A minimal CRISPR-Cas3 system for genome engineering [preprint]

CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an “all-in-one” vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an “anti-anti-CRISPR” strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function

Use of Coronary Computed Tomographic Angiography to guide management of patients with coronary disease

Background In a prospective, multicenter, randomized controlled trial, 4,146 patients were randomized to receive standard care or standard care plus coronary computed tomography angiography (CCTA). Objectives The purpose of this study was to explore the consequences of CCTA-assisted diagnosis on invasive coronary angiography, preventive treatments, and clinical outcomes. Methods In post hoc analyses, we assessed changes in invasive coronary angiography, preventive treatments, and clinical outcomes using national electronic health records. Results Despite similar overall rates (409 vs. 401; p = 0.451), invasive angiography was less likely to demonstrate normal coronary arteries (20 vs. 56; hazard ratios [HRs]: 0.39 [95% confidence interval (CI): 0.23 to 0.68]; p < 0.001) but more likely to show obstructive coronary artery disease (283 vs. 230; HR: 1.29 [95% CI: 1.08 to 1.55]; p = 0.005) in those allocated to CCTA. More preventive therapies (283 vs. 74; HR: 4.03 [95% CI: 3.12 to 5.20]; p < 0.001) were initiated after CCTA, with each drug commencing at a median of 48 to 52 days after clinic attendance. From the median time for preventive therapy initiation (50 days), fatal and nonfatal myocardial infarction was halved in patients allocated to CCTA compared with those assigned to standard care (17 vs. 34; HR: 0.50 [95% CI: 0.28 to 0.88]; p = 0.020). Cumulative 6-month costs were slightly higher with CCTA: difference $462 (95$303 to $621). Conclusions In patients with suspected angina due to coronary heart disease, CCTA leads to more appropriate use of invasive angiography and alterations in preventive therapies that were associated with a halving of fatal and non-fatal myocardial infarction. (Scottish COmputed Tomography of the HEART Trial [SCOT-HEART]; NCT01149590

Ripple oscillations in the left temporal neocortex are associated with impaired verbal episodic memory encoding

Background: We sought to determine if ripple oscillations (80-120Hz), detected in intracranial EEG (iEEG) recordings of epilepsy patients, correlate with an enhancement or disruption of verbal episodic memory encoding. Methods: We defined ripple and spike events in depth iEEG recordings during list learning in 107 patients with focal epilepsy. We used logistic regression models (LRMs) to investigate the relationship between the occurrence of ripple and spike events during word presentation and the odds of successful word recall following a distractor epoch, and included the seizure onset zone (SOZ) as a covariate in the LRMs. Results: We detected events during 58,312 word presentation trials from 7,630 unique electrode sites. The probability of ripple on spike (RonS) events was increased in the seizure onset zone (SOZ, p<0.04). In the left temporal neocortex RonS events during word presentation corresponded with a decrease in the odds ratio (OR) of successful recall, however this effect only met significance in the SOZ (OR of word recall 0.71, 95% CI: 0.59-0.85, n=158 events, adaptive Hochberg p<0.01). Ripple on oscillation events (RonO) that occurred in the left temporal neocortex non-SOZ also correlated with decreased odds of successful recall (OR 0.52, 95% CI: 0.34-0.80, n=140, adaptive Hochberg , p<0.01). Spikes and RonS that occurred during word presentation in the left middle temporal gyrus during word presentation correlated with the most significant decrease in the odds of successful recall, irrespective of the location of the SOZ (adaptive Hochberg, p<0.01). Conclusion: Ripples and spikes generated in left temporal neocortex are associated with impaired verbal episodic memory encoding

Bacteriophage application restores ethanol fermentation characteristics disrupted by Lactobacillusfermentum

BACKGROUND: Contamination of corn mash by lactic acid bacteria (LAB) reduces the efficiency of the ethanol fermentation process. The industry relies heavily on antibiotics for contamination control and there is a need to develop alternative methods. The goals of this study were to determine the diversity and abundance of bacteria contaminating commercial ethanol fermentations, and to evaluate the potential of anti-LAB bacteriophages in controlling production losses. RESULTS: Bacterial populations in 27 corn mash samples collected from nine different commercial plants were determined by pyrosequencing of 16S rRNA amplicons. The results showed that the most abundant bacteria (>50 % of total population) in 24 of the 27 samples included LAB genera such as Lactobacillus, Streptococcus, Lactococcus, Weissella, Enterococcus, and Pediococcus. Lactobacillus was identified as the most prevalent genus at all fermentation stages in all plants, accounting for between 2.3 and 93.7 % of each population and constituting the major genus (>50 %) in nine samples from five plants and the most abundant genus in five other samples. Lactobacillus species, including L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri were the most well-represented species. Two bacteriophages that target L. fermentum strains from ethanol plants, vB_LfeS_EcoSau and vB_LfeM_EcoInf (EcoSau and EcoInf), were isolated and characterized as a siphophage and a myophage, respectively. Analysis of the 31,703 bp genome of EcoSau revealed its similarity to the P335-like phage group, and the 106,701 bp genome of phage EcoInf was determined to be a novel phage type despite its distant relationship to the SPO1-like phages. Addition of phages EcoSau and EcoInf to L. fermentum-contaminated corn mash fermentation models restored the yields of ethanol and reduced levels of residual glucose, lactic acid, and acetic acid to that comparable to the infection-free control. CONCLUSIONS: This study provides detailed insight into the microbiota contaminating commercial ethanol fermentations, and highlights the abundance of LAB, especially L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri, in the process. This study suggests that phages with broad coverage of major LAB species can be applied directly to corn mash for antibiotic-free control of contamination in the ethanol fermentation industry. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-015-0325-9) contains supplementary material, which is available to authorized users

A quantum Monte Carlo study of the one-dimensional ionic Hubbard model

Quantum Monte Carlo methods are used to study a quantum phase transition in a 1D Hubbard model with a staggered ionic potential (D). Using recently formulated methods, the electronic polarization and localization are determined directly from the correlated ground state wavefunction and compared to results of previous work using exact diagonalization and Hartree-Fock. We find that the model undergoes a thermodynamic transition from a band insulator (BI) to a broken-symmetry bond ordered (BO) phase as the ratio of U/D is increased. Since it is known that at D = 0 the usual Hubbard model is a Mott insulator (MI) with no long-range order, we have searched for a second transition to this state by (i) increasing U at fixed ionic potential (D) and (ii) decreasing D at fixed U. We find no transition from the BO to MI state, and we propose that the MI state in 1D is unstable to bond ordering under the addition of any finite ionic potential. In real 1D systems the symmetric MI phase is never stable and the transition is from a symmetric BI phase to a dimerized BO phase, with a metallic point at the transition

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans

Minimally Invasive Surgical Approaches and Traditional Total Hip Arthroplasty: A Meta-Analysis of Radiological and Complications Outcomes

BACKGROUND: Minimally invasive total hip arthroplasty (MITHA) remains considerably controversial. Limited visibility and prosthesis malposition increase the risk of post-surgical complications compared to those of the traditional method. METHODS: A meta-analysis was undertaken of all published databases up to May 2011. The studies were divided into four subgroups according to the surgical approach taken. The radiological outcomes and complications of minimally invasive surgery were compared to traditional total hip arthroplasty (TTHA) using risk ratio, mean difference, and standardized mean difference statistics. RESULTS: In five studies involving the posterolateral approach, no significant differences were found between the MITHA groups and the TTHA groups in the acetabular cup abduction angle (p = 0.41), acetabular anteversion (p = 0.96), and femoral prosthesis position (p = 0.83). However, the femoral offset was significantly increased (WMD = 3.00; 95% CI, 0.40-5.60; p = 0.02). Additionally, there were no significant differences among the complications in both the groups (dislocations, nerve injury, infection, deep vein thrombosis, proximal femoral fracture) and revision rate (p>0.05). In three studies involving the posterior approach, there were no significant differences in radiological outcomes or all other complications between MITHA or TTHA groups (p>0.05). Three studies involved anterolateral approach, while 2 studies used the lateral approach. However, the information from imaging and complications was not adequate for statistical analysis. CONCLUSIONS: Posterior MITHA seems to be a safe surgical procedure, without the increased risk of post-operative complication rates and component malposition rates. The posterolateral approach THA may lead to increased femoral offset. The current data are not enough to reach a positive conclusion that lateral and anterolateral approaches will result in increased risks of adverse effects and complications at the prosthesis site

Executive summary: heart disease and stroke statistics--2013 update: a report from the American Heart Association.

Each year, the American Heart Association (AHA), in conjunction with the Centers for Disease Control and Prevention, the National Institutes of Health, and other government agencies, brings together the most up-to-date statistics on heart disease, stroke, other vascular diseases, and their risk factors and presents them in its Heart Disease and Stroke Statistical Update*The Statistical Update is a valuable resource for researchers, clinicians, healthcare policy makers, media professionals, the lay public, and many others who seek the best national data available on heart disease, stroke, and other cardiovascular disease-related morbidity and mortality and the risks, quality of care, medical procedures and operations, and costs associated with the management of these diseases in a single document*Indeed, since 1999, the Statistical Update has been cited \u3e10 500 times in the literature, based on citations of all annual versions*In 2011 alone, the various Statistical Updates were cited ≈1500 times (data from ISI Web of Science)*In recent years, the Statistical Update has undergone some major changes with the addition of new chapters and major updates across multiple areas, as well as increasing the number of ways to access and use the information assembled*For this year\u27s edition, the Statistics Committee, which produces the document for the AHA, updated all of the current chapters with the most recent nationally representative data and inclusion of relevant articles from the literature over the past year*This year\u27s edition also implements a new chapter organization to reflect the spectrum of cardiovascular health behaviors and health factors and risks, as well as subsequent complicating conditions, disease states, and outcomes*Also, the 2013 Statistical Update contains new data on the monitoring and benefits of cardiovascular health in the population, with additional new focus on evidence-based approaches to changing behaviors, implementation strategies, and implications of the AHA\u27s 2020 Impact Goals*Below are a few highlights from this year\u27s Update . © 2013 American Heart Association, Inc

The Caulobacter crescentus phage phiCbK: genomics of a canonical phage

BACKGROUND: The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives. RESULTS: Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK) to 280 kb encoding 448 proteins (CcrColossus), and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell’s replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level. CONCLUSIONS: Due to their lack of any apparent relationship to other described phages, this group is proposed as the founding cohort of a new phage type, the phiCbK-like phages. This work will serve as a foundation for future studies on morphogenesis, infection and phage-host interactions in C. crescentus