Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR.

The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs).

Pleiotropic Effects of PhaR Regulator in Microaerobic Metabolism.

can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium.

Surface Plasmon Resonance as a Tool to Elucidate the Molecular Determinants of Key Transcriptional Regulators Controlling Rhizobial Lifestyles.

Bacteria must be provided with a battery of tools integrated into regulatory networks, in order to respond and, consequently, adapt their physiology to changing environments. Within these networks, transcription factors finely orchestrate the expression of genes in response to a variety of signals, by recognizing specific DNA sequences at their promoter regions. Rhizobia are host-interacting soil bacteria that face severe changes to adapt their physiology from free-living conditions to the nitrogen-fixing endosymbiotic state inside root nodules associated with leguminous plants.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis.

Fine-Tuning Modulation of Oxidation-Mediated Posttranslational Control of FixK Transcription Factor.

FixK is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont . Aside from the balanced expression of the gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK, we replaced C183 for aspartic acid.

Effect of Copper on Expression of Functional Genes and Proteins Associated with Denitrification.

Nitrous oxide (NO) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of NO in soils. The soybean endosymbiont is a model for rhizobial denitrification studies since, in addition to fixing N, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway.

Dissection of FixK protein-DNA interaction unveils new insights into Bradyrhizobium diazoefficiens lifestyles control.

The FixK protein plays a pivotal role in a complex regulatory network, which controls genes for microoxic, denitrifying, and symbiotic nitrogen-fixing lifestyles in Bradyrhizobium diazoefficiens. Among the microoxic-responsive FixK -activated genes are the fixNOQP operon, indispensable for respiration in symbiosis, and the nnrR regulatory gene needed for the nitric-oxide dependent induction of the norCBQD genes encoding the denitrifying nitric oxide reductase. FixK is a CRP/FNR-type transcription factor, which recognizes a 14 bp-palindrome (FixK box) at the regulated promoters through three residues (L195, E196, and R200) within a C-terminal helix-turn-helix motif.

Bacterial nitric oxide metabolism: Recent insights in rhizobia.

Nitric oxide (NO) is a reactive gaseous molecule that has several functions in biological systems depending on its concentration. At low concentrations, NO acts as a signaling molecule, while at high concentrations, it becomes very toxic due to its ability to react with multiple cellular targets. Soil bacteria, commonly known as rhizobia, have the capacity to establish a N-fixing symbiosis with legumes inducing the formation of nodules in their roots.

Expanding the Regulon of the NnrR Transcription Factor: New Insights Into the Denitrification Pathway.

Denitrification in the soybean endosymbiont is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK and NnrR exert disparate control on expression of core denitrifying systems encoded by , , , and genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in , we compared the transcriptional profile of an mutant with that of the wild type, both grown under anoxic denitrifying conditions.

An Integrated Systems Approach Unveils New Aspects of Microoxia-Mediated Regulation in .

The adaptation of rhizobia from the free-living state in soil to the endosymbiotic state comprises several physiological changes in order to cope with the extremely low oxygen availability (microoxia) within nodules. To uncover cellular functions required for bacterial adaptation to microoxia directly at the protein level, we applied a systems biology approach on the key rhizobial model and soybean endosymbiont USDA 110 (formerly USDA 110). As a first step, the complete genome of 1104, the model strain used in most prior functional genomics studies, was sequenced revealing a deletion of a ~202 kb fragment harboring 223 genes and several additional differences, compared to strain USDA 110.

FixK Is the Main Transcriptional Activator of Genes in Response to Low Oxygen.

The powerful greenhouse gas, nitrous oxide (NO) has a strong potential to drive climate change. Soils are the major source of NO and microbial nitrification and denitrification the main processes involved. The soybean endosymbiont is considered a model to study rhizobial denitrification, which depends on the , and genes.

Dissecting the role of NtrC and RpoN in the expression of assimilatory nitrate and nitrite reductases in Bradyrhizobium diazoefficiens.

Bradyrhizobium diazoefficiens, a nitrogen-fixing endosymbiont of soybeans, is a model strain for studying rhizobial denitrification. This bacterium can also use nitrate as the sole nitrogen (N) source during aerobic growth by inducing an assimilatory nitrate reductase encoded by nasC located within the narK-bjgb-flp-nasC operon along with a nitrite reductase encoded by nirA at a different chromosomal locus. The global nitrogen two-component regulatory system NtrBC has been reported to coordinate the expression of key enzymes in nitrogen metabolism in several bacteria.

