Experiments and comprehensive simulations of the formation of a helical turn

We consider the kinetics and thermodynamics of a helical turn formation in the peptide Ac-WAAAH-NH2. NMR measurements indicate that the peptide has significant tendency to form a structure of a helical turn, while temperature dependent CD establishes the helix fraction at different temperatures. Molecular Dynamics and Milestoning simulations agree with experimental observables and suggests an atomically detailed picture for the turn formation. Using a network representation two alternative mechanisms of folding are identified: (i) a direct cooperative mechanism from the unfolded to the folded state without intermediate formation of hydrogen bonds and (ii) an indirect mechanism with structural intermediates with two residues in a helical conformation. This picture is consistent with kinetic measurements that reveal two experimental time scales of sub nanosecond and several nanoseconds

ВEНТИЛЯЦИОННАЯ ПОДДEРЖКА НEПРEРЫВНЫМ ПОТОКОМ (VPKP) (клиничeский опыт)

Background. The world literature contains no reports on the clinical application of continuous flow ventilatory support by an insufflation catheter. Despite the use of different forms of ventilatory support, disconnection of patients from artificial ventilation is unsuccessful in 10—30% of cases despite the fact that the clinical and biochemical criteria are met.Objective: to discuss the efficiency of the new ventilation regime — continuous flow ventilatory support in the clinical setting.Methods: continuous flow ventilatory support with an original licensed multi-jet insufflation catheter or a terminal one-orifice catheter nasally inserted into the trachea was applied to 70 patients. It was used in a subgroup of 64 patients with chronic obstructive lung disease (COLD) due to the occurrence of global respiratory insufficiency caused by infectious complications and in a group of 6 patients as a ventilatory regime for their disconnection from long-term artificial ventilation, whose disconnection other ventilatory regimens being used were unsuccessful.Results. None patient with COLD should be intubated, and just 30 minutes after the initiation of ventilatory support with a multi-jet catheter, there were decreases in the mean respiration rate from 33±2.8 to 27±2.5 cycles/min and in paCo2 from 11.9±1.7 to 10.8±1.6 kPa and an increase in paCo2 from 5.7±1.1 to 6.8±1.3 kPa at FiO2 =0.3. Within 24 hours after the initiation of ventilatory support, blood gas levels changed in response to the values typical of partial respiratory insufficiency. The spontaneous ventilation rate decreased to 20±2.2, paCO2 reduced to 6.4±1.2  kPa  and  pO2 continuously  increased  up  to  the  value  8.9±1.4  kPa  (FiO2 =0.3)  at  hour  24  of  ventilatory  support. Ventilatory support lasted an average of 5 days. Statistical comparison of the study parameters showed a significant improvement (p<0.05) just 6 hours after ventilatory support and a marked improvement of the parameters (p<0.01) following 72 hours. In the other group of patients, continuous flow ventilatory support was used due to failing disconnection of the patients from long-term artificial ventilation. After extubation and 30 minutes after the initiation of continuous flow ventilatory support, the ventilation rated decreased to 27±2.5 cycles/min, there was a continuous reduction in paCO2 to 3.9±0.9 kPa as a manifestation of hyperventilation that had been likely to be induced by a continuous decrease of paCO2 to 8.8±1.4 kPa. Only 60 minutes after the initiation of ventilatory support, with the equal ventilation rate, the values of blood gases (paO2 =9.9±1.5 kPa, paCO2=5.2±1.1 kPa) increased, as did VT (0.38±0.30), which permitted one to proceed with continuous flow ventilatory support that could be interrupted following 48 hours.Conclusion. The findings lead to the conclusion that continuous flow ventilatory support is an effective ventilation regimen that is applicable to patients with chronic obstructive lung disease in global respiratory insufficiency and makes it possible to overcome the period of, for example, infectious complications without  intubation  and  artificial  ventilation.  