Juan L. Lequerica

Research and development of a new RF-assisted device for bloodless rapid transection of the liver: Computational modeling and in vivo experiments

BackgroundEfficient and safe transection of biological tissue in liver surgery is strongly dependent on the ability to address both parenchymal division and hemostasis simultaneously. In addition to the conventional clamp crushing or finger fracture methods other techniques based on radiofrequency (RF) currents have been extensively employed to reduce intraoperative blood loss. In this paper we present our broad research plan for a new RF-assisted device for bloodless, rapid resection of the liver.MethodsOur research plan includes computer modeling and in vivo studies. Computer modeling was based on the Finite Element Method (FEM) and allowed us to estimate the distribution of electrical power deposited in the tissue, along with assessing the effect of the characteristics of the device on the temperature profiles. Studies based on in vivo pig liver models provided a comparison of the performance of the new device with other techniques (saline-linked technology) currently employed in clinical practice. Finally, the plan includes a pilot clinical trial, in which both the new device and the accessory equipment are seen to comply with all safety requirements.ResultsThe FEM results showed a high electrical gradient around the tip of the blade, responsible for the maximal increase of temperature at that point, where temperature reached 100°C in only 3.85 s. Other hot points with lower temperatures were located at the proximal edge of the device. Additional simulations with an electrically insulated blade produced more uniform and larger lesions (assessed as the 55°C isotherm) than the electrically conducting blade. The in vivo study, in turn, showed greater transection speed (3 ± 0 and 3 ± 1 cm2/min for the new device in the open and laparoscopic approaches respectively) and also lower blood loss (70 ± 74 and 26 ± 34 mL) during transection of the liver, as compared to saline-linked technology (2 ± 1 cm2/min with P = 0.002, and 527 ± 273 mL with P = 0.001).ConclusionA new RF-assisted device for bloodless, rapid liver resection was designed, built and tested. The results demonstrate the potential advantages of this device over others currently employed.

Lactose utilization by Saccharomyces cerevisiae strains expressing Kluyveromyces lactis LAC genes.

Whey generated in cheese manufacture continues being an industrial problem without a satisfactory solution. Genetic modification of the yeast S. cerevisiae to obtain strains able to utilize lactose, is a prerequisite for the utilization of this yeast to convert cheese whey into useful fermentation products (i.e. biomass, heterologous protein and other recombinant products). Although the construction of S. cerevisiae Lac(+) strains has been achieved by different strategies, most of these strains have unsuitable characteristics, such as genetic instability of the Lac phenotype or diauxic growth. In previous communications we have described the construction of genetically stable strains of S. cerevisiae that assimilate lactose with a high efficiency. These strains carry multiple copies of Kluyveromyces lactis LAC4 and LAC12 genes, which code for a beta-galactosidase and a lactose permease, respectively. In this work we report additional results about the effect of gene dosage, and analyze the performance of a selected strain in the bioconversion of cheese whey. Additionally, we describe the construction of a new strain, which combines the Lac(+) phenotype with additional properties of biotechnological interest: flocculence, and the ability to hydrolyze starch.

Fluidized bed anaerobic biodegradation of food industry wastewaters

Abstract Anaerobic fluidized bed reactors were used to reduce the COD of low-strength food industry wastewaters. Soluble organic removal efficiencies of 75%, 80% and 50% were obtained for hog slaughterhouse, dairy and brewery wastewaters, respectively, at 35°C and 8 h hydraulic retention time. Removal efficiencies decreased with decreasing temperature (35°C to 20°C); no detrimental effect of temperature was observed when treating the slaughterhouse wasterwater. Methane production rate was only relevant on brewery wastewater treatment.

Assessment of Hyperbolic Heat Transfer Equation in Theoretical Modeling for Radiofrequency Heating Techniques

Theoretical modeling is a technique widely used to study the electrical-thermal performance of different surgical procedures based on tissue heating by use of radiofrequency (RF) currents. Most models employ a parabolic heat transfer equation (PHTE) based on Fourier’s theory, which assumes an infinite propagation speed of thermal energy. We recently proposed a one-dimensional model in which the electrical-thermal coupled problem was analytically solved by using a hyperbolic heat transfer equation (HHTE), i.e. by considering a non zero thermal relaxation time. In this study, we particularized this solution to three typical examples of RF heating of biological tissues: heating of the cornea for refractive surgery, cardiac ablation for eliminating arrhythmias, and hepatic ablation for destroying tumors. A comparison was made of the PHTE and HHTE solutions. The differences between their temperature profiles were found to be higher for lower times and shorter distances from the electrode surface. Our results therefore suggest that HHTE should be considered for RF heating of the cornea (which requires very small electrodes and a heating time of 0.6 s), and for rapid ablations in cardiac tissue (less than 30 s).

A halocin acting on Na+/H+ exchanger of Haloarchaea as a new type of inhibitor in NHE of mammals

The capability of halocin H6 (a bacteriocin-like protein produced by haloarchaeaHaloferax gibbonsii) to inhibit Na+/H+ exchange (NHE) in mammalian cells and its cardio-protective efficacy on the ischemic and reperfused myocardium were evaluated in the present study. H6 inhibits NHE activity (measured by a flow cytometry method) in a dose-dependent form of cell lines of mammalian origin (HEK293, NIH3T3, Jurkat and HL-1) as well as in primary cell culture from human skeletal muscle (myocytes and fibroblasts).In vivo, an ischemia-reperfusion model in dogs by coronary arterial occlusion was used (two hours of regional ischemia and three hours of reperfusion). In animals treated with halocin H6 there was a significant reduction of premature ventricular ectopic beats and infarct size, whereas blood pressure and heart rate remained unchanged. Up to date, halocin H6 is the only described biological molecule that exerts a, specific inhibitory activity in NHE of eukaryotic cells.ResumenEn el presente trabajo se evalúa la capacidad de la halocina H6 (una proteína tipo bacteriocina producida por la haloarchaeaHaloferax gibbonsii) para inhibir el intercambiador Na+/H+ (NHE) de céludas de mamífero y su posible eficacia cardioprotectora frente a los daños causados por isquemia-reperfusión del miocardio. En experimentosin vitro H6 inhibe la actividad de NHE (determinada por citometría de flujo) de forma dosis-dependiente tanto en líneas celulares de mamíferos (HEK293, NIH3T3, Jurkat y HL-1) como en cultivos primarios de miocitos y fibroblastos aislados de músculo esquelético humano. En experimentosin vivo se utilizó un modelo de isquemia-reperfusión en perros por oclusión de la arteria coronaria (dos horas de isquemia y tres de reperfusión). En animales tratados con halocina H6 se produjo una disminución significativa a nivel estadístico, tanto del número de latidos ectópicos ventriculares como del tamaño del infarto, mientras que no se produjeron cambios tanto en la presión sanguínea como en el ritmo cardíaco. Hasta la fecha la halocina H6 es la única molécula biológica descrita que ejerce una actividad inhibidora específica sobre el NHE de células eucariotas.

Esophageal temperature monitoring during radiofrequency catheter ablation: experimental study based on an agar phantom model

Although previous studies have established the feasibility of monitoring esophageal temperature during radiofrequency cardiac ablation using an esophageal temperature probe (ETP), some questions remain regarding its efficacy. The aims of this study were to study the effect of the location of the ETP on the temperature reached, and to test the characteristics of ETP as used in clinical practice. We constructed an agar phantom to model the thermal and electrical characteristics of the biological tissues (left atrium, esophagus and connective tissue). The ETP was positioned at 6.5 mm from an ablation electrode and at distances of 0, 5, 10, 15, 20 mm from the catheter axis. A thermocouple was located on the probe to measure the actual temperature of the external esophageal layer during the ablations (55 degrees C, 60 s). The mean temperatures reached at the thermocouple were significantly higher than those measured by the ETP (48.3 +/- 1.9 degrees C versus 39.6 +/- 1.1 degrees C). The temperature values measured with the ETP were significantly lower when the probe was located further from the catheter axis (up to 2.5 degrees C lower when the distance from the probe-catheter axis was 2 cm). The dynamic calibration of the ETP showed a mean value for the time constant of 8 s. In conclusion, the temperature measured by the ETP always underestimates the temperature reached in the thermocouple. This fact can be explained by the distance gap between the thermocouple and probe and by the dynamic response of the ETP. The longer the distance between the ETP and catheter axis, the higher is the temperature difference.

Fluidized bed biomethanation of acetic acid

SummaryThe kinetics of acetate biomethanation was studied in a high recycle ratio biological fluidized bed reactor behaving in practice as a completely mixed reactor. The active biofilm consisted of bacteria from a methane fermenter that after spontaneous immobilization on the bed particles (sand) were adapted to acetate as the only carbon source. The effects of temperature (13°, 20°, 25° and 35°C), substrate concentration (500, 1000 and 1500 mg chemical oxygen demand (COD) l-1) and hydraulic retention time θ (1 to 8 h) on substrate consumption were studied. Maximum substrate consumption (as % COD reduction) amounted from 25% (13°C, 1500 mg COD l-1) to 93% (35°C, 500 mg COD l-1). At 35°C the concentration of attached biomass presented a weakly increase with reactor substrate concentration (from 3.10 g VS l-1 to 4.54 g VS l-1 for 32 and 1150 mg COD l-1 respectively). On the other hand when reducing ∩, a sharp incrase in biomass loss coefficient was observed showing that excess biofilm growth was continuously removed by shearing forces. Thus in the assayed conditions the attached biomass concentration was basically determined by the bed superficial velocity. Result show that diffusional resistances are negligible. Data are fairly well correlated by a variable order kinetic model. The apparent reaction order is a function of temperature and increases from 0.27 to 0.7 when temperature decreases from 35° C to 13°C.

A cooled water-irrigated intraesophageal balloon to prevent thermal injury during cardiac ablation: experimental study based on an agar phantom

A great deal of current research is directed to finding a way to minimize thermal injury in the esophagus during radiofrequency catheter ablation of the atrium. A recent clinical study employing a cooling intraesophageal balloon reported a reduction of the temperature in the esophageal lumen. However, it could not be determined whether the deeper muscular layer of the esophagus was cooled enough to prevent injury. We built a model based on an agar phantom in order to experimentally study the thermal behavior of this balloon by measuring the temperature not only on the balloon, but also at a hypothetical point between the esophageal lumen and myocardium (2 mm distant). Controlled temperature (55 degrees C) ablations were conducted for 120 s. The results showed that (1) the cooling balloon provides a reduction in the final temperature reached, both on the balloon surface and at a distance of 2 mm; (2) coolant temperature has a significant effect on the temperature measured at 2 mm from the esophageal lumen (it has a less effect on the temperature measured on the balloon surface) and (3) the pre-cooling period has a significant effect on the temperature measured on the balloon surface (the effect on the temperature measured 2 mm away is small). The results were in good agreement with those obtained in a previous clinical study. The study suggests that the cooling balloon gives thermal protection to the esophagus when a minimum pre-cooling period of 2 min is programmed at a coolant temperature of 5 degrees C or less.

Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells

The Na+/H+ exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H+ ion in exchange for extracellular Na+ and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s.

Esophagus histological analysis after hyperthermia-induced injury: Implications for cardiac ablation

Purpose: To evaluate and numerically score histological alterations observed in the acute phase in the esophagus after being exposed to a hyperthermic dosage and subsequently to correlate the scores obtained with the hyperthermic treatment parameters (i.e. temperature (T) and time (t)). Material and methods: Esophagus samples obtained from New Zealand white rabbits were immersed in a temperature-controlled saline bath at 40, 50, 60 and 70°C for 30, 60 and 90 s. Samples were then processed for histological analysis (Masson Trichrome technique), and evaluated by searching for objective heat-damage signs. A numerical value was assigned to each sample for each finding. Results: In general, all the layers were affected by the treatment, however, the greatest alterations were found in the epithelium and deeper muscular layers (circular and longitudinal). We found no damage (i.e. no differences to control) in all of the samples treated at 40°C, and severe damage in treatments at 60 and 70°C, regardless of exposure time. On the other hand, samples treated at 50°C did show different results related to time: no damage for 30 s, light damage for 60 s, and moderate damage for 90 s. We assigned a score value to each hyperthermic dosage, and obtained the fitted equation based on a logarithmic transformation of the Arrhenius equation: Score = 130.7 – 40,851/(T + 273) + log t, (R2 = 0.9326, P < 0.0001). Conclusions: Hyperthermic treatment mainly affects the epithelium and deeper muscular layers. The results suggest a damage threshold of 50°C for treatments of 30–90 s. The proposed scoring system provides a good fit with the hyperthermic parameters.