Recurring renal lithiasis due to Dent's disease.

Dent's disease is a rare cause of hypercalciuria and recurring urolithiasis. Patients with this disease have elevated bone resorption due to the presence of parathormone (PTH) in the urine. We describe the case of a 21-year-old male with hypercalciuria, elevated bone resorption and recurring bilateral urolithiasis that achieves radiological and clinical stability with percutaneous nephrolithotomy (PNL) and medical treatment with hydrochlorothiazide, potassium-citrate and phytate.

Stabilization of pre-existing neurotensin receptor conformational states by ß-arrestin-1 and the biased allosteric modulator ML314.

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13CεH3-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor's intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs - without dramatically altering the structural ensemble. ß-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A ß-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

Modulation of Functional Phosphorylation Sites by Basic Residues in the Unique Domain of c-Src.

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.

Targeting the Src N-terminal regulatory element in cancer.

The signaling pathways displayed by cancer cells are often composed by the same components than the physiological ones, yet the overall result is a pathological deregulation. The non-receptor protein tyrosine kinase Src is a good example. Src is the first described proto-oncogene and a demonstrated player in cancer progression, as it affects proliferation, invasion, survival, cancer stemness, and drug resistance. Src activation is linked to poor prognosis in many cancer types, yet mutations in this protein are rarely observed. In addition, being a demonstrated cancer target, unspecific inhibition of the kinase activity has proven inefficient in clinics since the inhibition of Src in non-cancerous cells results in unacceptable toxicity. Thus, there is a need for new target regions in Src that could inhibit Src activity only in certain cell types, e.g., cancer cells, while maintaining the normal physiological activity in healthy cells. The Src N-terminal regulatory element (SNRE) includes the poorly studied intrinsically disordered region with unique sequences for each of the members of the Src family. In this perspective, we discuss the non-canonical regulatory mechanisms involving the SNRE and their potential use as oncotargets.

Implementation of clinical assistants in a pediatric oncology department: An impact analysis.

Bureaucratic and administrative tasks associated with health care provision have historically fallen on health care professionals, which is one among the factors contributing to low job satisfaction and lower productivity. Incorporating new professional roles that help to better respond to the needs of both patients and professionals can increase the quality and efficiency of service provision. This article aims to evaluate the impact of the clinical assistant's introduction in the Sant Joan de Déu Barcelona Children's Hospital's pediatric oncology department, in terms of (i) displacement of activity loads carried out by this new professional role and the consequent time freed up for physicians, (ii) physicians' satisfaction and (iii) efficiency of the new care model. This is an observational and retrospective study using administrative data based on the type of activity performed by clinical assistants and the measurement of the time freed up in favor of the physicians. The potential skill mix productivity increase, survey of physicians' satisfaction, and reduction in costs with the new model was analyzed. During the first year of its implementation in the pediatric oncology department, clinical assistants have performed 13,553 requests (69% of the total), representing a total saving of 266.83 hours or 6.67 workweeks of 40 hours. They performed 74% of outpatient surgical requests in the oncology department, 87% of day hospital requests and 54% of total requests in the outpatient consultations area. Physicians are overall satisfied with the new role and think they can use the time gained to do other things such as research or improving the quality of care. The role change allows reducing the cost per request by 56% in relation to the conventional model. In conclusion, the introduction of clinical assistants in the oncology department could be efficient to the extent that it displaces a significant part of the bureaucratic and administrative tasks previously performed by health care professionals and thus enables to reduce the cost of these processes. This delegation allows them to work more closely to the maximum of their competences and the physicians to have more time for higher added value clinical tasks and increase professional satisfaction.

Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS1).

Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use 13Cε-methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS1). We establish that the NTS1 solution ensemble includes substates with lifetimes on several, discrete timescales. The longest-lived states reflect those captured in agonist- and inverse agonist-bound crystal structures, separated by large energy barriers. We observe that the rapid fluctuations of individual methionine residues, superimposed on these long-lived states, respond collectively with the degree of fast, global dynamics correlating with ligand pharmacology. This approach lends confidence to interpreting spectra in terms of local structure and methyl dihedral angle geometry. The results suggest a role for sub-microsecond dynamics and conformational entropy in GPCR ligand discrimination.

A FRET-Based Biosensor for the Src N-Terminal Regulatory Element.

