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The 2019 coronavirus disease (COVID-19) pandemic has demonstrated the importance of predicting, identifying, and tracking mutations throughout a pandemic event. As the COVID-19 global pandemic surpassed one year, several variants had emerged resulting in increased severity and transmissibility. Here, we used PCR as a surrogate for viral load and consequent severity to evaluate the real-world capabilities of a genome-based clinical severity predictive algorithm. Using a previously published algorithm, we compared the viral genome-based severity predictions to clinically derived PCR-based viral load of 716 viral genomes. For those samples predicted to be "severe" (probability of severe illness >0.5), we observed an average cycle threshold (Ct) of 18.3, whereas those in in the "mild" category (severity probability <0.5) had an average Ct of 20.4 (P = 0.0017). We also found a nontrivial correlation between predicted severity probability and cycle threshold (r = -0.199). Finally, when divided into severity probability quartiles, the group most likely to experience severe illness (=75% probability) had a Ct of 16.6 (n = 10), whereas the group least likely to experience severe illness (<25% probability) had a Ct of 21.4 (n = 350) (P = 0.0045). Taken together, our results suggest that the severity predicted by a genome-based algorithm can be related to clinical diagnostic tests and that relative severity may be inferred from diagnostic values. [ABSTRACT FROM AUTHOR]

Efforts to develop effective and safe drugs for treatment of tuberculosis require preclinical evaluation in animal models. Alongside efficacy testing of novel therapies, effects on pulmonary pathology and disease progression are monitored by using histopathology images from these infected animals. To compare the severity of disease across treatment cohorts, pathologists have historically assigned a semi-quantitative histopathology score that may be subjective in terms of their training, experience, and personal bias. Manual histopathology therefore has limitations regarding reproducibility between studies and pathologists, potentially masking successful treatments. This report describes a pathologist-assistive software tool that reduces these user limitations, while providing a rapid, quantitative scoring system for digital histopathology image analysis. The software, called 'Lesion Image Recognition and Analysis' (LIRA), employs convolutional neural networks to classify seven different pathology features, including three different lesion types from pulmonary tissues of the C3HeB/FeJ tuberculosis mouse model. LIRA was developed to improve the efficiency of histopathology analysis for mouse tuberculosis infection models, this approach has also broader applications to other disease models and tissues. The full source code and documentation is available from https://Github.com/TB-imaging/LIRA. [ABSTRACT FROM AUTHOR]

Problematic pornography viewing (PPV) is a growing concern. Based on a masculine gender role strain framework, individuals endorsing traditional masculinity ideology (TMI) may be especially drawn to pornography. However, relatively few studies have explored how TMI is related to PPV. Furthermore, no known studies have explored how these connections differ in men and women. To address these gaps, we conducted a large survey of 310 men and 469 women in the United States assessing multiple PPV and TMI dimensions. A bifactor structural equation model was used to regress PPV domains onto global and specific TMI factors. Invariance testing further examined the moderating effects of participants' gender in the model. Results indicated that global TMI was unrelated to men's PPV. However, men's dominance ideologies predicted greater functional problems and excessive pornography use. Men's restrictive emotionality and heterosexist ideologies predicted control difficulties with pornography use and using pornography to escape negative emotions. Additionally, men's avoidance of femininity ideology predicted excessive pornography use and control difficulties. For women, only global TMI was associated with functional problems. Invariance testing suggested the observed gender differences were not due to underlying discrepancies in the measurement of TMI or PPV. Clinical interventions for PPV that incorporate gender role themes are recommended. [ABSTRACT FROM AUTHOR]

Background Although randomization is considered essential for causal inference, it is often not possible to randomize in nutrition and obesity research. To address this, we develop a framework for an experimental design--packet randomized experiments (PREs), which improves causal inferences when randomization on a single treatment variable is not possible. This situation arises when subjects are randomly assigned to a condition (such as a new roommate) which varies in one characteristic of interest (such as weight), but also varies across many others. There has been no general discussion of this experimental design, including its strengths, limitations, and statistical properties. As such, researchers are left to develop and apply PREs on an ad hoc basis, limiting its potential to improve causal inferences among nutrition and obesity researchers. Methods We introduce PREs as an intermediary design between randomized controlled trials and observational studies. We review previous research that used the PRE design and describe its application in obesity-related research, including random roommate assignments, heterochronic parabiosis, and the quasi-random assignment of subjects to geographic areas. We then provide a statistical framework to control for potential packet-level confounders not accounted for by randomization. Results Packet randomized experiments have successfully been used to improve causal estimates of the effect of roommates, altitude, and breastfeeding on weight outcomes. When certain assumptions are met, PREs can asymptotically control for packet-level characteristics. This has the potential to statistically estimate the effect of a single treatment even when randomization to a single treatment did not occur. Conclusions Applying PREs to obesity-related research will improve decisions about clinical, public health, and policy actions insofar as it offers researchers new insight into cause and effect relationships among variables. [ABSTRACT FROM AUTHOR]

