Spontaneous Unfolding and Refolding of FNIII Domains Assayed by Thiol Exchange

Fibronectin (FN) is a large extracellular matrix (ECM) protein that is made up of

type I (FNI), type II (FNII), & type III (FNIII) domains. It assembles into an insoluble

supra-­‐‑molecular structure: the fibrillar FN matrix. FN fibrillogenesis is a cell‐‑mediated process, which is initiated when FN binds to integrins on the cell surface. The FN matrix plays an important role in cell migration, proliferation, signaling & adhesion. Despite decades of research, the FN matrix is one of the least understood supra-­‐‑molecular protein assemblies. There have been several attempts to elucidate the exact mechanism of matrix assembly resulting in significant progress in the field but it is still unclear as to what are FN-­‐‑FN interactions, the nature of these interactions and the domains of FN that

are in contact with each other. FN matrix fibrils are elastic in nature. Two models have been proposed to explain the elasticity of the fibrils. The first model: the ‘domain unfolding’ model postulates that the unraveling of FNIII domains under tension explains fibril elasticity.

The second model relies on the conformational change of FN from compact to extended to explain fibril elasticity. FN contain 15 FNIII domains, each a 7-­‐‑strand beta sandwich. Earlier work from our lab used the technique of labeling a buried Cys to study the ‘domain unfolding’ model. They used mutant FNs containing a buried Cys in a single FNIII domain and found that 6 of the 15 FNIII domains label in matrix fibrils. Domain unfolding due to tension, matrix associated conformational changes or spontaneous folding and unfolding are all possible explanation for labeling of the buried Cys. The present study also uses the technique of labeling a buried Cys to address whether it is spontaneous folding and unfolding that labels FNIII domains in cell culture. We used thiol reactive DTNB to measure the kinetics of labeling of buried Cys in eleven FN III domains over a wide range of urea concentrations (0-­‐‑9M). The kinetics data were globally fit using Mathematica. The results are equivalent to those of H-­‐‑D exchange, and

provide a comprehensive analysis of stability and unfolding/folding kinetics of each

domain. For two of the six domains spontaneous folding and unfolding is possibly the reason for labeling in cell culture. For the rest of the four domains it is probably matrix associated conformational changes or tension induced unfolding.

A long-­‐‑standing debate in the protein-­‐‑folding field is whether unfolding rate

constants or folding rate constants correlate to the stability of a protein. FNIII domains all have the same ß sandwich structure but very different stabilities and amino acid sequences. Our study analyzed the kinetics of unfolding and folding and stabilities of eleven FNIII domains and our results show that folding rate constants for FNIII domains are relatively similar and the unfolding rates vary widely and correlate to stability. FN forms a fibrillar matrix and the FN-­‐‑FN interactions during matrix fibril formation are not known. FNI 1-­‐‑9 or the N-­‐‑ terminal region is indispensible for matrix formation and its major binding partner has been shown to be FNIII 2. Earlier work from our lab, using FRET analysis showed that the interaction of FNI 1-­‐‑9 with a destabilized FNIII 2 (missing the G strand, FNIII 2ΔG) reduces the FRET efficiency. This efficiency is restored in the presence of FUD (bacterial adhesion from S. pyogenes) that has been known to interact with FNI 1-­‐‑9 via a tandem ß zipper. In the present study we

use FRET analysis and a series of deletion mutants of FNIII 2ΔG to study the shortest fragment of FNIII 2ΔG that is required to bind FNI 1-­‐‑9. Our results presented here are qualitative and show that FNIII 2ΔC’EFG is the shortest fragment required to bind FNI 1-­‐‑9. Deletion of one more strand abolishes the interaction with FNI 1-­‐‑9.

Variation in the type and frequency of postoperative invasive Staphylococcus aureus infections according to type of surgical procedure.