In signaling proteins, intrinsically disordered regions often represent regulatory elements, which are sensitive to environmental effects, ligand binding, and post-translational modifications. The conformational space sampled by disordered regions can be affected by environmental stimuli and these changes trigger, vis a vis effector domain, downstream processes. The disordered nature of these regulatory elements enables signal integration and graded responses but prevents the application of classical approaches for drug screening based on the existence of a fixed three-dimensional structure. We have designed a genetically encodable biosensor for the N-terminal regulatory element of the c-Src kinase, the first discovered protooncogene and lead representative of the Src family of kinases. The biosensor is formed by two fluorescent proteins forming a FRET pair fused at the two extremes of a construct including the SH4, unique and SH3 domains of Src. An internal control is provided by an engineered proteolytic site allowing the generation of an identical mixture of the disconnected fluorophores. We show FRET variations induced by ligand binding. The biosensor has been used for a high-throughput screening of a library of 1669 compounds with seven hits confirmed by NMR.

Regulation of Src tumor activity by its N-terminal intrinsically disordered region.

The membrane-anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid-Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src's oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

Combinatorial characterization of a certain class of words and a conjectured connection with general subclasses of phylogenetic tree-child networks.

The combinatorial study of phylogenetic networks has attracted much attention in recent times. In particular, one class of them, the so-called tree-child networks, are becoming the most prominent ones. However, their combinatorial properties are largely unknown. In this paper we address the problem of exactly counting them. We conjecture a relationship with the cardinality of a certain class of words. By solving the counting problem for the words, and on the basis of the conjecture, several simple recurrence formulas for general cases arise. Moreover, a precise asymptotic analysis is provided. Our results coincide with all current formulas in the literature for particular subclasses of tree-child networks, as well as with numerical results obtained for small networks. We expect that the study of the relationship between the newly defined words and the networks will lead to further combinatoric characterizations of this class of phylogenetic networks.

Abdominal metastatic melanoma invading the transverse colon and stomach.

Gastrointestinal melanoma metastases are not uncommon, with the jejunum and ileum being the most common locations (58 %), followed by the stomach (26 %), colon (22 %), duodenum (12 %), and rectum (5 %).

A Methionine Chemical Shift Based Order Parameter Characterizing Global Protein Dynamics.

Coupling of side chain dynamics over long distances is an important component of allostery. Methionine side chains show the largest intrinsic flexibility among methyl-containing residues but the actual degree of conformational averaging depends on the proximity and mobility of neighboring residues. The 13 C NMR chemical shifts of the methyl groups of methionine residues located at long distances in the same protein show a similar scaling with respect to the values predicted from the static X-ray structure by quantum methods. This results in a good linear correlation between calculated and observed chemical shifts. The slope is protein dependent and ranges from zero for the highly flexible calmodulin to 0.7 for the much more rigid calcineurin catalytic domain. The linear correlation is indicative of a similar level of side-chain conformational averaging over long distances, and the slope of the correlation line can be interpreted as an order parameter of the global side-chain flexibility.

Revisión de estrategias para promover el envejecimiento activo en Iberoamérica / Review of strategies to promote active aging in Ibero-America

RESUMEN En Iberoamérica se reconocen variadas experiencias que favorecen un envejecimiento activo, y que pueden ser replicadas en diversas comunidades, como estrategias orientadas a una mejor calidad de vida. La presente revisión tiene como objetivo identificar intervenciones o recomendaciones orientadas al fomento de un envejecimiento activo en países de Iberoamérica. La búsqueda de evidencias se efectuó en publicaciones de artículos disponibles entre los años 2010 y 2019, utilizando las bases de datos Medline, Scopus, Scielo, Science-Direct, Biblioteca Virtual de Salud, MEDES y Dialnet. Las recomendaciones y experiencias realizadas para este grupo etario incluyen actividades clásicas como la realización de actividad física o talleres de educación en salud, pero también acciones culturales, turísticas, intergeneracionales, de fortaleciendo cognitivo, actividades intersectoriales, historias de vida, lugar de residencia o el uso de tecnologías de información. Futuras experiencias de promoción destinadas a este grupo etario deben planificarse con una mirada integradora de actividades que favorezcan la vida sana a través de la creación, la cultura y la vida saludable.

