The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation.

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI +] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes

Validation and implementation of a custom 21-gene panel next-generation sequencing assay for myeloid neoplasms

Rapid and reliable mutational analysis of myeloid neoplasms is increasingly important for diagnostic, prognostic and therapeutic reasons. In this article we describe the development and validation of a custom next-generation sequencing (NGS) assay that reliably tests across a broad range of myeloid neoplasms, including AML, MDS, and myeloproliferative neoplasms. The lllumina TruSeq Custom Amplicon panel was designed to detect variants in 21 genes. The validation protocol included sequencing of cell lines (n=3) and patient samples (n=36) on an Illumina MiSeq platform. A read depth ≥100x was observed for &gt;97% of targeted bases. After filtering for artifacts, a specificity of 100% was obtained. A detection limit of ≤5% was observed for variants present in cell lines. On average two reportable variants were present in samples from patients with a myeloid neoplasm. In conclusion, the custom NGS assay provides an adequate routine assay for genetic analysis of variants present in myeloid neoplasms. Practical considerations on choice of targeted genes, type of assay and method of data analysis are provided in this report.</p

Validation and implementation of a custom 21-gene panel next-generation sequencing assay for myeloid neoplasms

Rapid and reliable mutational analysis of myeloid neoplasms is increasingly important for diagnostic, prognostic and therapeutic reasons. In this article we describe the development and validation of a custom next-generation sequencing (NGS) assay that reliably tests across a broad range of myeloid neoplasms, including AML, MDS, and myeloproliferative neoplasms. The lllumina TruSeq Custom Amplicon panel was designed to detect variants in 21 genes. The validation protocol included sequencing of cell lines (n=3) and patient samples (n=36) on an Illumina MiSeq platform. A read depth ≥100x was observed for &gt;97% of targeted bases. After filtering for artifacts, a specificity of 100% was obtained. A detection limit of ≤5% was observed for variants present in cell lines. On average two reportable variants were present in samples from patients with a myeloid neoplasm. In conclusion, the custom NGS assay provides an adequate routine assay for genetic analysis of variants present in myeloid neoplasms. Practical considerations on choice of targeted genes, type of assay and method of data analysis are provided in this report.</p

Genetic risk factors for clozapine-induced neutropenia and agranulocytosis in a Dutch psychiatric population

Personalised Therapeutic

A Novel Rho-Like Protein TbRHP Is Involved in Spindle Formation and Mitosis in Trypanosomes

Background: In animals and fungi Rho subfamily small GTPases are involved in signal transduction, cytoskeletal function and cellular proliferation. These organisms typically possess multiple Rho paralogues and numerous downstream effectors, consistent with the highly complex contributions of Rho proteins to cellular physiology. By contrast, trypanosomatids have a much simpler Rho-signaling system, and the Trypanosoma brucei genome contains only a single divergent Rho-related gene, TbRHP (Tb927.10.6240). Further, only a single RhoGAP-like protein (Tb09.160.4180) is annotated, contrasting with the.70 Rho GAP proteins from Homo sapiens. We wished to establish the function(s) of TbRHP and if Tb09.160.4180 is a potential GAP for this protein. Methods/Findings: TbRHP represents an evolutionarily restricted member of the Rho GTPase clade and is likely trypanosomatid restricted. TbRHP is expressed in both mammalian and insect dwelling stages of T. brucei and presents with a diffuse cytoplasmic location and is excluded from the nucleus. RNAi ablation of TbRHP results in major cell cycle defects and accumulation of multi-nucleated cells, coinciding with a loss of detectable mitotic spindles. Using yeast two hybrid analysis we find that TbRHP interacts with both Tb11.01.3180 (TbRACK), a homolog of Rho-kinase, and the sole trypanosome RhoGAP protein Tb09.160.4180, which is related to human OCRL. Conclusions: Despite minimization of the Rho pathway, TbRHP retains an important role in spindle formation, and henc

The Ordered Extension of Pseudopodia by Amoeboid Cells in the Absence of External Cues

Eukaryotic cells extend pseudopodia for movement. In the absence of external cues, cells move in random directions, but with a strong element of persistence that keeps them moving in the same direction Persistence allows cells to disperse over larger areas and is instrumental to enter new environments where spatial cues can lead the cell. Here we explore cell movement by analyzing the direction, size and timing of ∼2000 pseudopodia that are extended by Dictyostelium cells. The results show that pseudpopod are extended perpendicular to the surface curvature at the place where they emerge. The location of new pseudopods is not random but highly ordered. Two types of pseudopodia may be formed: frequent splitting of an existing pseudopod, or the occasional extension of a de novo pseudopod at regions devoid of recent pseudopod activity. Split-pseudopodia are extended at ∼60 degrees relative to the previous pseudopod, mostly as alternating Right/Left/Right steps leading to relatively straight zigzag runs. De novo pseudopodia are extended in nearly random directions thereby interrupting the zigzag runs. Persistence of cell movement is based on the ratio of split versus de novo pseudopodia. We identify PLA2 and cGMP signaling pathways that modulate this ratio of splitting and de novo pseudopodia, and thereby regulate the dispersal of cells. The observed ordered extension of pseudopodia in the absence of external cues provides a fundamental insight into the coordinated movement of cells, and might form the basis for movement that is directed by internal or external cues