Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum.

Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed. Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) produced using T7 RNA polymerase (T7 RNAP) efficiently edit the Plasmodium falciparum genome. Targeting the genes encoding native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175), we achieved high (≥ 50-100%) gene disruption frequencies within the usual time frame for generating transgenic parasites.

A Four-Year Experience of Symptomatic Intracranial Hemorrhage Following Intravenous Tissue Plasminogen Activator at a Comprehensive Stroke Center.

BACKGROUND: To describe the 4-year experience of symptomatic intracranial hemorrhage (sICH) rate at a high-volume comprehensive stroke center. METHODS: All admitted adult (≥18 years) patients presenting with an ischemic stroke from 2010 to 2013 were included in this study. The primary outcome was sICH, defined as any hemorrhage with neurological deterioration (change in National Institutes of Health Stroke Scale score ≥4) within 36 hours of intravenous tissue plasminogen activator (IV-tPA) treatment, or any hemorrhage resulting in death. Secondary outcomes were in-hospital mortality and having a favorable modified Rankin Scale (mRS) score (≤2). RESULTS: A total of 1925 did not receive intravascular (IV) or intra-arterial (IA) therapy; only 451 received IV therapy; and 175 received both IV and IA therapies. In IV-only patients, the overall rate of sICH was 2.2%; in IV and IA patients, the rate was 5.7%; and in patients who received no therapy, the rate was .4%. The IV-only group had an sICH rate of .9% in 2013. There were no differences in the adjusted odds of dying in the hospital between the study groups. IV-only treatment offered significantly better odds of achieving a favorable functional outcome, compared to no therapy, among patients with moderate stroke severity, whereas IV and IA treatments offered significantly better odds among patients with severe strokes. The odds of achieving a favorable functional outcome by discharge were decreased by 97% if patients suffered an sICH (OR = .03, 95%CI = .004, .19). CONCLUSIONS: Despite an increased risk of sICH with IV-tPA, treatment with IV-tPA continues to be associated with increased odds of a favorable discharge mRS.

Age ≥80 Years Is Not a Contraindication for Intra-Arterial Therapy after Ischemic Stroke.

BACKGROUND: Clinical trials confirmed the safety and efficacy of intra-arterial therapy (IAT) in the management of ischemic stroke. At a community hospital, we compared outcomes in patients aged ≥80 and patients in the age range 55-79 years receiving IAT following ischemic stroke. METHODS: Data were retrospectively abstracted for ischemic stroke patients ≥55 years treated with IAT at an urban comprehensive stroke center between 2010 and 2013. Baseline demographics, incidence of symptomatic intracranial hemorrhage (sICH), in-hospital mortality, discharge modified Rankin scale (mRS) score (favorable ≤2) and improvement in National Institutes of Health Stroke Scale Score (NIHSS; decreased score at discharge) were compared between patients in the age range 55-79 and patients ≥80 years. Data were analyzed using univariate analyses and multivariate logistic regression. RESULTS: IAT was performed in 239 patients with ischemic stroke; 63 (26.4%) were ≥80 years. When compared to patients aged 55-79, the elderly patients were more often female and non-smokers, with a history of atrial fibrillation. No differences were observed in those ≥80 years compared to patients in the age range 55-79 years for sICH (10 vs. 5%, p = 0.23), mortality (24 vs. 18%, p = 0.28), favorable discharge mRS score (13 vs. 19%, p = 0.27), or improvement in NIHSS (83 vs. 92%, p = 0.10). The nonsignificant association of age with the outcomes persisted after adjusting for covariates and when analyzing the subset of patients who received IAT only. CONCLUSIONS: These findings suggest that in a cohort not subject to the criteria of a clinical trial, age ≥80 years should not be a contraindication to IAT.

An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum.

BACKGROUND: The construction of plasmid vectors for transgene expression in the malaria parasite, Plasmodium falciparum, presents major technical hurdles. Traditional molecular cloning by restriction and ligation often yields deletions and re-arrangements when assembling low-complexity (A + T)-rich parasite DNA. Furthermore, the use of large 5'- and 3'- untranslated regions of DNA sequence (UTRs) to drive transgene transcription limits the number of expression cassettes that can be incorporated into plasmid vectors. METHODS: To address these challenges, two high fidelity cloning strategies, namely yeast homologous recombination and the Gibson assembly method, were evaluated for constructing P. falciparum vectors. Additionally, some general rules for reliably using the viral 2A-like peptide to express multiple proteins from a single expression cassette while preserving their proper trafficking to various subcellular compartments were assessed. RESULTS: Yeast homologous recombination and Gibson assembly were found to be effective strategies for successfully constructing P. falciparum plasmid vectors. Using these cloning methods, a validated family of expression vectors that provide a flexible starting point for user-specific applications was created. These vectors are also compatible with traditional cloning by restriction and ligation, and contain useful combinations of commonly used features for enhancing plasmid segregation and site-specific integration in P. falciparum. Additionally, application of a 2A-like peptide for the synthesis of multiple proteins from a single expression cassette, and some rules for combinatorially directing proteins to discrete subcellular compartments were established. CONCLUSIONS: A set of freely available, sequence-verified and functionally validated parts that offer greater flexibility for constructing P. falciparum vectors having expanded expression capacity is provided.

Development of an Engineered Mycobacterium tuberculosis Strain for a Safe and Effective Tuberculosis Human Challenge Model.

Human challenge experiments could greatly accelerate the development of a tuberculosis (TB) vaccine. Human challenge for tuberculosis requires a strain that can both replicate in the host and be reliably cleared. To accomplish this, we designed Mycobacterium tuberculosis (Mtb) strains featuring up to three orthogonal kill switches, tightly regulated by exogenous tetracyclines and trimethoprim. The resultant strains displayed immunogenicity and antibiotic susceptibility similar to wild-type Mtb under permissive conditions. In the absence of supplementary exogenous compounds, the strains were rapidly killed in axenic culture, mice and nonhuman primates. Notably, the strain that contained three kill switches had an escape rate of less than 10 -10 per genome per generation and displayed no relapse in a SCID mouse model. Collectively, these findings suggest that this engineered Mtb strain could be a safe and effective candidate for a human challenge model.

An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum.

Establishing robust genome engineering methods in the malarial parasite, Plasmodium falciparum, has the potential to substantially improve the efficiency with which we gain understanding of this pathogen's biology to propel treatment and elimination efforts. Methods for manipulating gene expression and engineering the P. falciparum genome have been validated. However, a significant barrier to fully leveraging these advances is the difficulty associated with assembling the extremely high AT content DNA constructs required for modifying the P. falciparum genome. These are frequently unstable in commonly-used circular plasmids. We address this bottleneck by devising a DNA assembly framework leveraging the improved reliability with which large AT-rich regions can be efficiently manipulated in linear plasmids. This framework integrates several key functional genetics outcomes via CRISPR/Cas9 and other methods from a common, validated framework. Overall, this molecular toolkit enables P. falciparum genetics broadly and facilitates deeper interrogation of parasite genes involved in diverse biological processes.

Di-adenosine tetraphosphate (Ap4A) metabolism impacts biofilm formation by Pseudomonas fluorescens via modulation of c-di-GMP-dependent pathways.

Dinucleoside tetraphosphates are common constituents of the cell and are thought to play diverse biological roles in organisms ranging from bacteria to humans. In this study we characterized two independent mechanisms by which di-adenosine tetraphosphate (Ap4A) metabolism impacts biofilm formation by Pseudomonas fluorescens. Null mutations in apaH, the gene encoding nucleoside tetraphosphate hydrolase, resulted in a marked increase in the cellular level of Ap4A. Concomitant with this increase, Pho regulon activation in low-inorganic-phosphate (P(i)) conditions was severely compromised. As a consequence, an apaH mutant was not sensitive to Pho regulon-dependent inhibition of biofilm formation. In addition, we characterized a Pho-independent role for Ap4A metabolism in regulation of biofilm formation. In P(i)-replete conditions Ap4A metabolism was found to impact expression and localization of LapA, the major adhesin regulating surface commitment by P. fluorescens. Increases in the level of c-di-GMP in the apaH mutant provided a likely explanation for increased localization of LapA to the outer membrane in response to elevated Ap4A concentrations. Increased levels of c-di-GMP in the apaH mutant were associated with increases in the level of GTP, suggesting that elevated levels of Ap4A may promote de novo purine biosynthesis. In support of this suggestion, supplementation with adenine could partially suppress the biofilm and c-di-GMP phenotypes of the apaH mutant. We hypothesize that changes in the substrate (GTP) concentration mediated by altered flux through nucleotide biosynthetic pathways may be a significant point of regulation for c-di-GMP biosynthesis and regulation of biofilm formation.

Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa.

Bacteriophage infection has profound effects on bacterial biology. Clustered regular interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) genes are found in most archaea and many bacteria and have been reported to play a role in resistance to bacteriophage infection. We observed that lysogenic infection of Pseudomonas aeruginosa PA14 with bacteriophage DMS3 inhibits biofilm formation and swarming motility, both important bacterial group behaviors. This inhibition requires the CRISPR region in the host. Mutation or deletion of five of the six cas genes and one of the two CRISPRs in this region restored biofilm formation and swarming to DMS3 lysogenized strains. Our observations suggest a role for CRISPR regions in modifying the effects of lysogeny on P. aeruginosa.

Plasma Oxidized Albumin in Acute Ischemic Stroke Is Associated With Better Outcomes.

Introduction: Plasma oxidized human serum albumin (OxHSA) is evidence of an active antioxidant mechanism as measured by oxidized species of HSA. CXCL-10 is a pro-inflammatory chemokine associated with ischemic conditions. Accordingly, we examined the relationship of admission OxHSA and CXCL-10 with discharge mRS in acute ischemic stroke (AIS). Methods: Plasma samples and clinical data were collected prospectively at a Comprehensive Stroke Center. Admission biomarkers of oxidative stress, CXCL-10 and %OxHSA, were measured. We examined if CXCL-10 or %OxHSA correlated with age, admission NIHSS score, and discharge mRS score using Spearman's Rank correlation. Logistic regression was performed to identify independent predictors of a favorable discharge mRS (≤2). Results: In 106 consecutive AIS patients, the median age was 73 (IQR 61-84), 47% were male, and the median admission NIHSS score was 11 (IQR 5-19). %OxHSA and CXCL-10 were significantly correlated (r = 0.23, p = 0.02). Both biomarkers were significantly correlated with age: %OxHSA (r = 0.44, p < 0.001) and CXCL-10 (r = 0.32, p = 0.001). Neither biomarker was correlated with admission NIHSS. There was a borderline significant correlation with discharge mRS and %OxHSA (r = -0.17, p = 0.08), where higher %OxHSA correlated with lower discharge mRS scores. For every 1% increase in %OxHSA, the odds of a favorable discharge mRS increased 11%. The odds of a favorable discharge mRS decreased 18% for every 1-point increase in the initial NIHSS. Conclusions: OxHSA, the result of an oxidative environment and evidence of the strong antioxidant buffering capacity of HSA, correlated with CXCL-10 and discharge mRS, implying that strong antioxidant activity of albumin may confer better outcomes.

Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria.

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape.

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.

How a CT-Direct Protocol at an American Comprehensive Stroke Center Led to Door-to-Needle Times Less Than 30 Minutes.

BACKGROUND AND PURPOSE: The safety and efficacy of intravenous tissue plasminogen activator (IV tPA) following acute ischemic stroke (AIS) is dependent on its timely administration. In 2014, our Comprehensive Stroke Center designed and implemented a computed tomography-Direct protocol to streamline the evaluation process of suspected patients with AIS, with the aim of reducing door-to-needle (DTN) times. The objectives of our study were to describe the protocol development and implementation process, and to compare DTN times and symptomatic intracranial hemorrhage (sICH) rates before and after protocol implementation. METHODS: Data were prospectively collected for patients with AIS receiving IV tPA between January 1, 2010, and May 31, 2015. The DTN times, examined as median times and time treatment windows, and sICH rates were compared pre- and postimplementation. RESULTS: Two hundred ninety-five patients were included in the study. After protocol implementation, median DTN times were significantly reduced (38 vs 28 minutes; P < .001). The distribution of patients treated in the three time treatment windows described below changed significantly, with an increase in patients with DTN times of 30 minutes or less, and a decrease in patients with DTN times 31 to 60 minutes and over 60 minutes (P < .001). There were two cases of sICH prior to implementation and one sICH case postimplementation. CONCLUSIONS: The implementation of a protocol that streamlined the processing of suspected patients with AIS significantly reduced DTN time without negatively impacting patient safety.

Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction.

The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important outcome given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum.

HIV infection presenting with motor axonal variant of Guillain-Barré Syndrome.

OBJECTIVES: We present a case of acute motor axonal neuropathy in a patient with previously unrecognized human immunodeficiency virus (HIV) infection. METHODS: A 46-year-old male graduate student from Mali, Africa presented with a 3-week history of progressive weakness that began during a visit home from Utah. The symptoms stabilized by 6 weeks and gradually improved without treatment. RESULTS: Electrodiagnostic studies revealed widespread fibrillation potentials and positive sharp waves with normal sensory amplitudes and no demyelinating features. Cerebrospinal fluid pleocytosis and elevated protein prompted a test for HIV infection, which returned positive. CONCLUSIONS: To our knowledge, this is the first case of acute motor axonal neuropathy in HIV outside of a seroconversion reaction.

Thrombolysis in young adults.

Obstacles to thrombolytic treatment could be unique in young stroke patients and identifying and addressing them would potentially enhance outcomes. However, it has not been well studied how age affects the time to presentation, diagnosis or treatment of acute strokes. Although studies suggest that younger patients may be more aware of stroke symptoms and signs than older ones, they may be less likely to use emergency medical services. Importantly, for strokes of similar severity, younger patients have more favorable outcomes with thrombolysis than older patients. On the other hand, young patients who experience extensive middle cerebral artery strokes are more likely to develop fatal brain edema than older patients. Current data support the use of thrombolytics for all ischemic stroke etiologies, including entities such as cervical artery dissection that are more common in young patients.