Engineering of a critical membrane-anchored enzyme for high solubility and catalytic activity.

Membrane-associated proteins carry out a wide range of essential cellular functions but the structural characterization needed to understand these functions is dramatically underrepresented in the Protein Data Bank. Producing a soluble, stable and active form of a membrane-associated protein presents formidable challenges, as evidenced by the variety of approaches that have been attempted with a multitude of different membrane proteins to achieve this goal. Aspartate N-acetyltransferase (ANAT) is a membrane-anchored enzyme that performs a critical function, the synthesis of N-acetyl-l-aspartate (NAA), the second most abundant amino acid in the brain. This amino acid is a precursor for a neurotransmitter, and alterations in brain NAA levels have been implicated as a causative effect in Canavan disease and has been suggested to be involved in other neurological disorders. Numerous prior attempts have failed to produce a soluble form of ANAT that is amenable for functional and structural investigations. Through the application of a range of different approaches, including fusion partner constructs, linker modifications, membrane-anchor modifications, and domain truncations, a highly soluble, stable and fully active form of ANAT has now been obtained. Producing this modified enzyme form will accelerate studies aimed at structural characterization and structure-guided inhibitor development.

Mitogen and Stress-Activated Kinases 1 and 2 Mediate Endothelial Dysfunction.

Inflammation promotes endothelial dysfunction, but the underlying mechanisms remain poorly defined in vivo. Using translational vascular function testing in myocardial infarction patients, a situation where inflammation is prevalent, and knock-out (KO) mouse models we demonstrate a role for mitogen-activated-protein-kinases (MAPKs) in endothelial dysfunction. Myocardial infarction significantly lowers mitogen and stress kinase 1/2 (MSK1/2) expression in peripheral blood mononuclear cells and diminished endothelial function. To further understand the role of MSK1/2 in vascular function we developed in vivo animal models to assess vascular responses to vasoactive drugs using laser Doppler imaging. Genetic deficiency of MSK1/2 in mice increased plasma levels of pro-inflammatory cytokines and promoted endothelial dysfunction, through attenuated production of nitric oxide (NO), which were further exacerbated by cholesterol feeding. MSK1/2 are activated by toll-like receptors through MyD88. MyD88 KO mice showed preserved endothelial function and reduced plasma cytokine expression, despite significant hypercholesterolemia. MSK1/2 kinases interact with MAPK-activated proteins 2/3 (MAPKAP2/3), which limit cytokine synthesis. Cholesterol-fed MAPKAP2/3 KO mice showed reduced plasma cytokine expression and preservation of endothelial function. MSK1/2 plays a significant role in the development of endothelial dysfunction and may provide a novel target for intervention to reduce vascular inflammation. Activation of MSK1/2 could reduce pro-inflammatory responses and preserve endothelial vasodilator function before development of significant vascular disease.

A randomized controlled trial of metformin on left ventricular hypertrophy in patients with coronary artery disease without diabetes: the MET-REMODEL trial.

AIM: We tested the hypothesis that metformin may regress left ventricular hypertrophy (LVH) in patients who have coronary artery disease (CAD), with insulin resistance (IR) and/or pre-diabetes. METHODS AND RESULTS: We randomly assigned 68 patients (mean age 65 ± 8 years) without diabetes who have CAD with IR and/or pre-diabetes to receive either metformin XL (2000 mg daily dose) or placebo for 12 months. Primary endpoint was change in left ventricular mass indexed to height1.7 (LVMI), assessed by magnetic resonance imaging. In the modified intention-to-treat analysis (n = 63), metformin treatment significantly reduced LVMI compared with placebo group (absolute mean difference -1.37 (95% confidence interval: -2.63 to -0.12, P = 0.033). Metformin also significantly reduced other secondary study endpoints such as: LVM (P = 0.032), body weight (P = 0.001), subcutaneous adipose tissue (P = 0.024), office systolic blood pressure (BP, P = 0.022) and concentration of thiobarbituric acid reactive substances, a biomarker for oxidative stress (P = 0.04). The glycated haemoglobin A1C concentration and fasting IR index did not differ between study groups at the end of the study. CONCLUSION: Metformin treatment significantly reduced LVMI, LVM, office systolic BP, body weight, and oxidative stress. Although LVH is a good surrogate marker of cardiovascular (CV) outcome, conclusive evidence for the cardio-protective role of metformin is required from large CV outcomes trials.