It  may  also  be  used  as  a  non-invasive  ventilation  regime  in  the  disconnection  of patients from long-term artificial ventilation. Its application in acute respiratory failure (acute respiratory failure, acute respiratory distress syndrome) requires further prospective studies.Проблема. В мировой литературе нe было описано клиничeскоe примeнeниe вeнтиляционной поддeржки нeпрeрывным потоком посрeдством инсуффляционного катeтера. Нeсмотря на примeнeниe разных форм вeнтиляционной поддeржки, в 10—30% отсоeдинeниe пациeнтов от искусствeнной вeнтиляции лeгких являeтся нeудачным, нeсмотря на выполнeниe клиничeских и биохимичeских критeриeв.Цeль. В клиничeских условиях обсудить эффeктивность нового вeнтиляционного рeжима — вeнтиляционной поддeржки нeпрeрывным потоком.Мeтоды. Вeнтиляционная поддeржка нeпрeрывным потоком с оригинальным, защищeнным патeнтом многоструйным инсуффляционным катeтером или катeтером с одним тeрминальным отвeрстиeм, ввeдeнным назально в трахeю было примeнeно у 70 пациeнтов. В подгруппe из 64 пациeнтов с хроничeской обструктивной болeзнью лeгких (COPC) она была примeнeна из-за возникновeния глобальной рeспирационной инсуффициeнции вслeдствиe инфeкционных осложнeний и в подгруппe из 6 пациeнтов она примeнялась как вeнтиляционный  рeжим  отсоeдинeния  пациeнтов  от  долгосрочной  искусствeнной  вeнтиляции  лeгких,  у  которых  нe  были успeшными другиe вeнтиляционныe рeжимы, примeняeмыe при отсоeдинeнии.Рeзультаты. Ни один пациeнт с COPC нe должeн был быть интубированным и ужe 30 минут с начала вeнтиляционной поддeржки многоструйным катeтером понизилась срeдняя частота дыхания с 33±2,8 до 27±2,5 циклов/мин, понизилось paCO2 с 11,9±1,7 до 10,8±1,6 кPa и повысилось paO2 с 5,7±1,1 до 6,8±1,3 кPa при FiO2=0,3. В тeчeниe 24 часов с начала вeнтиляционной поддeржки измeнился уровeнь газов в крови на значeния, которыe характeрны для парциальной рeспирационной инсуффициeнции. Частота самостоятeльной  вeнтиляции  снизилась  до  20±2,2,  paCO2 снизилось  до  6,4±1,2  кPa  и  paO2 нeпрeрывно  повышалось  до значeния 8,9±1,4 кРа (FiO2 =0,3) в 24-ом часу вeнтиляционной поддeржки. Вeнтиляционная поддeржка длилась в срeднeм 5 днeй. Статистичeскоe сравнeниe исслeдуeмых парамeтров ужe через 6 часов вeнтиляционной поддeржки показываeт значитeльноe улучшeниe (p<0,05) и чeрeз 72 часов выраженное улучшeниe парамeтров (p<0,01). В другой группe пациeнтов была вeнтиляционная поддeржка нeпрeрывным потоком примeнeна из-за нeудачи при отсоeдинeнии от долгосрочной искусствeнной вeнтиляции лeгких. Послe экстубации и чeрeз 30 минут послe начала вeнтиляционной поддeржки нeпрeрывным потоком частота вeнтиляции снизилась до 27±2,5 циклов/мин, в значeниях газов крови было отмeчeно продолжающeeся снижeниe paCO2 до 3,9±0,9 кPa как проявлeниe гипeрвeнтиляции, которая была вeроятно вызвана продолжающимся снижeниeм paO2 до значeния 8,8±1,4 кPa. Только чeрeз 60 минут с начала вeнтиляционной поддeржки, при одинаковой частотe вeнтиляции значeния газов в крови повысились (paO2 =9,9±1,5 кPa, paCO2 =5,2±1,1 кPa) и повысился и VT (0,38±0,30), что позволило продолжать вeнтиляционную поддeржку нeпрeрывным потоком, которую можно было прeрвать чeрeз 48 часов.Заключeниe. На основe получeнных рeзультатов можно сдeлать заключeниe, что вeнтиляционная  поддeржка  нeпрeрывным  потоком  прeдставляeт  эффeктивный  вeнтиляционный  рeжим,  который  примeним  у пациeнтов с хроничeской обструктивной болeзнью лeгких при глобальной рeспирационной инсуффициeции и позволяeт прeодолeть пeриод, напримeр, инфeкционных осложнeний бeз интубации и искусствeнной вeнтиляции лeгких. В качeствe нeинвазивного вeнтиляционного рeжима можно eго примeнить такжe при отсоeдинeнии пациeнтов от долгосрочной ИВЛ. Еe  примeнeниe  при  остром  рeспирационном  отказe  (ARF,  ARDS)  трeбуeт  дальнeйшего  проспeктивного  изучения

Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/-mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host

Biallelic loss-of-function mutation in NIK causes a primary immunodeficiency with multifaceted aberrant lymphoid immunity

Primary immunodeficiency disorders enable identification of genes with crucial roles in the human immune system. Here we study patients suffering from recurrent bacterial, viral and Cryptosporidium infections, and identify a biallelic mutation in the MAP3K14 gene encoding NIK (NF- B-inducing kinase). Loss of kinase activity of mutant NIK, predicted by in silico analysis and confirmed by functional assays, leads to defective activation of both canonical and non-canonical NF- B signalling. Patients with mutated NIK exhibit B-cell lymphopenia, decreased frequencies of class-switched memory B cells and hypogammaglobulinemia due to impaired B-cell survival, and impaired ICOSL expression. Although overall T-cell numbers are normal, both follicular helper and memory T cells are perturbed. Natural killer (NK) cells are decreased and exhibit defective activation, leading to impaired formation of NK-cell immunological synapses. Collectively, our data illustrate the non-redundant role for NIK in human immune responses, demonstrating that loss-of-function mutations in NIK can cause multiple aberrations of lymphoid immunity

Allosteric Transitions of Supramolecular Systems Explored by Network Models: Application to Chaperonin GroEL

Identification of pathways involved in the structural transitions of biomolecular systems is often complicated by the transient nature of the conformations visited across energy barriers and the multiplicity of paths accessible in the multidimensional energy landscape. This task becomes even more challenging in exploring molecular systems on the order of megadaltons. Coarse-grained models that lend themselves to analytical solutions appear to be the only possible means of approaching such cases. Motivated by the utility of elastic network models for describing the collective dynamics of biomolecular systems and by the growing theoretical and experimental evidence in support of the intrinsic accessibility of functional substates, we introduce a new method, adaptive anisotropic network model (aANM), for exploring functional transitions. Application to bacterial chaperonin GroEL and comparisons with experimental data, results from action minimization algorithm, and previous simulations support the utility of aANM as a computationally efficient, yet physically plausible, tool for unraveling potential transition pathways sampled by large complexes/assemblies. An important outcome is the assessment of the critical inter-residue interactions formed/broken near the transition state(s), most of which involve conserved residues

Increasing Incidence of Geomyces destructans Fungus in Bats from the Czech Republic and Slovakia

BACKGROUND: White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval. CONCLUSIONS/SIGNIFICANCE: G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme

Metagenomic Antimicrobial Susceptibility Testing from Simulated Native Patient Samples

Genomic antimicrobial susceptibility testing (AST) has been shown to be accurate for many pathogens and antimicrobials. However, these methods have not been systematically evaluated for clinical metagenomic data. We investigate the performance of in-silico AST from clinical metagenomes (MG-AST). Using isolate sequencing data from a multi-center study on antimicrobial resistance (AMR) as well as shotgun-sequenced septic urine samples, we simulate over 2000 complicated urinary tract infection (cUTI) metagenomes with known resistance phenotype to 5 antimicrobials. Applying rule-based and machine learning-based genomic AST classifiers, we explore the impact of sequencing depth and technology, metagenome complexity, and bioinformatics processing approaches on AST accuracy. By using an optimized metagenomics assembly and binning workflow, MG-AST achieved balanced accuracy within 5.1% of isolate-derived genomic AST. For poly-microbial infections, taxonomic sample complexity and relatedness of taxa in the sample is a key factor influencing metagenomic binning and downstream MG-AST accuracy. We show that the reassignment of putative plasmid contigs by their predicted host range and investigation of whole resistome capabilities improved MG-AST performance on poly-microbial samples. We further demonstrate that machine learning-based methods enable MG-AST with superior accuracy compared to rule-based approaches on simulated native patient samples

Genome-Wide Mutation Scoring for Machine-Learning-Based Antimicrobial Resistance Prediction

The prediction of antimicrobial resistance (AMR) based on genomic information can improve patient outcomes. Genetic mechanisms have been shown to explain AMR with accuracies in line with standard microbiology laboratory testing. To translate genetic mechanisms into phenotypic AMR, machine learning has been successfully applied. AMR machine learning models typically use nucleotide k-mer counts to represent genomic sequences. While k-mer representation efficiently captures sequence variation, it also results in high-dimensional and sparse data. With limited training data available, achieving acceptable model performance or model interpretability is challenging. In this study, we explore the utility of feature engineering with several biologically relevant signals. We propose to predict the functional impact of observed mutations with PROVEAN to use the predicted impact as a new feature for each protein in an organism’s proteome. The addition of the new features was tested on a total of 19,521 isolates across nine clinically relevant pathogens and 30 different antibiotics. The new features significantly improved the predictive performance of trained AMR models for Pseudomonas aeruginosa, Citrobacter freundii, and Escherichia coli. The balanced accuracy of the respective models of those three pathogens improved by 6.0% on average

Effects of Alanyl-Glutamine Treatment on the Peritoneal Dialysis Effluent Proteome Reveal Pathomechanism-Associated Molecular Signatures*

Peritoneal dialysis (PD) is a modality of renal replacement therapy in which the high volumes of available PD effluent (PDE) represents a rich source of biomarkers for monitoring disease and therapy. Although this information could help guide the management of PD patients, little is known about the potential of PDE to define pathomechanism-associated molecular signatures in PD. We therefore subjected PDE to a high-performance multiplex proteomic analysis after depletion of highly-abundant plasma proteins and enrichment of low-abundance proteins. A combination of label-free and isobaric labeling strategies was applied to PDE samples from PD patients (n = 20) treated in an open-label, randomized, two-period, cross-over clinical trial with standard PD fluid or with a novel PD fluid supplemented with alanyl-glutamine (AlaGln). With this workflow we identified 2506 unique proteins in the PDE proteome, greatly increasing coverage beyond the 171 previously-reported proteins. The proteins identified range from high abundance plasma proteins to low abundance cellular proteins, and are linked to larger numbers of biological processes and pathways, some of which are novel for PDE. Interestingly, proteins linked to membrane remodeling and fibrosis are overrepresented in PDE compared with plasma, whereas the proteins underrepresented in PDE suggest decreases in host defense, immune-competence and response to stress. Treatment with AlaGln-supplemented PD fluid is associated with reduced activity of membrane injury-associated mechanisms and with restoration of biological processes involved in stress responses and host defense. Our study represents the first application of the PDE proteome in a randomized controlled prospective clinical trial of PD. This novel proteomic workflow allowed detection of low abundance biomarkers to define pathomechanism-associated molecular signatures in PD and their alterations by a novel therapeutic intervention