Characterization of novel human intragenic antimicrobial peptides, incorporation and release studies from ureasil-polyether hybrid matrix.

Intragenic antimicrobial peptides (IAPs) are internal sequences of proteins with physicochemical similarities to Antimicrobial Peptides (AMPs) that, once identified and synthesized as individual entities, present antimicrobial activity. Many mature proteins encoded by the genomes of virtually any organism may be regarded as inner reservoirs of IAPs, conferring them ample biotechnological potential. However, IAPs may also share shortcomings with AMPs, such as low half-life in biological media and non-specific adsorption in eukaryotic cells. The present manuscript reports a translational approach that encompasses the uncovering of two novel IAPs from human proteins as well as the first results concerning the incorporation and sustained release of one of these peptides from ureasil-polyether hybrid polymeric films. For such, the software Kamal was used to scan putative IAPs in the human proteome, and two peptides, named Hs05 and Hs06, were identified, synthesized, and tested as antimicrobials. Biophysical assays were conducted using model phospholipid vesicles and 1H NMR solution structures in phospholipid micelles were obtained for the IAP Hs05. This peptide was incorporated in a polymeric matrix composed of the ureasil/PPO-PEO-PPO triblock copolymer, and the resulting films were evaluated by atomic force microscopy and imaging mass spectrometry. The release rate of Hs05 from the polymeric matrix was assessed and the antimicrobial activity of Hs05-loaded hybrid polymeric films was evaluated against the bacterium Escherichia coli. This study represents the first steps towards the development of polymeric films enriched with IAPs obtained from the human proteome as sustained release devices for topical application.

Relative quantification of plasma N-glycans in type II congenital disorder of glycosylation patients by mass spectrometry.

BACKGROUND: Type II Congenital Disorders of Glycosylation (CDG-II) are a group of diseases with challenging diagnostics characterized by defects in the processing of glycans in the Golgi apparatus. Mass Spectrometry (MS) has been a valuable tool in the definition of CDG-II subtypes. While some CDG-II subtypes are associated with specific N-glycan structures, others only produce changes in relative levels, reinforcing the demand for quantification methods. METHODS: Plasma samples from control individuals were pooled, derivatized with deuterated iodomethane (I-CD3), and used as internal standards for controls and patients whose glycans were derivatized with iodomethane (I-CH3), followed by MALDI MS, LC-MS and -MS/MS analyses. RESULTS: Total N-glycans from fifteen CDG-II patients were evaluated, and 4 cases with molecular diagnosis were considered in detail: 2ATP6V0A2-CDG siblings, and 2 MAN1B1-CDG patients, one of them carrying a previously undescribed p.Gly536Val mutation. CONCLUSIONS: Our methodology offers a feasible alternative to the current methods for CDG-II diagnosis by MS, which quantify glycan structures as fractions of the total summed signal across a mass spectrum, a strategy that lowers the variability of minor components. Moreover, given its sensitivity for less concentrated yet biologically relevant structures, it might assist the uncovering of novel diagnostic glycans in other CDG-II subtypes.

Protocolo com nove dias de progesterona para inseminação artificial em tempo fixo em vacas taurinas adaptadas ao clima tropical / Protocol with nine days of progesterone for fixed-time artificial insemination in Bos taurus cows adapted to the tropical weather

Três experimentos foram realizados para adaptar um protocolo de sincronização de estro e da ovulação para ser utilizado em programas de inseminação artificial em tempo fixo (IATF) em vacas taurinas tropicalmente adaptadas. No Exp. 1 (crossover), vacas pluríparas Curraleiro Pé-Duro (n= 12) receberam um dispositivo intravaginal contendo 1g de P4 por oito dias e 2mg de BE intramuscular (IM) no momento da inserção do dispositivo (dia 0). No dia da remoção do dispositivo (dia 8), as fêmeas receberam 150µg de D-cloprostenol sódico e 300UI de gonadotrofina coriônica equina (eCG) IM, sendo, então, divididas aleatoriamente para receber 1mg de BE no dia 8 (BE8) ou 1mg de BE no dia 9 (BE9). A aplicação de BE no D9 atrasou a ovulação em aproximadamente 15 horas (P<0,05). No Experimento 2, foram avaliados protocolos com oito (P4D8) e nove dias (P4D9) de exposição à progesterona, resultando em parâmetros de desenvolvimento folicular e luteal semelhantes entre os tratamentos (P>0,05). No Experimento 3, os protocolos hormonais de IATF BE8 e P4D9 foram testados para a taxa de prenhez, alcançando 23% (10/43) e 20% (9/45), respectivamente (P>0,05). Embora o grupo P4D9 tenha mostrado avanço na proporção de animais que responderam ao protocolo quando comparado ao protocolo BE8, este não se refletiu em melhora na taxa de prenhez.(AU)

