Histologic, metabolomic, and transcriptomic differences in fir trees from a peri-urban forest under chronic ozone exposure.

Urbanization modifies ecosystem conditions and evolutionary processes. This includes air pollution, mostly as tropospheric ozone (O3), which contributes to the decline of urban and peri-urban forests. A notable case are fir (Abies religiosa) forests in the peripheral mountains southwest of Mexico City, which have been severely affected by O3 pollution since the 1970s. Interestingly, some young individuals exhibiting minimal O3-related damage have been observed within a zone of significant O3 exposure. Using this setting as a natural experiment, we compared asymptomatic and symptomatic individuals of similar age (≤15 years old; n = 10) using histologic, metabolomic, and transcriptomic approaches. Plants were sampled during days of high (170 ppb) and moderate (87 ppb) O3 concentration. Given that there have been reforestation efforts in the region, with plants from different source populations, we first confirmed that all analyzed individuals clustered within the local genetic group when compared to a species-wide panel (Admixture analysis with ~1.5K SNPs). We observed thicker epidermis and more collapsed cells in the palisade parenchyma of needles from symptomatic individuals than from their asymptomatic counterparts, with differences increasing with needle age. Furthermore, symptomatic individuals exhibited lower concentrations of various terpenes (ß-pinene, ß-caryophylene oxide, α-caryophylene, and ß-α-cubebene) than asymptomatic trees, as evidenced through GC-MS. Finally, transcriptomic analyses revealed differential expression for 13 genes related to carbohydrate metabolism, plant defense, and gene regulation. Our results indicate a rapid and contrasting phenotypic response among trees, likely influenced by standing genetic variation and/or plastic mechanisms. They open the door to future evolutionary studies for understanding how O3 tolerance develops in urban environments, and how this knowledge could contribute to forest restoration.

La urbanización altera tanto las condiciones del ecosistema como los procesos evolutivos, siendo la contaminación del aire, principalmente el ozono troposférico (O3), un factor que contribuye al declive de los bosques urbanos y periurbanos. Un ejemplo destacado son los bosques de oyamel (Abies religiosa) en las montañas periféricas al suroeste de la Ciudad de México, que han sufrido graves afectaciones por la contaminación de O3 desde la década de 1970. Resulta curioso observar que algunos individuos jóvenes presentan un daño mínimo relacionado con el O3 dentro de zonas con una exposición significativa a este contaminante. Aprovechando este entorno como un experimento natural, hemos comparado individuos asintomáticos y sintomáticos de edad similar (≤15 años; n = 10) mediante enfoques histológicos, metabolómicos y transcriptómicos. Las muestras de plantas se recolectaron durante días con concentraciones altas (170 ppb) y moderadas (87 ppb) de O3. Dado que se han llevado a cabo esfuerzos de reforestación en la región con plantas de diferentes poblaciones, primero confirmamos que todos los individuos analizados se organizaron dentro del grupo genético local en comparación con un amplio panel poblacional de esta misma especie (Análisis de Admixture con ~1.5 K SNPs). Observamos una epidermis más gruesa y más células colapsadas en el parénquima en empalizada de las agujas de los individuos sintomáticos que de sus contrapartes asintomáticas, y estas diferencias aumentaban con la edad de la aguja. Además, los individuos sintomáticos exhibieron concentraciones más bajas de varios terpenos (ß­pineno, óxido de ß­cariofileno, α­cariofileno y ß­α­cubebeno) que los árboles asintomáticos, según se evidenció mediante GC­MS. Por último, los análisis transcriptómicos revelaron una expresión diferencial para trece genes relacionados con el metabolismo de carbohidratos, la defensa de plantas y la regulación génica. Nuestros resultados indican una respuesta fenotípica rápida y contrastante entre los árboles, probablemente influenciada por la variación genética presente y/o mecanismos plásticos. Estos hallazgos abren la puerta a futuros estudios evolutivos para comprender cómo se desarrolla la tolerancia al O3 en entornos urbanos y cómo este conocimiento podría contribuir a la restauración forestal.

Modular Synthesis of PEG-Dendritic Block Copolymers by Thermal Azide-Alkyne Cycloaddition with Internal Alkynes and Evaluation of their Self-Assembly for Drug Delivery Applications.

Linear-dendritic block copolymers assemble in solution due to differences in the solubility or charge properties of the blocks. The monodispersity and multivalency of the dendritic block provide unparalleled control for the design of drug delivery systems when incorporating poly(ethylene glycol) (PEG) as a linear block. An accelerated synthesis of PEG-dendritic block copolymers based on the click and green chemistry pillars is described. The tandem composed of the thermal azide-alkyne cycloaddition with internal alkynes and azide substitution is revealed as a flexible, reliable, atom-economical, and user-friendly strategy for the synthesis and functionalization of biodegradable (polyester) PEG-dendritic block copolymers. The high orthogonality of the sequence has been exploited for the preparation of heterolayered copolymers with terminal alkenes and alkynes, which are amenable for subsequent functionalization by thiol-ene and thiol-yne click reactions. Copolymers with tunable solubility and charge were so obtained for the preparation of various types of nanoassemblies with promising applications in drug delivery.

Prevalence of HIV, Treponema pallidum and Their Coinfection in Men Who Have Sex with Men, Medellín-Colombia.

Introduction: In Colombia, HIV and gestational syphilis are notifiable events; however, they are poorly investigated infections in men who have sex with men (MSM). Objective: To determine the prevalence of HIV, Treponema pallidum, and their co-infection in MSM treated at a Health Services Provider Institution (HSPI) specialized in infectious diseases from Medellín. Methods: Cross-sectional study with 3454 MSM. Prevalence was determined with its 95% confidence interval; associated factors were identified using Fisher's Exact test, Pearson's Chi-square, and trend Chi-square. Multivariate adjustment was performed using logistic regression. Analyses were performed using SPSS 29.0. Results: The prevalence of HIV was 5.7%, T. pallidum 0.7%, and co-infection 0.6%. The prevalence of HIV was higher in MSM aged between 24-40 years (7.5%), with technical or university studies (10.0%), without health insurance affiliation (12.4%), and those who have had a sexual partner with HIV (36.2%). T. pallidum was higher in MSM without health insurance affiliation (3.4%), who had sexual relations with people diagnosed with an STI (5.9%), and a sexual partner with HIV (12.1%). Co-infection was higher in MSM without health insurance affiliation (2.7%), and those who had a partner with HIV (11.2%). Conclusion: Compared with the general Colombian population, MSM have a higher risk of HIV, but are similar to T. pallidum. The identification of the main associated factors in each infection demonstrates the need to prioritize subgroups of MSM that show greater vulnerability to these events. This research demonstrates the urgency of implementing health education strategies in MSM who have a sexual partner with HIV or other STIs. Large gaps were also evident in the magnitude of the three events according to the health insurance affiliation regime, which demonstrates problems of social and health injustice, especially with MSM without health insurance affiliation.

Combining anaerobic digestion slurry and different biochars to develop a biochar-based slow-release NPK fertilizer.