Given the high prevalence of Chagas disease in the Americas, we targeted the unique arginyl-tRNA synthetase of its causative agent Trypanosoma cruzi. Among their many possible uses, naphthalene-derived fluorescent ligands, such as ANS and bis-ANS, may be employed in pharmacokinetic research. Although ANS and bis-ANS have become prominent fluorescent probes for protein characterization, the structural and spectroscopic characteristics of protein-ANS/bis-ANS complexes remain largely unknown. Both fluorescent dyes bind to either the folded or partially folded hydrophobic regions of proteins. Additionally, they serve to identify molten globule-like intermediates. These probes have been used to study the folding problems of protein structures and the mechanisms of protein-protein interactions. ANS and bis-ANS exhibited significant enhancement and blue shift in their emission spectra upon binding to TcArgRS, the primary enzyme responsible for attaching l-arginine to its corresponding tRNA. Through fluorescence spectroscopy and computational studies, we concluded that bis-ANS binds more tightly to TcArgRS and that ATP affects bis-ANS fluorescence signal. Thus, these probes are useful resources for studying the intricate intermolecular relationships between proteins in terms of their structure, function, and mechanism. Our study provides a framework for identifying the hydrophobic regions present in TcArgRS. The utilization of hydrophobic patches on proteins for drug targeting is noteworthy because they can assist in identifying regions on the surface of proteins that are likely to interact with ligands. These patches help identify hotspot residues that play a vital role in determining binding affinity. Drugs are mainly small and hydrophobic in nature, and they target protein surfaces which have complementary properties. In this study, we elucidated the potential of TcArgRS as a target for combating trypanosomal diseases and extending life expectancy., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: The coronavirus disease 2019 (COVID-19) pandemic is a global public health emergency causing a disparate burden of death and disability around the world. The viral genetic variants associated with outcome severity are still being discovered., Methods: We downloaded 155 958 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from GISAID. Of these genomes, 3637 samples included useable metadata on patient outcomes. Using this subset, we evaluated whether SARS-CoV-2 viral genomic variants improved prediction of reported severity beyond age and region. First, we established whether including genomic variants as model features meaningfully increased the predictive power of our model. Next, we evaluated specific variants in order to determine the magnitude of association with severity and the frequency of these variants among SARS-CoV-2 genomes., Results: Logistic regression models that included viral genomic variants outperformed other models (area under the curve = 0.91 as compared with 0.68 for age and gender alone; P  < 0.001). We found 84 variants with odds ratios greater than 2 for outcome severity (17 and 67 for higher and lower severity, respectively). The median frequency of associated variants was 0.15% (interquartile range 0.09-0.45%). Altogether 85% of genomes had at least one variant associated with patient outcome., Conclusion: Numerous SARS-CoV-2 variants have 2-fold or greater association with odds of mild or severe outcome and collectively, these variants are common. In addition to comprehensive mitigation efforts, public health measures should be prioritized to control the more severe manifestations of COVID-19 and the transmission chains linked to these severe cases. Lay summary: This study explores which, if any, SARS-CoV-2 viral genomic variants are associated with mild or severe COVID-19 patient outcomes. Our results suggest that there are common genomic variants in SARS-CoV-2 that are more often associated with negative patient outcomes, which may impact downstream public health measures., (Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health 2021. This work is written by a US Government employee and is in the public domain in the US.)

Abstract: Although largely benign, sickle cell trait (SCT) has been associated with exertion-related events, to include sudden death. In 2011, a summit on SCT introduced the term exercise collapse associated with SCT (ECAST). A series of ECAST deaths in military personnel in 2019 prompted reevaluation of current efforts and led to a second summit in October 2019 hosted by the Consortium for Health and Military Performance of the Uniformed Services University in Bethesda, MD. The goals were to (1) review current service policies on SCT screening, (2) develop draft procedural instructions for executing current policy on SCT within the Department of Defense, (3) develop draft clinical practice guidelines for management of ECAST, (4) establish a framework for education on SCT and ECAST, and (5) prepare a research agenda to address identified gaps., (Copyright © 2021 by the American College of Sports Medicine.)

