and pancreas (40.0%). Median MTV was 3.8 mL and median TLG was 19.9. Dichotomization formed identical cohorts regardless of whether MTV or TLG were used. Median OS was 72 months; OS did not differ based on MTV/TLG high versus low (47.4 months vs not reached; hazard ratio, 0.43; 95% confidence interval [CI], 0.18-1.04; P = 0.0594). Median PFS was 30.4 months; PFS differed based on MTV/TLG high versus low (21.6 months vs 45.7 months; hazard ratio, 0.35; 95% CI, 0.19-0.64; P = 0.007). CONCLUSIONS: Low MTV/TLG on pre-treatment FDG-PET was associated with longer PFS in metastatic NEN patients receiving PRRT.

Exploring Ligand Effects on Structure, Bonding, and Photolytic Hydride Transfer of Cationic Gold(I) Bridging Hydride Complexes of Molybdocene and Tungstenocene.

A diverse family of heterobimetallic bridging hydride adducts of the type [LAu(µ-H)2MCp2][X] (L = 1,3-bis(2,6-diisopropylphenyl)imidazole-2-ylidene, IPr; 1,3-bis(1-adamantyl)imidazole-2-ylidene, IAd; 1,3-bis(2,6-di-iso-propylphenyl)-5,5-dimethyl-4,6-diketopyrimidinyl-2-ylidene, DippDAC; triphenylphosphine, PPh3; 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl, tBuXPhos; X = SbF6-, BF4- or TfO-) was synthesized by reacting group VI metallocene dihydrides Cp2MH2 (Cp = cyclopentadienyl anion; M = Mo, W) with cationic gold(I) complexes [LAu(NCMe)][X]. Trimetallic [L'Au2(µ-H)2WCp2][X]2 and tetrametallic [L'Au2{(µ-H)2WCp2}2] [X]2 complexes (L' = rac-2,2'-bis(diphenylphosphino)-1,1'-binaphthalene or bis(diphenylphosphinomethane)) were obtained by reacting digold [L'{Au(NCMe)}2][X]2 with Cp2WH2 in a 1:1 and a 1:2 stoichiometry. Accessing such a broad structural diversity allowed us to pinpoint roles played by the ancillary ligands and group VI metals on the bonding properties of this family of bridging hydrides. In particular, a clear effect of the ligand on the interaction energy and electronic structure was observed, with important implications on photolytic reactivity. UV or visible light irradiation, indeed, leads to the selective cleavage of the heterobimetallic Au(µ-H)2M arrangement and formation of molecular gold hydrides. The photolysis was found to be chromoselective (wavelength-dependent), which can be ascribed to different charge redistributions upon excitation to the first (Kasha's reactivity) and higher (anti-Kasha's reactivity) excited states.

The Open Force Field Initiative: Open Software and Open Science for Molecular Modeling.

Force fields are a key component of physics-based molecular modeling, describing the energies and forces in a molecular system as a function of the positions of the atoms and molecules involved. Here, we provide a review and scientific status report on the work of the Open Force Field (OpenFF) Initiative, which focuses on the science, infrastructure and data required to build the next generation of biomolecular force fields. We introduce the OpenFF Initiative and the related OpenFF Consortium, describe its approach to force field development and software, and discuss accomplishments to date as well as future plans. OpenFF releases both software and data under open and permissive licensing agreements to enable rapid application, validation, extension, and modification of its force fields and software tools. We discuss lessons learned to date in this new approach to force field development. We also highlight ways that other force field researchers can get involved, as well as some recent successes of outside researchers taking advantage of OpenFF tools and data.

Hepatitis C-related knowledge and attitude among adults on probation in a large US city.

BACKGROUND: Hepatitis C virus (HCV) continues to cause significant morbidity and mortality within the US, and disproportionately impacts those involved with the criminal justice system. Despite this, knowledge and attitudes regarding HCV treatment among adults on probation have not been well studied. We conducted a cross-sectional survey of adults on probation accessing on-site HCV testing and linkage services at the adult probation department in Denver, Colorado. The survey assessed general knowledge of HCV and HCV treatment, as well as attitudes surrounding HCV treatment that might reflect medical mistrust. We used bivariate and multivariable logistic regression to identify factors associated with previous HCV testing, previous HCV treatment, and HCV antibody positivity at the time the survey was conducted. RESULTS: A total of 402 participants completed all or a portion of the survey. 69% of the participants were cis-gender men; 29% were white, 27% were Black, and 30% were Hispanic/Latinx. Fewer than half of participants correctly identified that HCV infection is commonly asymptomatic (46%), that there is currently no vaccine that prevents HCV (19%), and that reinfection after treatment is possible (47%). Very few participants felt that side-effects (9%) or cost of treatment (10%) were barriers to care. Many participants believed that racial disparities exist in the treatment of HCV (59%). The belief that people who use substances are treated inequitably by health care providers was also commonly reported (35% of participants). Self-reported injection drug use and higher HCV-related knowledge were positively associated with previous testing for HCV. Higher HCV-related knowledge was positively associated with HCV antibody positivity at the time of survey completion, though the magnitude of the association was small. CONCLUSION: Interventions are needed to increase knowledge of HCV, to improve access to HCV testing and treatment, and to reduce bias associated with HCV and substance use within the probation population.

Qualitative evaluation of a multidisciplinary master of cancer sciences: impacts on graduates and influencing curricular factors.

