Biosensors based on zinc oxide thin-film transistors using recyclable plastic substrates as an alternative for real-time pathogen detection.

The fabrication of biosensors has different future applications mainly from the perspective of eco-friendly technologies. Label-free strategies, recyclable materials and low-temperature processing are parameters to consider for the development of a new generation of biosensor devices. In this work, Zinc oxide (ZnO) Thin-film Transistors (TFTs) using recyclable plastic substrates were used for real-time enteropathogenic Escherichia coli detection as an approach for biosensing (bio-TFTs). Fourier Transform Infrared Spectroscopy was used to verify the characteristic absorption peaks at the different steps of the bio-TFT assembly process. The bio-TFTs are ready to observe the bacterial detection by electrical characterization. Finally, detection was validated by a coupled strategy that fuses the genomic DNA extraction from bacteria attached in situ over bio-TFTs surface and, the development of the Polymerase Chain Reaction to amplify specific genes from enteropathogenic Escherichia coli.

Brain-derived neurotrophic factor expression in granulosa lutein cells.

Brain-derived neurotrophic factor (BDNF) is thought to play a role in follicle activation and oocyte maturation. It is postulated that BDNF and its receptor, tyrosine kinase receptor B (TrkB), may also play a role in maintaining the corpus luteum. Therefore,human granulosa lutein cells (GLC) were obtained from women undergoing ovulation induction and treated with increasing concentrations of cAMP (0, 125, 500 and 1000 µmol/l). BDNF and progesterone concentrations were quantified by enzyme-linked immunosorbent assay. cAMP treatment significantly increased progesterone output but had no effect on BDNF concentration in the spent media. However, the BDNF concentration was significantly increased in GLC lysates. To assess the expression of BDNF and TrkB in active versus regressing corpora lutea, ovaries from adult female BALBc mice (n = 4) from each day of the oestrous cycle were processed for immunohistochemistry. Two markers of luteal activity were used (3b-hydroxysteroid dehydrogenase and tenascin-X). There was a trend towards higher BDNF and TrkB H-scores in active versus regressing corpus lutea. In conclusion, intracellular BNDF concentrations were dose-dependently increased by cAMP but treatments had no effect on BDNF output. It is speculated that BDNF contributes in an autocrine manner to GLC survival in the active corpus luteum.

Endometrial microbiota is more diverse in people with endometriosis than symptomatic controls.

Endometriosis is a chronic, estrogen-dependent gynecological condition affecting approximately 10% of reproductive age women. The most widely accepted theory of its etiology includes retrograde menstruation. Recent reports suggest the uterus is not sterile. Thus, the refluxed menstrual effluent may carry bacteria, and contribute to inflammation, the establishment and growth of endometriotic lesions. Here, we compared and contrasted uterine bacteria (endometrial microbiota) in people with surgically confirmed presence (N = 12) or absence of endometriosis (N = 9) using next-generation 16S rRNA gene sequencing. We obtained an average of > 9000 sequence reads per endometrial biopsy, and found the endometrial microbiota of people with endometriosis was more diverse (greater Shannon Diversity Index and proportion of 'Other' taxa) than symptomatic controls (with pelvic pain, surgically confirmed absence of endometriosis; diagnosed with other benign gynecological conditions). The relative abundance of bacterial taxa enriched in the endometrial microbiota of people with endometriosis belonged to the Actinobacteria phylum (Gram-positive), Oxalobacteraceae (Gram-negative) and Streptococcaceae (Gram-positive) families, and Tepidimonas (Gram-negative) genus, while those enriched in the symptomatic controls belonged to the Burkholderiaceae (Gram-negative) family, and Ralstonia (Gram-negative) genus. Taken together, results suggest the endometrial microbiota is perturbed in people with endometriosis.

Daily running trials increase sprint speed in endangered lizards (Gallotia simonyi).

Due to increasing number of animal species in danger of extinction, captive breeding of individuals has become a necessary procedure for many recovery programs. As specimens born and raised in captivity during several generations may not develop some behavioral and performance aptitudes properly, several types of training are useful to apply before releasing them into the wild. We present here the results of experiments aiming to detect the effect of daily running trials in young males of the endangered lizard (Gallotia simonyi) from El Hierro (Canary Islands). We made individuals run in a racetrack twice every day, for five days a week between the end of July and the end of September. We filmed all running trials and calculated running speed for each individual dividing the distance run by the time used. Running speed did not correlate with body condition of the lizards but there was variation in running speeds of some individuals with similar body conditions. Running speed of lizards used in the experiments significantly increased along the whole trial period. By contrast, mean running speed did not change significantly in a control group, participating twice in running trials, one at the beginning and the other at the end of the experimental period. From these results we suggest that locomotor training contributed to increasing final running speeds of experimental lizards.

