Characterization of two putative potassium channels in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Potassium channels are essential for cell survival and participate in the regulation of cell membrane potential and electrochemical gradients. During its lifecycle, <it>Plasmodium falciparum </it>parasites must successfully traverse widely diverse environmental milieus, in which K⁺channel function is likely to be critical. Dramatically differing conditions will be presented to the parasite in the mosquito mid-gut, red blood cell (RBC) cytosol and the human circulatory system.</p> <p>Methods</p> <p><it>In silico </it>sequence analyses identified two open-reading frames in the <it>P. falciparum </it>genome that are predicted to encode for proteins with high homology to K⁺channels. To further analyse these putative channels, specific antisera were generated and used in immunoblot and immunofluorescence analyses of <it>P. falciparum</it>-infected RBCs. Recombinant genome methods in cultured <it>P. falciparum </it>were used to create genetic knock outs of each K⁺channel gene to asses the importance of their expression.</p> <p>Results</p> <p>Immunoblot and IFA analyses confirmed the expression of the two putative <it>P. falciparum </it>K⁺channels (PfK1 and PfK2). PfK1 is expressed in all asexual stage parasites, predominantly in late stages and localizes to the RBC membrane. Conversely, PfK2 is predominantly expressed in late schizont and merozoite stage parasites and remains primarily localized to the parasite. Repeated attempts to knockout PfK1 and PfK2 expression by targeted gene disruption proved unsuccessful despite evidence of recombinant gene integration, indicating that <it>pfk1 </it>and <it>pfk2 </it>are apparently refractory to genetic disruption.</p> <p>Conclusion</p> <p>Putative K⁺channel proteins PfK1 and PfK2 are expressed in cultured <it>P. falciparum </it>parasites with differing spatial and temporal patterns. Eventual functional characterization of these channels may reveal future pharmacological targets.</p

Crotalus atrox venom preconditioning increases plasma fibrinogen and reduces perioperative hemorrhage in a rat model of surgical brain injury.

Perioperative bleeding is a potentially devastating complication in neurosurgical patients, and plasma fibrinogen concentration has been identified as a potential modifiable risk factor for perioperative bleeding. The aim of this study was to evaluate preconditioning with Crotalus atrox venom (Cv-PC) as potential preventive therapy for reducing perioperative hemorrhage in the rodent model of surgical brain injury (SBI). C. atrox venom contains snake venom metalloproteinases that cleave fibrinogen into fibrin split products without inducing clotting. Separately, fibrinogen split products induce fibrinogen production, thereby elevating plasma fibrinogen levels. Thus, the hypothesis was that preconditioning with C. atrox venom will produce fibrinogen spilt products, thereby upregulating fibrinogen levels, ultimately improving perioperative hemostasis during SBI. We observed that Cv-PC SBI animals had significantly reduced intraoperative hemorrhage and postoperative hematoma volumes compared to those of vehicle preconditioned SBI animals. Cv-PC animals were also found to have higher levels of plasma fibrinogen at the time of surgery, with unchanged prothrombin time. Cv-PC studies with fractions of C. atrox venom suggest that snake venom metalloproteinases are largely responsible for the improved hemostasis by Cv-PC. Our findings indicate that Cv-PC increases plasma fibrinogen levels and may provide a promising therapy for reducing perioperative hemorrhage in elective surgeries

Use of a targeted, combinatorial next-generation sequencing approach for the study of bicuspid aortic valve

