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The World Health Organization goal of universal drug susceptibility testing for patients with tuberculosis is most likely to be achieved through molecular diagnostics; however, to date these have focused largely on first-line drugs, and always on predicting binary susceptibilities. Here, we used whole genome sequencing and a quantitative microtiter plate assay to relate genomic mutations to minimum inhibitory concentration in 15,211 Mycobacterium tuberculosis patient isolates from 27 countries across five continents.This work identifies 449 unique MIC-elevating genetic determinants across thirteen drugs, as well as 91 mutations resulting in hypersensitivity for eleven drugs. Our results provide a guide for further implementation of personalized medicine for the treatment of tuberculosis using genetics-based diagnostics and can serve as a training set for novel approaches to predict drug resistance.

Demographic histories are largely understood to be a product of their environment, as populations expand or contract in response to major environmental changes. Deep-pelagic fishes inhabit one of the most temporally and spatially stable habitats on the planet, so they may be resistant to the demographic instability commonly reported in other marine habitats, but their demographic histories are poorly understood. We reconstructed the demographic histories of thirteen species of deep-pelagic fishes using mitochondrial and nuclear DNA sequence data. We uncovered widespread evidence of demographic expansion in our study species, a counterintuitive result bases on the nature of the deep-pelagic. The frequency-based methods detected potential demographic changes in eleven species, while the Extended Bayesian Skyline Plots were more conservative and identified population expansion in five species. The dates of expansion largely coincide with periods of warm sea-surface temperature at the northern and southern boundaries for the ranges these species inhabit. We suggest that this is the result of the pelagic larval phase shared by most deep-pelagic fishes, where the larvae inhabit the upper 200 meters. Changes in sea surface conditions likely alter the suitability of the habitat in a given region for the larval phase, affecting the species range and in turn population size. These results are critical to our understanding of how the deep-pelagic fish community will respond to future climatic changes.

Bedaquiline (BDQ) and clofazimine (CFZ) are core drugs for treatment of multidrug resistant tuberculosis (MDR-TB), however, our understanding of the resistance mechanisms for these drugs is sparse which is hampering rapid molecular diagnostics. To address this, we employed a unique approach using experimental evolution, protein modelling, genome sequencing, and minimum inhibitory concentration data combined with genomes from a global strain collection of over 14,151Mycobacterium tuberculosiscomplex isolates and an extensive literature review. Overall, 230 genomic variants causing elevated BDQ and/or CFZ MICs could be discerned, with 201 (87.4%) variants affecting the transcriptional repressor (Rv0678) of an efflux system (mmpS5-mmpL5). Structural modelling of Rv0678 suggests four major mechanisms that confer resistance: impairment of DNA binding, reduction in protein stability, disruption of protein dimerization, and alteration in affinity for its fatty acid ligand. These modelling and experimental techniques will improve personalized medicine in an impending drug resistant era.

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here we use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.

Purpose/objectives To describe and examine the relationship between caregiver burden and the affective disorders anxiety and depression in caregivers of patients with brain metastases. . Design Cross-sectional, descriptive, correlational. . Setting Moores Cancer Center at the University of California, San Diego. . Sample 56 family caregivers of patients with brain metastases from solid tumors at other primary sites. . Methods Self-administered survey. . Main research variables Caregiver burden, anxiety, and depression. . Findings With the exception of caregiver esteem, no statistically significant relationships were noted between impact on schedule, a dimension of caregiver burden, and screening positive for affective disorders. . Conclusions Findings from this study support previous reports indicating that the odds of having anxiety and depressive symptoms are greater in family caregivers who report higher levels of caregiver burden. . Implications for nursing The identification and management of caregiver burden are important considerations for a comprehensive cancer care program. Addressing the needs of the cancer caregiver, who is at heightened risk for various psychological, physical, financial, and social problems, is increasingly vital.

