Native mass spectrometry approaches to study zinc-binding plasma proteins

Human serum albumin (HSA) is a plasma protein that fulfils a wide range of biological functions and is thought to be the major Zn2+ transporter in blood plasma. The high affinity Zn2+ binding site (Site A) has recently been characterised and is located at an interdomain site. In addition to metal binding, HSA is also important in the transport of fatty acids. Previous work has shown that the binding of Zn2+ at Site A and the binding of myristate at fatty acid site 2 are mutually exclusive. It has been predicted that upon fatty acid binding, a conformational change occurs that can disrupt the residues that form Site A. This allosteric interaction could have an impact on the Zn2+ dependent activities of histidine-rich glycoprotein (HRG), a plasma protein involved in blood coagulation. The purpose of this work was to investigate the metal-binding properties of HSA and a peptide derived from HRG using a native MS approach. Furthermore, the possible Zn2+ transfer between the proteins was explored and also whether fatty acids influenced the Zn2+ distribution. Native ESI-MS was able to detect Zn2+ ions associating with HSA although the interactions with fatty acids appeared to be broken upon entering the gas phase. No apparent loss of Zn2+ from HSA was observed by ESI-MS following incubation with myristate which was confirmed by elemental analysis in solution. Travelling wave ion mobility-MS showed no significant conformational changes between apo-HSA and holo-HSA although Zn2+ appears to have a role in stabilising the domain I/II interface. HSA incubated with myristate showed a larger collisional cross section that is in agreement with the X-ray crystal structures. A peptide mimicking the His-rich region of HRG, HRGP330, was found to bind up to 5 Zn2+ ions by ESI-MS and evidence from a combination of circular dichroism spectroscopy, ion mobility and top-down MS/MS indicated that a conformational change occurs upon Zn2+ binding. During CID and ETD, Zn2+-binding fragments were able to be detected in order to map which residues Zn2+ was bound to. However, numerous fragments were detected and so it would appear that several possible binding sites in HRGP330 have a similar binding affinity for Zn2+. Complementary ESIMS and elemental analysis showed that up to 90% of Zn2+ was transferred from HSA to HRGP330 even in the absence of fatty acid. Cu2+ also preferentially bound to HRGP330 over the N-terminal peptide mimic of HSA. Overall this could have implications for how these metal ions are transported in blood plasma as it would appear from this evidence that HRG is a significant competitor for metal ions bound to HSA

When Can You Fold a Map?

We explore the following problem: given a collection of creases on a piece of paper, each assigned a folding direction of mountain or valley, is there a flat folding by a sequence of simple folds? There are several models of simple folds; the simplest one-layer simple fold rotates a portion of paper about a crease in the paper by +-180 degrees. We first consider the analogous questions in one dimension lower -- bending a segment into a flat object -- which lead to interesting problems on strings. We develop efficient algorithms for the recognition of simply foldable 1D crease patterns, and reconstruction of a sequence of simple folds. Indeed, we prove that a 1D crease pattern is flat-foldable by any means precisely if it is by a sequence of one-layer simple folds. Next we explore simple foldability in two dimensions, and find a surprising contrast: ``map'' folding and variants are polynomial, but slight generalizations are NP-complete. Specifically, we develop a linear-time algorithm for deciding foldability of an orthogonal crease pattern on a rectangular piece of paper, and prove that it is (weakly) NP-complete to decide foldability of (1) an orthogonal crease pattern on a orthogonal piece of paper, (2) a crease pattern of axis-parallel and diagonal (45-degree) creases on a square piece of paper, and (3) crease patterns without a mountain/valley assignment.Comment: 24 pages, 19 figures. Version 3 includes several improvements thanks to referees, including formal definitions of simple folds, more figures, table summarizing results, new open problems, and additional reference

3D‐Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4‐Hydroxystilbene

Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio‐ and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose‐based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor‐free phenacrylate decarboxylase enzymes suitable for the production of 4‐vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk‐shaped agarose‐based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate‐catalysed Heck reaction, 4‐hydroxystilbene was synthesized from p‐coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches

Allosteric modulation of zinc speciation by fatty acids

Background: Serum albumin is the major protein component of blood plasma and is responsible for the circulatory transport of a range of small molecules that include fatty acids, hormones, metal ions and drugs. Studies examining the ligand-binding properties of albumin make up a large proportion of the literature. However, many of these studies do not address the fact that albumin carries multiple ligands (including metal ions) simultaneously in vivo. Thus the binding of a particular ligand may influence both the affinity and dynamics of albumin interactions with another. Scope of review: Here we review the Zn2 + and fatty acid transport properties of albumin and highlight an important interplay that exists between them. Also the impact of this dynamic interaction upon the distribution of plasma Zn2 +, its effect upon cellular Zn2 + uptake and its importance in the diagnosis of myocardial ischemia are considered. Major conclusions: We previously identified the major binding site for Zn2 + on albumin. Furthermore, we revealed that Zn2 +-binding at this site and fatty acid-binding at the FA2 site are interdependent. This suggests that the binding of fatty acids to albumin may serve as an allosteric switch to modulate Zn2 +-binding to albumin in blood plasma. General significance: Fatty acid levels in the blood are dynamic and chronic elevation of plasma fatty acid levels is associated with some metabolic disorders such as cardiovascular disease and diabetes. Since the binding of Zn2 + to albumin is important for the control of circulatory/cellular Zn2 + dynamics, this relationship is likely to have important physiological and pathological implications. This article is part of a Special Issue entitled Serum Albumin

3D-Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4-Hydroxystilbene

Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio- and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose-based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor-free phenacrylate decarboxylase enzymes suitable for the production of 4-vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk-shaped agarose-based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate-catalysed Heck reaction, 4-hydroxystilbene was synthesized from p-coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches

A national survey of clinical practice for the management of whiplash-associated disorders in UK emergency departments

Objective: To undertake a national survey to determine current practice for the management of whiplash injuries in UK emergency departments (ED). Methods: Postal questionnaire survey. 316 lead consultants from all UK ED with annual new attendances of over 50 000 people were asked to indicate the use of a range of treatments and the frequency with which these treatments were used. Samples of written advice were requested and content analysis was conducted and compared with survey responses. Results: The response rate was 79% (251/316). The intervention most frequently used was verbal advice to exercise, reported by 84% of respondents for most or all cases, and advice against the use of a collar (83%). Other treatments reported as being used frequently were written advice and anti-inflammatory medication. 106 consultants (42%) provided a sample of written materials. Reference to expected recovery and encouragement for early return to activities were included in less than 6%. Nearly 50% of written materials contained information on how to use a soft collar and 61% contained information on solicitors and pursuing a personal injury claim. There were important differences between reported verbal behaviours and written advice. Conclusion: Verbal advice is the primary method for managing whiplash injuries in ED and is usually supplemented by written advice. Within individual hospitals there is a lack of consistency between verbal and written advice. The promotion of personal injury claims is a common feature of written advice. Research is required to develop effective and consistent models of advice

What are effects of a spaced activation of virtual patients in a pediatric course?

BACKGROUND: Virtual patients (VPs) have a long tradition in the curriculum of the medical faculty at the Ludwig-Maximilians-University (LMU) Munich. However, the pediatric VPs were not well integrated into the curriculum and hardly used by students. METHODS: Therefore we created and implemented a self-contained E-learning module based on virtual patients (VPs), which was embedded into the pediatric curriculum. Students taking this course were divided into two groups. For Group A the virtual patients were activated in a timed order (“spaced activation”), whereas Group B could work on all VPs from the beginning. We investigated the performance of these two groups concerning usage pattern including number of sessions and session duration, score on questions integrated into the VP and results of the intermediate exam. RESULTS: The integration of the VPs into the pediatric course was successful for both groups. The usage pattern for the spaced activation turned out to be more balanced, however we did not find any significant differences in the results of the intermediate exam, the score on questions included in the VPs nor in the time students spent working on the VPs. CONCLUSIONS: Our study showed that the spaced activation led to a more balanced VP usage pattern with a lower peak of sessions at the end of the course. Further studies will have to investigate whether a spaced activation of VPs leads to favorable long-term learning outcomes

Characterization of <i>Aedes aegypti</i> innate-immune pathways that limit Chikungunya virus replication

