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Importance: Microsatellite (MS) instability (MSI-H) occurs frequently in Lynch syndrome (LS)-associated tumors and is associated with response to immune checkpoint blockade (ICB) therapy. MSI-H is conferred by germline or somatic variants in mismatch repair genes. The contribution of somatic oncogenesis to MSI-H in pancreatic cancer (PC) is unknown., Objective: To evaluate an LS-related PC cohort to define clinicogenomic features, describe somatic MSI-H cases (germline negative), characterize response to ICB, and guide preferred MS testing methods., Design, Setting, and Participants: This single-institution, retrospective analysis was conducted from March 2012 to July 2023 at Memorial Sloan Kettering Cancer Center and included 55 patients with PC and either an LS germline pathogenic variant (gPV) or somatic mismatch repair (MMR) variant., Main Outcomes and Measures: Composite MMR and MS status determined using orthogonal methods. An artificial intelligence classifier was used to account for low-cellularity specimens. Demographic and clinical data were abstracted from medical record. Zygosity status and somatic comutation landscape analyzed., Results: Fifty-five patients (23 women [42%]) had PC and an MMR variant: 32 (58%) had LS (LS cohort) and 23 (42%) had a somatic MMR variant (no germline pathogenic variant, somatic MMR cohort). In the LS cohort, 10 (31%) had gMSH2, 9 (28%) gMSH6, 8 (25%) gPMS2, 4 (13%) gMLH1, 1 (3%) gEPCAM. The median age at diagnosis was 68 years (range, 45-88 years). For composite MS status, 17 (59%) were MSI-H, 12 (41%) MS stable, and 3 MS unknown. Five cases were reclassified as MSI-H by the artificial intelligence classifier. In the somatic MMR cohort, 11 (48%) had MSH6, 7 (30%) MLH1, 3 (13%) MSH2, and 2 (9%) PMS2. The median age at diagnosis was 72 years (range, 66-85 years). For composite MS status, 10 (43%) were MSI-H, 11 (48%) MS stable, and 2 (9%) MS indeterminate. Six cases were reclassified as MSI-H by the artificial intelligence classifier. For the LS and somatic MMR cohorts, 20 received ICB (n = 17 MSI-H). The median ICB duration was 27.7 months (95% CI, 11.5 to not reached); the disease control rate was 80%., Conclusion: The results of this cross-sectional study suggest that MSI-H occurs due to LS or somatic oncogenesis in PC. Orthogonal MS testing is key in PC; the artificial intelligence classifier reclassified approximately 20% of cases, most of which were low cellularity. ICB for patients with LS or somatic MSI-H PC provided significant benefit.

Background: Systematic reviews of Randomized Controlled Trials (RCTs) are an important part of the evidence-based medicine paradigm. However, the creation of such systematic reviews by clinical experts is costly as well as time-consuming, and results can get quickly outdated after publication. Most RCTs are structured based on the Patient, Intervention, Comparison, Outcomes (PICO) framework and there exist many approaches which aim to extract PICO elements automatically. The automatic extraction of PICO information from RCTs has the potential to significantly speed up the creation process of systematic reviews and this way also benefit the field of evidence-based medicine., Results: Previous work has addressed the extraction of PICO elements as the task of identifying relevant text spans or sentences, but without populating a structured representation of a trial. In contrast, in this work, we treat PICO elements as structured templates with slots to do justice to the complex nature of the information they represent. We present two different approaches to extract this structured information from the abstracts of RCTs. The first approach is an extractive approach based on our previous work that is extended to capture full document representations as well as by a clustering step to infer the number of instances of each template type. The second approach is a generative approach based on a seq2seq model that encodes the abstract describing the RCT and uses a decoder to infer a structured representation of a trial including its arms, treatments, endpoints and outcomes. Both approaches are evaluated with different base models on a manually annotated dataset consisting of RCT abstracts on an existing dataset comprising 211 annotated clinical trial abstracts for Type 2 Diabetes and Glaucoma. For both diseases, the extractive approach (with flan-t5-base) reached the best F 1 score, i.e. 0.547 ( ± 0.006 ) for type 2 diabetes and 0.636 ( ± 0.006 ) for glaucoma. Generally, the F 1 scores were higher for glaucoma than for type 2 diabetes and the standard deviation was higher for the generative approach., Conclusion: In our experiments, both approaches show promising performance extracting structured PICO information from RCTs, especially considering that most related work focuses on the far easier task of predicting less structured objects. In our experimental results, the extractive approach performs best in both cases, although the lead is greater for glaucoma than for type 2 diabetes. For future work, it remains to be investigated how the base model size affects the performance of both approaches in comparison. Although the extractive approach currently leaves more room for direct improvements, the generative approach might benefit from larger models., (© 2024. The Author(s).)

To fully appreciate genetics, one must understand the link between genotype (DNA sequence) and phenotype (observable characteristics). Advances in high-throughput genomic sequencing technologies and applications, so-called "-omics," have made genetic sequencing readily available across fields in biology from applications in non-traditional study organisms to precision medicine. Thus, understanding these tools is critical for any biologist, especially those early in their career. This comprehensive review discusses the chronological development of different sequencing methods, the bioinformatics steps to analyzing this data, and social and ethical issues raised by these techniques that must be discussed and evaluated, including anticipatory guides and discussion questions for active engagement in the classroom. Additionally, the Supporting Information includes a case study to apply technical and ethical concepts from the text., (© 2021 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Disrupting a gene to determine its effect on an organism's phenotype is an indispensable tool in molecular biology. Such techniques are critical for understanding how a gene product contributes to the development and cellular identity of organisms. The explosion of genomic sequencing technologies combined with recent advances in genome-editing techniques has elevated the possibilities of genetic manipulations in numerous organisms in which these experiments were previously not readily accessible or possible. Introducing the next generation of molecular biologists to these emerging techniques is key in the modern biology classroom. This comprehensive review introduces undergraduates to CRISPR/Cas9 editing and its uses in genetic studies. The goals of this review are to explain how CRISPR functions as a prokaryotic immune system, describe how researchers generate mutations with CRISPR/Cas9, highlight how Cas9 has been adapted for new functions, and discuss ethical considerations of genome editing. Additionally, anticipatory guides and questions for discussion are posed throughout the review to encourage active exploration of these topics in the classroom. Finally, the supplement includes a study guide and practical suggestions to incorporate CRISPR/Cas9 experiments into lab courses at the undergraduate level. © 2018 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 46(2):195-205, 2018., (© 2018 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology.)