INTRODUCTION : Aerial ports are being modernized with automated technologies, but the impact on musculoskeletal injury (MSKI) is unknown. METHODS : In this retrospective cohort study of U.S. Air Force aerial port technicians and traffic management technicians, we compared reported injury rates from January 2006-December 2016 and Veterans Benefits Administration disability compensation claims awarded from January 2001-March 2017. Ton-adjusted injury rates, associated lost/affected duty time, and percent risk attributable to lack of automation were compared at Dover Air Force Base (which features base-specific automation), Travis Air Force Base, Ramstein Air Base, and Yokota Air Base. RESULTS : Injuries most often occurred during aircraft/flight line activities and were typically sprains/strains, with extremities being most affected. Among aerial port technicians there were 8.0 injury reports per 1000 person-years compared to 5.2 per 1000 among traffic management technicians (incidence rate ratio = 1.5; 95% CI: 0.9, 3.0). Of the aerial port technicians with a compensation award, 70.7% included an MSKI component, whereas 75.7% of traffic management awards included an MSKI component. Aerial port technicians at Dover AFB experienced 1.4 injury reports per 1000 personnel per 1000 cargo-tons per year, lower than the other ports: 3.2 (Travis); 3.7 (Ramstein); and 7.6 (Yokota). Overall, 56% of injuries at Travis, 62% at Ramstein, and 82% at Yokota could be attributed to absence of Dover-like automation. However, mean lost/affected duty days at Dover (12.4) far exceeded those at the other bases (range: 4.5-8.6). DISCUSSION : Automating aerial ports may reduce injury rates, but the impact on lost/affected duty time requires further investigation. Bylsma VFH, Webber BJ, Erich RA, Voss JD. Musculoskeletal injuries and automation in aerial port operations . Aerosp Med Hum Perform. 2020; 91(8):669-673.

The purpose of the present study was to evaluate whether phonemic and semantic verbal fluency were more related to aspects of language processing than executive functioning (EF). An exploratory factor analysis was performed on a college-aged sample of 320 healthy participants using principle axis factoring and promax rotation on nine measures of EF. The first three factors, labeled: working memory, fluid reasoning, and shifting/updating, were extracted and used as latent executive variables. Participants were also split into low, medium, and high phonemic and semantic verbal fluency ability groups. Phonemic and semantic fluency correlated similarly across all three extracted EF factors and word knowledge. Using one-way analysis of variance (ANOVAs), there was a main effect for both phonemic and semantic verbal fluency groups and all outcome variables (i.e., the EF factors and word knowledge). Tukey HSD post hoc analyses showed that those in the low verbal fluency ability groups had significantly lower scores across all outcome measures compared to the high verbal fluency ability groups. Across all analyses, semantic fluency had stronger relations with the EF factors, signifying a large executive component involved in the task. Both phonemic and semantic fluency were similarly related to multiple dimensions of EF and word knowledge and should be considered executive language tasks.

Previous studies suggest that genetic variants within genes affecting the circadian rhythm influence the development of posttraumatic stress symptoms (PTSS). In the present study, we used data from three emergency care-based cohorts to search genetic variants in circadian pathway genes previously associated with neuropsychiatric disorders for variants that influence PTSS severity. The three cohorts used included a discovery cohort of African American men and women enrolled following motor vehicle collision ( n = 907) and two replication cohorts: one of multi-ethnic women enrolled following sexual assault ( n = 274) and one of multi-ethnic men and women enrolled following major thermal burn injury ( n = 68). DNA and RNA were collected from trauma survivors at the time of initial assessment. Validated questionnaires were used to assess peritraumatic distress severity and to assess PTSS severity 6 weeks, 6 months, and 1 year following trauma exposure. Thirty-one genetic variants from circadian rhythm genes were selected for analyses, and main effect and potential gene * stress and gene * sex interactions were evaluated. Secondary analyses assessed whether associated genetic variants affected mRNA expression levels. We found that six genetic variants across five circadian rhythm-associated genes predicted PTSS outcomes following motor vehicle collision ( p < 0.05), but only two of these variants survived adjustment for multiple comparisons (False Discovery Rate < 5%). The strongest of these associations, an interaction between the PAR-zip transcription factor, thyrotroph embryonic factor ( TEF ) variant rs5758324 and peritraumatic distress, predicted PTSS development in all three cohorts. Further analysis of genetic variants in the genetic region surrounding TEF rs5758324 (±125,000 nucleotides) indicated that this allele showed the strongest association. Further, TEF RNA expression levels (determined via RNA-seq) were positively associated with PTSS severity in distressed individuals with at least one copy of the TEF rs5758324 minor allele. These results suggest that rs5758324 genetic variant in TEF , a regulator of clock-controlled genes and key mediator of the core circadian rhythm, influence PTSS severity in a stress-dependent manner.