Interhospital Transfer Before Thrombectomy Is Associated With Delayed Treatment and Worse Outcome in the STRATIS Registry (Systematic Evaluation of Patients Treated With Neurothrombectomy Devices for Acute Ischemic Stroke).

BACKGROUND: Endovascular treatment with mechanical thrombectomy (MT) is beneficial for patients with acute stroke suffering a large-vessel occlusion, although treatment efficacy is highly time-dependent. We hypothesized that interhospital transfer to endovascular-capable centers would result in treatment delays and worse clinical outcomes compared with direct presentation. METHODS: STRATIS (Systematic Evaluation of Patients Treated With Neurothrombectomy Devices for Acute Ischemic Stroke) was a prospective, multicenter, observational, single-arm study of real-world MT for acute stroke because of anterior-circulation large-vessel occlusion performed at 55 sites over 2 years, including 1000 patients with severe stroke and treated within 8 hours. Patients underwent MT with or without intravenous tissue plasminogen activator and were admitted to endovascular-capable centers via either interhospital transfer or direct presentation. The primary clinical outcome was functional independence (modified Rankin Score 0-2) at 90 days. We assessed (1) real-world time metrics of stroke care delivery, (2) outcome differences between direct and transfer patients undergoing MT, and (3) the potential impact of local hospital bypass. RESULTS: A total of 984 patients were analyzed. Median onset-to-revascularization time was 202.0 minutes for direct versus 311.5 minutes for transfer patients (P<0.001). Clinical outcomes were better in the direct group, with 60.0% (299/498) achieving functional independence compared with 52.2% (213/408) in the transfer group (odds ratio, 1.38; 95% confidence interval, 1.06-1.79; P=0.02). Likewise, excellent outcome (modified Rankin Score 0-1) was achieved in 47.4% (236/498) of direct patients versus 38.0% (155/408) of transfer patients (odds ratio, 1.47; 95% confidence interval, 1.13-1.92; P=0.005). Mortality did not differ between the 2 groups (15.1% for direct, 13.7% for transfer; P=0.55). Intravenous tissue plasminogen activator did not impact outcomes. Hypothetical bypass modeling for all transferred patients suggested that intravenous tissue plasminogen activator would be delayed by 12 minutes, but MT would be performed 91 minutes sooner if patients were routed directly to endovascular-capable centers. If bypass is limited to a 20-mile radius from onset, then intravenous tissue plasminogen activator would be delayed by 7 minutes and MT performed 94 minutes earlier. CONCLUSIONS: In this large, real-world study, interhospital transfer was associated with significant treatment delays and lower chance of good outcome. Strategies to facilitate more rapid identification of large-vessel occlusion and direct routing to endovascular-capable centers for patients with severe stroke may improve outcomes. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02239640.

Systematic Evaluation of Patients Treated With Neurothrombectomy Devices for Acute Ischemic Stroke: Primary Results of the STRATIS Registry.

BACKGROUND AND PURPOSE: Mechanical thrombectomy with stent retrievers has become standard of care for treatment of acute ischemic stroke patients because of large vessel occlusion. The STRATIS registry (Systematic Evaluation of Patients Treated With Neurothrombectomy Devices for Acute Ischemic Stroke) aimed to assess whether similar process timelines, technical, and functional outcomes could be achieved in a large real world cohort as in the randomized trials. METHODS: STRATIS was designed to prospectively enroll patients treated in the United States with a Solitaire Revascularization Device and Mindframe Capture Low Profile Revascularization Device within 8 hours from symptom onset. The STRATIS cohort was compared with the interventional cohort of a previously published SEER patient-level meta-analysis. RESULTS: A total of 984 patients treated at 55 sites were analyzed. The mean National Institutes of Health Stroke Scale score was 17.3. Intravenous tissue-type plasminogen activator was administered in 64.0%. The median time from onset to arrival in the enrolling hospital, door to puncture, and puncture to reperfusion were 138, 72, and 36 minutes, respectively. The Core lab-adjudicated modified Thrombolysis in Cerebral Infarction ≥2b was achieved in 87.9% of patients. At 90 days, 56.5% achieved a modified Rankin Scale score of 0 to 2, all-cause mortality was 14.4%, and 1.4% suffered a symptomatic intracranial hemorrhage. The median time from emergency medical services scene arrival to puncture was 152 minutes, and each hour delay in this interval was associated with a 5.5% absolute decline in the likelihood of achieving modified Rankin Scale score 0 to 2. CONCLUSIONS: This largest-to-date Solitaire registry documents that the results of the randomized trials can be reproduced in the community. The decrease of clinical benefit over time warrants optimization of the system of care. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02239640.