BACKGROUND: Evaluations of continuing professional development programs typically focus on short-term knowledge and skill acquisition. There is a need for more comprehensive program evaluation methods that assess a broader range of impacts and can elicit how and why these outcomes occurred. We conducted a qualitative study to investigate the impacts of a multidisciplinary, online health professional postgraduate degree and to gain insights into the factors that led to these impacts. METHODS: Participants were graduates of the University of Melbourne's Master of Cancer Sciences who could participate in an online interview. Semi-structured, qualitative interviews were conducted exploring a broad range of impacts, including changes in professional practice and career trajectory since graduation, and how the degree influenced these impacts. Data were analysed inductively. RESULTS: Fifteen participants (female: 80%, 31-50 years old: 67%) from a range of professions were interviewed. A number of major themes were uncovered. Impacts on career trajectory included expanded career horizons (e.g. increased role diversity and complexity), and increased confidence in their professional identity. Impacts on professional practice included individual improvements in patient care and research, as well as changes in organisational practice. Factors identified as leading to these impacts were: (i) active, interactive and interprofessional learning; (ii) networking, informal mentoring, and role-modelling; and (iii) support at multiple levels. CONCLUSION: This study provides preliminary evidence of the positive impact of a Master of Cancer Sciences on graduate career trajectory and professional practice. In addition, the inductive methodology enabled identification of the curricular features (both planned and emergent) that influenced these impacts, facilitating potential transferability of learnings to other teaching programs.

Quantitative Measurement of HER2 Expression in Non-Small Cell Lung Cancer with a High Sensitivity Assay.

Recently, low HER2 protein expression has been proposed as a predictive biomarker for response to antibody-drug conjugate trastuzumab deruxtecan (T-DXd) in metastatic breast cancer. HER2 expression in non-small cell lung cancer (NSCLC) patients has never been carefully measured, and little is known about the frequency of cases with unamplified but detectable levels of the protein. Although some HER2-targeted therapies have been studied in NSCLC patients, they have been restricted to those with genomic ERBB2 gene alterations, which only represent relatively rare cases of NSCLC. Still, emerging investigations of T-DXd in NSCLC have shown promise in patients with unamplified HER2. Taken together, we hypothesize that there may be many cases of NSCLC with levels of HER2 protein expression comparable to levels seen in breast cancer who benefit from T-DXd. Here, we used a previously validated, analytic, quantitative immunofluorescence (QIF) assay that is more sensitive than legacy clinical HER2 immunohistochemistry assays. We measured HER2 protein levels in NSCLC cases to determine the proportion of cases with detectable HER2 expression. Using cell line calibration microarrays alongside our QIF method enabled us to convert HER2 signal into units of attomoles per mm2. We found that over 63% of the 741 analyzed NSCLC cases exhibited HER2 expression above the limit of detection, with more than 17% of them exceeding the lower limit of quantification. While the threshold for response to T-DXd in breast cancer is still unknown, many cases of NSCLC have expression in a range comparable to breast cancer cases with immunohistochemistry scores of 1+ or 2+. Our assay could potentially select NSCLC cases with detectable target (i.e., HER2) that might benefit from HER2 antibody-drug conjugates, irrespective of ERBB2 genomic alterations.

Research to Confront Climate Change Complexity: Intersectionality, Integration, and Innovative Governance.

Climate impacts increasingly unfold in interlinked systems of people, nature, and infrastructure. The cascading consequences are revealing sometimes surprising connections across sectors and regions, and prospects for climate responses also depend on complex, difficult-to-understand interactions. In this commentary, we build on the innovations of the United States Fifth National Climate Assessment to suggest a framework for understanding and responding to complex climate challenges. This approach involves: (a) integration of disciplines and expertise to understand how intersectionality shapes complex climate impacts and the wide-ranging effects of climate responses, (b) collaborations among diverse knowledge holders to improve responses and better encompass intersectionality, and (c) sustained experimentation with and learning about governance approaches capable of handling the complexity of climate change. Together, these three pillars underscore that usability of climate-relevant knowledge requires transdisciplinary coordination of research and practice. We outline actionable steps for climate research to incorporate intersectionality, integration, and innovative governance, as is increasingly necessary for confronting climate complexity and sustaining equitable, ideally vibrant climate futures.

Oxidative stress across multiple tissues in house sparrows (Passer domesticus) acclimated to warm, stable cold, and unpredictable cold thermal treatments.

With climate change increasing not just mean temperatures but the frequency of cold snaps and heat waves, animals occupying thermally variable areas may be faced with thermal conditions for which they are not prepared. Studies of physiological adaptations of temperate resident birds to such thermal variability are largely lacking in the literature. To address this gap, we acclimated winter-phenotype house sparrows (Passer domesticus) to stable warm, stable cold, and fluctuating cold temperatures. We then measured several metrics of the oxidative stress (OS) system, including enzymatic and non-enzymatic antioxidants and lipid oxidative damage, in brain (post-mitotic), kidney (mitotic), liver (mitotic) and pectoralis muscle (post-mitotic). We predicted that high metabolic flexibility could be linked to increases in reactive oxygen damage. Alternatively, if variation in ROS production is not associated with metabolic flexibility, then we predict no antioxidant compensation with thermal variation. Our data suggest that ROS production is not associated with metabolic flexibility, as we found no differences across thermal treatment groups. However, we did find differences across tissues. Brain catalase activity demonstrated the lowest values compared with kidney, liver and muscle. In contrast, brain glutathione peroxidase (GPx) activities were higher than those in kidney and liver. Muscle GPx activities were intermediate to brain and kidney/liver. Lipid peroxidation damage was lowest in the kidney and highest in muscle tissue.