85 research outputs found
Novel Patient Cell-Based HTS Assay for Identification of Small Molecules for a Lysosomal Storage Disease
Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets
Expression of the 5T4 oncofoetal antigen in renal cell carcinoma: a potential target for T-cell-based immunotherapy
The 5T4 oncofoetal antigen is a heavily glycosylated cell surface protein found on human placental trophoblast and on diverse types of human cancer but is not expressed at significant levels on adult human tissues in health. It therefore satisfies the criteria for a tumour-associated antigen and is an ideal target for the immunotherapy of cancer. We report here that 5T4 is strongly expressed on the majority of renal cell carcinomas and therefore this population of patients is suitable for trials of 5T4-targeted therapies. In particular, we have shown that T cells from renal cell carcinoma patients can be genetically modified to kill 5T4 expressing renal cancer cell lines by introduction of a chimeric-signalling protein. This protein consists of a single chain antibody fragment capable of binding antigen directly at the cell surface and then activating the T cell by virtue of a CD3ζ-signalling domain. This is a powerful tool that bypasses a number of mechanisms that allow tumours to escape T-cell killing and can be readily scaled up for clinical use
Enzymatic degradation of granular potato starch by Microbacterium aurum strain B8.A
Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules
Human keratinocytes are vanilloid resistant
BACKGROUND: Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca(2+)-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. METHODS: To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. RESULTS: Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca(2+)-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1-50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca(2+)-cytotoxicity ([RTX]>15 microM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca(2+)-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. CONCLUSION: TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials
A phase II study of a 5T4 oncofoetal antigen tumour-targeted superantigen (ABR-214936) therapy in patients with advanced renal cell carcinoma
In a phase II study, 43 renal cell carcinoma patients were treated with individualised doses of ABR-214936; a fusion of a Fab recognising the antigen 5T4, and Staphylococcal enterotoxin A. Drug was given intravenously on 4 consecutive days, treatment was repeated 1 month later. Treatment was associated with moderate fever and nausea, but well tolerated. Of 40 evaluable patients, 28 had disease control at 2 months, and at 4 months, one patient showed partial response (PR) and 16 patients stable disease. Median survival, with minimum follow-up of 26 months was 19.7 months with 13 patients alive to date. Stratification by the Motzer's prognostic criteria highlights prolonged survival compared to published expectation. Patients receiving higher drug exposure had greater disease control and lived almost twice as long as expected, whereas the low-exposure patients survived as expected. Sustained interleukin-2 (IL-2) production after a repeated injection appears to be a biomarker for clinical effect, as the induced-IL-2 level on the day 2 of treatment correlated with survival. The high degree of disease control and the prolonged survival suggest that this treatment can be effective. These findings will be used in the trial design for the next generation of drug, with reduced antigenicity and toxicity
Molecular Dynamics of Mesophilic-Like Mutants of a Cold-Adapted Enzyme: Insights into Distal Effects Induced by the Mutations
Networks and clusters of intramolecular interactions, as well as their “communication” across the three-dimensional architecture have a prominent role in determining protein stability and function. Special attention has been dedicated to their role in thermal adaptation. In the present contribution, seven previously experimentally characterized mutants of a cold-adapted α-amylase, featuring mesophilic-like behavior, have been investigated by multiple molecular dynamics simulations, essential dynamics and analyses of correlated motions and electrostatic interactions. Our data elucidate the molecular mechanisms underlying the ability of single and multiple mutations to globally modulate dynamic properties of the cold-adapted α-amylase, including both local and complex unpredictable distal effects. Our investigation also shows, in agreement with the experimental data, that the conversion of the cold-adapted enzyme in a warm-adapted variant cannot be completely achieved by the introduction of few mutations, also providing the rationale behind these effects. Moreover, pivotal residues, which are likely to mediate the effects induced by the mutations, have been identified from our analyses, as well as a group of suitable candidates for protein engineering. In fact, a subset of residues here identified (as an isoleucine, or networks of mesophilic-like salt bridges in the proximity of the catalytic site) should be considered, in experimental studies, to get a more efficient modification of the features of the cold-adapted enzyme
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An integrated bioinformatics analysis reveals divergent evolutionary pattern of oil biosynthesis in high- and low-oil plants
Seed oils provide a renewable source of food, biofuel and industrial raw materials that is important for humans. Although many genes and pathways for acyl-lipid metabolism have been identified, little is known about whether there is a specific mechanism for high-oil content in high-oil plants. Based on the distinct differences in seed oil content between four high-oil dicots (20~50%) and three low-oil grasses (<3%), comparative genome, transcriptome and differential expression analyses were used to investigate this mechanism. Among 4,051 dicot-specific soybean genes identified from 252,443 genes in the seven species, 54 genes were shown to directly participate in acyl-lipid metabolism, and 93 genes were found to be associated with acyl-lipid metabolism. Among the 93 dicot-specific genes, 42 and 27 genes, including CBM20-like SBDs and GPT2, participate in carbohydrate degradation and transport, respectively. 40 genes highly up-regulated during seed oil rapid accumulation period are mainly involved in initial fatty acid synthesis, triacylglyceride assembly and oil-body formation, for example, ACCase, PP, DGAT1, PDAT1, OLEs and STEROs, which were also found to be differentially expressed between high- and low-oil soybean accessions. Phylogenetic analysis revealed distinct differences of oleosin in patterns of gene duplication and loss between high-oil dicots and low-oil grasses. In addition, seed-specific GmGRF5, ABI5 and GmTZF4 were predicted to be candidate regulators in seed oil accumulation. This study facilitates future research on lipid biosynthesis and potential genetic improvement of seed oil content
Long-term exposure to PGE2 causes homologous desensitization of receptor-mediated activation of protein kinase A
How Disorder-Specific are Depressive Attributions? A Comparison of Individuals with Depression, Post-Traumatic Stress Disorder and Healthy Controls
Diving into the vertical dimension of elasmobranch movement ecology
This is the final version. Available on open access from the American Association for the Advancement of Science via the DOI in this recordData and materials availability: Processed data and code used in the analysis are accessible from the Zenodo Repository: 10.5281/zenodo.6885455Knowledge of the three-dimensional movement patterns of elasmobranchs is vital to understand their ecological roles and exposure to anthropogenic pressures. To date, comparative studies among species at global scales have mostly focused on horizontal movements. Our study addresses the knowledge gap of vertical movements by compiling the first global synthesis of vertical habitat use by elasmobranchs from data obtained by deployment of 989 biotelemetry tags on 38 elasmobranch species. Elasmobranchs displayed high intra- and interspecific variability in vertical movement patterns. Substantial vertical overlap was observed for many epipelagic elasmobranchs, indicating an increased likelihood to display spatial overlap, biologically interact, and share similar risk to anthropogenic threats that vary on a vertical gradient. We highlight the critical next steps toward incorporating vertical movement into global management and monitoring strategies for elasmobranchs, emphasizing the need to address geographic and taxonomic biases in deployments and to concurrently consider both horizontal and vertical movements.Bertarelli FoundationResearch EnglandMoore FoundationPackard FoundationInstituto Politecnico NacionalDarwin InitiativeGeorgia AquariumRolex Awards for EnterpriseWhitley Fund for Natur
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