Laboratory of Molecular Diagnostics

Rio de Janeiro, Brazil

Laboratory of Molecular Diagnostics

Rio de Janeiro, Brazil

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Sikorska K.,Medical University of Gdańsk | Bernat A.,Laboratory of Molecular Diagnostics | Wroblewska A.,Laboratory of Molecular Diagnostics
Hepatobiliary and Pancreatic Diseases International | Year: 2016

Background The liver, as the main iron storage compartment and the place of hepcidin synthesis, is the central organ involved in maintaining iron homeostasis in the body. Excessive accumulation of iron is an important risk factor in liver disease progression to cirrhosis and hepatocellular carcinoma. Here, we review the literature on the molecular pathogenesis of iron overload and its clinical consequences in chronic liver diseases. Data Sources PubMed was searched for English-language articles on molecular genesis of primary and secondary iron overload, as well as on their association with liver disease progression. We have also included literature on adjuvant therapeutic interventions aiming to alleviate detrimental effects of excessive body iron load in liver cirrhosis. Results Excess of free, unbound iron induces oxidative stress, increases cell sensitivity to other detrimental factors, and can directly affect cellular signaling pathways, resulting in accelerated liver disease progression. Diagnosis of liver cirrhosis is, in turn, often associated with the identification of a pathological accumulation of iron, even in the absence of genetic background of hereditary hemochromatosis. Iron depletion and adjuvant therapy with antioxidants are shown to cause significant improvement of liver functions in patients with iron overload. Phlebotomy can have beneficial effects on liver histology in patients with excessive iron accumulation combined with compensated liver cirrhosis of different etiology. Conclusion Excessive accumulation of body iron in liver cirrhosis is an important predictor of liver failure and available data suggest that it can be considered as target for adjuvant therapy in this condition. © 2016 The Editorial Board of Hepatobiliary & Pancreatic Diseases International


De Sa L.R.V.,Brazilian National Institute of Technology | De Sa L.R.V.,Federal University of Rio de Janeiro | Cammarota M.C.,Federal University of Rio de Janeiro | De Oliveira T.C.,Laboratory of Molecular Diagnostics | And 4 more authors.
International Journal of Hydrogen Energy | Year: 2013

Biofuels production in Brazil is a traditional activity, and it has becoming more important each year. Within this context, biohydrogen production could exploit residual streams from first generation ethanol (ethanol 1G), second generation ethanol (ethanol 2G) and biodiesel production. Therefore hexoses, pentoses and glycerin were tested as substrates for hydrogen production. Firstly, the effects of different inoculum pretreatments (acid, alkaline and heat) on bacterial communities' performance were evaluated through the levels of Clostridium hydrogenase expression. The heat pretreated inoculum provided the highest yield of H2 (4.62 mol H2/mol sucrose) and also the highest level of hydrogenase expression, 64 times higher when compared with untreated inoculum after 72 h. Then C5 and C6 sugars and also glycerin were tested for H2 production (35 °C and pH 5.5), which resulted in promising yields of H2: sucrose (4.24 mol H 2/mol sucrose), glucose (2.19 mol H2/mol glucose), fructose (2.09 mol H2/mol fructose), xylose (1.88 mol H 2/mol xylose) and glycerin (0.80 mol H2/mol glycerin).


De Sa L.R.V.,Brazilian National Institute of Technology | De Sa L.R.V.,Federal University of Rio de Janeiro | De Oliveira T.C.,Laboratory of Molecular Diagnostics | Dos Santos T.F.,Iguau University | And 4 more authors.
International Journal of Hydrogen Energy | Year: 2011