Objectives: We aim to understand how the second epidemiologic transition unfolded on the island of Newfoundland during the early 20th century. The focus is on changes in predominant causes of mortality throughout this period, urban and rural differences, and comparisons with other Western nations. We explore factors driving these patterns including the economy, nutrition, sanitation, and access to healthcare and discuss their relevance to understanding epidemiologic transitions in other developing regions., Methods: We examined official provincial death records (n = 65,394) and census materials from 1900 to 1939 for three rural districts (Burgeo-La Poile, Bonavista, and Twillingate) and the large city of St. John's. Life expectancies, infant mortality rates, survival curves, and proportionate mortality from communicable and non-communicable diseases (NCDs) were calculated., Results: In all districts, old age mortality increased while infant and childhood mortality decreased, with corresponding increases in life expectancy. Proportionate mortality from communicable causes decreased while deaths from NCDs increased. These changes occurred earlier in urban St. John's than in outlying districts, suggesting rural-urban differences played a significant role in Newfoundland's second epidemiologic transition. However, the transition was significantly delayed relative to other Western nations., Conclusions: We suggest that observed mortality patterns were the result of complex interactions between the poor economy, malnutrition, high prevalence of tuberculosis, and limited health and social services. These factors contribute to the delayed onset of the second epidemiologic transition in Newfoundland relative to other Western nations and the earlier onset in St. John's than in rural areas., (© 2017 Wiley Periodicals, Inc.)

As the only catalytic member of the Sir-protein gene-silencing complex, Sir2's catalytic activity is necessary for silencing. The only known role for Sir2's catalytic activity in Saccharomyces cerevisiae silencing is to deacetylate N-terminal tails of histones H3 and H4, creating high-affinity binding sites for the Sir-protein complex, resulting in association of Sir proteins across the silenced domain. This histone deacetylation model makes the simple prediction that preemptively removing Sir2's H3 and H4 acetyl substrates, by mutating these lysines to unacetylatable arginines, or removing the acetyl transferase responsible for their acetylation, should restore silencing in the Sir2 catalytic mutant. However, this was not the case. We conducted a genetic screen to explore what aspect of Sir2's catalytic activity has not been accounted for in silencing. Mutation of a nonsirtuin histone deacetylase, Rpd3, restored Sir-protein-based silencing in the absence of Sir2's catalytic activity. Moreover, this antagonism could be mediated by either the large or the small Rpd3-containing complex. Interestingly, this restoration of silencing appeared independent of any known histone H3 or H4 substrates of Rpd3 Investigation of Sir-protein association in the Rpd3 mutant revealed that the restoration of silencing was correlated with an increased association of Sir proteins at the silencers, suggesting that Rpd3 was an antagonist of Sir2's function in nucleation of Sir proteins to the silencer. Additionally, restoration of silencing by Rpd3 was dependent on another sirtuin family member, Hst3, indicating multiple antagonistic roles for deacetylases in S. cerevisiae silencing., (Copyright © 2016 by the Genetics Society of America.)

Background: Increasing numbers of survivors of critical illness are at risk for physical, cognitive, and/or mental health impairments that may persist for months or years after hospital discharge. The post-intensive care syndrome framework encompassing these multidimensional morbidities was developed at the 2010 Society of Critical Care Medicine conference on improving long-term outcomes after critical illness for survivors and their families., Objectives: To report on engagement with non-critical care providers and survivors during the 2012 Society of Critical Care Medicine post-intensive care syndrome stakeholder conference. Task groups developed strategies and resources required for raising awareness and education, understanding and addressing barriers to clinical practice, and identifying research gaps and resources, aimed at improving patient and family outcomes., Participants: Representatives from 21 professional associations or health systems involved in the provision of both critical care and rehabilitation of ICU survivors in the United States and ICU survivors and family members., Design: Stakeholder consensus meeting. Researchers presented summaries on morbidities for survivors and their families, whereas survivors presented their own experiences., Meeting Outcomes: Future steps were planned regarding 1) recognizing, preventing, and treating post-intensive care syndrome, 2) building strategies for institutional capacity to support and partner with survivors and families, and 3) understanding and addressing barriers to practice. There was recognition of the need for systematic and frequent assessment for post-intensive care syndrome across the continuum of care, including explicit "functional reconciliation" (assessing gaps between a patient's pre-ICU and current functional ability at all intra- and interinstitutional transitions of care). Future post-intensive care syndrome research topic areas were identified across the continuum of recovery: characterization of at-risk patients (including recognizing risk factors, mechanisms of injury, and optimal screening instruments), prevention and treatment interventions, and outcomes research for patients and families., Conclusions: Raising awareness of post-intensive care syndrome for the public and both critical care and non-critical care clinicians will inform a more coordinated approach to treatment and support during recovery after critical illness. Continued conceptual development and engagement with additional stakeholders is required.

