Patterning biomolecules using AFM charge-writing on polymeric and silicon dioxide substrates

Electrostatic interactions are common-place in biological processes with biomolecules typically charged in solution. In this thesis electrostatic forces were used for the spatial manipulation of biomolecules by driving their attachment to surfaces patterned with localised charges. Charge-patterns can be created on dielectric materials using Atomic Force Microscope Charge-Writing and imaged by Kelvin-probe Force Microscopy with a resolution of one hundred nanometres. There are a wide variety of materials that can be used for charge-patterning including polymers such as poly (methyl methacrylate) (PMMA) and polystyrene (PS), which are also commonly used in nanofabrication and in biological environments. Silicon dioxide is also well known for its charge storage capabilities. Since it is a key material for microfabrication and electronic devices, achieving controlled attachment of biomolecules on its surface would have far-reaching applications for the development of novel biosensors. The charge-pattern stability on PMMA, PS and Si02 was investigated regarding their preparation and the surrounding media. The charge-patterns were subsequently used to localise biomolecular attachment. The biomolecules were enclosed in water droplets dispersed in a dielectric oil to pre- vent the rapid decay of charge-patterns in an aqueous environment. The droplets were attracted electrostatically to the charge-patterns. After removing the liquid, the contents of the droplets remained localised on the surface and a resolution of approximately 1 fim was achieved. To illustrate the repeatability of the process, different types of biomolecules commonly used in immunoassays were patterned on silicon dioxide: mouse immunoglobulin G, rabbit immunoglobulin G and biotinylated bovine serum albumin. The multiprotein array thus produced wTas tested by immersing it in a solution containing three different fluorescently labelled biomolecules, each exhibiting specific, biomolecular interaction to the deposited ones. The discrimination from the mixture of these secondary biomolecules by antibody-antibody or biotin-avidin interactions confirmed the functionality and high selectivity of the patterned array

Fluorescent poly(vinylpyrrolidone)-supported copper nanoclusters in miniaturized analytical systems for iodine sensing

Poly(vinylpyrrolidone)-supported copper nanoclusters (CuNCs) are employed in the present work as luminescent probes for iodide determination in water samples. The method involves the combination of two miniaturized analytical systems, namely nanoparticle-enhanced liquid-phase microextraction and microvolume fluorospectrometry. The proposed method is based on the in situ generation of iodine and trapping of the evolved volatile into a CuNCs-containing aqueous microdrop, thus leading to fluorescence quenching. The fluorescence quenching mechanism for iodine sensing can be presumably ascribed to a ‘sphere of action’ static quenching model. Instrumental conditions, as well as a number of experimental parameters affecting extractant phase composition, iodine generation conditions and mass transfer of the volatile, have been evaluated. An outstanding enrichment factor of ca. 1100 was achieved under optimal conditions, yielding limits of detection and quantification of 1.0 ng/mL and 3.4 ng/mL, respectively. The repeatability, expressed as relative standard deviation, was found to be 7.4% (N = 7). The method was validated against a certified reference material and successfully applied to the analysis of different water samples. Furthermore, paper-based analytical devices containing CuNCs have been evaluated for the non-instrumental sensing of iodine generated in situ, showing promise as an inexpensive and portable alternative for iodide determination.Agencia Estatal de Investigación | Ref. RTI2018-093697-B-I00Xunta de Galici

Detection of gases and organic vapors by cellulose-based sensors

The growing interest in the development of cost-effective, straightforward, and rapid analytical systems has found cellulose-based materials, including cellulose derivatives, cellulose-based gels, nanocellulosic materials, and the corresponding (nano)cellulose-based composites, to be valuable platforms for sensor development. The present work presents recent advances in the development of cellulose-based sensors for the determination of volatile analytes and derivatives of analytical relevance. In particular, strategies described in the literature for the fabrication and modification of cellulose-based substrates with responsive materials are summarized. In addition, selected contributions reported in the field of paper-based volatile sensors are discussed, with a particular emphasis on quick response (QR) code paper-based platforms, intelligent films for food freshness monitoring, and sensor arrays for volatile discrimination purposes. Furthermore, analytical strategies devised for the determination of ionic species by in situ generation of volatile derivatives in both paper-based analytical devices (PADs) and microfluidic PADs will also be described.Universidade de Vigo/CISUGAgencia Estatal de Investigación | Ref. RTI2018-093697-B-I0