Three experiments were performed to adapt a synchronization protocol of estrus synchronization and ovulation to be used in fixed time artificial insemination programs (FTAI) in tropically adapted Bos taurus cows. In Exp. 1 (crossover) multiparous Curraleiro Pé-Duro cows (n= 12) received an intravaginal device containing 1g of P4 for 8 days and 2mg of EB at the time of device insertion (Day 0). On the P4 device removal (Day 8) females received 150g of D-cloprostenol Sodic and 300IU of equine chorionic gonadotropin (eCG). Then, they were randomly divided to receive 1mg of EB on Day 8 (EB8) or on Day 9 (EB9). EB9 delayed ovulation approximately 15 hours (P<0.05). In Exp. 2, protocols using progesterone for eight (P4D8) or nine days (P4D9) were evaluated, resulting in similar parameters of folicular and luteal development (P>0.05). In Exp. 3, EB8 and P4D9 protocols were used to evaluate the pregnancy rate, reaching 23% (10/43) and 20% (9/45), respectively (P>0.05). Although P4D9 protocol has shown improvement in proportion of animals that responded to the protocol when compared to EB8 protocol, it was not able to improve pregnancy rate.(AU)

Diffractive refractometer for liquid characterization and transient processes monitoring.

A simple refractometer using a reflective diffraction grating immersed in the test liquid is developed and its performance is studied. Due to the dependence of the light wavelength on the refractive index, determining the angle of the diffracted beam provides the refractive index of the liquid. The glass cell containing the test liquid is cylindrical, and the grating plane is parallel to the cylinder symmetry axis. The light beam normally impinges on the cell front wall and reaches the center of the grating so that the diffracted beam leaves the cell without being deviated by refraction. It is demonstrated that this characteristic of the optical setup minimizes important error sources due to undesired beam deviations and enables real-time refractive index measurement of liquids in transient processes. Moreover, the performances of the diffractive refractometer and of a commercial Abbe refractometer are compared in the measurement of the refractive indexes of aqueous NaCl solutions. A He-Ne laser at 632.8 nm is used as a light source, and the diffraction grating has 1200 lines/mm. Measurement precisions of the order of 8 × 10-4 are achieved.

Proteomic analysis of seminal plasma from locally-adapted "Curraleiro Pé-Duro bulls" (Bos taurus): identifying biomarkers involved in sperm physiology in endangered animals for conservation of biodiversity.

The present study was aimed at evaluating the seminal plasma proteins and sperm parameters of Curraleiro Pé-Duro bulls. Semen was collected from 10 bulls by electroejaculation, and sperm parameters were evaluated in fresh and frozen-thawed semen. Seminal plasma proteins were analyzed by 2-D SDS-PAGE and mass spectrophotometry. Tools in computational biology were used to generate bioinformatic knowledge and evaluate gene ontology, protein-protein interactions, phylogenetic trees and multiple sequence alignments. Sperm motility in fresh and frozen-thawed semen was 78.8±1.8% and 21.2±1.6%, respectively. Pearson's correlations were evaluated (p<0.05). Sperm motility and vigor in fresh semen were correlated with clusterin, TIMP2 and cathepsin S (r=0.64-0.71) and sperm defects were related to inhibitor of carbonic anhydrase and BSP 5 (r=0.78-0.80). Clusterin, BSP 5, alpha-enolase, creatine kinase M-type, glyceraldehyde-3-phosphate dehydrogenase, BSP 3, albumin, and 5'-nucleotidase and legumain were correlated with acrosome intact live sperm (r=0.80-0.64). Associations were detected between sperm vigor and spermadhesin 1 (r=-0.89), and between sperm defects in fresh semen and spermadhesin 1 and clusterin (r=-0.81). Sperm motility in frozen-thawed semen was associated with BSP 1, spermadhesin 1, clusterin and spermadhesin Z13 (r=0.64-0.85). The percent of motile sperm after freeze-thawing was negatively correlated (r=-0.64) with the amount of spermadhesin 1 in the seminal plasma. Based on in silico analysis, TIMP2 interacted with BSP1, BSP3, BSP5 and metalloproteinases. Molecular functions of proteins associated with sperm parameters were binding, catalytic activity and enzymatic regulation. Amino acid sequences of spermadhesin 1 and BSP 1 from Bos taurus, and other domestic species were similar. Phylogenetic tree analysis demonstrated that clusterin from Bos taurus was related to Ovis aries and domains of clusterin, spermadhesin 1, BSP 1 and inhibitor of carbonic anhydrase were conserved as well. In summary, specific seminal proteins are associated with sperm parameters of locally-adapted bulls. Use of the endangered mammalian as a model may assist in understanding aspects of evolutionary adaptations and could improve assisted reproductive biotechnologies.

Immersed diffraction grating refractometers of liquids.

In this work, we report the development, construction, and the performance of two liquid refractometers that use a reflective diffraction grating immersed in a test liquid. The liquid is contained in a transparent glass cell with a rectangular cross section. The grating is oriented in such a way that the propagation directions of the incident beam and the beam diffracted by the lower part of the grating immersed in the liquid are perpendicular. In this configuration, the refractive index is determined by measuring the angle of the zeroth-order diffraction beam coming from the upper part of the grating, which is in contact with air. The diffractive refractometers (DR-1 and DR-2) have different angle measurement procedures and different light detection systems, and their advantages and drawbacks are pointed out. In the experiments, precisions of the order of 10-5 and 10-4 for DR-1 and DR-2 are achieved, respectively. The performances of both systems are compared with the performance of a commercial Abbe refractometer in the measurement of sugar and NaCl aqueous solutions.

Quality improvement and geographical indication of cachaça (Brazilian spirit) by using locally selected yeast strains.

AIMS: In order to improve the quality and to create a biological basis for obtainment of the protected denomination of origin (PDO), indigenous yeast were isolated and characterized for use in Salinas city (the Brazilian region of quality cachaça production). MATERIAL AND METHODS: Seven thousand and two hundred yeast colonies from 15 Salinas city distilleries were screened based on their fermentative behaviour and the physicochemical composition of cachaça. Molecular polymorphic analyses were performed to characterize these isolates. RESULTS: Two Saccharomyces cerevisiae strains (nos. 678 and 680) showed appropriate characteristics to use in the cachaça production: low levels of acetaldehyde and methanol, and high ethyl lactate/ethyl acetate ratio respectively. They also presented polymorphic characteristics more closely related between themselves even when compared to other strains from Salinas. CONCLUSIONS: The application of selected yeast to cachaça production can contribute for the improvement of the quality product as well as be used as a natural marker for PDO. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the use of selected yeast strains could contribute to obtain a cachaça similar to those produced traditionally, while getting wide acceptation in the market, yet presenting more homogeneous organoleptic characteristics, and thus contributing to the PDO implementation.

Modification of trout sperm membranes associated with activation and cryopreservation. Implications for fertilizing potential.

We investigated the effects of two trout sperm activation solutions on sperm physiology and membrane organization prior to and following cryopreservation using flow cytometry and investigated their impact on in vitro fertility. Overall, frozen-thawed samples had greater phospholipid disorder when compared with fresh samples (high plasma membrane fluidity; P < 0.0001) and sperm activated with water also had high plasma membrane fluidity when compared to sperm activated with Lahnsteiner solution (LAS; P < 0.0001). Following cryopreservation water activated samples had membranes with greater membrane protein disorganization compared with LAS but the membrane protein organization of LAS samples was similar to samples prior to freezing (P < 0.0001). Post-thaw water activation resulted in significant increases in intracellular calcium compared to LAS (P < 0.002). In vitro fertility trials with frozen-thawed milt and LAS activation resulted in greater fertility (45%) compared to water activated samples (10%; P < 0.0001). Higher fertility rates correlated with lower intracellular calcium with water (R(2) = -0.9; P = 0.01) and LAS (R(2) = -0.85; P = 0.03) activation. Greater plasma membrane phospholipid (R(2) = -0.89; P = 0.02) and protein (R(2) = -0.84; P = 0.04) disorder correlated with lower water activation fertility rates. These membrane organization characteristics only approached significance with LAS activation in vitro fertility (P = 0.09, P = 0.06, respectively). Potentially the understanding of sperm membrane reorganizations and the physiology associated with activation following cryopreservation may enable users in a repository or hatchery setting to estimate the fertilizing potential of a sample and determine its value.

Caracterização da curva de crescimento de fêmeas Crioulas Lageanas criadas em condições naturais / Growth curve characterization of Crioula Lagena cows raised under natural conditions

Morphometry and ultrasound of carcasses were used to estimate the growth curve of Criola Lageana cows, raised under natural conditions. A total of 111 cows, raised under extensively production system in Central Plateau of Santa Catarina, were evaluated. Morphometrics and ultrasound measurements were related with the age of the animals using the targeted univariate regression model, assigning the variable response to gamma distribution. The inflection points of the growth curve were 24 and 25 months for withers height and hip height, between 27 and 29 for weight, body length, rump length, thorax perimeter, depth and distance between ilea. For ribeye area (REA), inflection point was 18 months. The Crioula Lageana cows presented greater growth velocity around 27 months, showing good productive performance under the natural conditions of the Central Plateau of Santa Catarina.(AU)

SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains.

Subinhibitory concentrations (subMICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of a subMIC (⅛ MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell-surface hydrophobicity (CSH) and biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell-surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox(-) strain; CDC-E8392 and 241 tox(+) strains). All micro-organisms tested were susceptible to PEN and ERY. Growth in the presence of PEN induced bacterial filamentation, whereas subMIC of ERY caused cell-size reduction of strains 241 and CDC-E8392. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while CSH was increased by a subMIC of both antibiotics in bacterial adherence to n-hexadecane assays. Conversely, antibiotic inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only strain 241 increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by ⅛ MIC of ERY. After growth in the presence of both antimicrobial agents, strains 241 and CDC-E8392 exhibited anionic surface charges with focal distribution. In conclusion, subMICs of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell-surface hydrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.

Simple and versatile heterodyne whole-field interferometer for phase optics characterization.

A wavefront sensor for thermally induced lens and passive lens characterization based on low-coherence digital speckle interferometry was developed and studied. By illuminating the optical setup with two slightly detuned red diode lasers, whole-field contour interference fringes were generated according to the resulting synthetic wavelength. For fringe pattern visualization the optical setup used the light transmitted through a ground glass plate as object beam. The performance of the sensor was investigated and its versatility was demonstrated by measuring the thermal lens induced in an Er-doped glass sample pumped by a 1.76-W diode laser emitting at 976 nm and by evaluating the wavefront distortion introduced by an ophthalmic progressive lens.

Design, construction, and performance of a real-time holographic refractometry prototype for liquid analysis.

The development and the performance of a portable holographic refractometer prototype for liquid measurement employing multimode diode lasers with emission centered at 662 nm as light sources is reported. Due to the multiwavelength character of the holographic recording, a synthetic wavelength was generated, and the diffracted wave intensity was thus modulated as a function of the optical path difference between the reference and the object beams. The transparent test cell containing the liquid was placed at the reference-beam arm of the optical setup, while the contour interferogram generated on the holographic image of a flat object was used for fringe counting. A change ΔL on the liquid column length is proportional to the Δp running fringes on the object image, and from this relation the refractive index of the test liquid was obtained. The holograms were recorded on a photorefractive Bi(12)TiO(20) crystal whether using a single multimode diode laser or by combining two diode lasers. In the latter configuration the synthetic wavelength can be varied in order to enhance the measurement sensitivity and∕or to allow the analysis of turbid liquids. The size of the whole prototype is 54 × 22 × 14 cm(3). The refractive indexes of ethanol∕water mixtures with different concentrations were measured, as well as the NaCl concentrations in aqueous solutions were determined upon comparison with an empirical curve. In both cases the results were compared with the ones obtained through an Abbe refractometer.

Association of HDL cholesterol and triglycerides with mortality in patients with heart failure

It has been demonstrated that there is an association between serum lipoproteins and survival rate in patients with ischemic cardiomyopathy, as well as in patients with non-ischemic causes of heart failure. We tested the hypothesis of an association between serum lipoprotein levels and prognosis in a cohort of outpatients with heart failure, including Chagas' heart disease. The lipid profile of 833 outpatients with heart failure in functional classes III and IV of the New York Heart Association, with a mean age of 46.9 ± 10.6 years, 655 (78.6 percent) men and 178 (21.4 percent) women, was studied from April 1991 to June 2003. The survival rate was estimated by the Kaplan-Meyer's method and the Cox proportional hazards models. Etiology of heart failure was ischemic cardiomyopathy in 171 (21 percent) patients, Chagas' heart disease in 144 (17 percent), hypertensive cardiomyopathy in 136 (16 percent), and other etiologies in 83 (10 percent). In 299 (36 percent) patients, heart failure was ascribed to idiopathic dilated cardiomyopathy. Variables significantly associated with mortality were age (hazard ratio, HR = 1.02; 95 percentCI = 1.01-1.03; P = 0.0074), male gender (HR = 1.77; 95 percentCI = 1.2-2.62; P = 0.004), idiopathic dilated cardiomyopathy (HR = 1.81; 95 percentCI = 1.16-2.82; P = 0.0085), serum triglycerides (HR = 0.97; 95 percentCI = 0.96-0.98; P < 0.0001), and HDL cholesterol (HR = 0.99; 95 percentCI = 0.99-1.0; P = 0.0280). Therefore, higher serum HDL cholesterol and higher serum triglycerides were associated with lower mortality in this cohort of outpatients with heart failure.

Association of HDL cholesterol and triglycerides with mortality in patients with heart failure.

It has been demonstrated that there is an association between serum lipoproteins and survival rate in patients with ischemic cardiomyopathy, as well as in patients with non-ischemic causes of heart failure. We tested the hypothesis of an association between serum lipoprotein levels and prognosis in a cohort of outpatients with heart failure, including Chagas' heart disease. The lipid profile of 833 outpatients with heart failure in functional classes III and IV of the New York Heart Association, with a mean age of 46.9 +/- 10.6 years, 655 (78.6%) men and 178 (21.4%) women, was studied from April 1991 to June 2003. The survival rate was estimated by the Kaplan-Meyer's method and the Cox proportional hazards models. Etiology of heart failure was ischemic cardiomyopathy in 171 (21%) patients, Chagas' heart disease in 144 (17%), hypertensive cardiomyopathy in 136 (16%), and other etiologies in 83 (10%). In 299 (36%) patients, heart failure was ascribed to idiopathic dilated cardiomyopathy. Variables significantly associated with mortality were age (hazard ratio, HR = 1.02; 95%CI = 1.01-1.03; P = 0.0074), male gender (HR = 1.77; 95%CI = 1.2-2.62; P = 0.004), idiopathic dilated cardiomyopathy (HR = 1.81; 95%CI = 1.16-2.82; P = 0.0085), serum triglycerides (HR = 0.97; 95%CI = 0.96-0.98; P < 0.0001), and HDL cholesterol (HR = 0.99; 95%CI = 0.99-1.0; P = 0.0280). Therefore, higher serum HDL cholesterol and higher serum triglycerides were associated with lower mortality in this cohort of outpatients with heart failure.

Phase-compensated holographic recording based on anisotropic photorefractive diffraction.

We report a simple actively stabilized setup for holographic recording on sillenite photorefractive crystals that is based on the anisotropic diffraction properties of these materials. The method is much simpler than previously published ones, requires neither phase modulation nor synchronous phase-sensitive detection, and needs no external reference. We describe the successful operation of this stabilization procedure for a Bi(12)TiO(20) crystal in a practical holographic interferometry setup, using the 632.8-nm wavelength of a He-Ne laser.