Identification of novel Cryptosporidium genotypes from the Czech Republic

Isolates of Cryptosporidium from the Czech Republic were characterized from a variety of different hosts using sequence and phylogenetic analysis of the 18S ribosomal DNA and the heat-shock (HSP-70) gene. Analysis expanded the host range of accepted species and identified several novel genotypes, including horse, Eurasian woodcock, rabbit, and cervid genotypes.     

A predictive aggregate transport model for microfiltration of combined macromolecular solutions and poly-disperse suspensions: testing model with transgenic goat milk

To meet the technical challenge of recovering human IgG fusion protein from transgenic whole goat milk at reasonable cost with high purity and yield, a predictive aggregate transport model for microfiltration has been developed (Baruah and Belfort, 2003). Here, to test the model's predictability of permeate flux and mass transport, a comprehensive series of experiments with varying wall shear rate, feed temperature, feed concentration, and module design are presented. A very good fit was obtained between the model predictions and measurements for a wide variety of experimental conditions. For microfiltration module design comparison, a linear hollow fiber module (representing current commercial technologies) gave lower permeation flux and higher yield than a helical hollow fiber module (representing the latest self-cleaning methodology). These results are easily explained with the model that is now being used to define operating conditions for maximizing performance. The procedure described by the model is generalizable and can be used to obtain optimal filtration performance for applications other than milk.     

A predictive aggregate transport model for microfiltration of combined macromolecular solutions and poly-disperse suspensions: model development

A methodology, called the aggregate transport model, is presented that can a priori predict both the pressure-independent permeation flux and yield of target species for the microfiltration of poly-disperse solutions. The model captures the phenomenon of critical shear rate. Beyond the critical shear rate (expressed as a ratio of shear rate to permeation flux), the transmission of proteins drops sharply as a result of cake classification. The widely reported benefits of operating at uniform transmembrane pressure and constant wall concentration follow from this method. The methodology is general in nature and can be used predictively to obtain an optimal balance between flux and yield of target species during the microfiltration of many commercial poly-disperse suspensions. In the accompanying paper we test this model for microfiltration of transgenic whole goat milk.     

Glioma inhibition by HGF/NK2, an antagonist of scatter factor/hepatocyte growth factor

Strategies that antagonize growth factor signaling are attractive candidates for the biological therapy of brain tumors. HGF/NK2 is a secreted truncated splicing variant and potential antagonist of scatter factor/hepatocyte growth factor (SF/HGF), a multifunctional cytokine involved in the malignant progression of solid tumors including glioblastoma. U87 human malignant glioma cells that express an autocrine SF/HGF stimulatory loop were transfected with the human HGF/NK2 cDNA and clonal cell lines that secrete high levels of HGF/NK2 protein (U87-NK2) were isolated. The effects of HGF/NK2 gene transfer on the U87 malignant phenotype were examined. HGF/NK2 gene transfer had no effect on 2-dimensional anchorage-dependent cell growth. In contrast, U87-NK2 cell lines were approximately 20-fold less clonogenic in soft agar and approximately 4-fold less migratory than control-transfected cell lines. Intracranial tumor xenografts derived from U87-NK2 cells grew much slower than controls. U87-NK2 tumors were approximately 50-fold smaller than controls at 21 days post-implantation and HGF/NK2 gene transfer resulted in a trend toward diminished tumorigenicity. This report shows that the predominant effect of transgenic HGF/NK2 overexpression by glioma cells that are autocrine for SF/HGF stimulation is to inhibit their malignant phenotype.     

Phylogenetic relationships of Cryptosporidium parasites based on the 70-kilodalton heat shock protein (HSP70) gene

We have characterized the nucleotide sequences of the 70-kDa heat shock protein (HSP70) genes of Cryptosporidium baileyi, C. felis, C. meleagridis, C. muris, C. serpentis, C. wrairi, and C. parvum from various animals. Results of the phylogenetic analysis revealed the presence of several genetically distinct species in the genus Cryptosporidium and eight distinct genotypes within the species C. parvum. Some of the latter may represent cryptic species. The phylogenetic tree constructed from these sequences is in agreement with our previous results based on the small-subunit rRNA genes of Cryptosporidium parasites. The Cryptosporidium species formed two major clades: isolates of C. muris and C. serpentis formed the first major group, while isolates of C. felis, C. meleagridis, C. wrairi, and eight genotypes of C. parvum formed the second major group. Sequence variations were also observed between C. muris isolates from ruminants and rodents. The HSP70 gene provides another useful locus for phylogenetic analysis of the genus Cryptosporidium.     

Cryptosporidium spp. in domestic dogs: the "dog" genotype

Genetic and phylogenetic characterization of Cryptosporidium isolates at two loci (18S rRNA gene and heat shock gene) from both Australian and United States dogs demonstrated that dog-derived Cryptosporidium isolates had a distinct genotype which is conserved across geographic areas. Phylogenetic analysis provided support for the idea that the "dog" genotype is, in fact, a valid species.     

Gene discovery using the maize genome database ZmDB

Zea mays DataBase (ZmDB) is a repository and analysis tool for sequence, expression and phenotype data of the major crop plant maize. The data accessible in ZmDB are mostly generated in a large collaborative project of maize gene discovery, sequencing and phenotypic analysis using a transposon tagging strategy and expressed sequence tag (EST) sequencing. ESTs constitute most of the current content. Database search tools, convenient links to external databases, and novel sequence analysis programs for spliced alignment are provided and together serve as an efficient protocol for gene discovery by sequence inspection. ZmDB can be accessed at http://zmdb. iastate.edu. ZmDB also provides web-based ordering of materials generated in the project, including EST and genomic DNA clones, seeds of mutant plants and microarrays of amplified EST and genomic DNA sequences.     

Molecular characterization of a Cryptosporidium isolate from a black bear

To further validate the observation of the existence of host-adapted strains of Cryptosporidium parvum, we genetically characterized an isolate of Cryptosporidium parasite from a black bear. Sequence analysis of the ribosomal RNA small subunit and the 70-kDa heat shock protein (HSP70) showed that this parasite represents a new genotype of C. parvum and is related to the C. parvum dog genotype. This finding is helpful for clarifying Cryptosporidium taxonomy.     

The importance of homologous recombination in the generation of large deletions in hybrid plasmids in Amycolatopsis mediterranei

The whole nucleotide sequence of pT3.2I, the smallest plasmid of the acidophilic bacterium Thiobacillus T3.2, has been determined. pT3.2I is 15,390 bp long with a 53.7% GC content. Different regions can be defined in it: one 2569-bp putative insertion sequence similar to other insertion sequences of some Agrobacterium Ti plasmids; and a longer sequence, which occurs in two almost identical copies, differing only in a 1-bp deletion (6406 and 6405 bp). Several open reading frames and some smaller sequences were found in this duplicated region: ORFA and ORFG, encoding a putative polyol dehydrogenase and a putative RepA replication protein, respectively, an 83-bp sequence which could code for an antisense RNA, and a 36-bp region highly homologous to ori sequences of ColE2- and ColE3-related plasmids. Another putative gene, ORFH, is only present in the longer copy of this region (it is deleted in the short copy) and might encode a 90-amino-acid polypeptide which could act as a second replication protein, RepB. Based on sequence comparisons, pT3. 2I can be related to plasmids in the pColE2-CA42 incB incompatibility group.     

Best practice BioBanking of human heart tissue

This review provides a guide to researchers who wish to establish a biobank. It also gives practical advice to investigators seeking access to samples of healthy or diseased human hearts. We begin with a brief history of the Sydney Heart Bank (SHB) from when it began in 1989, including the pivotal role played by the late Victor Chang. We discuss our standard operating procedures for tissue collection which include cryopreservation and the quality assurance needed to maintain the long-term molecular and cellular integrity of the samples. The SHB now contains about 16,000 heart samples derived from over 450 patients who underwent isotopic heart transplant procedures and from over 100 healthy organ donors. These enable us to provide samples from a wide range of categories of heart failure. So far, we have delivered heart samples to more than 50 laboratories over two decades, and we answer their most frequently asked questions. Other SHB services include the development of tissue microarrays (TMA). These enable end users to perform preliminary examinations of the expression and localisation of target molecules in diseased or aging donor hearts, all in a single section of the TMA. Finally, the processes involved in managing tissue requests from external users and logistics considerations for the shipment of human tissue are discussed in detail.

Electronic supplementary material

The online version of this article (doi:10.1007/s12551-015-0182-6) contains supplementary material, which is available to authorized users.