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The application of two-photon excitation (TPE) in the study of light-responsive materials holds immense potential due to its deeper penetration and reduced photodamage. Despite these benefits, TPE has been underutilised in the investigation of the photoinduced spin crossover (SCO) phenomenon. Here, we employ TPE to delve into the out-of-equilibrium dynamics of a SCO FeII dimer of the form [FeII(HL)2]2(BF4)4·2MeCN (HL = 3,5-bis{6-(2,2'-bipyridyl)}pyrazole). Optical transient absorption (OTA) spectroscopy in solution proves that the same dynamics take place under both one-photon excitation (OPE) and TPE. The results show the emergence of the photoinduced high spin state in less than 2 ps and with a lifetime of 147 ns. Time-resolved photocrystallography (TRXRD) reveals a single molecular reorganisation within the first 500 ps following TPE. Additionally, variable temperature single crystal X-ray diffraction (VTSCXRD) and magnetic susceptibility measurements confirm that the thermal transition is silenced by the solvent. While the results of the OTA and TRXRD utilising TPE are intriguing, the high pump fluencies required to excite enough metal centres to the high spin state may impair its practical application. Nonetheless, this study sheds light on the potential of TPE for the investigation of the out-of-equilibrium dynamics of SCO complexes.
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Time-resolved X-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data.
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DJ-1 (PARK7) is an intensively studied protein whose cytoprotective activities are dysregulated in multiple diseases. DJ-1 has been reported as having two distinct enzymatic activities in defense against reactive carbonyl species that are difficult to distinguish in conventional biochemical experiments. Here, we establish the mechanism of DJ-1 using a synchrotron-compatible version of mix-and-inject-serial crystallography (MISC), which was previously performed only at XFELs, to directly observe DJ-1 catalysis. We designed and used new diffusive mixers to collect time-resolved Laue diffraction data of DJ-1 catalysis at a pink beam synchrotron beamline. Analysis of structurally similar methylglyoxal-derived intermediates formed through the DJ-1 catalytic cycle shows that the enzyme catalyzes nearly two turnovers in the crystal and defines key aspects of its glyoxalase mechanism. In addition, DJ-1 shows allosteric communication between a distal site at the dimer interface and the active site that changes during catalysis. Our results rule out the widely cited deglycase mechanism for DJ-1 action and provide an explanation for how DJ-1 produces L-lactate with high chiral purity.
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The unfolding dynamics of ubiquitin were studied using a combination of x-ray solution scattering (XSS) and molecular dynamics (MD) simulations. The kinetic analysis of the XSS ubiquitin signals showed that the protein unfolds through a two-state process, independent of the presence of destabilizing salts. In order to characterize the ensemble of unfolded states in atomic detail, the experimental XSS results were used as a constraint in the MD simulations through the incorporation of x-ray scattering derived potential to drive the folded ubiquitin structure toward sampling unfolded states consistent with the XSS signals. We detail how biased MD simulations provide insight into unfolded states that are otherwise difficult to resolve and underscore how experimental XSS data can be combined with MD to efficiently sample structures away from the native state. Our results indicate that ubiquitin samples unfolded in states with a high degree of loss in secondary structure yet without a collapse to a molten globule or fully solvated extended chain. Finally, we propose how using biased-MD can significantly decrease the computational time and resources required to sample experimentally relevant nonequilibrium states.
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Simulación de Dinámica Molecular , Desplegamiento Proteico , Ubiquitina , Ubiquitina/química , Difracción de Rayos X , CinéticaRESUMEN
Understanding protein structure and kinetics under physiological conditions is crucial for elucidating complex biological processes. While time-resolved (TR) techniques have advanced to track molecular actions, their practical application in biological reactions is often confined to reversible photoreactions within limited experimental parameters due to inefficient sample utilization and inflexibility of experimental setups. Here, we introduce serial X-ray liquidography (SXL), a technique that combines time-resolved X-ray liquidography with a fixed target of serially arranged microchambers. SXL breaks through the previously mentioned barriers, enabling microgram-scale TR studies of both irreversible and reversible reactions of even a non-photoactive protein. We demonstrate its versatility in studying a wide range of biological reactions, highlighting its potential as a flexible and multi-dimensional assay framework for kinetic and structural characterization. Leveraging X-ray free-electron lasers and micro-focused X-ray pulses promises further enhancements in both temporal resolution and minimizing sample quantity. SXL offers unprecedented insights into the structural and kinetic landscapes of molecular actions, paving the way for a deeper understanding of complex biological processes.
