Analytical tools for the multiplex rapid detection of SARS-CoV-2

There is a high demand for analytical tools that can readily be applied to effectively diagnose the Covid-19 but also to carry out screening and surveillance detection with enough frequency to get the transmission rates under control and thus help to timely tackling the disease. On the one hand, high throughput analytical benchtop approaches are still highly demanding for accelerating diagnostics. Such platforms are required to show multiplexed capabilities while in turn reducing the turnaround times of currently applied techniques such as the RT-PCR gold standard. On the other hand, massive screening and surveillance protocols still require for effective tools at the point of need that could reliably detect the virus in individuals after being exposed or the likelihood of being immunized after suffering from the disease. The rapid detection of coronavirus biomarkers, including RNA as well as spike and nucleocapsid proteins in nasopharyngeal and oropharyngeal samples, together with host biomarkers such as immunoglobulins and cytokines in serum has been addressed in this work. We aim to produce tools that provided with a global response to the diagnosis, prognosis and follow-up of the disease (Figure 1). All the biocomponents and corresponding bioassay protocols required for measuring these biomarkers have been tailored made and implemented in three different platforms. A calorimetric device based on a lateral flow assay format [1, 2] and a multiplex electrochemical platform comprising an electrochemical transducer array and a paper microfluidic component [3] have been adapted to produce tools to be used at the point of care. Likewise, a fluorescence microarray platform has been set up with the potential for high-throughput screening by recording molecular signatures thanks to the its multiplexing and miniaturization capabilities. REFERENCES [1] E. Polo et al. PCT, ES2013/070549 [2] E. Polo et al. Chem. Commun., 49 (2013) 3676 [3] C. Fernández-Sánchez et al. Application no. EP20382721.

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Interaction Testing and Polygenic Risk Scoring to Estimate the Association of Common Genetic Variants with Treatment Resistance in Schizophrenia

Importance: About 20% to 30% of people with schizophrenia have psychotic symptoms that do not respond adequately to first-line antipsychotic treatment. This clinical presentation, chronic and highly disabling, is known as treatment-resistant schizophrenia (TRS). The causes of treatment resistance and their relationships with causes underlying schizophrenia are largely unknown. Adequately powered genetic studies of TRS are scarce because of the difficulty in collecting data from well-characterized TRS cohorts. Objective: To examine the genetic architecture of TRS through the reassessment of genetic data from schizophrenia studies and its validation in carefully ascertained clinical samples. Design, Setting, and Participants: Two case-control genome-wide association studies (GWASs) of schizophrenia were performed in which the case samples were defined as individuals with TRS (n = 10501) and individuals with non-TRS (n = 20325). The differences in effect sizes for allelic associations were then determined between both studies, the reasoning being such differences reflect treatment resistance instead of schizophrenia. Genotype data were retrieved from the CLOZUK and Psychiatric Genomics Consortium (PGC) schizophrenia studies. The output was validated using polygenic risk score (PRS) profiling of 2 independent schizophrenia cohorts with TRS and non-TRS: a prevalence sample with 817 individuals (Cardiff Cognition in Schizophrenia [CardiffCOGS]) and an incidence sample with 563 individuals (Genetics Workstream of the Schizophrenia Treatment Resistance and Therapeutic Advances [STRATA-G]). Main Outcomes and Measures: GWAS of treatment resistance in schizophrenia. The results of the GWAS were compared with complex polygenic traits through a genetic correlation approach and were used for PRS analysis on the independent validation cohorts using the same TRS definition. Results: The study included a total of 85490 participants (48635 [56.9%] male) in its GWAS stage and 1380 participants (859 [62.2%] male) in its PRS validation stage. Treatment resistance in schizophrenia emerged as a polygenic trait with detectable heritability (1% to 4%), and several traits related to intelligence and cognition were found to be genetically correlated with it (genetic correlation, 0.41-0.69). PRS analysis in the CardiffCOGS prevalence sample showed a positive association between TRS and a history of taking clozapine (r2 = 2.03%; P =.001), which was replicated in the STRATA-G incidence sample (r2 = 1.09%; P =.04). Conclusions and Relevance: In this GWAS, common genetic variants were differentially associated with TRS, and these associations may have been obscured through the amalgamation of large GWAS samples in previous studies of broadly defined schizophrenia. Findings of this study suggest the validity of meta-analytic approaches for studies on patient outcomes, including treatment resistance

Padrões de condução de impulsos nervosos, determinados eletronicamente, para análise de comportamento de nervos "in vivo"

Padrões de condução de impulsos nervosos foram determinados eletrônicamente, in vivo, nos nervos ulnar (13), hipoglosso (16) e cutâneo medial do braço (7) de cães anestesiados. Os traçados obtidos confirmaram, em linhas gerais, os descritos na literatura. Todavia, demonstraram um fato novo, adicional, isto é, que fibras motoras e sensitivas, integrantes de nervos periféricos, apresentam uma condução específica, unidirecional (centrífuga nas fibras motoras e centrípeta nas sensitivas), desde que mantidas praticamente intactas em seus respectivos leitos conectivos naturais. Embora este tipo de condução específica corresponda ao que se admite e aceita como comportamento normal, biológico, dessas fibras nervosas, e constitua ainda, base do exame neurológico, esta evidência nunca foi demonstrada anteriormente em experiências e preparações neurofisiológicas

NO1, a New Sigma 2 Receptor/TMEM97 Fluorescent Ligand, Downregulates SOCE and Promotes Apoptosis in the Triple Negative Breast Cancer Cell Lines

(1) Background: The structure of the Sigma 2 receptor/TMEM97 (&sigma;2RTMEM97) has recently been reported. (2, 3) Methods and results: We used genetic and biochemical approaches to identify the molecular mechanism downstream of &sigma;2R/TMEM97. The novel &sigma;2R/TMEM97 fluorescent ligand, NO1, reduced the proliferation and survival of the triple negative breast cancer cell lines (TNBC: MDA-MB-231 and MDA-MB-468 cell lines), due to NO1-induced apoptosis. Greater bioaccumulation and faster uptake of NO1 in MDA-MB-231 cells compared to MCF10A or MCF7 cell lines were also shown. Accordingly, elevated &sigma;2R/TMEM97 expression was confirmed by Western blotting. In contrast to NO1, other &sigma;2R/TMEM97 ligands, such as SM21 and PB28, enhanced MDA-MB-231 cell proliferation and migration. Store-operated calcium entry (SOCE) is crucial for different cancer hallmarks. Here, we show that NO1, but not other &sigma;2R/TMEM97 ligands, reduced SOCE in MDA-MB-231 cells. Similarly, TMEM97 silencing in MDA-MB-231 cells also impaired SOCE. NO1 administration downregulated STIM1-Orai1 interaction, probably by impairing the positive regulatory effect of &sigma;2R/TMEM97 on STIM1, as we were unable to detect interaction with Orai1. (4) Conclusion: &sigma;2R/TMEM97 is a key protein for the survival of triple negative breast cancer cells by promoting SOCE; therefore, NO1 may become a good pharmacological tool to avoid their proliferation

NO1, a New Sigma 2 Receptor/TMEM97 Fluorescent Ligand, Downregulates SOCE and Promotes Apoptosis in the Triple Negative Breast Cancer Cell Lines

ANTECEDENTES: Recientemente se ha publicado la estructura del receptor Sigma 2/TMEM97 (σ2RTMEM97). (2, 3) MÉTODOS Y RESULTADOS: Utilizamos enfoques genéticos y bioquímicos para identificar el mecanismo molecular aguas abajo de σ2R/TMEM97. El nuevo ligando fluorescente σ2R/TMEM97, NO1, redujo la proliferación y la supervivencia de las líneas celulares de cáncer de mama triple negativo (TNBC: líneas celulares MDA-MB-231 y MDA-MB-468), debido a la apoptosis inducida por NO1. También se demostró una mayor bioacumulación y una captación más rápida de NO1 en las células MDA-MB-231 en comparación con las líneas celulares MCF10A o MCF7. En consecuencia, la elevada expresión de σ2R/TMEM97 se confirmó mediante Western blotting. A diferencia de NO1, otros ligandos σ2R/TMEM97, como SM21 y PB28, potenciaron la proliferación y migración de las células MDA-MB-231. La entrada de calcio operada por almacenamiento (SOCE) es crucial para diferentes características del cáncer. Aquí demostramos que NO1, pero no otros ligandos σ2R/TMEM97, redujo la SOCE en células MDA-MB-231. Del mismo modo, el silenciamiento de TMEM97 en células MDA-MB-231 también afectó a SOCE. La administración de NO1 redujo la interacción STIM1-Orai1, probablemente al afectar al efecto regulador positivo de σ2R/TMEM97 sobre STIM1, ya que no pudimos detectar interacción con Orai1. (4) CONCLUSIONES: σ2R/TMEM97 es una proteína clave para la supervivencia de las células de cáncer de mama triple negativo mediante la promoción de SOCE; por lo tanto, NO1 puede convertirse en una buena herramienta farmacológica para evitar su proliferación.BACKGROUND: The structure of the Sigma 2 receptor/TMEM97 (σ2RTMEM97) has recently been reported. (2, 3) METHODS AND RESULTS: We used genetic and biochemical approaches to identify the molecular mechanism downstream of σ2R/TMEM97. The novel σ2R/TMEM97 fluorescent ligand, NO1, reduced the proliferation and survival of the triple negative breast cancer cell lines (TNBC: MDA-MB-231 and MDA-MB-468 cell lines), due to NO1-induced apoptosis. Greater bioaccumulation and faster uptake of NO1 in MDA-MB-231 cells compared to MCF10A or MCF7 cell lines were also shown. Accordingly, elevated σ2R/TMEM97 expression was confirmed by Western blotting. In contrast to NO1, other σ2R/TMEM97 ligands, such as SM21 and PB28, enhanced MDA-MB-231 cell proliferation and migration. Store-operated calcium entry (SOCE) is crucial for different cancer hallmarks. Here, we show that NO1, but not other σ2R/TMEM97 ligands, reduced SOCE in MDA-MB-231 cells. Similarly, TMEM97 silencing in MDA-MB-231 cells also impaired SOCE. NO1 administration downregulated STIM1-Orai1 interaction, probably by impairing the positive regulatory effect of σ2R/TMEM97 on STIM1, as we were unable to detect interaction with Orai1. (4) CONCLUSION: σ2R/TMEM97 is a key protein for the survival of triple negative breast cancer cells by promoting SOCE; therefore, NO1 may become a good pharmacological tool to avoid their proliferation.• Ministerio de Industria, Economía y Competitividad. Proyectos BFU2013-45564C2-1-P y BFU2016-74932-C2-1-P • Junta de Extremadura y Fondos FEDER. Subvenciones GR18061 e IB16046 • Junta de Extremadura. Beca predoctoral PD16072, para Carlos Cantonero ChamorropeerReviewe