Purpose: Communication in the intensive care unit (ICU) is an important component of quality ICU care. In this report, we evaluate the long-term effects of a quality improvement (QI) initiative, based on the VALUE communication strategy, designed to improve communication with family members of critically ill patients., Materials and Methods: We implemented a multifaceted intervention to improve communication in the ICU and measured processes of care. Quality improvement components included posted VALUE placards, templated progress note inclusive of communication documentation, and a daily rounding checklist prompt. We evaluated care for all patients cared for by the intensivists during three separate 3 week periods, pre, post, and 3 years following the initial intervention., Results: Care delivery was assessed in 38 patients and their families in the pre-intervention sample, 27 in the post-intervention period, and 41 in follow-up. Process measures of communication showed improvement across the evaluation periods, for example, daily updates increased from pre 62% to post 76% to current 84% of opportunities., Conclusions: Our evaluation of this quality improvement project suggests persistence and continued improvements in the delivery of measured aspects of ICU family communication. Maintenance with point-of-care-tools may account for some of the persistence and continued improvements., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To compare estimation of glomerular filtration rate determined via conventional methods (ie, scintigraphy and plasma clearance of technetium Tc 99m pentetate) and dynamic single-slice computed tomography (CT)., Animals: 8 healthy adult cats., Procedures: Scintigraphy, plasma clearance testing, and dynamic CT were performed on each cat on the same day; order of examinations was randomized. Separate observers performed GFR calculations for scintigraphy, plasma clearance testing, or dynamic CT. Methods were compared via Bland-Altman plots and considered interchangeable and acceptable when the 95% limits of agreement (mean difference between methods ± 1.96 SD of the differences) were ≤ 0.7 mL/min/kg., Results: Global GFR differed < 0.7 mL/min/kg in 5 of 8 cats when comparing plasma clearance testing and dynamic CT; the limits of agreement were 1.4 and -1.7 mL/min/kg. The mean ± SD difference was -0.2 ± 0.8 mL/min/kg, and the maximum difference was 1.6 mL/min/kg. The mean ± SD difference (absolute value) for percentage filtration by individual kidneys was 2.4 ± 10.5% when comparing scintigraphy and dynamic CT; the maximum difference was 20%, and the limits of agreement were 18% and 23% (absolute value)., Conclusions and Clinical Relevance: GFR estimation via dynamic CT exceeded the definition for acceptable clinical use, compared with results for conventional methods, which was likely attributable to sample size and preventable technical complications. Because 5 of 8 cats had comparable values between methods, further investigation of dynamic CT in a larger sample population with a wide range of GFR values should be performed.

Background: Millions of patients are discharged from intensive care units annually. These intensive care survivors and their families frequently report a wide range of impairments in their health status which may last for months and years after hospital discharge., Objectives: To report on a 2-day Society of Critical Care Medicine conference aimed at improving the long-term outcomes after critical illness for patients and their families., Participants: Thirty-one invited stakeholders participated in the conference. Stakeholders represented key professional organizations and groups, predominantly from North America, which are involved in the care of intensive care survivors after hospital discharge., Design: Invited experts and Society of Critical Care Medicine members presented a summary of existing data regarding the potential long-term physical, cognitive and mental health problems after intensive care and the results from studies of postintensive care unit interventions to address these problems. Stakeholders provided reactions, perspectives, concerns and strategies aimed at improving care and mitigating these long-term health problems., Measurements and Main Results: Three major themes emerged from the conference regarding: (1) raising awareness and education, (2) understanding and addressing barriers to practice, and (3) identifying research gaps and resources. Postintensive care syndrome was agreed upon as the recommended term to describe new or worsening problems in physical, cognitive, or mental health status arising after a critical illness and persisting beyond acute care hospitalization. The term could be applied to either a survivor or family member., Conclusions: Improving care for intensive care survivors and their families requires collaboration between practitioners and researchers in both the inpatient and outpatient settings. Strategies were developed to address the major themes arising from the conference to improve outcomes for survivors and families.

The mechanism of oxidation of a peptide substrate by the flavoprotein lysine-specific demethylase (LSD1) has been examined using the effects of pH and isotopic substitution on steady-state and rapid-reaction kinetic parameters. The substrate contained the 21 N-terminal residues of histone H3, with a dimethylated lysyl residue at position 4. At pH 7.5, the rate constant for flavin reduction, k(red), equals k(cat), establishing the reductive half-reaction as rate-limiting at physiological pH. Deuteration of the lysyl methyls results in identical kinetic isotope effects of 3.1 +/- 0.2 on the k(red), k(cat), and k(cat)/K(m) values for the peptide, establishing C-H bond cleavage as rate-limiting with this substrate. No intermediates between oxidized and reduced flavin can be detected by stopped-flow spectroscopy, consistent with the expectation for a direct hydride transfer mechanism. The k(cat)/K(m) value for the peptide is bell-shaped, consistent with a requirement that the nitrogen at the site of oxidation be uncharged and that at least one of the other lysyl residues be charged for catalysis. The (D)(k(cat)/K(m)) value for the peptide is pH-independent, suggesting that the observed value is the intrinsic deuterium kinetic isotope effect for oxidation of this substrate.

A facile synthetic route to substituted trans-2-arylcyclopropylamines was developed to provide access to mechanism-based inhibitors of the human flavoenzyme oxidase lysine-specific histone demethylase LSD1 and related enzyme family members such as monoamine oxidases A and B.

A number of brain imaging techniques have been developed in order to investigate brain function and to develop diagnostic tools for various brain disorders. Each modality has strengths as well as weaknesses compared to the others. Recent work has explored how multiple modalities can be integrated effectively so that they complement one another while maintaining their individual strengths. Bayesian inference employing Markov Chain Monte Carlo (MCMC) techniques provides a straightforward way to combine disparate forms of information while dealing with the uncertainty in each. In this paper we introduce methods of Bayesian inference as a way to integrate different forms of brain imaging data in a probabilistic framework. We formulate Bayesian integration of magnetoencephalography (MEG) data and functional magnetic resonance imaging (fMRI) data by incorporating fMRI data into a spatial prior. The usefulness and feasibility of the method are verified through testing with both simulated and empirical data.