ABSTRACT In Latin America, various experiences that promote active aging are recognized, and can be replicated in various communities, as strategies aimed at a better quality of life. The objective of this review is to identify interventions or recommendations aimed at promoting active aging in Ibero-American countries. The search for evidence was carried out in publications of articles available between 2010 and 2019, using the Medline, Scopus, Scielo, Science-Direct, Virtual Health Library, MEDES and Dialnet databases. Recommendations and experiences carried out for this age group include classic activities such as physical activity or health education workshops, but also cultural, tourist, intergenerational, cognitive strengthening actions, intersectoral activities, life stories, place of residence or use of information technologies. Future promotional experiences for this age group should be planned with an integrating perspective of activities that promote healthy living through creation, culture and healthy living.

Integrating disorder in globular multidomain proteins: Fuzzy sensors and the role of SH3 domains.

Intrinsically disordered proteins represent about one third of eukaryotic proteins. An additional third correspond to proteins containing folded domains as well as large intrinsically disordered regions (IDR). While IDRs may represent functionally autonomous domains, in some instances it has become clear that they provide a new layer of regulation for the activity displayed by the folded domains. The sensitivity of the conformational ensembles defining the properties of IDR to small changes in the cellular environment and the capacity to modulate this response through post-translational modifications makes IDR ideal sensors enabling continuous, integrative responses to complex cellular inputs. Folded domains (FD), on the other hand, are ideal effectors, e.g. by catalyzing enzymatic reactions or participating in binary on/off switches. In this perspective review we discuss the possible role of intramolecular fuzzy complexes to integrate the very different dynamic scales of IDR and FD, inspired on the recent observations of such dynamic complexes in Src family kinases, and we explore the possible general role of the SH3 domains connecting IDRs and FD.

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region.

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compaction.

The disordered boundary of the cell: emerging properties of membrane-bound intrinsically disordered proteins.

We define the disordered boundary of the cell (DBC) as the system formed by membrane tethered intrinsically disordered protein regions, dynamically coupled to the underlying membrane. The emerging properties of the DBC makes it a global system of study, which cannot be understood from the individual properties of their components. Similarly, the properties of lipid bilayers cannot be understood from just the sum of the properties of individual lipid molecules. The highly anisotropic confined environment, restricting the position and orientation of interacting sites, is affecting the properties of individual disordered proteins. In fact, the collective effect caused by high concentrations of disordered proteins extend beyond the sum of individual effects. Examples of emerging properties of the DBC include enhanced protein-protein interactions, protein-driven phase separations, Z-compartmentalization, and protein modulated electrostatics.

A Myristoyl-Binding Site in the SH3 Domain Modulates c-Src Membrane Anchoring.

The c-Src oncogene is anchored to the cytoplasmic membrane through its N-terminal myristoylated SH4 domain. This domain is part of an intramolecular fuzzy complex with the SH3 and Unique domains. Here we show that the N-terminal myristoyl group binds to the SH3 domain in the proximity of the RT loop, when Src is not anchored to a lipid membrane. Residues in the so-called Unique Lipid Binding Region modulate this interaction. In the presence of lipids, the myristoyl group is released from the SH3 domain and inserts into the lipid membrane. The fuzzy complex with the SH4 and Unique domains is retained in the membrane-bound form, placing the SH3 domain close to the membrane surface and restricting its orientation. The apparent affinity of myristoylated proteins containing the SH4, Unique, and SH3 domains is modulated by these intramolecular interactions, suggesting a mechanism linking c-Src activation and membrane anchoring.

Cis-trans proline isomers in the catalytic domain of calcineurin.

Calcineurin is an essential calcium-activated serine/threonine phosphatase. The six NMR-observable methionine methyl groups in the catalytic domain of human calcineurin Aα (CNA) were assigned and used as reporters of the presence of potential cis-trans isomers in solution. Proline 84 is found in the cis conformation in most calcineurin X-ray structures, and proline 309, which is part of a highly conserved motif in phosphoprotein phosphatases, was modeled with a cis peptide bond in one of the two molecules present in the asymmetric unit of CNA. We mutated each of the two prolines to alanine to force the trans conformation. Solution NMR shows that the P84A CNA mutant exists in two forms, compatible with cis-trans isomers, while the P309A mutant is predominantly in the trans conformation. DATABASE: PDB depositions mentioned PDB 5C1V and 2JOG.