Insights Arising from Gene Expression Profiling in Amyotrophic Lateral Sclerosis

Public health & preventive medicin

Biotechnological Transformation of Hydrocortisone into 16α-Hydroxyprednisolone by Coupling Arthrobacter simplex and Streptomyces roseochromogenes

16α-Hydroxyprednisolone, an anti-inflammatory drug, could be potentially obtained from hydrocortisone bioconversion by combining a 1,2-dehydrogenation reaction performed by Arthrobacter simplexATCC31652 with a 16α-hydroxylation reaction by Streptomyces roseochromogenes ATCC13400. In this study we tested, for the first time, potential approaches to couple the two reactions using similar pH and temperature conditions for hydrocortisone bioconversion by the two strains. The A. simplex capability to 1,2-dehydrogenate the 16α-hydroxyhydrocortisone, the product of S. roseochromogenes transformation of hydrocortisone, and vice versa the capability of S. roseochromogenes to 16α-hydroxylate the prednisolone were assessed. Bioconversions were studied in shake flasks and strain morphology changes were observed by SEM. Whole cell experiments were set up to perform the two reactions in a sequential mode in alternate order or contemporarily at diverse temperature conditions. A. simplex catalyzed either the dehydrogenation of hydrocortisone into prednisolone efficiently or of 16α-hydroxyhydrocortisone into 16α-hydroxyprednisolone in 24 h (up to 93.9%). Surprisingly S. roseochromogenes partially converted prednisolone back to hydrocortisone. A 68.8% maximum of 16α-hydroxyprednisolone was obtained in 120-h bioconversion by coupling whole cells of the two strains at pH 6.0 and 26 °C. High bioconversion of hydrocortisone into 16α-hydroxyprednisolone was obtained for the first time by coupling A. simplex and S. roseochromogenes

The cyclin-like protein Spy1/RINGO promotes mammary transformation and is elevated in human breast cancer

Abstract Background Spy1 is a novel \u27cyclin-like\u27 activator of the G1/S transition capable of enhancing cell proliferation as well as inhibiting apoptosis. Spy1 protein levels are tightly regulated during normal mammary development and forced overexpression in mammary mouse models accelerates mammary tumorigenesis. Methods Using human tissue samples, cell culture models and in vivo analysis we study the implications of Spy1 as a mediator of mammary transformation and breast cancer proliferation. Results We demonstrate that this protein can facilitate transformation in a manner dependent upon the activation of the G2/M Cdk, Cdk1, and the subsequent inhibition of the anti-apoptotic regulator FOXO1. Importantly, we show for the first time that enhanced levels of Spy1 protein are found in a large number of human breast cancers and that knockdown of Spy1 impairs breast cancer cell proliferation. Conclusions Collectively, this work supports that Spy1 is a unique activator of Cdk1 in breast cancer cells and may represent a valuable drug target and/or a prognostic marker for subsets of breast cancers

Investigation of Superconducting Molybdenum Silicide Nanostrips and Microstrips for Single Photon Detectors

Superconducting nanostrip single photon detectors have emerged as the highest performing single-photon detectors; however, the possibility to use superconducting microstrip as single photon detectors is very appealing both to use them as larger areas detectors and for an easier technology in the manufacturing. The aim of this work is to test the photoresponse in liquid helium dewar of 9 nm thick MoSi covered with a very thin (2 nm) layer of Al, in two different configurations: nanomeanders and microstrips. We demonstrate that MoSi/Al microstrips can work as photodetectors also at T = 4.2 K. We also compare the dark count rate of the microstrip and the nanowire, confirming the lower noise for the microstrips also at 4.2 K

Mutations in CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS)

Background: Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3-10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis. Methodology/Principal Findings: Sequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p. Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B. Conclusions/Significance: We conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS. We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype

Phosphatase and tensin homologue/protein kinase B pathway linked to motor neuron survival in human superoxide dismutase 1-related amyotrophic lateral sclerosis

Gene expression profiling has been used previously with spinal cord homogenates and laser capture microdissected motor neurons to determine the mechanisms involved in neurodegeneration in amyotrophic lateral sclerosis. However, while cellular and animal model work has focused on superoxide dismutase 1-related amyotrophic lateral sclerosis, the transcriptional profile of human mutant superoxide dismutase 1 motor neurons has remained undiscovered. The aim of this study was to apply gene expression profiling to laser captured motor neurons from human superoxide dismutase 1-related amyotrophic lateral sclerosis and neurologically normal control cases, in order to determine those pathways dysregulated in human superoxide dismutase 1-related neurodegeneration and to establish potential pathways suitable for therapeutic intervention. Identified targets were then validated in cultured cell models using lentiviral vectors to manipulate the expression of key genes. Microarray analysis identified 1170 differentially expressed genes in spinal cord motor neurons from superoxide dismutase 1-related amyotrophic lateral sclerosis, compared with controls. These genes encoded for proteins in multiple functional categories, including those involved in cell survival and cell death. Further analysis determined that multiple genes involved in the phosphatidylinositol-3 kinase signalling cascade were differentially expressed in motor neurons that survived the disease process. Functional experiments in cultured cells and primary motor neurons demonstrate that manipulating this pathway by reducing the expression of a single upstream target, the negative phosphatidylinositol-3 kinase regulator phosphatase and tensin homology, promotes a marked pro-survival effect. Therefore, these data indicate that proteins in the phosphatidylinositol-3 kinase pathway could represent a target for therapeutic manipulation in motor neuron degeneration

Differential expression of glucose transporters and hexokinases in prostate cancer with a neuroendocrine gene signature: A mechanistic perspective for 18 F-FDG imaging of PSMA-suppressed tumors