Source localization by electroencephalography (EEG) requires an accurate model of head geometry and tissue conductivity. The estimation of source time courses from EEG or from EEG in conjunction with magnetoencephalography (MEG) requires a forward model consistent with true activity for the best outcome. Although MRI provides an excellent description of soft tissue anatomy, a high resolution model of the skull (the dominant resistive component of the head) requires CT, which is not justified for routine physiological studies. Although a number of techniques have been employed to estimate tissue conductivity, no present techniques provide the noninvasive 3D tomographic mapping of conductivity that would be desirable. We introduce a formalism for probabilistic forward modeling that allows the propagation of uncertainties in model parameters into possible errors in source localization. We consider uncertainties in the conductivity profile of the skull, but the approach is general and can be extended to other kinds of uncertainties in the forward model. We and others have previously suggested the possibility of extracting conductivity of the skull from measured electroencephalography data by simultaneously optimizing over dipole parameters and the conductivity values required by the forward model. Using Cramer-Rao bounds, we demonstrate that this approach does not improve localization results nor does it produce reliable conductivity estimates. We conclude that the conductivity of the skull has to be either accurately measured by an independent technique, or that the uncertainties in the conductivity values should be reflected in uncertainty in the source location estimates.

The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee, M. G., et al. (2006) Chem. Biol. 13, 563-567], demonstrating that trans-2-phenylcyclopropylamine (2-PCPA, tranylcypromine, Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1, with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A, respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo.

We propose a new model to approximate spatiotemporal noise covariance for use in neural electromagnetic source analysis, which better captures temporal variability in background activity. As with other existing formalisms, our model employs a Kronecker product of matrices representing temporal and spatial covariance. In our model, spatial components are allowed to have differing temporal covariances. Variability is represented as a series of Kronecker products of spatial component covariances and corresponding temporal covariances. Unlike previous attempts to model covariance through a sum of Kronecker products, our model is designed to have a computationally manageable inverse. Despite increased descriptive power, inversion of the model is fast, making it useful in source analysis. We have explored two versions of the model. One is estimated based on the assumption that spatial components of background noise have uncorrelated time courses. Another version, which gives closer approximation, is based on the assumption that time courses are statistically independent. The accuracy of the structural approximation is compared to an existing model, based on a single Kronecker product, using both Frobenius norm of the difference between spatiotemporal sample covariance and a model, and scatter plots. Performance of ours and previous models is compared in source analysis of a large number of single dipole problems with simulated time courses and with background from authentic magnetoencephalography data.

The performance of parametric magnetoencephalography (MEG) and electroencephalography (EEG) source localization approaches can be degraded by the use of poor background noise covariance estimates. In general, estimation of the noise covariance for spatiotemporal analysis is difficult mainly due to the limited noise information available. Furthermore, its estimation requires a large amount of storage and a one-time but very large (and sometimes intractable) calculation or its inverse. To overcome these difficulties, noise covariance models consisting of one pair or a sum of multi-pairs of Kronecker products of spatial covariance and temporal covariance have been proposed. However, these approaches cannot be applied when the noise information is very limited, i.e., the amount of noise information is less than the degrees of freedom of the noise covariance models. A common example of this is when only averaged noise data are available for a limited prestimulus region (typically at most a few hundred milliseconds duration). For such cases, a diagonal spatiotemporal noise covariance model consisting of sensor variances with no spatial or temporal correlation has been the common choice for spatiotemporal analysis. In this work, we propose a different noise covariance model which consists of diagonal spatial noise covariance and Toeplitz temporal noise covariance. It can easily be estimated from limited noise information, and no time-consuming optimization and data-processing are required. Thus, it can be used as an alternative choice when one-pair or multi-pair noise covariance models cannot be estimated due to lack of noise information. To verify its capability we used Bayesian inference dipole analysis and a number of simulated and empirical datasets. We compared this covariance model with other existing covariance models such as conventional diagonal covariance, one-pair and multi-pair noise covariance models, when noise information is sufficient to estimate them. We found that our proposed noise covariance model yields better localization performance than a diagonal noise covariance, while it performs slightly worse than one-pair or multi-pair noise covariance models - although these require much more noise information. Finally, we present some localization results on median nerve stimulus empirical MEG data for our proposed noise covariance model.

Demethylation of histone H3 lysine 4 is carried out by BHC110/LSD1, an enzyme with close homology to monoamine oxidases (MAO). Monoamine oxidase A or B are frequent targets of selective and nonselective small molecular inhibitors used for treatment of depression. Here we show that in contrast to selective monoamine oxidase inhibitors such as pargyline, nonselective monoamine oxidase inhibitors potently inhibit nucleosomal demethylation of histone H3 lysine 4. Tranylcypromine (brand name Parnate) displayed the best inhibitory activity with an IC50 of less than 2 microM. Treatment of P19 embryonal carcinoma cells with tranylcypromine resulted in global increase in H3K4 methylation as well as transcriptional derepression of two BHC110 target genes, Egr1 and the pluripotent stem cell marker Oct4. These results attest to the effectiveness of tranylcypromine as a small molecular inhibitor of histone demethylation.

Most existing spatiotemporal multi-dipole approaches for MEG/EEG source localization assume that the dipoles are active for the full time range being analysed. If the actual time range of activity of sources is significantly shorter than the time range being analysed, the detectability, localization and time-course determination of such sources may be adversely affected, especially for weak sources. In order to improve detectability and reconstruction of such sources, it is natural to add active time range information (starting time point and ending time point of source activation) for each candidate source as unknown parameters in the analysis. However, this adds additional nonlinear free parameters that could burden the analysis and could be unfeasible for some methods. Recently, we described a spatiotemporal Bayesian inference multi-dipole analysis for the MEG/EEG inverse problem. This approach treated the number of dipoles as a free parameter, produced realistic uncertainty estimates using a Markov chain Monte Carlo numerical sampling of the posterior distribution and included a method to reduce the unwanted effects of local minima. In this paper, our spatiotemporal Bayesian inference multi-dipole analysis is extended to incorporate active time range parameters of starting and stopping time points. The properties of this analysis in comparison to the previous one without active time range parameters are demonstrated through extensive studies using both simulated and empirical MEG data.