Purpose of Review: Obesity is a multifactorial disease that is now endemic throughout most of the world. Although addressing proximate causes of obesity (excess energy intake and reduced energy expenditure) have been longstanding global health priorities, the problem has continued to worsen at the global level., Recent Findings: Numerous microbial agents cause obesity in various experimental models-a phenomena known as infectobesity. Several of the same agents alter metabolic function in human cells and are associated with human obesity or metabolic dysfunction in humans. We address the evidence for a role in the genesis of obesity for viral agents in five broad categories: adenoviridae, herpesviridae, phages, transmissible spongiform encephalopathies (slow virus), and other encephalitides and hepatitides. Despite the importance of this topic area, there are many persistent knowledge gaps that need to be resolved. We discuss factors motivating further research and recommend that future infectobesity investigation should be more comprehensive, leveraged, interventional, and patient-centered.

Mild traumatic injuries to the brain (e.g., concussion) are common and have been recognized since antiquity, although definitions have varied historically. Nonetheless, studying the epidemiology of concussion helps clarify the overall importance, risk factors, and at-risk populations for this injury. The present review will focus on recent findings related to the epidemiology of concussion including definition controversies, incidence, and patterns in the population overall and in the military and athlete populations specifically. Finally, as this is an area of active research, we will discuss how future epidemiologic observations hold promise for gaining greater clarity about concussion and mild traumatic brain injury.

Context: Obesity is a public health priority, which also threatens national security. Adenovirus 36 (Adv36) increases adiposity in animals and Adv36 antibody status is associated with human obesity, but it is unknown whether infection predicts the development of human adiposity., Objective: The objective of the study was to assess infection status and subsequent weight gain., Design: The study had a retrospective cohort design., Setting: The study was conducted at Air Force fitness testing and clinical encounters., Participants: PARTICIPANTS included Air Force male enlistees, aged 18-22 years, with a baseline body mass index (BMI) of 20-30 kg/m(2) followed up from enlistment (beginning in 1995) until 2012 or separation from the Air Force., Exposure: EXPOSURE included Adv36 infection status at the time of entry., Main Outcome Measure: Follow-up BMI, the primary outcome, and diagnosis of overweight/obesity by the International Classification of Diseases, ninth revision V85.25+ and 278.0* series (secondary outcome) were recorded., Results: The last recorded follow-up BMI was similar among infected and uninfected, 26.4 and 27.2 kg/m(2), respectively (P > .05). However, infected individuals had a higher hazard of a medical provider's diagnosis of overweight/obese over time (hazard ratio 1.8, 95% confidence interval 1.0-3.1, P = .04), adjusted for baseline BMI. Additionally, infected individuals who were lean at baseline (BMI of 22.5 kg/m(2)) had a 3.9 times greater hazard of developing an overweight/obese clinical diagnosis (95% confidence interval 1.5-9.7, P = .004) compared with uninfected lean individuals after adjusting for interaction (P = .03) between infection and baseline BMI., Conclusions: The presence of Adv36 antibodies was not associated with higher BMI at baseline or follow-up within this military population. However, being infected was associated with developing a clinical diagnosis of overweight/obesity, especially among those lean at baseline.

We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a "cycle of knowledge" strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Multiple models of human metabolism have been reconstructed, but each represents only a subset of our knowledge. Here we describe Recon 2, a community-driven, consensus 'metabolic reconstruction', which is the most comprehensive representation of human metabolism that is applicable to computational modeling. Compared with its predecessors, the reconstruction has improved topological and functional features, including ∼2× more reactions and ∼1.7× more unique metabolites. Using Recon 2 we predicted changes in metabolite biomarkers for 49 inborn errors of metabolism with 77% accuracy when compared to experimental data. Mapping metabolomic data and drug information onto Recon 2 demonstrates its potential for integrating and analyzing diverse data types. Using protein expression data, we automatically generated a compendium of 65 cell type-specific models, providing a basis for manual curation or investigation of cell-specific metabolic properties. Recon 2 will facilitate many future biomedical studies and is freely available at http://humanmetabolism.org/.

We describe limitations in the use of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to examine unfolding intermediates associated with guanidinium chloride (GuHCl)-induced protein denaturation. Several studies have used alterations in fluorescence emission of bis-ANS to quantify the population of "molten globule" states. Our findings indicate that the observed changes in bis-ANS spectroscopic properties could originate from the interactions of bis-ANS and GuHCl and the aggregation of the dye at higher GuHCl concentrations. We posit that in the absence of additional complementary structural or spectroscopic measurements, the use of bis-ANS emission alone to monitor protein conformations can be misleading., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Absolute protein concentration determination is becoming increasingly important in a number of fields including diagnostics, biomarker discovery and systems biology modeling. The recently introduced quantification concatamer methodology provides a novel approach to performing such determinations, and it has been applied to both microbial and mammalian systems. While a number of software tools exist for performing analyses of quantitative data generated by related methodologies such as SILAC, there is currently no analysis package dedicated to the quantification concatamer approach. Furthermore, most tools that are currently available in the field of quantitative proteomics do not manage storage and dissemination of such data sets., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: To date, several genome-scale network reconstructions have been used to describe the metabolism of the yeast Saccharomyces cerevisiae, each differing in scope and content. The recent community-driven reconstruction, while rigorously evidenced and well annotated, under-represented metabolite transport, lipid metabolism and other pathways, and was not amenable to constraint-based analyses because of lack of pathway connectivity., Results: We have expanded the yeast network reconstruction to incorporate many new reactions from the literature and represented these in a well-annotated and standards-compliant manner. The new reconstruction comprises 1102 unique metabolic reactions involving 924 unique metabolites--significantly larger in scope than any previous reconstruction. The representation of lipid metabolism in particular has improved, with 234 out of 268 enzymes linked to lipid metabolism now present in at least one reaction. Connectivity is emphatically improved, with more than 90% of metabolites now reachable from the growth medium constituents. The present updates allow constraint-based analyses to be performed; viability predictions of single knockouts are comparable to results from in vivo experiments and to those of previous reconstructions., Conclusions: We report the development of the most complete reconstruction of yeast metabolism to date that is based upon reliable literature evidence and richly annotated according to MIRIAM standards. The reconstruction is available in the Systems Biology Markup Language (SBML) and via a publicly accessible database http://www.comp-sys-bio.org/yeastnet/.

Distributed aperture synthesis is an exciting technique for recovering high-resolution images from an array of small telescopes. Such a system requires optical field values measured at individual apertures to be phased together so that a single, high-resolution image can be synthesized. This paper describes the application of sharpness metrics to the process of phasing multiple coherent imaging systems into a single high-resolution system. Furthermore, this paper will discuss hardware and present the results of simulations and experiments which will illustrate how aperture synthesis is performed., ((c) 2010 Optical Society of America.)

The Schwartz Center Educational Rounds, the first Schwartz Center Rounds held at an educational institution, emphasize the overall theme of difficult conversations faced by students and professionals with patients, families, and other members of the health care team. Students in the health care professions do not have sufficient opportunities to practice the communication and decision making skills integral to compassionate caregiving. Such practice enhances communication with patients and families, as well as with other members of the interdisciplinary team. This article presents the underlying assumptions for this change-oriented project and describes the development, implementation, and evaluation of the Rounds for an interdisciplinary group of graduate students during a 4-year period.

Ladar-based vibrometry has been shown to be a powerful technique in enabling the plant identification of machines. Rather than sensing the geometric shape of a target laser vibrometers sense motions of the target induced by moving parts within the system. Since the target need not be spatially resolved, vibration can be sensed reliably and provide positive identification at ranges beyond the imaging limits of the aperture. However, as the range of observation increases, the diffraction-limited beam size on the target increases as well, and may encompass multiple vibrational modes on the target's surface. As a result, vibration estimates formed from large laser footprints illuminating multiple modes on a vibrating target will experience a degradation. This degradation is manifest as a spatial low-pass filtering effect: high-order mode shapes, associated with high-frequency vibrations, will be averaged out while low-frequency vibrations will be affected less. A model to predict this phenomenology is proposed for both pulse-pair and cw vibrometry systems. The cw model is compared to results obtained using an off-the-shelf laser vibrometry system.

Nile Red, a dye that fluoresces at defined wavelengths depending upon the polarity of the surrounding medium, has been proposed to determine the neutral lipid content of microalgal cells. Herein we communicate modifications to this technique that facilitate its use as a high-throughput screening technology, as well as improving its accuracy and versatility.