Although the incidence of de novo neuroendocrine prostate cancer (PC) is rare, recent data suggest that low expression of prostatespecific membrane antigen (PSMA) is associated with a spectrum of neuroendocrine hallmarks and androgen receptor (AR) suppression in PC. Previous clinical reports indicate that PCs with a phenotype similar to neuroendocrine tumors can be more amenable to imaging by 18F-FDG than by PSMA-targeting radioligands. In this study, we evaluated the association between neuroendocrine gene signature and 18F-FDG uptake-associated genes including glucose transporters (GLUTs) and hexokinases, with the goal of providing a genomic signature to explain the reported 18F-FDG avidity of PSMA suppressed tumors. Methods: Data-mining approaches, cell lines, and patient-derived xenograft models were used to study the levels of 14 members of the SLC2A family (encoding GLUT proteins), 4 members of the hexokinase family (genes HK1-HK3 and GCK), and PSMA (FOLH1 gene) after AR inhibition and in correlation with neuroendocrine hallmarks. Also, we characterize a neuroendocrine-like PC (NELPC) subset among a cohort of primary and metastatic PC samples with no neuroendocrine histopathology. We measured glucose uptake in a neuroendocrine-induced in vitro model and a zebrafish model by nonradioactive imaging of glucose uptake using a fluorescent glucose bioprobe, GB2-Cy3. Results: This work demonstrated that a neuroendocrine gene signature associates with differential expression of genes encoding GLUT and hexokinase proteins. In NELPC, elevated expression of GCK (encoding glucokinase protein) and decreased expression of SLC2A12 correlated with earlier biochemical recurrence. In tumors treated with AR inhibitors, high expression of GCK and low expression of SLC2A12 correlated with neuroendocrine histopathology and PSMA gene suppression. GLUT12 suppression and upregulation of glucokinase were observed in neuroendocrine- induced PC cell lines and patient-derived xenograft models. A higher glucose uptake was confirmed in low-PSMA tumors using a GB2-Cy3 probe in a zebrafish model. Conclusion: A neuroendocrine gene signature in neuroendocrine PC and NELPC associates with a distinct transcriptional profile of GLUTs and hexokinases. PSMA suppression correlates with GLUT12 suppression and glucokinase upregulation. Alteration of 18F-FDG uptake-associated genes correlated positively with higher glucose uptake in AR- and PSMA-suppressed tumors. Zebrafish xenograft tumor models are an accurate and efficient preclinical method for monitoring nonradioactive glucose uptake

Astrocytic C–X–C motif chemokine ligand-1 mediates β-amyloid-induced synaptotoxicity

Background Pathological interactions between β-amyloid (Aβ) and tau drive synapse loss and cognitive decline in Alzheimer’s disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to Aβ-induced synaptotoxicity in AD is not well understood. Methods We stimulated mouse and human astrocytes with conditioned medium containing concentrations and species of human Aβ that mimic those in human AD brain. Medium from stimulated astrocytes was collected and immunodepleted of Aβ before being added to naïve rodent or human neuron cultures. A cytokine, identified in unbiased screens of stimulated astrocyte media and in postmortem human AD brain lysates was also applied to neurons, including those pre-treated with a chemokine receptor antagonist. Tau mislocalisation, synaptic markers and dendritic spine numbers were measured in cultured neurons and organotypic brain slice cultures. Results We found that conditioned medium from stimulated astrocytes induces exaggerated synaptotoxicity that is recapitulated following spiking of neuron culture medium with recombinant C–X–C motif chemokine ligand-1 (CXCL1), a chemokine upregulated in AD brain. Antagonism of neuronal C–X–C motif chemokine receptor 2 (CXCR2) prevented synaptotoxicity in response to CXCL1 and Aβ-stimulated astrocyte secretions. Conclusions Our data indicate that astrocytes exacerbate the synaptotoxic effects of Aβ via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokine–receptor pair as a novel target for therapeutic intervention in AD

Integrated Epigenome Profiling of Repressive Histone Modifications, DNA Methylation and Gene Expression in Normal and Malignant Urothelial Cells

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20–30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5–10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer

A Monoclonal Antibody against p53 Cross-Reacts with Processing Bodies

The p53 tumor suppressor protein is an important regulator of cell proliferation and apoptosis. p53 can be found in the nucleus and in the cytosol, and the subcellular location is key to control p53 function. In this work, we found that a widely used monoclonal antibody against p53, termed Pab 1801 (Pan antibody 1801) yields a remarkable punctate signal in the cytoplasm of several cell lines of human origin. Surprisingly, these puncta were also observed in two independent p53-null cell lines. Moreover, the foci stained with the Pab 1801 were present in rat cells, although Pab 1801 recognizes an epitope that is not conserved in rodent p53. In contrast, the Pab 1801 nuclear staining corresponded to genuine p53, as it was upregulated by p53-stimulating drugs and absent in p53-null cells. We identified the Pab 1801 cytoplasmic puncta as P Bodies (PBs), which are involved in mRNA regulation. We found that, in several cell lines, including U2OS, WI38, SK-N-SH and HCT116, the Pab 1801 puncta strictly colocalize with PBs identified with specific antibodies against the PB components Hedls, Dcp1a, Xrn1 or Rck/p54. PBs are highly dynamic and accordingly, the Pab 1801 puncta vanished when PBs dissolved upon treatment with cycloheximide, a drug that causes polysome stabilization and PB disruption. In addition, the knockdown of specific PB components that affect PB integrity simultaneously caused PB dissolution and the disappearance of the Pab 1801 puncta. Our results reveal a strong cross-reactivity of the Pab 1801 with unknown PB component(s). This was observed upon distinct immunostaining protocols, thus meaning a major limitation on the use of this antibody for p53 imaging in the cytoplasm of most cell types of human or rodent origin