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To improve assessment of risks associated with pharmaceutical contamination of the environment, it is crucial to understand effects and mode of action of drugs in non-target species. The evidence is accumulating that species with well-conserved drug targets are prone to be at risk when exposed to pharmaceuticals. An interesting group of pharmaceuticals released into the environment is imidazoles, antifungal agents with inhibition of ergosterol synthesis as a primary mode of action in fungi. However, imidazoles have also been identified as competitive antagonists of calmodulin (CaM), a calcium-binding protein with phylogenetically conserved structure and function. Therefore, imidazoles would act as CaM inhibitors in various organisms, including those with limited capacity to synthesize sterols, such as arthropods. We hypothesized that effects observed in crustaceans exposed to imidazoles are related to the CaM inhibition and CaM-dependent nitric oxide (NO) synthesis. To test this hypothesis, we measured (i) CaM levels and its gene expression, (ii) NO accumulation and (iii) gene expression of NO synthase (NOS1 and NOS2), in the cladoceran Daphnia magna exposed to miconazole, a model imidazole drug. Whereas significantly increased CaM gene expression and its cellular allocation were observed, supporting the hypothesized mode of action, no changes occurred in either NO synthase expression or NO levels in the exposed animals. These findings suggest that CaM inhibition by miconazole leads to protein overexpression that compensates for the loss in the protein activity, with no measurable downstream effects on NO pathways. The inhibition of CaM in D. magna may have implications for effect assessment of exposure to mixtures of imidazoles in aquatic non-target species.

Significance: Gene expression analysis is an important fundamental area of biomedical research. However, live gene expression imaging has proven challenging due to constraints in conventional optical devices and fluorescent reporters. Aim: Our aim is to develop smaller, more cost-effective, and versatile imaging capabilities compared with conventional devices. Bioluminescence reporter-based gene expression analysis was targeted due to its advantages over fluorescence-based imaging. Approach: We created a small compact imaging system using micro-CMOS image sensors (μCIS). The μCIS model had an improved pixel design and a patterned absorption filter array to detect the low light intensity of bioluminescence. Results: The device demonstrated lower dark current, lower temporal noise, and higher sensitivity compared with previous designs. The filter array enabled us to subtract dark current drift and attain a clearer light signal. These improvements allowed us to measure bioluminescence reporter-based gene expression in living mammalian cells. Conclusion: Using our μCIS system for bioluminescence imaging in the future, the device can be implanted in vivo for simultaneous gene expression imaging, behavioral analysis, and optogenetic modulation.

Sirtuin 1 (SIRT1), the NAD-dependent histone deacetylase, has been extensively investigated due to its cognitive protective effect. Studies suggest microRNAs (miRNAs) and histone modifications are key epigenetic regulators of gene expression and play important role in brain development. We previously showed that cognitive impairment by lead (Pb) was associated with downregulation of SIRT1, but the epigenetic role of this is unclear. Thus, we exposed 4-week-old male mice to 0.2% lead acetate solution for three months, and subsequently extracted brain homogenate from mice cortex and hippocampus at the age of 1, 4, and 16 months, respectively. In this study, we found that the protein level of SIRT1 was inhibited in the hippocampus and cortex of 16-month-old aged mice exposed to Pb. Moreover, changes in the levels of miR-138-5p and miR-141-3p, which were considered to the mechanistic target of SIRT1 by bioinformatic analysis, were negative correlations SIRT1 protein expression. We also found miR-34c-3p expression was increased in the cortex of mice at the age of 16 months. Collectively, our results showed the expression of neural SIRT1 and three selected microRNAs at different age nodes of mice for the first time of following Pb exposure. Our results suggest that additional efforts should focus on the consequences of early Pb exposure from an epigenetic perspective.

Cadmium (Cd) exposure may induce chronic intoxication, but the harm of cadmium in rice to human at chronic low-level Cd exposure remains unclear. This study employed a mouse model to investigate the toxicity and mechanism of cadmium in rice and CdCl2. After 8-week exposure to Cd (CdCl2 and Cd-contaminated rice), the biochemical indicators and oxidation indicators in the serum and liver of mice were determined, and used mRNA sequencing to investigate the mechanism of different forms of Cd. Results showed that the cadmium concentration of the liver in the CdCl2 + Rice-N group (CdCl2 mixed with feed and normal rice, 0.4mg/kg.bw) was higher than that in the Rice-H group (0.4mg/kg.bw). However, the cadmium concentration of the kidneys in the Rice-H group was higher than that in the CdCl2 + Rice-N group. Our study demonstrated that Cd-treated (Cd in rice and CdCl2) ICR mice generated obviously tissues injury, such as the increased biochemical studies, the activity of antioxidant enzymes debasement. Simultaneously, our data also indicated that there existed difference of the hepatic toxicity between Cd in rice and CdCl2. By means of transcriptomics, we discovered that CdCl2 and Cd in rice may affect different gene expression at the molecular level. We hope to provide some theoretical basis for the revision of food security standards.

Ursolic acid is a natural compound possessing several therapeutic properties including anticancer potential. In present study, cytotoxic and antimetastatic properties of ursolic acid were investigated in intestinal cancer cell lines INT-407 and HCT-116. The cells growth and number were decreased in a dose- and time-dependent manner in both the cell lines. It also increases reactive oxygen species levels in the cells in order to induce apoptosis. Ursolic acid was found to be a significant inhibitor of cancer cells migration and gene expression of migration markers FN1, CDH2, CTNNB1 and TWIST was also downregulated. Ursolic acid treatment downregulated the gene expression of survival factors BCL-2, SURVIVIN, NFKB and SP1, while upregulated the growth-restricting genes BAX, P21 and P53. These results indicate that ursolic acid has anticancer and antimetastatic properties against intestinal cancer. These properties could be beneficial in cancer treatment and could be used as complementary medicine.

