PAM-Engineered Toehold Switches as Input-Responsive Activators of CRISPR-Cas12a for Sensing Applications.

The RNA-programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio-engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used to control Cas12a in response to molecular inputs. Reconfiguring the Toehold Switch DNA from a hairpin to a duplex conformation through a strand displacement reaction provides an effective means to modulate the accessibility of the PAM, thereby controlling the binding and cleavage activities of Cas12a. Through this approach, we showcase the potential to trigger downstream Cas12a activity by leveraging proximity-based strand displacement reactions in response to target binding. By utilizing the trans-cleavage activity of Cas12a as a signal transduction method, we demonstrate the versatility of our approach for sensing applications. Our system enables rapid, one-pot detection of IgG antibodies and small molecules with high sensitivity and specificity even within complex matrices. Besides the bioanalytical applications, the switchable PAM-engineered Toehold Switches serve as programmable tools capable of regulating Cas12a-based targeting and DNA processing in response to molecular inputs and hold promise for a wide array of biotechnological applications.

Protein kinase B (AKT) upregulation and Thy-1-αvß3 integrin-induced phosphorylation of Connexin43 by activated AKT in astrogliosis.

BACKGROUND: In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvß3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. METHODS: Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). RESULTS: The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. CONCLUSIONS: Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.

Encapsulation of Gold Nanostructures and Oil-in-Water Nanocarriers in Microgels with Biomedical Potential.

Here we report the incorporation of gold nanostructures (nanospheres or nanorods, functionalized with carboxylate-end PEG) and curcumin oil-in-water (O/W) nanoemulsions (CurNem) into alginate microgels using the dripping technique. While gold nanostructures are promising nanomaterials for photothermal therapy applications, CurNem possess important pharmacological activities as reported here. In this sense, we evaluated the effect of CurNem on cell viability of both cancerous and non-cancerous cell lines (AGS and HEK293T, respectively), demonstrating preferential toxicity in cancer cells and safety for the non-cancerous cells. After incorporating gold nanostructures and CurNem together into the microgels, microstructures with diameters of 220 and 540 µm were obtained. When stimulating microgels with a laser, the plasmon effect promoted a significant rise in the temperature of the medium; the temperature increase was higher for those containing gold nanorods (11⁻12 °C) than nanospheres (1⁻2 °C). Interestingly, the incorporation of both nanosystems in the microgels maintains the photothermal properties of the gold nanostructures unmodified and retains with high efficiency the curcumin nanocarriers. We conclude that these results will be of interest to design hydrogel formulations with therapeutic applications.

Rationally Designed DNA-Based Scaffolds and Switching Probes for Protein Sensing.

The detection of a protein analyte and use of this type of information for disease diagnosis and physiological monitoring requires methods with high sensitivity and specificity that have to be also easy to use, rapid and, ideally, single step. In the last 10 years, a number of DNA-based sensing methods and sensors have been developed in order to achieve quantitative readout of protein biomarkers. Inspired by the speed, specificity, and versatility of naturally occurring chemosensors based on structure-switching biomolecules, significant efforts have been done to reproduce these mechanisms into the fabrication of artificial biosensors for protein detection. As an alternative, in scaffold DNA biosensors, different recognition elements (e.g., peptides, proteins, small molecules, and antibodies) can be conjugated to the DNA scaffold with high accuracy and precision in order to specifically interact with the target protein with high affinity and specificity. They have several advantages and potential, especially because the transduction signal can be drastically enhanced. Our aim here is to provide an overview of the best examples of structure switching-based and scaffold DNA sensors, as well as to introduce the reader to the rational design of innovative sensing mechanisms and strategies based on programmable functional DNA systems for protein detection.

Magnetic resonance imaging-based criteria to differentiate dysferlinopathy from other genetic muscle diseases.

The identification of disease-characteristic patterns of muscle fatty replacement in magnetic resonance imaging (MRI) is helpful for diagnosing neuromuscular diseases. In the Clinical Outcome Study of Dysferlinopathy, eight diagnostic rules were described based on MRI findings. Our aim is to confirm that they are useful to differentiate dysferlinopathy (DYSF) from other genetic muscle diseases (GMD). The rules were applied to 182 MRIs of dysferlinopathy patients and 1000 MRIs of patients with 10 other GMD. We calculated sensitivity (S), specificity (Sp), positive and negative predictive values (PPV/NPV) and accuracy (Ac) for each rule. Five of the rules were more frequently met by the DYSF group. Patterns observed in patients with FKRP, ANO5 and CAPN3 myopathies were similar to the DYSF pattern, whereas patterns observed in patients with OPMD, laminopathy and dystrophinopathy were clearly different. We built a model using the five criteria more frequently met by DYSF patients that obtained a S 95.9%, Sp 46.1%, Ac 66.8%, PPV 56% and NPV 94% to distinguish dysferlinopathies from other diseases. Our findings support the use of MRI in the diagnosis of dysferlinopathy, but also identify the need to externally validate "disease-specific" MRI-based diagnostic criteria using MRIs of other GMD patients.

