Evolution of Drug Resistance: Insight on TEM β-Lactamases Structure and Activity and β-Lactam Antibiotics

Since the discovery of the first penicillin bacterial resistance to β-lactam antibiotics has spread and evolved promoting new resistances to pathogens. The most common mechanism of resistance is the production of β-lactamases that have spread thorough nature and evolve to complex phenotypes like CMT type enzymes. New antibiotics have been introduced in clinical practice, and therefore it becomes necessary a concise summary about their molecular targets, specific use and other properties. β-lactamases are still a major medical concern and they have been extensively studied and described in the scientific literature. Several authors agree that Glu166 should be the general base and Ser70 should perform the nucleophilic attack to the carbon of the carbonyl group of the β-lactam ring. Nevertheless there still is controversy on their catalytic mechanism. TEMs evolve at incredible pace presenting more complex phenotypes due to their tolerance to mutations. These mutations lead to an increasing need of novel, stronger and more specific and stable antibiotics. The present review summarizes key structural, molecular and functional aspects of ESBL, IRT and CMT TEM β-lactamases properties and up to date diagrams of the TEM variants with defined phenotype. The activity and structural characteristics of several available TEMs in the NCBI-PDB are presented, as well as the relation of the various mutated residues and their specific properties and some previously proposed catalytic mechanisms.

Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

Electrophoretic protein patterns and numerical analysis of Candida albicans from the oral cavities of healthy children

The aim of this research was to evaluate the protein polymorphism degree among seventy-five C. albicans strains from healthy children oral cavities of five socioeconomic categories from eight schools (private and public) in Piracicaba city, São Paulo State, in order to identify C. albicans subspecies and their similarities in infantile population groups and to establish their possible dissemination route. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed with cold saline solution. The whole-cell proteins were extracted by cell disruption, using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrix and dendrogram were generated by using the Dice similarity coefficient and UPGMA algorithm, respectively, which made it possible to evaluate the similarity or intra-specific polymorphism degrees, based on whole-cell protein fingerprinting of C. albicans oral isolates. A total of 13 major phenons (clusters) were analyzed, according to their homogeneous (socioeconomic category and/or same school) and heterogeneous (distinct socioeconomic categories and/or schools) characteristics. Regarding to the social epidemiological aspect, the cluster composition showed higher similarities (0.788 < S D < 1.0) among C. albicans strains isolated from healthy children independent of their socioeconomic bases (high, medium, or low). Isolates of high similarity were not found in oral cavities from healthy children of social stratum A and D, B and D, or C and E. This may be explained by an absence of a dissemination route among these children. Geographically, some healthy children among identical and different schools (private and public) also are carriers of similar strains but such similarity was not found among other isolates from children from certain schools. These data may reflect a restricted dissemination route of these microorganisms in some groups of healthy scholars, which may be dependent of either socioeconomic categories or geographic site of each child. In contrast to the higher similarity, the lower similarity or higher polymorphism degree (0.499 < S D < 0.788) of protein profiles was shown in 23 (30.6%) C. albicans oral isolates. Considering the social epidemiological aspect, 42.1%, 41.7%, 26.6%, 23.5%, and 16.7% were isolates from children concerning to socioeconomic categories A, D, C, B, and E, respectively, and geographically, 63.6%, 50%, 33.3%, 33.3%, 30%, 25%, and 14.3% were isolates from children from schools LAE (Liceu Colégio Albert Einstein), MA (E.E.P.S.G. "Prof. Elias de Melo Ayres"), CS (E.E.P.G. "Prof. Carlos Sodero"), AV (Alphaville), HF (E.E.P.S.G. "Honorato Faustino), FMC (E.E.P.G. "Prof. Francisco Mariano da Costa"), and MEP (E.E.P.S.G. "Prof. Manasses Ephraim Pereira), respectively. Such results suggest a higher protein polymorphism degree among some strains isolated from healthy children independent of their socioeconomic strata or geographic sites. Complementary studies, involving healthy students and their families, teachers, servants, hygiene and nutritional habits must be done in order to establish the sources of such colonization patterns in population groups of healthy children. The whole-cell protein profile obtained by SDS-PAGE associated with computer-assisted numerical analysis may provide additional criteria for the taxonomic and epidemiological studies of C. albicans.

X-ray crystal structure and EPR spectra of "arsenite-inhibited" Desulfovibriogigas aldehyde dehydrogenase: a member of the xanthine oxidase family

J. Am. Chem. Soc., 2004, 126 (28), pp 8614–8615 DOI: 10.1021/ja0490222

Development and Characterization of bioactive, edible whey protein films and coatings to improve quality and safety of Food products

Dissertação para a obtenção de grau de doutor em Ciências da Engenharia e Tecnologia

Liver and serum soluble protein changes and pathomorphology in undernourished mice with acute Schistosomiasis mansoni

Body, liver and spleen weights; histopathology of the liver, spleen and intestines; hepatic and serum soluble proteins changes were the parameters studied in undernourished Swiss albino mice experimentally infected with S. mansoni. Non-infected deficient animab had lower liver/body weight and spleen/body weight ratios as compared to the controls (22.60% casein group). Infected mice showed higher values regardless the type of diet. Undernourished infected subgroup showed a persistent exudative periovular reaction in the liver. Soluble hepatic proteins content and serum protein fractions appeared to be lower in the deficient infected mice. A significant difference was detected in the gammaglobulin fraction between infected and non-infected animals fed the control diet with higher values for the former. Our data suggest that the effects of malnutrition, per se, are sometimes more detrimental to the host than those due to Manson 's schistosomiasis.

Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens

The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors. NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals. To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed. The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences. Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.

Does the existing Corporate Governance structure and strict Legal Framework, affect the strategic decisionmaking of CEOs?

This thesis is a case study on Corporate Governance and Business Ethics, using the Portuguese Corporate Law as a general setting. The thesis was conducted in Portugal with illustrations on past cases under the Business Judgment Rule of the State of Delaware, U.SA along with illustrations on current cases in Portugal under the Portuguese Judicial setting, along with a comparative analysis between both. A debate is being considered among scholars and executives; a debate on best practices within corporate governance and corporate law, associated with recent discoveries of unlawful investments that lead to the bankruptcy of leading institutions and an aggravation of the crisis in Portugal. The study aimed at learning possible reasons and causes for the current situation of the country’s corporations along with attempts to discover the best way to move forward. From the interviews and analysis conducted, this paper concluded that the corporate governance structure and legal frameworks in Portugal were not the sole influencers behind the actions and decisions of Corporate Executives, nor were they the main triggers for the recent corporate mishaps. But it is rather a combination of different factors that played a significant role, such as cultural and ethical aspects, individual personalities, and others all of which created gray areas beyond the legal structure, which in turn accelerated and aggravated the corporate governance crisis in the country.

Structure and composition of the ground-herb community in a terra-firme Central Amazonian forest

The herb community of tropical forests is very little known, with few studies addressing its structure quantitatively. Even with this scarce body of information, it is clear that the ground herbs are a rich group, comprising 14 to 40% of the species found in total species counts in tropical forests. The present study had the objective of increasing the knowledge about the structure and composition of the ground-herb community and to compare the sites for which there are similar studies. The study was conducted in a tropical non-inundated and evergreen forest 90 km north of Manaus, AM. Ground herbs were surveyed in 22 transects of 40 m², distributed in five plots of 4 ha. The inventoried community was composed of 35 species, distributed in 24 genera and 18 families. Angiosperms were represented by 8 families and Pteridophytes by 10 families. Marantaceae (12 sp) and Cyperaceae (4 sp) were the richest families. Marantaceae and Poaceae were the families with greatest abundance and cover. Marantaceae, Poaceae, Heliconiaceae and Pteridophytes summed 96% of total herb cover, and therefore were responsible for almost all the cover of the community. The 10 most important species had 83.7% of the individuals. In general, the most abundant species were also the most frequent. Richness per transect varied from 7 to 19 species, and abundance varied from 30 to 114 individuals. The community structure was quite similar to 3 other sites in South America and one site in Asia.

Relationship between galactomannan structure and physicochemical properties of films produced thereof

In this work five sources of galactomannans, Adenanthera pavonina, Cyamopsis tetragonolobus, Caesalpinia pulcherrima, Ceratonia siliqua and Sophora japonica, presenting mannose/galactose ratios of 1.3, 1.7, 2.9, 3.4 and 5.6, respectively, were used to produce galactomannan-based films. These films were characterized in terms of: water vapour, oxygen and carbon dioxide permeabilities (WVP, O 2 P and CO 2 P); moisture content, water solubility, contact angle, elongation-at-break (EB), tensile strength (TS) and glass transition temperature (T g ). Results showed that films properties vary according to the galactomannan source (different galactose distribution) and their mannose/galactose ratio. Water affinity of mannan and galactose chains and the intermolecular interactions of mannose backbone should also be considered being factors that affect films properties. This work has shown that knowing mannose/galactose ratio of galactomannans is possible to foresee galactomannan-based edible films properties.

Influence of glucose concentration on the structure and quantity of biofilms formed by Candida parapsilosis

Candida parapsilosis is nowadays an emerging opportunistic pathogen and its increasing incidence is part related to the capacity to produce biofilm. In addition, one of the most important C. parapsilosis pathogenic risk factors includes the organisms\textquoteright selective growth capabilities in hyper alimentation solutions. Thus, in this study, we investigated the role of glucose in C. parapsilosis biofilm modulation, by studying biofilm formation, matrix composition and structure. Moreover, the expression of biofilm-related genes (BCR1, FKS1 and OLE1) were analyzed in the presence of different glucose percentages. The results demonstrated the importance of glucose in the modulation of C. parapsilosis biofilm. The concentration of glucose had direct implications on the C. parapsilosis transition of yeast cells to pseudohyphae. Additionally, it was demonstrated that biofilm related genes BCR1, FKS1 and OLE1 are involved in biofilm modulation by glucose. The mechanism by which glucose enhances biofilm formation is not fully understood, however with this study we were able to demonstrate that C. parapsilosis respond to stress conditions caused by elevated levels of glucose by up-regulating genes related to biofilm formation (BCR1, FKS1 and OLE1).

Effect of butadiene/styrene ratio, block structure and carbon nanotube content on the mechanical and electrical properties of thermoplastic elastomers after UV ageing

Thermoplastic elastomers based on a triblock copolymer styrene-butadiene-styrene (SBS) with different butadiene/styrene ratios, block structure and carbon nanotube (CNT) content were submitted to accelerated weathering in a Xenontest set up, in order to evaluate their stability to UV ageing. It was concluded that ageing mainly depends on butadiene/styrene ratio and block structure, with radial block structures exhibiting a faster ageing than linear block structures. Moreover, the presence of carbon nanotubes in the SBS copolymer slows down the ageing of the copolymer. The evaluation of the influence of ageing on the mechanical and electrical properties demonstrates that the mechanical degradation is higher for the C401 sample, which is the SBS sample with the largest butadiene content and a radial block structure. On the other hand, a copolymer derivate from SBS, the styrene-ethylene/butadiene-styrene (SEBS) sample, retains a maximum deformation of ~1000% after 80 h of accelerated ageing. The hydrophobicity of the samples decreases with increasing ageing time, the effect being larger for the samples with higher butadiene content. It is also verified that cytotoxicity increases with increasing UV ageing with the exception of SEBS, which remains not cytotoxic up to 80 h of accelerated ageing time, demonstrating its potential for applications involving exposition to environmental conditions.

An evaluation of fibrous structure and physical characteristics of Cutia nut (Couepia edulis Prance) shell

The Cutia-nut (Couepia edulis Prance), a species originally from the Amazon region, has a kernel with reasonable nutritional value and a hard and thick woody shell that constitute most of the fruit. After the kernel removal, the shells are regarded as waste. The possibility of using such shells, as raw material for burning or charcoal production, as well as milled residue for structural reinforcement materials is quite feasible, considering environmental and economical aspects. There is, however, a complete lack of characterization of the Cutia-nut shell and other similar species which can aggregate desirable qualities for application as engineering material. In this study some analyses are presented aiming at providing information for potential uses of these residues. In general, the shells follow a regular shape with certain dimensional proportionality to the kernel. The shell is a fibrous material with high lignin content, present low water absorption and high resistance to natural degradation.