BACKGROUND: Bicuspid aortic valve (BAV) is the most common type of congenital heart disease with a population prevalence of 1-2%. While BAV is known to be highly heritable, mutations in single genes (such as GATA5 and NOTCH1) have been reported in few human BAV cases. Traditional gene sequencing methods are time and labor intensive, while next-generation high throughput sequencing remains costly for large patient cohorts and requires extensive bioinformatics processing. Here we describe an approach to targeted multi-gene sequencing with combinatorial pooling of samples from BAV patients. METHODS: We studied a previously described cohort of 78 unrelated subjects with echocardiogram-identified BAV. Subjects were identified as having isolated BAV or BAV associated with coarctation of aorta (BAV-CoA). BAV cusp fusion morphology was defined as right-left cusp fusion, right non-coronary cusp fusion, or left non-coronary cusp fusion. Samples were combined into 19 pools using a uniquely overlapping combinatorial design; a given mutation could be attributed to a single individual on the basis of which pools contained the mutation. A custom gene capture of 97 candidate genes was sequenced on the Illumina HiSeq 2000. Multistep bioinformatics processing was performed for base calling, variant identification, and in-silico analysis of putative disease-causing variants. RESULTS: Targeted capture identified 42 rare, non-synonymous, exonic variants involving 35 of the 97 candidate genes. Among these variants, in-silico analysis classified 33 of these variants as putative disease-causing changes. Sanger sequencing confirmed thirty-one of these variants, found among 16 individuals. There were no significant differences in variant burden among BAV fusion phenotypes or isolated BAV versus BAV-CoA. Pathway analysis suggests a role for the WNT signaling pathway in human BAV. CONCLUSION: We successfully developed a pooling and targeted capture strategy that enabled rapid and cost effective next generation sequencing of target genes in a large patient cohort. This approach identified a large number of putative disease-causing variants in a cohort of patients with BAV, including variants in 26 genes not previously associated with human BAV. The data suggest that BAV heritability is complex and polygenic. Our pooling approach saved over $39,350 compared to an unpooled, targeted capture sequencing strategy

Rationale for the Cytogenomics of Cardiovascular Malformations Consortium: A Phenotype Intensive Registry Based Approach

Cardiovascular malformations (CVMs) are the most common birth defect, occurring in 1%-5% of all live births. Although the genetic contribution to CVMs is well recognized, the genetic causes of human CVMs are identified infrequently. In addition, a failure of systematic deep phenotyping of CVMs, resulting from the complexity and heterogeneity of malformations, has obscured genotype-phenotype correlations and contributed to a lack of understanding of disease mechanisms. To address these knowledge gaps, we have developed the Cytogenomics of Cardiovascular Malformations (CCVM) Consortium, a multi-site alliance of geneticists and cardiologists, contributing to a database registry of submicroscopic genetic copy number variants (CNVs) based on clinical chromosome microarray testing in individuals with CVMs using detailed classification schemes. Cardiac classification is performed using a modification to the National Birth Defects Prevention Study approach, and non-cardiac diagnoses are captured through ICD-9 and ICD-10 codes. By combining a comprehensive approach to clinically relevant genetic analyses with precise phenotyping, the Consortium goal is to identify novel genomic regions that cause or increase susceptibility to CVMs and to correlate the findings with clinical phenotype. This registry will provide critical insights into genetic architecture, facilitate genotype-phenotype correlations, and provide a valuable resource for the medical community

Characterization of a spark ignition system for flameholding cavities

This paper presents an experimental investigation of a capacitive-discharge spark ignition system designed to promote ignition in CH- and CH-fuelled supersonic combustors. The purpose of this study is the characterization of the ignition system and the plasma generated in the discharge. Schlieren and luminescence imaging are used to visualize the temporal evolution of the spark plasma. Transient voltages and currents across the primary-side of the ignition coil and input-side of the ignition unit are recorded using a high-speed data acquisition system. Three different ignition coils are tested with two different spark plug gaps in an attempt to increase the performance of the ignition system which is evaluated through spatially and temporally integrated luminescence recordings as well as temporally integrated photo diode signals. The data suggests that an increase in performance of a factor of 4-5 over the baseline setup can be achieved. A capacitive ignition lead is used to assess whether or not any capacitance on the coil secondary side can increase the performance of the ignition system. The experiments have also shown that the ignition system parameters can be set to cause sufficient heating of the electrodes to support ignition from a combined glow-spark plug setup

Genetic Evaluation of Cardiomyopathy - a Heart Failure Society of America Practice Guideline

This guideline describes the approach and expertise needed for the genetic evaluation of cardiomyopathy. First published in 2009 by the Heart Failure Society of America (HFSA), this guidance has now been updated in collaboration with the American College of Medical Genetics and Genomics (ACMG). The writing group, composed of cardiologists and genetics professionals with expertise in adult and pediatric cardiomyopathy, reflects the emergence and increased clinical activity devoted to cardiovascular genetic medicine. The genetic evaluation of cardiomyopathy is a rapidly emerging key clinical priority, as high throughput sequencing is now feasible for clinical testing, and conventional interventions can improve survival, reduce morbidity, and enhance quality of life. Moreover, specific interventions may be guided by genetic analysis. A systematic approach is recommended: always a comprehensive family history; an expert phenotypic evaluation of the proband and at-risk family members to confirm a diagnosis and guide genetic test selection and interpretation; referral to expert centers as needed; genetic testing, with pre- and post-test genetic counseling; and specific guidance as indicated for drug and device therapies. The evaluation of infants and children demands special expertise. The approach to manage secondary and incidental sequence findings as recommended by the ACMG is provided

