The filamentous type III secretion translocon of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (TTSS) to inject effector proteins into the plasma membrane and cytosol of infected cells. To translocate proteins, EPEC, like Salmonella and Shigella , is believed to assemble a macromolecular complex (type III secreton) that spans both bacterial membranes and has a short needle-like projection. However, there is a special interest in studying the EPEC TTSS owing to the fact that one of the secreted proteins, EspA, is assembled into a unique filamentous structure also required for protein translocation. In this report we present electron micrographs of EspA filaments which reveal a regular segmented substructure. Recently we have shown that deletion of the putative structural needle protein, EscF, abolished protein secretion and formation of EspA filaments. Moreover, we demonstrated that EspA can bind directly to EscF, suggesting that EspA filaments are physically linked to the EPEC needle complex. In this paper we provide direct evidence for the association between an EPEC bacterial membrane needle complex and EspA filaments, defining a new class of filamentous TTSS.     

Genetic analysis of Brugada syndrome in Israel: two novel mutations and possible genetic heterogeneity

Idiopathic ventricular fibrillation in patients with an electrocardiogram (ECG) pattern of right bundle branch block and ST-segment elevation in leads V1 to V3 (now frequently called Brugada syndrome) is associated with a high incidence of syncopal episodes or sudden death. The disease is inherited as an autosomal dominant trait. Mutations in SCN5A, a cardiac sodium channel gene, have been recently associated with Brugada syndrome. We have analyzed 7 patients from Israel affected with Brugada syndrome. The families of these patients are characterized by a small number of symptomatic members. Sequencing analysis of SCN5A revealed two novel mutations, G35S and R104Q, in two Brugada patients, and a possible R34C polymorphism in two unrelated controls. No mutations were detected in 5 other patients, suggesting genetic heterogeneity. Low penetrance is probably the cause for the small number of symptomatic members in the two families positive for the SCN5A mutations.     

Mutation and haplotype studies of familial Mediterranean fever reveal new ancestral relationships and evidence for a high carrier frequency with reduced penetrance in the Ashkenazi Jewish population

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever with serositis or synovitis. The FMF gene (MEFV) was cloned recently, and four missense mutations were identified. Here we present data from non-Ashkenazi Jewish and Arab patients in whom we had not originally found mutations and from a new, more ethnically diverse panel. Among 90 symptomatic mutation-positive individuals, 11 mutations accounted for 79% of carrier chromosomes. Of the two mutations that are novel, one alters the same residue (680) as a previously known mutation, and the other (P369S) is located in exon 3. Consistent with another recent report, the E148Q mutation was observed in patients of several ethnicities and on multiple microsatellite haplotypes, but haplotype data indicate an ancestral relationships between non-Jewish Italian and Ashkenazi Jewish patients with FMF and other affected populations. Among approximately 200 anonymous Ashkenazi Jewish DNA samples, the MEFV carrier frequency was 21%, with E148Q the most common mutation. Several lines of evidence indicate reduced penetrance among Ashkenazi Jews, especially for E148Q, P369S, and K695R. Nevertheless, E148Q helps account for recessive inheritance in an Ashkenazi family previously reported as an unusual case of dominantly inherited FMF. The presence of three frequent MEFV mutations in multiple Mediterranean populations strongly suggests a heterozygote advantage in this geographic region.     

Megalencephalic leukoencephalopathy with subcortical cysts; a founder effect in Israeli patients and a higher than expected carrier rate among Libyan Jews

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a progressive inherited neurological disorder characterized by macrocephaly, deterioration in motor functions and cerebellar ataxia. In Israel the disease is found in an increased frequency among Libyan Jews. The disease is caused by mutations in the MLC1 gene, which encodes a putative CNS membrane transporter. We describe three novel mutations (p.G59E, p.P92S, and 134_136insC) in seven MLC families. One of these mutations, p.G59E, was found in the vast majority of MLC patients in Israel. Screening of 200 normal Libyan Jewish individuals for the p.G59E mutation, revealed a carrier rate of 1/40 compared with an expected carrier rate of 1/81. Several explanations could account for this difference the most likely one is an admixture of the Libyan Jewish population.     