Identification and functional characterization of polymorphisms in promoter sequences of porcine NOD1 and NOD2 genes.

NOD-like receptors (NLRs) play a key role in the innate immune system, acting as a second line of surveillance against pathogens. NLRs detect particular bacteria that have gained access to the cytoplasm, evading recognition by other pattern recognition receptors, such as Toll-like receptors. It has been demonstrated that coding sequence-single nucleotide polymorphisms may alter the ligand recognition ability of NLRs, affecting their pathogen-sensing function. However, there have been no data relating to the identification and functional analysis of SNPs in porcine NLR promoters. We examined the promoter sequences of the porcine NOD1 and NOD2 genes with the aim to identify and to evaluate the effect of genetic variations on promoter activity. Six SNPs in NOD1 and three SNPs in NOD2 were identified. Luciferase reporter gene assays showed significant differences in promoter activity between allele variants of NOD1 -920G>A (NC_010460.4:g.42431413G>A) and NOD2 -1670G>A (NC_010448.4:g.34169122T>C) SNPs. The results suggest that promoter polymorphisms could modify the expression levels of porcine NOD1 and NOD2 genes.

Bisphenol A concentration-dependently increases human granulosa-lutein cell matrix metalloproteinase-9 (MMP-9) enzyme output.

Bisphenol A (BPA) is an estrogenic contaminant that has been quantified at higher levels in the follicular fluid of women with polycystic ovarian syndrome (PCOS) compared to healthy fertile controls. However, the effect of BPA on granulosa cell function is unknown. Therefore, the objective of the present study was to quantify the effect of BPA on granulosa cell progesterone (P4) output and matrix metalloproteinase (MMP)-2, and -9 output and activity. Granulosa-lutein cells (GLCs) were collected from women undergoing oocyte retrieval in an academic in vitro fertilization (IVF) program. Granulosa-lutein cells were treated with increasing log concentrations of BPA (1-10,000 ng/ml) or 17beta-estradiol (E2, 272 pg/ml or 1.0 nM) and treatment effects on MMP-2 and -9 activity and output, cell viability and cell proliferation were measured by commercial gelatin zymography, MMP-ELISA, MTS and BrdU incorporation assays, respectively. Granulosa-lutein cells in culture secrete MMP-2 and MMP-9. Bisphenol A treatment concentration-dependently increased MMP-9 output by GLCs with a maximal effect observed at 1000 ng/ml. Cell viability/proliferation was unaffected by BPA treatment at concentrations

To What Extent Does Radiotherapy Improve the Quality of Life of Patients With Bone Metastasis?: A Prospective, Single-Institutional Study.

PURPOSE/OBJECTIVES: Radiation therapy (RT) is an effective method of palliating painful bone metastases and improves the quality of life (QoL) of these patients. The purpose of this trial is 2-fold: to quantify the impact of RT in the QoL of patients with bone metastasis and to compare the QoL results between the most used schemes of RT at our Centre. MATERIALS AND METHODS: A consecutive sample of patients with bone metastasis treated with RT in the Complejo Hospitalario de Navarra, Spain, was addressed between January 2011 and November 2012. The QoL was measured with the Quality of Life Questionnaire-C15-Palliative questionnaire, a short version of the European Organisation for Research and Treatment of Cancer-Quality of Life Questionnaire-C30 for palliative care. Two assessments were proposed for each patient: one on the first day of the treatment and the other one a month after the end of the radiotherapy sessions. One hundred and sixteen patients completed the first questionnaire and 75 completed the second one (65%). RESULTS: Significant differences appeared in 9 domains, with better QoL in the second assessment. Five areas (physical functioning, global, fatigue, nausea, dyspnea, and constipation) showed little change (between 5 and 9 points), 3 (emotional functioning, insomnia, and appetite loss) showed moderate change (10 to 20 points), and 1 (pain) showed a very positive change (>30 points).When we compare the QoL scores between the 2 most used schemes of RT (30 Gy/10 fractions vs. 20 Gy/4 to 5 fractions), there are no significant differences in any QoL areas (and in 2 areas P was near 0.05). CONCLUSIONS: Palliative RT is a very active treatment for patients with bone metastasis regardless of age, location, primary tumor, or RT scheme. RT significantly improves the QoL, fundamentally by controlling pain and reducing analgesic use. Shorter schemes of RT produce at least-if not better-the same effect on QL than longer schemes (30 Gy).