Replication of arboviruses in their arthropod vectors is controlled by innate immune responses. The RNA sequence-specific break down mechanism, RNA interference (RNAi), has been shown to be an important innate antiviral response in mosquitoes. In addition, immune signaling pathways have been reported to mediate arbovirus infections in mosquitoes; namely the JAK/STAT, immune deficiency (IMD) and Toll pathways. Very little is known about these pathways in response to chikungunya virus (CHIKV) infection, a mosquito-borne alphavirus (Togaviridae) transmitted by aedine species to humans resulting in a febrile and arthralgic disease. In this study, the contribution of several innate immune responses to control CHIKV replication was investigated. In vitro experiments identified the RNAi pathway as a key antiviral pathway. CHIKV was shown to repress the activity of the Toll signaling pathway in vitro but neither JAK/STAT, IMD nor Toll pathways were found to mediate antiviral activities. In vivo data further confirmed our in vitro identification of the vital role of RNAi in antiviral defence. Taken together these results indicate a complex interaction between CHIKV replication and mosquito innate immune responses and demonstrate similarities as well as differences in the control of alphaviruses and other arboviruses by mosquito immune pathways

Diet quality and depressive symptoms in adolescence: no cross-sectional or prospective associations following adjustment for covariates.

OBJECTIVE: Adolescence is a critical period for development of depression and understanding of behavioural risk factors is needed to support appropriate preventive strategies. We examined associations between adolescent diet quality and depressive symptoms, cross-sectionally and prospectively, in a large community cohort, adjusting for behavioural and psychosocial covariates. DESIGN: Prospective community-based cohort study (ROOTS). SETTING: Secondary schools in Cambridgeshire and Suffolk, UK. SUBJECTS: Study participants (n 603) who completed 4 d diet diaries at age 14 years and reported depressive symptoms (Moods and Feelings Questionnaire (MFQ)) at 14 and 17 years of age. RESULTS: Diet data were processed to derive a Mediterranean diet score (MDS) and daily servings of fruit and vegetables, and fish. At age 14 years, a negative association between fruit and vegetable intake and MFQ score was seen in the unadjusted cross-sectional regression model (β=-0·40; 95 % CI -0·71,-0·10), but adjustment for behavioural covariates, including smoking and alcohol consumption, attenuated this association. Fish intake and MDS were not cross-sectionally associated with MFQ score. No prospective associations were found between MDS, fruit and vegetable intake or fish intake and later MFQ score. CONCLUSIONS: Diet quality was not associated with depressive symptoms in mid-adolescence. Previously reported associations in this age range may be due to confounding. Further longitudinal studies are needed that investigate associations between adolescent diet and depression across different time frames and populations, ensuring appropriate adjustment for covariates

Implant removal after internal fixation of a femoral neck fracture: Effects on physical functioning

Objectives: The effect of implant removal after internal fixation of a femoral neck fracture on physical functioning was analyzed. Characteristics of patients who had their implant removed were studied, as it is currently unknown from which type of patients implants are removed and what effect removal has on function. Design: Secondary cohort study alongside a randomized controlled trial. Setting: Multicenter study in 14 hospitals. Patients and Intervention: Patients who had their implant removed after internal fixation of a femoral neck fracture are compared with patients who did not. Main outcome measurements: Patient characteristics and quality of life (Short Form 12, Western Ontario McMaster Osteoarthritis Index) were compared. Matched pairs were selected based on patient/fracture characteristics and prefracture physical functioning. Results: Of 162 patients, 37 (23%) had their implant removed. These patients were younger (median age: 67 vs. 72 years, P 0.024) and more often independently ambulatory prefracture (100% vs. 84%, P 0.008) than patients who did not. They more often had evident implant back-out on x-rays (54% vs. 34%, P 0.035), possibly related to a higher rate of Pauwels 3 fractures (41% vs. 22%, P 0.032). In time, quality of life improved more in implant removal patients [+2 vs. -4 points, Short Form 12 (physical component), P 0.024; +9 vs. 0 points, Western Ontario McMaster Osteoarthritis Index, P 0.019]. Conclusions: Implant removal after internal fixation of a femoral neck fracture positively influenced quality of life. Implant removal patients were younger and more often independently ambulatory prefracture, more often had a Pauwels 3 fracture, and an evident implant back-out. Implant removal should be considered liberally for these patients if pain persists or functional recovery is unsatisfactory. Level of Evidence: Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence