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Alfonso M Durán,1 Lorena M Salto,1 Justin Câmara,1 Anamika Basu,1 Ivette Paquien,1 W Lawrence Beeson,1,2 Anthony Firek,3 Zaida Cordero-MacIntyre,1,2 Marino De León1 1Center for Health Disparities and Molecular Medicine, Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, USA; 2Center for Nutrition, Healthy Lifestyle and Disease Prevention, School of Public Health, Loma Linda University, Loma Linda, CA, USA; 3Comparative Effectiveness and Clinical Outcomes Research Center, Riverside University Health System Medical Center, Moreno Valley, CA, USA Purpose: To determine whether dietary supplementation with omega-3 polyunsaturated fatty acids (PUFAs) reduces neuropathic pain symptoms in Mexican-Americans with type 2 diabetes.Methods: Forty volunteers with type 2 diabetes enrolled in the “En Balance-PLUS” program, which provided weekly nutrition–diabetes education and daily supplementation with 1,000 mg docosahexaenoic acid (DHA)–200 mg eicosapentaenoic acid over 3 months. The study assessed self-reported neuropathic pain symptoms pre/postintervention using the short-form McGill Pain Questionnaire (SF-MPQ), monitored clinical laboratory values at baseline and 3 months, and performed baseline and 3-month metabolomic analysis of plasma samples.Results: A total of 26 participants self-reported neuropathic pain symptoms at baseline. After 3 months of omega-3 PUFA supplementation, participants reported significant improvement in SF-MPQ scores (sensory, affective, and visual analogue scale; P

Infectious diseases account for nine percent of annual human deaths, and the widespread emergence of antimicrobial resistances threatens to significantly increase this number in the coming decades. The prospect of antimicrobial peptides (AMPs) derived from venomous animals presents an interesting alternative for developing novel active pharmaceutical ingredients (APIs). Small, cationic and amphiphilic peptides were predicted from the venom gland transcriptome of Pamphobeteus verdolaga using a custom database of the arthropod's AMPs. Ninety-four candidates were chemically synthesized and screened against ATCC ® strains of Escherichia coli and Staphylococcus aureus . Among them, one AMP, named PvAMP66, showed broad-spectrum antimicrobial properties with selectivity towards Gram-negative bacteria. It also exhibited activity against Pseudomonas aeruginosa , as well as both an ATCC ® and a clinically isolated multidrug-resistant (MDR) strain of K. pneumoniae . The scanning electron microscopy analysis revealed that PvAMP66 induced morphological changes of the MDR K. pneumoniae strain suggesting a potential "carpet model" mechanism of action. The isobologram analysis showed an additive interaction between PvAMP66 and gentamicin in inhibiting the growth of MDR K. pneumoniae , leading to a ten-fold reduction in gentamicin's effective concentration. A cytotoxicity against erythrocytes or peripheral blood mononuclear cells was observed at concentrations three to thirteen-fold higher than those exhibited against the evaluated bacterial strains. This evidence suggests that PvAMP66 can serve as a template for the development of AMPs with enhanced activity and deserves further pre-clinical studies as an API in combination therapy.

Background: The Minnesota Lab Appropriateness (MLAB) criteria were developed for assessing appropriateness of complete blood counts (CBCs) and serum electrolyte panels (SEPs) ordered for adult inpatients., Methods: Two independent raters used the MLAB criteria to rate appropriateness of labs ordered during 50 hospitalizations through retrospective medical record review., Results: Evaluation of 208 CBCs and 253 SEPs on a 2-category scale (appropriate/inappropriate) resulted in an inappropriate lab rate of 24% and 25% for CBCs and SEPs, respectively. Using a 3-category Likert scale that included an "equivocal" rating to allow for clinical uncertainty, 17% of CBCs and 20% of SEPs were considered inappropriate. Interrater reliability was "substantial" using the dichotomous scale for both CBCs and SEPs. Using the 3-category Likert scale, reliability was "substantial" for CBCs and "moderate" for SEPs., Conclusion: The MLAB criteria identified inappropriate labs at a rate consistent with published figures, with good interrater reliability.

The voltage-gated potassium channel KCNQ1 (KV7.1) assembles with the KCNE1 accessory protein to generate the slow delayed rectifier current, IKS, which is critical for membrane repolarization as part of the cardiac action potential. Loss-of-function (LOF) mutations in KCNQ1 are the most common cause of congenital long QT syndrome (LQTS), type 1 LQTS, an inherited genetic predisposition to cardiac arrhythmia and sudden cardiac death. A detailed structural understanding of KCNQ1 is needed to elucidate the molecular basis for KCNQ1 LOF in disease and to enable structure-guided design of new anti-arrhythmic drugs. In this work, advanced structural models of human KCNQ1 in the resting/closed and activated/open states were developed by Rosetta homology modeling guided by newly available experimentally-based templates: X. leavis KCNQ1 and various resting voltage sensor structures. Using molecular dynamics (MD) simulations, the capacity of the models to describe experimentally established channel properties including state-dependent voltage sensor gating charge interactions and pore conformations, PIP2 binding sites, and voltage sensor-pore domain interactions were validated. Rosetta energy calculations were applied to assess the utility of each model in interpreting mutation-evoked KCNQ1 dysfunction by predicting the change in protein thermodynamic stability for 50 experimentally characterized KCNQ1 variants with mutations located in the voltage-sensing domain. Energetic destabilization was successfully predicted for folding-defective KCNQ1 LOF mutants whereas wild type-like mutants exhibited no significant energetic frustrations, which supports growing evidence that mutation-induced protein destabilization is an especially common cause of KCNQ1 dysfunction. The new KCNQ1 Rosetta models provide helpful tools in the study of the structural basis for KCNQ1 function and can be used to generate hypotheses to explain KCNQ1 dysfunction., Competing Interests: The authors have declared that no competing interests exist.

This study evaluated the antimicrobial activity (i.e., against Bacillus cereus and Staphylococcus aureus ) and the antioxidant activity (i.e., ABTS, FRAP, and DPPH) of annatto seeds extract obtained by ultrasound-assisted extraction. A response surface design with three levels such as pH (2-11), solvent concentration (50-96 %), seed-to-solvent ratio (1:2-1:10), and treatment time (0-30 min) was employed to determine the optimal experimental conditions. Thus, a pH of 7.0, seed-to-solvent ratio of 1:7, and treatment time of 20 min were selected as optimal rendering an extract having a 0.62% of bixin, 3.81 mg gallic acid/mg equivalent of polyphenol compounds (ABTS 1035.7, FRAP 424.7, and DPPH 1161.5 μ M trolox/L), and a minimal inhibitory concentration against Bacillus cereus and Staphylococcus aureus of 32 and 16 mg/L, respectively. Further, the main bioactive compounds identified by LC/ESI-MS were bixin and catechin, chlorogenic acid, chrysin, butein, hypolaetin, licochalcone A, and xanthohumol., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter., Competing Interests: The authors declare no conflict of interest.

Mutations that induce loss of function (LOF) or dysfunction of the human KCNQ1 channel are responsible for susceptibility to a life-threatening heart rhythm disorder, the congenital long QT syndrome (LQTS). Hundreds of KCNQ1 mutations have been identified, but the molecular mechanisms responsible for impaired function are poorly understood. We investigated the impact of 51 KCNQ1 variants with mutations located within the voltage sensor domain (VSD), with an emphasis on elucidating effects on cell surface expression, protein folding, and structure. For each variant, the efficiency of trafficking to the plasma membrane, the impact of proteasome inhibition, and protein stability were assayed. The results of these experiments combined with channel functional data provided the basis for classifying each mutation into one of six mechanistic categories, highlighting heterogeneity in the mechanisms resulting in channel dysfunction or LOF. More than half of the KCNQ1 LOF mutations examined were seen to destabilize the structure of the VSD, generally accompanied by mistrafficking and degradation by the proteasome, an observation that underscores the growing appreciation that mutation-induced destabilization of membrane proteins may be a common human disease mechanism. Finally, we observed that five of the folding-defective LQTS mutant sites are located in the VSD S0 helix, where they interact with a number of other LOF mutation sites in other segments of the VSD. These observations reveal a critical role for the S0 helix as a central scaffold to help organize and stabilize the KCNQ1 VSD and, most likely, the corresponding domain of many other ion channels.

Computational membrane protein design is challenging due to the small number of high-resolution structures available to elucidate the physical basis of membrane protein structure, multiple functionally important conformational states, and a limited number of high-throughput biophysical assays to monitor function. However, structural determination of membrane proteins has made tremendous progress in the past years. Concurrently the field of soluble computational design has made impressive inroads. These developments allow us to tackle the formidable challenge of designing functional membrane proteins. Herein, Rosetta is benchmarked for membrane protein design. We evaluate strategies to cope with the often reduced quality of experimental membrane protein structures. Further, we test the usage of symmetry in design protocols, which is particularly important as many membrane proteins exist as homo-oligomers. We compare a soluble scoring function with a scoring function optimized for membrane proteins, RosettaMembrane. Both scoring functions recovered around half of the native sequence when completely redesigning membrane proteins. However, RosettaMembrane recovered the most native-like amino acid property composition. While leucine was overrepresented in the inner and outer-hydrophobic regions of RosettaMembrane designs, it resulted in a native-like surface hydrophobicity indicating that it is currently the best option for designing membrane proteins with Rosetta., (© 2017 The Protein Society.)

There is a compelling and growing need to accurately predict the impact of amino acid mutations on protein stability for problems in personalized medicine and other applications. Here the ability of 10 computational tools to accurately predict mutation-induced perturbation of folding stability (ΔΔG) for membrane proteins of known structure was assessed. All methods for predicting ΔΔG values performed significantly worse when applied to membrane proteins than when applied to soluble proteins, yielding estimated concordance, Pearson, and Spearman correlation coefficients of <0.4 for membrane proteins. Rosetta and PROVEAN showed a modest ability to classify mutations as destabilizing (ΔΔG < -0.5 kcal/mol), with a 7 in 10 chance of correctly discriminating a randomly chosen destabilizing variant from a randomly chosen stabilizing variant. However, even this performance is significantly worse than for soluble proteins. This study highlights the need for further development of reliable and reproducible methods for predicting thermodynamic folding stability in membrane proteins.

Previously, we published an article providing an overview of the Rosetta suite of biomacromolecular modeling software and a series of step-by-step tutorials [Kaufmann, K. W., et al. (2010) Biochemistry 49, 2987-2998]. The overwhelming positive response to this publication we received motivates us to here share the next iteration of these tutorials that feature de novo folding, comparative modeling, loop construction, protein docking, small molecule docking, and protein design. This updated and expanded set of tutorials is needed, as since 2010 Rosetta has been fully redesigned into an object-oriented protein modeling program Rosetta3. Notable improvements include a substantially improved energy function, an XML-like language termed "RosettaScripts" for flexibly specifying modeling task, new analysis tools, the addition of the TopologyBroker to control conformational sampling, and support for multiple templates in comparative modeling. Rosetta's ability to model systems with symmetric proteins, membrane proteins, noncanonical amino acids, and RNA has also been greatly expanded and improved.

Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design.

Background: Regular physical activity plays an important role in the amelioration of several mental health disorders; however, its relationship with burnout has not yet been clarified., Objective: To determine the association between achievement of national physical activity guidelines and burnout in internal medicine resident physicians., Methods: A Web-based survey of internal medicine resident physicians at the University of Minnesota and Hennepin County Medical Center was conducted from September to October 2012. Survey measures included the Maslach Burnout Inventory-Human Services Survey and the International Physical Activity Questionnaire., Results: Of 149 eligible residents, 76 (51.0%) completed surveys, which were used in the analysis. Burnout prevalence, determined by the Maslach Burnout Inventory, was 53.9% (41 of 76). Prevalence of failure to achieve US Department of Health and Human Services physical activity guidelines was 40.8% (31 of 76), and 78.9% (60 of 76) of residents reported that their level of physical activity has decreased since they began medical training. Residents who were able to meet physical activity guidelines were less likely to be burned out than their fellow residents (OR, 0.38, 95% CI 0.147-0.99)., Conclusions: Among internal medicine resident physicians, achievement of national physical activity guidelines appears to be inversely associated with burnout. Given the high national prevalence of burnout and inactivity, additional investigation of this relationship appears warranted.

Helical membrane proteins such as transporters, receptors, or channels often exhibit structural symmetry. Symmetry is perfect in homo-oligomers consisting of two or more copies of the same protein chain. Intriguingly, in single chain membrane proteins, often internal pseudo-symmetry is observed, in particular in transporters and channels. In several cases single chain proteins with pseudo-symmetry exist, that share the fold with homo-oligomers suggesting evolutionary pathways that involve gene duplication and fusion. It has been hypothesized that such evolutionary pathways allow for the rapid development of large proteins with novel functionality. At the same time symmetry can be leveraged to recognize highly symmetric substrates such as ions. Here we review helical transporter proteins with an inverted two-fold pseudo-symmetry. In this special scenario the symmetry axis lies in the membrane plane. As a result, the putative ancestral monomeric protein would insert in both directions into the membrane and its open-to-the-inside and open-to-the-outside conformations would be structurally identical and iso-energetic, giving a possible evolutionary pathway to create a transporter protein that needs to flip between the two states.

Unlabelled: The power of music to relieve anxiety or pain has been widely used throughout history., Objective: To evaluate effects of music on anxiety and pain in patients on invasive mechanical ventilation., Design: A randomized controlled trial with repeated measures., Material and Method: This was a randomized, experimental prospective study in a tertiary hospital conducted from January 2009 to June 2010. The sample was made up of 44 participants. Intervention consisted in a 30-minute musical session in which the subject used a headset and was in an individual room. For the control group, the usual setting of an intensive care unit was maintained unchanged. Each patient underwent a minimum of 3 and maximum of 5 sessions. The patient per se selected the music from among a selection prepared by the investigator team. Anxiety and pain and hemodynamic variables of heart rate, respiratory rate systolic and diastolic blood pressure were measured at baseline, after the music session and then one-hour later., Results: Music therapy significantly decreased anxiety score (P=.000) when measured with the State-Trait Anxiety Inventory (STAI) scale. There were no differences in pain in the experimental group (P=.157) when measured with the visual analogue scale. No summative effects were demonstrated during multiple sessions., Conclusion: Music reduces anxiety in patients with invasive mechanical ventilation. Invasive mechanical ventilation can be established as a non-pharmacologic tool added to the available therapeutic options., (Copyright © 2012 Elsevier España, S.L. y SEEIUC. All rights reserved.)

Presentation of an exceptional case of priapism in an 82-year-old male, secondary to primary bilateral abscess corpus cavernosum. Diagnosis was reached through aspiration puncture and pus drainage, which also allowed the resolution of the condition. After a literature review, no similar case was found. This was a painful, long-lasting erection that resulted from corpus cavernosum repletion with purulent material instead of blood.

Objectives: The diagnostic and therapeutic aspects of endometriosis of the bladder is reviewed. Although it is relatively uncommon, it should be taken into account when making the differential diagnosis in young females presenting with dysuria., Methods/results: A young female patient with dysuria and hypogastric pain caused by endometriosis of the bladder is described., Conclusions: Diagnosis is basically by cystoscopic exploration and histological analyses of the specimens obtained by electrofulguration. Hormone therapy with Danazol may be effective in some cases. The most effective treatment is excision of the lesion by TUR or conventional surgery. Furthermore, laser could be useful in the treatment of this pathology.

We report the case of a 54-year-old female patient, diagnosed of nasal polyposis and intrinsic corticodependent bronchial asthma, who since a year has developed episodes of asthma when exposed to vapours from cooking some kinds of legumes (peas, chick-peas, beans, lentils) and an oral allergy syndrome with peanuts. We prepared extract with these legumes. The skin tests were clearly positive for legumes but negative for pneumoallergens. Specific IgE by CAP was strongly positive for legumes. CAP inhibition was preformed and the results show the presence of cross-reactivity among legumes.

Pregnancy outcomes of 1707 women, who enrolled for care between 1971 and 1989 with a home birth service run by lay midwives in rural Tennessee, were compared with outcomes from 14,033 physician-attended hospital deliveries derived from the 1980 US National Natality/National Fetal Mortality Survey. Based on rates of perinatal death, of low 5-minute Apgar scores, of a composite index of labor complications, and of use of assisted delivery, the results suggest that, under certain circumstances, home births attended by lay midwives can be accomplished as safely as, and with less intervention than, physician-attended hospital deliveries.