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Proteínas , Cinética , Rayos X , Proteínas/química , Conformación Proteica , Rayos Láser , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismoRESUMEN
Hibernating animals demonstrate a remarkable ability to withstand extreme physiological brain changes without triggering adverse neuroinflammatory responses. While hibernators may offer valuable insights into the neuroprotective mechanisms inherent to hibernation, studies using such species are constrained by the limited availability of molecular tools. Laboratory mice may serve as an alternative, entering states of hypometabolism and hypothermia similar to the torpor observed in hibernation when faced with energy shortage. Notably, prolonged calorie restriction (CR) induces serial daily torpor patterns in mice, comparable to species that utilize daily hibernation. Here, we examined the neuroinflammatory response in the hippocampus of male C57BL/6 mice undergoing serial daily torpor induced by a 30% CR for 4 weeks. During daily torpor episodes, CR mice exhibited transient increases in TNF-α mRNA expression, which normalized upon arousal. Concurrently, the CA1 region of the hippocampus showed persistent morphological changes in microglia, characterized by reduced cell branching, decreased cell complexity and altered shape. Importantly, these morphological changes were not accompanied by evident signs of astrogliosis or oxidative stress, typically associated with detrimental neuroinflammation. Collectively, the adaptive nature of the brain's inflammatory response to CR-induced torpor in mice parallels observations in hibernators, highlighting its value for studying the mechanisms of brain resilience during torpor. Such insights could pave the way for novel therapeutic interventions in stroke and neurodegenerative disorders in humans.
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There are currently 5 million active high school, collegiate, professional, and master athletes in the United States. Regular intense exercise by these athletes can promote structural, electrical and functional remodeling of the heart, which is termed the "athlete's heart." In addition, regular intense exercise can lead to pathological adaptions that promote or worsen cardiac disease. Many of the athletes in the United States seek medical care. Consequently, physicians must be aware of the normal cardiac anatomy and physiology of the athlete, the differentiation of the normal athlete heart from the athlete with cardiomyopathy, and the contemporary care of the athlete with a cardiomyopathy. In athletes with persistent cardiovascular symptoms, investigations should include a detailed history and physical examination, an ECG, a transthoracic echocardiogram, and in athletes in whom the diagnosis is uncertain, a maximal exercise stress test or a continuous ECG recording, and cardiac magnetic resonance imaging or cardiac computed tomography angiography when definition of the coronary anatomy or characterization of the aorta and the aortic great vessels is indicated. This article discusses the differentiation of the normal athlete with physiologic cardiac remodeling from the athlete with hypertrophic, dilated or arrhythmogenic ventricular cardiomyopathy (ACM). The ECG changes in trained athletes that are considered normal, borderline, or abnormal are listed. In addition, the normal echocardiographic measurements for athletes who consistently participate in endurance, power, combined or heterogeneous sports are enumerated and discussed. Algorithms are listed that are useful in the diagnosis of trained athletes with borderline or abnormal echocardiographic measurements suggestive of cardiomyopathies along with the major and minor criteria for the diagnosis of ACM in athletes. Thereafter, the treatment of athletes with hypertrophic, dilated, and arrhythmogenic right ventricular cardiomyopathies are reviewed. The distinction between physiologic changes and pathologic changes in the hearts of athletes has important therapeutic and prognostic implications. Failure by the physician to correctly diagnose an athlete with hypertrophic cardiomyopathy, dilated cardiomyopathy, or ACM, can lead to the sudden cardiac arrest and death of the athlete during training or sports competition. Conversely, an incorrect diagnosis by a physician of cardiac pathology in a normal athlete can lead to an unnecessary restriction of athlete training and competition with resultant significant emotional, psychological, financial, and long-term health consequences in the athlete.
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Atletas , Cardiomiopatías , Electrocardiografía , Remodelación Ventricular , Humanos , Remodelación Ventricular/fisiología , Cardiomiopatías/diagnóstico , Cardiomiopatías/fisiopatología , Diagnóstico Diferencial , Cardiomegalia Inducida por el Ejercicio/fisiología , Ecocardiografía/métodosRESUMEN
A major goal in biomedical science is to move beyond static images of proteins and other biological macromolecules to the internal dynamics underlying their function. This level of study is necessary to understand how these molecules work and to engineer new functions and modulators of function. Stemming from a visionary commitment to this problem by Keith Moffat decades ago, a community of structural biologists has now enabled a set of x-ray scattering technologies for observing intramolecular dynamics in biological macromolecules at atomic resolution and over the broad range of timescales over which motions are functionally relevant. Many of these techniques are provided by BioCARS, a cutting-edge synchrotron radiation facility built under Moffat leadership and located at the Advanced Photon Source at Argonne National Laboratory. BioCARS enables experimental studies of molecular dynamics with time resolutions spanning from 100 ps to seconds and provides both time-resolved x-ray crystallography and small- and wide-angle x-ray scattering. Structural changes can be initiated by several methods-UV/Vis pumping with tunable picosecond and nanosecond laser pulses, substrate diffusion, and global perturbations, such as electric field and temperature jumps. Studies of dynamics typically involve subtle perturbations to molecular structures, requiring specialized computational techniques for data processing and interpretation. In this review, we present the challenges in experimental macromolecular dynamics and describe the current state of experimental capabilities at this facility. As Moffat imagined years ago, BioCARS is now positioned to catalyze the scientific community to make fundamental advances in understanding proteins and other complex biological macromolecules.
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Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to poor experimental access. This shortcoming is evident with Escherichia coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we describe ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments to map the conformational dynamics of the Michaelis complex of DHFR. We resolve coupled global and local motions and find that these motions are engaged by the protonated substrate to promote efficient catalysis. This result suggests a fundamental design principle for multistep enzymes in which pre-existing dynamics enable intermediates to drive rapid electrostatic reorganization to facilitate subsequent chemical steps.
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Aminoácidos , Electricidad , Catálisis , Escherichia coli , Conformación Molecular , Tetrahidrofolato DeshidrogenasaRESUMEN
Health Improvement Through Employee Control (HITEC) is a 16-year program directed toward the health of corrections personnel and developed through the application of the principles of Participatory Action Research (PAR) and participatory ergonomics. Its impetus has always been the adverse health status of the corrections workforce: early mortality, depression, obesity, and hypertension. The HITEC program trained small "Design Teams" (DTs) of front-line personnel in participatory methods for intervention design for health improvement and organizational change in line with the Total Worker Health® principles. Periodic surveys and physical testing were introduced for longitudinal assessments. Comparative interventions at comparable sites included DTs without a priori assignation, problem-focused kaizen effectiveness teams (KETs), and bargaining unit-centered DTs. DT resilience and the replacement of members who transferred facilities or retired was aided by novel cooperative administrative structures. DT-generated interventions included stress lounges, changes in critical event report writing, a joint program with trained inmates to improve air quality, and training in staff mental health and sleep behavior. A specialized peer-to-peer Health Mentoring Program (HMP) paired new officers with trained peers. Many interventions and program features were institutionalized, thus improving prospects for self-supporting program longevity. Participatory interventions designed and supported by the corrections workforce were found to be both feasible and exceptionally effective.
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Salud Laboral , Humanos , Connecticut , Evaluación de Programas y Proyectos de Salud/métodos , Lugar de Trabajo , Promoción de la Salud/métodosRESUMEN
BACKGROUND: Sepsis is a life-threatening syndrome characterized by acute loss of organ function due to infection. Sepsis survivors are at risk for long-term comorbidities, have a reduced Quality of Life (QoL), and are prone to increased long-term mortality. The societal impact of sepsis includes its disease burden and indirect economic costs. However, these societal costs of sepsis are not fully understood. This study assessed sepsis's disease-related and indirect economic costs in the Netherlands. METHODS: Sepsis prevalence, incidence, sepsis-related mortality, hospitalizations, life expectancy, QoL population norms, QoL reduction after sepsis, and healthcare use post-sepsis were obtained from previous literature and Statistics Netherlands. We used these data to estimate annual Quality-adjusted Life Years (QALYs), productivity loss, and increase in healthcare use post-sepsis. A sensitivity analysis was performed to analyze the burden and indirect economic costs of sepsis under alternative assumptions, resulting in a baseline, low, and high estimated burden. The results are presented as a baseline (low-high burden) estimate. RESULTS: The annual disease burden of sepsis is approximately 57,304 (24,398-96,244; low-high burden) QALYs. Of this, mortality accounts for 26,898 (23,166-31,577) QALYs, QoL decrease post-sepsis accounts for 30,406 (1232-64,667) QALYs. The indirect economic burden, attributed to lost productivity and increased healthcare expenditure, is estimated at 416.1 (147.1-610.7) million utilizing the friction cost approach and 3.1 (0.4-5.7) billion using the human capital method. Cumulatively, the combined disease and indirect economic burdens range from 3.8 billion (friction method) to 6.5 billion (human capital method) annually within the Netherlands. CONCLUSIONS: Sepsis and its complications pose a substantial disease and indirect economic burden to the Netherlands, with an indirect economic burden due to production loss that is potentially larger than the burden due to coronary heart disease or stroke. Our results emphasize the need for future studies to prevent sepsis, saving downstream costs and decreasing the economic burden.
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Calidad de Vida , Sepsis , Humanos , Países Bajos/epidemiología , Sepsis/epidemiología , Costo de Enfermedad , HospitalizaciónRESUMEN
Cardiac disease is the leading cause of death in women. Among women with recurrent chest pain, abnormal electrocardiograms, and/or stress tests who undergo coronary angiography, as many as 50% have normal or <50% coronary artery obstructive disease. Pharmacologic stress assessment of coronary artery flow reserve in these women frequently demonstrates an inability to increase blood flow to >2.5 times normal flow. Contributory factors include abnormal epicardial or microvascular reactivity, microvascular remodeling or rarefaction, autonomic dysfunction, or coronary plaque rupture/erosion. Assessment is necessary of serum biomarkers and coronary artery flow reserve, fractional flow reserve, microvascular resistance, and epicardial/microvascular spasm. Aggressive treatment of women with positive tests is necessary because these women have an increased incidence of recurrent chest pain, repeated hospitalizations and coronary angiograms, and cardiac death.
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Enfermedad de la Arteria Coronaria , Reserva del Flujo Fraccional Miocárdico , Humanos , Femenino , Vasos Coronarios/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/diagnóstico , Enfermedad de la Arteria Coronaria/terapia , Dolor en el Pecho , Angiografía Coronaria , Isquemia , Circulación Coronaria/fisiologíaRESUMEN
Air pollution is responsible worldwide for 9-12 million deaths annually. The major contributor to air pollution is particulate matter ≤2.5 µg per cubic meter of air (PM2.5) from vehicles, industrial emissions, and wildfire smoke. United States ambient air standards recommend annual average PM2.5 concentrations of ≤12 µg/m³ while European standards allow an average annual PM2.5 concentration of ≤20 µg/m3. However, significant PM2.5 cardiovascular and pulmonary health risks exist below these concentrations. Chronic PM2.5 exposure significantly increases major cardiovascular and pulmonary event risks in Americans by 8 to more than 20% for each 10-µg/m3 increase in PM2.5. PM2.5-induced increases in lipid peroxidation, induction of vascular inflammation and endothelial cell injury initiate and propagate respiratory diseases, coronary and carotid atherosclerosis. PM2.5 can cause atherosclerotic vascular plaque rupture and myocardial infarction and stroke by activating metalloproteinases. This article discusses PM2.5 effects on the cardiovascular and pulmonary systems, specific PM2.5 pathophysiologic mechanisms contributing to cardiopulmonary disease, and preventive measures to limit the cardiovascular and pulmonary effects of PM2.5.
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Contaminantes Atmosféricos , Contaminación del Aire , Enfermedades Cardiovasculares , Placa Aterosclerótica , Humanos , Estados Unidos/epidemiología , Material Particulado/efectos adversos , Material Particulado/análisis , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/etiología , Contaminantes Atmosféricos/efectos adversos , Contaminantes Atmosféricos/análisis , Contaminación del Aire/efectos adversos , Contaminación del Aire/análisis , Factores de Riesgo , Placa Aterosclerótica/etiologíaRESUMEN
Background: Type 2 diabetes disproportionately affects individuals of non-White ethnicity through a complex interaction of multiple factors. Therefore, early disease detection and prediction are essential and require tools that can be deployed on a large scale. We aimed to tackle this problem by developing questionnaire-based prediction models for type 2 diabetes prevalence and incidence for multiple ethnicities. Methods: In this proof of principle analysis, logistic regression models to predict type 2 diabetes prevalence and incidence, using questionnaire-only variables reflecting health state and lifestyle, were trained on the White population of the UK Biobank (n = 472,696 total, aged 37-73 years, data collected 2006-2010) and validated in five other ethnicities (n = 29,811 total) and externally in Lifelines (n = 168,205 total, aged 0-93 years, collected between 2006 and 2013). In total, 631,748 individuals were included for prevalence prediction and 67,083 individuals for the eight-year incidence prediction. Type 2 diabetes prevalence in the UK Biobank ranged between 6% in the White population to 23.3% in the South Asian population, while in Lifelines, the prevalence was 1.9%. Predictive accuracy was evaluated using the area under the receiver operating characteristic curve (AUC), and a detailed sensitivity analysis was conducted to assess potential clinical utility. We compared the questionnaire-only models to models containing physical measurements and biomarkers as well as to clinical non-laboratory type 2 diabetes risk tools and conducted a reclassification analysis. Findings: Our algorithms accurately predicted type 2 diabetes prevalence (AUC = 0.901) and eight-year incidence (AUC = 0.873) in the White UK Biobank population. Both models replicated well in the Lifelines external validation, with AUCs of 0.917 and 0.817 for prevalence and incidence, respectively. Both models performed consistently well across different ethnicities, with AUCs of 0.855-0.894 for prevalence and 0.819-0.883 for incidence. These models generally outperformed two clinically validated non-laboratory tools and correctly reclassified >3,000 additional cases. Model performance improved with the addition of blood biomarkers but not with the addition of physical measurements. Interpretation: Our findings suggest that easy-to-implement, questionnaire-based models could be used to predict prevalent and incident type 2 diabetes with high accuracy across several ethnicities, providing a highly scalable solution for population-wide risk stratification. Future work should determine the effectiveness of these models in identifying undiagnosed type 2 diabetes, validated in cohorts of different populations and ethnic representation. Funding: University Medical Center Groningen.
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The structural evolution of spin crossover (SCO) complexes during their spin transition at equilibrium and out-of-equilibrium conditions needs to be understood to enable their successful utilisation in displays, actuators and memory components. In this study, diffraction techniques were employed to study the structural changes accompanying the temperature increase and the light irradiation of a defect [2 × 2] triiron(II) metallogrid of the form [FeII3LH2(HLH)2](BF4)4·4MeCN (FE3), LH = 3,5-bis{6-(2,2'-bipyridyl)}pyrazole. Although a multi-temperature crystallographic investigation on single crystals evidenced that the compound does not exhibit a thermal spin transition, the structural analysis of the defect grid suggests that the flexibility of the grid, provided by a metal-devoid vertex, leads to interesting characteristics that can be used for intermolecular cooperativity in related thermally responsive systems. Time-resolved photocrystallography results reveal that upon excitation with a ps laser pulse, the defect grid shows the first two steps of the out-of-equilibrium process, namely the photoinduced and elastic steps, occurring at the ps and ns time scales, respectively. Similar to a previously reported [2 × 2] tetrairon(II) metallogrid, FE3 exhibits a local distortion of the entire grid during the photoinduced step and a long-range distortion of the lattice during the elastic step. Although the lifetime of the pure photoinduced high spin (HS) state is longer in the tetranuclear grid than in the defect grid, suggesting that the global nuclearity plays a crucial role for the lifetime of the photoinduced species, the influence of the co-crystalising solvent on the lifetime of the photoinduced HS state remains unknown. This study sheds light on the out-of-equilibrium dynamics of a thermally silent defect triiron SCO metallogrid.
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The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservation limits, yet it remains unclear how exactly organ preservation solutions exert their cold protection. Thus, we explored cooling effects on lipid peroxidation and adenosine triphosphate (ATP) levels and the actions of blockers of apoptosis and ferroptosis, and of compounds enhancing mitochondrial function. Cooling and rewarming experiments were performed in a cellular transplantation model using Human Embryonic Kidney (HEK) 293 cells. Cell viability was assessed by neutral red assay. Lipid peroxidation levels were measured by Western blot against 4-Hydroxy-Nonenal (4HNE) and the determination of Malondialdehyde (MDA). ATP was measured by luciferase assay. Cooling beyond 5 h in Dulbecco's Modified Eagle Medium (DMEM) induced complete cell death in HEK293, whereas cooling in UW preserved ~60% of the cells, with a gradual decline afterwards. Cooling-induced cell death was not precluded by inhibiting apoptosis. In contrast, the blocking of ferroptosis by Ferrostatin-1 or maintaining of mitochondrial function by the 6-chromanol SUL150 completely inhibited cell death both in DMEM- and UW-cooled cells. Cooling for 24 h in UW followed by rewarming for 15 min induced a ~50% increase in MDA, while concomitantly lowering ATP by >90%. Treatment with SUL150 of cooled and rewarmed HEK293 effectively precluded the increase in MDA and preserved normal ATP in both DMEM- and UW-cooled cells. Likewise, treatment with Ferrostatin-1 blocked the MDA increase and preserved the ATP of rewarmed UW HEK293 cells. Cooling-induced HEK293 cell death from hypothermia and/or rewarming was caused by ferroptosis rather than apoptosis. UW slowed down ferroptosis during hypothermia, but lipid peroxidation and ATP depletion rapidly ensued upon rewarming, ultimately resulting in complete cell death. Treatment throughout UW cooling with small-molecule Ferrostatin-1 or the 6-chromanol SUL150 effectively prevented ferroptosis, maintained ATP, and limited lipid peroxidation in UW-cooled cells. Counteracting ferroptosis during cooling in UW-based preservation solutions may provide a simple method to improve graft survival following cold static cooling.
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Ferroptosis , Hipotermia , Humanos , Células HEK293 , Recalentamiento , Universidades , Wisconsin , Adenosina Trifosfato/metabolismo , Frío , Alopurinol/farmacología , Glutatión/farmacología , Insulina/farmacología , Preservación de ÓrganosRESUMEN
Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to lack of experimental access. This shortcoming is evident with E. coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we present ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments that enable identification of coupled conformational changes in DHFR. We identify a global hinge motion and local networks of structural rearrangements that are engaged by substrate protonation to regulate solvent access and promote efficient catalysis. The resulting mechanism shows that DHFR's two-step catalytic mechanism is guided by a dynamic free energy landscape responsive to the state of the substrate.
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Hibernating mammals have developed many physiological adaptations to accommodate their decreased metabolism, body temperature, heart rate and prolonged immobility without suffering organ injury. During hibernation, the animals must suppress blood clotting to survive prolonged periods of immobility and decreased blood flow that could otherwise lead to the formation of potentially lethal clots. Conversely, upon arousal hibernators must be able to quickly restore normal clotting activity to avoid bleeding. Studies in multiple species of hibernating mammals have shown reversible decreases in circulating platelets, cells involved in hemostasis, as well as in protein coagulation factors during torpor. Hibernator platelets themselves also have adaptations that allow them to survive in the cold, while those from non-hibernating mammals undergo lesions during cold exposure that lead to their rapid clearance from circulation when re-transfused. While platelets lack a nucleus with DNA, they contain RNA and other organelles including mitochondria, in which metabolic adaptations may play a role in hibernator's platelet resistance to cold induced lesions. Finally, the breakdown of clots, fibrinolysis, is accelerated during torpor. Collectively, these reversible physiological and metabolic adaptations allow hibernating mammals to survive low blood flow, low body temperature, and immobility without the formation of clots during torpor, yet have normal hemostasis when not hibernating. In this review we summarize blood clotting changes and the underlying mechanisms in multiple species of hibernating mammals. We also discuss possible medical applications to improve cold preservation of platelets and antithrombotic therapy.
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More than 1.2 million people in the United States have Human Immunodeficiency Virus (HIV) infections but 13% of these people are unaware of their HIV infection. Current combination antiretroviral therapy (ART) does not cure HIV infection but rather suppresses the infection with the virus persisting indefinitely in latent reservoirs in the body. As a consequence of ART, HIV infection has changed from a fatal disease in the past to a chronic disease today. Currently in the United States, more than 45% of HIV+ individuals are greater than 50 years of age and 25% will be greater than 65 years of age by 2030. Atherosclerotic cardiovascular disease (CVD), including myocardial infarction, stroke, and cardiomyopathy, is now the major cause of death in HIV+ individuals. Novel risk factors, including chronic immune activation and inflammation in the body, antiretroviral therapy, and traditional CVD risk factors, such as tobacco and illicit drug use, hyperlipidemia, the metabolic syndrome, diabetes mellitus, hypertension, and chronic renal disease, contribute to cardiovascular atherosclerosis. This article discusses the complex interactions involving HIV infection, the novel and traditional risk factors for CVD, and the antiretroviral HIV therapies which can contribute to CVD in HIV-infected people. In addition, the treatment of HIV+ patients with acute myocardial infarction, stroke, and cardiomyopathy/heart failure are discussed. Current recommended ART and their major side effects are summarized in table format. All medical personnel must be aware of the increasing incidence of CVD on the morbidity and mortality in HIV infected patients and must be watchful for the presence of CVD in their patients with HIV.