The canonical mode of action (MOA) of microcystins (MC) is the inhibition of protein phosphatases, but complete characterization of toxicity pathways is lacking. The existence of over 200 MC congeners complicates risk estimates worldwide. This work employed RNA-seq to provide an unbiased and comprehensive characterization of cellular targets and impacted cellular processes of hepatocytes exposed to either MC-LR or MC-RR congeners. The human hepatocyte cell line, HepaRG, was treated with three concentrations of MC-LR or -RR for 2 h. Significant reduction in cell survival was observed in LR1000 and LR100 treatments whereas no acute toxicity was observed in any MR-RR treatment. RNA-seq was performed on all treatments of MC-LR and -RR. Differentially expressed genes and pathways associated with oxidative and endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) were highly enriched by both congeners as were inflammatory pathways. Genes associated with both apoptotic and inflammatory pathways were enriched in LR1000. We present a model of MC toxicity that immediately causes oxidative stress and leads to ER stress and the activation of the UPR. Differential activation of the three arms of the UPR and the kinetics of JNK activation ultimately determine whether cell survival or apoptosis is favored. Extracellular exosomes were enrichment of by both congeners, suggesting a previously unidentified mechanism for MC-dependent extracellular signaling. The complement system was enriched only in MC-RR treatments, suggesting congener-specific differences in cellular effects. This study provided an unbiased snapshot of the early systemic hepatocyte response to MC-LR and MC-RR congeners and may explain differences in toxicity among MC congeners., Highlights • Microcystin-LR and microcystin-RR have similar transcriptional responses. • Genes associated with oxidative stress and the unfolded protein response were enriched by congeners. • Genes associated with extracellular exosomes were enriched, suggesting a potential new mechanism for cell signaling. • Complement associated genes were strongly enriched only by microcystin-RR. • Identified a potential molecular mechanism underlying the cellular fate of hepatocyte.

Biclustering is a powerful data mining technique that allows clustering of rows and columns, simultaneously, in a matrix-format data set. It was first applied to gene expression data in 2000, aiming to identify co-expressed genes under a subset of all the conditions/samples. During the past 17 years, tens of biclustering algorithms and tools have been developed to enhance the ability to make sense out of large data sets generated in the wake of high-throughput omics technologies. These algorithms and tools have been applied to a wide variety of data types, including but not limited to, genomes, transcriptomes, exomes, epigenomes, phenomes and pharmacogenomes. However, there is still a considerable gap between biclustering methodology development and comprehensive data interpretation, mainly because of the lack of knowledge for the selection of appropriate biclustering tools and further supporting computational techniques in specific studies. Here, we first deliver a brief introduction to the existing biclustering algorithms and tools in public domain, and then systematically summarize the basic applications of biclustering for biological data and more advanced applications of biclustering for biomedical data. This review will assist researchers to effectively analyze their big data and generate valuable biological knowledge and novel insights with higher efficiency.

open access article Food contact materials (FCM) are any type of item intended to come into contact with foods and thus represent a potential source for human exposure to chemicals. Regarding FCMs made of paper and board, information pertaining to their chemical constituents and the potential impacts on human health remains scarce, which hampers safety evaluation. We describe an effect-directed strategy to identify and characterize emerging chemicals in paper and board FCMs. Twenty FCMs were tested in eight reporter gene assays, including assays for the AR, ER, AhR, PPARγ, Nrf2 and p53, as well as mutagenicity. All FCMs exhibited activities in at least one assay. As proof-of-principle, FCM samples obtained from a sandwich wrapper and a pizza box were carried through a complete step-by-step multi-tiered approach. The pizza box exhibited ER activity, likely caused by the presence of bisphenol A, dibutyl phthalate, and benzylbutyl phthalate. The sandwich wrapper exhibited AR antagonism, likely caused by abietic acid and dehydroabietic acid. Migration studies confirmed that the active chemicals can transfer from FCMs to food simulants. In conclusion, we report an effect-directed strategy that can identify hazards posed by FCMs made from paper and board, including the identification of the chemical(s) responsible for the observed activity.

© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com Bacterial pathogens subvert host cells by manipulating cellular pathways for survival and replication; in turn, host cells respond to the invading pathogen through cascading changes in gene expression. Deciphering these complex temporal and spatial dynamics to identify novel bacterial virulence factors or host response pathways is crucial for improved diagnostics and therapeutics. Dual RNA sequencing (dRNA-Seq) has recently been developed to simultaneously capture host and bacterial transcriptomes from an infected cell. This approach builds on the high sensitivity and resolution of RNA sequencing technology and is applicable to any bacteria that interact with eukaryotic cells, encompassing parasitic, commensal or mu-tualistic lifestyles. Several laboratory protocols have been presented that outline the collection, extraction and sequencing of total RNA for dRNA-Seq experiments, but there is relatively little guidance available for the detailed bioinformatic analyses required. This protocol outlines a typical dRNA-Seq experiment, based on a Chlamydia trachomatis-infected host cell, with a detailed description of the necessary bioinformatic analyses with currently available software tools.

Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.

Although effluent treatment systems within pulp and paper mills remove many toxicants and improve wastewater quality, there is a need to understand and quantify the effectiveness of the treatment process. At a combined news and kraft pulp and paper mill in northwestern Ontario, Canada, fathead minnow (FHM) reproduction and physiology were examined before, during, and after a short-term (6-d) exposure to 10% (v/v) untreated kraft mill effluent (UTK), 25% (v/v) secondary treated kraft mill effluent (TK), and 100% (v/v) combined mill outfall (CMO). Although UTK exposure significantly decreased egg production, neither TK nor CMO caused any reproductive changes. The expression of six genes responsive to endocrine-disrupting compounds, stress, or metals was then examined in livers of these fish using real-time polymerase chain reaction. In female FHMs, none of the three effluents induced significant expression changes in any genes investigated. By contrast, in males there were significant increases in the mRNA levels of androgen receptor, estrogen receptor (ER) β, and cytochrome P4501A (CYP1A) upon UTK and TK exposure but no changes in ERα or vitellogenin (VTG) gene expression, whereas CMO exposure significantly increased the mRNA levels of ERα, VTG, and CYP1A. Together, these results suggest that kraft effluent before and after biological treatment contained compounds able to induce androgenic effects in FHMs, and that combination of kraft and newsmill effluents eliminated the androgenic compounds while inducing distinct and significant patterns of gene expression changes that were likely due to estrogenic compounds produced by the newsmill. Environ. Toxicol. Chem. 2010;29:430–439. © 2009 SETAC

The purpose of this study was to produce recombinant xylanase in transgenic plants and to test its potential application for pulp bleaching. The xynII xylanase gene from Trichoderma reesei was inserted into the Arabidopsis genome. Many transgenic plants produced biologically active XYNII and accumulated in leaves at level of 1.4-3.2% of total soluble proteins. The bleaching ability of XYNII on Kraft pulp was demonstrated by a reduction in the kappa number and the residual lignin contents. The bleaching efficiency of transgenic plant produced XYNII was similar to commercial xylanase on unbleached Kraft pulp. The effect of xylanase treatment on Kraft pulp was also investigated by SEM. Clear physical change on the pulp fiber surface was observed and was related to the amount xylan removed and microfibrils were visible on the fiber surface. This report demonstrates the potential application of plant produced recombinant xylanase for pulp and paper bleaching.

Background: Laing early onset distal myopathy (MPD1) is an autosomal dominant myopathy caused by mutations within the slow skeletal muscle fibre myosin heavy chain gene, MYH7. It is allelic with myosin storage myopathy, with the commonest form of familial hypertrophic cardiomyopathy, and with one form of dilated cardiomyopathy. However, the clinical picture of MPD1 is distinct from these three conditions. Objective: To collate and discuss the histological features reported in the muscle biopsies of MPD1 patients and to outline the clinical features. Results: The phenotype of MPD1 was consistent, with initial weakness of great toe/ankle dorsiflexion, and later development of weakness of finger extension and neck flexion. Age of onset was the only variable, being from birth up to the 20s, but progression was always very slow. The pathological features were variable. In this retrospective series, there were no pathognomonic diagnostic features, although atrophic type I fibres were found in half the families. Rimmed vacuoles are consistently seen in all other distal myopathies with the exception of Myoshi distal myopathy. However, they were found in a minority of patients with MPD1, and were not prominent when present. Immunohistochemical staining for slow and fast myosin showed co-expression of slow and fast myosin in some type I fibres, possibly indicating a switch to type II status. This may be a useful aid to diagnosis. Conclusions: The pathological findings in MPD1 are variable and appear to be affected by factors such as the specific muscle biopsied, the age of the patient at biopsy, and the duration of disease manifestations.

Background: It has been proposed that, independent of the e4 allele, APOE promoter polymorphisms (−491 A/T and −219 G/T) may be risks factor for Alzheimer’s disease by modulating APOE expression. Objective: To measure the level of APOE expression in Alzheimer’s disease. Methods: Brains were obtained at necropsy from 114 patients with early and late onset sporadic Alzheimer’s disease in Greater Manchester (UK) during years 1986 to 2001. Total RNA was extracted from 84 brains. Purified lymphocytes were obtained from fresh blood from 16 probable Alzheimer cases from Lille (France). APOE and β-actin gene expression was determined by reverse transcriptase polymerase chain reaction in brain and lymphocytes. Results: An inverse correlation between APOE expression level and Aβ loads was observed. As previously described and extended to 114 cases here, an association between the −219 TT genotype and a higher level of parenchymal Aβ deposition was found, irrespective of APOE e4 allele status. This effect was more pronounced in older individuals, whereas higher Aβ load appeared more closely related to e4 in the younger age group (cut off point at the median age at death (72.5 years)). The −219 TT genotype was associated with a decrease in APOE expression. There was a 60% decrease in APOE expression in lymphocytes from probable Alzheimer cases v controls (p = 0.01). Conclusions: In the oldest individuals, reduced APOE expression, modulated in part by −219 G/T polymorphism, may influence risk and constitute a determinant Aβ load in Alzheimer’s disease.

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes ( tfdA and tfdC ) from populations of JMP134 as low as 10 5 cells/ml was clearly detected against a background of 10 8 cells/ml. Induction of two others ( tfdB and tfdE ) was detected from populations of 10 6 cells/ml in the same background; however, the last gene, tfdF , showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.

Pathotyping and subtyping of influenza A virus were performed with a packaged paper fluidic-based analytical microdevice (PFAM) after one-step reverse transcription-polymerase chain reaction (RT-PCR). The PFAM contains two test lines: one for detecting M gene to identify the influenza A virus and another for haemagglutinin subtyping to determine the viral strain among H1N1, H3N2, and H5N1. The M gene and the haemagglutinin gene (H1, H3, and H5 genes) were amplified by using the Digoxigenin and the Texas Red modified primers, respectively, in the multiplex RT-PCR. The amplicon products were loaded in the conjugate pad of the PFAM in which the streptavidin coated gold nanoparticles were linked with the biotin moieties that were incorporated in the middle of the DNA strands, and then captured by the anti-Digoxigenin and anti-Texas Red immobilized on the test lines. Influenza A H1N1, H3N2, and H5N1 could be identified with a limit of detection of 10(2) copies of RNA templates in 10 min. Pathotyping and subtyping of the clinical nasopharyngeal swab samples were also analyzed whose results were confirmed by real-time RT-PCR.