Effects of dietary salt on gene and protein expression in brain tissue of a model of sporadic small vessel disease

Background: The effect of salt on cerebral small vessel disease (SVD) is poorly understood. We assessed the effect of dietary salt on the cerebral tissue of the strokeprone spontaneously hypertensive rat (SHRSP) - a relevant model of sporadic SVD - at both the gene and protein level. Methods: Brains from 21 week old SHRSP and Wistar-Kyoto rats, half additionally salt-loaded (via a 3 week regime of 1% NaCl in drinking water) were split into 2 hemispheres and sectioned coronally – one hemisphere for mRNA microarray and qRT-PCR, the other for immunohistochemistry using a panel of antibodies targeting components of the neurovascular unit. Results: We observed differences in gene and protein expression affecting the acute phase pathway and oxidative stress (ALB, AMBP, APOH, AHSG and LOC100129193, up-regulated in salt-loaded WKY versus WKY, &gt;2-fold), active microglia (increased Iba-1 protein expression in salt-loaded SHRSP versus saltloaded WKY, p&lt;0.05), vascular structure (ACTB &amp; CTNNB, up-regulated in saltloaded SHRSP versus SHRSP, &gt;3-fold; CLDN-11,VEGF and VGF downregulated &gt;- 2-fold in salt-loaded SHRSP versus SHRSP) and myelin integrity (MBP downregulated in salt loaded WKY rats versus WKY, &gt;2.5-fold). Changes of salt-loading were more pronounced in SHRSP and occurred without an increase in blood pressure in WKY rats. Conclusion: Salt exposure induced changes in gene and protein expression in an experimental model of SVD and its parent rat strain in multiple pathways involving components of the glio-vascular unit. Further studies in pertinent experimental models at different ages would help clarify the short and long-term effect of dietary salt in SVD

Adaptive Low-level Storage of Very Large Knowledge Graphs

The increasing availability and usage of Knowledge Graphs (KGs) on the Web calls for scalable and general-purpose solutions to store this type of data structures. We propose Trident, a novel storage architecture for very large KGs on centralized systems. Trident uses several interlinked data structures to provide fast access to nodes and edges, with the physical storage changing depending on the topology of the graph to reduce the memory footprint. In contrast to single architectures designed for single tasks, our approach offers an interface with few low-level and general-purpose primitives that can be used to implement tasks like SPARQL query answering, reasoning, or graph analytics. Our experiments show that Trident can handle graphs with 10^11 edges using inexpensive hardware, delivering competitive performance on multiple workloads.Comment: Accepted WWW 202

Familial co-occurrence of congenital heart defects follows distinct patterns

Aims Congenital heart defects (CHD) affect almost 1% of all live born children and the number of adults with CHD is increasing. In families where CHD has occurred previously, estimates of recurrence risk, and the type of recurring malformation are important for counselling and clinical decision-making, but the recurrence patterns in families are poorly understood. We aimed to determine recurrence patterns, by investigating the co-occurrences of CHD in 1163 families with known malformations, comprising 3080 individuals with clinically confirmed diagnosis. Methods and results We calculated rates of concordance and discordance for 41 specific types of malformations, observing a high variability in the rates of concordance and discordance. By calculating odds ratios for each of 1640 pairs of discordant lesions observed between affected family members, we were able to identify 178 pairs of malformations that co-occurred significantly more or less often than expected in families. The data show that distinct groups of cardiac malformations co-occur in families, suggesting influence from underlying developmental mechanisms. Analysis of human and mouse susceptibility genes showed that they were shared in 19% and 20% of pairs of co-occurring discordant malformations, respectively, but none of malformations that rarely co-occur, suggesting that a significant proportion of co-occurring lesions in families is caused by overlapping susceptibility genes. Conclusion Familial CHD follow specific patterns of recurrence, suggesting a strong influence from genetically regulated developmental mechanisms. Co-occurrence of malformations in families is caused by shared susceptibility genes