An Alternative Paper Based Tissue Washing Method for Mass Spectrometry Imaging: Localized Washing and Fragile Tissue Analysis

Surface treatment of biological tissue sections improves detection of peptides and proteins for mass spectrometry imaging. However, liquid surface treatments can result in diffusion of surface analytes and fragile tissue sections can be easily damaged by typical washing solvents. Here, we present a new surface washing procedure for mass spectrometry imaging. This procedure uses solvent wetted fiber-free paper to enable local washing of tissue sections for mass spectrometry imaging and tissue profiling experiments. In addition, the method allows fragile tissues that cannot be treated by conventional washing techniques to be analyzed by mass spectrometry imaging

Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV Core DII protein

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV

Mathematical modelling of polyamine metabolism in bloodstream-form trypanosoma brucei: An application to drug target identification

© 2013 Gu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis article has been made available through the Brunel Open Access Publishing Fund.We present the first computational kinetic model of polyamine metabolism in bloodstream-form Trypanosoma brucei, the causative agent of human African trypanosomiasis. We systematically extracted the polyamine pathway from the complete metabolic network while still maintaining the predictive capability of the pathway. The kinetic model is constructed on the basis of information gleaned from the experimental biology literature and defined as a set of ordinary differential equations. We applied Michaelis-Menten kinetics featuring regulatory factors to describe enzymatic activities that are well defined. Uncharacterised enzyme kinetics were approximated and justified with available physiological properties of the system. Optimisation-based dynamic simulations were performed to train the model with experimental data and inconsistent predictions prompted an iterative procedure of model refinement. Good agreement between simulation results and measured data reported in various experimental conditions shows that the model has good applicability in spite of there being gaps in the required data. With this kinetic model, the relative importance of the individual pathway enzymes was assessed. We observed that, at low-to-moderate levels of inhibition, enzymes catalysing reactions of de novo AdoMet (MAT) and ornithine production (OrnPt) have more efficient inhibitory effect on total trypanothione content in comparison to other enzymes in the pathway. In our model, prozyme and TSHSyn (the production catalyst of total trypanothione) were also found to exhibit potent control on total trypanothione content but only when they were strongly inhibited. Different chemotherapeutic strategies against T. brucei were investigated using this model and interruption of polyamine synthesis via joint inhibition of MAT or OrnPt together with other polyamine enzymes was identified as an optimal therapeutic strategy.The work was carried out under a PhD programme partly funded by Prof. Ray Welland, School of Computing Science, University of Glasgo

Prospects for the Improvement of Energy Performance in Agroindustry Using Phase Change Materials

This work was partially supported by the Fundação para a Ciência e Tecnologia, UIDB/00066/2020 (CTS – Center of Technology and Systems).The use of Phase Change Materials (PCMs), able to store latent heat, represents an opportunity to improve energy efficiency in the agroindustry by means of thermal energy storage. PCMs provide higher energy density then sensible heat storage mediums, thus paving the way to multiple applications, like supporting the integration of renewables or allowing for new storage architectures, decentralized and directly installed in the chain production equipment, creating e.g. the opportunity to recover and value low-grade operational heat sub-products. Such new and decentralized architecture, not currently applied in agroindustry, is proposed in this work. A chocolate tempering machine using an organic PCM is conceived and analyzed using ANSYS Fluent software for computational fluid dynamics simulations, comparing the main aspects in the storage capacity and discharging process with a conventional sensitive heat storage solution that uses water. PCMs allows improving the stored energy, keeping the chocolate in the working temperature after being tempered for more than four times longer than using only hot water. If the PCMs are charged by renewables, the self-consumption ratio can be improved while providing energy flexibility to the user.authorsversionpublishe

Expression of CXCL10 is associated with response to radiotherapy and overall survival in squamous cell carcinoma of the tongue

Five-year survival for patients with oral cancer has been disappointingly stable during the last decades, creating a demand for new biomarkers and treatment targets. Lately, much focus has been set on immunomodulation as a possible treatment or an adjuvant increasing sensitivity to conventional treatments. The objective of this study was to evaluate the prognostic importance of response to radiotherapy in tongue carcinoma patients as well as the expression of the CXC-chemokines in correlation to radiation response in the same group of tumours. Thirty-eight patients with tongue carcinoma that had received radiotherapy followed by surgery were included. The prognostic impact of pathological response to radiotherapy, N-status, T-stage, age and gender was evaluated using Cox's regression models, Kaplan-Meier survival curves and chi-square test. The expression of 23 CXC-chemokine ligands and their receptors were evaluated in all patients using microarray and qPCR and correlated with response to treatment using logistic regression. Pathological response to radiotherapy was independently associated to overall survival with a 2-year survival probability of 81 % for patients showing a complete pathological response, while patients with a non-complete response only had a probability of 42 % to survive for 2 years (p = 0.016). The expression of one CXC-chemokine, CXCL10, was significantly associated with response to radiotherapy and the group of patients with the highest CXCL10 expression responded, especially poorly (p = 0.01). CXCL10 is a potential marker for response to radiotherapy and overall survival in patients with squamous cell carcinoma of the tongue

A Small Genomic Region Containing Several Loci Required for Gastrulation in Drosophila

