First structures of an active bacterial tyrosinase reveal copper plasticity

Tyrosinase is a member of the type 3 copper enzyme family that is involved in the production of melanin in a wide range of organisms. The crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 2.0-2.3 Å. The enzyme crystallized as a dimer in the asymmetric unit and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding “caddie” protein. Two Cu(II) ions, serving as the major cofactors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions shows varying occupancies and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Additionally, we suggest that residues R209 and V218, situated in a second shell of residues surrounding the active site, play a role in substrate binding orientation based on their flexibility and position. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site. Comparison of wild-type structures with the structure of the site-specific variant R209H, which possesses a higher monophenolase/diphenolase activity ratio, lends further support to a previously suggested mechanism by which monophenolic substrates dock mainly to CuA.     

How well can post-traumatic stress disorder be predicted from pre-trauma risk factors? An exploratory study in the WHO World Mental Health Surveys

Post‐traumatic stress disorder (PTSD) should be one of the most preventable mental disorders, since many people exposed to traumatic experiences (TEs) could be targeted in first response settings in the immediate aftermath of exposure for preventive intervention. However, these interventions are costly and the proportion of TE‐exposed people who develop PTSD is small. To be cost‐effective, risk prediction rules are needed to target high‐risk people in the immediate aftermath of a TE. Although a number of studies have been carried out to examine prospective predictors of PTSD among people recently exposed to TEs, most were either small or focused on a narrow sample, making it unclear how well PTSD can be predicted in the total population of people exposed to TEs. The current report investigates this issue in a large sample based on the World Health Organization (WHO)'s World Mental Health Surveys. Retrospective reports were obtained on the predictors of PTSD associated with 47,466 TE exposures in representative community surveys carried out in 24 countries. Machine learning methods (random forests, penalized regression, super learner) were used to develop a model predicting PTSD from information about TE type, socio‐demographics, and prior histories of cumulative TE exposure and DSM‐IV disorders. DSM‐IV PTSD prevalence was 4.0% across the 47,466 TE exposures. 95.6% of these PTSD cases were associated with the 10.0% of exposures (i.e., 4,747) classified by machine learning algorithm as having highest predicted PTSD risk. The 47,466 exposures were divided into 20 ventiles (20 groups of equal size) ranked by predicted PTSD risk. PTSD occurred after 56.3% of the TEs in the highest‐risk ventile, 20.0% of the TEs in the second highest ventile, and 0.0‐1.3% of the TEs in the 18 remaining ventiles. These patterns of differential risk were quite stable across demographic‐geographic sub‐samples. These results demonstrate that a sensitive risk algorithm can be created using data collected in the immediate aftermath of TE exposure to target people at highest risk of PTSD. However, validation of the algorithm is needed in prospective samples, and additional work is warranted to refine the algorithm both in terms of determining a minimum required predictor set and developing a practical administration and scoring protocol that can be used in routine clinical practice.     

Prospective Study of Police Officer Spouse/Partners: A New Pathway to Secondary Trauma and Relationship Violence?

Introduction

It has been reported that posttraumatic stress disorder (PTSD) is associated with secondary spouse/partner (S/P) emotional distress and relationship violence.

Objective

To investigate the relationships between PTSD, S/P emotional distress and relationship violence among police recruits using a prospective design.

Methods

Two hypotheses were tested in 71 S/Ps: (1) Police officer reports of greater PTSD symptoms after 12 months of police service will be associated with greater secondary trauma symptoms among S/Ps; (2) Greater secondary trauma symptoms among S/Ps at 12 months will be associated with S/P reports of greater relationship violence.

Methods

71 police recruits and their S/Ps were assessed at baseline and 12 months after the start of police officer duty. Using linear and logistic regression, we analyzed explanatory variables for 12 month S/P secondary traumatic stress symptoms and couple violence, including baseline S/P variables and couple violence, as well as exposure and PTSD reports from both S/P and officer.

Results

S/P perception of officer PTSD symptoms predicted S/P secondary traumatic stress. OS/P secondary trauma was significantly associated with both total couple violence (.34, p = .004) and S/P to officer violence (.35, p = .003).