Evolution of mitochondrial genes is far from clock-like. The substitution rate varies considerably between species, and there are many species that have a significantly increased rate with respect to their close relatives. There is also considerable variation among species in the rate of gene order rearrangement. Using a set of 55 complete arthropod mitochondrial genomes, we estimate the evolutionary distance from the common ancestor to each species using protein sequences, tRNA sequences, and breakpoint distances (a measure of the degree of genome rearrangement). All these distance measures are correlated. We use relative rate tests to compare pairs of related species in several animal phyla. In the majority of cases, the species with the more highly rearranged genome also has a significantly higher rate of sequence evolution. Species with higher amino acid substitution rates in mitochondria also have more variable amino acid composition in response to mutation pressure. We discuss the possible causes of variation in rates of sequence evolution and gene rearrangement among species and the possible reasons for the observed correlation between the two rates.

Animal mitochondrial genomes usually have two transfer RNAs for leucine: one, with anticodon UAG, translates the four-codon family CUN, while the other, with anticodon UAA, translates the two-codon family UUR. These two genes must differ at the third anticodon position, but in some species the genes differ at many additional sites, indicating that these genes have been independent for a long time. Duplication and deletion of genes in mitochondrial genomes occur frequently during the evolution of the Metazoa. If a tRNA-Leu gene were duplicated and a substitution occurred in the anticodon, this would effectively turn one type of tRNA into the other. The original copy of the second tRNA type might then be lost by a deletion elsewhere in the genome. There are several groups of species in which the two tRNA-Leu genes occur next to one another (or very close) on the genome, which suggests that tandem duplication has occurred. Here we use RNA-specific phylogenetic methods to determine evolutionary trees for both genes. We present evidence that the process of duplication, anticodon mutation, and deletion of tRNA-Leu genes has occurred at least five times during the evolution of the metazoa-once in the common ancestor of all protostomes, once in the common ancestor of echinoderms and hemichordates, once in the hermit crab, and twice independently in mollusks.

The interaction of dynamin II with giant unilamellar vesicles was studied using two-photon fluorescence microscopy. Dynamin II, labeled with fluorescein, was injected into a microscope chamber containing giant unilamellar vesicles, which were composed of either pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Binding of the fluorescent dynamin II to giant unilamellar vesicles, in the presence and absence of PI(4,5)P2, was directly observed using two-photon fluorescence microscopy. This binding was also visualized using the fluorescent N-methylanthraniloyl guanosine 5'-[gamma-thio]triphosphate analogue. The membrane probe 6-dodecanoyl-2-dimethylamine-naphthalene was used to monitor the physical state of the lipid in the giant unilamellar vesicles in the absence and presence of dynamin. A surprising finding was the fact that dynamin II bound to vesicles in the absence of PI(4,5)P2. Activation of the GTPase activity of dynamin II by pure POPC was then shown.

The influence of two different di(1-pyrazolyl)alkane ligands on the rate constant of aqua ligand substitution of ruthenium(II) complexes with the formula [Ru(H2O)(L2)(tpmm)]2+ (L2 = di(1-pyrazolyl)methane (DPMet) or 2,2-di(1-pyrazolyl)propane (DPPro)) was investigated. A 9.4 x 10(5)-fold increase in the rate constant of ligand substitution at pH = 6.86 was observed when DPMet was replaced with DPPro. This remarkable increase was unexpected, considering that these bidentate ligands appear quite similar. To help lend insight into this dramatic spectator ligand effect, the activation parameters for the ligand substitution reactions were determined, and single-crystal X-ray data were collected on the structurally analogous (chloro)ruthenium(II) complexes, [Ru(Cl)(L2)(tpmm)]+. These results are discussed in the context of a heteroscorpionate effect exerted by the DPPro ligand.

Purpose: This study was undertaken to characterize the fracture resistance and mode of fracture of endodontically treated incisors restored with cast post-and-core, prefabricated stainless steel post, or carbon fiber-reinforced composite post systems., Materials and Methods: Ten endodontically treated teeth restored with each technique were subjected to a compressive load delivered at a 130-degree angle to the long axis until the first sign of failure was noted. The fracture load and the mode of fracture were recorded., Results: The failure loads registered in the three groups were not significantly different. Between 70%, and 80% of teeth from any of the three groups displayed fractures that were located above the simulated bone level., Conclusion: The use of carbon fiber-reinforced composite posts did not change the fracture resistance or the failure mode of endodontically treated central incisors compared to the use of metallic posts.

