Isothermal amplification of long DNA fragments at low temperature by improved strand displacement amplification.

Strand displacement amplification (SDA) is an isothermal amplification technique wherein amplification of a nucleic acid is initiated by nicking enzyme activity at sites flanking the target. Diagnostic SDA is very fast but requires precise optimization and is limited to very short amplicons. Here we report an enhanced approach by addition of single-stranded DNA binding protein, crowding agents and dUTP to enable amplification of kilobase-length products at low temperatures.

A Meta-Analysis of Transcriptomics Reveals a Stage-Specific Transcriptional Response Shared Across Different Hosts.

is a genus containing obligate, intracellular endosymbionts with arthropod and nematode hosts. Numerous studies have identified differentially expressed transcripts in endosymbionts that potentially inform the biological interplay between these endosymbionts and their hosts, albeit with discordant results. Here, we re-analyze previously published RNA-Seq transcriptomics data sets using a single workflow consisting of the most up-to-date algorithms and techniques, with the aim of identifying trends or patterns in the pan- transcriptional response.

Computational and experimental analysis of the glycophosphatidylinositol-anchored proteome of the human parasitic nematode Brugia malayi.

Further characterization of essential systems in the parasitic filarial nematode Brugia malayi is needed to better understand its biology, its interaction with its hosts, and to identify critical components that can be exploited to develop novel treatments. The production of glycophosphatidylinositol-anchored proteins (GPI-APs) is essential for eukaryotic cellular and physiological function. In addition, GPI-APs perform many important roles for cells.

Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™.

Efficient transcriptomic sequencing of microbial mRNA derived from host-microbe associations is often compromised by the much lower relative abundance of microbial RNA in the mixed total RNA sample. One solution to this problem is to perform extensive sequencing until an acceptable level of transcriptome coverage is obtained. More cost-effective methods include use of prokaryotic and/or eukaryotic rRNA depletion strategies, sometimes in conjunction with depletion of polyadenylated eukaryotic mRNA.

Heme acquisition in the parasitic filarial nematode Brugia malayi.

Nematodes lack a heme biosynthetic pathway and must acquire heme from exogenous sources. Given the indispensable role of heme, this auxotrophy may be exploited to develop drugs that interfere with heme uptake in parasites. Although multiple heme-responsive genes (HRGs) have been characterized within the free-living nematode Caenorhabditis elegans, we have undertaken the first study of heme transport in Brugia malayi, a causative agent of lymphatic filariasis.

Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi.

A homologue of the ecdysone receptor has been identified and shown to be responsive to 20-hydroxyecdysone in Brugia malayi. However, the role of this master regulator of insect development has not been delineated in filarial nematodes. Gravid adult female B.

Tissue-specific transcriptomics and proteomics of a filarial nematode and its Wolbachia endosymbiont.

Background: Filarial nematodes cause debilitating human diseases. While treatable, recent evidence suggests drug resistance is developing, necessitating the development of novel targets and new treatment options. Although transcriptomic and proteomic studies around the nematode life cycle have greatly enhanced our knowledge, whole organism approaches have not provided spatial resolution of gene expression, which can be gained by examining individual tissues.

Exploring the Fe(III) binding sites of human serum transferrin with EPR at 275 GHz.

We report 275 GHz EPR spectra of human serum transferrin. At this high microwave frequency the zero-field splitting between the magnetic sublevels of the high-spin [Formula: see text] sites can be accurately determined. We find the zero-field splitting to be a sensitive probe of the structure of the transferrin iron-binding sites.

Concurrent transcriptional profiling of Dirofilaria immitis and its Wolbachia endosymbiont throughout the nematode life cycle reveals coordinated gene expression.

Background: Dirofilaria immitis, or canine heartworm, is a filarial nematode parasite that infects dogs and other mammals worldwide. Current disease control relies on regular administration of anthelmintic preventives, however, relatively poor compliance and evidence of developing drug resistance could warrant alternative measures against D. immitis and related human filarial infections be taken.

Wolbachia endosymbionts and human disease control.

Most human filarial nematode parasites and arthropods are hosts for a bacterial endosymbiont, Wolbachia. In filaria, Wolbachia are required for normal development, fertility and survival, whereas in arthropods, they are largely parasitic and can influence development and reproduction, but are generally not required for host survival. Due to their obligate nature in filarial parasites, Wolbachia have been a target for drug discovery initiatives using several approaches including diversity and focused library screening and genomic sequence analysis.

Human serum transferrin: is there a link among autism, high oxalate levels, and iron deficiency anemia?

It has been previously suggested that large amounts of oxalate in plasma could play a role in autism by binding to the bilobal iron transport protein transferrin (hTF), thereby interfering with iron metabolism by inhibiting the delivery of iron to cells. By examining the effect of the substitution of oxalate for the physiologically utilized synergistic carbonate anion in each lobe of hTF, we sought to provide a molecular basis for or against such a role. Our work clearly shows both qualitatively (6 M urea gels) and quantitatively (kinetic analysis by stopped-flow spectrofluorimetry) that the presence of oxalate in place of carbonate in each binding site of hTF does indeed greatly interfere with the removal of iron from each lobe (in the absence and presence of the specific hTF receptor).

Structure and dynamics of drug carriers and their interaction with cellular receptors: focus on serum transferrin.

Highly proliferative cells have a dramatically increased need for iron which results in the expression of an increased number of transferrin receptors (TFR). This insight makes the transferrin receptor on these cells an excellent candidate for targeted therapeutics. In this regard, it is critical to understand at a molecular level exactly how the TFR interacts with its ligand, hTF.

Transferrin-mediated cellular iron delivery.

Essential to iron homeostasis is the transport of iron by the bilobal protein human serum transferrin (hTF). Each lobe (N- and C-lobe) of hTF forms a deep cleft which binds a single Fe(3+). Iron-bearing hTF in the blood binds tightly to the specific transferrin receptor (TFR), a homodimeric transmembrane protein.