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

Plastic strain arrangement in copper single crystals in sliding

Deformation of tribologically loaded contact zone is one of the wear mechanisms in spite of the fact that no mass loss may occur during this process. Generation of optimal crystallographic orientations of the grains in a polycrystalline materials (texturing) may cause hardening and reducing the deformation wear. To reveal the orientation dependence of an individual gain and simplify the task we use copper single crystals with the orientations of the compression axis along [11 1] and [110]. The plastic deformation was investigated by means of optical, scanning electron microscopy and EBSD techniques. It was established that at least four different zones were generated in the course of sliding test, such as non-deformed base metal, plastic deformation layer sliding, crystalline lattice reorientation layer and subsurface grain structure layer. The maximum plastic strain penetration depth was observed on [110]-single crystals. The minimum stability of [11 1]-crystals with respect to rotation deformation mode as well as activation of shear in the sliding contact plane provide for rotation deformation localization below the worn surface. The high-rate accumulation of misorientations and less strain penetration depth was observed on [11 1]-crystals as compared to those of [110]-oriented ones

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

Agave negatively regulates YAP and TAZ transcriptionally and post-translationally in osteosarcoma cell lines

Osteosarcoma (OS) is the most aggressive type of primary solid tumor that develops in bone. Whilst conventional chemotherapy can improve survival rates, the outcome for patients with metastatic or recurrent OS remains poor, so novel treatment agents and strategies are required. Research into new anticancer therapies has paved the way for the utilisation of natural compounds as they are typically less expensive and less toxic compared to conventional chemotherapeutics. Previously published works indicate that Agave exhibits anticancer properties, however potential molecular mechanisms remain poorly understood. In the present study, we investigate the anticancer effects of Agave leaf extract in OS cells suggesting that Agave inhibits cell viability, colony formation, and cell migration, and can induce apoptosis in OS cell lines. Moreover, Agave sensitizes OS cells to cisplatin (CDDP) and radiation, to overcome chemo- and radio-resistance. We demonstrate that Agave extract induces a marked decrease of Yes Associated Protein (YAP) and Tafazzin (TAZ) mRNA and protein expression upon treatment. We propose an initial mechanism of action in which Agave induces YAP/TAZ protein degradation, followed by a secondary event whereby Agave inhibits YAP/TAZ transcription, effectively deregulating the Nuclear Factor kappa B (NF-\u3baB) p65:p50 heterodimers responsible for transcriptional induction of YAP and TAZ

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

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

Amyotrophic lateral sclerosis-associated mutant VAPBP56S perturbs calcium homeostasis to disrupt axonal transport of mitochondria

A proline-to-serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes some dominantly inherited familial forms of motor neuron disease, including amyotrophic lateral sclerosis (ALS) type-8. Here, we show that expression of ALS mutant VAPBP56S but not wild-type VAPB in neurons selectively disrupts anterograde axonal transport of mitochondria. VAPBP56S-induced disruption of mitochondrial transport involved reductions in the frequency, velocity and persistence of anterograde mitochondrial movement. Anterograde axonal transport of mitochondria is mediated by the microtubule-based molecular motor kinesin-1. Attachment of kinesin-1 to mitochondria involves the outer mitochondrial membrane protein mitochondrial Rho GTPase-1 (Miro1) which acts as a sensor for cytosolic calcium levels ([Ca2+]c); elevated [Ca2+]c disrupts mitochondrial transport via an effect on Miro1. To gain insight into the mechanisms underlying the VAPBP56S effect on mitochondrial transport, we monitored [Ca2+]c levels in VAPBP56S-expressing neurons. Expression of VAPBP56S but not VAPB increased resting [Ca2+]c and this was associated with a reduction in the amounts of tubulin but not kinesin-1 that were associated with Miro1. Moreover, expression of a Ca2+ insensitive mutant of Miro1 rescued defective mitochondrial axonal transport and restored the amounts of tubulin associated with the Miro1/kinesin-1 complex to normal in VAPBP56S-expressing cells. Our results suggest that ALS mutant VAPBP56S perturbs anterograde mitochondrial axonal transport by disrupting Ca2+ homeostasis and effecting the Miro1/kinesin-1 interaction with tubulin

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