Aims—Differential display reverse transcription polymerase chain reaction (RT-PCR) was performed to identify genes associated with the invasive potential of human epithelioid sarcoma. Methods—Two different clonal subpopulations, GRU-1A and GRU-1B, derived from the same human epithelioid sarcoma cell line GRU-1 and known to differ greatly in their invasive potential were compared by means of mRNA fingerprinting. Results—Using a set of 10 arbitrary upstream primers and nine anchored oligo-dT primers, 22 candidate gene fragments were identified; differential expression was confirmed in four of these fragments by northern blot analysis. At the mRNA level, apoferritin light chain was predominantly expressed by the highly invasive cell line GRU-1A. In contrast, the mitochondrial gene M1, encoding cytochrome c oxidase I, and the TI-227H gene were expressed more strongly by the low invasive cell line GRU-1B. Furthermore, a novel gene fragment was identified and cloned that was preferentially expressed in the low invasive cell line GRU-1B, and therefore might have an inhibitory role in invasion. Consequently, this gene fragment was designated as expressed in low invasive sarcoma cells (ELISC-1). Conclusions—A novel gene fragment (ELISC-1) and three known genes were identified as potential regulators of tumour invasiveness. Cloning of the entire sequence of ELISC-1 and subsequent investigations are required to establish its biological role.

Although the role of transforming growth factor beta (TGFbeta) in initiating fibrosis is well established, the role that TGFbeta plays in maintaining fibrosis is unclear. The gene encoding connective tissue growth factor (ccn2; ctgf), which promotes fibrosis, is not normally expressed in dermal fibroblasts unless TGFbeta is present. However, in dermal fibroblasts cultured from lesional areas of scleroderma, ccn2 (ctgf) is expressed constitutively. The contribution of several elements in the ccn2 (ctgf) promoter to basal and TGFbeta induced ccn2 (ctgf) expression in normal and scleroderma fibroblasts has been investigated. A functional SMAD binding site in the ccn2 (ctgf) promoter that is necessary for the TGFbeta mediated induction of this gene has been identified. The previously termed TGFbeta responsive enhancer (TGFbetaRE) in the ccn2 (ctgf) promoter has been found to be necessary for basal promoter activity in normal fibroblasts. The SMAD element is not necessary for the high ccn2 (ctgf) promoter activity seen in scleroderma fibroblasts. However, mutation of the previously termed TGFbetaRE reduces ccn2 (ctgf) promoter activity in scleroderma fibroblasts to that seen in normal fibroblasts. Thus, the maintenance of the scleroderma phenotype, as assessed by a high degree of ccn2 (ctgf) promoter activity, appears to be relatively independent of SMAD action and seems to reflect increased basal promoter activity.

Aims—Microarray technology has recently led to the identification of molecular prognostic subgroups in non-Hodgkin's lymphomas. To determine the usefulness of ready made macroarrays as routine diagnostic tools in haematopathology, lymph node biopsies were analysed using a cDNA macroarray containing genes involved in apoptosis, including caspases. Methods—Nine biopsy specimens were analysed using total frozen tissues: four samples of B cell follicular lymphoma, two of B cell diffuse large cell lymphoma, and three of non-neoplastic lymph nodes from benign lymphadenitis. Nine cell populations were sorted from fresh tissues: malignant B cells from two patients with follicular lymphoma and two with diffuse large cell lymphoma, reactive B cells from two benign lymph nodes, reactive T cells from one benign lymph node, and virgin (mantle zone) B cells and germinal centre B cells from benign tonsils. Immunohistochemistry (IHC) on paraffin wax sections was performed for the localisation of caspases 2, 3, 4, 7, 8, and 9. Results—In the clustered array data, sorted cells from samples sharing common histological lesions were grouped together, whereas the array/histology correlation was less satisfactory for tissues. The expression profiles of both the array and IHC methods correlated for most caspases and samples. Conclusions—Variations in array profiles of sorted cell populations can be associated with specific histological features, suggesting a possible diagnostic application of ready made apoptosis macroarrays in haematopathology.

Juvenile whitefish (Coregonus lavaretus L. s.l.) were exposed by caging in the field to diluted effluents from three operating pulp, paper, and paperboard mills in Southern Lake Saimaa, Finland. The expression of the vitellogenin gene, used as a biomarker of estrogenic contamination of effluents, was measured using a Northern blotting method. Increased mRNA levels, the most specific and reliable evidence for estrogen receptor-mediated actions in vivo, were found in fish caged in the vicinity of one of three mills studied. This mill was found to discharge wood-derived compounds, such as sterols and resin acids, into Lake Saimaa in amounts considerably exceeding those from the other two mills. The increased vitellogenin gene expression suggests that the effluent is a source of estrogenic contaminants.

Some endocytic proteins have recently been shown to play a role in tumorigenesis. In this study, we demonstrate that APPL2, an adapter protein with known endocytic functions, is upregulated in 40% cases of glioblastoma multiforme, the most common and aggressive cancer of the central nervous system. The silencing of APPL2 expression by small interfering RNAs (siRNAs) in glioma cells markedly reduces cell survival under conditions of low growth factor availability and enhances apoptosis (measured by executor caspase activity). Long‐term depletion of APPL2 by short hairpin RNAs (shRNAs), under regular growth factor availability, suppresses the cell transformation abilities, assessed by inhibited colony formation in soft agar and by reduced xenograft tumor growth in vivo. At the molecular level, the negative effect of APPL2 knockdown on cell survival is not due to the alterations in AKT or GSK3β activities which were reported to be modulated by APPL proteins. Instead, we attribute the reduced cell survival upon APPL2 depletion to the changes in gene expression, in particular to the upregulation of apoptosis‐related genes, such as UNC5B (a proapoptotic dependence receptor) and HRK (harakiri, an activator of apoptosis, which antagonizes anti‐apoptotic function of Bcl2). In support of this notion, the loss of glioma cell survival upon APPL2 knockdown can be rescued either by an excess of netrin‐1, the prosurvival ligand of UNC5B or by simultaneous silencing of HRK. Consistently, APPL2 overexpression reduces expression of HRK and caspase activation in cells treated with apoptosis inducers, resulting in the enhancement of cell viability. This prosurvival activity of APPL2 is independent of its endosomal localization. Cumulatively, our data indicate that a high level of APPL2 protein might enhance glioblastoma growth by maintaining low expression level of genes responsible for cell death induction., Highlights ► APPL2 protein levels are elevated in 40% cases of glioblastoma multiforme.► Overexpression of APPL2 exhibits cytoprotective effects in glioma cells.► APPL2 depletion reduces survival and transformation abilities of glioma cells.► Silencing of APPL2 promotes expression of proapoptotic genes HRK and UNC5B.

A gene expressing xylanase activity was isolated from a genomic library of Thermotoga sp. strain FjSS3-B.1. The sequence of the gene shows that it encodes a single domain, family 10 xylanase. The recombinant enzyme has extremely high thermal stability, activity over a relatively broad pH range, and activity on Pinus radiata kraft pulp.

Genomic sequencing analysis and previous studies have shown that there are eight genes in Streptomyces coelicolor A3(2) encoding putative cellulases. One of these genes, sco6548 , was cloned into the Streptomyces / Escherichia coli shuttle vector pUWL201PW. The recombinant protein was successfully overexpressed in Streptomyces lividans TK24 under the control of the strong ermE promoter. Sco6548 was 1,740 bp in length, and encoded a 579-amino acid-, 60.8-kDa protein with strong hydrolyzing activity toward Avicel and filter paper, yielding cellobiose as the final product. SCO6548 showed optimal activity at 50°C and pH 5. The Km values of SCO6548 toward Avicel and filter paper were 15.38 mg/mL and 16.1 mg/mL, respectively. The V max values toward Avicel and filter paper were 0.432 μM/min and 0.084 μM/min, respectively. EDTA did not affect cellulase activity; however, several divalent cations, including Co2+, Cu2+, Ni2+, and Mn2+ (at 10 mM) had severe inhibitory effects on enzyme activity. Our analysis showed that SCO6548 is a cellulose 1,4-β-cellobiosidase that hydrolyzes cellulose into cellobiose.

The toxicity of pulp and paper mill effluents (PPMEs) has been greatly decreased, yet some continue to negatively affect fish reproduction. We hypothesized that PPMEs are affecting the brain resulting in decreased reproductive performance. Our goal was to use gene expression profiling to test the hypothesis that PPMEs are having an effect on neural systems in the fathead minnow (FHM; Pimephales promelas) in vivo. Sexually mature male and female FHM were exposed to 100% final biotreated PPMEs from 5 different sources for 5 days. Using an oligo-array (15K genes) we examined the effect of PPMEs on gene expression in the hypothalamus of female fish. We validated selected genes (cholecystokinin, RevErbbeta2, and urotensin I) that were identified by microarray analysis using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We compared the FHM microarray dataset to multiple microarray datasets from experiments conducted with goldfish injected with different dopaminergic pharmaceuticals to examine whether PPMEs could be affecting the dopamine system. Exposure of FHM to PPMEs resulted in varying degrees of spawning inhibition. Microarray analysis revealed surprisingly few genes in the brain that were commonly affected by the different PPMEs. Real-time RT-PCR confirmed the changes in expression for cholecystokinin, RevErbbeta2, and urotensin I. Comparison of the FHM and goldfish microarray datasets suggest that some PPMEs may be acting on the dopamine system. We show that PPMEs are neuroactive in fish and may be acting through some of the pathways in a manner similar to dopamine.

MicroRNAs (miRNAs) are small 21–25 nucleotide-long non-coding RNAs that have emerged as key negative post-transcriptional regulators of gene expression [1, 2]. Currently there are more than 700 mammalian miRNAs that can potentially target up to one-third of protein-coding human genes [1] involved in diverse physiological and pathological processes, including cancer [3, 4]. Indeed, aberrant levels of miRNAs have been reported in all major human malignancies [5, 6]. In tumors, altered expression of miRNAs has been demonstrated to inhibit tumor suppressor genes or inappropriately activate oncogenes and has been associated with every aspect of tumor biology, including tumor progression, invasiveness, metastasis, and acquisition of resistance by malignant cells to chemotherapeutic agents [3, 4, 7, 8]. These observations lead to the suggestion that aberrant expression of miRNAs may contribute to tumorigenesis [9]. However, most of the tumor-miRNA-related studies are based on expression analysis of miRNAs in tumors in comparison with corresponding adjacent normal tissues [4, 5, 6]. The altered expression of any given miRNA in neoplastic cells is not sufficient to address conclusively the role of these changes in tumorigenesis [10]. Additionally, despite the established biological significance of miRNA dysregulation in neoplastic cells, there is a lack of knowledge on the role of miRNAs during early stages of tumor development, especially if variations in the expression of specific miRNAs are associated with differences in the susceptibility to tumorigenesis.

Expansin is a plant protein family that induces plant cell wall-loosening and cellulose disruption without exerting cellulose-hydrolytic activity. Expansin-like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a beta-expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose-binding and cellulose-weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7-fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non-eukaryotic source.

The formation of alpha-synuclein aggregates may be a critical event in the pathogenesis of multiple system atrophy (MSA). However, the role of this gene in the aetiology of MSA is unknown and untested.The linkage disequilibrium (LD) structure of the alpha-synuclein gene was established and LD patterns were used to identify a set of tagging single nucleotide polymorphisms (SNPs) that represent 95% of the haplotype diversity across the entire gene. The effect of polymorphisms on the pathological expression of MSA in pathologically confirmed cases was also evaluated.In 253 Gilman probable or definite MSA patients, 457 possible, probable, and definite MSA cases and 1472 controls, a frequency difference for the individual tagging SNPs or tag-defined haplotypes was not detected. No effect was observed of polymorphisms on the pathological expression of MSA in pathologically confirmed cases.

Contains fulltext : 49888.pdf (Publisher’s version ) (Closed access) BACKGROUND: Segawa syndrome due to GTP cyclohydrolase deficiency is an autosomal dominant disorder with variable expression, that is clinically characterised by l-dopa responsive, diurnally fluctuating dystonia and parkinsonian symptoms. OBJECTIVE: To delineate the neurological and psychiatric phenotype in all affected individuals of three extended families. METHODS: GTP cyclohydrolase deficiency was documented by biochemical analyses, enzymatic measurements in fibroblasts, and molecular investigations. All affected individuals were examined neurologically, and psychiatric data were systematically reviewed. RESULTS: Eighteen affected patients from three families with proven GTP cyclohydrolase deficiency were identified. Eight patients presenting at less than 20 years of age had typical motor symptoms of dystonia with diurnal variation. Five family members had late-presenting mild dopa-responsive symptoms of rigidity, frequent falls, and tendonitis. Among mutation carriers older than 20 years of age, major depressive disorder, often recurrent, and obsessive-compulsive disorder were strikingly more frequent than observed in the general population. Patients responded well to medication increasing serotonergic neurotransmission and to l-dopa substitution. Sleep disorders including difficulty in sleep onset and maintenance, excessive sleepiness, and frequent disturbing nightmares were present in 55% of patients. CONCLUSION: Physicians should be aware of this expanded phenotype in affected members of families with GTP cyclohydrolase deficiency.

Background: MxA gene expression is one of the most appropriate markers of biological activity of exogenous interferon (IFN) beta. Methods: We quantified MxA mRNA for five consecutive days in 62 patients treated with IFN beta (16, Avonex; 10, Betaferon; 24, Rebif 22; 12, Rebif 44), by quantitative-competitive polymerase chain reaction. Every three months, IFN beta induced neutralising antibodies (NAbs) were evaluated in sera using a cytopathic effect assay. Results: Two categories of patients were identified: one group (49/62) had a sharp post-injection increase in MxA expression (defined as "IFN beta biological responder"), whereas the other group (13/62) had no MxA induction after IFN beta administrations (defined as "IFN beta biological non-responder"). In 11/13 biological non-responders, the persistent presence of NAbs correlated with abolished biological activity, independently of treatment regimen. The two remaining IFN beta biological non-responders were NAb–. Among the 49 IFN beta biological responders, biological activity was comparable between the four preparations on day 2 and 3 (+12 and +36 hours post-injection), but it was greater in Betaferon and both Rebif preparations on day 1, 4, and 5. In biological responders treated three times a week, only 82% (59/72) of injections were considered effective, compared with 100% (13/13) of Avonex injections. Conclusion: Our results suggest that an optimal IFN beta regimen is not yet available: Avonex, given once a week, shows lower cumulative biological activity. On the other hand, both Betaferon and Rebif, given three times a week, show 18% biologically ineffective injections and higher risk of developing NAbs, which abolish biological activity.

1. Bi-directional GABA-transport was studied by performing uptake and superfusion experiments in human embryonic kidney 293 cells stably expressing the rat GABA transporter rGAT-1. 2. K(M) and V(max) values for [(3)H]-GABA uptake were 11.7+/-1.8 microM and 403+/-55 pmol min(-1) 10(-6) cells (n=9), respectively. 3. Kinetic analysis of outward transport was performed by pre-labelling the cells with increasing concentrations of [(3)H]-GABA and triggering outward transport with 333 microM GABA. Approximate apparent K(M) and V(max) values were 12 mM and 50 pmol min(-1) 10(-6) cells, respectively. 4. GABA re-uptake inhibitors (RI; e.g. tiagabine), as well as, substrates of the rGAT-1 (e.g. GABA, nipecotic acid) concentration dependently decreased [(3)H]-GABA uptake and increased efflux of [(3)H]-GABA from pre-labelled cells. The IC(50) values for inhibiting uptake and the EC(50) values for increasing efflux were significantly correlated (r(2)=0.99). 5. On superfusion, RI antagonized the efflux-enhancing effect of the substrates. The effect of the latter was markedly augmented in the presence of ouabain (100 microM), whereas the effect of RI remained unchanged. The most likely explanation for the release enhancing effect of RI is interruption of ongoing re-uptake. 6. The structural GABA-analogue 2,4-diamino-n-butyric acid (DABA) exhibited a bell-shaped concentration response curve on [(3)H]-GABA efflux with the maximum at 1 mM, and displayed a deviation from the sigmoidal inhibition curve in uptake experiments in the same concentration range. At concentrations below 1 mM, DABA inhibited [(3)H]-GABA uptake non-competitively, while at 1 mM and above the inhibition of uptake followed a competitive manner. 7. The results provide information of GABA inward and outward transport, and document a complex interaction of the rGAT-1 with its substrate DABA.

