Properties of purified actin depolymerizing factor from chick brain

Actin depolymerizing factor (ADF) from 19-day embryonic chick brains has been purified to greater than 98% homogeneity with a yield of 7.2 mg/100 g of brain. Quantitative immunoblotting with a monospecific antibody to ADF indicated that ADF comprises 0.3% of the total brain protein, resulting in an actual purification yield of about 20%. Brain ADF migrates as a single polypeptide of 19,000 kDa on SDS-containing polyacrylamide gels. The molecular weight of the native protein determined from sedimentation equilibrium in buffers containing from 50 to 200 mM KCl is 20,000. The secondary structure of ADF calculated from the circular dichroic spectrum consists of about 22% alpha-helix, 24% beta-sheet, and 18% beta-turn. ADF contains a blocked N-terminus, a single tryptophan residue located about one-third of the way from one end of the protein, and six cysteine residues (all in reduced form in the native protein). All six cysteine residues could be chemically modified with eosinylmaleimide under nondenaturing conditions; however, ADF activity was lost when more than one cysteine residue was modified. ADF microheterogeneity has been observed upon nonequilibrium pH gradient electrophoresis in polyacrylamide gels containing 9 M urea, the major isoform having a pI of congruent to 7.9-8.0. ADF can interact with either monomeric or filamentous actin to give a complex which can be isolated by gel filtration chromatography. Both major and minor isoforms of the ADF are found in the complex. Assembly-competent actin and active ADF can both be recovered from the complex by chromatography on ATP-saturated DEAE-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simulation of Daily Snapshot Rhythm Monitoring to Identify Atrial Fibrillation in Continuously Monitored Patients with Stroke Risk Factors

Background

New technologies are diffusing into medical practice swiftly. Hand-held devices such as smartphones can record short-duration (e.g., 1-minute) ECGs, but their effectiveness in identifying patients with paroxysmal atrial fibrillation (AF) is unknown.

Methods

We used data from the TRENDS study, which included 370 patients (mean age 71 years, 71% men, CHADS₂ score≥1 point: mean 2.3 points) who had no documentation of atrial tachycardia (AT)/AF or antiarrhythmic or anticoagulant drug use at baseline. All were subsequently newly diagnosed with AT/AF by a cardiac implantable electronic device (CIED) over one year of follow-up. Using a computer simulation approach (5,000 repetitions), we estimated the detection rate for paroxysmal AT/AF via daily snapshot ECG monitoring over various periods, with the probability of detection equal to the percent AT/AF burden on each day.

Results

The estimated AT/AF detection rates with snapshot monitoring periods of 14, 28, 56, 112, and 365 days were 10%, 15%, 21%, 28%, and 50% respectively. The detection rate over 365 days of monitoring was higher in those with CHADS₂ scores ≥2 than in those with CHADS₂ scores of 1 (53% vs. 38%), and was higher in those with AT/AF burden ≥0.044 hours/day compared to those with AT/AF burden <0.044 hours/day (91% vs. 14%; both P<0.05).

Conclusions

Daily snapshot ECG monitoring over 365 days detects half of patients who developed AT/AF as detected by CIED, and shorter intervals of monitoring detected fewer AT/AF patients. The detection rate was associated with individual CHADS₂ score and AT/AF burden.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00279981","term_id":"NCT00279981"}}NCT00279981     

The Geographic Pathology of Coronary Atherosclerosis

The purpose of these studies was to find large groups with significantly less coronary atherosclerosis than New Yorkers and to investigate the possible reasons for these differences. Direct comparison of hearts and measurements of coronary artery wall thickness, autopsy series, and clinical diagnoses of outpatients and hospital admissions revealed that the amount of coronary atherosclerosis and the number of myocardial infarcts is significantly less in East and West Africans compared to New Yorkers matched for age and sex. The factors producing these differences are apparently operative in childhood. East Africans were found to have a shorter blood clot-lysis time, fewer venous (and arterial) thromboemboli and lower serum lipid levels, with a lower relative percentage of serum linoleates, than age-matched New Yorkers.     

Identification of novel candidate genes by exome sequencing in Tunisian familial male breast cancer patients

Molecular Biology Reports - Male Breast Cancer (MBC) is a rare and aggressive disease that is associated with genetic factors. Mutations in BRCA1&nbsp;and&nbsp;BRCA2&nbsp;account for...     