Pyrazinamide is one of four first-line antibiotics currently used to treat tuberculosis and has been included in newer treatment regimens undergoing clinical trials due to its unique sterilizing effects and synergy with newer drugs. However, phenotypic antibiotic susceptibility testing for pyrazinamide is problematic. Resistance to pyrazinamide is primarily driven by genetic variation in pncA, which encodes PncA, an enzyme that converts pyrazinamide into its active form. We curated a derivation dataset of 291 non-redundant, missense amino acid mutations in PncA with associated high-confidence phenotypes from studies of clinical isolates and in vitro/in vivo screening studies and then trained machine learning models to predict pyrazinamide resistance based on sequence- and structure-based features of each missense mutation. The clinical relevance of the models was tested by predicting the binary resistance phenotype of 2,292 clinical isolates harboring missense mutations in PncA to pyrazinamide. The probabilities of resistance predicted by the model were also compared with in vitro pyrazinamide minimum inhibitory concentrations of 27 isolates to determine whether the machine learning model could predict the degree of resistance. Finally, we predicted the effect on pyrazinamide resistance of the remaining 814 possible missense mutations caused by single nucleotide polymorphisms in PncA that have not yet been observed in public databases. Overall, this work offers an approach to improve the sensitivity and specificity of pyrazinamide resistance prediction in genetics-based clinical microbiology workflows for tuberculosis, highlights novel mutations for future biochemical investigation, and is a proof of concept for using this approach in other drugs such as bedaquiline.

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here we present the cryo-EM structure of the type I-F CRISPR RNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a Ȉ180-degree rotation of the C-terminal helical bundle on the “large” Cas8f subunit. We show that the dsDNA-induced conformational change in Cas8f exposes a Cas2/3 “nuclease recruitment helix” that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f “nuclease recruitment helix”, implying that cas genes may sometimes serve as genetic fodder for the evolution of anti-CRISPRs.

Summary Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the “large” Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 “nuclease recruitment helix” that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.

In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.

Brain metastases (BMs) are diagnosed in 10%-40% of all patients with cancer, and the incidence continues to increase along with the number of long-term survivors. When BMs occur, they are often associated with a myriad of symptoms, including neurologic dysfunction and functional decline; both are difficult to manage and can be distressing for patients and their caregivers. Although clinically significant findings have not kept up with the rapid pace of scientific breakthroughs in understanding the mechanisms of BMs, novel approaches that affect the prognosis of patients with BMs have been introduced in clinical practice. At a Glance • Screening for brain metastases (BMs) is not routinely performed in patients with no neurologic symptoms. However, screening is indicated in lung cancer and possibly in the context of high-risk cancers. • Individual differences in patients warrant a personalized approach in the management of BMs. • Whole brain radiation therapy and steroids are considered to be the cornerstones of treatment for BMs.

RNA-guided Cas9 endonucleases protect bacteria from viral infection and have been creatively repurposed as programmable molecular scalpels for surgical manipulation of DNA. Now, two papers in Cell (Pawluk et al. and Rauch et al.) identify viral proteins that suppress Cas9 and may function like molecular sheaths for the Cas9 scalpel.

The type I-F CRISPR adaptive immune system in Pseudomonas aeruginosa (PA14) consists of two CRISPR loci and six CRISPR-associated (cas) genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/32:Cas14) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1–2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity.

PURPOSE: Neurologic deficits that may be manifested as cognitive impairment contribute to the challenges faced by caregivers of patients with brain metastases. To better address their needs, we examined how caregivers respond to these challenges and explore the relationship between the patient’s cognitive impairment and caregiver resilience and coping. METHODS: We conducted a descriptive, cross-sectional study using self-reported data from 56 caregivers of patients with brain metastases. Study participants from a comprehensive cancer center were asked to complete a series of instruments that measured their perception of the patient’s cognitive dysfunction (revised memory and behavior problems checklist, RMBC), their own personal resilience (Resilience Scale, RS), and their utilization of a broad range of coping responses (COPE inventory and Emotional-Approach Coping scale). RESULTS: Caregivers reported that memory-related problems occurred more frequently in the patients they cared for compared to depression and disruptive behavior (mean scores 3.52 vs 2.34 vs. 1.32, respectively). Coping strategies most frequently used by caregivers were acceptance (3.28), planning (3.08), and positive reinterpretation and growth (2.95). Most caregivers scored moderate to high on the RS (77%). The coping strategy acceptance correlated significantly with the memory and disruptive behavior subscales of the RMBC. CONCLUSIONS: Given the protective effect of problem-focused coping and the high rate of caregivers utilizing less effective coping strategies in instances of worsening cognitive dysfunction, healthcare professionals need to systematically assess the coping strategies of caregivers and deliver a more personalized approach to enhance effective coping among caregivers of patients with brain metastases.

Bacteria and archaea have evolved different CRISPR based adaptive immune system for defense against phage infections. Pseudomonas aeruginosa has type I-F CRISPR-cas defense system, where the cas-crRNA surveillance complex (csy-complex) binds to foreign DNA and recruits the cas-2/3 nuclease for degradation of the foreign DNA. Bacteriophages have evolved mechanisms of evading this CRISPR defense system by encoding different anti-CRISPR (ACR) proteins. ACR1F and ACR2F are two such ACRs that bind to specific regions of the csy-complex and suppress the immune system. We present here a 3.4A cryo-EM reconstruction of the ACR-bound csy-complex. This structure not only shows the overall organization of the type I-F csy-complex, but also reveals how ACRs interact with this complex and prevent the binding of phage DNA.

Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-mediated adaptive immune systems for protection from viral infection and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here we report the cryo-electron microscopy structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.

A foreign-DNA–destroying machine Bacteria have an adaptive immune system, called CRISPR, that identifies invading viruses through their DNA or RNA sequences and cuts them up (see the Perspective by Zhang and Sontheimer). Jackson et al. and Mulepati et al. have determined the structure of the large protein complex, called Cascade, that targets the invading nucleic acids and does the cutting. The seahorse-shaped structure reveals how the 11 subcomponents of Cascade assemble into the final protein complex. The structure also shows how Cascade presents the short CRISPR-derived RNAs so that they can bind and target foreign DNA. Science , this issue p. 1473 and p. 1479 ; see also p. 1452

Hydroxymethyl resorcinol (HMR) is an effective adhesion promoter (primer) for wood bonding; it dramatically improves adhesion and enhances bond durability against moisture exposure. In an effort to improve understanding of the HMR mechanism of action, this work compared HMR with two other chemical treatments investigated as wood primers: alkyl-HMR (a-HMR), an HMR variant having reduced crosslink density, and a 5% solution of polymeric methylenebis(phenylisocyanate) in N-methylpyrrolidone (solution referred to as "pMDI"). The experimental system was red oak (Quercus rubra) bonded with a moisture-cure polyurethane adhesive (PUR). The objective was to document wood rheological changes induced by the three primers, and determine if these changes correlated to primer efficacy in PUR-bonded red oak. Adhesion was tested in mode-I (opening) fracture using dual cantilever beam specimens. HMR and a-HMR proved to be highly effective primers for PUR-bonded red oak; both primers dramatically improved bondline toughness and durability. Relative to HMR, the reduced crosslink density in a-HMR did not impair primer efficacy. In contrast, the pMDI primer was ineffective; it reduced bondline toughness and durability. Solvent-submersion, torsional dynamic mechanical analysis (DMA) was conducted on primer-treated red oak (with specimens immersed in dimethylformamide). Using all three grain orientations, the lignin glass/rubber transition was carefully studied with attention directed towards primer-induced changes in stiffness (storage modulus), the glass transition temperature (Tg), the associated damping (tan ° maximum intensity), and the breadth of tan ° transition. It was found that primer effectiveness correlated with a reduction in damping intensity, and also with a Tg increase greater than 5°C. Determination of these correlations was complicated by grain dependency, and also by rheological changes caused by solvent treatments that were used as primer control treatments. Master of Science

Hydrodynamics generated by moving deep-draft vessels create significant impacts to navigation safety and other users in the waterway. An integrated numerical modeling system has been developed to assist Ports in evaluating navigation issues such as squat effects on the maneuvering and berthed vessels, forces on berthed vessels and shoreside structures, and other impacts such as shoreline erosion and water quality. Vessels maneuvering in confined waterways of arbitrary configurations subject port structures and other vessels to varying forces and moments, which change in time due to speed and location in the waterway, as well as changes in waterway configuration. Accurate prediction of hydrodynamics and forces in real waterways with variable depths, channel widths, and ambient currents are essential to improve port efficiency and for optimizing navigation channel and mooring design. The Vessel Hydrodynamics Longwave Unsteady (VH-LU) modeling system was developed as part of a complete coastal engineering modeling system, and incorporates into the vessel hydrodynamics code a wide array of coastal ambient forces and processes which provide an integrated evaluation of navigation processes and impacts in real port settings. The modeling system has been previously validated for hydrodynamic calculations using field water level and velocity measurements as well as for berthed vessel loading using laboratory measurements. The present paper presents two test cases of evaluation of passing vessel forces on berthed vessels from the Port of Los Angeles, CA and Port of Tampa, FL. In both cases, numerical modeling and analysis results reveal completely different results than empirical methods due to irregular passing vessel and waterway conditions. This paper also describes model enhancements for prediction of moving and berthed vessel sinkage (squat) in arbitrary channels, with validation performed using empirical methods. Squat calculations are compared with empirical/laboratory data for a wide variety of conditions and example squat calculations of transit through complex channels are provided as an example of the variability of squat during arrival and departure into ports. Squat example calculations are also provided for situations where vessels pass each other in complex waterways. Copyright 2010 ASCE.

Helicases utilize NTPs to modulate their binding to nucleic acids and many of these enzymes also unwind DNA or RNA duplexes in an NTP-dependent fashion. These proteins are phylogenetically related but functionally diverse, with essential roles in virtually all aspects of nucleic acid metabolism. A new class of helicases associated with RNA-guided adaptive immune systems in bacteria and archaea has recently been identified. Prokaryotes acquire resistance to invading genetic parasites by integrating short fragments of foreign nucleic acids into repetitive loci in the host chromosome known as CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR-associated gene 3 (cas3) encodes a conserved helicase protein that is essential for phage defense. Here we review recent advances in Cas3 biology, and provide a new phylogenetic framework that positions Cas3 in the helicase family tree. We anticipate that this Cas3 phylogeny will guide future biochemical and structural studies.

Bacteria and archaea have evolved sophisticated adaptive immune systems that reply on CRISPR loci and a diverse cassette of Cas genes that are classified into three main types and at least eleven subtypes. All CRISPR-Cas immune systems operate through three main stages: acquisition, biogenesis, and interference. This SnapShot summarizes our current knowledge of these fascinating immune systems. To view this SnapShot, open or download the PDF.

11 págs, 4 figuras y fórmulas matemáticas. Material suplementario en: http://dx.doi.org/10.1093/molbev/msaa069, The dN/dS ratio provides evidence of adaptation or functional constraint in protein-coding genes by quantifying the relative excess or deficit of amino acid-replacing versus silent nucleotide variation. Inexpensive sequencing promises a better understanding of parameters, such as dN/dS, but analyzing very large data sets poses a major statistical challenge. Here, I introduce genomegaMap for estimating within-species genome-wide variation in dN/dS, and I apply it to 3,979 genes across 10,209 tuberculosis genomes to characterize the selection pressures shaping this global pathogen. GenomegaMap is a phylogeny-free method that addresses two major problems with existing approaches: 1) It is fast no matter how large the sample size and 2) it is robust to recombination, which causes phylogenetic methods to report artefactual signals of adaptation. GenomegaMap uses population genetics theory to approximate the distribution of allele frequencies under general, parent-dependent mutation models. Coalescent simulations show that substitution parameters are well estimated even when genomegaMap's simplifying assumption of independence among sites is violated. I demonstrate the ability of genomegaMap to detect genuine signatures of selection at antimicrobial resistance-conferring substitutions in Mycobacterium tuberculosis and describe a novel signature of selection in the cold-shock DEAD-box protein A gene deaD/csdA. The genomegaMap approach helps accelerate the exploitation of big data for gaining new insights into evolution within species., D.J.W. is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (grant no. 101237/Z/13/B) and is a Big Data Institute Robertson Fellow. The CRyPTIC Consortium was supported by grants from the Bill and Melinda Gates Foundation (OPP1133541) and a Wellcome Trust/Newton Fund-MRC Collaborative Award (200205/Z/15/Z). F.A.D. was supported by the Imperial Biomedical Research Centre