Fab fragments derived from ten different IgG populations of hyperimmune rabbit polyclonal anti-fluorescein antibodies were further resolved into subfractions based on differences in time-dependent dissociation from an FITC-adsorbent in the presence of 0.1 M fluorescein at 4 degrees C. Fab fragments separated into subpopulations based on specific dissociation times of 0.1 day, 1.0 day, 10 days and 100 days from the adsorbent. Finally, after the 100 days elution step incubation with 6.0 M guanidine-HCl was included to determine total protein concentration of specific anti-fluorescein Fab fragments. Yields of specifically eluted Fab fragments ranged from 12.7 to 84.1% of the total Fab population originally incubated with the adsorbent. All Fab polyclonal populations and subpopulations analyzed quenched the fluorescence of the bound ligand by 90% or greater. None of the plots of protein concentration versus percent yield of the total specific antibody obtained for each of the five resolved fractions constituting a specific polyclonal population conformed to Gaussian distributions. All resolved Fab subpopulations retained bound fluorescein ligand that exhibited significant bathochromic shifts in absorbancy. Based on the extent of the red-shift the antibodies segregated into one of two general spectral families showing either a peak shift to 505-507 nm or to 518-520 nm. The red-shift to 518-520 nm appeared unique to rabbit anti-fluorescein antibodies, since corresponding large shifts have not been observed with antibodies derived from other species (e.g. mouse, rat, chicken, etc.). K(d) values determined for the resolved fractions confirmed a continuous progression in affinity from the 0.1day through the 100 days elution. Preliminary isoelectric focusing analyses revealed progressive selection for relatively more homogeneous fractions, especially in the 100 days resolved fraction.

Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.

Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.

Quantitative determinations of the dissociation constants of biomolecular interactions, in particular protein-protein interactions, are essential for a detailed understanding of the molecular basis of their specificities. Fluorescence spectroscopy is particularly well suited for such studies. This article highlights the theoretical and practical aspects of fluorescence polarization and its application to the study of protein-protein interactions. Consideration is given to the nature of the different types of fluorescence probes available and the probe characteristics appropriate for the system under investigation. Several examples from the literature are discussed that illustrate different practical aspects of the technique applied to diverse systems., (Copyright 1999 Academic Press.)

The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G(0), the level of MAPKK activity was observed to affect the kinetics of progression through both the G(1) and G(2) phases of the cell cycle in NIH 3T3 cells. Ectopic expression of dominant-negative forms of MAPKK1, which was previously shown to inhibit G(0)/G(1) progression, was found to also delay progression of cells through G(2). In addition, treatment of cells with the specific MAPKK inhibitor PD 98059 during a synchronous S phase arrested the cells in the following G(2) phase. These data demonstrate a novel role for the MAPK cascade in progression from G(2) into mitosis in NIH 3T3 cells.

Filipin is a pentaene macrolide antibiotic which was previously shown to incorporate more extensively into DPPC bilayers below the main phase transition temperature than above this temperature. This result was extremely unusual because drugs tend to be expelled from ordered gel phases. However, such results could not be safely attributed to the phase change of the bilayer itself because the temperature was changing concomitantly. In this work we changed the bilayer phase isothermally (53 degrees C) by hydrostatic pressure variation and discovered that filipin has a slightly more extensive incorporation in the pure DPPC gel phase (P>ca. 54.4 MPa): Kp,lc approximately 3x10(3) vs. Kp,gel approximately 6x10(3). The presence of sterols (45% molar ergosterol or cholesterol) caused an increase in the partition coefficients, regardless of pressure, ergosterol having a more pronounced effect (Kp approximately 2x10(4)-6x10(4)). Kp was pressure dependent in both cases, but mainly with cholesterol (Kp approximately 2x10(3)-2x10(4)). At variance with cholesterol, when ergosterol was used, no phase transition was detected. This difference cannot be due to a more extended uptake of filipin by cholesterol-containing membranes, and so must be due to specific interactions with cholesterol. In agreement with this finding, we discovered that filipin is more tightly packed (lower partial molar volume) in the cholesterol-rich phase than in the ergosterol-rich phase. Our results also point to a 2:1 DPPC:cholesterol stoichiometry in the cholesterol-rich phase (17% molar cholesterol). All partition coefficients were calculated from steady-state fluorescence anisotropy measurements.

The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized by a tetramer/monomer equilibrium with an equilibrium constant of 1.67 x 10(17) M(-3). Stopped-flow fluorescence experiments show that the association rate constant for 2'(3')-O-N-methylanthraniloyl (mant)GTP is 7.0 x 10(-5) M(-1) s(-1) and the dissociation rate constant is 2.1 s(-1), whereas the dissociation rate constant for mantdeoxyGDP is 93 s(-1). We also demonstrate the cooperativity of dynamin binding and GTPase activation on a microtubule lattice. Our results indicate that dynamin self-association is not a sufficient condition for the expression of maximal GTPase activity, which suggests that dynamin molecules must be in the proper conformation or orientation if they are to form an active oligomer.