Next-generation sequencing for diagnosis of rare diseases in the neonatal intensive care unit

Background:Rare diseases often present in the first days and weeks of life and may require complex management in the setting of a neonatal intensive care unit (NICU). Exhaustive consultations and traditional genetic or metabolic investigations are costly and often fail to arrive at a final diagnosis when no recognizable syndrome is suspected. For this pilot project, we assessed the feasibility of next-generation sequencing as a tool to improve the diagnosis of rare diseases in newborns in the NICU.Methods:We retrospectively identified and prospectively recruited newborns and infants admitted to the NICU of the Children’s Hospital of Eastern Ontario and the Ottawa Hospital, General Campus, who had been referred to the medical genetics or metabolics inpatient consult service and had features suggesting an underlying genetic or metabolic condition. DNA from the newborns and parents was enriched for a panel of clinically relevant genes and sequenced on a MiSeq sequencing platform (Illumina Inc.). The data were interpreted with a standard informatics pipeline and reported to care providers, who assessed the importance of genotype–phenotype correlations.Results:Of 20 newborns studied, 8 received a diagnosis on the basis of next-generation sequencing (diagnostic rate 40%). The diagnoses were renal tubular dysgenesis, SCN1A-related encephalopathy syndrome, myotubular myopathy, FTO deficiency syndrome, cranioectodermal dysplasia, congenital myasthenic syndrome, autosomal dominant intellectual disability syndrome type 7 and Denys–Drash syndrome.Interpretation:This pilot study highlighted the potential of next-generation sequencing to deliver molecular diagnoses rapidly with a high success rate. With broader use, this approach has the potential to alter health care delivery in the NICU.A rare disease is defined by a prevalence of less than 1 in 2000 individuals.¹ However, when considered in aggregate, 1%–2% of Canadians will manifest a rare disease in their lifetime.^2,3 These disorders can present in the newborn period, and a third of these young children will succumb to the disease in their first year of life.^3–5 Newborns who present with rare diseases typically require admission to a neonatal intensive care unit (NICU), where the standard of care includes exhaustive consultations and investigations to determine a molecular diagnosis. Reaching such a diagnosis is a challenge, given the considerable clinical and genetic heterogeneity associated with rare diseases; diagnosis is also confounded by the early stage of presentation, which is further accentuated in premature newborns. As a result, traditional genetic or metabolic investigations can be lengthy and expensive, and they often fail to arrive at a diagnosis in a timely manner.⁶The current approach during a medical genetics consultation begins with a clinical assessment, followed by diagnostic testing that usually includes sequential testing of one or more candidate genes or panels of candidate genes. This step often requires approval for out-of-country testing, as only a limited number of gene tests are available for clinical testing in Canada. If the result of the first test is negative, the clinician may consider testing the next most likely candidate gene, frequently with diminishing returns. This approach can take months or years and can be a frustrating process for the patient, family and clinicians providing care.⁷ The inability to arrive at a timely and efficient diagnosis represents a substantial lost opportunity, as a diagnosis can limit or even halt further invasive, and at times futile, investigations for the neonate. Importantly, an accurate diagnosis informs prognosis and may guide management decisions.The advent of next-generation sequencing has greatly advanced the ability to rapidly identify the novel genes responsible for disease.⁸ Whole-exome sequencing (sequencing of the coding portion of the genome) is beginning to be used on a clinical basis in tertiary care centres.^9,10 In these initial clinical cohort studies, a molecular diagnosis was provided by whole-exome sequencing for about 25% of families. The proportion increased to 31% when the patient’s parents were also analyzed.⁹ Another study used retrospective whole-genome sequencing to make a diagnosis in 57% of 35 children from the intensive care setting.¹¹Although whole-exome and whole-genome sequencing are powerful tools, important conditions are required for translation of these methods to the clinic or hospital setting. The availability of high-throughput sequencers, complex and costly infrastructure, and personnel with bioinformatics expertise are prerequisites. These resources may not be broadly available within some health care systems, and other strategies may be more relevant and effective.Another attractive alternative is analysis based on next-generation sequencing that focuses only on the clinically relevant genes with known associated clinical phenotypes.¹² This strategy offers several advantages over whole-exome or whole-genome sequencing — interpretation of variants may be more straight-forward, a higher depth of coverage can be readily achieved, and less infrastructure and fewer personnel are required — all of which contribute to a more rapid return of results.For this pilot study, we evaluated the performance of a targeted next-generation sequencing panel that included 4813 “disease-relevant” genes in a cohort of newborns with rare disease in the NICU and assessed the effectiveness of this method to accurately diagnose these critically ill babies.     