Comparison of aerobic and photosynthetic Rhodobacter sphaeroides 2.4.1 proteomes.

The analysis of proteomes from aerobic and photosynthetic Rhodobacter sphaeroides 2.4.1 cell cultures by liquid chromatography-mass spectrometry yielded approximately 6,500 high confidence peptides representing 1,675 gene products (39% of the predicted proteins). The identified proteins corresponded primarily to open reading frames (ORFs) contained within the two chromosomal elements of this bacterium, but a significant number were also observed from ORFs associated with 5 naturally occurring plasmids. Using the accurate mass and time (AMT) tag approach, comparative studies showed that a number of proteins were uniquely detected within the photosynthetic cell culture. The estimated abundances of proteins observed in both aerobic respiratory and photosynthetic grown cultures were compared to provide insights into bioenergetic models for both modes of growth. Additional emphasis was placed on gene products annotated as hypothetical to gain information as to their potential roles within these two growth conditions. Where possible, transcriptome and proteome data for R. sphaeroides obtained under the same culture conditions were also compared.

Deciphering the interface between a CD40 receptor and borrelial ligand OspA.

Neuroborreliosis is serious sequelae of Lyme borreliosis. Neuroinvasion is largely relied on successful translocation of Borrelia across the blood-brain barrier. Adherence of Borrelia to brain microvascular endothelial cell (BMEC) seems to be critical for translocation. Here we unfold the interface between OspA and CD40 molecules, major ligand and receptor, that are involved in adhesion of Borrelia to BMECs. We found that a region between Asn127 and Asp205 of OspA forms the CD40-receptor binding site. This region encompasses human umbilical vein endothelial cell (HUVEC) binding domain and contains a potential ligand-binding pocket lined by three amino acid residues: Arg139, Glu160 and Lys189. Disruption of this pocket (by truncation of the HUVEC binding domain) caused complete abrogation of its ability to bind CD40. To identify the amino acid residues within the HUVEC binding domain involved in the CD40 binding, site-directed mutagenesis and binding assays were performed. Results showed that Asp149, Phe165, Ala172, Val186 and Leu192 might form interface with CD40 molecule. Other side of the interface was also identified with the help of a ligand-binding assay with OspA and truncated CD40 fragments. Results exposed that cysteine rich domain 2 (CRD2) of CD40 might be the site for OspA binding. Precise knowledge of the molecular basis of the ligand-receptor interactions is essential in order to understand mechanisms of pathogenesis and could help in the development of novel therapeutics and vaccines.

Identification and functional characterization of novel genetic variations in porcine TLR5 promoter.

Toll-like receptors (TLRs) play a critical role in the immune process acting as innate sensors of pathogens. Toll-like receptor 5 (TLR5) is especially relevant in those tissues maintaining close contact with microorganisms, not only because it prevents infections but also due to its involvement in the regulation of host-commensal interactions. Recent studies suggest that the occurrence of genetic polymorphisms may impair TLR function, consequently increasing or decreasing the individual susceptibility to infectious diseases. In this study, the promoter sequence of the porcine TLR5 gene was scanned with the aim of identifying mutations with potential effects on gene expression. Two Indel variations in the predicted promoter sequence and seven single-nucleotide polymorphisms were identified. The luciferase reporter gene assay indicated that one Indel, consisting in a 23-bp insertion at the -581 to -559 nucleotide position, creates an additional STAT binding site, and it is associated with an increase of the promoter activity. This finding suggests that genetic variation in the TLR5 promoter could alter the expression of the gene, and may be used as a molecular marker to define pathogen susceptibility or resistance patterns in pigs.

Universal genetic assay for engineering extracellular protein expression.

A variety of strategies now exist for the extracellular expression of recombinant proteins using laboratory strains of Escherichia coli . However, secreted proteins often accumulate in the culture medium at levels that are too low to be practically useful for most synthetic biology and metabolic engineering applications. The situation is compounded by the lack of generalized screening tools for optimizing the secretion process. To address this challenge, we developed a genetic approach for studying and engineering protein-secretion pathways in E. coli . Using the YebF pathway as a model, we demonstrate that direct fluorescent labeling of tetracysteine-motif-tagged secretory proteins with the biarsenical compound FlAsH is possible in situ without the need to recover the cell-free supernatant. High-throughput screening of a bacterial strain library yielded superior YebF expression hosts capable of secreting higher titers of YebF and YebF-fusion proteins into the culture medium. We also show that the method can be easily extended to other secretory pathways, including type II and type III secretion, directly in E. coli . Thus, our FlAsH-tetracysteine-based genetic assay provides a convenient, high-throughput tool that can be applied generally to diverse secretory pathways. This platform should help to shed light on poorly understood aspects of these processes as well as to further assist in the construction of engineered E. coli strains for efficient secretory-protein production.