In this research, we developed a biochar-based fertilizer using biogas slurry and biochar derived from lignocellulosic agro-residues. Biogas slurry was obtained through the anaerobic digestion of the organic fraction of municipal solid waste (fresh vegetable biomass and/or prepared food), while biochars were derived from residues from quinoa, maize, rice, and sugarcane. The biochar-based fertilizers were prepared using an impregnation process, where the biogas slurry was mixed with each of the raw biochars. Subsequently, we characterized the N, P and K concentrations of the obtained biochar-based fertilizers. Additionally, we analyzed their surface properties using SEM/EDS and FTIR and conducted a slow-release test on these biochar-based fertilizers to assess their capability to gradually release nutrients. Lastly, a bioassay using cucumber plants was conducted to determine the N, P, and K bioavailability. Our findings revealed a significant correlation (r > 0.67) between the atomic O/C ratio, H/C ratio, cation exchange capacity, surface area, and the base cations concentration with N, P, and/or K adsorption on biochar. These properties, in turn, were linked to the capability of the biochar-based fertilizer to release nutrients in a controlled manner. The biochar-based fertilizer derived from corn residues showed <15 % release of N, P and K at 24 h. Utilization of these biochar-based fertilizers had a positive impact on the mineral nutrition of cucumber plants, resulting in an average increase of 61 % in N, 32 % in P, and 19 % in K concentrations. Our results underscore the potential of biochar-based fertilizers in controlled nutrient release and enhanced plant nutrition. Integration of biochar and biogas slurry offers a promising and sustainable approach for NPK recovery and fertilizer production in agriculture. This study presents an innovative and sustainable approach combining the use of biochar for NPK recovery from biogas slurry and its use as a biochar-based fertilizer in agriculture.

Stochastic processes and changes in evolutionary rate are associated with diversification in a lineage of tropical hard pines (Pinus).

The study of the patterns of polymorphism and molecular evolution among closely related species is key to understanding the evolutionary forces involved in the diversification of lineages. This point is a big challenge in species with slow evolutionary rates, long life cycles, and ancient, shared polymorphisms such as conifers. Under the premise of divergence in a stepwise migration process, we expect clinal geographical patterns of purifying selection efficiency, and genetic structure related to latitude or longitude. If migration is accompanied by changes in the environment, we could further expect a role of positive selection in driving species divergence. Here, we infer patterns of polymorphism, efficiency of purifying selection, and molecular evolution using a dataset of 161 nuclear genes (∼71 Kb) in a lineage of hard pines from North America, the Caribbean, Mexico, and Central America presumed to have migrated from North America toward lower latitudes with tropical conditions. Under the premise of differences in selective pressures, we also look for possible signals of positive selection. To test our hypothesis, first we estimated different indices to infer patterns of polymorphism and efficiency of purifying selection (Ka, Ks, Ka/Ks, dN, dS, dN/dS, and dxy) and compared these metrics across five clades. Also, we investigated possible clinal patterns in these indices and morphological traits (needle length and cone length). Then we inferred genetic structure and environmental differences among species to test for possible signals of positive selection using phylogenetic methods in specific clades. We found differences among clades using Ka, Ks, and Ka/Ks with a relaxation of purifying selection, especially in the Elliotti and Patula clades. We also found environmental differences related to geographic distance, and among clades suggesting differences in selective pressures. The indices Ks, dxy, and needle length had relationships with geography but not ovulate cone length. Finally, we found that most analyzed genes are under purifying selection, but there was an exception of faster evolutionary rate in some pine species, suggesting the possible action of positive selection in divergence. Our study indicated that stochastic processes have played a key role in the diversification of the group, with a possible input of positive selection in pines from Mexico and Central America.

Whoever wants better healthcare simply pays more: citizens' perception about voluntary private health insurance in Colombia.

OBJECTIVES: To explore the perceptions that Colombians have about voluntary private health insurance plans (VPHI) in the health system to identify the tensions that exist between the public and private systems. METHODS: A qualitative case study approach with 46 semi structured interviews of patients, healthcare workers, healthcare administrators, decision-makers, and citizens. Interviews were recorded, transcribed, anonymized, digitally stored, and analyzed following grounded theory guidelines. RESULTS: We developed a paradigmatic matrix that explores how, in a context mediated by both the commodification of health and social stratification, perceptions about the failures in the public health system related to lack of timely care, extensive administrative procedures, and the search for privileged care led to positioning VPHI as a solution to these failures. The interviewees identified three consequences of using VPHI: first, the worsening of problems of timely access to care in the public system; second, higher costs for citizens translated into double payment for technologies and services to which they are entitled; third, the widening of inequity gaps in access to health services between people with similar needs but different payment capacities. CONCLUSIONS: These findings can help decision makers to understand citizens´ perceptions about the implications that VPHI may have in worsening equity gaps in the Colombian health system. It also shows, how VPHI is perceived as a double payment for services covered within social security plans and suggests that the perceived lack of timely access to care in the public systems and the fear that citizens have for themselves or their family members when using suboptimal healthcare are important drivers to purchase these private insurances.

