Effect of intestinal pressure on fistula closure during vacuum assisted treatment: A computational approach

AbstractBackgroundEnterocutaneous fistulae, pathological communications between the intestinal lumen and the abdominal skin, can arise as serious complication of gastrointestinal surgery. A current non-surgical treatment for this pathology involves topical application of sub-atmospheric pressure, also known as vacuum assisted closure (VAC). While this technique appears to be promising, surgeons report a number of cases in which its application fails to achieve fistula closure. Here, we evaluate the fistula’s physical properties during the vacuum assisted closure process in a computational approach exploring the relevance of intraluminal intestinal pressure.MethodsA mathematical model formulated by differential equations based on tissue elasticity properties and principles of fluid mechanics was created and forcing functions were integrated to mimic intestinal pressure dynamics. A software to solve equations and to fit the model to experimentally obtained data was developed. This enabled simulations of vacuum assisted fistula closure under different intestinal pressure.ResultsThe simulation output indicates conditions, in which fistula closure can or cannot be expected suggesting favoured or impeded healing, respectively. When modifications of intestinal pressure, as observed in fistula accompanying pathologies, are integrated, the outcome of fistula closure changes considerably. Rise of intestinal pressure is associated with delay of fistula closure and temporary fistula radius augmentation, while reduction of intestinal pressure during sub-atmospheric pressure treatment contributes to a faster and direct fistula closure.ConclusionFrom the model predictions, we conclude that administration of intestinal pressure decreasing compounds (e.g. butylscopolamine, glucagon) may improve VAC treatment, while intestinal pressure increasing drugs should be avoided

Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.Instituto de Física de Líquidos y Sistemas Biológico

Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.Instituto de Física de Líquidos y Sistemas Biológico

Cooperativity in binding processes: New insights from phenomenological modeling

Cooperative binding is one of the most interesting and not fully understood phenomena involved in control and regulation of biological processes. Here we analyze the simplest phenomenological model that can account for cooperativity (i.e. ligand binding to a macromolecule with two binding sites) by generating equilibrium binding isotherms from deterministically simulated binding time courses. We show that the Hill coefficients determined for cooperative binding, provide a good measure of the Gibbs free energy of interaction among binding sites, and that their values are independent of the free energy of association for empty sites. We also conclude that although negative cooperativity and different classes of binding sites cannot be distinguished at equilibrium, they can be kinetically differentiated. This feature highlights the usefulness of pre-equilibrium time-resolved strategies to explore binding models as a key complement of equilibrium experiments. Furthermore, our analysis shows that under conditions of strong negative cooperativity, the existence of some binding sites can be overlooked, and experiments at very high ligand concentrations can be a valuable tool to unmask such sites.Instituto de Física de Líquidos y Sistemas BiológicosFacultad de Ciencias Exacta

Effect of intestinal pressure on fistula closure during vacuum assisted treatment: A computational approach

Background: Enterocutaneous fistulae, pathological communications between the intestinal lumen and the abdominal skin, can arise as serious complication of gastrointestinal surgery. A current non-surgical treatment for this pathology involves topical application of sub-atmospheric pressure, also known as vacuum assisted closure (VAC). While this technique appears to be promising, surgeons report a number of cases in which its application fails to achieve fistula closure. Here, we evaluate the fistula's physical properties during the vacuum assisted closure process in a computational approach exploring the relevance of intraluminal intestinal pressure. Methods: A mathematical model formulated by differential equations based on tissue elasticity properties and principles of fluid mechanics was created and forcing functions were integrated to mimic intestinal pressure dynamics. A software to solve equations and to fit the model to experimentally obtained data was developed. This enabled simulations of vacuum assisted fistula closure under different intestinal pressure. Results: The simulation output indicates conditions, in which fistula closure can or cannot be expected suggesting favoured or impeded healing, respectively. When modifications of intestinal pressure, as observed in fistula accompanying pathologies, are integrated, the outcome of fistula closure changes considerably. Rise of intestinal pressure is associated with delay of fistula closure and temporary fistula radius augmentation, while reduction of intestinal pressure during sub-atmospheric pressure treatment contributes to a faster and direct fistula closure. Conclusion: From the model predictions, we conclude that administration of intestinal pressure decreasing compounds (e.g. butylscopolamine, glucagon) may improve VAC treatment, while intestinal pressure increasing drugs should be avoided.Facultad de Ciencias ExactasInstituto de Física de Líquidos y Sistemas Biológico

Cooperativity in binding processes: New insights from phenomenological modeling

Cooperative binding is one of the most interesting and not fully understood phenomena involved in control and regulation of biological processes. Here we analyze the simplest phenomenological model that can account for cooperativity (i.e. ligand binding to a macromolecule with two binding sites) by generating equilibrium binding isotherms from deterministically simulated binding time courses. We show that the Hill coefficients determined for cooperative binding, provide a good measure of the Gibbs free energy of interaction among binding sites, and that their values are independent of the free energy of association for empty sites. We also conclude that although negative cooperativity and different classes of binding sites cannot be distinguished at equilibrium, they can be kinetically differentiated. This feature highlights the usefulness of pre-equilibrium time-resolved strategies to explore binding models as a key complement of equilibrium experiments. Furthermore, our analysis shows that under conditions of strong negative cooperativity, the existence of some binding sites can be overlooked, and experiments at very high ligand concentrations can be a valuable tool to unmask such sites.Instituto de Física de Líquidos y Sistemas BiológicosFacultad de Ciencias Exacta

Effect of intestinal pressure on fistula closure during vacuum assisted treatment: A computational approach

Background: Enterocutaneous fistulae, pathological communications between the intestinal lumen and the abdominal skin, can arise as serious complication of gastrointestinal surgery. A current non-surgical treatment for this pathology involves topical application of sub-atmospheric pressure, also known as vacuum assisted closure (VAC). While this technique appears to be promising, surgeons report a number of cases in which its application fails to achieve fistula closure. Here, we evaluate the fistula's physical properties during the vacuum assisted closure process in a computational approach exploring the relevance of intraluminal intestinal pressure. Methods: A mathematical model formulated by differential equations based on tissue elasticity properties and principles of fluid mechanics was created and forcing functions were integrated to mimic intestinal pressure dynamics. A software to solve equations and to fit the model to experimentally obtained data was developed. This enabled simulations of vacuum assisted fistula closure under different intestinal pressure. Results: The simulation output indicates conditions, in which fistula closure can or cannot be expected suggesting favoured or impeded healing, respectively. When modifications of intestinal pressure, as observed in fistula accompanying pathologies, are integrated, the outcome of fistula closure changes considerably. Rise of intestinal pressure is associated with delay of fistula closure and temporary fistula radius augmentation, while reduction of intestinal pressure during sub-atmospheric pressure treatment contributes to a faster and direct fistula closure. Conclusion: From the model predictions, we conclude that administration of intestinal pressure decreasing compounds (e.g. butylscopolamine, glucagon) may improve VAC treatment, while intestinal pressure increasing drugs should be avoided.Facultad de Ciencias ExactasInstituto de Física de Líquidos y Sistemas Biológico

A Two-Stage Model for Lipid Modulation of the Activity of Integral Membrane Proteins

Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes

Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation

Abstract Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA