Biochar impact on the estimation of the colorimetric-based enzymatic assays of soil

This study was carried out in order to assess the influence of biochar applications on the estimation of colorimetric-based enzymatic assays and to verify the effectiveness of the most common methods. Since most methods used to determine enzymatic activities in the soil are based on colorimetry, biochar may absorb substrates and/ or coloured products thereby distorting the analytical result. Biochar was added to two soils, with different textures and cation exchangeable capacities, at a rate of 2% (w/w), and seven enzyme activities were determined following standard methods. The biochar amendment lowered the spectrophotometer reading of the activity of FDAase and dehydrogenase in the sandy soil. In the three enzymatic activities based on p-nitrophenol production (β-glucosidase, phosphatase and arylsulphatase), the addition of biochar did not change the enzyme assays. The biochar led to an overestimation in terms of the protease and urease activities in the sandy soil. In the clay loamy soil, biochar did not change the response of any of the enzyme activities tested. A biochar dose of up to 2% only guarantees the effectiveness of the most common spectrophotometric methods for not excessively sandy soils

Homovanillic acid in CSF of mild stage Parkinson's disease patients correlates with motor impairment.

In Parkinson's disease (PD), several efforts have been spent in order to find biochemical parameters able to identify the progression of the pathological processes at the basis of the disease. It is already known that advanced PD patients manifesting dyskinesia are featured by the high homovanillic acid (HVA)/dopamine (DA) ratio, suggesting the increased turnover of DA in these patients. Less clear is whether similar changes affect mild and moderate stages of the disease (between 1 and 2.5 of Hoehn & Yahr -H&Y- stage). Hence, here we tested whether cerebrospinal fluid (CSF) concentrations of DA and its major metabolites, either 3,4-dihydroxyphenylacetic acid (DOPAC) or HVA, correlate with motor performance in mild and moderate PD patients. CSF samples were collected after 2 days of anti-PD drugs washout, via lumbar puncture (LP) performed 130 min following administration of oral levodopa (LD) dose (200 mg). LP timing was determined in light of our previous tests clarifying that 2 h after oral LD administration CSF DA concentration reaches a plateau, which was un-respective of PD stage or duration. DA, DOPAC and HVA were assayed by high performance liquid chromatography in a group of 19 patients, distributed in two groups on the basis of the H&Y stage with a cut-off of 1.5. In these PD patients, HVA was correlated with DOPAC (R = 0,56, p < 0,01) and both HVA and DOPAC CSF levels increased in parallel with the motor impairment. More importantly, HVA correlated with motor impairment measured by the Unified Parkinson's Disease Score -III (UPDRS) (R = 0.61; p < 0.0001). The present findings showed the early alteration of the DA pre-synaptic machinery, as documented by the progressive increase of CSF HVA concentrations, which also correlated with PD motor impairment. Therefore, we suggest the potential use of measuring the CSF HVA level as a possible biomarker of PD stage changes in order to monitor the effectiveness of PD-modifying pharmacological therapies

The Hay Wells Syndrome-Derived TAp63aQ540L Mutant has Impaired Transcriptional and Cell Growth Regulatory Activity

p63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC (ectrodactyly, ectodermal dysplasia, clefting) syndrome, ADULT (acro, dermato, ungual, lacrimal, tooth) syndrome and AEC (ankyloblepharon, ectodermal dysplasia, clefting) syndrome (also called Hay-Wells syndrome). The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. In this paper we report a study on the TAp63alpha isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63alpha wt, DeltaNp63alpha or the TAp63alpha-Q540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs thetranscriptional activity of TAp63alpha and causes misregulation of genes involved in the control of cell growth and epidermal differentiation

Metagenes Associated with Survival in Non-Small Cell Lung Cancer

NSCLC (non-small cell lung cancer) comprises about 80% of all lung cancer cases worldwide. Surgery is most effective treatment for patients with early-stage disease. However, 30%–55% of these patients develop recurrence within 5 years. Therefore, markers that can be used to accurately classify early-stage NSCLC patients into different prognostic groups may be helpful in selecting patients who should receive specific therapies

RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization

Background RT-qPCR is a sensitive and increasingly used method for gene expression quantification. To normalize RT-qPCR measurements between samples, most laboratories use endogenous reference genes as internal controls. There is increasing evidence, however, that the expression of commonly used reference genes can vary significantly in certain contexts. Results Using the Genevestigator database of normalized and well-annotated microarray experiments, we describe the expression stability characteristics of the transciptomes of several organisms. The results show that a) no genes are universally stable, b) most commonly used reference genes yield very high transcript abundances as compared to the entire transcriptome, and c) for each biological context a subset of stable genes exists that has smaller variance than commonly used reference genes or genes that were selected for their stability across all conditions. Conclusion We therefore propose the normalization of RT-qPCR data using reference genes that are specifically chosen for the conditions under study. RefGenes is a community tool developed for that purpose. Validation RT-qPCR experiments across several organisms showed that the candidates proposed by RefGenes generally outperformed commonly used reference genes. RefGenes is available within Genevestigator at http://www.genevestigator.com

Selection of suitable reference genes for accurate normalization of gene expression profile studies in non-small cell lung cancer

BACKGROUND: In real-time RT quantitative PCR (qPCR) the accuracy of normalized data is highly dependent on the reliability of the reference genes (RGs). Failure to use an appropriate control gene for normalization of qPCR data may result in biased gene expression profiles, as well as low precision, so that only gross changes in expression level are declared statistically significant or patterns of expression are erroneously characterized. Therefore, it is essential to determine whether potential RGs are appropriate for specific experimental purposes. Aim of this study was to identify and validate RGs for use in the differentiation of normal and tumor lung expression profiles. METHODS: A meta-analysis of lung cancer transcription profiles generated with the GeneChip technology was used to identify five putative RGs. Their consistency and that of seven commonly used RGs was tested by using Taqman probes on 18 paired normal-tumor lung snap-frozen specimens obtained from non-small-cell lung cancer (NSCLC) patients during primary curative resection. RESULTS: The 12 RGs displayed showed a wide range of Ct values: except for rRNA18S (mean 9.8), the mean values of all the commercial RGs and ESD ranged from 19 to 26, whereas those of the microarray-selected RGs (BTF-3, YAP1, HIST1H2BC, RPL30) exceeded 26. RG expression stability within sample populations and under the experimental conditions (tumour versus normal lung specimens) was evaluated by: (1) descriptive statistic; (2) equivalence test; (3) GeNorm applet. All these approaches indicated that the most stable RGs were POLR2A, rRNA18S, YAP1 and ESD. CONCLUSION: These data suggest that POLR2A, rRNA18S, YAP1 and ESD are the most suitable RGs for gene expression profile studies in NSCLC. Furthermore, they highlight the limitations of commercial RGs and indicate that meta-data analysis of genome-wide transcription profiling studies may identify new RGs

Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80–90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes.American Society for Radiation Oncology (Junior Faculty Career Research Training Award)Harvard University. Joint Center for Radiation Therapy (Foundation Grant)Dana-Farber/Harvard Cancer Center (SPORE Developmental Research Project Award in Lung Cancer Research)National Cancer Institute (U.S.) (Award K08CA172354

Identification of Reference Genes across Physiological States for qRT-PCR through Microarray Meta-Analysis

The accuracy of quantitative real-time PCR (qRT-PCR) is highly dependent on reliable reference gene(s). Some housekeeping genes which are commonly used for normalization are widely recognized as inappropriate in many experimental conditions. This study aimed to identify reference genes for clinical studies through microarray meta-analysis of human clinical samples.After uniform data preprocessing and data quality control, 4,804 Affymetrix HU-133A arrays performed by clinical samples were classified into four physiological states with 13 organ/tissue types. We identified a list of reference genes for each organ/tissue types which exhibited stable expression across physiological states. Furthermore, 102 genes identified as reference gene candidates in multiple organ/tissue types were selected for further analysis. These genes have been frequently identified as housekeeping genes in previous studies, and approximately 71% of them fall into Gene Expression (GO:0010467) category in Gene Ontology.Based on microarray meta-analysis of human clinical sample arrays, we identified sets of reference gene candidates for various organ/tissue types and then examined the functions of these genes. Additionally, we found that many of the reference genes are functionally related to transcription, RNA processing and translation. According to our results, researchers could select single or multiple reference gene(s) for normalization of qRT-PCR in clinical studies