Performance of a novel Viresolve NFR virus filter

Mammalian cell-expressed therapeutic proteins are particularly vulnerable to contamination by endogenous retrovirus-like particles (RVLPs). The Viresolve NFR filter was designed to meet the critical requirement of manufacturing a safe and virus-free therapeutic by retaining RVLPs by a minimum of six log reduction value (LRV). The NFR designation refers to retrovirus removal in a normal flow format. To qualify the product, we tested two model viruses: the 78 nm diameter phi6 bacteriophage and the 80-110 nm diameter Xenotropic Murine Leukemia Virus (X-MuLV). Robust retention was demonstrated over a wide range of process parameters. Viresolve NFR filters also retain other model adventitious viruses including 70-85 nm diameter Reovirus 3 (Reo3), 70-90 nm diameter Adenovirus 2 (Ad2), and 53 nm diameter PR772 by >6 LRV. In addition to these model viruses, the filter retains >7 LRV of both the mycoplasma Acholeplasma laidlawii and the bacterium Brevundimonas diminuta. Protein passage is shown to be consistently high (95-100%) for a variety of therapeutic protein products, including monoclonal antibodies. Characterization of the filter in specific applications is made simple by availability of ultralow surface area (5 cm(2)) disks, which are shown to scale linearly to the manufacturing scale pleated-filters. Viresolve NFR filters provide consistent water permeability performance (34-37 LMH/psi) and show very little plugging for all feedstocks evaluated. The Viresolve NFR filter incorporates Retropore, a unique asymmetric polyethersulfone membrane, the surface of which has been modified to minimize protein binding.     

Plasmodium gallinaceum: transmission-blocking immunity in chickens. I. Comparative immunogenicity of gametocyte- and gamete-containing preparations.

Immunization of chickens by intravenous inoculation of preparations derived from blood infected with Plasmodium galinaceum led to reduced infectivity to mosquitoes (Aedes aegypti) during subsequently induced blood infection but had little effect on the course of asexual parasitemia. Immunization with preparations containing intracellular gametocytes was much less effective than immunization with preparations in which the extracellular gametes had been released during gametogenesis (emergence and exflagellation) prior to inoculation. By far the most effective material were preparations of partially purified gametes of both sexes. Three weekly inoculations of this material resulted in 99.99% suppression of infectivity to mosquitoes during subsequently induced blood infection. Preparations of purified gamete material from which gametes of one or another sex were absent were considerably less effective as transmission-blocking immunogens than the mixed gamete preparation. It is possible that the two sexes of gamete act synergistically to induce transmission-blocking immunity.     

Proteomic analysis of germinating urediniospores of Phakopsora pachyrhizi,causal agent of Asian soybean rust

Phakopsora pachyrhizi is an obligate pathogen that causes Asian soybean rust. Asian soybean rust has an unusually broad host range and infects by direct penetration through the leaf cuticle. In order to understand the early events in the infection process, it is important to identify and characterize proteins in P. pachyrhizi. Germination of the urediniospore is the first stage in the infection process and represents a critical life stage applicable to studies with this obligate pathogen. We have applied a 2‐DE and MS approach to identify 117 proteins from the National Center of Biotechnology Information nonredundant protein database and a custom database of Basidiomycota EST sequences. Proteins with roles in primary metabolism, energy transduction, stress, cellular regulation and signaling were identified in this study. This data set is accessible at http://world‐2dpage.expasy.org/repository/database=0018 .     

Nucleotide sequence and taxonomic value of the major outer membrane protein gene of Chlamydia pneumoniae IOL-207

Chlamydia pneumoniae IOL-207 genomic DNA was hybridized with a 1.5 kb labelled DNA probe containing the 3' region of the coding sequence for the major outer membrane protein (MOMP) of C. trachomatis serovar L1. An 8.5 kb Bg/II fragment containing the complete MOMP gene was cloned into lambda EMBL3. Two hybridizing EcoRI fragments were sub-cloned into the lambda ZAP II cloning vector and the resulting plasmids were used as templates for sequencing both strands of the C. pneumoniae MOMP gene. Computer taxonomic studies using the nucleotide and inferred amino acid sequence of the MOMP of C. pneumoniae IOL-207 and all known chlamydial MOMP sequences supported the designation of C. pneumoniae as a new species, but electron microscope studies suggested that the presence of pear-shaped elementary bodies (EBs) may not be a reliable taxonomic criterion.     

Resistance and Resistant Reaction of Gossypium arboreum to the Reniform, Nematode,Rotylenchulus reniformis

Gossypium arboreum ''Nanking CB 1402'' possessed a high level of resistance to Rotylenchulus reniformis. Within 16 h, the nematode penetrated roots of resistant and susceptible cottons equally. After 36 h, significantly fewer nematodes were found in resistant roots. Larvae fed in either an endodermal or pericyclic cell and had no specificity for root tissue of a particular age. In roots of resistant G. arboreum ''1402,'' wall breakdown of pericyclic cells was evident after 3 d, endodermal and cortical cells collapsed, and the hypertrophied pericyclic cells disintegrated within 12 d. Cell walls immediately adjacent to the nematode''s head were thickened and more safranin positive in resistant than in susceptible cotton cultivars. Several other cultivars of G. arboreum were also resistant to R. reniformis, based on nematode fecundity and percent egg reduction.     

An inbred 129SvEv GFPCre transgenic mouse that deletes loxP-flanked genes in all tissues

A common method for generating mice with subtle genetic manipulations uses homologous recombination (HR) in embryonic stem (ES) cells to replace a wild-type gene with a slightly modified one. Generally, a drug resistance gene is inserted with the modified gene to select correctly targeted clones. Often, however, the presence of this drug resistance gene interferes with the normal locus and creates a null or hypomorphic allele. Flanking of the selectable marker by loxP sites followed by Cre-mediated deletion after drug selection can overcome this problem. The simplest method used to remove a loxP-flanked selectable marker is to breed an animal carrying a loxP-flanked drug resistance gene to an animal that expresses Cre recombinase in the germline. To date only outbred transgenic mice are available for this purpose. This can be problematic for phenotypic analysis in many organ systems, including the brain, and for the analysis of behavior. While attempting to make 129S6/SvEvTac inbred background (isogenic to our ES cells) mice that express Cre under the control of several tissue-specific promoters, we serendipitously generated a line that excises loxP-flanked drug resistance genes in all tissues, including the germline. This reagent allows deletion of loxP-flanked sequences while maintaining the mutation on an inbred background.     

Living with myotonic dystrophy; what can be learned from couples? a qualitative study

Background  

Myotonic dystrophy type 1 (MD1) is one of the most prevalent neuromuscular diseases, yet very little is known about how MD1 affects the lives of couples and how they themselves manage individually and together. To better match health care to their problems, concerns and needs, it is important to understand their perspective of living with this hereditary, systemic disease.     

Assessment of the functionality of genome‐wide canine SNP arrays and implications for canine disease association studies

Domestic dogs share a wide range of important disease conditions with humans, including cancers, diabetes and epilepsy. Many of these conditions have similar or identical underlying pathologies to their human counterparts and thus dogs represent physiologically relevant natural models of human disorders. Comparative genomic approaches whereby disease genes can be identified in dog diseases and then mapped onto the human genome are now recognized as a valid method and are increasing in popularity. The majority of dog breeds have been created over the past few hundred years and, as a consequence, the dog genome is characterized by extensive linkage disequilibrium (LD), extending usually from hundreds of kilobases to several megabases within a breed, rather than tens of kilobases observed in the human genome. Genome‐wide canine SNP arrays have been developed, and increasing success of using these arrays to map disease loci in dogs is emerging. No equivalent of the human HapMap currently exists for different canine breeds, and the LD structure for such breeds is far less understood than for humans. This study is a dedicated large‐scale assessment of the functionalities (LD and SNP tagging performance) of canine genome‐wide SNP arrays in multiple domestic dog breeds. We have used genotype data from 18 breeds as well as wolves and coyotes genotyped by the Illumina 22K canine SNP array and Affymetrix 50K canine SNP array. As expected, high tagging performance was observed with most of the breeds using both Illumina and Affymetrix arrays when multi‐marker tagging was applied. In contrast, however, large differences in population structure, LD coverage and pairwise tagging performance were found between breeds, suggesting that study designs should be carefully assessed for individual breeds before undertaking genome‐wide association studies (GWAS).     

Replacement of fish oil with thraustochytrid Schizochytrium sp. L oil in Atlantic salmon parr (Salmo salar L) diets

Replacing fish oil with that from a docosahexaenoic acid (22:6omega3, DHA) rich single cell micro-organism, thraustochytrid Schizochytrium sp. L, in diets for Atlantic salmon (Salmo salar) was investigated. Four experimental diets containing 100% thraustochytrid oil (TO), 100% palm oil (PO) and a 4:1 palm and thraustochytrid oil mixture (MX) were compared to a fish oil (FO) diet over 9 weeks. A saltwater transfer challenge occurred at the end of the trial for 14 days to test the diet treatments on the ability of salmon to smolt. There were no significant differences in the feed consumption of the diets or the digestibility of the omega3 or omega6 PUFA, indicating no differences in the digestibility of fatty acids between diets. No significant differences were noted between the growth of fish on the four diet treatments. Significant differences were noted in the fatty acid profiles of the fish muscle tissues between all diets. Fish on the TO diet had a significantly greater percentage of DHA in muscle tissue compared with fish on all other diets. Blood osmolarity, which is inversely related to the ability of salmon to smolt, from the TO and FO fed fish was significantly lower than that of fish on the PO diet. This study showed that thraustochytrid oil can be used to replace fish oil in Atlantic salmon diets without detriment to the growth of parr. Including thraustochytrid oil in fish diets significantly increases the amount of DHA in Atlantic salmon muscle and therefore is a candidate for use in oil blends for salmon diets. Thraustochytrid oil provides a renewable source of essential fatty acids, in particular DHA, for aquafeeds.