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

Comparison of multiple techniques for endobronchial ultrasound-transbronchial needle aspiration specimen preparation in a single institution experience.

The optimal method for specimen preparation of endobronchial ultrasound-transbronchial needle aspiration (EBUS-TBNA) is still controversial. This study aims to compare several techniques available for EBUS-TBNA specimen acquisition and processing, in order to identify the best performing technique. We retrospectively reviewed the data of 199 consecutive patients [male, 73%; median age, 64 years (IQR: 52-74 years)] undergoing EBUS-TBNA at our institution from 2012 through 2014 for diagnosis of hilar-mediastinal lymph node enlargement suspect of neoplastic (n=139) or granulomatous (n=60) disease. All procedures were performed by two experienced bronchoscopists, under conscious sedation and local anaesthesia, using 21/22-Gauge (G) needle, without rapid on-site evaluation (ROSE). Five specimen-processing techniques were used: cytology slides in 42 cases (21%); cell-block in 25 (13%); core-tissue in 60 (30%); combination of cytology slides and core-tissue in 51 (26%); combination of cytology slides and cell-block in 21 (10%). To assess the diagnostic accuracy of each tissue-processing technique we compared the EBUS-TBNA results to those obtained with surgical lymphadenectomy, or 1-year follow-up in non-operated patients. Diagnostic yield, accuracy and area under the curve (AUC) were as follows. Cytology slides: 81%, 80%, 0.90; cell-block: 48%, 33%, 0.67; core-tissue: 87%, 99%, 0.96; cytology slides + core-tissue: 80%, 100%, 1.00; cytology slides + cell-block: 86%, 100%, 1.00. Cytology slides and core-tissue method showed non-significantly different diagnostic yield (P=0.435) and AUC (P=0.152). In our single-institution experience, cytology slides and core-tissue preparations demonstrated high and similar diagnostic performance. Cytology slides combination with core-tissue or cell-block showed the highest performance, however these combination methods were more resource-consuming

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

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

Risk factors for endocrine complications in transfusion-dependent thalassemia patients on chelation therapy with deferasirox: a risk assessment study from a multicentre nation-wide cohort

Transfusion-dependent patients typically develop iron-induced cardiomyopathy, liver disease, and endocrine complications. We aimed to estimate the incidence of endocrine disorders in transfusion-dependent thalassemia (TDT) patients during long-term iron-chelation therapy with deferasirox (DFX).We developed a multicentre follow-up study of 426 TDT patients treated with once-daily DFX for a median duration of 8 years, up to 18.5 years. At baseline, 118, 121, and 187 patients had 0, 1, or ≥2 endocrine diseases respectively. 104 additional endocrine diseases were developed during the follow-up. The overall risk of developing a new endocrine complication within 5 years was 9.7% (95%CI=6.3-13.1). Multiple Cox regression analysis identified 3 key predictors: age showed a positive log-linear effect (adjusted HR for 50% increase=1.2, 95%CI=1.1-1.3, P=0.005), the serum concentration of thyrotropin (TSH) showed a positive linear effect (adjusted HR for 1 mIU/L increase=1.3, 95%CI=1.1-1.4, P

Mode-matching in multiresonant plasmonic nanoantennas for enhanced second harmonic generation

Boosting nonlinear frequency conversion in extremely confined volumes remains a key challenge in nano-optics, nanomedicine, photocatalysis, and background-free biosensing. To this aim, field enhancements in plasmonic nanostructures are often exploited to effectively compensate for the lack of phase-matching at the nanoscale. Second harmonic generation (SHG) is, however, strongly quenched by the high degree of symmetry in plasmonic materials at the atomic scale and in nanoantenna designs. Here, we devise a plasmonic nanoantenna lacking axial symmetry, which exhibits spatial and frequency mode overlap at both the excitation and the SHG wavelengths. The effective combination of these features in a single device allows obtaining unprecedented SHG conversion efficiency. Our results shed new light on the optimization of SHG at the nanoscale, paving the way to new classes of nanoscale coherent light sources and molecular sensing devices based on nonlinear plasmonic platforms.Comment: 14 pages, 4 figure