Modest Influence of FRET Chromophores on the Properties of Unfolded Proteins

AbstractSingle-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor κ2, has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances

Flea beetles (Coleoptera: Chrysomelidae: Alticinae) collected by malaise trap method in Gölcük Natural Park (Isparta, Turkey), with a new record for Turkish fauna

This study is based on Alticinae (Coleoptera: Chrysomelidae) material collected by Malaise trapping which is different from other standardized collecting methods. A total of 19 flea beetle species belonging to 6 genera were collected from Gölcük Natural Park, Isparta (Turkey) during 2009. The species are listed in a table together with distributional data in Turkey. Among them, Longitarsus curtus (Allard, 1860) is recorded for the first time in Turkey. L. monticola Kutschera, 1863 and L. curtus are recently separated synonyms and thus all data referring to the distribution of both species are currently important. Hence, the zoogeographical distribution of the new record is reviewed with some remarks; habitus and genitalia are illustrated

Site Saturation Mutagenesis Applications on <i>Candida methylica</i> Formate Dehydrogenase

In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH) is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM) which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6×104-fold and 5×104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH

Thrombotic risk assessment in antiphospholipid syndrome: do noncriteria antibodies contribute?

BACKGROUND/AIM: In this cross-sectional study, it was aimed to test the predictive value of noncriteria antiphospholipid antibodies (aPL) in addition to the global antiphospholipid syndrome score (GAPSS) in predicting vascular thrombosis (VT) in a cohort of patients with APS and aPL (+) systemic lupus erythematosus (SLE). MATERIAL AND METHODS: This study included 50 patients with primary APS, 68 with SLE/APS, and 52 with aPL (+) SLE who were classified according to VT as VT ± pregnancy morbidity (PM), PM only or aPL (+) SLE. Antiphospholipid serology consisting of lupus anticoagulant (LA), anticardiolipin (aCL) immunoglobulin G (IgG)/IgM/IgA, antibeta2 glycoprotein I (aβ2GPI) IgG/IgM/IgA, antiphosphatidylserine/prothrombin (aPS/PT) IgG/IgM and antidomain-I (aDI) IgG was determined for each patient. The GAPSS and adjusted GAPSS (aGAPSS) were calculated for each patient, as previously defined. Logistic regression analysis was carried out with thrombosis as the dependent variable and high GAPSS, aCL IgA, aβ2GPI IgA, and aDI IgG as independent variables. RESULTS: The mean GAPSS and aGAPSS of the study population were 11.6 ± 4.4 and 9.6 ± 3.8. Both the VT ± PM APS (n = 105) and PM only APS (n = 13) groups had significantly higher GAPSS and aGAPSS values compared to the aPL (+) SLE (n = 52) group. The patients with recurrent thrombosis had higher aGAPSS but not GAPSS than those with a single thrombotic event. The computed area under the receiver operating characteristic curve demonstrated that a GAPSS ≥13 and aGAPSS ≥10 had the best predictive values for thrombosis. Logistic regression analysis including a GAPSS ≥13, aCL IgA, aβ2GPI IgA, and aDI IgG showed that none of the factors other than a GAPSS ≥13 could predict thrombosis. CONCLUSION: Both the GAPSS and aGAPSS successfully predict the thrombotic risk in aPL (+) patients and aCL IgA, aβ2GPI IgA, and aDI IgG do not contribute to high a GAPSS or aGAPSS

Automated detection of e-scooter helmet use with deep learning

E-scooter riders have an increased crash risk compared to cyclists [1 ]. Hospital data finds increasing numbers of injured e-scooter riders, with head injuries as one of the most common injury types [2]. To decrease this high prevalence of head injuries, the use of e-scooter helmets could present a potential countermeasure [3]. Despite this, studies show a generally low rate of helmet use rates in countries without mandatory helmet use laws [4][5][6]. In countries with mandatory helmet use laws for e-scooter riders, helmet use rates are higher, but generally remain lower than bicycle use rates [7]. As the helmet use rate is a central factor for the safety of e-scooter riders in case of a crash and a key performance indicator in the European Commission's Road Safety Policy Framework 2021-2030 [8], efficient e-Scooter helmet use data collection methods are needed. However, currently, human observers are used to register e-scooter helmet use either in direct roadside observations or in indirect video-based observation, which is time-consuming and costly. In this study, a deep learning-based method for the automated detection of e-scooter helmet use in video data was developed and tested, with the aim to provide an efficient data collection tool for road safety researchers and practitioners

Survey of small intestinal and systemic immune responses following murine Arcobacter butzleri infection

Background Arcobacter (A.) butzleri has been described as causative agent for sporadic cases of human gastroenteritis with abdominal pain and acute or prolonged watery diarrhea. In vitro studies revealed distinct adhesive, invasive and cytotoxic properties of A. butzleri. Information about the underlying immunopathological mechanisms of infection in vivo, however, are scarce. The aim of this study was to investigate the immunopathological properties of two different A. butzleri strains in a well-established murine infection model. Results Gnotobiotic IL-10 −/− mice, in which the intestinal microbiota was depleted by broad-spectrum antibiotic treatment, were perorally infected with two different A. butzleri strains isolated from a diseased patient (CCUG 30485) or fresh chicken meat (C1), respectively. Eventhough bacteria of either strain could stably colonize the intestinal tract at day 6 and day 16 postinfection (p.i.), mice did not exert infection induced symptoms such as diarrhea or wasting. In small intestines of infected mice, however, increased numbers of apoptotic cells could be detected at day 16, but not day 6 following infection with either strain. A strain-dependent influx of distinct immune cell populations such as T and B cells as well as of regulatory T cells could be observed upon A. butzleri infection which was accompanied by increased small intestinal concentrations of pro-inflammatory cytokines such as TNF, IFN-γ, MCP-1 and IL-6. Remarkably, inflammatory responses following A. butzleri infection were not restricted to the intestinal tract, given that the CCUG 30485 strain induced systemic immune responses as indicated by increased IFN-γ concentrations in spleens at day 6, but not day 16 following infection. Conclusion Upon peroral infection A. butzleri stably colonized the intestinal tract of gnotobiotic IL-10 −/− mice. The dynamics of distinct local and systemic inflammatory responses could be observed in a strain-dependent fashion pointing towards an immunopathogenic potential of A. butzleri in vivo. These results indicate that gnotobiotic IL-10 −/− mice are well suited to further investigate the molecular mechanisms underlying arcobacteriosis in vivo

Arcobacter butzleri Induce Colonic, Extra-Intestinal and Systemic Inflammatory Responses in Gnotobiotic IL-10 Deficient Mice in a Strain-Dependent Manner

BACKGROUND: The immunopathological impact of human Arcobacter (A.) infections is under current debate. Episodes of gastroenteritis with abdominal pain and acute or prolonged watery diarrhea were reported for A. butzleri infected patients. Whereas adhesive, invasive and cytotoxic capacities have been described for A. butzleri in vitro, only limited information is available about the immunopathogenic potential and mechanisms of infection in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Gnotobiotic IL-10-/- mice were generated by broad-spectrum antibiotic treatment and perorally infected with the A. butzleri strains CCUG 30485 and C1 shown to be invasive in cell culture assays. Bacterial colonization capacities, clinical conditions, intestinal, extra-intestinal and systemic immune responses were monitored at day six and 16 postinfection (p.i.). Despite stable intestinal A. butzleri colonization at high loads, gnotobiotic IL-10-/- mice were virtually unaffected and did not display any overt symptoms at either time point. Notably, A. butzleri infection induced apoptosis of colonic epithelial cells which was paralleled by increased abundance of proliferating cells. Furthermore A. butzleri infection caused a significant increase of distinct immune cell populations such as T and B cells, regulatory T cells, macrophages and monocytes in the colon which was accompanied by elevated colonic TNF, IFN-γ, nitric oxide (NO), IL-6, IL-12p70 and MCP-1 concentrations. Strikingly, A. butzleri induced extra-intestinal and systemic immune responses as indicated by higher NO concentrations in kidney and increased TNF, IFN-γ, IL-12p70 and IL-6 levels in serum samples of infected as compared to naive mice. Overall, inflammatory responses could be observed earlier in the course of infection by the CCUG 30485 as compared to the C1 strain. CONCLUSION/SIGNIFICANCE: Peroral A. butzleri infection induced not only intestinal but also extra-intestinal and systemic immune responses in gnotobiotic IL-10-/- mice in a strain-dependent manner. These findings point towards an immunopathogenic potential of A. butzleri in vertebrate hosts

Kinetic and thermodynamic properties of the folding and assembly of formate dehydrogenase

AbstractThe folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10−13M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate (k1) of about 2×10−3s−1 (by deduction k−1 is about10−4s−1) and assembles into the active dimeric state with a bimolecular rate constant (k2) of about 2×104M−1s−1. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k−2∼3×10−7s−1)

Developing a TRIZ-based Design for Flexibility Tool for Manufacturing Facilities

As manufacturers evaluate assets and long-term production plans, they struggle with how best to meet complex building requirements that maximize building flexibility and minimize costs. Research shows that manufacturers highly prioritize facility flexibility. However, infusing flexibility into facility design can be complex and achieving it can be costly. These issues could be mitigated with a dedicated tool for addressing flexibility in facility design. TRIZ (Theory of Inventive Problem Solving) is a problem-solving method that exploits information contained in millions of patents to identify solution genres and standard contradictions to drive inventive design principles. This userfriendly, decision support tool can efficiently reduce the complexity of incorporating flexibility into manufacturing facility design. Using this tool as a platform and incorporating information from fifteen case studies, constructionspecific terms were mapped to TRIZ parameters and principles to create a construction industry specific TRIZ contradiction matrix. This paper describes basic TRIZ theory and previous uses in the construction industry. It then discusses industry input and case studies that helped make it construction-specific. Finally, it addresses the modified TRIZ tool’s potential benefits to the construction industry regarding flexibility considerations