Dna methylation changes in fibromyalgia suggest the role of the immune‐inflammatory response and central sensitization

Fibromyalgia (FM) has been explained as a result of gene‐environment interactions. The present study aims to verify DNA methylation differences in eleven candidate genome regions previously associated to FM, evaluating DNA methylation patterns as potential disease biomarkers. DNA methylation was analyzed through bisulfite sequencing, comparing 42 FM women and their 42 healthy sisters. The associations between the level of methylation in these regions were further explored through a network analysis. Lastly, a logistic regression model investigated the regions potentially associated with FM, when controlling for sociodemographic variables and depressive symptoms. The analysis highlighted significant differences in the GCSAML region methylation between patients and controls. Moreover, seventeen single CpGs, belonging to other genes, were significantly different, however, only one cytosine related to GCSAML survived the correction for multiple comparisons. The network structure of methylation sites was different for each group; GRM2 methylation represented a central node only for FM patients. Logistic regression revealed that depressive symptoms and DNA methylation in the GRM2 region were significantly associated with FM risk. Our study encourages better exploration of GCSAML and GRM2 functions and their possible role in FM affecting immune, inflammatory response, and central sensitization of pain

Intercambios metodológicos aplicados a los estudios de postgrado en género, etnomusicología y atropología

Memoria ID-164. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2019-2020

Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening?closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA

Quality and reporting of clinical guidelines for breast cancer treatment: A systematic review

Background: High-quality, well-reported clinical practice guidelines (CPGs) and consensus statements (CSs) underpinned by systematic reviews are needed. We appraised the quality and reporting of CPGs and CSs for breast cancer (BC) treatment. Methods: Following protocol registration (Prospero no: CRD42020164801), CPGs and CSs on BC treatment were identified, without language restrictions, through a systematic search of bibliographic databases (MEDLINE, EMBASE, Web of Science, Scopus, CDSR) and online sources (12 guideline databases and 51 professional society websites) from January 2017 to June 2020. Data were extracted in duplicate assessing overall quality using AGREE II (% of maximum score) and reporting compliance using RIGHT (% of total 35 items); reviewer agreement was 98% and 96% respectively. Results: There were 59 relevant guidance documents (43 CPGs, 16 CSs), of which 20 used systematic reviews for evidence synthesis. The median overall quality was 54.0% (IQR 35.9e74.3) and the median overall reporting compliance was 60.9% (IQR 44.5e84.4). The correlation between quality and reporting was 0.9. Compared to CSs, CPGs had better quality (55.4% vs 44.2%; p ¼ 0.032) and reporting (67.18% vs 44.5%; p ¼ 0.005). Compared to subjective methods of evidence analysis, guidance documents that used systematic reviews had better quality (76.3% vs 51.4%; p ¼ 0.001) and reporting (87.1% vs 59.4%; p ¼ 0.001). Conclusion: The quality and reporting of CPGs and CSs in BC treatment were moderately strong. Systematic reviews should be used to improve the quality and reporting of CPGs and CSs.Beatriz Galindo (senor modality) Program by the Ministry of Science, Innovation, and Universities of the Spanish Governmen

Using jasmonates and salicylates to reduce losses within the fruit supply chain

The fresh produce industry is constantly growing, due to increasing consumer demand. The shelf-life of some fruit, however, is relatively short, limited by microbial contamination or visual, textural and nutritional quality loss. Thus, techniques for reducing undesired microbial contamination, spoilage and decay, as well as maintaining product’s visual, textural and nutritional quality are in high demand at all steps within the supply chain. The postharvest use of signalling molecules, i.e. jasmonates and salicylates seems to have unexplored potential. The focus of this review is on the effects of treatment with jasmonates and salicylates on the fresh produce quality, defined by decay incidence and severity, chilling injury, maintenance of texture, visual quality, taste and aroma, and nutritional content. Postharvest treatments with jasmonates and salicylates have the ability to reduce decay by increasing fruit resistance to diseases and reducing chilling injury in numerous products. These treatments also possess the ability to improve other quality characteristics, i.e. appearance, texture maintenance and nutritional content. Furthermore, they can easily be combined with other treatments, e.g. heat treatment, ultrasound treatment. A good understanding of all the benefits and limitations related to the postharvest use of jasmonates and salicylates is needed, and relevant information has been reviewed in this paper