An integrated biochemical system for nitrate assimilation and nitric oxide detoxification in Bradyrhizobium japonicum.

Rhizobia are recognized to establish N2-fixing symbiotic interactions with legume plants. Bradyrhizobium japonicum, the symbiont of soybeans, can denitrify and grow under free-living conditions with nitrate (NO3 (-)) or nitrite (NO2 (-)) as sole nitrogen source. Unlike related bacteria that assimilate NO3 (-), genes encoding the assimilatory NO3 (-) reductase (nasC) and NO2 (-) reductase (nirA) in B.

The value of thyroperoxidase as a prognostic factor for differentiated thyroid cancer -- a long-term follow-up study.

Background: Thyroperoxidase (TPO) is a membrane-bound protein essential for the production of thyroid hormones; because of this, TPO expression may be impaired in selected thyroid diseases. The goal of this study is to analyze TPO immune expression in differentiated thyroid cancer, and to determine whether TPO has any prognostic value.

Methods: A total of 139 patients who required surgery due to a thyroid nodule with signs or symptoms suspicious for malignancy during their physical, ultrasound and/or cytology examination were consecutively selected for the study.

Late bone metastasis from an apparently benign oncocytic follicular thyroid tumor.

Unlabelled: A man underwent total thyroidectomy for goiter when he was 62 years old. The pathology report informed on a 5.5 cm oncocytic follicular adenoma and a 3.

The nitric oxide response in plant-associated endosymbiotic bacteria.

Nitric oxide (NO) is a gaseous signalling molecule which becomes very toxic due to its ability to react with multiple cellular targets in biological systems. Bacterial cells protect against NO through the expression of enzymes that detoxify this molecule by oxidizing it to nitrate or reducing it to nitrous oxide or ammonia. These enzymes are haemoglobins, c-type nitric oxide reductase, flavorubredoxins and the cytochrome c respiratory nitrite reductase.

Nitric oxide detoxification in the rhizobia-legume symbiosis.

NO (nitric oxide) is a signal molecule involved in diverse physiological processes in cells which can become very toxic under certain conditions determined by its rate of production and diffusion. Several studies have clearly shown the production of NO in early stages of rhizobia-legume symbiosis and in mature nodules. In functioning nodules, it has been demonstrated that NO, which has been reported as a potent inhibitor of nitrogenase activity, can bind Lb (leghaemoglobin) to form LbNOs (nitrosyl-leghaemoglobin complexes).

Short alleles of both GGN and CAG repeats at the exon-1 of the androgen receptor gene are associated to increased PSA staining and a higher Gleason score in human prostatic cancer.

The exon 1 of the human androgen receptor (AR) gene contains two length polymorphisms of CAG (polyglutamine) and GGN (polyglycine). "In vitro" experiments suggest that the larger GGN repeats provide a lower AR-protein yield, whereas the larger CAG repeats decrease the AR transcriptional activity, both decreasing the AR signalling intensity. Here we have tested such possibilities in human prostatic cancer (CaP) specimens.

Evaluation of acute hepatotoxic effects exerted by environmental estrogens nonylphenol and 4-octylphenol in immature male rats.

Nonylphenol (NP) and 4-Octylphenol (4OP) have shown estrogenic properties both in vivo and in vitro. Researchers have known for years that estrogens induce a wide number of hepatotoxic actions in rodents. In order to study the acute hepatic effects exerted by NP and 4OP on rat liver the following endpoints were evaluated: relative liver weight (RLW), morphology, cell cycle and ploidy status, monooxygenase enzymes content and levels of both, cytosolic estrogen receptor (cER) and microsomal binding sites for estrogens (mEBS).

Steroid hormone progesterone induces cell proliferation and abnormal mitotic processes in rat liver.

The aim of this study was to evaluate the acute hepatic effects exerted by the steroid hormone progesterone (PR) in the rat. Although the liver is not a target tissue for this hormone, a number of hepatic actions of PR have been described, and, furthermore, a specific binding site for PR (PBS) exists in rat liver microsomes. Immature male rats were treated intraperitoneally with 60 mg/kg PR per day for 1, 5 or 10 days, and different parameters were evaluated in order to detect possible alterations in liver cells.