RESUMEN: OBJETIVOS: Explorar las percepciones que tienen los colombianos sobre los planes de seguro de salud privados voluntarios (VPHI) en el sistema de salud para identificar las tensiones que existen entre los sistemas público y privado. MéTODOS: Un estudio cualitativo de caso con 46 entrevistas semiestructuradas a pacientes, trabajadores de la salud, administradores de salud, tomadores de decisiones y ciudadanos. Las entrevistas se grabaron, transcribieron y almacenaron de manera anónima. El análisis se hizo siguiendo conceptos de la teoría fundamentada. RESULTADOS: Desarrollamos una matriz paradigmática que explora cómo, en un contexto mediado tanto por la mercantilización de la salud como por la estratificación social, las percepciones sobre las fallas en el sistema de salud público relacionadas con la falta de atención oportuna, procedimientos administrativos extensos y la búsqueda de atención privilegiada llevaron a posicionar los VPHI como una solución a estas fallas. Los entrevistados identificaron tres consecuencias del uso de los VPHI: primero, el empeoramiento de los problemas de acceso oportuno a la atención en el sistema público; segundo, mayores costos para los ciudadanos, traducidos en un pago doble por tecnologías y servicios a los que tienen derecho; tercero, el aumento de las brechas de equidad en el acceso a los servicios de salud entre personas con necesidades similares pero diferentes capacidades de pago. CONCLUSIONES: Estos hallazgos pueden ayudar a los tomadores de decisiones a comprender las percepciones de los ciudadanos sobre las implicaciones que el VPHI puede tener en el empeoramiento de las brechas de equidad en el sistema de salud colombiano. También muestra cómo el VPHI se percibe como un pago doble por servicios cubiertos dentro de los planes de seguridad social y sugiere que la falta percibida de acceso oportuno a la atención en los sistemas públicos y el miedo que los ciudadanos tienen por sí mismos o por sus familiares cuando utilizan una atención sanitaria subóptima son factores importantes para adquirir estos seguros privados.

A low total metabolic tumor volume independently predicts for a longer time to first treatment in initially observed, low tumor burden follicular lymphoma.

Watchful waiting is an acceptable management strategy for advanced-stage, low tumor burden (LTB) patients with follicular lymphoma (FL). However, the prediction of how long this treatment-free observation period will last remains imperfect. We explored whether total metabolic tumor volume (TMTV) and other positron emission tomography parameters were predictive of time to first treatment (TTFT). We analyzed 97 grade 1-3A advanced-stage LTB FL patients and found that a high TMTV was associated with other tumor burden features at diagnosis. Patients with a TMTV above our established cutoff of 50 mL had a significantly shorter median duration of observation (2.6 vs. 8.8 years; p = 0.001). At 5 years, 77% of patients with a high TMTV and 46% of patients with a low TMTV required treatment. In the multivariable analysis, a high TMTV was the only independent factor predicting TTFT (hazard ratio = 2.09; p = 0.017). Overall, TMTV is a strong predictor of the duration of observation in LTB FL patients. Upon validation of our cutoff in external series and standardization of the methodology, the TMTV could become an additional factor to consider deferring or initiating treatment in otherwise LTB patients.

The academic point-of-care anti-CD19 chimeric antigen receptor T-cell product varnimcabtagene autoleucel (ARI-0001 cells) shows efficacy and safety in the treatment of relapsed/refractory B-cell non-Hodgkin lymphoma.

Varnimcabtagene autoleucel (var-cel) is an academic anti-CD19 chimeric antigen receptor (CAR) product used for the treatment of non-Hodgkin lymphoma (NHL) in the CART19-BE-01 trial. Here we report updated outcomes of patients with NHL treated with var-cel. B-cell recovery was compared with patients with acute lymphoblastic leukaemia (ALL). Forty-five patients with NHL were treated. Cytokine release syndrome (any grade) occurred in 84% of patients (4% grade ≥3) and neurotoxicity in 7% (2% grade ≥3). The objective response rate was 73% at Day +100, and the 3-year duration of response was 56%. The 3-year progression-free and overall survival were 40% and 52% respectively. High lactate dehydrogenase was the only covariate with an impact on progression-free survival. The 3-year incidence of B-cell recovery was lower in patients with NHL compared to ALL (25% vs. 60%). In conclusion, in patients with NHL, the toxicity of var-cel was manageable, while B-cell recovery was significantly prolonged compared to ALL. This trial was registered as NCT03144583.

Promoting Reciprocal Relations across Subfields of Behavior Analysis via Collaborations.

Several barriers may inhibit the growth of behavior analysis as a more integrated and collaborative field. Two such barriers are siloed environments that reinforce a basic-applied distinction, and a lack of translational research pathways. We describe the perils of silos, and elaborate on potential solutions to increase reciprocal relations among subfields in behavior analysis. We promote a five-tiered system to classify research in behavior analysis, and discuss literature on cultivating effective intra and cross-disciplinary collaborations, including using the framework of metacontingencies to understand collaborations. We also propose quantitative and qualitative measures to examine whether the potential solutions increase intra and interdisciplinary interactions. These measures include bibliometric (e.g., citations across fields), sociometric (e.g., social network analysis), and narrative analysis. We apply some of these measures to publications from 2011-2022 from the Journal of the Experimental Analysis of Behavior and Journal of Applied Behavior Analysis, and argue that behavior analysis overall may benefit from a more collaborative approach. Supplementary Information: The online version contains supplementary material available at 10.1007/s40614-023-00386-x.

GPCR molecular dynamics forecasting using recurrent neural networks.

G protein-coupled receptors (GPCRs) are a large superfamily of cell membrane proteins that play an important physiological role as transmitters of extracellular signals. Signal transmission through the cell membrane depends on conformational changes in the transmembrane region of the receptor, which makes the investigation of the dynamics in these regions particularly relevant. Molecular dynamics (MD) simulations provide a wealth of data about the structure, dynamics, and physiological function of biological macromolecules by modelling the interactions between their atomic constituents. In this study, a Recurrent and Convolutional Neural Network (RNN) model, namely Long Short-Term Memory (LSTM), is used to predict the dynamics of two GPCR states and three specific simulations of each one, through their activation path and focussing on specific receptor regions. Active and inactive states of the GPCRs are analysed in six scenarios involving APO, Full Agonist (BI 167107) and Partial Inverse Agonist (carazolol) of the receptor. Four Machine Learning models with increasing complexity in terms of neural network architecture are evaluated, and their results discussed. The best method achieves an overall RMSD lower than 0.139 Å and the transmembrane helices are the regions showing the minimum prediction errors and minimum relative movements of the protein.

The evolving landscape of COVID-19 and post-COVID condition in patients with chronic lymphocytic leukemia: A study by ERIC, the European research initiative on CLL.

In this retrospective international multicenter study, we describe the clinical characteristics and outcomes of patients with chronic lymphocytic leukemia (CLL) and related disorders (small lymphocytic lymphoma and high-count monoclonal B lymphocytosis) infected by SARS-CoV-2, including the development of post-COVID condition. Data from 1540 patients with CLL infected by SARS-CoV-2 from January 2020 to May 2022 were included in the analysis and assigned to four phases based on cases disposition and SARS-CoV-2 variants emergence. Post-COVID condition was defined according to the WHO criteria. Patients infected during the most recent phases of the pandemic, though carrying a higher comorbidity burden, were less often hospitalized, rarely needed intensive care unit admission, or died compared to patients infected during the initial phases. The 4-month overall survival (OS) improved through the phases, from 68% to 83%, p = .0015. Age, comorbidity, CLL-directed treatment, but not vaccination status, emerged as risk factors for mortality. Among survivors, 6.65% patients had a reinfection, usually milder than the initial one, and 16.5% developed post-COVID condition. The latter was characterized by fatigue, dyspnea, lasting cough, and impaired concentration. Infection severity was the only risk factor for developing post-COVID. The median time to resolution of the post-COVID condition was 4.7 months. OS in patients with CLL improved during the different phases of the pandemic, likely due to the improvement of prophylactic and therapeutic measures against SARS-CoV-2 as well as the emergence of milder variants. However, mortality remained relevant and a significant number of patients developed post-COVID conditions, warranting further investigations.

Analysis of the Stress Field in Photoelasticity Used to Evaluate the Residual Stresses of a Plastic Injection-Molded Part.

The degree of quality of thermoplastic injection-molded parts can be established based on their weight, appearance, and defects. However, the conditions of the injection process may induce effects on the mechanical performance of the injected parts, and the residual stresses can cause cracks or early failures when an external load or force is applied. To evaluate these mechanical behaviors, different experimental techniques have been reported in the literature, where digital photoelasticity has stood out both for being a non-contact technique and for achieving quantitative results through sophisticated computational algorithms. Against this background, our proposal consists of analyzing the overall residual stress distribution of parts injected under different molding conditions by using digital photoelasticity. In this case, the specimens are subjected to bending strength tests to identify possible effects of the injection process conditions. The findings show that, at mold temperatures of 80 °C, flow-induced residual stresses increase with packing pressure. However, these internal stress levels do not affect the external load applied by the mechanical bending test, while the mass injected at higher levels of packing pressure helps to increase the bending strength of the injected part. At lower mold temperatures (50 °C), the mechanical strength of the injected part is slightly reduced, possibly due to a lower effect of the packing pressure.

Cross-Modal Musical Expectancy in Complex Sound Music: A Grounded Theory.

Expectancy is a core mechanism for constructing affective and cognitive experiences of music. However, research on musical expectations has been largely founded upon the perception of tonal music. Therefore, it is still to be determined how this mechanism explains the cognition of sound-based acoustic and electroacoustic music, such as complex sound music (CSM). Additionally, the dominant methodologies have consisted of well-controlled experimental designs with low ecological validity that have overlooked the listening experience as described by the listeners. This paper presents results concerning musical expectancy from a qualitative research project that investigated the listening experiences of 15 participants accustomed to CSM listening. Corbin and Strauss' (2015) grounded theory was used to triangulate data from interviews along with musical analyses of the pieces chosen by the participants to describe their listening experiences. Cross-modal musical expectancy (CMME) emerged from the data as a subcategory that explained prediction through the interaction of multimodal elements beyond just the acoustic properties of music. The results led to hypothesise that multimodal information coming from sounds, performance gestures, and indexical, iconic, and conceptual associations re-enact cross-modal schemata and episodic memories where real and imagined sounds, objects, actions, and narratives interrelate to give rise to CMME processes. This construct emphasises the effect of CSM's subversive acoustic features and performance practices on the listening experience. Further, it reveals the multiplicity of factors involved in musical expectancy, such as cultural values, subjective musical and non-musical experiences, music structure, listening situation, and psychological mechanisms. Following these ideas, CMME is conceived as a grounded cognition process.

Evaluating pollution-related damage and restoration success in urban forests with participatory monitoring and digital tools.

Peri-urban forest monitoring requires indicators of vegetation damage. An example is the sacred fir (Abies religiosa) forests surrounding Mexico City, which have been heavily exposed to tropospheric ozone, a harmful pollutant, for over 4 decades. We developed a participatory monitoring system with which local community members and scientists generated data on ozone tree damage. Santa Rosa Xochiac rangers (13) used the digital tool KoboToolBox to record ozone damage to trees, tree height, tree ages, tree condition, tree position, and whether the tree had been planted. Thirty-five percent of the trees (n = 1765) had ozone damage. Younger trees had a lower percentage of foliage damaged by ozone than older trees (p < 0.0001), and asymptomatic trees tended to be younger (p < 0.0001). Symptomatic trees were taller than asymptomatic trees of the same age (R2 c  = 0.43, R2 m  = 0.27). Involving local communities facilitated forest monitoring and using digital technology improved data quality. This participatory system can be used to monitor forest condition change over time and thus aids restoration efforts driven by government or local communities' interests, facilitating local decision-making.

