Hit Snooze: An Imperiled Hibernator Assesses Spring Snow Conditions to Decide Whether to Terminate Hibernation or Reenter Torpor.

AbstractMany animals follow annual cycles wherein physiology and behavior change seasonally. Hibernating mammals undergo one of the most drastic seasonal alterations of physiology and behavior, the timing of which can have significant fitness consequences. The environmental cues regulating these profound phenotypic changes will heavily influence whether hibernators acclimate and ultimately adapt to climate change. Hence, identifying the cues and proximate mechanisms responsible for hibernation termination timing is critical. Northern Idaho ground squirrels (Urocitellus brunneus)-a rare, endemic species threatened with extinction-exhibit substantial variation in hibernation termination phenology, but it is unclear what causes this variation. We attached geolocators to free-ranging squirrels to test the hypothesis that squirrels assess surface conditions in spring before deciding whether to terminate seasonal heterothermy or reenter torpor. Northern Idaho ground squirrels frequently reentered torpor following a brief initial emergence from hibernacula and were more likely to do so earlier in spring or when challenged by residual snowpack. Female squirrels reentered torpor when confronted with relatively shallow snowpack upon emergence, whereas male squirrels reentered torpor in response to deeper spring snowpack. This novel behavior was previously assumed to be physiologically constrained in male ground squirrels by testosterone production required for spermatogenesis and activated by the circannual clock. Assessing surface conditions to decide when to terminate hibernation may help buffer these threatened squirrels against climate change. Documenting the extent to which other hibernators can facultatively alter emergence timing by reentering torpor after emergence will help identify which species are most likely to persist under climate change.

Weather influences survival probability in two coexisting mammals directly and indirectly via competitive asymmetry.

Ecologists have studied the role of interspecific competition in structuring ecological communities for decades. Differential weather effects on animal competitors may be a particularly important factor contributing to the outcome of competitive interactions, though few studies have tested this hypothesis in free-ranging animals. Specifically, weather might influence competitive dynamics by altering competitor densities and/or per-capita competitive effects on demographic vital rates. We used a 9-year data set of marked individuals to test for direct and interactive effects of weather and competitor density on survival probability in two coexisting mammalian congeners: Columbian ground squirrels (Urocitellus columbianus) and northern Idaho ground squirrels (Urocitellus brunneus). Ambient temperature and precipitation influenced survival probability in both species, but the effects of weather differed between the two species. Moreover, density of the larger Columbian ground squirrel negatively impacted survival probability in the smaller northern Idaho ground squirrel (but not vice versa), and the strength of the negative effect was exacerbated by precipitation. That is, cooler, wetter conditions benefited the larger competitor to the detriment of the smaller species. Our results suggest weather-driven environmental variation influences the competitive equilibrium between ecologically similar mammals of differential body size. Whether future climate change leads to the competitive exclusion of either species will likely depend on the mechanism(s) explaining the coexistence of these competing species. Divergent body size and, hence, differences in thermal tolerance and giving up densities offer potential explanations for the weather-dependent competitive asymmetry we documented, especially if the larger species competitively excludes the smaller species from habitat patches of shared preference via interference.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, the use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Furthermore, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. We conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

Mass spectrometers as cryoEM grid preparation instruments.

Structure determination by single-particle cryoEM has matured into a core structural biology technique. Despite many methodological advancements, most cryoEM grids are still prepared using the plunge-freezing method developed ∼40 years ago. Embedding samples in thin films and exposing them to the air-water interface often leads to sample damage and preferential orientation of the particles. Using native mass spectrometry to create cryoEM samples, potentially avoids these problems and allows the use of mass spectrometry sample isolation techniques during EM grid creation. We review the recent publications that have demonstrated protein complexes can be ionized, flown through the mass spectrometer, gently landed onto EM grids, imaged, and reconstructed in 3D. Although many uncertainties and challenges remain, the combination of cryoEM and MS has great potential.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes. Further, landing under cryogenic conditions can increase the diversity of particle orientations, allowing for improved 3D structural interpretation. Finally, we conclude that this approach allows for the direct coupling of mass spectrometry with cryo-electron microscopy.

Onto Grid Purification and 3D Reconstruction of Protein Complexes Using Matrix-Landing Native Mass Spectrometry.