Genetic screens in Drosophila designed to search for loci involved in gastrulation have identified four regions of the genome that are required zygotically for the formation of the ventral furrow. For three of these, the genes responsible for the mutant phenotypes have been found. We now describe a genetic characterization of the fourth region, which encompasses the cytogenetic interval 24C3-25B, and the mapping of genes involved in gastrulation in this region. We have determined the precise breakpoints of several existing deficiencies and have generated new deficiencies. Our results show that the region contains at least three different loci associated with gastrulation effects. One maternal effect gene involved in ventral furrow formation maps at 24F but could not be identified. For a second maternal effect gene which is required for germ band extension, we identify a candidate gene, CG31660, which encodes a G protein coupled receptor. Finally, one gene acts zygotically in ventral furrow formation and we identify it as Traf4

Identifying and Characterizing Alternative Molecular Markers for the Symbiotic and Free-Living Dinoflagellate Genus Symbiodinium

Dinoflagellates in the genus Symbiodinium are best known as endosymbionts of corals and other invertebrate as well as protist hosts, but also exist free-living in coastal environments. Despite their importance in marine ecosystems, less than 10 loci have been used to explore phylogenetic relationships in this group, and only the multi-copy nuclear ribosomal Internal Transcribed Spacer (ITS) regions 1 and 2 have been used to characterize fine-scale genetic diversity within the nine clades (A–I) that comprise the genus. Here, we describe a three-step molecular approach focused on 1) identifying new candidate genes for phylogenetic analysis of Symbiodinium spp., 2) characterizing the phylogenetic relationship of these candidate genes from DNA samples spanning eight Symbiodinium clades (A–H), and 3) conducting in-depth phylogenetic analyses of candidate genes displaying genetic divergences equal or higher than those within the ITS-2 of Symbiodinium clade C. To this end, we used bioinformatics tools and reciprocal comparisons to identify homologous genes from 55,551 cDNA sequences representing two Symbiodinium and six additional dinoflagellate EST libraries. Of the 84 candidate genes identified, 7 Symbiodinium genes (elf2, coI, coIII, cob, calmodulin, rad24, and actin) were characterized by sequencing 23 DNA samples spanning eight Symbiodinium clades (A–H). Four genes displaying higher rates of genetic divergences than ITS-2 within clade C were selected for in-depth phylogenetic analyses, which revealed that calmodulin has limited taxonomic utility but that coI, rad24, and actin behave predictably with respect to Symbiodinium lineage C and are potential candidates as new markers for this group. The approach for targeting candidate genes described here can serve as a model for future studies aimed at identifying and testing new phylogenetically informative genes for taxa where transcriptomic and genomics data are available

Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism

Cyclin D1 and mammary carcinoma: new insights from transgenic mouse models

Cyclin D1 is one of the most commonly overexpressed oncogenes in breast cancer, with 45–50% of primary ductal carcinomas overexpressing this oncoprotein. Targeted deletion of the gene encoding cyclin D1 demonstrates an essential role in normal mammary gland development while transgenic studies provide evidence that cyclin D1 is a weak oncogene in mammary epithelium. In a recent exciting development, Yu et al. demonstrate that cyclin D1-deficient mice are resistant to mammary carcinomas induced by c-neu and v-Ha-ras, but not those induced by c-myc or Wnt-1. These findings define a pivotal role for cyclin D1 in a subset of mammary cancers in mice and imply a functional role for cyclin D1 overexpression in human breast cancer

Viral Load Levels Measured at Set-Point Have Risen Over the Last Decade of the HIV Epidemic in the Netherlands

HIV-1 RNA plasma concentration at viral set-point is associated not only with disease outcome but also with the transmission dynamics of HIV-1. We investigated whether plasma HIV-1 RNA concentration and CD4 cell count at viral set-point have changed over time in the HIV epidemic in the Netherlands.We selected 906 therapy-naïve patients with at least one plasma HIV-1 RNA concentration measured 9 to 27 months after estimated seroconversion. Changes in HIV-1 RNA and CD4 cell count at viral set-point over time were analysed using linear regression models. The ATHENA national observational cohort contributed all patients who seroconverted in or after 1996; the Amsterdam Cohort Studies (ACS) contributed seroconverters before 1996. The mean of the first HIV-1 RNA concentration measured 9-27 months after seroconversion was 4.30 log(10) copies/ml (95% CI 4.17-4.42) for seroconverters from 1984 through 1995 (n = 163); 4.27 (4.16-4.37) for seroconverters 1996-2002 (n = 232), and 4.59 (4.52-4.66) for seroconverters 2003-2007 (n = 511). Compared to patients seroconverting between 2003-2007, the adjusted mean HIV-1 RNA concentration at set-point was 0.28 log(10) copies/ml (95% CI 0.16-0.40; p<0.0001) and 0.26 (0.11-0.41; p = 0.0006) lower for those seroconverting between 1996-2002 and 1984-1995, respectively. Results were robust regardless of type of HIV-1 RNA assay, HIV-1 subtype, and interval between measurement and seroconversion. CD4 cell count at viral set-point declined over calendar time at approximately 5 cells/mm(3)/year.The HIV-1 RNA plasma concentration at viral set-point has increased over the last decade of the HIV epidemic in the Netherlands. This is accompanied by a decreasing CD4 cell count over the period 1984-2007 and may have implications for both the course of the HIV infection and the epidemic