Allele-Specific Suppression of Mutant Huntingtin Using Antisense Oligonucleotides: Providing a Therapeutic Option for All Huntington Disease Patients

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.     

PfPKB, a protein kinase B-like enzyme from Plasmodium falciparum: II. Identification of calcium/calmodulin as its upstream activator and dissection of a novel signaling pathway

Intracellular cell signaling cascades of protozoan parasite Plasmodium falciparum are not clearly understood. We have reported previously (Kumar, A., Vaid, A., Syin, C., and Sharma, P. (2004) J. Biol. Chem. 279, 24255-24264) the identification and characterization of a protein kinase B-like enzyme in P. falciparum (PfPKB). PfPKB lacks the phosphoinositide-interacting pleckstrin homology domain present in mammalian protein kinase B. Therefore, the mechanism of PfPKB regulation was expected to be different from that of the host and had remained unknown. We have identified calmodulin (CaM) as the regulator of PfPKB activity. A CaM binding domain was mapped in the N-terminal region of PfPKB. CaM, in a calcium-dependent manner, interacts with this domain and activates PfPKB. CaM associates with PfPKB in the parasite and regulates its activity. Furthermore phospholipase C acts as an upstream regulator of this cascade as it facilitates the release of calcium from intracellular stores. This is one of the first multicomponent signaling pathways to be dissected in the malaria parasite.     

Extracting absolute titanium-alkyl and-hydride bond enthalpies from relative D(TiR(H)) in (silox)2(Bu3SiNH)TiR: electronegativity and ECT models

Relative D(Ti-R)_rel have been measured previously for (silox)₂(^tBu₃SiNH)TiR (1-R). Pauling and Matcha equations of D(MR) that contain elements of electronegativity, D(MM), D(HH) and D(RH) were used to estimate absolute D(TiR) in 1 with minimal success. The electronegativity approaches need a variety of substituents with a wide spread in χ_R, hence examination of 1-H and 1-R are heavily and incorrectly weighted by D(TiH)-D(TiR). Absolute D(TiR) generated in this manner approach those of related systems only when χX_Ti = 2.9, a value shown to be irrational. Drago's ECT parameters were used in the hope that the three parameter fit would compensate for inadequacies in electronegativity-based methods. The limited set of alkyl parameters available, and a lack of distinguishing parameters that can significantly differentiate one alkyl from another, render this potentially useful method imprecise.     

Modified solvent partitioning scheme providing increased specificity and rapidity of immunoassay for indole-3-acetic acid

Based on the distribution constant of IAA, the efficiency of solvent partitioning has been improved by modifying the proportions of the solvents. IAA is recovered almost quantitatively by this method which also renders further sample reduction superfluous. Selective IAA recovery is supported by the distribution of immunoreactive materials on chromatograms. This modified scheme simplifies prepurification of samples for more reliable immunoassay.     

Patterns of Biofilm Succession on a Sheltered Rocky Shore in Hong Kong

Successional patterns are dependent on the nature of the substratum, water flow, concentrations of organics as well as the availability of bacteria, algal spores and invertebrate larvae in the coastal environment. Bacteria play an especially important role in biofilm formation as they are generally the earliest colonizers. In the present study, both winter and summer biofilm succession patterns were examined on glass coverslips inverted on experimental racks attached at two tidal levels on a sheltered shore in Hong Kong. In the succession, bacteria were followed by diatoms and cyanobacteria. Encrusting algae appeared in the late stages of the experiment (day 80 in summer and day 60 in winter). Colonization by bacteria was much slower in summer and their density remained low throughout the experimental period. The first appearance of diatoms and cyanobacteria, however, was more rapid in the summer. Bacteria and diatoms on the low-shore surfaces also had a faster succession rate than on the high-shore surfaces, suggesting that desiccation/aerial temperature are the causal factors for such differences.     

