Permanent draft genome sequence of the gliding predator Saprospira grandis strain Sa g1 (= HR1)

Saprospira grandis Gross 1911 is a member of the Saprospiraceae, a family in the class 'Sphingobacteria' that remains poorly characterized at the genomic level. The species is known for preying on other marine bacteria via 'ixotrophy'. S. grandis strain Sa g1 was isolated from decaying crab carapace in France and was selected for genome sequencing because of its isolated location in the tree of life. Only one type strain genome has been published so far from the Saprospiraceae, while the sequence of strain Sa g1 represents the second genome to be published from a non-type strain of S. grandis. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,495,250 bp long Improved-High-Quality draft of the genome with its 3,536 protein-coding and 62 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Tsukamurella paurometabola type strain (no. 33)

Tsukamurella paurometabola corrig. (Steinhaus 1941) Collins et al. 1988 is the type species of the genus Tsukamurella, which is the type genus to the family Tsukamurellaceae. The species is not only of interest because of its isolated phylogenetic location, but also because it is a human opportunistic pathogen with some strains of the species reported to cause lung infection, lethal meningitis, and necrotizing tenosynovitis. This is the first completed genome sequence of a member of the genus Tsukamurella and the first genome sequence of a member of the family Tsukamurellaceae. The 4,479,724 bp long genome contains a 99,806 bp long plasmid and a total of 4,335 protein-coding and 56 RNA genes, and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41 °C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH).It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.     

Multimembrane dot-blotting: a cost-effective tool for proteome analysis

The molecular profiles of protein expression from hundreds of cell lysates can be determined in a high-throughput manner by using fluorescent bead technologies, enzyme-linked immunosorbent assays (ELISAs), and protein microarrays. Although powerful, these tools are costly and technically challenging and thus have limited accessibility for many research groups. We propose a modification of traditional dot blotting that increases throughput of this approach and provides a simple and cost-effective technique for profiling multiple samples. In contrast to traditional blotting that uses a single membrane, we introduce blotting onto a stack of novel, thin, sieve-like membranes. These membranes have a high affinity for binding proteins, but have a lower capacity of protein binding compared to traditional (nitrocellulose) membranes. We compare the linear binding capacity and variability of these novel membranes with nitrocellulose membranes. Also, we describe the use of these membranes in a multilayer dot blot format for profiling mitogen-mediated signal transduction pathways in T cells.     

Dynamic lateral organization of opioid receptors (kappa,muwt and muN40D) in the plasma membrane at the nanoscale level

Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOP_wt), and its naturally occurring isoform (MOP_N40D) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble‐averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside‐enriched domains and partial association with cholesterol‐enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor‐specific. KOP‐containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOP_N40D. Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOP_wt, whereas this effect was not observed for MOP_N40D.      

Recruitment of cellular clathrin to viral factories and disruption of clathrin-dependent trafficking

The viral factories of mammalian reovirus (MRV) are cytoplasmic structures that serve as sites of viral genome replication and particle assembly. A 721-aa MRV non-structural protein, μNS, forms the factory matrix and recruits other viral proteins to these structures. In this report, we show that μNS contains a conserved C-proximal sequence (711-LIDFS-715) that is similar to known clathrin-box motifs and is required for recruitment of clathrin to viral factories. Clathrin recruitment by μNS occurs independently of infecting MRV particles or other MRV proteins. Ala substitution for a single Leu residue (mutation L711A) within the putative clathrin-binding motif of μNS inhibits clathrin recruitment, but does not prevent formation or expansion of viral factories. Notably, clathrin-dependent cellular functions, including both endocytosis and secretion, are disrupted in cells infected with MRV expressing wild-type, but not L711A, μNS. These results identify μNS as a novel adaptor-like protein that recruits cellular clathrin to viral factories, disrupting normal functions of clathrin in cellular membrane trafficking. To our knowledge, this is the only viral or bacterial protein yet shown to interfere with clathrin functions in this manner. The results additionally establish a new approach for studies of clathrin functions, based on μNS-mediated sequestration.     

WHO/KFDA joint workshop on implementing WHO guidelines on evaluating similar biotherapeutic products, Seoul, Republic of Korea 24-26 August, 2010

In August 2010, the World Health Organization and the Korea Food & Drug Administration jointly organized the first implementation workshop of WHO guidelines on evaluating similar biotherapeutic products (SBPs) at the global level. The objective of the Workshop was to facilitate implementation of the newly adopted WHO Guidelines into the practice of national regulatory authorities (NRAs). WHO Guidelines were recognized by the workshop participants as a tool for harmonizing regulatory requirements worldwide. By reviewing and practicing several case studies, better understanding and consensus on the principles of clinical trial designs were reached. However, variations in terms of the national requirements for quality, safety and efficacy of these products revealed diversity in the regulatory expectations in different countries and regions. In addition, lack of terminology for the products developed as copy products (so called "me too" products) with a partial comparability to an RBP, led to a great diversity in evaluating as well as naming these products. The workshop participants proposed the following actions: a) NRAs should make efforts to build their capacities for regulation of SBPs; b) WHO should revise WHO Guidelines for assuring the quality of products prepared by recombinant DNA technology (WHO TRS 814) and continue monitoring progress with the implementation of the Guidelines on evaluating SBPs. Publication of the outcome of the Workshop was recognized as another action that WHO should coordinate.