Using a noise covariance model based on a single Kronecker product of spatial and temporal covariance in the spatiotemporal analysis of MEG data was demonstrated to provide improvement in the results over that of the commonly used diagonal noise covariance model. In this paper we present a model that is a generalization of all of the above models. It describes models based on a single Kronecker product of spatial and temporal covariance as well as more complicated multi-pair models together with any intermediate form expressed as a sum of Kronecker products of spatial component matrices of reduced rank and their corresponding temporal covariance matrices. The model provides a framework for controlling the tradeoff between the described complexity of the background and computational demand for the analysis using this model. Ways to estimate the value of the parameter controlling this tradeoff are also discussed.

Recently, we described a Bayesian inference approach to the MEG/EEG inverse problem that used numerical techniques to estimate the full posterior probability distributions of likely solutions upon which all inferences were based [Schmidt, D.M., George, J.S., Wood, C.C., 1999. Bayesian inference applied to the electromagnetic inverse problem. Human Brain Mapping 7, 195; Schmidt, D.M., George, J.S., Ranken, D.M., Wood, C.C., 2001. Spatial-temporal bayesian inference for MEG/EEG. In: Nenonen, J., Ilmoniemi, R. J., Katila, T. (Eds.), Biomag 2000: 12th International Conference on Biomagnetism. Espoo, Norway, p. 671]. Schmidt et al. (1999) focused on the analysis of data at a single point in time employing an extended region source model. They subsequently extended their work to a spatiotemporal Bayesian inference analysis of the full spatiotemporal MEG/EEG data set. Here, we formulate spatiotemporal Bayesian inference analysis using a multi-dipole model of neural activity. This approach is faster than the extended region model, does not require use of the subject's anatomical information, does not require prior determination of the number of dipoles, and yields quantitative probabilistic inferences. In addition, we have incorporated the ability to handle much more complex and realistic estimates of the background noise, which may be represented as a sum of Kronecker products of temporal and spatial noise covariance components. This reduces the effects of undermodeling noise. In order to reduce the rigidity of the multi-dipole formulation which commonly causes problems due to multiple local minima, we treat the given covariance of the background as uncertain and marginalize over it in the analysis. Markov Chain Monte Carlo (MCMC) was used to sample the many possible likely solutions. The spatiotemporal Bayesian dipole analysis is demonstrated using simulated and empirical whole-head MEG data.

The repertoire of reactions in the mechanistically diverse enolase superfamily is the result of divergent evolution that conserved enolization of a carboxylate anion substrate but allowed different overall reactions using different substrates. Details of the pathways for the natural evolutionary process are unknown, but the events reasonably involve (1) incremental increases in the level of the "new" reaction that would provide a selective advantage and (2) an accompanying loss of the "old" reaction catalyzed by the progenitor. In an effort to better understand the molecular processes of divergent evolution, the D297G mutant of the l-Ala-d/l-Glu epimerase (AEE) from Escherichia coli was designed so that it could bind the substrate for the o-succinylbenzoate synthase (OSBS) reaction and, as a result, catalyze that reaction [Schmidt, D. M. Z., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Biochemistry 42, 8387-8393]. The AEE progenitor did not catalyze the OSBS reaction, but the D297G mutant catalyzed a low level of the OSBS reaction (k(cat), 0.013 s(-)(1); K(m), 1.8 mM; k(cat)/K(m), 7.4 M(-)(1) s(-)(1)) that was sufficient to permit anaerobic growth by an OSBS-deficient strain of E. coli; the level of the progenitor's natural AEE reaction was significantly diminished. Using random mutagenesis and an anaerobic metabolic selection, we now have identified the I19F substitution as an additional mutation that enhances both growth of the OSBS-deficient strain and the kinetic constants for the OSBS reaction (k(cat), 0.031 s(-)(1); K(m), 0.34 mM; k(cat)/K(m), 90 M(-)(1) s(-)(1)). Several other substitutions for Ile 19 also enhanced the level of the OSBS reaction. All of the substitutions substantially decreased the level of the AEE reaction from that possessed by the D297G progenitor. The changes in the kinetic constants for both the OSBS and AEE reactions are attributed to a readjustment of substrate specificity so that the substrate for the OSBS reaction is more productively presented to the conserved acid/base catalysts in the active site. These observations support our hypothesis that evolution of "new" functions in the enolase superfamily can occur simply by changes in specificity-determining residues.

The members of the mechanistically diverse enolase superfamily share a bidomain structure formed from a (beta/alpha)7beta-barrel domain [a modified (beta/alpha)8- or TIM-barrel] and a capping domain formed from N- and C-terminal segments of the polypeptide. The active sites are located at the interface between the C-terminal ends of the beta-strands in the barrel domain and two flexible loops in the capping domain. Within this structure, the acid/base chemistry responsible for formation and stabilization of an enediolate intermediate derived from a carboxylate anion substrate and the processing of it to product is "hard-wired" by functional groups at the C-terminal ends of the beta-strands in the barrel domain; the identity of the substrate is determined in part by the identities of residues located at the end of the eighth beta-strand in the barrel domain and two mobile loops in the capping domain. On the basis of the identities of the acid/base functional groups at the ends of the beta-strands, the currently available structure-function relationships derived from functionally characterized members are often sufficient for "deciphering" the identity of the chemical reaction catalyzed by sequence-divergent members discovered in genome projects. However, insufficient structural information for liganded complexes for specifying the identity of the substrate is available. In this paper, the structure of the complex of L-Ala-L-Glu with the L-Ala-D/L-Glu epimerase from Bacillus subtilis is reported. As expected for the 1,1-proton transfer reaction catalyzed by this enzyme, the alpha-carbon of the substrate is located between Lys 162 and Lys 268 at the ends of the second and sixth beta-strands in the barrel domain. The alpha-ammonium group of the l-Ala moiety is hydrogen bonded to both Asp 321 and Asp 323 at the end of the eighth beta-strand, revealing a novel strategy for substrate recognition in the superfamily. The delta-carboxylate group of the Glu moiety is hydrogen bonded to Arg 24 in one of the flexible loops in the capping domain, thereby providing a structural explanation for the restricted substrate specificity of this epimerase [Schmidt, D. M., Hubbard, B. K., and Gerlt, J. A. (2001) Biochemistry 40, 15707-15715]. These studies provide important new information about the structural bases for substrate specificity in the enolase superfamily.

