Purified Argonaute2 and an siRNA form recombinant human RISC

Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5' phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.     

Wolf Lethal Control and Livestock Depredations: Counter-Evidence from Respecified Models

We replicated the study conducted by Wielgus and Peebles (2014) on the effect of wolf mortality on livestock depredations in Montana, Wyoming and Idaho states in the US. Their best models were found to be misspecified due to the omission of the time index and incorrect functional form. When we respecified the models, this replication failed to confirm the magnitude, direction and often the very existence of the original results. Wielgus and Peebles (2014) reported that the increase in the number of wolves culled the previous year would increase the expected number of livestock killed this year by 4 to 6%. But our results showed that the culling of one wolf the previous year would decrease the expected number of cattle killed this year by 1.9%, and the expected number of sheep killed by 3.4%. However, for every wolf killed there is a corresponding 2.2% increase in the expected number of sheep killed in the same year. The increase in sheep depredation appears to be a short term phenomenon.     

ISHAN: sequence homology analysis package

Sequence based homology studies play an important role in evolutionary tracing and classification of proteins. Various methods are available to analyze biological sequence information. However, with the advent of proteomics era, there is a growing demand for analysis of huge amount of biological sequence information, and it has become necessary to have programs that would provide speedy analysis. ISHAN has been developed as a homology analysis package, built on various sequence analysis tools viz FASTA, ALIGN, CLUSTALW, PHYLIP and CODONW (for DNA sequences). This JAVA application offers the user choice of analysis tools. For testing, ISHAN was applied to perform phylogenetic analysis for sets of Caspase 3 DNA sequences and NF-kappaB p105 amino acid sequences. By integrating several tools it has made analysis much faster and reduced manual intervention.     

ALLOPHYCOCYANIN FROM A LOCAL ISOLATE GEITLERINEMA SP. A28DM (CYANOBACTERIA): A SIMPLE AND EFFICIENT PURIFICATION PROCESS1

Allophycocyanin (APC) is the least‐studied cyanobacterial bile‐pigment invariably present within the phycobilisome core of cyanobacteria. In the present study, we describe a simple, cost‐effective, and reproducible method for the purification of APC from a local isolate, Geitlerinema sp. A28DM. The pigment was extracted from the algal biomass and precipitated with 0.25% aqueous solution of the highly aromatic cationic dye “ethodin.” The precipitated APC was then subjected to a single size‐exclusion chromatographic step using Sephadex G‐100. Pure cyanobacterial APC (C‐APC) (A₆₅₂/A₂₈₀ of 3.2) was obtained and characterized by its absorption spectrum with maximum at 652 nm and a shoulder at 620 nm, and by SDS‐PAGE, showing two bands with molecular masses of 15 and 17.5 kDa, corresponding to α and β subunits of the biliprotein. The final yield of C‐APC was 66% from its content in the crude extract. The procedure appears to be promising for wider applications and larger production of APC.     

Structural and functional conservation profiles of novel cathepsin L-like proteins identified in the Drosophila melanogaster genome

Cathepsin L is a cysteine protease which degrades connective tissue proteins including collagen, elastin, and fibronectin. In this study, five well-characterized cathepsin L proteins from different arthropods were used as query sequences for the Drosophila genome database. The search yielded 10 cathepsin L-like sequences, of which eight putatively represent novel cathepsin L-like proteins. To understand the phylogenetic relationship among these cathepsin L-like proteins, a phylogenetic tree was constructed based on their sequences. In addition, models of the tertiary structures of cathepsin L were constructed using homology modeling methods and subjected to molecular dynamics simulations to obtain reasonable structure to understand its dynamical behavior. Our findings demonstrate that all of the potential Drosophila cathepsin L-like proteins contain at least one cathepsin propeptide inhibitor domain. Multiple sequence alignment and homology models clearly highlight the conservation of active site residues, disulfide bonds, and amino acid residues critical for inhibitor binding. Furthermore, comparative modeling indicates that the sequence/structure/function profiles and active site architectures are conserved.     

Microfluidic cell culture models for tissue engineering

Microfluidic systems have emerged as revolutionary new platform technologies for a range of applications, from consumer products such as inkjet printer cartridges to lab-on-a-chip diagnostic systems. Recent developments have opened the door to a new set of opportunities for microfluidic systems, in the field of tissue and organ engineering. Advances in the design of physiologically relevant structures and networks, fabrication processes for biomaterials suitable for in vivo use, and techniques for scaling towards large, three-dimensional constructs, are converging towards therapeutic applications of microfluidic technologies in engineering complex tissues and organs. These advances herald a new generation of microfluidics-based approaches designed for specific tissue and organ applications, incorporating microvascular networks, structures for transport and filtration, and a three-dimensional microenvironment suitable for supporting phenotypic cell behavior, tissue function, and implantation and host integration.