AL-12182, a novel 11-oxa prostaglandin analog with topical ocular hypotensive activity in the monkey

A series of 11-oxa prostaglandin analogs was evaluated for FP receptor binding and activation. Several compounds having aryloxy-terminated lower chains were found to be potent agonists. Topical ocular dosing of AL-12182, the isopropyl ester prodrug of the potent agonist 13, lowered intraocular pressure in the monkey by 40% accompanied by minimal conjunctival hyperemia in the rabbit. AL-12182 was synthesized on multigram scale starting with D-sorbitol.     

Stomoxys calcitrans,mechanical vector of virulent Besnoitia besnoiti from chronically infected cattle to susceptible rabbit

Cattle besnoitiosis caused by Besnoitia besnoiti (Eucoccidiorida: Sarcocystidae) is a re‐emerging disease in Europe. Its mechanical transmission by biting flies has not been investigated since the 1960s. The aim of this study was to re‐examine the ability of Stomoxys calcitrans (Diptera: Muscidae) to transmit virulent B. besnoiti bradyzoites from chronically infected cows to susceptible rabbits. Three batches of 300 stable flies were allowed to take an interrupted bloodmeal on chronically infected cows, followed by an immediate bloodmeal on three rabbits (Group B). A control group of rabbits and a group exposed to the bites of non‐infected S. calcitrans were included in the study. Blood quantitative polymerase chain reaction (qPCR) analyses, and clinical, serological and haematological surveys were performed in the three groups over 152 days until the rabbits were killed. Quantitative PCR analyses and histological examinations were performed in 24 tissue samples per rabbit. Only one rabbit in Group B exhibited clinical signs of the acute phase of besnoitiosis (hyperthermia, weight loss, regenerative anaemia and transient positive qPCR in blood) and was seroconverted. Parasite DNA was detected in four tissue samples from this rabbit, but no cysts were observed on histological examination. These findings indicate that S. calcitrans may act as a mechanical vector of B. besnoiti more efficiently than was previously considered.     

Molecular Epidemiology of Influenza A(H1N1)pdm09 Viruses from Pakistan in 2009-2010

Background

In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses.

Methods/Results

The first case of human infection with A(H1N1)pdm09 in Pakistan was detected on 18^th June 2009. Since then, 262 laboratory-confirmed cases have been detected during various outbreaks with 29 deaths (as of 31^st August 2010). The peak of the epidemic was observed in December with over 51% of total respiratory cases positive for influenza. Representative isolates from Pakistan viruses were sequenced and analyzed antigenically. Sequence analysis of genes coding for surface glycoproteins HA and NA showed high degree of high levels of sequence identity with corresponding genes of regional viruses circulating South East Asia. All tested viruses were sensitive to Oseltamivir in the Neuraminidase Inhibition assays.

Conclusions

Influenza A(H1N1)pdm09 viruses from Pakistan form a homogenous group of viruses. Their HA genes belong to clade 7 and show antigenic profile similar to the vaccine strain A/California/07/2009. These isolates do not show any amino acid changes indicative of high pathogenicity and virulence. It is imperative to continue monitoring of these viruses for identification of potential variants of high virulence or drug resistance.     

Synthesis of chicken major histocompatibility complex class II oligomers using a baculovirus expression system

Chicken major histocompatibility complex (MHC) B21 and B19 haplotypes are associated with resistance and susceptibility to Marek's disease (MD), respectively. T-cell-mediated immune response is crucial in coordinating protection against Marek's disease virus (MDV) infection, but it has been difficult to identify and characterize antigen-specific T-cells. MHC class II tetramers and oligomers have been widely used for characterization of antigen-specific T-cells in the context of infectious and autoimmune diseases. Thus, the objective of this study was to synthesize chicken MHC class II oligomers of B21 and B19 haplotypes for the future identification of antigen-specific T-cells. To achieve this objective, full-length coding sequences of chicken MHC class II B21 and B19 molecules were amplified and the molecules were expressed as fusion proteins, carrying Fos and Jun leucine zipper (LZ), histidine-tag and biotin ligase recognition site sequences, using a baculovirus expression system. Recombinant MHC-II were loaded with self-peptides, which stabilized the heterodimer in SDS-PAGE and allowed the detection of these molecules in Western blots with a conformation-specific anti-chicken MHC class II antibody. Biotinylated MHC molecules were conjugated to streptavidin to form oligomers, which were resolved under the transmission electron microscope through immuno-gold labelling, thus confirming success of oligomerization. In conclusion, chicken MHC class II oligomers may be used in the future to study the antigen-specific CD4+ T-cell compartment.     

Structural basis of TRAPPIII‐mediated Rab1 activation

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α‐helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.     

Analysis of glioma cell platinum response by metacomparison of two-dimensional chromatographic proteome profiles

Successful clinical development of cancer treatments is aided by the development of molecular markers that allow the identification of patients likely to respond. In the case of broadly cytotoxic drugs, such as the multinuclear series of platinum chemotherapeutic agents that we are evaluating for the treatment of glioma, one route to marker identification is proteomic profiling. We are using the two-dimensional chromatography system, the ProteomeLab PF2D, to compare proteomic profiles of glioma cells in culture before and after drug treatment. The existing software tools allowed the rapid identification of peaks increased by treatment of a given drug as compared with control untreated cells. To compare across these pairs, we developed new software, called the MetaComparison Tool (MCT). The MCT uses the chromatographic characteristics of peaks as identifiers, an approach that was validated by mass spectrometry of two independent isolations of a peak, from cells that were treated with two different platinum compounds. The MCT made it possible to rapidly query whether a given peak responded to more than one treatment and so allowed the identification of peaks that were specific to a given drug. As a result, this analysis greatly reduced the list of peaks whose isolation and downstream analysis by mass spectrometry is warranted, accelerating the search for protein markers of response.