The oral histone deacetylase inhibitor ITF2357 reduces cytokines and protects islet β cells in vivo and in vitro

In type 1 diabetes, inflammatory and immunocompetent cells enter the islet and produce proinflammatory cytokines such as interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); each contribute to β-cell destruction, mediated in part by nitric oxide. Inhibitors of histone deacetylases (HDAC) are used commonly in humans but also possess antiinflammatory and cytokine-suppressing properties. Here we show that oral administration of the HDAC inhibitor ITF2357 to mice normalized streptozotocin (STZ)-induced hyperglycemia at the clinically relevant doses of 1.25-2.5 mg/kg. Serum nitrite levels returned to nondiabetic values, islet function improved and glucose clearance increased from 14% (STZ) to 50% (STZ + ITF2357). In vitro, at 25 and 250 nmol/L, ITF2357 increased islet cell viability, enhanced insulin secretion, inhibited MIP-1α and MIP-2 release, reduced nitric oxide production and decreased apoptosis rates from 14.3% (vehicle) to 2.6% (ITF2357). Inducible nitric oxide synthase (iNOS) levels decreased in association with reduced islet-derived nitrite levels. In peritoneal macrophages and splenocytes, ITF2357 inhibited the production of nitrite, as well as that of TNFα and IFNγ at an IC(50) of 25-50 nmol/L. In the insulin-producing INS cells challenged with the combination of IL-1β plus IFNγ, apoptosis was reduced by 50% (P < 0.01). Thus at clinically relevant doses, the orally active HDAC inhibitor ITF2357 favors β-cell survival during inflammatory conditions.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Bacterial surface display library screening by target enzyme labeling: Identification of new human cathepsin G inhibitors

The aim of this study was to establish a new tool for screening surface displayed peptide libraries based on the idea that cells expressing an enzyme inhibitor at the surface can be specifically labeled by the target enzyme. For this purpose peptide P15, exhibiting a K(i) value of 0.25 microM toward human cathepsin G, was expressed on the Escherichia coli cell surface by the use of Autodisplay. Purified cathepsin G was coupled to biotin and incubated with cells expressing the inhibitor. After addition of streptavidin-fluorescein isothiocyanate, these cells could be clearly differentiated from control cells by whole-cell fluorescence using flow cytometer analysis. To determine whether this protocol can be used for the sorting of single cells, a mixed population of cells with and without inhibitor was treated accordingly. Single cells were selected by increased fluorescence and sorted using fluorescence-activated cell sorting (FACS). Single cell clones were obtained and subjected to DNA sequence analysis. It turned out that the bacteria selected by this protocol displayed the correct peptide inhibitor at the cell surface. The protocol was then used to screen random peptide libraries, expressed at the cell surface, and a new lead structure for human cathepsin G (IC50 = 11.7 microM) was identified. The new drug discovery tool presented here consists of three steps: (a) surface display of peptide libraries, (b) selection of single cells with inhibiting structures by using the inherent affinity of the target enzyme, and (c) sorting of single cells, which were labeled by FACS.     

Skeletal formation in the modern but ultraconservative chaetetid spongeSpirastrella (Acanthochaetetes) wellsi (demospongiae,porifera)

Summary The modern hadromerid coralline spongeSpirastrella (Acanthochaetetes) wellsi exhibits a unique secondary high-Mg calcite (>19 mol % MgCO₃) basal skeleton. The basal skeleton is constructed of bundles of elongated crystals more or less tangentially orientated. The initial formation of these crystals is controlled by soluble highly acidic aspartic and glutamic-rich (40%) macromolecules. The skeletal mineralization occurs in four different loci: in the top of the calicles, at the tabulae, on collagenous anchor fibres, and within closed spaces between the tabulae. The clicle walls are formed on the uppermost top of the basal skeleton as a continuous process. Based on long term stainings with Ca²⁺-chelating fluorochroms (calcein, chlorotetracyclines) the growth rate of this sponge is extremely low with ca. 50–100μm/a. The skeletal formation takes places outside the sponge, within a narrow zone (300–500 nm) between the basopinacoderm and the mature basal skeleton. The sponge produces thread-like folded templates (‘spaghetti fibres’) of 0,5–2 μm size, the shape controlling insoluble organic matrix. These templates become mineralized in a first step as MgCO₃, then are stretched. A soluble organic matrix is also secreted, and remains are included inside the mineralized skeleton. This organic matrix consists of in a complex mixture containing small very acidic proteins (5, 13, 31 KD; 40% Asp and Glu and therefore most probably Ca²⁺-binding) and high molecular weight glycoproteins among several other organic compounds. The mature crystals are high-Mg calcites. During calcification large cells with large reserve granules (LCG) are always present in a tight connection with the basopinacoderm. These cells form also the collagenous anchor fibres. Primary tabulae are formed by a non-collagenous organic sheet. Calcification happens only when LCG cells are enriched on the organic sheet. Randomly oriented high-Mg calcite crystals are growing on the collagenous anchor fibres. The same type of the mineralization is observed within the spaces of the tabulae. This particular case of mineralization is controlled by decaying sponge tissue (ammonification). The δ¹³C values are in equilibrium with the ambient sea water and vary between +3.2 and +2.8 ‰. The mode of mineralization of the basal skeleton can be described as biologically induced resp. matrix mediated.     

Single-Stranded Annealing Induced by Re-Initiation of Replication Origins Provides a Novel and Efficient Mechanism for Generating Copy Number Expansion via Non-Allelic Homologous Recombination

Copy number expansions such as amplifications and duplications contribute to human phenotypic variation, promote molecular diversification during evolution, and drive the initiation and/or progression of various cancers. The mechanisms underlying these copy number changes are still incompletely understood, however. We recently demonstrated that transient, limited re-replication from a single origin in Saccharomyces cerevisiae efficiently induces segmental amplification of the re-replicated region. Structural analyses of such re-replication induced gene amplifications (RRIGA) suggested that RRIGA could provide a new mechanism for generating copy number variation by non-allelic homologous recombination (NAHR). Here we elucidate this new mechanism and provide insight into why it is so efficient. We establish that sequence homology is both necessary and sufficient for repetitive elements to participate in RRIGA and show that their recombination occurs by a single-strand annealing (SSA) mechanism. We also find that re-replication forks are prone to breakage, accounting for the widespread DNA damage associated with deregulation of replication proteins. These breaks appear to stimulate NAHR between re-replicated repeat sequences flanking a re-initiating replication origin. Our results support a RRIGA model where the expansion of a re-replication bubble beyond flanking homologous sequences followed by breakage at both forks in trans provides an ideal structural context for SSA–mediated NAHR to form a head-to-tail duplication. Given the remarkable efficiency of RRIGA, we suggest it may be an unappreciated contributor to copy number expansions in both disease and evolution.