A missense mutation in the PISA domain of HsSAS-6 causes autosomal recessive primary microcephaly in a large consanguineous Pakistani family.

Asymmetric cell division is essential for normal human brain development. Mutations in several genes encoding centrosomal proteins that participate in accurate cell division have been reported to cause autosomal recessive primary microcephaly (MCPH). By homozygosity mapping including three affected individuals from a consanguineous MCPH family from Pakistan, we delineated a critical region of 18.53 Mb on Chromosome 1p21.3-1p13.1. This region contains the gene encoding HsSAS-6, a centrosomal protein primordial for seeding the formation of new centrioles during the cell cycle. Both next-generation and Sanger sequencing revealed a homozygous c.185T>C missense mutation in the HsSAS-6 gene, resulting in a p.Ile62Thr substitution within a highly conserved region of the PISA domain of HsSAS-6. This variant is neither present in any single-nucleotide polymorphism or exome sequencing databases nor in a Pakistani control cohort. Experiments in tissue culture cells revealed that the Ile62Thr mutant of HsSAS-6 is substantially less efficient than the wild-type protein in sustaining centriole formation. Together, our findings demonstrate a dramatic impact of the mutation p.Ile62Thr on HsSAS-6 function and add this component to the list of genes mutated in primary microcephaly.

The Combination of Clinical Features, Transcranial Doppler, and Alberta Stroke Program Early Computed Tomography Score (Computed Tomography Angiography) in Predicting Outcome in Intravenous Recombinant Tissue Plasminogen Activator-Treated Patients.

BACKGROUND: Little data exist on using combined baseline clinical neuroimaging and transcranial Doppler (TCD) information in predicting clinical outcome in stroke patients treated with intravenous (IV) thrombolysis. METHODS: Stroke patients received IV recombinant tissue plasminogen activator (rt-PA) and had diagnostic TCD within 3 hours of symptom onset. The TCD result was interpreted using the thrombolysis in brain ischemia (TIBI) flow grading system. Following multiple regression analysis, a grading system was created with 1 point for each of the following: National Institutes of Health Stroke Scale (NIHSS) score of 16 or higher, TIBI score of 1 or lower, and Alberta Stroke Program Early CT Score (ASPECTS) of 6 or lower. The patients' scores were compared to modified Rankin Scale (mRS) scores at 90 days. RESULTS: A total of 349 patients were included. In unvaried analysis, age of 80 years or older (P = .002), an ASPECTS of 6 or lower (P < .001), an NIHSS score of 16 or higher (P < .001), a TIBI score of 1 or lower (P < .001), and a glucose level ≥ 200 mg/dl (P = .04) were associated with poor outcome (mRS score > 2). In the multiple regression analysis, age of 80 years or older, an ASPECTS of 6 or lower, an NIHSS score of 16 or higher, and hyperglycemia were predictors of poor outcome (P < .05). Based on our scoring system, the patients' odds ratios for poor outcome were 7 (95% confidence interval [CI]: 2-23, P = .003), 8 (95% CI: 3-25, P < .001), and 24 (95% CI: 4-151, P = .001) for scores of 1, 2, and 3, respectively, after adjustment for common stroke risk factors. The mean time to recanalization increased as the score increased (score of 0: 160 ± 45 minutes versus score of 3: 186 ± 38 (P = .70). CONCLUSION: A multimodal grading system is useful in predicting outcome in patients treated with IV rt-PA. Those withhigher scores might be candidates for interventional therapy.

CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly.

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference, reduction in the size of the cerebral cortex with otherwise grossly normal brain structure and variable intellectual disability. MCPH is caused by mutations of 11 different genes which code for proteins implicated in cell division and cell cycle regulation. We studied a consanguineous eight-generation family from Pakistan with ten microcephalic children using homozygosity mapping and found a new MCPH locus at HSA 7q21.11-q21.3. Sanger sequencing of the most relevant candidate genes in this region revealed a homozygous single nucleotide substitution c.589G>A in CDK6, which encodes cyclin-dependent kinase 6. The mutation changes a highly conserved alanine at position 197 into threonine (p.Ala197Thr). Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. CDK6 is an important protein for the control of the cell cycle and differentiation of various cell types. We show here for the first time that CDK6 associates with the centrosome during mitosis; however, this was not observed in patient fibroblasts. Moreover, the mutant primary fibroblasts exhibited supernumerary centrosomes, disorganized microtubules and mitotic spindles, an increased centrosome nucleus distance, reduced cell proliferation and impaired cell motility and polarity. Upon ectopic expression of the mutant protein and knockdown of CDK6 through shRNA, we noted similar effects. We propose that the identified CDK6 mutation leads to reduced cell proliferation and impairs the correct functioning of the centrosome in microtubule organization and its positioning near the nucleus which are key determinants during neurogenesis.

Meeting report - building a centrosome.

Located in the 16th century Wiston House in West Sussex, UK, the 'Building a Centrosome' Workshop was organised by The Company of Biologists and chaired by Fanni Gergely and David Glover (University of Cambridge). Held in March 2013, the Workshop gathered together many of the leaders in the field of centrosome biology, as well as postdocs and students who were given the opportunity to meet and interact with many of the scientists who inspired their early careers. The diverse range of speakers provided a multi-disciplinary forum for the exchange of ideas, and gave fresh impetus to tackling outstanding questions related to centrosome biology. Here, we provide an overview of the meeting and highlight the main themes that were discussed.

Solitary C1 spinal osteochondroma causing vertebral artery compression and acute cerebellar infarct.

Osteochondroma is a common benign bone lesion, usually involving the long bones. Spinal involvement is rare. The clinical presentation of spinal osteochondroma varies according to the site of the lesion. The most common reported clinical presentation is secondary to encroachment of the lesion on the spinal canal or nerve roots. Less common presentations such as a palpable neck mass, dysphagia, sleep apnea, paralysis of left vocal cord or acute respiratory distress have been reported when the lesions compress the anatomic structures anteriorly. We describe a rare case of a young patient who presented with an emergent critical condition of acute cerebellar infarct as a result of vertebral artery compression caused by a solitary C1 spinal osteochondroma.

Safety and Hemodynamic Profile of Propofol and Dexmedetomidine Anesthesia during Intra-arterial Acute Stroke Therapy.

BACKGROUND: There is limited data on the safety, hemodynamic profile, and outcome of patients undergoing intra-arterial therapy (IAT) for acute ischemic stroke (AIS) under sedation with dexmedetomidine (DEX) versus propofol (PROP). METHODS: Retrospective study of patients with anterior circulation AIS, who underwent IAT without intubation, and received either DEX or PROP between January 2008 and December 2012, was performed. Demographics, stroke treatments, time metrics, anesthesia, intraprocedural hemodynamics, vasopressor use, infarct volumes, recanalization status, and intracerebral hemorrhage were collected. RESULTS: Seventy-two patients met inclusion criteria, of which 35 received DEX and 37 PROP. There was no difference in baseline demographics, stroke treatments, successful recanalization, hemorrhages, infarct volume growth, good clinical outcome (mRS ≤ 2 [19% versus 22%, P = .742]), or in-hospital mortality (18% versus 8%, P = .225) between DEX and PROP. The DEX group had lower intraprocedural minimum systolic blood pressure (103 ± 27 versus 114 ± 18 mm Hg, P = .032) and minimum mean arterial pressure (MAP; 67 ± 17 versus 77 ± 10 mm Hg, P = .006). More patients in the DEX group experienced episodes of hypotension (MAP < 60 mm Hg; 24% versus 3%; P = .010) and had higher vasopressor requirement (phenylephrine: 1825 ± 2390 versus 491 ± 884 mcg, P = .007) compared to PROP. CONCLUSIONS: There was no difference in good clinical outcome or in-hospital mortality in patients undergoing IAT for AIS using DEX versus PROP sedation. However, hemodynamic instability and vasopressor requirement were significantly higher in the DEX group. DEX should be cautiously utilized in IAT.

Predictors of infarct growth after endovascular therapy for acute ischemic stroke.