Imaging mass cytometry analysis of Becker muscular dystrophy muscle samples reveals different stages of muscle degeneration.

Becker muscular dystrophy (BMD) is characterised by fiber loss and expansion of fibrotic and adipose tissue. Several cells interact locally in what is known as the degenerative niche. We analysed muscle biopsies of controls and BMD patients at early, moderate and advanced stages of progression using Hyperion imaging mass cytometry (IMC) by labelling single sections with 17 markers identifying different components of the muscle. We developed a software for analysing IMC images and studied changes in the muscle composition and spatial correlations between markers across disease progression. We found a strong correlation between collagen-I and the area of stroma, collagen-VI, adipose tissue, and M2-macrophages number. There was a negative correlation between the area of collagen-I and the number of satellite cells (SCs), fibres and blood vessels. The comparison between fibrotic and non-fibrotic areas allowed to study the disease process in detail. We found structural differences among non-fibrotic areas from control and patients, being these latter characterized by increase in CTGF and in M2-macrophages and decrease in fibers and blood vessels. IMC enables to study of changes in tissue structure along disease progression, spatio-temporal correlations and opening the door to better understand new potential pathogenic pathways in human samples.

Trade-offs between parameter constraints and model realism: a case study.

Tightly constraint parameter ranges are seen as an important goal in constructing hydrological models, a difficult task in complex models. However, many studies show that complex models are often good at capturing the behaviour of a river. Therefore, this study explores the trade-offs between tightly constrained parameters and the ability to predict hydrological signatures, that capture the behaviour of a river. To accomplish this we built five models of differing complexity, ranging from a simple lumped model to a semi-lumped model with eight spatial subdivisions. All models are built within the same modelling framework, use the same data, and are calibrated with the same algorithm. We also consider two different methods for the potential evapotranspiration. We found that that there is a clear trade-off along the axis of complexity. While the more simple models can constrain their parameters quite well, they fail to get the hydrological signatures right. It is the other way around for the more complex models. The method of evapotranspiration only influences the parameters directly related to it. This study highlights that it is important to focus not only on parametric uncertainty. Tightly constrained parameters can be misguiding as they give credibility to oversimplified model structures.

Nanochannel array device operating through Prussian blue nanoparticles for sensitive label-free immunodetection of a cancer biomarker.

A novel nanochannel array (NC) device that operates through Prussian blue nanoparticles (PBNPs) as redox indicator for sensitive label free immunodetection of a cancer biomarker is presented. Stable and narrow-sized (around 4 nm) PBNPs, protected by polyvinylpyrrolidone, exhibited a well-defined and reproducible redox behavior and were successfully applied for the voltammetric evaluation of the nanochannels (20 nm pore sized) blockage due to the immunocomplex formation. The bigger size of the PBNPs compared with ionic indicators such as the [Fe(CN)6](4-/3-) system leads to an increase in the steric effects hindering their diffusion toward the signaling electrode which in turn is transduced to an improvement of the detection limit from 200 µg mL(-1) to 34 pg human IgG mL(-1). This novel and effective PBNPs-NC technology for the detection of small proteins captured inside the nanochannels is successfully applied for the quantification of a cancer biomarker (parathyroid hormone-related protein, PTHrP) in a real clinical scenario such as cell culture medium. The achieved label-free detection of PTHrP at levels of 50 ng mL(-1) is with great interest to study relevant functions that this protein exerts in normal tissues and cancer.

Disfunción erectiva: revisión clínica y actualización / Erectile dysfunction: clinical review and update

Este trabajo pretende entregar una visión actualizada de la disfunción erectiva. Para esto se revisa la fisiología de la erección señalando la contribución de diversos factores en la impotencia; se analiza el problema del diagnóstico diferencial entre disfunciones de etiología orgánica y psicógenas, y se señalan la necesidad de la evaluación de ambos tipos de factores. Finalmente se trata distintos conceptos y visiones respecto a la disfunción erectiva psicógena, así como sus indicaciones terapéuticas. Se analiza la interacción entre disfunción erectiva y conflicto de pareja. Se presenta algunos problemas clínicos y sugerencias de tratamiento

Expresión verbal de ansiedad en pacientes intentadoras de suicidios / Verbal expression of anxiety in attempters suicide patients

Se investigó la expresión verbal de ansiedad mediante análisis de contenido, en 46 mujeres intentadoras de suicidios de nivel socioeconómico bajo. La ansiedad de separación fue la más frecuente, y su mayor prevalencia se observó en pacientes con diagnósticos de depresión mayor y de trastorno de adaptación con ánimo depresivo. Casi la mitad de las pacientes con distimia evidenciaron niveles elevados de ansiedad de muerte. Otras formas, como ansiedad de culpa y difusa, fueron más evidentes en los trastornos de adaptación con rasgos emocionales mixtos y ansiosos. Los hallazgos corroboran la relevancia de los eventos de pérdida y de la ansiedad de separación en sujetos que han intentado suicidarse