Addressing mixtures and heterogeneity in structural biology requires approaches that can differentiate and separate structures based on mass and conformation. Mass spectrometry (MS) provides tools for measuring and isolating gas-phase ions. The development of native MS including electrospray ionization allowed for manipulation and analysis of intact noncovalent biomolecules as ions in the gas phase, leading to detailed measurements of structural heterogeneity. Conversely, transmission electron microscopy (TEM) generates detailed images of biomolecular complexes that show an overall structure. Our matrix-landing approach uses native MS to probe and select biomolecular ions of interest for subsequent TEM imaging, thus unifying information on mass, stoichiometry, heterogeneity, etc., available via native MS with TEM images. Here, we prepare TEM grids of protein complexes purified via quadrupolar isolation and matrix-landing and generate 3D reconstructions of the isolated complexes. Our results show that these complexes maintain their structure through gas-phase isolation.

Matrix-Landing Mass Spectrometry for Electron Microscopy Imaging of Native Protein Complexes.

Recently, we described the use of a chemical matrix for landing and preserving the cations of protein-protein complexes within a mass spectrometer (MS) instrument. By use of a glycerol-landing matrix, we used negative stain transmission electron microscopy (TEM) to obtain a three-dimensional (3D) reconstruction of landed GroEL complexes. Here, we investigate the utilities of other chemical matrices for their abilities to land, preserve, and allow for direct imaging of these cationic particles using TEM. We report here that poly(propylene) glycol (PPG) offers superior performance over glycerol for matrix landing. We demonstrated the utility of the PPG matrix landing using three protein-protein complexes─GroEL, the 20S proteasome core particle, and ß-galactosidase─and obtained a 3D reconstruction of each complex from matrix-landed particles. These structures have no detectable differences from the structures obtained using conventional preparation methods, suggesting the structures are well preserved at least to the resolution limit of the reconstructions (∼20 Å). We conclude that matrix landing offers a direct approach to couple native MS with TEM for protein structure determination.

Development of a novel radiofluorinated riboflavin probe for riboflavin receptor-targeting PET imaging.

Riboflavin receptor 3 (RFVT3) is a key protein in energetic metabolism reprogramming and is overexpressed in multiple cancers involved in malignant proliferation, angiogenesis, chemotherapy resistance, and immunosuppression. To enable non-invasive real-time quantification of RFVT3 in tumors, we sought to develop a suitable PET probe that would allow specific and selective RFVT3 imaging in vivo. A novel radiofluorinated riboflavin probe (18F-RFTA) based on riboflavin was synthesized and characterized in terms of radiochemical purity, hydrophilicity, binding affinity, and stability. Positron emission tomography (PET) imaging of 18F-RFTA was performed in U87MG tumor-bearing mice. Immunohistochemistry staining was carried out to determine the expression of RFVT3 in U87MG tumors. 18F-RFTA was characterized by high radiochemical purity and RFVT3 binding affinity, and remarkable stability in vitro and in vivo. Small-animal PET imaging with 18F-RFTA revealed significantly higher uptake in RFVT3-expressing U87MG tumors than in muscle. In conclusion, we have developed the first radiofluorinated riboflavin-based PET probe that is suitable for imaging RFVT3-positive tumors. The new target/probe system can be leveraged for extensive use in the diagnosis and treatment of RFVT3 overexpressing diseases, such as oncologic, cardiovascular, and neurodegenerative diseases.

Defining mitochondrial protein functions through deep multiomic profiling.

Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved5. Here, to establish a more complete functional compendium of human mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into protein function. Guided by these data, we discovered that PIGY upstream open reading frame (PYURF) is an S-adenosylmethionine-dependent methyltransferase chaperone that supports both complex I assembly and coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic mitochondrial disorder. We further linked the putative zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause cerebrofaciothoracic dysplasia. Our data, which can be explored through the interactive online MITOMICS.app resource, suggest biological roles for many other orphan mitochondrial proteins that still lack robust functional characterization and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases.

Three-dimensional structure determination of protein complexes using matrix-landing mass spectrometry.

Native mass spectrometry (MS) is increasingly used to provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids. We further describe the use of a chemical landing matrix that preserves the structural integrity of the deposited particles. With this system we obtain a three-dimensional reconstruction of the 800 kDa protein complex GroEL from gas-phase deposited GroEL ions. These data provide direct evidence that non-covalent protein complexes can indeed retain their condensed-phase structures following ionization and vaporization. Finally, we describe how further developments of this technology could pave the way to an integrated MS-EM technology with promise to provide improved cryo-EM sample preparation over conventional plunge-freezing techniques.

Recent advances and applications of microspheres and nanoparticles in transarterial chemoembolization for hepatocellular carcinoma.