Social Network and Vulnerability: A Clear Link in Bedouin Society (Egypt)

In the last 60 years, the livelihoods of agro-pastoral and pastoral families in the arid and semi-arid zones in North Africa and the Middle East have undergone major changes caused by significant incentives to adopt a sedentary lifestyle and the increasing intensity of drought events. Such changes have also been influenced by land reclamation projects accompanied by the construction of reservoirs and dikes in the dry lands as well as the extension of irrigation canals in the desert in the Coastal Zone of the Western Desert, Egypt. To understand the changes in the traditional social organization of this desert society, and how these social changes have affected families’ ability to adapt to external shocks such as the recent 15-year drought, we developed a typological approach to investigate the link between family livelihoods and social capital in Bedouin society. We showed a clear link between physical assets (mainly land and animals), the nature and intensity of social links within the traditional society, and level of education. The analysis revealed also some new wealth accumulation processes in link with the socio-political influence of urban zones and the increasing level of education in the zone.     

Inhibition of neutrophil apoptosis by TLR agonists in whole blood: involvement of the phosphoinositide 3-kinase/Akt and NF-kappaB signaling pathways, leading to increased levels of Mcl-1, A1, and phosphorylated Bad

Using flow cytometry, we investigated the effect of TLR agonists on human polymorphonuclear neutrophil (PMN) apoptosis in whole blood. LPS (TLR4), peptidoglycan (TLR2), R-848 (TLR7/8), and CpG-DNA (TLR9) were equally effective at delaying spontaneous apoptosis of PMN, while PamCSK4 (TLR1/2), macrophage-activating lipopeptide-2 (TLR2/6), flagellin (TLR5), and loxoribine (TLR7) were less effective or inactive. TLR agonists found to delay apoptosis also extended the functional life span of PMN. Analysis of signaling pathways revealed that the antiapoptotic effect of TLR agonists required NF-kappaB and PI3K activation. Furthermore, analysis of intact cells by flow cytometry showed that TLR agonists delaying PMN apoptosis increased phosphorylation of Akt, a major target of PI3K. This effect was associated with a PI3K-dependent increase in heat shock protein 27 phosphorylation, which has been reported to play a key role in PMN survival. Finally, the TLR-induced delay in PMN apoptosis was associated with increased levels of Mcl-1 and A1, which are antiapoptotic members of the Bcl-2 family. These effects were reversed by PI3K and NF-kappaB inhibitors, respectively. TLR activation also led to PI3K-dependent phosphorylation of the proapoptotic protein Bad. Taken together, our results strongly suggest a role of NF-kappaB and PI3K in TLR-induced PMN survival, leading to modulation of Bcl-2 family molecules.     

Comparative studies on simple lipids ofSclerotium cepivorum,the causal agent of white rot of onion,and other fungi of the class Deuteromycetes

Summary The simple lipids ofSclerotium cepivorum, the causal agent of white rot of onion and nine other fungal species of the same class were investigated. The fatty acid composition of the simple lipids of these fungi were determined by GLC. The main fatty acids common to these fungal species were C₁₆ (saturated) and C₁₈ (unsaturated) acids. The sterol fraction was isolated by column chromatography and its components were detected by GLC and mass spectrometry. Ergosterol and γ-Ergostenol were found mostly in all fungal species under investigation. However, two fungal species namelyAlternaria alternata andScolecobasidium constrictum showed no Ergosterol.     

Oral-dental health status in patients with infectious endocarditis

Mouth and dental examination is a systematic act in the etiologic follow up of an infectious endocarditis. Our study concerned 42 patients collected during 5 years period, starting july 1995 until July 2000, in the dental medical service of Hedi Chaker university hospital of Sfax oriented by the cardiovascular diseases service looking for a buccodental entry of bacteria. Our patients group is divided into 25 men and 17 women of 36.1 years mean age. Dental or parodontal lesions were observed in 25 patients. Panoramic X-rays showed periapical lesions of granuloma or cystic type in 15 patients. Among the etiological factors encountered we noticed dental acts accomplished without antibioprophylaxis and dental bacterial collection medically treated without extraction. Buccodental lesions were a frequent cause of the infectious endocarditis in 59.5% of the patients studied in this report, and were due more to defectious hygienic conditions than to a disabled dental act.