Michael acceptors have long been recognized as reactive functionalities that may link a biologically active molecule to its cellular target. 1,2-Dehydro amino acids are potential Michael acceptors present in a large number of natural products, but their reactivity is modulated by the deactivating nature of the alpha-amino group engaged in an amide bond. We describe here the preparation of 3-fluoro-1,2-dehydroalanine moieties within peptides that significantly enhance the reactivity of the Michael acceptor. Two different routes were designed to access these compounds, one relying on chemical means to introduce the desired functionality and the second taking advantage of a peptide epimerase. In the chemical approach, the fluoro-Pummerer reaction of cysteine derivatives afforded 3-fluorocysteine residues that were oxidized to the corresponding sulfoxides, followed by thermolytic elimination to provide the desired 3-fluorodehydroalanines. The mechanism of the fluoro-Pummerer reaction was investigated and several possible pathways were ruled out. The enzymatic approach utilized the dipeptide epimerase YcjG from Escherichia coli. Difluorinated alanine was incorporated at the C terminus of a dipeptide by chemical means. The resulting peptide proved to be a substrate for YcjG, which catalyzed fluoride elimination to provide the 3-fluorodehydroalanine-containing peptide. Mechanistic investigations showed that fluoride elimination occurred faster than epimerization and at a rate close to that of epimerization of Ala-Ala.

Purpose: Recurrent urinary tract infections (UTIs) in susceptible women remain a common urological condition. With an increasing number of UTIs being caused by antibiotic resistant bacteria there is a need for alternatives to antibiotics. We determined whether multiple doses of a vaginal mucosal vaccine are effective for increasing long-term resistance to recurrent UTIs., Materials and Methods: A total of 54 women were entered into a double-blind, placebo controlled, phase 2 clinical trial using a vaginal vaccine containing 10 heat killed uropathogenic bacteria. Patients were withdrawn from prophylactic antibiotics and randomly assigned to 1 of 3 treatment groups, namely placebo only, primary immunization or primary plus booster immunizations. Subjects received treatments at 0, 1, 2, 6, 10 and 14 weeks. Placebo treated patients received suppositories without bacteria. The primary immunization group received vaccine suppositories, followed by 3 doses of placebo. Patients receiving booster immunizations were given 6 vaccine suppositories. All women were followed for 6 months to determine the time until first recurrence, number of infections and adverse reactions., Results: Women receiving 6 vaccine doses remained free of infections for a significantly longer period than those receiving placebo or primary immunizations. Of patients receiving 6 immunizations 55% did not experience an infection, whereas 89% of placebo treated women had UTIs. No women had significant adverse effects., Conclusions: This study demonstrates that vaginal mucosal vaccine given for a 14-week period increased the time to re-infection in UTI susceptible women. The infrequent, minimal adverse reactions confirm previous observations on the safety of this vaginal mucosal immunization regimen.

The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction. The molecular pathway for natural divergent evolution of function in the superfamily is unknown. We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.

The members of the enolase superfamily catalyze different overall reactions, yet share a partial reaction that involves Mg(2+)-assisted enolization of the substrate carboxylate anion. The fate of the resulting enolate intermediate is determined by the active site of each enzyme. Several members of this superfamily have been structurally characterized to permit an understanding of the evolutionary strategy for using a common structural motif to catalyze different overall reactions. In the preceding paper, two new members of the superfamily were identified that catalyze the epimerization of the glutamate residue in L-Ala-D/L-Glu. These enzymes belong to the muconate lactonizing enzyme subgroup of the enolase superfamily, and their sequences are only 31% identical. The structure of YcjG, the epimerase from Escherichia coli, was determined by MAD phasing using both the SeMet-labeled protein and a heavy atom derivative. The structure of YkfB, the epimerase from Bacillus subtilis, was determined by molecular replacement using the muconate lactonizing enzyme as a search model. In this paper, we report the three-dimensional structures of these enzymes and compare them to the structure of o-succinylbenzoate synthase, another member of the muconate lactonizing enzyme subgroup.

The members of the mechanistically diverse enolase superfamily catalyze different overall reactions by using a common catalytic strategy and structural scaffold. In the muconate lactonizing enzyme (MLE) subgroup of the superfamily, abstraction of a proton adjacent to a carboxylate group initiates reactions, including cycloisomerization (MLE), dehydration [o-succinylbenzoate synthase (OSBS)], and 1,1-proton transfer (catalyzed by an OSBS that also catalyzes a promiscuous N-acylamino acid racemase reaction). The realization that a member of the MLE subgroup could catalyze a 1,1-proton transfer reaction, albeit poorly, led to a search for other enzymes which might catalyze a 1,1-proton transfer as their physiological reaction. YcjG from Escherichia coli and YkfB from Bacillus subtilis, proteins of previously unknown function, were discovered to be L-Ala-D/L-Glu epimerases, although they also catalyze the epimerization of other dipeptides. The values of k(cat)/K(M) for L-Ala-D/L-Glu for both proteins are approximately 10(4) M(-1) s(-1). The genomic context and the substrate specificity of both YcjG and YkfB suggest roles in the metabolism of the murein peptide, of which L-Ala-D-Glu is a component. Homologues possessing L-Ala-D/L-Glu epimerase activity have been identified in at least two other organisms.