BACKGROUND: Intra-arterial (IA) thrombectomy for acute ischemic stroke has an excellent recanalization rate but variable outcomes. The core infarct also grows at a variable rate despite recanalization. We aim to study the factors that are associated with infarct growth after IA therapy. METHODS: We reviewed the hyperacute ischemic stroke imaging database at Cleveland Clinic for those undergoing endovascular thrombectomy of anterior circulation from 2009 to 2012. Patients with both pretreatment and follow-up magnetic resonance imaging were included. Seventy-six patients were stratified into quartiles by infarct volume growth from initial to follow-up diffusion-weighted imaging (DWI) measure by a region of interest demarcation. RESULTS: The median infarct growth of each quartile was .6 cm(3) (no-growth group), 13.8, 37, and 160.2 cm(3) (large-growth group). Pretreatment stroke severity was comparable among groups. Compared with the no-growth group, the large-growth group had larger initial infarct defined by computed tomography (CT) Alberta Stroke Program Early CT score (median 10 versus 8, P = .032) and DWI volume (mean 13.8 versus 29.2 cm(3), P = .034), lack of full collateral vessels on CT angiography (36.8% versus 0%, P = .003), and a lower recanalization rate (thrombolysis in cerebral infarction ≥2b, P = .044). The increase in infarct growth is associated with decrease in favorable outcomes defined by a modified Rankin Scale score of 0-2 at 30 days: 57.9%, 42.1%, 21.1%, and 5.3%, respectively (P < .001). DWI reversal was observed in 11 of 76 patients, translating to 82% favorable outcome. CONCLUSIONS: Infarct evolution after endovascular thrombectomy is associated with an outcome. DWI reversal or no growth translated to a favorable outcome. Small initial ischemic core, good collateral support, and better recanalization grades predict the smaller infarct growth and favorable outcome after endovascular thrombectomy.

Intra-arterial therapy for acute ischemic stroke under general anesthesia versus monitored anesthesia care.

BACKGROUND: Recent studies have shown that intra-arterial recanalization therapy (IAT) for acute ischemic stroke (AIS) is associated with worse clinical outcomes when performed under general anesthesia (GA) compared to local anesthesia, with or without conscious sedation. The reasons for this association have not been systematically studied. METHODS: We retrospectively reviewed 190 patients who underwent IAT for anterior circulation AIS from January 2008 to December 2012 at our institution. Baseline demographics, vessels involved, acute stroke treatment including intravenous tissue type plasminogen activator (tPA) use, use of GA vs. monitored anesthesia care (MAC), location of thrombus, recanalization grade, radiologic post-procedural intracerebral hemorrhage, and 30-day outcomes were collected. Relevant clinical time points were recorded. Detailed intra-procedural hemodynamics including maximum/minimum heart rate, systolic blood pressure (BP), diastolic BP, mean BP, use of pressors and episodes of hypotension were collected. Our study's outcomes were as follows: in-hospital mortality, 30-day good outcome (mRS ≤2), successful recanalization and radiologic post-procedural intracerebral hemorrhage. RESULTS: Ninety-one patients received GA and 99 patients received MAC. There was no significant difference in the NIHSS score between the two groups but the GA group had a higher number of ICA occlusions (31.9 vs. 18.2%, p = 0.043). The time from the start of anesthesia to incision (23.0 ± 12.5 min vs. 18.7 ± 11.3 min, p = 0.020) and the time from the start of anesthesia to recanalization (110 ± 57.2 vs. 92.3 ± 43.0, p = 0.045) was longer in the GA group. The time from incision to recanalization was not significantly different between the two groups. mRS 0-2 was achieved in 22.8% of patients in the MAC group compared to 14.9% in GA (p = 0.293). Higher mortality was seen in the GA group (25.8 vs. 13.3%, p = 0.040). Successful recanalization (TICI 2b-3) was similar between the GA and MAC (57.8 vs. 48.5%, p = 0.182) groups, but GA had a higher number of parenchymal hematomas (26.3 vs. 10.1%, p = 0.003). There was no difference in the intra-procedural hemodynamic variables between the GA and MAC groups. Anesthesia type was an independent predictor for mortality (along with age and initial NIHSS), and the only independent predictor for parenchymal hematomas, with MAC being protective for both. CONCLUSION: Our study has confirmed previous findings of GA being associated with poorer outcomes and higher mortality in patients undergoing IAT for AIS. Detailed analysis of intra-procedural hemodynamics did not reveal any significant difference between the two groups. Parenchymal hematoma was the major driver of the difference in outcomes.