The conversion of organic compounds into H2 has received increasing attention. Enrichment of inocula by heat pretreatment eliminates non-spore forming hydrogen consuming microorganisms and promotes spore germination in genus Clostridium, which is known as one of the key hydrogen producers. Useful information about metabolic pathway is provided by some intermediate metabolites, such as: acetic, propionic, butyric and formic acids. The increase of acetic/butyric acids ratio indicates H2 production in heat pretreated inoculum when compared to untreated inoculum in the same cultivation conditions. The effect of heat pretreatment on inocula and consequently on the performance of bacterial communities responsible for H 2 production was monitored through the measurement of the level of hydrogenase gene expression, as well as through the content and distribution of volatile fatty acids. The acetic acid type fermentation was followed by the microorganisms presented in untreated and heat pretreated sludge. The medium containing untreated sludge presented a ratio of acetic/butyric acid of approximately 4, the same parameter was 7 when heat pretreated sludge was employed. The level of hydrogenase gene expression tripled when heat pretreated inoculum was used, indicating a higher production of H2. © 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights.


PubMed | Medical University of Gdańsk and Laboratory of Molecular Diagnostics
Type: | Journal: Clinical and experimental medicine | Year: 2016

Single nucleotide polymorphisms (SNPs) within DNA region containing interferon lambda 3 (IFNL3) and IFNL4 genes are prognostic factors of treatment response in chronic hepatitis C (CHC). Iron overload, frequently diagnosed in CHC, is associated with unfavorable disease course and a risk of carcinogenesis. Its etiology and relationship with the immune response in CHC are not fully explained. Our aim was to determine whether IFNL polymorphisms in CHC patients associate with body iron indices, and whether they are linked with hepatic expression of genes involved in iron homeostasis and IFN signaling. For 192 CHC patients, four SNPs within IFNL3-IFNL4 region (rs12979860, rs368234815, rs8099917, rs12980275) were genotyped. In 185 liver biopsies, histopathological analyses were performed. Expression of five mRNAs and three long non-coding RNAs (lncRNAs) was determined with qRT-PCR in 105 liver samples. Rs12979860 TT or rs8099917 GG genotypes as well as markers of serum and hepatocyte iron overload associated with higher activity of gamma-glutamyl transpeptidase and liver steatosis. The presence of two minor alleles in any of the tested SNPs predisposed to abnormally high serum iron concentration and correlated with higher hepatic expression of lncRNA NRIR. On the other hand, homozygosity in any major allele associated with higher viral load. Patients bearing rs12979860 CC genotype had lower hepatic expression of hepcidin (HAMP; P=0.03). HAMP mRNA level positively correlated with serum iron indices and degree of hepatocyte iron deposits. IFNL polymorphisms influence regulatory pathways of cellular response to IFN and affect body iron balance in chronic hepatitis C virus infection.


Krychowiak M.,Medical University of Gdańsk | Grinholc M.,Laboratory of Molecular Diagnostics | Banasiuk R.,Medical University of Gdańsk | Krauze-Baranowska M.,Medical University of Gdańsk | And 3 more authors.
PLoS ONE | Year: 2014

Staphylococcus aureus is the most common infectious agent involved in the development of skin infections that are associated with antibiotic resistance, such as burn wounds. As drug resistance is a growing problem it is essential to establish novel antimicrobials. Currently, antibiotic resistance in bacteria is successfully controlled by multi-drug therapies. Here we demonstrate that secondary metabolites present in the extract obtained from Drosera binata in vitro cultures are effective antibacterial agents against S. aureus grown in planktonic culture and in biofilm. Moreover, this is the first report demonstrating the synergistic interaction between the D. binata extract and silver nanoparticles (AgNPs), which results in the spectacular enhancement of the observed bactericidal activity, while having no cytotoxic effects on human keratinocytes. Simultaneous use of these two agents in significantly reduced quantities produces the same effect, i.e. by killing 99.9% of bacteria in inoculum or eradicating the staphylococcal biofilm, as higher amounts of the agents used individually. Our data indicates that combining AgNPs with either the D. binata extract or with its pure compound (3-chloroplumbagin) may provide a safe and highly effective alternative to commonly used antibiotics, which are ineffective towards the antibiotic-resistant S. aureus. © 2014 Krychowiak et al.


Palmirotta R.,Laboratory of Molecular Diagnostics | De Marchis M.L.,Laboratory of Molecular Diagnostics | Ludovici G.,Laboratory of Molecular Diagnostics | Leone B.,Laboratory of Molecular Diagnostics | And 8 more authors.
International Journal of Biological Markers | Year: 2012

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR). Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications. © 2012 Wichtig Editore.


Palmirotta R.,Laboratory of Molecular Diagnostics | De Marchis M.L.,Laboratory of Molecular Diagnostics | Ludovici G.,Laboratory of Molecular Diagnostics | Leone B.,Laboratory of Molecular Diagnostics | And 6 more authors.
Human Mutation | Year: 2012

Familial adenomatous polyposis (FAP) is an autosomal-dominant conditionmainly due to amutation of the adenomatous polyposis coli (APC) gene. The present study reports evidence of a technical issue occurring during the mutational analysis of APC exon 4. Genetic conventional direct sequence analysis of a repetitive AT-rich region in the splice acceptor site of APC intron 3 could be misinterpreted as a pathogenetic frameshift result. However, this potential bias may be bypassed adopting a method for random mutagenesis of DNA based on the use of a triphosphate nucleoside analogues mixture. Using this method as a second-level analysis, we also demonstrated the nonpathogenic nature of the variant in the poly A trait in APC exon 4 region (c.423-4delA) that do not result in aberrant splicing of APC exons 3-4; conversely, we did not find a previously reported T deletion/insertion polymorphism. © 2012 Wiley Periodicals, Inc.


Van Lint P.,Laboratory of Molecular Diagnostics | Rossen J.W.,St Elisabeth Hospital | Vermeiren S.,Laboratory of Molecular Diagnostics | Ver Elst K.,Laboratory of Molecular Diagnostics | And 5 more authors.
Acta Clinica Belgica | Year: 2013

Diagnosis of intestinal parasites in stool samples is generally still carried out by microscopy; however, this technique is known to suffer from a low sensitivity and is unable to discriminate between certain protozoa. In order to overcome these limitations, a real-time multiplex PCR was evaluated as an alternative approach for diagnosing Giardia lamblia, Cryptosporidium spp. and Entamoeba histolytica in stool samples. Therefore, a total of 631 faecal samples were analysed both by microscopy as well as by real-time PCR following automated DNA extraction. Results showed that real-time PCR exhibited sensitivity and specificity of both 100%, whereas traditional microscopy exhibited sensitivity and specificity of 37.5% and 99.8% respectively. As real-time PCR provides simple, sensitive and specific detection of these three important pathogenic protozoan parasites, this technique, rather than microscopy, has become our diagnostic method of choice for the detection of enteric protozoan parasites for the majority of patients.


PubMed | Laboratory of Molecular Diagnostics
Type: Journal Article | Journal: Human mutation | Year: 2012

Familial adenomatous polyposis (FAP) is an autosomal-dominant condition mainly due to a mutation of the adenomatous polyposis coli (APC) gene. The present study reports evidence of a technical issue occurring during the mutational analysis of APC exon 4. Genetic conventional direct sequence analysis of a repetitive AT-rich region in the splice acceptor site of APC intron 3 could be misinterpreted as a pathogenetic frameshift result. However, this potential bias may be bypassed adopting a method for random mutagenesis of DNA based on the use of a triphosphate nucleoside analogues mixture. Using this method as a second-level analysis, we also demonstrated the nonpathogenic nature of the variant in the poly A trait in APC exon 4 region (c.423-4delA) that do not result in aberrant splicing of APC exons 3-4; conversely, we did not find a previously reported T deletion/insertion polymorphism.


PubMed | Unit of Medical Oncology 2, Clinical Epidemiology Unit, Unit of Pathology, Cellular Biology Unit and 5 more.
Type: | Journal: Journal of translational medicine | Year: 2015

Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab.Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcRIIIA 158V>F and FcRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by (51)Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab.We found that the FcRIIIA 158F and/or the FcRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006).Although this study was performed in a limited number of patients, it would indicate a correlation of FcR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory.

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