The BvgA-BvgS two-component signal transduction system regulates expression of virulence factors in Bordetella pertussis. The BvgA response regulator activates transcription by binding to target promoters, which include those for the genes encoding filamentous hemagglutinin (fha) and pertussis toxin (ptx). We have previously shown that at both promoters the phosphorylated form of BvgA binds multiple high- and low-affinity sites. Specifically, at the fha promoter, we proposed that there may be high- and a low-affinity binding sites for the BvgA dimer. In our present investigation, we used DNA binding analyses and in vitro and in vivo assays of promoters with substitutions and deletions to support and extend this hypothesis. Our observations indicate that (i) binding of BvgA approximately P to a primary (high-affinity) site and a secondary binding region (lower affinity) is cooperative, (ii) although both the primary binding site and the secondary binding region are required for full activity of the wild-type (undeleted) promoter, deletion of two helical turns within the secondary binding region can produce a fully active or hyperactive promoter, and (iii) BvgA binding to the secondary binding region shows limited DNA sequence specificity.

Recent approaches to the problem of inferring a continuous probability distribution from a finite set of data have used a scalar field theory for the form of the prior probability distribution. This paper presents a more general form for the prior distribution that has a geometrical interpretation and can improve the specificity of likely solutions. It is also demonstrated that a numerical sampling of the posterior probability distribution can be used as an alternative to a histogram for visualization and to make probabilistic inferences from the data.

We present a new approach to the electromagnetic inverse problem that explicitly addresses the ambiguity associated with its ill-posed character. Rather than calculating a single "best" solution according to some criterion, our approach produces a large number of likely solutions that both fit the data and any prior information that is used. Whereas the range of the different likely results is representative of the ambiguity in the inverse problem even with prior information present, features that are common across a large number of the different solutions can be identified and are associated with a high degree of probability. This approach is implemented and quantified within the formalism of Bayesian inference, which combines prior information with that of measurement in a common framework using a single measure. To demonstrate this approach, a general neural activation model is constructed that includes a variable number of extended regions of activation and can incorporate a great deal of prior information on neural current such as information on location, orientation, strength, and spatial smoothness. Taken together, this activation model and the Bayesian inferential approach yield estimates of the probability distributions for the number, location, and extent of active regions. Both simulated MEG data and data from a visual evoked response experiment are used to demonstrate the capabilities of this approach.

Case Report: A 28-year-old male ingested the herbicide diquat. The patient rapidly developed severe gastrointestinal and metabolic disturbances, airway compromise, respiratory failure, renal failure, hemodynamic collapse, and seizures. We describe multiple metabolic abnormalities, an apparent artifact introduced by diquat in the laboratory assay for serum creatinine, serum diquat levels, and the need for emergency airway management.

We used MRI of hyperpolarized 3He to demonstrate some novel aspects of gas diffusion. Two different techniques were used. First, a slice was burned into a one-dimensional image by inverting the spins in the slice and diffusion was studied by measuring the magnetization as it filled the depleted slice. A diffusion coefficient was determined by the fit of these data. Second, one-dimensional diffusion images were made using a Stejskal-Tanner PGSE method. This was done with and without a temperature gradient present, showing that the effect of temperature can be dynamically monitored by such diffusion images. Copyright 1997 Academic Press. Copyright 1997Academic Press

To determine the pulmonary complications in HIV-1-infected patients in Dar es Salaam, Tanzania, and to evaluate the diagnostic utility of bronchoscopy and bronchoalveolar lavage, we carried out a prospective study of 237 patients with acute respiratory disease who were hospitalized at Muhimbili Medical Center (MMC). Diagnoses were made using well-defined criteria. Of the total, 127 (54%) were HIV-1-seropositive and 110 (46%) were seronegative. Tuberculosis was the most common diagnosis occurring in 95 (75%) HIV-1-seropositive and 87 (79%) seronegative patients. Bacterial pneumonia was the next most common diagnosis occurring in 18 (14%) HIV-1-seropositive and 17 (15%) seronegative patients. Pneumocystis carinii pneumonia was diagnosed in one and Kaposi's sarcoma was seen in only two HIV-1-seropositive patients. Bronchoscopy with bronchoalveolar lavage was the sole source of a diagnosis in nine (8%) seropositive and six (5%) seronegative patients. We conclude that the HIV seroprevalence rate among patients hospitalized for acute respiratory disease at MMC is extremely high. Tuberculosis was the most common cause of pulmonary disease, regardless of HIV serostatus, and other HIV-associated opportunistic pulmonary infections were unusual. Bronchoscopy with bronchoalveolar lavage added little to the diagnosis and thus should not be high-priority procedures for the routine workup in resource-poor areas where tuberculosis is endemic.

A rare case of cryptococcal infection presenting as a neck mass in an otherwise healthy individual is reported. The mass resulted in lytic destruction of portions of the cervical vertebrae and produced a focal neurologic deficit in one of the upper extremities. Although other lesions in the skull, femur, and humerus were identified, no involvement of the lungs or central nervous system could be detected. The patient was initially treated with amphotericin B and flucytosine, but eventually also required fluconazole and surgical debridement for complete resolution of the infection. The diagnosis, treatment, and manifestations of cryptococcosis in the head and neck are discussed.