Combined use of micro-preparative gel electrophoresis and reversed-phase high-performance liquid chromatography for purification of amyloid beta peptides deposited in brains of Alzheimer's disease patients

A new micro-technique is developed for purification of amyloid beta peptides (A beta) extracted from brain tissues of patients with Alzheimer's disease (AD). It includes SDS-polyacrylamide gel electrophoresis of the extracted brain tissue material, electroblotting onto supporting membranes, and reversed-phase HPLC of the proteins eluted from membranes. By this technique, the extracted A beta are first separated electrophoretically from the higher and lower molecular mass tissue components, and then purified by reversed-phase HPLC from the contaminants having similar molecular masses, but different retention times on the column. In contrast to the common large-scale isolation procedures employing density gradient centrifugation, enzymatic digestions and size-exclusion chromatography, the developed micro-technique might be applied for biochemical analysis of A beta contained in small AD brain tissue specimens.     

"Beyond 'broke'": three steps to improve how your hospital buys, uses, and repairs equipment

By approaching problems in the hospital as an observational researcher, biomeds can begin to think about the problems in a different way, which may point toward opportunities to solve these problems. Of all of the people in the hospital, biomeds are in the best position to address these issues because they see how the products are used and what goes wrong. These three steps are just a starting point for moving beyond product repair and maintenance so biomeds can improve product use in their hospitals.     

A Late Phase of Long-Term Synaptic Depression in Cerebellar Purkinje Cells Requires Activation of MEF2

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The cellular modifier MOAG‐4/SERF drives amyloid formation through charge complementation

While aggregation‐prone proteins are known to accelerate aging and cause age‐related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG‐4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid‐promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG‐4 to neutralize charge. Our data indicate that MOAG‐4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation‐prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age‐related protein toxicity.     

A High-Resolution Genetic Map of the Familial Mediterranean Fever Candidate Region Allows Identification of Haplotype-Sharing among Ethnic Groups

Familial Mediterranean fever (FMF) is a recessive disorder of inflammation caused by mutations in a gene (designatedMEFV) on chromosome 16p13.3. We have recently constructed a 1-Mb cosmid contig that includes the FMF critical region. Here we show genotype data for 12 markers from our physical map, including 5 newly identified microsatellites, in FMF families. Intrafamilial recombinations placedMEFVin the ∼285 kb betweenD16S468/D16S3070andD16S3376.We observed significant linkage disequilibrium in the North African Jewish population, and historical recombinants in the founder haplotype placedMEFVbetweenD16S3082andD16S3373(∼200 kb). In smaller panels of Iraqi Jewish, Arab, and Armenian families, there were significant allelic associations only forD16S3370andD16S2617among the Armenians. A sizable minority of Iraqi Jewish and Armenian carrier chromosomes appeared to be derived from the North African Jewish ancestral haplotype. We observed a unique FMF haplotype common to Iraqi Jews, Arabs, and Armenians and two other haplotypes restricted to either the Iraqi Jewish or the Armenian population. These data support the view that a few major mutations account for a large percentage of the cases of FMF and suggest that some of these mutations arose before the affected Middle Eastern populations diverged from one another.     

Transmembrane Current Imaging in the Heart during Pacing and Fibrillation

Recently, we described a method to quantify the time course of total transmembrane current (I_m) and the relative role of its two components, a capacitive current (I_c) and a resistive current (I_ion), corresponding to the cardiac action potential during stable propagation. That approach involved recording high-fidelity (200 kHz) transmembrane potential (V_m) signals with glass microelectrodes at one site using a spatiotemporal coordinate transformation via measured conduction velocity. Here we extend our method to compute these transmembrane currents during stable and unstable propagation from fluorescence signals of V_m at thousands of sites (3 kHz), thereby introducing transmembrane current imaging. In contrast to commonly used linear Laplacians of extracellular potential (V_e) to compute I_m, we utilized nonlinear image processing to compute the required second spatial derivatives of V_m. We quantified the dynamic spatial patterns of current density of I_m and I_ion for both depolarization and repolarization during pacing (including nonplanar patterns) by calibrating data with the microelectrode signals. Compared to planar propagation, we found that the magnitude of I_ion was significantly reduced at sites of wave collision during depolarization but not repolarization. Finally, we present uncalibrated dynamic patterns of I_m during ventricular fibrillation and show that I_m at singularity sites was monophasic and positive with a significant nonzero charge (I_m integrated over 10 ms) in contrast with nonsingularity sites. Our approach should greatly enhance the understanding of the relative roles of functional (e.g., rate-dependent membrane dynamics and propagation patterns) and static spatial heterogeneities (e.g., spatial differences in tissue resistance) via recordings during normal and compromised propagation, including arrhythmias.