OBJECTIVE: To determine the epidemiological characteristics of postoperative invasive Staphylococcus aureus infection following 4 types of major surgical procedures.design. Retrospective cohort study. SETTING: Eleven hospitals (9 community hospitals and 2 tertiary care hospitals) in North Carolina and Virginia. PATIENTS: Adults undergoing orthopedic, neurosurgical, cardiothoracic, and plastic surgical procedures. METHODS: We used previously validated, prospectively collected surgical surveillance data for surgical site infection and microbiological data for bloodstream infection. The study period was 2003 through 2006. We defined invasive S. aureus infection as either nonsuperficial incisional surgical site infection or bloodstream infection. Nonparametric bootstrapping was used to generate 95% confidence intervals (CIs). P values were generated using the Pearson chi2 test, Student t test, or Wilcoxon rank-sum test, as appropriate. RESULTS: In total, 81,267 patients underwent 96,455 procedures during the study period. The overall incidence of invasive S. aureus infection was 0.47 infections per 100 procedures (95% CI, 0.43-0.52); 227 (51%) of 446 infections were due to methicillin-resistant S.aureus. Invasive S. aureus infection was more common after cardiothoracic procedures (incidence, 0.79 infections per 100 procedures [95%CI, 0.62-0.97]) than after orthopedic procedures (0.37 infections per 100 procedures [95% CI, 0.32-0.42]), neurosurgical procedures (0.62 infections per 100 procedures [95% CI, 0.53-0.72]), or plastic surgical procedures (0.32 infections per 100 procedures [95% CI, 0.17-0.47]) (P < .001). Similarly, S. aureus bloodstream infection was most common after cardiothoracic procedures (incidence, 0.57 infections per 100 procedures [95% CI, 0.43-0.72]; P < .001, compared with other procedure types), comprising almost three-quarters of the invasive S. aureus infections after these procedures. The highest rate of surgical site infection was observed after neurosurgical procedures (incidence, 0.50 infections per 100 procedures [95% CI, 0.42-0.59]; P < .001, compared with other procedure types), comprising 80% of invasive S.aureus infections after these procedures. CONCLUSION: The frequency and type of postoperative invasive S. aureus infection varied significantly across procedure types. The highest risk procedures, such as cardiothoracic procedures, should be targeted for ongoing preventative interventions.

Impact of gene variants on sex-specific regulation of human Scavenger receptor class B type 1 (SR-BI) expression in liver and association with lipid levels in a population-based study.

BACKGROUND: Several studies have noted that genetic variants of SCARB1, a lipoprotein receptor involved in reverse cholesterol transport, are associated with serum lipid levels in a sex-dependent fashion. However, the mechanism underlying this gene by sex interaction has not been explored. METHODS: We utilized both epidemiological and molecular methods to study how estrogen and gene variants interact to influence SCARB1 expression and lipid levels. Interaction between 35 SCARB1 haplotype-tagged polymorphisms and endogenous estradiol levels was assessed in 498 postmenopausal Caucasian women from the population-based Rancho Bernardo Study. We further examined associated variants with overall and SCARB1 splice variant (SR-BI and SR-BII) expression in 91 human liver tissues using quantitative real-time PCR. RESULTS: Several variants on a haplotype block spanning intron 11 to intron 12 of SCARB1 showed significant gene by estradiol interaction affecting serum lipid levels, the strongest for rs838895 with HDL-cholesterol (p=9.2x10(-4)) and triglycerides (p=1.3x10(-3)) and the triglyceride:HDL cholesterol ratio (p=2.7x10(-4)). These same variants were associated with expression of the SR-BI isoform in a sex-specific fashion, with the strongest association found among liver tissue from 52 young women<45 years old (p=0.002). CONCLUSIONS: Estrogen and SCARB1 genotype may act synergistically to regulate expression of SCARB1 isoforms and impact serum levels of HDL cholesterol and triglycerides. This work highlights the importance of considering sex-dependent effects of gene variants on serum lipid levels.

A genome-wide association study identifies susceptibility variants for type 2 diabetes in Han Chinese.

To investigate the underlying mechanisms of T2D pathogenesis, we looked for diabetes susceptibility genes that increase the risk of type 2 diabetes (T2D) in a Han Chinese population. A two-stage genome-wide association (GWA) study was conducted, in which 995 patients and 894 controls were genotyped using the Illumina HumanHap550-Duo BeadChip for the first genome scan stage. This was further replicated in 1,803 patients and 1,473 controls in stage 2. We found two loci not previously associated with diabetes susceptibility in and around the genes protein tyrosine phosphatase receptor type D (PTPRD) (P = 8.54x10(-10); odds ratio [OR] = 1.57; 95% confidence interval [CI] = 1.36-1.82), and serine racemase (SRR) (P = 3.06x10(-9); OR = 1.28; 95% CI = 1.18-1.39). We also confirmed that variants in KCNQ1 were associated with T2D risk, with the strongest signal at rs2237895 (P = 9.65x10(-10); OR = 1.29, 95% CI = 1.19-1.40). By identifying two novel genetic susceptibility loci in a Han Chinese population and confirming the involvement of KCNQ1, which was previously reported to be associated with T2D in Japanese and European descent populations, our results may lead to a better understanding of differences in the molecular pathogenesis of T2D among various populations.

Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli.

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.

Sociocultural and socioeconomic influences on type 2 diabetes risk in overweight/obese African-American and Latino-American children and adolescents.