Integrated analyses of human anatomical and functional measurements offer a powerful paradigm for human brain mapping. Magnetoencephalography (MEG) and EEG provide excellent temporal resolution of neural population dynamics as well as capabilities for source localization. Anatomical magnetic resonance imaging (MRI) provides excellent spatial resolution of head and brain anatomy, whereas functional MRI (fMRI) techniques provide an alternative measure of neural activation based on associated hemodynamic changes. These methodologies constrain and complement each other and can thereby improve our interpretation of functional neural organization. We have developed a number of computational tools and techniques for the visualization, comparison, and integrated analysis of multiple neuroimaging techniques. Construction of geometric anatomical models from volumetric MRI data allows improved models of the head volume conductor and can provide powerful constraints for neural electromagnetic source modeling. These approaches, coupled to enhanced algorithmic strategies for the inverse problem, can significantly enhance the accuracy of source-localization procedures. We have begun to apply these techniques for studies of the functional organization of the human visual system. Such studies have demonstrated multiple, functionally distinct visual areas that can be resolved on the basis of their locations, temporal dynamics, and differential sensitivity to stimulus parameters. Our studies have also produced evidence of internal retinotopic organization in both striate and extrastriate visual areas but have disclosed organizational departures from classical models. Comparative studies of MEG and fMRI suggest a reasonable but imperfect correlation between electrophysiological and hemodynamic responses. We have demonstrated a method for the integrated analysis of fMRI and MEG, and we outline strategies for improvement of these methods. By combining multiple measurement techniques, we can exploit the complementary strengths and transcend the limitations of the individual neuro-imaging methods.

Site-directed mutagenesis was used to introduce mutations into the gene for the iron protein (IP) of succinate dehydrogenase (SDH) of Saccharomyces cerevisiae. Specifically, three mutations were examined which caused the synthesis of truncated IP peptides missing four, seven, or 17 amino acids from the C-terminus, respectively. The deletion of seven or more amino acids includes the loss of two lysine residues, which appear to have been highly conserved in evolution. While the deletion of four amino acids had no effect on the assembly of complex II and on its activity, the deletion including the two lysines abolished SDS activity completely and led to the failure of the imported IP peptide to be incorporated into a stable complex II or SDH complex. Replacement of one of the lysines by threonine had no effect, but replacement of both by threonine affected the specific activity of complex II but not its assembly and stability.

Tethering of the spinal cord is frequently associated with myelomeningocele. However, orthopaedic surgeons must be familiar with the primary tethered cord, which is associated not with an overt myelomeningocele but with more subtle clinical findings, particularly in the pediatric patient. The charts of patients with tethered spinal cords from the Honolulu Unit of the Shriners Hospital for Crippled Children are reviewed to highlight potential diagnostic and treatment problems. Tethered spinal cord should be considered in the differential diagnosis of patients who present with isolated lower extremity deformities and/or subtle neurologic findings. Appropriate evaluation may require magnetic resonance imaging, ultrasonography, or metrizamide computed tomography. Urologic function must also be evaluated and monitored. The literature supports an aggressive diagnostic and, where indicated, surgical approach to the tethered spinal cord. Diagnosis and treatment at a young age has been shown to correlate with optimal results.

Replantation of fingers is a well established procedure following traumatic amputation. Several studies have documented the functional results of replanted digits. Studies have not documented the long-term (greater than 5 yrs) patency of the microvascular anastomosis in the replanted digit. Duplex imaging, a combination of B-mode ultrasound and bidirectional Doppler ultrasound has been shown to visualize vessels 1 mm in diameter. Color enhancement of the ultrasound image is now possible. We evaluated 18 patients with 24 replanted fingers to assess vascular patency. A minimum of five years follow-up was required for inclusion in the study group. Examination showed a patent anastomosis in every case and a Digital-Brachial Index within normal limits in 71% of the digits. Microvascular anastomoses in vessels as small as 1 mm in diameter remain open as long as 11 years following completion. The sophisticated techniques of color enhanced Duplex imaging makes long-term evaluation of these procedures possible in a non-invasive and accurate fashion. The use of such imaging modalities will have an important role in the evaluation of replanted digits and microvascular procedures in general.

Muscarinic receptors found in the N1E-115 mouse neuroblastoma cell line were tested for their ability to mediate stimulation of phosphatidylinositol (PI) turnover. This study was facilitated by the development of a new solvent system (acetone:butanol:acetic acid:water, 3:5:1:1) for the rapid and consistent separation of PI by one-dimensional thin-layer chromatography. Cholinergic stimulation caused as much as a 680% increase in the incorporation of 32P into PI. Enhanced incorporation of 32P into PI could be measured as early as 4 min after stimulation began. By 20 min, the rate of incorporation by stimulated cells had decreased to that of unstimulated cells, indicating desensitization. The magnitude of the response was dependent on the extent of receptor occupancy, and the response elicited by a saturating dose of carbamylcholine was blocked completely by 10(-7) M atropine, a specific muscarinic antagonist. Chronic stimulation, known to cause a loss of receptor binding sites, led to a 90% decrease in the maximum response even after a 40-min withdrawal period. Replacement of Na+ ions in the medium with choline or K+ severely impaired the ability of the cells to incorporate added 32P into PI (90 and 50%, respectively). Removal of the putative second messenger Ca2+ for short periods of time by the addition of excess EGTA did not alter either basal or muscarinic-stimulated PI turnover.

Murine tumor cells frequently express retroviral protein p15E, a protein with antiinflammatory activity. This has led to the hypothesis that p15E expression allows nascent tumor cells to escape host immunologic defenses. To evaluate the role of p15E expression in tumorigenesis, NIH3T3 cells transformed by various oncogenes and BALB/c lines transformed by carcinogens or SV40 were examined for p15E expression and tumorigenicity. All of the NIH3T3 transformants and most of the BALB/c transformants did not express p15E, indicating that transformation per se does not inevitably induce the expression of p15E. Although not expressing p15E, some of these transformants were capable of forming tumors in immune competent hosts, indicating that p15E is not universally required for tumor growth. Four of the transformed cell lines negative for p15E expression and deficient in tumor-forming capacity were transfected with a gene coding for Moloney retroviral p15E. Despite the expression of p15E, there was no augmentation of their tumorigenic capacity, showing that p15E is not sufficient to ensure tumor formation by a transformed cell. These results argue against a general role for retroviral p15E expression in tumorigenesis.