Development of an automated platform for high-throughput P1-phage transduction of Escherichia coli.

Synthetic biology depends on the ability to rapidly produce strains with improved phenotypes but is limited by the ability to rapidly produce strain collections with directed mutations. Here, we present a system capable of overcoming this limitation through automated P1-phage transductions of Escherichia coli. By combining the Keio collection of single-gene deletion E. coli mutants with P1-phage, it is possible to generate an engineered host-strain collection consisting of every possible gene deletion mutant. This strategy was tested by transducing 355 genetic markers from the Keio collection into five different host strains, and it achieved a 98% success rate. This method offers an improved mechanism for rapidly engineering collections of microbes and provides one method for rapidly deploying a broader synthetic biology effort.

PITASOR epidemiological study: prevalence, incidence and treatment of anaemia in radiation therapy oncology departments in Spain.

INTRODUCTION: Anemia is the most common haematological complication in cancer patients. OBJECTIVE: Analysis of the incidence, prevalence and treatment of anemia in oncologic patients treated in Radiation Oncology Departments in Spain (ROD) and monitoring of the existing recommendations for the treatment of anemia. MATERIAL AND METHODS: Observational, prospective, multicenter study which involved 19 Spanish ROD. The study was approved by the CEIC Central Defense Hospital. 477 patients with solid tumors, subsidiary of RT with radical intent referred to such centers within a period of one month (5/5/09 to 5/6/09) and gave their consent to participate in the study. We gathered the main characteristics of patients and their oncologic disease. All patients underwent a determination of Hb levels before RT, upon reaching 25-35 Gy and at the end treatment. In patients with anemia we assessed the existence of related symptoms and its treatment. RESULTS: Basal situation: The prevalence of anemia was 34.8% (166 patients). Mean Hb in patients with anemia was 11.17 ± 1.07 g/dl. Anemia-related symptoms were present in 34% of the patients. Anemia predisposing factors were: stage of the disease, previously received chemotherapy, and hormonal therapy. 39% (66 patients) received anemia treatment, with a mean Hb of 10.43 ± 1.04 g/dl. During RT: The prevalence of anemia was 38.9% (182 patients) with a mean Hb of 11.24 ± 1.21 g/dl. Predisposing factors for anemia during RT treatment were: age, male sex, chemotherapy prior to RT, basal anemia and chemotherapy during RT. 36.3% (66 patients) had anemia-related symptoms. 34.6% (63 patients) with a mean Hb of 10.5 ± 1.37 g/dl received treatment for anemia. The prevalence of anemia at the end of the RT was 38.1% (177 patients) with a mean Hb of 11.19 ± 1.18 g/dl. The predisposing factors for the appearance of anemia at the end of RT were: male sex, anemia at basal situation and during treatment and chemotherapy during RT. 34% (61 patients) had anemia-related symptoms and 73 patients (41.2%) with a mean Hb of 10.5 ± 1.22 g/dl received treatment for anemia. The presence of anemia-related symptoms was significantly correlated with the beginning of treatment for anemia. The incidence of anemia (new cases) during radiotherapy was 17.5%. CONCLUSION: The prevalence of anemia in basal situation, during RT and at the end of RT is 34.8%, 38.9% and 38.1%. During RT the incidence of anemia is 17.5%. 39.8%-41.2% of patients with anemia and 64.2%-68% of patients with anemia-related symptoms received treatment. Treatment of anemia starts with Hb<11 g/dl and the goal is to achieve Hb 12 g/dl. In our Radiotherapy Oncology Departments, the treatment of anemia complies with the current recommendations and guidelines in use.

Application of the accurate mass and time tag approach to the proteome analysis of sub-cellular fractions obtained from Rhodobacter sphaeroides 2.4.1. Aerobic and photosynthetic cell cultures.

The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8,300 peptides were identified with high confidence from at least one subcellular fraction from either cell culture. These peptides were derived from 1,514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single subcellular fraction and their localization was compared to in silico predictions. However, the majority of proteins were observed in multiple subcellular fractions, and the most likely subcellular localization for these proteins was investigated using a Z-score analysis of estimated protein abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical.