Genome Sequences of Marichromatium gracile HOL-1 and Its Purple Photosynthetic Coisolate, Afifella sp. H1R.

We sequenced the genomes of both the purple sulfur gammaproteobacterium Marichromatium gracile HOL-1 and another purple photosynthetic organism, strain H1R, that was originally isolated as an unidentified contaminant. Through genome sequencing, we have now identified organism H1R as a species of Afifella. A whole-genome-based phylogenetic analysis of both species is provided.

CoeViz 2: Protein Graphs Derived from Amino Acid Covariance.

Proteins by and large carry out their molecular functions in a folded state when residues, distant in sequence, assemble together in 3D space to bind a ligand, catalyze a reaction, form a channel, or exert another concerted macromolecular interaction. It has been long recognized that covariance of amino acids between distant positions within a protein sequence allows for the inference of long range contacts to facilitate 3D structure modeling. In this work, we investigated whether covariance analysis may reveal residues involved in the same molecular function. Building upon our previous work, CoeViz, we have conducted a large scale covariance analysis among 7595 non-redundant proteins with resolved 3D structures to assess (1) whether the residues with the same function coevolve, (2) which covariance metric captures such couplings better, and (3) how different molecular functions compare in this context. We found that the chi-squared metric is the most informative for the identification of coevolving functional sites, followed by the Pearson correlation-based, whereas mutual information is the least informative. Of the seven categories of the most common natural ligands, including coenzyme A, dinucleotide, DNA/RNA, heme, metal, nucleoside, and sugar, the trace metal binding residues display the most prominent coupling, followed by the sugar binding sites. We also developed a web-based tool, CoeViz 2, that enables the interactive visualization of covarying residues as cliques from a larger protein graph. CoeViz 2 is publicly available at https://research.cchmc.org/CoevLab/.

CoeViz: A Web-Based Integrative Platform for Interactive Visualization of Large Similarity and Distance Matrices.

Similarity and distance matrices are general data structures that describe reciprocal relationships between the objects within a given dataset. Commonly used methods for representation of these matrices include heatmaps, hierarchical trees, dimensionality reduction, and various types of networks. However, despite a well-developed foundation for the visualization of such representations, the challenge of creating an interactive view that would allow for quick data navigation and interpretation remains largely unaddressed. This problem becomes especially evident for large matrices with hundreds or thousands objects. In this work, we present a web-based platform for the interactive analysis of large (dis-)similarity matrices. It consists of four major interconnected and synchronized components: a zoomable heatmap, interactive hierarchical tree, scalable circular relationship diagram, and 3D multi-dimensional scaling (MDS) scatterplot. We demonstrate the use of the platform for the analysis of amino acid covariance data in proteins as part of our previously developed CoeViz tool. The web-platform enables quick and focused analysis of protein features, such as structural domains and functional sites.

A Quantitative Model to Estimate Drug Resistance in Pathogens.

Pneumocystis pneumonia (PCP) is an opportunistic infection that occurs in humans and other mammals with debilitated immune systems. These infections are caused by fungi in the genus Pneumocystis, which are not susceptible to standard antifungal agents. Despite decades of research and drug development, the primary treatment and prophylaxis for PCP remains a combination of trimethoprim (TMP) and sulfamethoxazole (SMX) that targets two enzymes in folic acid biosynthesis, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), respectively. There is growing evidence of emerging resistance by Pneumocystis jirovecii (the species that infects humans) to TMP-SMX associated with mutations in the targeted enzymes. In the present study, we report the development of an accurate quantitative model to predict changes in the binding affinity of inhibitors (Ki, IC50) to the mutated proteins. The model is based on evolutionary information and amino acid covariance analysis. Predicted changes in binding affinity upon mutations highly correlate with the experimentally measured data. While trained on Pneumocystis jirovecii DHFR/TMP data, the model shows similar or better performance when evaluated on the resistance data for a different inhibitor of PjDFHR, another drug/target pair (PjDHPS/SMX) and another organism (Staphylococcus aureus DHFR/TMP). Therefore, we anticipate that the developed prediction model will be useful in the evaluation of possible resistance of the newly sequenced variants of the pathogen and can be extended to other drug targets and organisms.

CoeViz: a web-based tool for coevolution analysis of protein residues.