Conclusions

Although results from this relatively small study of young police officers and their S/Ps must be confirmed by larger studies in general populations, findings suggest that S/P perception of PTSD symptoms may play a key role in the spread of traumatic stress symptoms across intimate partner relationships and intimate partner violence in the context of PTSD.     

Development and validation of an isoform-independent monoclonal antibody–based ELISA for measurement of lipoprotein(a)

The study aims were to develop a new isoform-independent enzyme-linked immunoassay (ELISA) for the measurement of lipoprotein(a) [Lp(a)], validate its performance characteristics, and demonstrate its accuracy by comparison with the gold-standard ELISA method and an LC-MS/MS candidate reference method, both developed at the University of Washington. The principle of the new assay is the capture of Lp(a) with monoclonal antibody LPA4 primarily directed to an epitope in apolipoprotein(a) KIV₂ and its detection with monoclonal antibody LPA-KIV9 directed to a single antigenic site present on KIV₉. Validation studies were performed following the guidelines of the Clinical Laboratory Improvement Amendments and the College of American Pathologists. The analytical measuring range of the LPA4/LPA-KIV9 ELISA is 0.27–1,402 nmol/L, and the method meets stringent criteria for precision, linearity, spike and recovery, dilutability, comparison of plasma versus serum, and accuracy. Method comparison with both the gold-standard ELISA and the LC-MS/MS method performed in 64 samples with known apolipoprotein(a) isoforms resulted in excellent correlation with both methods (r=0.987 and r=0.976, respectively). Additionally, the variation in apolipoprotein(a) size accounted for only 0.2% and 2.2% of the bias variation, respectively, indicating that the LPA4/LPA-KIV9 ELISA is not affected by apolipoprotein(a) size polymorphism. Peptide mapping and competition experiments demonstrated that the measuring monoclonal antibodies used in the gold-standard ELISA (a-40) and in the newly developed ELISA (LPA-KIV9) are directed to the same epitope, ⁴⁰⁷⁶LETPTVV⁴⁰⁸², on KIV₉. In conclusion, no statistically or clinically significant bias was observed between Lp(a) measurements obtained by the LPA4/LPA-KIV9 ELISA and those obtained by the gold-standard ELISA or LC-MS/MS, and therefore, the methods are considered equivalent.     

Dual‐specificity tyrosine phosphorylation‐regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner

Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign‐body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion‐founder cells that fuse to fusion‐follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells.     

Noncellulosomal cohesin- and dockerin-like modules in the three domains of life

The high-affinity cohesin–dockerin interaction was originally discovered as modular components, which mediate the assembly of the various subunits of the multienzyme cellulosome complex that characterizes some cellulolytic bacteria. Until recently, the presence of cohesins and dockerins within a bacterial proteome was considered a definitive signature of a cellulosome-producing bacterium. Widespread genome sequencing has since revealed a wealth of putative cohesin- and dockerin-containing proteins in Bacteria, Archaea, and in primitive eukaryotes. The newly identified modules appear to serve diverse functions that are clearly distinct from the classical cellulosome archetype, and the vast majority of parent proteins are not predicted glycoside hydrolases. In most cases, only a few such genes have been identified in a given microorganism, which encode proteins containing but a single cohesin and/or dockerin. In some cases, one or the other module appears to be missing from a given species, and in other cases both modules occur within the same protein. This review provides a bioinformatics-based survey of the current status of cohesin- and dockerin-like sequences in species from the Bacteria, Archaea, and Eukarya. Surprisingly, many identified modules and their parent proteins are clearly unrelated to cellulosomes. The cellulosome paradigm may thus be the exception rather than the rule for bacterial, archaeal, and eukaryotic employment of cohesin and dockerin modules.     

Human Saliva-Derived Exosomes: Comparing Methods of Isolation

ExoQuick-TC^TM (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC.     

A multiscale modeling framework for studying the mechanobiology of sarcopenic obesity

Biomechanics and Modeling in Mechanobiology - An inactive sedentary lifestyle is a common risk factor contributing to sarcopenic obesity. At the cell scale, sustained mechanical deformations of the...