334 research outputs found

    Stable Isotope-Resolved Metabolomics Shows Metabolic Resistance to Anti-Cancer Selenite in 3D Spheroids Versus 2D Cell Cultures

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    Conventional two-dimensional (2D) cell cultures are grown on rigid plastic substrates with unrealistic concentration gradients of O2, nutrients, and treatment agents. More importantly, 2D cultures lack cell–cell and cell–extracellular matrix (ECM) interactions, which are critical for regulating cell behavior and functions. There are several three-dimensional (3D) cell culture systems such as Matrigel, hydrogels, micropatterned plates, and hanging drop that overcome these drawbacks but they suffer from technical challenges including long spheroid formation times, difficult handling for high throughput assays, and/or matrix contamination for metabolic studies. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles that enable spheroid formation and growth via magnetizing cells. M3DB spheroids have been shown to emulate tissue and tumor microenvironments while exhibiting higher resistance to toxic agents than their 2D counterparts. It is, however, unclear if and how such 3D systems impact cellular metabolic networks, which may determine altered toxic responses in cells. We employed a Stable Isotope-Resolved Metabolomics (SIRM) approach with 13C6-glucose as tracer to map central metabolic networks both in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells without or with an anti-cancer agent (sodium selenite). We found that the extent of 13C-label incorporation into metabolites of glycolysis, the Krebs cycle, the pentose phosphate pathway, and purine/pyrimidine nucleotide synthesis was largely comparable between 2D and M3DB culture systems for both cell lines. The exceptions were the reduced capacity for de novo synthesis of pyrimidine and sugar nucleotides in M3DB than 2D cultures of A549 and PANC1 cells as well as the presence of gluconeogenic activity in M3DB spheroids of PANC1 cells but not in the 2D counterpart. More strikingly, selenite induced much less perturbation of these pathways in the spheroids relative to the 2D counterparts in both cell lines, which is consistent with the corresponding lesser effects on morphology and growth. Thus, the increased resistance of cancer cell spheroids to selenite may be linked to the reduced capacity of selenite to perturb these metabolic pathways necessary for growth and survival

    Defects in the CAPN1 Gene Result in Alterations in Cerebellar Development and Cerebellar Ataxia in Mice and Humans

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    A CAPN1 missense mutation in Parson Russell Terrier dogs is associated with spinocerebellar ataxia. We now report that homozygous or heterozygous CAPN1-null mutations in humans result in cerebellar ataxia and limb spasticity in four independent pedigrees. Calpain-1 knockout (KO) mice also exhibit a mild form of ataxia due to abnormal cerebellar development, including enhanced neuronal apoptosis, decreased number of cerebellar granule cells, and altered synaptic transmission. Enhanced apoptosis is due to absence of calpain-1-mediated cleavage of PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), which results in inhibition of the Akt pro-survival pathway in developing granule cells. Injection of neonatal mice with the indirect Akt activator, bisperoxovanadium, or crossing calpain-1 KO mice with PHLPP1 KO mice prevented increased postnatal cerebellar granule cell apoptosis and restored granule cell density and motor coordination in adult mice. Thus, mutations in CAPN1 are an additional cause of ataxia in mammals, including humans

    Bi-allelic variants in RNF170 are associated with hereditary spastic paraplegia.

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    Alterations of Ca2+ homeostasis have been implicated in a wide range of neurodegenerative diseases. Ca2+ efflux from the endoplasmic reticulum into the cytoplasm is controlled by binding of inositol 1,4,5-trisphosphate to its receptor. Activated inositol 1,4,5-trisphosphate receptors are then rapidly degraded by the endoplasmic reticulum-associated degradation pathway. Mutations in genes encoding the neuronal isoform of the inositol 1,4,5-trisphosphate receptor (ITPR1) and genes involved in inositol 1,4,5-trisphosphate receptor degradation (ERLIN1, ERLIN2) are known to cause hereditary spastic paraplegia (HSP) and cerebellar ataxia. We provide evidence that mutations in the ubiquitin E3 ligase gene RNF170, which targets inositol 1,4,5-trisphosphate receptors for degradation, are the likely cause of autosomal recessive HSP in four unrelated families and functionally evaluate the consequences of mutations in patient fibroblasts, mutant SH-SY5Y cells and by gene knockdown in zebrafish. Our findings highlight inositol 1,4,5-trisphosphate signaling as a candidate key pathway for hereditary spastic paraplegias and cerebellar ataxias and thus prioritize this pathway for therapeutic interventions

    Clinical and Molecular Characterization of Ataxia with Oculomotor Apraxia Patients In Saudi Arabia

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    <p>Abstract</p> <p>Background</p> <p>Autosomal recessive ataxias represent a group of clinically overlapping disorders. These include ataxia with oculomotor apraxia type1 (AOA1), ataxia with oculomotor apraxia type 2 (AOA2) and ataxia-telangiectasia-like disease (ATLD). Patients are mainly characterized by cerebellar ataxia and oculomotor apraxia. Although these forms are not quite distinctive phenotypically, different genes have been linked to these disorders. Mutations in the <it>APTX </it>gene were reported in AOA1 patients, mutations in <it>SETX </it>gene were reported in patients with AOA2 and mutations in <it>MRE11 </it>were identified in ATLD patients. In the present study we describe in detail the clinical features and results of genetic analysis of 9 patients from 4 Saudi families with ataxia and oculomotor apraxia.</p> <p>Methods</p> <p>This study was conducted in the period between 2005-2010 to clinically and molecularly characterize patients with AOA phenotype. Comprehensive sequencing of all coding exons of previously reported genes related to this disorder (<it>APTX</it>, <it>SETX </it>and <it>MRE11</it>).</p> <p>Results</p> <p>A novel nonsense truncating mutation c.6859 C > T, R2287X in <it>SETX </it>gene was identified in patients from one family with AOA2. The previously reported missense mutation W210C in <it>MRE11 </it>gene was identified in two families with autosomal recessive ataxia and oculomotor apraxia.</p> <p>Conclusion</p> <p>Mutations in <it>APTX </it>, <it>SETX </it>and <it>MRE11 </it>are common in patients with autosomal recessive ataxia and oculomotor apraxia. The results of the comprehensive screening of these genes in 4 Saudi families identified mutations in <it>SETX </it>and <it>MRE11 </it>genes but failed to identify mutations in <it>APTX </it>gene.</p

    Mapping the internal recognition surface of an octanuclear coordination cage using guest libraries

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    Size and shape criteria for guest binding inside the cavity of an octanuclear cubic coordination cage in water have been established using a new fluorescence displacement assay to quantify guest binding. For aliphatic cyclic ketones of increasing size (from C5 to C11), there is a linear relationship between ΔG for guest binding and the guest’s surface area: the change in ΔG for binding is 0.3 kJ mol–1 Å–2, corresponding to 5 kJ mol–1 for each additional CH2 group in the guest, in good agreement with expectations based on hydrophobic desolvation. The highest association constant is K = 1.2 × 106 M–1 for cycloundecanone, whose volume is approximately 50% of the cavity volume; for larger C12 and C13 cyclic ketones, the association constant progressively decreases as the guests become too large. For a series of C10 aliphatic ketones differing in shape but not size, ΔG for guest binding showed no correlation with surface area. These guests are close to the volume limit of the cavity (cf. Rebek’s 55% rule), so the association constant is sensitive to shape complementarity, with small changes in guest structure resulting in large changes in binding affinity. The most flexible members of this series (linear aliphatic ketones) did not bind, whereas the more preorganized cyclic ketones all have association constants of 104–105 M–1. A crystal structure of the cage·cycloundecanone complex shows that the guest carbonyl oxygen is directed into a binding pocket defined by a convergent set of CH groups, which act as weak hydrogen-bond donors, and also shows close contacts between the exterior surface of the disc-shaped guest and the interior surface of the pseudospherical cage cavity despite the slight mismatch in shape

    Multilocus microsatellite analysis of European and African Candida glabrata isolates

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    This study aimed to elucidate the genetic relatedness and epidemiology of 127 clinical and environmental Candida glabrata isolates from Europe and Africa using multilocus microsatellite analysis. Each isolate was first identified using phenotypic and molecular methods and subsequently, six unlinked microsatellite loci were analyzed using automated fluorescent genotyping. Genetic relationships were estimated using the minimum-spanning tree (MStree) method. Microsatellite analyses revealed the existence of 47 different genotypes. The fungal population showed an irregular distribution owing to the over-representation of genetically different infectious haplotypes. The most common genotype was MG-9, which was frequently found in both European and African isolates. In conclusion, the data reported here emphasize the role of specific C. glabrata genotypes in human infections for at least some decades and highlight the widespread distribution of some isolates, which seem to be more able to cause disease than others.This research was supported in part by the EU Mare Nostrum (EUMN-III Call) program of the European Union, grant agreement number 2011-4050/001-EMA2. Dr Sanae Rharmitt was the recipient of a scholarship (10 months) signed within the EUMN program for PhD students (F.S. 1.04.11.01 UORI) under the supervision of Prof Orazio Romeo.info:eu-repo/semantics/publishedVersio
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