BACKGROUND: Proteins generally perform their function in a folded state. Residues forming an active site, whether it is a catalytic center or interaction interface, are frequently distant in a protein sequence. Hence, traditional sequence-based prediction methods focusing on a single residue (or a short window of residues) at a time may have difficulties in identifying and clustering the residues constituting a functional site, especially when a protein has multiple functions. Evolutionary information encoded in multiple sequence alignments is known to greatly improve sequence-based predictions. Identification of coevolving residues further advances the protein structure and function annotation by revealing cooperative pairs and higher order groupings of residues. RESULTS: We present a new web-based tool (CoeViz) that provides a versatile analysis and visualization of pairwise coevolution of amino acid residues. The tool computes three covariance metrics: mutual information, chi-square statistic, Pearson correlation, and one conservation metric: joint Shannon entropy. Implemented adjustments of covariance scores include phylogeny correction, corrections for sequence dissimilarity and alignment gaps, and the average product correction. Visualization of residue relationships is enhanced by hierarchical cluster trees, heat maps, circular diagrams, and the residue highlighting in protein sequence and 3D structure. Unlike other existing tools, CoeViz is not limited to analyzing conserved domains or protein families and can process long, unstructured and multi-domain proteins thousands of residues long. Two examples are provided to illustrate the use of the tool for identification of residues (1) involved in enzymatic function, (2) forming short linear functional motifs, and (3) constituting a structural domain. CONCLUSIONS: CoeViz represents a practical resource for a quick sequence-based protein annotation for molecular biologists, e.g., for identifying putative functional clusters of residues and structural domains. CoeViz also can serve computational biologists as a resource of coevolution matrices, e.g., for developing machine learning-based prediction models. The presented tool is integrated in the POLYVIEW-2D server (http://polyview.cchmc.org/) and available from resulting pages of POLYVIEW-2D.

Telephone communications between diabetes clients and nurse educators.

The purpose of this study was to describe the use of telephone communications between diabetes nurse educators (DNEs) and their clients with diabetes. A questionnaire was designed to examine the use of the telephone with diabetes clients from the perspective of DNEs. A total of 465 DNEs across the US were selected using a systematic sample from the membership directory of the American Association of Diabetes Educators. A total of 247 were questionnaires completed and returned (55%). Ninety-one percent of DNEs reported using the phone with clients and averaged 15 phone calls per week. Over 90% frequently reported discussing the following topics with clients: home blood glucose monitoring, hyperglycemia, hypoglycemia, insulin use, and diet. Analysis of telephone users showed that DNE experience and diabetes educator certification were significant factors in the differences observed in the reported topics discussed over the telephone. These findings suggest the need for guidelines for telephone contact with diabetes clients.

The impact of an educational program on improving diabetes knowledge and changing behaviors of nurses in long-term care facilities.

Nursing staffs from two long-term care facilities attended a multisession educational program about the care of residents with diabetes (treatment group). A control group consisted of the nursing staffs from two other similar facilities who did not participate in the educational program. Both groups were given a knowledge pretest and posttest. A chart review also was conducted following the educational intervention to determine any changes in the diabetes care provided by the treatment group. Following the educational program, the treatment group had a significant increase in their mean score on the knowledge test compared with the control group. However, a review of the residents' charts revealed no significant increases in specific behaviors related to diabetes care. The findings suggest that, in addition to educational programs, more focused training concerning diabetes care is needed to improve the care of residents with diabetes in long-term care facilities. Implications for diabetes educators are discussed.

Skeletal muscle GLUT4 protein concentration and aging in humans.

The insulin resistance of aging has been attributed to a postreceptor defect in skeletal muscle. The present study examined whether a reduction in the concentration of the insulin-stimulated glucose transporter (GLUT4) in skeletal muscle was associated with advancing age in men (n = 55) and women (n = 29). Insulin sensitivity (minimal model) was negatively associated (P < 0.001) with age (range, 18-80 years) in men (r = -0.44) and women (r = -0.58). GLUT4 protein concentration in the vastus lateralis was also negatively associated (P < 0.05) with age (men, r = -0.28; women, r = -0.51). There was no relation (P > 0.15) between GLUT4 content in the gastrocnemius and age. GLUT4 concentration in the vastus lateralis was positively associated (P < 0.01) with insulin sensitivity in both sexes (r = 0.42); this relationship persisted in the men after adjusting for overall adiposity, regional adiposity, and cardiorespiratory fitness. These findings suggest that a decrement in GLUT4 protein concentration in skeletal muscle may at least partially contribute to the insulin resistance of aging in humans.

Liver glucokinase: decreased activity in patients with type II diabetes.

Because of the demonstration of a genetic linkage between glucokinase and Type II diabetes, and the central role of glucokinase on glucose metabolism, we studied glucokinase activity in the liver of patients with and without Type II diabetes. Glucokinase activity was decreased by about 50% in obese subjects with diabetes (n = 12) compared with (p < 0.01) lean controls (n = 9) and (p < 0.05) obese controls (n = 10). There was no difference between lean and obese controls. Fifty percent of subjects with diabetes had lower liver glucokinase activity than the lowest value of the controls. These data further support the important role that glucokinase plays in the pathogenesis of Type II diabetes.

Conceptual implicit memory: a developmental study.

The widely accepted standpoint that implicit memory emerges earlier in development than explicit memory, and is more stable from childhood to adult age, is based on experimental data essentially collected in perceptual tasks. The present study was aimed at investigating whether these findings still hold when a more conceptual task is used. We compared the performance of children at two age levels (2nd and 4th grades) on a category-exemplar generation task. Results showed that performances of the two groups were comparable when the target items were typical of their categories, as in Experiment 2, and for a subset of the items in Experiment 1. However, the older children outperformed the younger children in Experiment 1 when the items selected were atypical of their categories. Interpretations of these findings are discussed.

Utilizing content analysis of counseling sessions to identify psychosocial stressors among patients with type II diabetes.

This qualitative, descriptive study identified specific individual and family psychosocial stressors that impact on the ability of patients with type II, non-insulin-dependent diabetes mellitus to manage the self-care regimens recommended by healthcare professionals. A series of 2 to 12 counseling sessions was incorporated into a nationally recognized outpatient diabetes education program. Data collection consisted of in-depth interviews in which six patients with type II diabetes and their families discussed current problems associated with their diabetes. Content analysis identified the following themes and problem areas: patients' inner experience with diabetes, family stressors that affect patients with diabetes, coping strategies of type II diabetes patients, and psychopathology. Based on these findings, specific interventions that emphasize family involvement and support were developed for the healthcare team to use with patients with type II diabetes.

Circulating insulin-like growth factor I concentrations in clinically severe obese patients with and without NIDDM in response to weight loss.

Earlier studies have demonstrated decreased levels of circulating Insulin-Like Growth Factor-I (IGF-I) in patients with NIDDM and IDDM (Yde 1969; Rieu and Binoux 1985), with a return to normal in those diabetics who achieve improved metabolic control (Rieu and Binoux 1985; Ameil, Sherwin, Hintz, Gertner, Press and Tamborlane 1984) following insulin therapy. One method of improving metabolic control in clinically severe obese NIDDM patients is the gastric bypass procedure (GBP). This study revealed a significant decrease in serum IGF-I concentrations in clinically severe obese patients with NIDDM (obese NIDDM) (105 ng/dl +/- 11; n = 29) as compared with clinically severe obese patients with normal glucose tolerances (obese control) (143 +/- 11; n = 21) and lean controls (177 +/- 14; n = 19) (p < 0.001). Following a GBP, IGF-I levels increased in the NIDDM group (142 ng/dl +/- 13.0; n = 20) to the extent that no significant difference was seen between postoperative NIDDM, obese controls, and lean controls. Postoperative IGF-I levels in the obese controls (151 +/- 14; n = 9) revealed no difference from preoperative levels. Postoperative obese NIDDM and obese control had a 28% and 29% decrease, respectively, in weight, with no difference between the groups in respect to Body Mass Indices. The NIDDM postoperative group revealed reductions in levels of HbA1C, insulin, and glucose concurrent with elevations in IGF-I when compared with controls. We conclude that improvement in glucose control led to the increase in IGF-I levels.

Measuring the diabetes knowledge of nurses in long-term care facilities.

A knowledge test was developed by a local diabetes educators' group to evaluate the diabetes knowledge base of 59 nurses currently employed in four long-term care facilities in eastern North Carolina. The test contained items in the following categories: monitoring, medication, hypoglycemia, hyperglycemia, illness care, diet, exercise, foot care, and patient/family education. The results of the diabetes knowledge test were analyzed according to overall test scores as well as scores in each of the selected categories. The overall scores ranged from 42% to 89%, with a group mean of 67% (passing = 70% or higher). The scores in the individual categories ranged from 18.6% for monitoring and 23.8% for medications to 91.5% for patient/family education.

Weight loss in severely obese subjects prevents the progression of impaired glucose tolerance to type II diabetes. A longitudinal interventional study.

OBJECTIVE: To determine if weight loss may prevent conversion of impaired glucose tolerance (IGT) to diabetes, because weight loss reduces insulin resistance. The prevalence of IGT in the U.S. population is estimated at 11.2%, more than twice that of diabetes. Furthermore, because an oral glucose tolerance test is needed for its detection, most of these patients are undiagnosed. Screening for IGT would be meaningful if progression to diabetes could be delayed or prevented. RESEARCH DESIGN AND METHODS: For an average of 5.8 years (range 2-10 years), 136 individuals with IGT and clinically severe obesity (> 45 kg excess body weight) were followed. The experimental group included 109 patients with IGT who underwent bariatric surgery for weight loss. The control group was made up of 27 subjects with IGT who did not have bariatric surgery. The criteria of the World Health Organization was used to detect IGT and diabetes in this population. The main outcome measure of this nonrandomized control trial is the incidence density, or number of events (development of diabetes) divided by the time of exposure to risk. RESULTS: Of the 27 subjects in the control group, 6 developed diabetes during an average of 4.8 +/- 2.5 years of postdiagnosis follow-up, yielding a rate of conversion to diabetes of 4.72 cases per 100 person-years. The 109 individuals of the experimental group were followed for an average of 6.2 +/- 2.5 years postbariatric surgery. Based on the 95% confidence interval of the comparison group, we would expect to find that between 22 and 36 subjects in the experimental group developed diabetes over the follow-up period. Only 1 of the 109 experimental-group patients developed diabetes, resulting in a conversion rate of the experimental group of only 0.15 cases per 100 person-years, which is significantly lower (P < 0.0001) than the control group. CONCLUSIONS: Weight loss in patients with clinically severe obesity prevents the progression of IGT to diabetes by > 30-fold.

Exercise training increases GLUT-4 protein concentration in previously sedentary middle-aged men.

The purpose of this study was to determine if 14 wk of exercise training would increase insulin-sensitive glucose transporter protein (GLUT-4) concentration in skeletal muscle of previously sedentary middle-aged men (47.2 +/- 1.3 yr; n = 13). Muscle samples (lateral gastrocnemius) and insulin action [insulin sensitivity index (ISI), minimal model] were obtained in the sedentary condition and 48 h after the final training bout. GLUT-4 protein concentration increased (P < 0.001, 2,629 +/- 331 to 4,140 +/- 391 absorbance units/100 micrograms protein) with exercise training by 1.8-fold. ISI increased by twofold (P < 0.05, 2.1 +/- 0.5 to 3.4 +/- 0.7 SI x 10(5) min/pM) with training. The percentage of GLUT-4 rich type IIa muscle fibers increased by approximately 10% (P < 0.01), which may have contributed to the elevation in transporter protein. GLUT-4 concentration and citrate synthase activity (1.7-fold, P < 0.001) also increased by similar increments. These findings indicate that GLUT-4 protein concentration is elevated in middle-aged individuals with exercise training.

Restoration of insulin responsiveness in skeletal muscle of morbidly obese patients after weight loss. Effect on muscle glucose transport and glucose transporter GLUT4.

A major defect contributing to impaired insulin action in human obesity is reduced glucose transport activity in skeletal muscle. This study was designed to determine whether the improvement in whole body glucose disposal associated with weight reduction is related to a change in skeletal muscle glucose transport activity and levels of the glucose transporter protein GLUT4. Seven morbidly obese (body mass index = 45.8 +/- 2.5, mean +/- SE) patients, including four with non-insulin-dependent diabetes mellitus (NIDDM), underwent gastric bypass surgery for treatment of their obesity. In vivo glucose disposal during a euglycemic clamp at an insulin infusion rate of 40 mU/m2 per min was reduced to 27% of nonobese controls (P less than 0.01) and improved to 78% of normal after weight loss of 43.1 +/- 3.1 kg (P less than 0.01). Maximal insulin-stimulated glucose transport activity in incubated muscle fibers was reduced by approximately 50% in obese patients at the time of gastric bypass surgery but increased twofold (P less than 0.01) to 88% of normal in five separate patients after similar weight reduction. Muscle biopsies obtained from vastus lateralis before and after weight loss revealed no significant change in levels of GLUT4 glucose transporter protein. These data demonstrate conclusively that insulin resistance in skeletal muscle of mobidly obese patients with and without NIDDM cannot be causally related to the cellular content of GLUT4 protein. The results further suggest that morbid obesity contributes to whole body insulin resistance through a reversible defect in skeletal muscle glucose transport activity. The mechanism for this improvement may involve enhanced transporter translocation and/or activation.

Surgical treatment of obesity and its effect on diabetes: 10-y follow-up.

Since 1980 we have performed the identical Greenville gastric bypass (GGB) procedure on 479 morbidly obese patients with an acceptable morbidity and a mortality rate of 1.2%. The weight loss in the series was well maintained over the follow-up period of 10 y. The GGB can control non-insulin-dependent diabetes mellitus (NIDDM) in most patients. The group of 479 patients included 101 (21%) with NIDDM and another 62 (13%) who were glucose impaired. Of these 163 individuals, 141 reverted to normal and only 22 (5%) remained with inadequate control of their carbohydrate metabolism. Those patients who were older or whose diabetes was of longer duration were less likely to revert to normal values. The gastric bypass operation is an effective approach for the treatment of morbid obesity. Along with its control of weight, the operation also controls the hyperglycemia, hyperinsulinemia, and insulin resistance of the majority of patients with either glucose impairment or frank NIDDM.