siRNA carrying an (E)-vinylphosphonate moiety at the 5' end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2

Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5'- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5'- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5'-(E)-vinylphosphonate (5'-E-VP) guide RNA at 2.5-A resolution. The structure demonstrates how the 5' binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5'-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5'-E-VP -modified siRNA to human Argonaute-2 in-vitro, as well as the enhanced silencing in the context of the trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA

Site-specific C-terminal and internal loop labeling of proteins using sortase-mediated reactions

Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.National Institutes of Health (U.S.) (Grant RO1 AI08787

Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.National Institutes of Health (U.S.) (grant R01 AI087879

Stochastic motion of test particle implies that G varies with time

The aim of this letter is to propose a new description to the time varying gravitational constant problem, which naturally implements the Dirac's large numbers hypothesis in a new proposed holographic scenario for the origin of gravity as an entropic force. We survey the effect of the Stochastic motion of the test particle in Verlinde's scenario for gravity\cite{Verlinde}. Firstly we show that we must get the equipartition values for $t\rightarrow\infty$ which leads to the usual Newtonian gravitational constant. Secondly,the stochastic (Brownian) essence of the motion of the test particle, modifies the Newton's 2'nd law. The direct result is that the Newtonian constant has been time dependence in resemblance as \cite{Running}.Comment: Accepted in International Journal of Theoretical Physic

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

Transpeptidation catalyzed by ​sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of ​sortase A on a solid support (Sepharose beads). Immobilization of ​sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca²⁺-independent variant of ​sortase A with increased catalytic activity. This heptamutant variant of ​sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized ​sortase A takes 1–2 d. Batch reactions take 3–12 h and flow reactions proceed at 0.5 ml h⁻¹, depending on the geometry of the reactor used.United States. National Institutes of Health (RO1 AI087879

The potential of marginal coastal nursery habitats for the conservation of a culturally important Caribbean marine species

Aim: Identifying the potential of marginal habitats for species conservation is of key importance when their core high-quality habitats are under substantial disturbances and threats. However, there is currently a knowledge gap on how useful marine marginal habitats may be for conserving endangered marine species. Here, we investigate the potential of groundwater-fed coastal areas for the conservation of the queen conch, an economically and culturally important marine gastropod. Location: The inlet of Xel-Ha, typical of groundwater-fed coastal areas widely distributed along the Yucatan Peninsula coast in Mexico and partially protected by a network of marine protected areas. Methods: We tracked 66 queen conchs (Lobatus gigas) using acoustic telemetry over a period of 3.5 years. We investigated for ontogenetic niche shift using a network analysis and by modelling their growth. Results: The queen conchs exhibited the same ontogenetic niche shift required to complete their life cycle in this marginal habitat as they do in offshore core habitats. A total of 33 individuals departed the inlet and migrated from shallow groundwater-affected nursery grounds to deeper marine habitats more suitable for breeding aggregation. Main conclusions: As the broad-scale movement behaviour of queen conch in this inlet is similar to that observed on the overfished core habitats, our findings suggest that groundwater-fed coastal areas should be included in conservation planning for an effective management of this species within a network of marine protected areas

In planta expression of human polyQ-expanded huntingtin fragment reveals mechanisms to prevent disease-related protein aggregation

In humans, aggregation of polyglutamine repeat (polyQ) proteins causes disorders such as Huntington’s disease. Although plants express hundreds of polyQ-containing proteins, no pathologies arising from polyQ aggregation have been reported. To investigate this phenomenon, we expressed an aggregation-prone fragment of human huntingtin (HTT) with an expanded polyQ stretch (Q69) in Arabidopsis thaliana plants. In contrast to animal models, we find that Arabidopsis sp. suppresses Q69 aggregation through chloroplast proteostasis. Inhibition of chloroplast proteostasis diminishes the capacity of plants to prevent cytosolic Q69 aggregation. Moreover, endogenous polyQ-containing proteins also aggregate on chloroplast dysfunction. We find tha