Environmental factors influencing the inactivation of Cronobacter sakazakii by high hydrostatic pressure

The effect of High Hydrostatic Pressure (HHP) on the survival of Cronobacter sakazakii was investigated. Deviations from linearity were found on the survival curves and the Mafart equation accurately described the kinetics of inactivation. Comparisons between strains and treatments were made based on the time needed for a 5-log10 reduction in viable count. The ability of C. sakazakii to tolerate high pressure was straindependent with a 26-fold difference in resistance among four strains tested. Pressure resistance was greatest in the stationary growth phase and at the highest growth temperatures tested (30 and 37 °C). Cells treated in neutral pH buffer were 5-fold more resistant than those treated at pH 4.0, and 8-fold more sensitive than those treated in buffer with sucrose added (aw=0.98). Pressure resistance data obtained in buffer at the appropriate pH adequately estimated the resistance of C. sakazakii in chicken and vegetables soups. In contrast, a significant protective effect against high pressure was conferred by rehydrated powdered milk. As expected, treatment efficacy improved as pressure increased. z values of 112, 136 and 156 MPa were obtained for pH 4.0, pH 7.0 and aw=0.98 buffers, respectively. Cells with sublethal injury to their outer and cytoplasmic membranes were detected after HHP under all the conditions tested. The lower resistance of C. sakazakii cells when treated in media of pH 4.0 seemed to be due to a decreased barostability of the bacterial envelopes. Conversely, the higher resistance displayed in media of reduced water activity may relate to a higher stability of bacterial envelopes.

Effect of high-hydrostatic pressure and pH treatments on the emulsification properties of gum arabic

This study investigated the emulsification properties of the native gums and those treated at high pressure (800 MPa) both at their “natural” pH (4.49 and 4.58, respectively) and under “acidic and basic” pH (2.8 and 8.0) conditions. The emulsification behaviour of KLTA gum was found to be superior to that of the GCA gum. High pressure and pH treatment changed the emulsification properties of both gums. The acidic amino acids in gum arabic were shown to play an important role in their emulsification behaviour, and mechanisms of emulsification for the two gums were suggested to be different. The highly “branched” nature of the carbohydrate in GCA gum was also thought to be responsible for the “spreading” of droplet size distributions observed. Coomassie brilliant blue binding was used to indicate conformational changes in protein structure and Ellman’s assay was used to estimate any changes in levels of free thiols present.

Development of new methodologies in sample treatment for proteomics workflow based on enzymatic probe sonication technology and mass spectrometry

Thesis submitted to the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia, for the degree of Doctor of Philosophy in Biochemistry

Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study.

BACKGROUND:HIV-1-infected patients vary considerably by their response to antiretroviral treatment, drug concentrations in plasma, toxic events, and rate of immune recovery. This variability could have a genetic basis. We did a pharmacogenetics study to analyse the association between response to antiretroviral treatment and allelic variants of several genes. METHODS:In 123 patients, we did PCR analyses of the gene for the multidrug-resistance transporter (MDR1), which codes for P-glycoprotein, of genes coding for isoenzymes of cytochrome P450, CYP3A4, CYP3A5, CYP2D6, and CYP2C19, and of the gene for the chemokine receptor CCR5. We measured concentrations in plasma of the antiretroviral agents efavirenz and nelfinavir by high-performance liquid-chromatography, and measured levels of P-glycoprotein expression, CD4-cell count, and HIV-1 viraemia. FINDINGS: Median drug concentrations in patients with the MDR1 3435 TT, CT, and CC genotypes were at the 30th, 50th, and 75th percentiles, respectively (p=0.0001). In patients with CYP2D6 extensive-metaboliser or poor-metaboliser alleles, median drug concentrations were at percentiles 45 and 62.5, respectively (p=0.04). Patients with the MDR1 TT genotype 6 months after starting treatment had a greater rise in CD4-cell count (257 cells/microL) than patients with the CT (165 cells/microL) and CC (121 cells/microL) genotype (p=0.0048), and the best recovery of naïve CD4-cells. INTERPRETATION:The polymorphism MDR1 3435 C/T predicts immune recovery after initiation of antiretroviral treatment. This finding suggests that P-glycoprotein has an important role in admittance of antiretroviral drugs to restricted compartments in vivo.

Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods.

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Delta/cdr1Delta, cdr2Delta/cdr2Delta, flu1Delta/flu1Delta, and mdr1Delta/mdr1Delta) and calcineurin subunit A (cnaDelta/cnaDelta) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% +/- 5.0%/96.5% +/- 2.4% (HPLC) and 94.9% +/- 6.1%/94.7% +/- 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% +/- 1.5%/5.4% +/- 0.9% and 6.5% +/- 2.5%/4.0% +/- 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg x h/liter (range, 12.9 to 71.1) on the first and 27.4 mg x h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg x h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg x h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.

In vivo characterisation of a novel water-soluble Cyclosporine A prodrug for the treatment of dry eye disease.

Cyclosporine A (CsA) has been demonstrated to be effective for the treatment of a variety of ophthalmological conditions, including ocular surface disorders such as the dry eye disease (DED). Since CsA is characterised by its low water solubility, the development of a topical ophthalmic formulation represents an interesting pharmaceutical question. In the present study, two different strategies to address this challenge were studied and compared: (i) a water-soluble CsA prodrug formulated within an aqueous solution and (ii) a CsA oil-in-water emulsion (Restasis, Allergan Inc., Irvine, CA). First, the prodrug formulation was shown to have an excellent ocular tolerance as well as no influence on the basal tear production; maintaining the ocular surface properties remained unchanged. Then, in order to allow in vivo investigations, a specific analytical method based on ultra high pressure liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was developed and optimised to quantify CsA in ocular tissues and fluids. The CsA ocular kinetics in lachrymal fluid for both formulations were found to be similar between 15 min and 48 h. The CsA ocular distribution study evidenced the ability of the prodrug formulation to penetrate into the eye, achieving therapeutically active CsA levels in tissues of both the anterior and posterior segments. In addition, the detailed analysis of the in vivo data using a bicompartmental model pointed out a higher bioavailability and lower elimination rate for CsA when it is generated from the prodrug than after direct application as an emulsion. The interesting in vivo properties displayed by the prodrug solution make it a safe and suitable option for the treatment of DED.

Improved growth with bioabsorbable sutures in both high- and low-pressure zones.

BACKGROUND: Compromised growth after operation remains a significant problem in the cardiovascular field. Some benefit of absorbable suture materials has been demonstrated for arterial anastomoses. However, for the low-pressure zone, few data are available. METHODS: To assess growth in high- versus low-pressure zones we transected the abdominal aorta (high-pressure zone) as well as the inferior vena cava (low-pressure zone) in 10 young mongrel dogs using for reanastomosis 7-0 nonabsorbable versus absorbable running sutures in random order. RESULTS: All animals survived and were evaluated over 12 months including body weight (gain, 212% +/- 45% for nonabsorbable versus 218% +/- 8% for absorbable; not significant), angiography, and, after elective sacrifice, detailed studies of aorta and vena cava. Systematic complication of angiographic data at 12 months showed at the suture level an area of 13.8 mm2 for nonabsorbable versus 24.3 +/- 14.4 mm2 for absorbable sutures in the high-pressure zone as compared with 12.9 +/- 4.9 mm2 for nonabsorbable versus 25.3 +/- 15.4 mm2 for absorbable sutures in the low-pressure zone. Residual lumen, calculated as a function of the area above and below the suture, accounted for 35% +/- 10% for nonabsorbable versus 92% +/- 12% for absorbable sutures (p < 0.001) in the high-pressure zone as compared with 37% +/- 13% for nonabsorbable versus 75% +/- 15% for absorbable sutures (p < 0.003) in the low-pressure zone (high versus low, not significant). Poststenotic dilatation accounted for 199% +/- 22% for nonabsorbable versus 126% +/- 43% for absorbable sutures (p < 0.01) in the high-pressure zone. In the low-pressure zone, poststenotic dilatation remained below the inflow area, and the residual poststenotic lumen accounted for 52% +/- 14% for nonabsorbable versus 77% +/- 16% for absorbable sutures (p < 0.004). Macroscopic, light, and scanning electron microscopic studies confirmed different growth patterns in high- versus low-pressure zones. CONCLUSIONS: Aortic narrowing resulted in poststenotic dilatation and unrestricted outflow path (hourglass-type stenosis). Caval narrowing was followed by restriction of poststenotic outflow path (funnel-type stenosis). Absorbable suture material allows for superior growth in both high- and low-pressure zones.

Transscleral Coulomb-controlled iontophoresis of methylprednisolone into the rabbit eye: influence of duration of treatment, current intensity and drug concentration on ocular tissue and fluid levels.

The major problems associated with the use of corticosteroids for the treatment of ocular diseases are their poor intraocular penetration to the posterior segment when administered locally and their secondary side effects when given systemically. To circumvent these problems more efficient methods and techniques of local delivery are being developed. The purposes of this study were: (1) to investigate the pharmacokinetics of intraocular penetration of hemisuccinate methyl prednisolone (HMP) after its delivery using the transscleral Coulomb controlled iontophoresis (CCI) system applied to the eye or after intravenous (i.v.) injection in the rabbit, (2) to test the safety of the CCI system for the treated eyes and (3) to compare the pharmacokinetic profiles of HMP intraocular distribution after CCI delivery to i.v. injection. For each parameter evaluated, six rabbit eyes were used. For the CCI system, two concentrations of HMP (62.5 and 150mg ml(-1)), various intensities of current and duration of treatment were analyzed. In rabbits serving as controls the HMP was infused in the CCI device but without applied electric current. For the i.v. delivery, HMP at 10mg kg(-1)as a 62.5mg ml(-1)solution was used. The rabbits were observed clinically for evidence of ocular toxicity. At various time points after the administration of drug, rabbits were killed and intraocular fluids and tissues were sampled for methylprednisolone (MP) concentrations by high pressure liquid chromatography (HPLC). Histology examinations were performed on six eyes of each group. Among groups that received CCI, the concentrations of MP increased in all ocular tissues and fluids in relation to the intensities of current used (0.4, 1.0 and 2.0mA/0.5cm(2)) and its duration (4 and 10min). Sustained and highest levels of MP were achieved in the choroid and the retina of rabbit eyes treated with the highest current and 10min duration of CCI. No clinical toxicity or histological lesions were observed following CCI. Negligible amounts of MP were found in ocular tissues in the CCI control group without application of current. Compared to i.v. administration, CCI achieved higher and more sustained tissue concentrations with negligible systemic absorption. These data demonstrate that high levels of MP can be safely achieved in intraocular tissues and fluids of the rabbit eye, using CCI. With this system, intraocular tissues levels of MP are higher than those achieved after i.v. injection. Furthermore, if needed, the drug levels achieved with CCI can be modulated as a function of current intensity and duration of treatment. CCI could therefore be used as an alternative method for the delivery of high levels of MP to the intraocular tissues of both the anterior and posterior segments.

Assessment of high hydrostatic pressure and starter culture on the quality properties of low-acid fermented sausages

The addition of starter culture and high pressure processing after ripening improved the microbial quality of low-acid fermented sausages (fuet and chorizo). The use of Lactobacillus sakei CTC6626 and Staphylococcus xylosus CTC6013 as starter culture significantly reduced Enterobacteriaceae and Enterococcus levels in the finished sausages. Moreover, the addition of starter culture produced sausages of similar quality to traditional low-acid fermented sausages. Slightly lower pH values and higher cohesiveness were obtained for both fuet and chorizo with starter culture. Sensory analysis showed no differences between lots of chorizo whereas starter fuet was more acid and gummy. High pressure induced an additional reduction of Enterobacteriaceae in non-starter sausages. An increase of textural properties was observed after pressurization. No other differences were observed between non-treated and pressurized sausages.

Regenerated cellulose ultrafiltration membranes in the treatment of pulp and paper mill process waters

Ultrafiltration (UF) is widely applied in different separation processes in the pulp and paper industry. The growing need to protect the environment, a lack of pure water and an interest in producing high-value chemicals from compounds present in process waters will probably lead to an increase in the use of UF in the pulp and paper industry. The efficiency and cost-effectiveness of a UF process depends on the applied membrane. The membrane should have a high and stable filtration capacity, a particular selectivity and a long operational lifetime. To meet these requirements a membrane should have a low fouling tendency. In addition, it should withstand the prevailing operational and chemical conditions. This thesis evaluates the performance and applicability of the regenerated cellulose (RC) membranes 00030T and C2 in the treatment of pulp and paper mill process waters based on the requirements above. The results demonstrated that both the tested RC membranes fulfilled well the requirement of high filtration capacity. In addition, in the filtration of a paper mill clear filtrate (CF) the RC membranes were not as greatly affected by variations in the CF quality as a polysulphone membrane. Furthermore, due to their extreme hydrophilicity and weak charge the fouling tendency of the membranes can be expected to be low in pulp and paper mill filtration applications. It is, however, known that fouling cannot be totally avoided even when the membrane is chosen very carefully. This study indicated that carbohydrates influenced negatively on permeability and caused fouling in the filtration of groundwood mill circulation water. Thus, a pre-treatment effectively reducing the amount of carbohydrates might help to maintain a stable capacity. However, the results of the thesis also showed that the removal of some of the possible foulants might just increase the harmful effect of others. Multivariate examination was useful in the understanding of the complicated factors causing the unstable capacity. The thesis also revealed that the 00030T and C2 membranes can be used at high pressure (max. tested pressure 12 bar). The C2 membrane, having a sponge-like substructure, was more pressure resistant, and its performance was more stable at high pressure compared to the UCO30T membrane containing macrovoids in its substructure. Both tested membranes can, according to the results, also be used at temperatures as high as 70°C in acidic, neutral and alkaline conditions. However, the use at extreme conditions might cause faster ageing of the membranes compared to ageing in neutral conditions. The thesis proved that both the tested RC membranes are very suitable for pulp and paper mill applications and that the membranes can be utilised in processes operating in challenging conditions. Thus, they could be used in more demanding applications than supposed earlier.

The chemistry involved in the steam treatment of lignocellulosic materials

Pretreatment of lignocellulosic materials is essential for bioconversion because of the various physical and chemical barriers that greatly inhibit their susceptibility to bioprocesses such as hydrolysis and fermentation. The aim of this article is to review some of the most important pretreatment methods developed to date to enhance the conversion of lignocellulosics. Steam explosion, which precludes the treatment of biomass with high-pressure steam under optimal conditions, is presented as the pretreatment method of choice and its mode of action on lignocellulosics is discussed. The optimal pretreatment conditions for a given plant biomass are defined as those in which the best substrate for hydrolysis is obtained with the least amount of soluble sugars lost to side reactions such as dehydration. Therefore, pretreatment optimization results from a compromise between two opposite trends because hemicellulose recovery in acid hydrolysates can only be maximized at lower pretreatment severities, whereas the development of substrate accessibility requires more drastic pretreatment conditions in which sugar losses are inevitable. To account for this heterogeneity, the importance of several process-oriented parameters is discussed in detail, such as the pretreatment temperature, residence time into the steam reactor, use of an acid catalyst, susceptibility of the pretreated biomass to bioconversion, and process design.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC) and under high pressure conditions at low temperature (3.75 kbar, -13ºC). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Effect of high hydrostatic pressure on the barrier properties of polyamide-6 films

Little is known about the barrier properties of polymer films during high pressure processing of prepackaged foods. In order to learn more about this, we examined the influence of high hydrostatic pressure on the permeation of raspberry ketone (dissolved in ethanol/water) through polyamide-6 films at temperatures between 20 and 60ºC. Permeation was lowered by increasing pressure at all temperatures. At 23°C, the increasing pressure sequence 0.1, 50, 100, 150, and 200 MPa correlated with the decreasing permeation coefficients P/(10(9) cm² s-1) of 6.2, 3.8, 3.0, 2.2, and 1.6. Analysis of the permeation kinetics indicated that this effect was due to a reduced diffusion coefficient. Pressure and temperature acted antagonistically to each other. The decrease in permeation at 200 MPa was compensated for by a temperature increase of 20ºC. After release of pressure, the former permeation coefficients were recovered, which suggests that this `pressure effect' is reversible. Taken together, our data revealed no detrimental effects of high hydrostatic pressure on the barrier properties of polymer films.

Chemical and physical properties of aloe vera (Aloe barbadensis Miller) gel stored after high hydrostatic pressure processing

The aim of this study was to evaluate the influence of high hydrostatic pressure (150, 250, 350, 450, and 550 MPa), applied for 5 minutes, on antioxidant capacity, total phenolic content, color, firmness, rehydration ratio, and water holding capacity of aloe vera gel stored for 60 days at 4 °C. The analyzed properties of the pressurized gel showed significant changes after the storage period. The highest value of total phenolic content was found at 550 MPa. However, a decrease in the antioxidant capacity was observed for all pressurized gel samples when compared to the control sample (p < 0.05). The smallest changes in product color were observed at pressure levels between 150 and 250 MP. The application of high hydrostatic pressure resulted in lower gel firmness, and the lowest value was found at 150 MPa (p < 0.05). On the other hand, the untreated sample showed a greater decrease in firmness, indicating that high pressure processing preserves this property. The application of high hydrostatic pressure exhibited modifications in the food matrix, which were evaluated in terms of rehydration ratio and water holding capacity.

Thermal and high hydrostatic pressure inactivation of myrosinase from green cabbage: a kinetic study

Myrosinase, a family of enzymes which coexist with glucosinolates in all Brassica vegetables, catalyses the hydrolysis of glucosinolates to yield compounds that can have beneficial effects on human health. In this study, the thermal and pressure inactivation of myrosinase from green cabbage was kinetically investigated. Thermal inactivation started at 35 C and inactivation kinetics was studied in the temperature range 35–55 C. Thermal inactivation of green cabbage myrosinase followed the well known consecutive step model. Pressure inactivation started at 300 MPa, even at 10 C, and the consecutive step model effectively described pressure inactivation in the range 300–450 MPa at 10 C. The combined effects of applying various pressures and temperatures on myrosinase inactivation kinetics were studied in the ranges 35–50 C and, 100–400 MPa. The inactivation followed first-order kinetics at all of the applied combinations. This study demonstrates that myrosinase from green cabbage is highly susceptible to both thermal and high pressure processing. Furthermore, it is also noted that myrosinase stability during processing appears to vary widely between different Brassica species.