Transarterial chemoembolization (TACE) is a recommended treatment for patients suffering from intermediate and advanced hepatocellular carcinoma (HCC). As compared to the conventional TACE, drug-eluting bead TACE demonstrates several advantages in terms of survival, treatment response, and adverse effects. The selection of embolic agents is critical to the success of TACE. Many studies have been performed on the modification of the structure, size, homogeneity, biocompatibility, and biodegradability of embolic agents. Continuing efforts are focused on efficient loading of versatile chemotherapeutics, controlled sizes for sufficient occlusion, real-time detection intra- and post-procedure, and multimodality imaging-guided precise treatment. Here, we summarize recent advances and applications of microspheres and nanoparticles in TACE for HCC. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Molecular Imaging of Tumor Microenvironment to Assess the Effects of Locoregional Treatment for Hepatocellular Carcinoma.

Liver cancer is one of the leading causes of cancer deaths worldwide. Among all primary liver cancers, hepatocellular carcinoma (HCC) is the most common type, representing 75%-85% of all primary liver cancer cases. Median survival following diagnosis of HCC is approximately 6 to 20 months due to late diagnosis in its course and few effective treatment options. Interventional therapy with minimal invasiveness is recognized as a promising treatment for HCC. However, due to the heterogeneity of HCC and the complexity of the tumor microenvironment, the long-term efficacy of treatment for HCC remains a challenge in the clinic. Tumor microenvironment, including factors such as hypoxia, angiogenesis, low extracellular pH, interstitial fluid pressure, aerobic glycolysis, and various immune responses, has emerged as a key contributor to tumor residual and progression after locoregional treatment for HCC. New approaches to noninvasively assess the treatment response and assist in the clinical decision-making process are therefore urgently needed. Molecular imaging tools enabling such an assessment may significantly advance clinical practice by allowing real-time optimization of treatment protocols for the individual patient. This review discusses recent advances in the application of molecular imaging technologies for noninvasively assessing changes occurring in the microenvironment of HCC after locoregional treatment.

Selective binding of anions by rigidified nanojars: sulfate vs. carbonate.

Selective binding and transport of highly hydrophilic anions is ubiquitous in nature, as anion binding proteins can differentiate between similar anions with over a million-fold efficiency. While comparable selectivity has occasionally been achieved for certain anions using small, artificial receptors, the selective binding of certain anions, such as sulfate in the presence of carbonate, remains a very challenging task. Nanojars of the formula [anion⊂{Cu(OH)(pz)}n]2- (pz = pyrazolate; n = 27-33) are totally selective for either CO32- or SO42- over anions such as NO3-, ClO4-, BF4-, Cl-, Br- and I-, but cannot differentiate between the two. We hypothesized that rigidification of the nanojar outer shell by tethering pairs of pyrazole moieties together will restrict the possible orientations of the OH hydrogen-bond donor groups in the anion-binding cavity of nanojars, similarly to anion-binding proteins, and will lead to selectivity. Indeed, by using either homoleptic or heteroleptic nanojars of the general formula [anion⊂Cun(OH)n(L2-L6)y(pz)n-2y]2- (n = 26-31) based on a series of homologous ligands HpzCH2(CH2)xCH2pzH (x = 0-4; H2L2-H2L6), selectivity for carbonate (with L2 and with L4-L6/pz mixtures) or for sulfate (with L3) has been achieved. The synthesis of new ligands H2L3, H2L4 and H2L5, X-ray crystal structures of H2L4 and the tetrahydropyranyl-protected derivatives (THP)2L4 and (THP)2L5, synthesis and characterization by electrospray-ionization mass spectrometry (ESI-MS) of carbonate- and sulfate-nanojars derived from ligands H2L2-H2L6, as well as detailed selectivity studies for CO32-vs. SO42- using these novel nanojars are presented.

Objective structured clinical examination for pharmacy students in Qatar: cultural and contextual barriers to assessment.

This study aimed to evaluate the feasibility and psychometric defensibility of implementing a comprehensive objective structured clinical examination (OSCE) on the complete pharmacy programme for pharmacy students in a Middle Eastern context, and to identify facilitators and barriers to implementation within new settings. Eight cases were developed, validated, and had standards set according to a blueprint, and were assessed with graduating pharmacy students. Assessor reliability was evaluated using inter-class coefficients (ICCs). Concurrent validity was evaluated by comparing OSCE results to professional skills course grades. Field notes were maintained to generate recommendations for implementation in other contexts. The examination pass mark was 424 points out of 700 (60.6%). All 23 participants passed. Mean performance was 74.6%. Low to moderate inter-rater reliability was obtained for analytical and global components (average ICC 0.77 and 0.48, respectively). In conclusion, OSCE was feasible in Qatar but context-related validity and reliability concerns must be addressed prior to future iterations in Qatar and elsewhere.

Objective structured clinical examination for pharmacy students in Qatar: cultural and contextual barriers to assessment

This study aimed to evaluate the feasibility and psychometric defensibility of implementing a comprehensive objective structured clinical examination [OSCE] on the complete pharmacy programme for pharmacy students in a Middle Eastern context, and to identify facilitators and barriers to implementation within new settings. Eight cases were developed, validated, and had standards set according to a blueprint, and were assessed with graduating pharmacy students. Assessor reliability was evaluated using inter-class coefficients [ICCs]. Concurrent validity was evaluated by comparing OSCE results to professional skills course grades. Field notes were maintained to generate recommendations for implementation in other contexts. The examination pass mark was 424 points out of 700 [60.6%]. All 23 participants passed. Mean performance was 74.6%. Low to moderate inter-rater reliability was obtained for analytical and global components [average ICC 0.77 and 0.48, respectively]. In conclusion, OSCE was feasible in Qatar but context-related validity and reliability concerns must be addressed prior to future iterations in Qatar and elsewhere

La présente étude avait pour objectif d'évaluer la faisabilité et la solidité psychométrique de la mise en place d'un examen clinique objectif structuré [ECOS] du programme pharmaceutique complet pour les étudiants en pharmacie au Moyen-Orient, ainsi que d'identifier les leviers et les obstacles à sa mise en place dans les nouveaux établissements. Huit cas ont été élaborés, validés, se sont vus attribuer des normes en fonction d'un modèle, et ont ensuite été soumis à des étudiants en pharmacie pour évaluation. La fiabilité des examinateurs a été mesurée au moyen de coefficients intra-classe [CIC]. La validité concourante a été évaluée en comparant les résultats de l'ECOS aux notes finales de cours sur les compétences professionnelles. Des notes d'observation ont été conservées en vue de la production de recommandations pour la mise en place du test dans d'autres contextes. La note de passage de l'examen était de 424 points sur 700 [soit 60,6%]. Les 23 participants ont tous réussi l'examen. La performance moyenne était de 74,6%. Des taux de fiabilité intra-examinateur faible à moyen ont été obtenus pour les composantes analytiques et globales [CIC moyen de 0,77 et 0,48 respectivement]. Pour conclure, l'ECOS était réalisable au Qatar, mais les questions de validité et de fiabilité dépendant du contexte doivent être prises en compte avant toute reproduction du test au Qatar et dans d'autres pays

Integrating family medicine and pharmacy to advance primary care therapeutics.

The prevalence of suboptimal prescribing of medications is well documented. Patients are often undertreated or not offered therapeutic treatments that are likely to confer benefit. As a result, drug-related hospital admissions are common and often preventable. Improvements to the health-care system are clearly needed in order to maximize the benefits that can be derived from medications. Many countries are changing their primary health-care systems to improve the quality of health-care delivery. One main transformation is the use of multidisciplinary care teams to provide care in a coordinated manner often from the same location or by using the common medical record of the patients. It has been demonstrated that pharmacists can improve prescribing, reduce health-care utilization and medication costs, and contribute to clinical improvements in many chronic medical conditions, such as cardiovascular disease, diabetes, and psychiatric illness. However, the effect of integrating a pharmacist providing general services into a primary care group has not been extensively studied. The Integrating Family Medicine and Pharmacy to Advance Primary Care Therapeutics (IMPACT) project was designed to provide a real-world demonstration of the feasibility of integrating the pharmacist into primary care office practice. This article provides a description of the IMPACT project participants; the IMPACT practice model and the concepts incorporated in its development; some initial results from the program evaluation; sustainability of the model; and some reflections on the implementation of the practice model.

Professional students' perceptions of the value, role, and impact of science in clinical education.

Health sciences curricula are, by definition, built on a foundation of scientific knowledge and inquiry. Professional programs in fields such as medicine, pharmacy, nursing, physical/occupational therapy, and dentistry purport to provide students with the ability to translate scientific knowledge and understanding into clinical practice, for improving the health and well-being of patients. Indeed, the scientific underpinning of each health profession is a point of pride, a reason these roles exist in the first place and are accorded the prestige of being called a "profession".