Evaluación del daño relacionado a la contaminación y del éxito de la restauración de los bosques urbanos con un monitoreo participativo y herramientas digitales Resumen El monitoreo de los bosques periurbanos requiere indicadores de daños en la vegetación. Un ejemplo son los bosques de abeto (Abies religiosa) que rodean la Ciudad de México, pues desde hace más de 4 décadas han estado expuestos al ozono troposférico, un contaminante nocivo. Desarrollamos un sistema de monitoreo participativo con el que miembros de la comunidad local y científicos generaron datos sobre los daños causados por el ozono en los árboles. Los guardabosques de Santa Rosa Xochiac (133) utilizaron la herramienta digital KoboToolBox para registrar los daños causados por el ozono en los árboles, su altura, edad, estado, posición y si eran árboles plantados. El 35% de los árboles (n = 1,765) presentó daños por ozono. Los árboles más jóvenes tenían un menor porcentaje de follaje dañado por el ozono que los árboles más viejos (p<0.0001), y los árboles asintomáticos tendían a ser más jóvenes (p<0.0001). Los árboles sintomáticos eran más altos que los asintomáticos de la misma edad (R2 c = 0.43, R2 m = 0.27). La participación de las comunidades locales facilitó el monitoreo forestal y el uso de tecnología digital mejoró la calidad de los datos. Este sistema participativo puede utilizarse para monitorear los cambios en el estado de los bosques a lo largo del tiempo y contribuir a los esfuerzos de restauración impulsados por el gobierno o las comunidades locales, facilitando la toma de decisiones a nivel local.

Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger.

INTRODUCTION: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. METHODS: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. RESULTS: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. CONCLUSION: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.

Validación de una nueva estrategia para la identificación de variantes de SARS-CoV-2 mediante secuenciación del gen espiga por Sanger / Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger

Introducción: La aparición de múltiples variantes del SARS-CoV-2 durante la pandemia de COVID-19 es motivo de gran preocupación mundial. Hasta el momento, su análisis se ha centrado principalmente en la secuenciación de nueva generación. Sin embargo, esta técnica es costosa y requiere equipos sofisticados, largos tiempos de procesamiento y personal técnico altamente cualificado con experiencia en bioinformática. Para contribuir al análisis de variantes de interés y de preocupación, aumentar la capacidad diagnóstica y procesar muestras para realizar vigilancia genómica, proponemos una metodología rápida y fácil de aplicar, basada en la secuenciación Sanger de 3 fragmentos del gen que codifica para la proteína espiga. Métodos: Se secuenciaron 15 muestras positivas para SARS-CoV-2 con un valor de umbral de ciclo inferior a 25 por metodologías Sanger y secuenciación de nueva generación. Los datos obtenidos fueron analizados en las plataformas Nextstrain y PANGO Lineages. Resultados: Ambas metodologías permitieron identificar las variantes de interés reportadas por la OMS. Se identificaron 2 muestras como alfa, 3 gamma, una delta, tres mu, una ómicron y 5 cepas cercanas al aislado inicial del virus Wuhan-Hu-1. Según el análisis in silico, también se pueden detectar mutaciones clave para identificar y clasificar otras variantes no evaluadas en el estudio. Conclusión: Los diferentes linajes de interés y preocupación de SARS-CoV-2 se clasifican de forma rápida, ágil y fiable con la metodología de secuenciación de Sanger.(AU)

Introduction: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. Methods: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. Results: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. Conclusion: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.(AU)