In the present study, structural aspects of the two soluble transducers, HtrX and HtrXI, from the archaeon H. salinarum have been examined using UV circular dichroism and steady-state fluorescence spectroscopies. Circular dichroism (CD) data indicate that both HtrX and HtrXI exhibit salt-dependent protein folding. Under low-ionic-strength conditions (0.2 M NaCl or KCl) the CD spectra of HtrXI is similar to that of the Gdn-HCl- or urea-denatured forms and is indicative of random coil structure. In contrast, the CD spectrum of HtrX under low-ionic-strength conditions contains roughly 85% alpha-helical character, indicating a significant degree of folding. Addition of NaCl or KCl to solutions of HtrX or HtrXI results in CD features consistent with predominately alpha-helical character (>95%) for both proteins. In addition, the transition points (i.e., ionic strengths at which the protein converts from random coil to alpha-helical character) are quite distinct and dependent upon the type of salt present (i.e., either NaCl or KCl). Accessibility of tryptophan residues to the solvent was also examined for both HtrX and HtrXI in both folded and unfolded states using KI quenching. The Stem-Volmer constants obtained suggest that the tryptophans (Trp35 in HtrX and both Trp47 and Trp74 in HtrXI) are partially exposed to the solvent, indicating that they are located near the surface of the protein in all three cases. Furthermore, fluorescence quenching with the single Trp mutants Trp74AIa and Trp47AIa of HtrXI indicates different environments for these two residues.

The unfolding and refolding of apohorseradish peroxidase, as a function of guanidinium chloride concentration, were monitored by the intrinsic fluorescence intensity, polarization, and lifetime of the single tryptophan residue. The unfolding was reversible and characterized by at least three distinct stages-the intensity and lifetime data, for example, were both characterized by an initial increase followed by a decrease and then a plateau region. The lifetime data, in the absence and presence of guanidinium chloride, were heterogeneous and fit best to a model consisting of a major Gaussian distribution component and a minor, short discrete component. The observed increase in intensity in the initial stage of the unfolding process is attributed to the conversion of this short component into the longer, distributed component as the guanidinium chloride concentration increases. Our results clarify and amplify previous studies on the unfolding of apohorseradish peroxidase by guanidinium chloride.

We conducted a study in the mental health and behavioral sciences program at Hines Veterans Administration Hospital to determine if a recent reorganization of the mental health services had resulted in the efficient use of treatment resources according to patients' needs. The Brief Symptom Inventory was used to assess symptomatology when patients were admitted to the inpatient psychiatry, dual diagnosis, or substance abuse lodger units. The results indicated that the groups differ significantly from one another on eight of nine symptom dimensions and on all three global indices of psychopathology. The results also reinforced the supposition that patients are receiving appropriate treatment and that resources are being used efficiently.

Site-directed mutagenesis was utilized to construct mutants, containing one or two tryptophan residues, of the bifunctional enzyme fructose 6-phosphate,2-kinase-fructose 2,6-bisphosphatase. Two of the single-tryptophan mutants (W15 and W64) had the tryptophan residue located in the kinase domain, which is in the N-terminal half, and two (W299 and W320) had the tryptophan residue located in the phosphatase domain, which is in the C-terminal half. The double-tryptophan mutants were W15/W64, W15/W299, W64/W299, and W299/W320. Dynamic polarization data indicated that these tryptophan residues had varying degrees of local mobility. Steady-state polarization data revealed energy transfer between the tryptophan residues in the double mutant W299/W320 but not in the W15/W64, W15/W299, or W64/W299 mutants, indicating the proximity of the W299 and W320 residues. The binding of fructose-6-phosphate resulted in a significant increase in the anisotropy of the W15 mutants, but did not affect the anisotropies of any of the other single-tryptophan mutants. Binding of fructose-2,6-bisphosphate also significantly increased the anisotropy of W15. In the case of fructose-6-phosphate binding, the increased anisotropy was shown to be due to a restriction of the tryptophan residue's local mobility in the presence of bound ligand, which suggests that the N-terminus is located near the kinase active site. These increases in anisotropies were used to estimate the dissociation constants of fructose-6-phosphate and fructose-2,6-bisphosphate, which were 29 +/- 3 and 2.1 +/- 0.3 microM, respectively. These observations are considered in light of the recently published crystal structure for this bifunctional enzyme.

The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 +/- 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below 10(-9) M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium.