Towards the understanding of molecular motors and its relationship with local unfolding.

Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane-Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as 'cracking'. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme's integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.

Determination of protein-protein interactions at the single-molecule level using optical tweezers.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule (in singulo) experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based (in multiplo) methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems. OTs are based on optical trapping, where a laser is used to exert a force on a dielectric bead; and optically trap the bead at a controllable position in all three dimensions. Different experimental approaches have been developed to study protein­protein interactions using OTs, such as: (1) refolding and unfolding in trans interaction where one protein is tethered between the beads and the other protein is in the solution; (2) constant force in cis interaction where each protein is bound to a bead, and the tension is suddenly increased. The interaction may break after some time, giving information about the lifetime of the binding at that tension. And (3) force ramp in cis interaction where each protein is attached to a bead and a ramp force is applied until the interaction breaks. With these experiments, parameters such as kinetic constants (koff, kon), affinity values (KD), energy to the transition state ΔG≠, distance to the transition state Δx≠ can be obtained. These parameters characterize the energy landscape of the interaction. Some parameters such as distance to the transition state can only be obtained from force spectroscopy experiments such as those described here.

Functionalization of stable fluorescent nanodiamonds towards reliable detection of biomarkers for Alzheimer's disease.

BACKGROUND: Stable and non-toxic fluorescent markers are gaining attention in molecular diagnostics as powerful tools for enabling long and reliable biological studies. Such markers should not only have a long half-life under several assay conditions showing no photo bleaching or blinking but also, they must allow for their conjugation or functionalization as a crucial step for numerous applications such as cellular tracking, biomarker detection and drug delivery. RESULTS: We report the functionalization of stable fluorescent markers based on nanodiamonds (NDs) with a bifunctional peptide. This peptide is made of a cell penetrating peptide and a six amino acids long ß-sheet breaker peptide that is able to recognize amyloid ß (Aß) aggregates, a biomarker for the Alzheimer disease. Our results indicate that functionalized NDs (fNDs) are not cytotoxic and can be internalized by the cells. The fNDs allow ultrasensitive detection (at picomolar concentrations of NDs) of in vitro amyloid fibrils and amyloid aggregates in AD mice brains. CONCLUSIONS: The fluorescence of functionalized NDs is more stable than that of fluorescent markers commonly used to stain Aß aggregates such as Thioflavin T. These results pave the way for performing ultrasensitive and reliable detection of Aß aggregates involved in the pathogenesis of the Alzheimer disease.

Characterization of the interaction between the Sec61 translocon complex and ppαF using optical tweezers.

The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro-alpha-factor. The unbinding parameters including off-rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko-Hummer-Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off-rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.

Exogenous pigmentation by silver nitrate: Dermatological and toxicological aspects, case report / [Pigmentación exógena por nitrato de plata: aspectos dermatológicos y toxicológicos, a propósito de un caso].

Exogenous pigmentation by silver nitrate is a rare disease whose clinical manifestations appear even years after the contact, making its diagnosis difficult on occasions. It is characterized by the presence of blue-gray macules or plaques on the skin or mucosa in the contact area, sometimes very similar to melanocytic lesions and melanoma, which constitute the main differential diagnosis. We report the case of a male patient from Medellín, Colombia, with a family history of melanoma and the presence of these lesions throughout his body.

La pigmentación exógena por nitrato de plata es una enfermedad poco frecuente, cuyas manifestaciones clínicas pueden aparecer años después del contacto, lo que en ocasiones dificulta su diagnóstico. Se caracteriza por la presencia de máculas o placas azul-grisáceas en la piel o las mucosas de la zona de contacto que, en ocasiones, son muy similares a las lesiones melanocíticas y al melanoma, sus principales diagnósticos diferenciales. Se reporta el caso de un paciente de Medellín, Colombia, con antecedentes familiares de melanoma y presencia de estas lesiones en todo el cuerpo.

A Call to Action to Address the Health Needs and Support Systems to Care for Central American Migrant Families.

The ongoing immigration crisis at the U.S. southern border represents a challenge to society and institutions. The socio-political discussion of immigration has reached and is likely to stay in the position of a top news category. It is hard to ignore the health care implications and negative health outcomes aff ecting Central American migrant families through their long journey to the U.S.-Mexico border. Additionally, radical changes in refugee/asylum policies are affecting how new migrants are being processed and connected to resources, which is affecting their care while detained. As individuals with familial ties to Central America, we share our knowledge of and perspective on contextual factors affecting migrant health. As health care professionals and scholars, we aim to trigger conversation and present a "call to action" to all stakeholders to address the health care needs of vulnerable Central American migrant families, who deserve high-quality and humanitarian care.

Structural characterization of the saxitoxin-targeting APTSTX1 aptamer using optical tweezers and molecular dynamics simulations.

Optical tweezers have enabled the exploration of picoNewton forces and dynamics in single-molecule systems such as DNA and molecular motors. In this work, we used optical tweezers to study the folding/unfolding dynamics of the APTSTX1-aptamer, a single-stranded DNA molecule with high affinity for saxitoxin (STX), a lethal neurotoxin. By measuring the transition force during (un)folding processes, we were able to characterize and distinguish the conformational changes of this aptamer in the presence of magnesium ions and toxin. This work was supported by molecular dynamics (MD) simulations to propose an unfolding mechanism of the aptamer-Mg+2 complex. Our results are a step towards the development of new aptamer-based STX sensors that are potentially cheaper and more sensitive than current alternatives.

Identifying Chaotropic and Kosmotropic Agents by Nanorheology.

Chemical agents are classified into chaotropes (disorder inducing) and kosmotropes (order inducing) based on their ability to destabilize or stabilize, respectively, the structure of hydrated macromolecules and their solutions. Here, we examine the effect of such agents on the mechanical stiffness of the hydration layer of proteins, measured by nanorheology. We examine four different agents and conclude that chaotropic substances induce the overall softening of the protein-hydration layer system, whereas the kosmotropic substances induce stiffening. Specifically, with glucose and trifluoroethanol, two known kosmotropic agents, we observe the stiffening of the hydration layer. In contrast, with guanidine hydrochloride and urea, known kaotropic agents, we observe softening. Thus, the viscoelastic mechanics of the folded, hydrated protein provides an experimental measure of the effect that chaotropes and kosmotropes have on the dynamics.

Single-molecule measurements of the effect of force on Thy-1/αvß3-integrin interaction using nonpurified proteins.

Thy-1 and αvß3 integrin mediate bidirectional cell-to-cell communication between neurons and astrocytes. Thy-1/αvß3 interactions stimulate astrocyte migration and the retraction of neuronal prolongations, both processes in which internal forces are generated affecting the bimolecular interactions that maintain cell-cell adhesion. Nonetheless, how the Thy-1/αvß3 interactions respond to mechanical cues is an unresolved issue. In this study, optical tweezers were used as a single-molecule force transducer, and the Dudko-Hummer-Szabo model was applied to calculate the kinetic parameters of Thy-1/αvß3 dissociation. A novel experimental strategy was implemented to analyze the interaction of Thy-1-Fc with nonpurified αvß3-Fc integrin, whereby nonspecific rupture events were corrected by using a new mathematical approach. This methodology permitted accurately estimating specific rupture forces for Thy-1-Fc/αvß3-Fc dissociation and calculating the kinetic and transition state parameters. Force exponentially accelerated Thy-1/αvß3 dissociation, indicating slip bond behavior. Importantly, nonspecific interactions were detected even for purified proteins, highlighting the importance of correcting for such interactions. In conclusion, we describe a new strategy to characterize the response of bimolecular interactions to forces even in the presence of nonspecific binding events. By defining how force regulates Thy-1/αvß3 integrin binding, we provide an initial step towards understanding how the neuron-astrocyte pair senses and responds to mechanical cues.

Mechanical properties of BiP protein determined by nano-rheology.

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (KD ) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.

Pigmentación exógena por nitrato de plata: aspectos dermatológicos y toxicológicos, a propósito de un caso / Exogenous pigmentation by silver nitrate: Dermatological and toxicological aspects, case report

Resumen | La pigmentación exógena por nitrato de plata es una enfermedad poco frecuente, cuyas manifestaciones clínicas pueden aparecer años después del contacto, lo que en ocasiones dificulta su diagnóstico. Se caracteriza por la presencia de máculas o placas azul-grisáceas en la piel o las mucosas de la zona de contacto que, en ocasiones, son muy similares a las lesiones melanocíticas y al melanoma, sus principales diagnósticos diferenciales. Se reporta el caso de un paciente de Medellín, Colombia, con antecedentes familiares de melanoma y presencia de estas lesiones en todo el cuerpo.

Abstract | Exogenous pigmentation by silver nitrate is a rare disease whose clinical manifestations appear even years after the contact, making its diagnosis difficult on occasions. It is characterized by the presence of blue-gray macules or plaques on the skin or mucosa in the contact area, sometimes very similar to melanocytic lesions and melanoma, which constitute the main differential diagnosis. We report the case of a male patient from Medellín, Colombia, with a family history of melanoma and the presence of these lesions throughout his body.

Necrólisis epidérmica tóxica y lupus eritematoso sistémico / Toxic epidermal necrolysis and systemic lupus erythematosus

Resumen El lupus eritematoso sistémico con manifestación tipo necrólisis epidérmica tóxica es una entidad descrita recientemente y cada vez aparecen más reportes en la literatura. Se describe el caso de una paciente de 15 años con lupus eritematoso sistémico quien presentó una necrólisis epidérmica tóxica extensa, cuyas lesiones iniciales eran tipo eritema multiforme. Se discute el caso a la luz de la literatura actual sobre esta nueva entidad.

Abstract Toxic epidermal necrolysis (TEN)-like systemic lupus erythematosus is a recently described entity and more cases are being published in the literature today. We describe the case of a 15-year old patient with systemic lupus erythematosus who developed TEN that initially started with erythema multiforme (EM)-like lesiones. We discuss this case according to the published literature on this new entity.

Fotodermatosis y terapia dedesensibilización / Photodermatosis and desensitization therapy

Las fotodermatosis son un grupo de desórdenes cutáneos causados o agravados por la radiación ultravioleta o la luz visible. Se dividen en cuatro grupos: I, fotodermatosis mediadas inmunológicamente (anteriormente llamadas idiopáticas); II, fotosensibilidad inducida por drogas y químicos; III, desórdenes de la reparación de excisión de nucleótidos del ADN y, IV, dermatosis fotoagravadas. Para llegar al diagnóstico correcto de estas enfermedades es necesario realizar un enfoque clínico adecuado, que esté apoyado en un interrogatorio y examen físico completos, además de exámenes especializados como la fotoprueba y fotoparche. En esta revisión se hace una recopilación de los aspectos epidemiológicos, clínicos, de diagnóstico y tratamiento de las fotodermatosis mediadas inmunológicamente, con mayor énfasis en la terapia de desensibilización con luz ultravioleta (PUVA, UVA, UVB), la cual ha mostrado resultados positivos en estas enfermedades, prefiriéndose la UVB por la mayoría de los autores, a excepción de la urticaria solar, en la cual la PUVA ha demostrado mejores resultados que la UVB a largo plazo. Para esto es fundamental determinar primero la dosis de eritema mínimo (DEM) y la dosis de urticaria mínima en el caso de la urticaria solar. La principal desventaja de la terapia de fotodesensibilización es que sus efectos son temporales y que los pacientes requieren terapia de mantenimiento para lograr un control duradero de la enfermedad, con el riesgo de los efectos carcinogénicos de la luz ultravioleta. Además, hasta el momento hay pocos estudios que evalúen la eficacia y seguridad de la fototerapia de desensibilización y en algunos casos las muestras son muy pequeñas.

Photodermatoses are a group of skin disorders caused or aggravated by ultraviolet radiation or visible light. They are divided in four groups: I, immunologically mediated photodermatoses (previously called idiopathic); II, drug- and chemical- induced photosensitivity; III, defective DNA repair disorders; and IV, photoaggravated dermatoses. To reach the correct diagnosis of these pathologies is necessary to make an appropriate clinical approach supported by a comprehensive interview and physical examination as well as specialized tests like photopatch and phototest. This review is a compilation of the epidemiological, clinical, diagnosis and treatment of immunologically mediated photodermatoses, with greater focus on desensitization therapy with ultraviolet light (PUVA, UVA and UVB), which has shown positive results in this pathologies, being UVB preferred by most authors except in solar urticaria, in which PUVA has displayed better results than UVB in the long term. Firstable, is essential to determine the minimal erythema dose and minimal urticarial dose for solar urticaria. The main disadvantages of photodesensitization therapy is the short therm effects and requirement of maintenance therapy to reach lasting control of disease with the risk of carcinogenesis of UVL. Further, there are few trials that study efficacy and security of desensitization phototherapy and some of these include small sample of patients corroborate this information.

Lesiones cutáneas en caddies de golf y tenis: Club Campestre / Skins lesions in golf and tennis's caddies: Campestre Club

Objetivo: Describir los tipos de lesiones cutáneas que presentan los caddies de golf y de tenis en el Club el Campestre en sus sedes de Medellín y Llanogrande y comparar la frecuencia y severidad de las mismas entre ambas poblaciones. Métodos: Se realizó un estudio observacional de corte analítico a partir de dos grupos poblacionales laboralmente expuestos. El estudio se realizó con 164 personas de sexo masculino laboralmente expuestos a luz solar, en los grupos de caddies de golf y tenis en la ciudad de Medellín y el municipio de Rionegro, a quienes se les aplicó la evaluación de tiempo - respuesta y se subdividieron de acuerdo a su localización geográfica. Resultados: Se encontró un total de 243 lesiones, de las cuales el mayor porcentaje fueron de tipo acneiforme, seguido de las solares, y posiblemente asociadas a la edad de la población de estudio. Conclusión: Debido a que la población en estudio está laboralmente expuesta a la luz solar por largos periodos de tiempo, se encontró una exacerbación de las lesiones acneiformes propias de la etapa de la adolescencia y, en segunda instancia, lesiones solares que reflejan un daño actínico prematuro, como resultado de la exposición continúa a la luz solar, el inadecuado uso de bloqueador solar y la falta de medios físicos de protección, lo cual hace necesario implementar medidas preventivas para evitar el desarrollo de futuras lesiones cancerosas.

Introduction: When sun exposure is chronic it produces actinic damage of the skin that predisposes to melanoma, basal cell carcinoma, squamous cell carcinoma and other skin lesions considered pre-malign. This study searchs for the association between time and exposure and the skin lesions present in a group of golf and tennis caddies from a private club in Medellin and Rionegro cities. Methods: It was made an observational study in two groups who have had chronic excessive exposure to UV light due to their work. In the study an evaluation of time/response variable had place and a subdivision according to the geographical location was done. The study was done in 164 male patients, chronically exposed to sun light, among groups of golf and tennis caddies in Medellin City and Rionegro. Results: It was found a total of 243 skin lesions of which the highest percentage where acneiform or acne-like eruptions followed by sun lesions lesions. It was found a predominance of acneiform eruptions in patients who used sunscreen within creased frequency. Conclusion: It was found an exacerbation of acneiform eruptions, lesions that are characteristic of the adolescence period. On the other hand sun lesions proof premature actinically damaged skin as a result of the continuous sun exposure, inadequate sunscreen use and the lack of physical protection methods. The latter shows the need to establish or improve preventive steps to avoid further progression of this kind of lesions that can potentiate the development of pre-cancers and skin cancers.