Assays to monitor aggrephagy in Drosophila brain

Accumulation of ubiquitinated protein aggregates is a hallmark of most ageingrelated neurodegenerative disorders. Autophagy has been found to be involved in the selective clearance of these protein aggregates, and this process is called aggrephagy. Here we provide two protocols for the investigation of protein aggregation and their removal by autophagy using western blotting and immunofluorescence techniques in Drosophila brain. Investigating the role of aggrephagy at the cellular and organismal level is important for the development of therapeutic interventions against ageing-related diseases

MinION Analysis and Reference Consortium: Phase 1 data release and analysis

The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance

Species-specific differences in the Pro-Ala rich region of cardiac myosin binding protein-C

Cardiac myosin binding protein-C (cMyBP-C) is an accessory protein found in the A-bands of vertebrate sarcomeres and mutations in the cMyBP-C gene are a leading cause of familial hypertrophic cardiomyopathy. The regulatory functions of cMyBP-C have been attributed to the N-terminus of the protein, which is composed of tandem immunoglobulin (Ig)-like domains (C0, C1, and C2), a region rich in proline and alanine residues (the Pro-Ala rich region) that links C0 and C1, and a unique sequence referred to as the MyBP-C motif, or M-domain, that links C1 and C2. Recombinant proteins that contain various combinations of the N-terminal domains of cMyBP-C can activate actomyosin interactions in the absence of Ca2+, but the specific sequences required for these effects differ between species; the Pro-Ala region has been implicated in human cMyBP-C whereas the C1 and M-domains appear important in mouse cMyBP-C. To investigate whether species-specific differences in sequence can account for the observed differences in function, we compared sequences of the Pro-Ala rich region in cMyBP-C isoforms from different species. Here we report that the number of proline and alanine residues in the Pro-Ala rich region varies significantly between different species and that the number correlates directly with mammalian body size and inversely with heart rate. Thus, systematic sequence differences in the Pro-Ala rich region of cMyBP-C may contribute to observed functional differences in human versus mouse cMyBP-C isoforms and suggest that the Pro-Ala region may be important in matching contractile speed to cardiac function across species

A Systems Approach for Tumor Pharmacokinetics

Recent advances in genome inspired target discovery, small molecule screens, development of biological and nanotechnology have led to the introduction of a myriad of new differently sized agents into the clinic. The differences in small and large molecule delivery are becoming increasingly important in combination therapies as well as the use of drugs that modify the physiology of tumors such as anti-angiogenic treatment. The complexity of targeting has led to the development of mathematical models to facilitate understanding, but unfortunately, these studies are often only applicable to a particular molecule, making pharmacokinetic comparisons difficult. Here we develop and describe a framework for categorizing primary pharmacokinetics of drugs in tumors. For modeling purposes, we define drugs not by their mechanism of action but rather their rate-limiting step of delivery. Our simulations account for variations in perfusion, vascularization, interstitial transport, and non-linear local binding and metabolism. Based on a comparison of the fundamental rates determining uptake, drugs were classified into four categories depending on whether uptake is limited by blood flow, extravasation, interstitial diffusion, or local binding and metabolism. Simulations comparing small molecule versus macromolecular drugs show a sharp difference in distribution, which has implications for multi-drug therapies. The tissue-level distribution differs widely in tumors for small molecules versus macromolecular biologic drugs, and this should be considered in the design of agents and treatments. An example using antibodies in mouse xenografts illustrates the different in vivo behavior. This type of transport analysis can be used to aid in model development, experimental data analysis, and imaging and therapeutic agent design.National Institutes of Health (U.S.) (grant T32 CA079443

A review of bioinformatics tools for bio-prospecting from metagenomic sequence data

The microbiome can be defined as the community of microorganisms that live in a particular environment. Metagenomics is the practice of sequencing DNA from the genomes of all organisms present in a particular sample, and has become a common method for the study of microbiome population structure and function. Increasingly, researchers are finding novel genes encoded within metagenomes, many of which may be of interest to the biotechnology and pharmaceutical industries. However, such “bioprospecting” requires a suite of sophisticated bioinformatics tools to make sense of the data. This review summarizes the most commonly used bioinformatics tools for the assembly and annotation of metagenomic sequence data with the aim of discovering novel genes

f(R) theories

Over the past decade, f(R) theories have been extensively studied as one of the simplest modifications to General Relativity. In this article we review various applications of f(R) theories to cosmology and gravity - such as inflation, dark energy, local gravity constraints, cosmological perturbations, and spherically symmetric solutions in weak and strong gravitational backgrounds. We present a number of ways to distinguish those theories from General Relativity observationally and experimentally. We also discuss the extension to other modified gravity theories such as Brans-Dicke theory and Gauss-Bonnet gravity, and address models that can satisfy both cosmological and local gravity constraints.Comment: 156 pages, 14 figures, Invited review article in Living Reviews in Relativity, Published version, Comments are welcom

Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4+ Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4+ T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4+ Th cells upon activation, whereas the ‘standard’ proteasome is up-regulated in Tregs only upon activation

Aged garlic extract therapy for sickle cell anemia patients

BACKGROUND: Sickle cell anemia is one of the most prevalent hereditary disorders with prominent morbidity and mortality. With this disorder oxidative, phenomena play a significant role in its pathophysiology. One of the garlic (Allium sativum L.) formulations, aged garlic extract (AGE), has been reported to exert an anti-oxidant effect in vitro, we have evaluated the anti-oxidant effect of AGE on sickle red blood cells (RBC). METHODS: Five patients (two men and three women, mean age 40 ± 15 years, range 24–58 years) with sickle cell anemia participated in the study. AGE was administered at a dose of 5 ml a day. Whole blood samples were obtained at baseline and at 4 weeks for primarily Heinz body analysis. RESULTS: The data were consistent with our hypothesis. In all patients, the number of Heinz bodies decreased over the 4 week period (58.9 ± 20.0% at baseline to 29.8 ± 15.3% at follow-up, p = 0.03). CONCLUSIONS: These data suggest that there is a significant anti-oxidant activity of AGE on sickle RBC. AGE may be further evaluated as a potential therapeutic agent to ameliorate complications of sickle cell anemia

Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4+ Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4+ T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4+ Th cells upon activation, whereas the ‘standard’ proteasome is up-regulated in Tregs only upon activation