PURPOSE: It is unclear whether sociocultural and socioeconomic factors are directly linked to type 2 diabetes risk in overweight/obese ethnic minority children and adolescents. This study examines the relationships between sociocultural orientation, household social position, and type 2 diabetes risk in overweight/obese African-American (n = 43) and Latino-American (n = 113) children and adolescents. METHODS: Sociocultural orientation was assessed using the Acculturation, Habits, and Interests Multicultural Scale for Adolescents (AHIMSA) questionnaire. Household social position was calculated using the Hollingshead Two-Factor Index of Social Position. Insulin sensitivity (SI), acute insulin response (AIRG) and disposition index (DI) were derived from a frequently sampled intravenous glucose tolerance test (FSIGT). The relationships between AHIMSA subscales (i.e., integration, assimilation, separation, and marginalization), household social position and FSIGT parameters were assessed using multiple linear regression. RESULTS: For African-Americans, integration (integrating their family's culture with those of mainstream white-American culture) was positively associated with AIRG (β = 0.27 ± 0.09, r = 0.48, P < 0.01) and DI (β = 0.28 ± 0.09, r = 0.55, P < 0.01). For Latino-Americans, household social position was inversely associated with AIRG (β = -0.010 ± 0.004, r = -0.19, P = 0.02) and DI (β = -20.44 ± 7.50, r = -0.27, P < 0.01). CONCLUSIONS: Sociocultural orientation and household social position play distinct and opposing roles in shaping type 2 diabetes risk in African-American and Latino-American children and adolescents.

Using electronic health record data for substance use Screening, Brief Intervention, and Referral to Treatment among adults with type 2 diabetes: Design of a National Drug Abuse Treatment Clinical Trials Network study.

BACKGROUND: The Affordable Care Act encourages healthcare systems to integrate behavioral and medical healthcare, as well as to employ electronic health records (EHRs) for health information exchange and quality improvement. Pragmatic research paradigms that employ EHRs in research are needed to produce clinical evidence in real-world medical settings for informing learning healthcare systems. Adults with comorbid diabetes and substance use disorders (SUDs) tend to use costly inpatient treatments; however, there is a lack of empirical data on implementing behavioral healthcare to reduce health risk in adults with high-risk diabetes. Given the complexity of high-risk patients' medical problems and the cost of conducting randomized trials, a feasibility project is warranted to guide practical study designs. METHODS: We describe the study design, which explores the feasibility of implementing substance use Screening, Brief Intervention, and Referral to Treatment (SBIRT) among adults with high-risk type 2 diabetes mellitus (T2DM) within a home-based primary care setting. Our study includes the development of an integrated EHR datamart to identify eligible patients and collect diabetes healthcare data, and the use of a geographic health information system to understand the social context in patients' communities. Analysis will examine recruitment, proportion of patients receiving brief intervention and/or referrals, substance use, SUD treatment use, diabetes outcomes, and retention. DISCUSSION: By capitalizing on an existing T2DM project that uses home-based primary care, our study results will provide timely clinical information to inform the designs and implementation of future SBIRT studies among adults with multiple medical conditions.

Patient beliefs and behaviors about genomic risk for type 2 diabetes: Implications for prevention

Copyright © Taylor & Francis Group, LLC 2015.Type 2 diabetes is a major health burden in the United States, and population trends suggest this burden will increase. High interest in, and increased availability of, testing for genetic risk of type 2 diabetes presents a new opportunity for reducing type 2 diabetes risk for many patients; however, to date, there is little evidence that genetic testing positively affects type 2 diabetes prevention. Genetic information may not fit patients illness representations, which may reduce the chances of risk-reducing behavior changes. The present study aimed to examine illness representations in a clinical sample who are at risk for type 2 diabetes and interested in genetic testing. The authors used the Common Sense Model to analyze survey responses of 409 patients with type 2 diabetes risk factors. Patients were interested in genetic testing for type 2 diabetes risk and believed in its importance. Most patients believed that genetic factors are important to developing type 2 diabetes (67%), that diet and exercise are effective in preventing type 2 diabetes (95%), and that lifestyle changes are more effective than drugs (86%). Belief in genetic causality was not related to poorer self-reported health behaviors. These results suggest that patients interest in genetic testing for type 2 diabetes might produce a teachable moment that clinicians can use to counsel behavior change.

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.

Ptolemaic Mummy Stuffings, 2: A Comic Fragment and Grammatical Text in the Yale Collection (P. CtYBR inv. 5019, 5043)

This is the second installment of a three-part project to publish a group of ten Ptolemaic papyri purchased by Yale’s Beinecke Library in 1998 (acquisition “1998b”), which came to the Beinecke as three hard wads that were apparently the stuffing from the stomach cavity of a mummified animal. This article publishes: (1) P.CtYBR inv. 5019, a fragment of line ends in iambic tetrameter catalectic meter from an unknown comedy; the format suggests that this is a further example of certain type of Ptolemaic writing exercise. (2) P.CtYBR inv. 5043, a fragmentary grammatical text of uncertain import.