The RNA-binding protein Rrm4 is essential for efficient secretion of endochitinase Cts1

Long-distance transport of mRNAs is crucial in determining spatio-temporal gene expression in eukaryotes. The RNA-binding protein Rrm4 constitutes a key component of microtubule-dependent mRNA transport in filaments of Ustilago maydis. Although a number of potential target mRNAs could be identified, cellular processes that depend on Rrm4-mediated transport remain largely unknown. Here, we used differential proteomics to show that ribosomal, mitochondrial, and cell wall-remodeling proteins, including the bacterial-type endochitinase Cts1, are differentially regulated in rrm4Δ filaments. In vivo UV crosslinking and immunoprecipitation and fluorescence in situ hybridization revealed that cts1 mRNA represents a direct target of Rrm4. Filaments of cts1Δ mutants aggregate in liquid culture suggesting an altered cell surface. In wild type cells Cts1 localizes predominantly at the growth cone, whereas it accumulates at both poles in rrm4Δ filaments. The endochitinase is secreted and associates most likely with the cell wall of filaments. Secretion is drastically impaired in filaments lacking Rrm4 or conventional kinesin Kin1 as well as in filaments with disrupted microtubules. Thus, Rrm4-mediated mRNA transport appears to be essential for efficient export of active Cts1, uncovering a novel molecular link between mRNA transport and the mechanism of secretion.     

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors

BACKGROUND: Diffuse anaplastic Wilms tumor (DAWT) is a rare, high-risk subtype that is often missed on diagnostic needle biopsy. Somatic mutations in TP53 are associated with the development of anaplasia and with poorer survival, particularly in advanced-stage disease. Early identification of DAWT harboring TP53 abnormalities could improve risk stratification of initial therapy and monitoring for recurrence. METHODS: Droplet digital polymerase chain reaction (ddPCR) was used to evaluate 21 samples from 4 patients with DAWT. For each patient, we assessed TP53 status in frozen tumor, matched germline DNA, and circulating tumor DNA (ctDNA) from plasma, serum, and urine collected throughout treatment. RESULTS: Mutant TP53 was detectable in ctDNA from plasma and serum in all patients. We did not detect variant TP53 in the same volume (200 μl) of urine. One patient displayed heterogeneity of TP53 in the tumor despite both histological sections displaying anaplasia. Concentration of ctDNA from plasma/serum taken prenephrectomy varied significantly between patients, ranging from 0.44 (0.05-0.90) to 125.25 (109.75-140.25) copies/μl. We observed variation in ctDNA throughout treatment, and in all but one patient, ctDNA levels fell significantly following nephrectomy. CONCLUSION: We demonstrate for the first time that ddPCR is an effective method for detection of mutant TP53 in ctDNA from children with DAWT even when there is intratumoral somatic heterogeneity. This should be further explored in a larger cohort of patients, as early detection of circulating variant TP53 may have significant clinical impact on future risk stratification and surveillance.     

Cubic exact solutions for the estimation of pairwise haplotype frequencies: implications for linkage disequilibrium analyses and a web tool 'CubeX'

Background  

The frequency of a haplotype comprising one allele at each of two loci can be expressed as a cubic equation (the 'Hill equation'), the solution of which gives that frequency. Most haplotype and linkage disequilibrium analysis programs use iteration-based algorithms which substitute an estimate of haplotype frequency into the equation, producing a new estimate which is repeatedly fed back into the equation until the values converge to a maximum likelihood estimate (expectation-maximisation).     

Assessing the impact of peat erosion on growing season CO2 fluxes by comparing erosional peat pans and surrounding vegetated haggs

Wetlands Ecology and Management - Peatlands are recognised as an important but vulnerable ecological resource. Understanding the effects of existing damage, in this case erosion, enables more...     

Novel M4 positive allosteric modulators derived from questioning the role and impact of a presumed intramolecular hydrogen-bonding motif in β-amino carboxamide-harboring ligands

This letter describes a focused exercise to explore the role of the β-amino carboxamide moiety found in all of the first generation M₄ PAMs and question if the NH₂ group served solely to stabilize an intramolecular hydrogen bond (IMHB) and enforce planarity. To address this issue (and to potentially find a substitute for the β-amino carboxamide that engendered P-gp and contributed to solubility liabilities), we removed the NH₂, generating des-amino congeners and surveyed other functional groups in the β-position. These modifications led to weak M₄ PAMs with poor DMPK properties. Cyclization of the β-amino carboxamide moiety by virtue of a pyrazole ring re-enforced the IMHB, led to potent (and patented) M₄ PAMs, many as potent as the classical bicyclic β-amino carboxamide analogs, but with significant CYP1A2 inhibition. Overall, this exercise indicated that the β-amino carboxamide moiety most likely facilitates an IMHB, and is essential for M₄ PAM activity within classical bicyclic M₄ PAM scaffolds.     

The interaction of platelet factor 4 with heparins of different chain length

The interaction of platelet factor 4 with heparin of varying chain lengths has been investigated by labelling the heparin with fluorescein isothiocyanate and monitoring the change in anisotropy of fluorescence when the protein is added to a solution of the polysaccharide. The shape of the titration curve depends on the Mr of the heparin and chains of Mr greater than 10 000 showed a definite break when the concentration of polysaccharide and protein became equimolar. Evidence is presented to show that most of the fluorescein label is linked to residual serines on the heparin. Similar break-points were observed if total fluorescence or light-scattering was used to monitor the interaction. Unlabelled heparin was used for the latter method. These results together with those obtained in buffer of high ionic strength lead us to propose a model where the heparin is wrapped around the tetrameric protein.     

Low-resolution structure of the complex of human blood platelet factor 4 with heparin determined by small-angle neutron scattering

Small-angle neutron scattering was used to confirm that human platelet factor 4 was a compact tetrameric globular protein of radius of gyration 1.74 nm and indistinguishable from a sphere. The same technique, when applied to the 1:1 mol/mol complex of platelet factor and heparin of Mr 14000, revealed that the radius of gyration of the particle varied, depending on the relative proportion of 2H2O to H2O in the solvent. Analysis of this variation by the method of Ibel and Stuhrmann (Ibel, K. and Stuhrmann, H.B. (1975) J. Mol. Biol. 93, 255-266) revealed that in the complex the material of greatest neutron-scattering length (the highly sulphated polysaccharide heparin) was furthest from the centre of the particle. This confirms the postulate of Luscombe and Holbrook (Luscombe, M. and Holbrook, J.J. (1983) in Glycoconjugates (Chester, A.M., Heineg?rd, D., Lundblad, A. and Svensson, S., eds.), pp. 818-819, Secretariat, Lund) that the exact 1:1 mole ratio of heparin (Mr greater than 10 000) to platelet factor in this stable complex arises from the heparin winding around the outside of a globular protein core.     

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α³²P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.     

Revealing pancrustacean relationships: Phylogenetic analysis of ribosomal protein genes places Collembola (springtails) in a monophyletic Hexapoda and reinforces the discrepancy between mitochondrial and nuclear DNA markers

Background  

In recent years, several new hypotheses on phylogenetic relations among arthropods have been proposed on the basis of DNA sequences. One of the challenged hypotheses is the monophyly of hexapods. This discussion originated from analyses based on mitochondrial DNA datasets that, due to an unusual positioning of Collembola, suggested that the hexapod body plan evolved at least twice. Here, we re-evaluate the position of Collembola using ribosomal protein gene sequences.