The polypeptide components of the parasporal fibres of Pasteuria penetrans

A variety of treatments were tested for their ability to solubilize the parasporal fibres from Pasteuria penetrans, a parasite of some plant–parasitic nematodes. Selective solubilization of the parasporal fibres resulted from some of the extraction procedures tested. Subsequent acrylamide gel electrophoresis and Western blotting of the resolved polypeptides, using polyclonal sera against the spores, disclosed up to 15 distinct bands, ranging in size from 12 to 195 kDa. An N-terminal amino acid sequence was obtained from a 50 kDa polypeptide and an oligonucleotide primer deduced from it. A whole cell, fluorescent, primed in situ labelling (PRINS) technique was adapted to be applicable to spores of P. penetrans and P. ramosa, a parasite of water fleas. Positive responses were obtained using the parasporal fibre primer on spores of the former but not of the latter organism, implying that this 50 kDa polypeptide is produced by P. penetrans but not by P. ramosa.     

Traffic to the malaria parasite food vacuole: a novel pathway involving a phosphatidylinositol 3-phosphate-binding protein

Phosphatidylinositol 3-phosphate (PI3P) is a key ligand for recruitment of endosomal regulatory proteins in higher eukaryotes. Subsets of these endosomal proteins possess a highly selective PI3P binding zinc finger motif belonging to the FYVE domain family. We have identified a single FYVE domain-containing protein in Plasmodium falciparum which we term FCP. Expression and mutagenesis studies demonstrate that key residues are involved in specific binding to PI3P. In contrast to FYVE proteins in other organisms, endogenous FCP localizes to a lysosomal compartment, the malaria parasite food vacuole (FV), rather than to cytoplasmic endocytic organelles. Transfections of deletion mutants further indicate that FCP is essential for trophozoite and FV maturation and that it traffics to the FV via a novel constitutive cytoplasmic to vacuole targeting pathway. This newly discovered pathway excludes the secretory pathway and is directed by a C-terminal 44-amino acid peptide domain. We conclude that an FYVE protein that might be expected to participate in vesicle targeting in the parasite cytosol instead has a vital and functional role in the malaria parasite FV.     

The role of dynamin 3 in the testis

We report here that dynamin 3 in the testis is associated with structures termed tubulobulbar complexes that internalize intact intercellular junctions during sperm release and turnover of the blood-testis barrier. The protein lies adjacent to an actin-Arp2/3 network that cuffs the double plasma membrane tubular invagination at the core of each complex. To explore the possible relationship between dynamin 3 and nectin-based adhesion junctions, we transiently transfected DsRed-tagged dynamin 3 into MDCK cells stably transfected with eGFP-tagged nectin 2, one of the adhesion molecules known to be expressed in Sertoli cells at adhesion junctions. Cells transfected with the dynamin 3 construct had less uniformly distributed nectin 2 at intercellular contacts when compared to control cells expressing only nectin 2 or transfected with the DsRed plasmid alone. Significantly, tubular extensions positive for nectin 2 were visible projecting into the cells from regions of intercellular contact. Our findings are consistent with the conclusion that dynamin 3 is involved with tubulobulbar morphogenesis. Dynamin 3 also occurs in concentrated deposits around the capitulum and striated columns in the connecting piece of sperm tails suggesting that the protein in these cells may function to stabilize the base of the tail or serve as a reservoir for use during or after fertilization.     

Pathology of Equine Influenza virus (H3N8) in Murine Model

Equine influenza viruses (EIV)—H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/c mouse were explored for suitability for conducting pathogenecity studies for EIV. The BALB/c mice were inoculated intranasally @ 2×10^6.24 EID₅₀ with EIV (H3N8) belonging to Clade 2 of Florida sublineage and monitored for setting up of infection and associated parameters. All mice inoculated with EIV exhibited clinical signs viz. loss in body weights, lethargy, dyspnea, etc, between 3 and 5 days which commensurate with lesions observed in the respiratory tract including rhinitis, tracheitis, bronchitis, bronchiolitis, alveolitis and diffuse interstitial pneumonia. Transmission electron microscopy, immunohistochemistry, virus quantification through titration and qRT-PCR demonstrated active viral infection in the upper and lower respiratory tract. Serology revealed rise in serum lactate dehydrogenase levels along with sero-conversion. The pattern of disease progression, pathological lesions and virus recovery from nasal washings and lungs in the present investigations in mice were comparable to natural and experimental EIV infection in equines. The findings establish BALB/c mice as small animal model for studying EIV (H3N8) infection and will have immense potential for dissecting viral pathogenesis, vaccine efficacy studies, preliminary screening of vaccine candidates and antiviral therapeutics against EIV.