Posttreatment variables improve outcome prediction after intra-arterial therapy for acute ischemic stroke.

BACKGROUND: There are multiple clinical and radiographic factors that influence outcomes after endovascular reperfusion therapy (ERT) in acute ischemic stroke (AIS). We sought to derive and validate an outcome prediction score for AIS patients undergoing ERT based on readily available pretreatment and posttreatment factors. METHODS: The derivation cohort included 511 patients with anterior circulation AIS treated with ERT at 10 centers between September 2009 and July 2011. The prospective validation cohort included 223 patients with anterior circulation AIS treated in the North American Solitaire Acute Stroke registry. Multivariable logistic regression identified predictors of good outcome (modified Rankin score ≤2 at 3 months) in the derivation cohort; model ß coefficients were used to assign points and calculate a risk score. Discrimination was tested using C statistics with 95% confidence intervals (CIs) in the derivation and validation cohorts. Calibration was assessed using the Hosmer-Lemeshow test and plots of observed to expected outcomes. We assessed the net reclassification improvement for the derived score compared to the Totaled Health Risks in Vascular Events (THRIVE) score. Subgroup analysis in patients with pretreatment Alberta Stroke Program Early CT Score (ASPECTS) and posttreatment final infarct volume measurements was also performed to identify whether these radiographic predictors improved the model compared to simpler models. RESULTS: Good outcome was noted in 186 (36.4%) and 100 patients (44.8%) in the derivation and validation cohorts, respectively. Combining readily available pretreatment and posttreatment variables, we created a score (acronym: SNARL) based on the following parameters: symptomatic hemorrhage [2 points: none, hemorrhagic infarction (HI)1-2 or parenchymal hematoma (PH) type 1; 0 points: PH2], baseline National Institutes of Health Stroke Scale score (3 points: 0-10; 1 point: 11-20; 0 points: >20), age (2 points: <60 years; 1 point: 60-79 years; 0 points: >79 years), reperfusion (3 points: Thrombolysis In Cerebral Ischemia score 2b or 3) and location of clot (1 point: M2; 0 points: M1 or internal carotid artery). The SNARL score demonstrated good discrimination in the derivation (C statistic 0.79, 95% CI 0.75-0.83) and validation cohorts (C statistic 0.74, 95% CI 0.68-0.81) and was superior to the THRIVE score (derivation cohort: C statistic 0.65, 95% CI 0.60-0.70; validation cohort: C-statistic 0.59, 95% CI 0.52-0.67; p < 0.01 in both cohorts) but was inferior to a score that included age, ASPECTS, reperfusion status and final infarct volume (C statistic 0.86, 95% CI 0.82-0.91; p = 0.04). Compared with the THRIVE score, the SNARL score resulted in a net reclassification improvement of 34.8%. CONCLUSIONS: Among AIS patients treated with ERT, pretreatment scores such as the THRIVE score provide only fair prognostic information. Inclusion of posttreatment variables such as reperfusion and symptomatic hemorrhage greatly influences outcome and results in improved outcome prediction.

Does the sex of acute stroke patients influence the effectiveness of rt-PA?

BACKGROUND: Women have been reported to show more frequent recanalization and better recovery after intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment for acute stroke compared with men. To investigate this we studied a series of stroke patients receiving IV rt-PA and undergoing acute transcranial doppler (TCD) examination. METHODS: Acute stroke patients received IV rt-PA and had acute TCD examination within 4 hours of symptom onset at 4 major stroke centers. TCD findings were interpreted using the Thrombolysis in Brain Ischemia (TIBI) flow grading system. The recanalization rates, and poor 3-month outcomes (modified Rankin scale >2) of men and women were compared using the chi-square test. Multiple regression analysis was used to assess sex as a predictor of recanalization and poor 3-month outcome after controlling for age, baseline NIH Stroke Scale (NIHSS), time to treatment, hypertension, and blood glucose. RESULTS: 369 patients had TCD examinations before or during IV rt-PA treatment. The 199 (53.9%) men and 170 (46.1%) women had mean ages of 67 ± 13 and 70 ± 14 years, respectively. The sexes did not differ significantly in baseline stroke severity, time to TCD examination, or time to thrombolysis. Of the men, 68 (34.2%) had complete recanalization, 58 (29.1%) had partial recanalization, and 73 (36.6%) had no recanalization. Of the women, 53 (31.2%) had complete recanalization, 46 (27%) had partial recanalization, and 71 (41.8%) had no recanalization (p = 0.6). Multiple regression analyses showed no difference between the sexes in recanalization rate, time to recanalization, or clinical outcome at 3 months. CONCLUSIONS: In our study; sex is not a significant predictor of recanalization rate, time to recanalization or 3-month outcome in stroke patients following IV rt-PA. TRIAL REGISTRATION: Data from CLOTBUST trial Clinicaltrials.gov Identifier: NCT01240356.

Reversal of coagulopathy using prothrombin complex concentrates is associated with improved outcome compared to fresh frozen plasma in warfarin-associated intracranial hemorrhage.

BACKGROUND: There are no studies demonstrating that prothrombin complex concentrates (PCC) improves outcome compared FFP in patients with warfarin-associated intracranial hemorrhage. METHODS: A prospective, observational study was conducted of patients who received PCC (Bebulin VH), FFP, or PCC + FFP. All groups received vitamin K 10 mg IV. INR reversal (<1.4), adverse events (venous thromboembolism, myocardial infraction, pulmonary edema), major hemorrhage (new or worsened intracranial hemorrhage, anemia requiring transfusion or GI bleed), and 3-month functional outcome were compared between the groups using Chi squared and logistic regression analysis. RESULTS: Of 64 patients, PCC alone was used in 16 (mean dose 48 IU/kg), FFP alone in 25 (mean dose 12.5 ml/kg), and PCC + FFP in 23 (median doses 47.4 IU/kg and 11.4 ml/kg, respectively). INR correction occurred in 88, 84, and 70 %, respectively. There were no differences in time to INR correction or adverse events between the groups, but FFP alone was associated with more major hemorrhage after administration (52 %, OR 5.0, 95 % CI 1.6-15.4, P = 0.006) and PCC with less (6 %, OR 0.1, 95 % CI 0.01-0.8, P = 0.033). After adjusting for age, admission GCS, initial INR, and bleed type, the use of PCC was associated with a lower risk of death or severe disability at 3-months (adjusted OR 0.02, 95 % CI 0.001-0.8, P = 0.039), while FFP alone was associated with a higher risk (adjusted OR 51.6, 95 % CI 1.2-2163.1, P = 0.039). CONCLUSIONS: PCC adequately corrected INR without any increase in adverse events compared to FFP and was associated with less major hemorrhage and improved 3-month outcomes in patients with warfarin-associated intracranial hemorrhage.

Phospholipases A2: enzymatic assay for snake venom (Naja naja karachiensis) with their neutralization by medicinal plants of Pakistan.

Phospholipases A2 (PLA2) are the most lethal and noxious component of Naja naja karachiensis venom. They are engaged to induce severe toxicities after their penetration in victims. Present study was designed to highlight hydrolytic actions of PLA. in an egg yolk mixture and to encounter their deleterious effects via medicinal plants of Pakistan. PLA2 were found to produce free fatty acids in a dose dependent manner. Venom at concentration of 0.1 mg was found to liberate 26.6 pmoles of fatty acids with a decline in pH1 of 0.2 owing to the presence of PLA2 (133 Unit/mg). When quantity of venom was increased up to 8 mg, it caused to release 133 pmoles of free fatty acids with a decrease in 1.0 pH due to abundance in PLA, (665 Unit/mg). The rest of other doses of venom (0.3-4.0 mg) was found to liberate fatty acids between these two upper and lower limits. Twenty eight medicinal plants (0.1-0.6 mg) were tried to abort PLA, hydrolytic action, however, all were found useful (50-100%) against PLA,. Bauhinia variegate L., Citrus limon (L.). Burm.f. Enicostemnma hyssopifolium (Willd.) Verdoorn, Ocimum sanctum. Psoralea corylifolia L. and Stenolobium stans (L.) D. Don were found excellent in switching off 100% phospholipases A, at their lowest concentration (0.1 mg). Three plants extract were found useful only at lower concentration (0.1 mg), however, their higher doses were seemed to aggravate venom response. Eight medicinal plants failed to neutralize PLA, rather their higher doses were found effective. Standard antidote and rest of other plants extract were able to show maximum of 50% efficiencies. Therefore, it is necessary to identify and isolate bioactive constituent(s) from above cited six medicinal plants to eradicate the problem of snake bite in the future.

Predictive Value of Clinical, CSF and Vessel Wall MRI Variables in Diagnosing Primary Angiitis of the CNS.

Background and Objectives: Without brain biopsy, there are limited diagnostic predictors to differentiate primary angiitis of the CNS (PACNS) from intracranial atherosclerotic disease (ICAD). We examined the utility of clinical, CSF, and quantitative vessel wall magnetic resonance imaging (VWMRI) variables in predicting PACNS from ICAD. Methods: In this cross-sectional design, observational study, we reviewed electronic medical records to identify patients (18 years and older) who presented to our medical center between January 2015 and December 2021 for ischemic stroke due to intracranial vasculopathy. Patients with biopsy-proven PACNS, probable PACNS, or ICAD were included. Patients with secondary CNS vasculitis or no VWMRI data were excluded. On VWMRI, for each patient, a total of 20 vessel wall segments were analyzed for percent concentricity, percent irregularity, and concentricity to eccentricity (C/E) ratios. We also collected several clinical and CSF variables. Using logistic regression models, we assessed the diagnostic value of VWMRI, CSF, and clinical variables in predicting PACNS in patients with biopsy-proven disease. We then performed a sensitivity analysis to assess predictors of biopsy-proven and probable PACNS. Results: Thirty-two patients with ICAD (54.2%) and 27 patients with PACNS (45.8%) were included. Of the patients with PACNS, 21 (77.8%) were not biopsied and considered probable PACNS. Twenty-four patients with ICAD (75%) and 6 biopsy-proven patients with PACNS (22.2%) showed large vessel involvement and were included in the primary analysis. Encephalopathy (odds ratio [OR], 7.60; 95% CI 1.07-54.09) and seizure (OR 23.00; 95% CI 1.77-298.45) were significantly associated with PACNS. All patients were included in the sensitivity analysis, in which headache significantly predicted PACNS (OR 7.60; 95% CI 1.07-54.09). In the primary analysis, for every 1 white blood cell/µL increase in CSF, there was a 47% higher odds of PACNS (OR 1.47; 95% CI 1.04-2.07). On VWMRI, a C/E ratio >1 (OR 115.00; 95% CI 6.11-2165.95), percent concentricity ≥50% (OR 55.00; 95% CI 4.13-732.71), and percent irregularity <50% (OR 55.00; 95% CI 4.13-732.71) indicated significantly higher odds of PACNS compared with ICAD. Discussion: Our results suggest that quantitative VWMRI metrics, CSF pleocytosis, and clinical features of encephalopathy, seizure, and headache significantly predict a diagnosis of probable PACNS when compared with ICAD.

Covalent Fragment Inhibits RhoA Activation by Guanine Exchange Factors.

Ras homolog gene family member (RhoA) is a GTPase and a member of the RAS superfamily of GTPases. RhoA is a master regulator of the actin cytoskeleton. It inhibits axon growth preventing repair and recovery following spinal cord and traumatic brain injuries. Despite decades of research into the biological function of Rho GTPases, there exist no small-molecule Rho inhibitors. Here, we screen a library of cysteine electrophiles to explore whether covalent bond formation at Cys-107 leads to inhibition of RhoA activation by guanine exchange factor Trio. Two fragments, propiolamide 1 (ACR-895) and acrylamide 2 (ACR-917), inhibited RhoA nucleotide exchange by Trio in a time-dependent manner. The fragments formed a covalent bond with wild-type RhoA but not Cys107Ser RhoA mutant. Time- and concentration-dependent studies led to equilibrium constants KIs and reaction rates that correspond to t1/2 values in the single-digit hour range. One fragment was selective for RhoA over Rac1 GTPase and had no effect on KRAS nucleotide exchange by SOS1. The fragments did not inhibit RhoA binding to ROCK effector protein. This work establishes Cys-107 as a suitable site for Rho GTPase inhibition and provides fragment starting points for the future development of Rho GTPase covalent inhibitors that could have profound implications in the treatment of patients with injuries of the central nervous system.