International audience; The agronomic and pulping performance of transgenic trees with altered lignin has been evaluated in duplicated, long-term field trials. Poplars expressing cinnamyl alcohol dehydrogenase (CAD) or caffeate/5-hydroxy-ferulate O-methyltransferase (COMT) antisense transgenes were grown for four years at two sites, in France and England. The trees remained healthy throughout the trial. Growth indicators and interactions with insects were normal. No changes in soil microbial communities were detected beneath the transgenic trees. The expected modifications to lignin were maintained in the transgenics over four years, at both sites. Kraft pulping of tree trunks showed that the reduced-CAD lines had improved characteristics, allowing easier delignification, using smaller amounts of chemicals, while yielding more high-quality pulp. This work highlights the potential of engineering wood quality for more environmentally benign papermaking without interfering with tree growth or fitness.

Aims—To establish whether the ectopic expression of CCN3 (NOV) in glioma cells can interfere with their tumorigenic potential and assess its potential value in molecular medicine. Methods—Glioma cell lines were used to assess whether differences in the degree of intracellular communication induced by the expression of the gap junction protein connexin 43 (Cx43) is related to the differential expression of CCN3 (NOV). The antiproliferative activity of rat CCN3 (rCCN3; NOV) in glioma cells, has been assessed both in vitro and in vivo with glioma cell lines expressing different amounts of CCN3 (NOV). Results—Upon ectopic expression of Cx43, the growth of C6 glioma cells is decreased. An increase of CCN3 (NOV) expression matches the reduced tumorigenic potential of these transfected cells. The localisation of CCN3 (NOV) is affected by the increased expression of Cx43 in the Cx-13 transfected cells, in which it is detected at areas of cell–cell contact. In a xenograft model, CCN3 (NOV) transfected glioma cells were found to induce tumours to a lesser degree than their parental counterparts, which do not express detectable amounts of CCN3 (NOV). Conclusions—Previous observations had suggested an inverse relation between CCN3 (NOV) expression in glioma cells and their tumorigenicity. These results establish a direct association between the establishment of functional gap junctional intercellular communication and the expression of rCCN3 (NOV). In addition to a negative effect on murine and human cell growth, CCN3 (NOV) has antiproliferative activity on tumour cells in vivo. Thus, the antiproliferative activity of the CCN3 (NOV) protein might involve reorganisation of cellular contacts that play a crucial role in tumorigenesis. The antiproliferative activity of CCN3 (NOV) established in this work sets the stage for the potential use of CCN proteins in molecular oncology.

Aims—To investigate the expression of the human ccn3 (hccn3; nov) proto-oncogene, a member of the CCN family of proteins, in prostate epithelial cells and prostate tissue samples. Methods—Normal adult prostate luminal epithelial cells immortalised by SV40 large T (PNT1A and PNT1B), metastatic tumours (LNCaP, DU-145, and PC-3), and prostate tissue samples from patients with benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma were used. hccn3 (nov) mRNA was measured by the reverse transcription polymerase chain reaction (RT-PCR) and hCCN3 (NOV) protein expression was determined by immunochemistry. Results—hccn3 (nov) RNA values were higher in PC-3 cells than in the other prostate cell lines. The immortalised normal cell lines either did not express hccn3 (nov) RNA (PNT1B) or expressed very low amounts (PNT1A). BPH samples expressed variable amounts of hccn3 (nov) RNA. With the use of immunocytochemistry, all cell lines except PNT1A and PNT1B were shown to contain hCCN3 (NOV) protein. hCCN3 (NOV) was localised mainly in the epithelial compartment of BPH and adenocarcinoma samples, and there was evidence of luminal secretion. Conclusion—The overexpression of hccn3 (nov) RNA in cancer cell lines compared with other cell lines and its epithelial localisation in human prostate samples are consistent with a role for hCCN3 (NOV) in prostatic tumorigenesis.

Background/Aims—Loss of function of the retinoblastoma (Rb) tumour suppressor gene, located on chromosome 13, is common in many inherited and sporadic forms of cancer. Inactivation of its gene product by oncogenic human papillomaviruses (HPV) plays a key role in the genesis of cervical cancer. It has been shown previously that non-melanoma skin cancers of renal transplant recipients and immunocompetent patients with skin cancer also frequently harbour potentially oncogenic HPV types. This study aimed to examine the integrity of the Rb gene in histologically confirmed squamous cell carcinomas (SCCs) from renal transplant recipients and immunocompetent patients with skin cancer. Methods—Loss of heterozygosity (LOH) at the Rb locus was examined in 13 histologically confirmed SCCs using the D13S153 microsatellite marker, which is located in exon 2 of the Rb gene. Loss of a second marker, D13S118, distal telomerically to the Rb gene at 13q14.3 was also analysed. Results—Of the 13 HPV associated SCCs examined 11 were informative (two SCCs were homozygous for both microsatellite markers). LOH at the D13S153 locus was found in four of the 10 informative SCCs and LOH at the D13S118 locus was found in five of the 11 informative cases. Overall, seven of the 11 informative cases showed LOH at one or other locus. This represents a high degree of chromosomal instability in these tumours. The expression of the Rb gene product in the 11 informative cases was analysed immunohistochemically. Expression of Rb was detected in 10 of the 11 SCCs examined. No correlation between the HPV status of the tumours and the expression of Rb was found. Although the only SCC not to express Rb also demonstrated LOH at the D13S153 locus, the remaining SCCs that had LOH at 13q14 were able to express Rb. Conclusion—Another tumour suppressor gene located at 13q14 might be responsible for the genesis of these tumours.

Aims—To establish the expression pattern of ccn3 (nov) in the central nervous system (CNS) of adult rats and to determine whether spatiotemporal variations in the expression of ccn3 (nov) are related to specific developmental stages and/or specific CNS functions. Methods—The sites of ccn3 (nov) expression have been identified by in situ hybridisation using didoxigenin labelled cRNA and by the reverse transcription-polymerase chain reaction (RT-PCR). The rat CCN3 (NOV) protein was characterised by western blotting performed on brain extracts. The localisation of the CCN3 (NOV) protein in the brain was established by immunocytochemistry. Results—Increased expression of ccn3 (nov) was detected in the developing brain of rats after birth, as shown by RT-PCR and immunocytochemistry analysis performed on a series of samples taken between day 5 (P5) and day 300 (P300), with a pronounced peak between P15 and P150, suggesting that CCN3 (NOV) might play a role in the maintenance or establishment of specific brain functions. The relatively high amounts of an N-terminal truncated CCN3 (NOV) related protein detected both in the brain tissues and cerebrospinal fluid suggested that post translational processing of CCN3 (NOV) might be particularly prevalent in the brain. Such processing might be of biological importance in the light of the previously reported growth stimulatory effects of N-terminal truncated CCN3 (NOV) isoforms. Conclusions—The postnatal differential expression of ccn3 (nov) in the brain of developing rats suggests that CCN3 (NOV) might be involved in the acquisition of specific functions. The rat species provides an as yet unequalled system for these studies. Because the CCN3 (NOV) protein is detected in restricted areas of the brain, it will be interesting to establish whether variations of ccn3 (nov) expression are associated with normal cognitive processes and whether ccn3 (nov) expression is affected by aging. In addition, because CCN3 (NOV) is found in the spinal cord and along the axonal processes, it will be of interest to determine the expression of the normal and truncated isoforms of CCN3 (NOV) in various pathological conditions, such as neurodegenerative diseases.

Escherichia coli KO11 was previously constructed to produce ethanol from acid hydrolysates of hemicellulose (pentoses and hexoses) by the chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). Klebsiella oxytoca P2 was constructed in an analogous fashion for the simultaneous saccharification and fermentation of cellulose and contains PTS enzymes for cellobiose. In this study, KO11 was further engineered for the fermentation of cellulose by adding the K. oxytoca casAB genes encoding Enzyme IIcellobiose and phospho-beta-glucosidase. Although the two K. oxytoca genes were well expressed in cloning hosts such as DH5 alpha, both were expressed poorly in E. coli KO11, a derivative of E. coli B. Spontaneous mutants which exhibited more than 15-fold-higher specific activities for cellobiose metabolism were isolated. The mutations of these mutants resided in the plasmid rather than the host. Three mutants were characterized by sequence analysis. All contained similar internal deletions which eliminated the casAB promoter and operator regions and placed the lacZ Shine-Dalgarno region immediately upstream from the casA Shine-Dalgarno region. KO11 harboring mutant plasmids (pLOI1908, pLOI1909, or pLOI1910) rapidly fermented cellobiose to ethanol, and the yield was more than 90% of the theoretical yield. Two of these strains were used with commercial cellulase to ferment mixed-waste office paper to ethanol.

Yeast ENO1 promoter was prepared by a chemical synthetic method. Two variant promoters containing a pyrimidine-rich region (CT block), located between the TATA box and the transcription start site, either 32 or 34 base pairs (bp) longer than the native ENO1 promoter were isolated during the chemical synthesis. Gene expression of variant promoters was compared with that of the native promoter by measuring the amount of mRNA and the activity of beta-galactosidase by constructing ENO1-lacZ gene fusions. No significant differences were observed between the native and variant promoters in transcription levels. The start site of transcription was mapped on CAAG, a consensus sequence of transcription start site of yeast glycolytic genes. The results suggest a longer CT block in ENO1 promoter may not affect the expression of the yeast ENO1 gene. In addition, the level of ENO1 gene expression was found to be higher in stationary phase cells than in log phase cells.

Sensitive assays of histidase activity were used to follow the production of this enzyme as directed by a gene newly introduced into cells of Bacillus subtilis by transformation. Histidase activity can be detected in histidase-negative recipient cells within 1 hr after the addition of deoxyribonucleic acid extracted from histidase-positive donors. Enzyme production continues for one to two additional hours and then ceases. Histidase production in the transformed cells is fully sensitive to catabolite repression. Catabolite repression is rapidly established after transformation of recipient cells that are resistant to this form of regulation.

To report a new mutation in the MPZ gene which encodes myelin protein zero (P0), associated with an axonal form of Charcot-Marie-Tooth disease (CMT).Three patients from an Italian family with a mild, late onset axonal peripheral neuropathy are described clinically and electrophysiologically. To detect point mutation in MPZ gene the whole coding sequence was examined. The structure of the mutated protein was investigated using the three dimensional model of P0.All patients showed a relatively mild CMT phenotype characterised by late onset and heterogeneity of the clinical and electrophysiological features. Molecular analysis demonstrated a novel heterozygous T/A transversion in the exon 3 of MPZ gene that predicts an Asp109Glu amino acid substitution in the extracellular domain of the P0. Asp109 is found at the protein surface, on beta strand E, in the interior of the P0 tetramer.The identification of Asp109Glu mutation confirms the pivotal role of P0 in axonal neuropathies and stresses the phenotypic heterogeneity associated with MPZ mutations. This study suggests the value of screening for MPZ mutations in CMT family members with minor clinical and electrophysiological signs of peripheral neuropathy.