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Dessi M.,University of Rome Tor Vergata | Noce A.,University of Rome Tor Vergata | Dawood K.F.,University of Rome Tor Vergata | Galli F.,University of Perugia | And 10 more authors.
Amino Acids | Year: 2012

The erythrocyte glutathione S-transferase (e-GST) is a member of a superfamily of inducible enzymes involved in cell detoxification that shows an increased expression in chronic kidney disease (CKD) patients. We propose a new automated analysis procedure for e-GST activity that has been validated in 72 CKD patients and 62 maintenance hemodialysis patients (MHD). Regression analysis was carried out to assess association between e-GST activity data, main clinical variables, and plasma homocysteine (Hcy), a modified sulfur amino acid known as potential risk factor for cardiovascular disease that is increased above normal levels in more than 90% of the uremic patients. An increased e-GST activity was confirmed in MHD patients (N = 62; 10.2 ± 0.4 U/gHb) compared with healthy subjects (N = 80; 5.8 ± 0.4 U/gHb), and as an original finding, a significant increase of e-GST activity was observed in pre-dialysis CKD patients with a positive correlation with disease severity weighted according to the four stages of "Kidney Disease Outcomes Quality Initiative" classification (7.4 ± 0.5, 8 ± 1, 9.5 ± 0.6, 12 ± 1 U/gHb, respectively). No correlation was found between e-GST activity and hemoglobin, transferrin, blood iron and the markers of systemic inflammation and renal function such as alpha-1 acid glycoprotein and high-sensitive C-Reactive Protein, beta-2 microglobulin and the index of malnutrition-inflammation PINI, while a significant correlation was observed for the first time between plasma Hcy and e-GST activity (r 2 = 0.64, P < 0.0001) in MHD patients. Hcy, however, was not identified as an inhibitor of e-GST enzyme. The results in this study suggest the potential for automated e-GST analysis as a valuable tool to further explore phase II-related uremic toxicity in CKD and MHD patients. © 2011 Springer-Verlag.

Uboldi S.,Mario Negri Institute for Pharmacological Research | Bernasconi S.,Mario Negri Institute for Pharmacological Research | Romano M.,Mario Negri Institute for Pharmacological Research | Marchini S.,Mario Negri Institute for Pharmacological Research | And 20 more authors.
International Journal of Cancer | Year: 2012

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O 6-methylguanine-DNA- methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and β, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin. Copyright © 2011 UICC.

Walczak-Sztulpa J.,Max Planck Institute for Molecular Genetics | Walczak-Sztulpa J.,Poznan University of Medical Sciences | Eggenschwiler J.,Princeton University | Osborn D.,University College London | And 17 more authors.
American Journal of Human Genetics | Year: 2010

Cranioectodermal dysplasia (CED) is a disorder characterized by craniofacial, skeletal, and ectodermal abnormalities. Most cases reported to date are sporadic, but a few familial cases support an autosomal-recessive inheritance pattern. Aiming at the elucidation of the genetic basis of CED, we collected 13 patients with CED symptoms from 12 independent families. In one family with consanguineous parents two siblings were affected, permitting linkage analysis and homozygosity mapping. This revealed a single region of homozygosity with a significant LOD score (3.57) on chromosome 3q21-3q24. By sequencing candidate genes from this interval we found a homozygous missense mutation in the IFT122 (WDR10) gene that cosegregated with the disease. Examination of IFT122 in our patient cohort revealed one additional homozygous missense change in the patient from a second consanguineous family. In addition, we found compound heterozygosity for a donor splice-site change and a missense change in one sporadic patient. All mutations were absent in 340 control chromosomes. Because IFT122 plays an important role in the assembly and maintenance of eukaryotic cilia, we investigated patient fibroblasts and found significantly reduced frequency and length of primary cilia as compared to controls. Furthermore, we transiently knocked down ift122 in zebrafish embryos and observed the typical phenotype found in other models of ciliopathies. Because not all of our patients harbored mutations in IFT122, CED seems to be genetically heterogeneous. Still, by identifying CED as a ciliary disorder, our study suggests that the causative mutations in the unresolved cases most likely affect primary cilia function too. © 2010 The American Society of Human Genetics.

Meini G.,University of Siena | Balestrieri M.,University of Siena | Cianchino S.,University of Siena | Tacconi D.,Unit of Infectious Diseases | And 7 more authors.
AIDS Research and Human Retroviruses | Year: 2015

A total of 81 HIV-1 protease (PR) and reverse transcriptase (RT) sequences were obtained from 46 drug-naive and 35 pretreated individual HIV-1-infected orphaned children followed at a donor-funded rural pediatric clinic in Dodoma, Tanzania. PR and RT sequencing was performed by home-brew technology on 70 plasma samples and 11 dried blood spot specimens. Nucleoside RT inhibitor (NRTI) resistance mutations were detected in 2.2% of drug-naive and 82.9% of pretreated children. Nonnucleoside RT inhibitor (NNRTI) resistance mutations were detected in 69.6% of drug-naive and 91.4% of pretreated children. Resistance to protease inhibitors was rare (8.6% in pretreated children). Based on few complete treatment records, only around 20% of the treated children had undetectable plasma HIV-1 RNA. The rate of NRTI and NNRTI resistance in this donor-funded rural pediatric clinic was high and appeared to limit virological response to treatment. © Copyright 2015, Mary Ann Liebert, Inc. 2015.

Putignani L.,Research Institute Bambino Gesu | Del Chierico F.,Research Institute Bambino Gesu | Onori M.,Research Institute Bambino Gesu | Mancinelli L.,Research Institute Bambino Gesu | And 11 more authors.
Molecular BioSystems | Year: 2011

Proteomics is particularly suitable for characterising human pathogens with high life cycle complexity, such as fungi. Protein content and expression levels may be affected by growth states and life cycle morphs and correlate to species and strain variation. Identification and typing of fungi by conventional methods are often difficult, time-consuming and frequently, for unusual species, inconclusive. Proteomic phenotypes from MALDI-TOF MS were employed as analytical and typing expression profiling of yeast, yeast-like species and strain variants in order to achieve a microbial proteomics population study. Spectra from 303 clinical isolates were generated and processed by standard pattern matching with a MALDI-TOF Biotyper (MT). Identifications (IDs) were compared to a reference biochemical-based system (Vitek-2) and, when discordant, MT IDs were verified with genotyping IDs, obtained by sequencing the 25-28S rRNA hypervariable D2 region. Spectra were converted into virtual gel-like formats, and hierarchical clustering analysis was performed for 274 Candida profiles to investigate species and strain typing correlation. MT provided 257/303 IDs consistent with Vitek-2 ones. However, amongst 26/303 discordant MT IDs, only 5 appeared "true". No MT identification was achieved for 20/303 isolates for incompleteness of database species variants. Candida spectra clustering agreed with identified species and topology of Candida albicans and Candida parapsilosis specific dendrograms. MT IDs show a high analytical performance and profiling heterogeneity which seems to complement or even outclass existing typing tools. This variability reflects the high biological complexity of yeasts and may be properly exploited to provide epidemiological tracing and infection dispersion patterns. © The Royal Society of Chemistry 2011.

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