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The understanding of anti-tumor drug effects requires specific experimental settings which model clinical scenarios. We describe a protocol for 10-day treatment of lowly aggressive tumor cell lines with antineoplastic agents at concentrations which do not affect cell growth. We describe steps for seeding cells and treating cells with anti-tumor drugs. We then detail steps for cell sensitivity, cell proliferation, and mRNA and protein expression assays. We also detail assays to determine modifications in compound efflux. For complete details on the use and execution of this protocol, please refer to Rios Medrano et al.1.

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Entomopathogenic fungi have been considered potential biological control agents against the fall armyworm Spodoptera frugiperda (J. E. Smith), the world's most important pest of maize. In this study, we evaluated the natural infection, molecular characteristics, and biological activity of Metarhizium rileyi (Farlow) isolated from S. frugiperda larvae of this insect, collected from maize crops in five Mexican locations. Natural infection ranged from 23% to 90% across all locations analyzed. Twenty-four isolates were evaluated on S. frugiperda second instars at a concentration of 1.0 × 108 conidia/mL, causing 70% to 98.7% mortality and 60.5% to 98.7% sporulation. Isolates T9-21, Z30-21, PP48-21, and L8-22 were selected to determine their phylogenetic relationships by ß-tubulin gene analysis and to compare median lethal concentration (CL50), median lethal time (LT50), and larval survival. These isolates were grouped into three clades. The T9-21, PP48-21, and J10-22 isolates were closely related (clade A), but phylogenetically distant from Z30-21 (clade B) and L8-22 (clade C) isolates. These genetic differences were not always reflected in their pathogenicity characteristics since no differences were observed among the LC50 values. Furthermore, isolates T9-21, J10-22, and L8-22 were the fastest to kill S. frugiperda larvae, causing lower survival rates. We conclude that native M. rileyi isolates represent an important alternative for the biocontrol of S. frugiperda.

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Mitochondria are dynamic organelles that respond to cellular stress through changes in global mass, interconnection, and subcellular location. As mitochondria play an important role in tumor development and progression, alterations in energy metabolism allow tumor cells to survive and spread even in challenging conditions. Alterations in mitochondrial bioenergetics have been recently proposed as a hallmark of cancer, and positive regulation of lipid metabolism constitutes one of the most common metabolic changes observed in tumor cells. Acyl-CoA synthetase 4 (ACSL4) is an enzyme catalyzing the activation of long chain polyunsaturated fatty acids with a strong substrate preference for arachidonic acid (AA). High ACSL4 expression has been related to aggressive cancer phenotypes, including breast cancer, and its overexpression has been shown to positively regulate the mammalian Target of Rapamycin (mTOR) pathway, involved in the regulation of mitochondrial metabolism genes. However, little is known about the role of ACSL4 in the regulation of mitochondrial function and metabolism in cancer cells. In this context, our objective was to study whether mitochondrial function and metabolism, processes usually altered in tumors, are modulated by ACSL4 in breast cancer cells. Using ACSL4 overexpression in MCF-7 cells, we demonstrate that this enzyme can increase the mRNA and protein levels of essential mitochondrial regulatory proteins such as nuclear respiratory factor 1 (NRF-1), voltage-dependent anion channel 1 (VDAC1) and respiratory chain Complex III. Furthermore, respiratory parameters analysis revealed an increase in oxygen consumption rate (OCR) and in spare respiratory capacity (SRC), among others. ACSL4 knockdown in MDA-MB-231 cells led to the decrease in OCR and in SCR, supporting the role of ACSL4 in the regulation of mitochondrial bioenergetics. Moreover, ACSL4 overexpression induced an increase in glycolytic function, in keeping with an increase in mitochondrial respiratory activity. Finally, there was a decrease in mitochondrial mass detected in cells that overexpressed ACSL4, while the knockdown of ACSL4 expression in MDA-MB-231 cells showed the opposite effect. Altogether, these results unveil the role of ACSL4 in mitochondrial function and metabolism and expand the knowledge of ACSL4 participation in pathological processes such as breast cancer.

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For many years, research in the field of steroid synthesis has aimed to understand the regulation of the rate-limiting step of steroid synthesis, i.e. the transport of cholesterol from the outer to the inner mitochondrial membrane, and identify the protein involved in the conversion of cholesterol into pregnenolone. The extraordinary work by B Clark, J Wells, S R King, and D M Stocco eventually identified this protein and named it steroidogenic acute regulatory protein (StAR). The group's finding was also one of the milestones in understanding the mechanism of nonvesicular lipid transport between organelles. A notable feature of StAR is its high degree of phosphorylation. In fact, StAR phosphorylation in the acute phase is required for full steroid biosynthesis. As a contribution to this subject, our work has led to the characterization of StAR as a substrate of kinases and phosphatases and as an integral part of a mitochondrion-associated multiprotein complex, essential for StAR function and cholesterol binding and mitochondrial transport to yield maximum steroid production. Results allow us to postulate the existence of a specific cellular microenvironment where StAR protein synthesis and activation, along with steroid synthesis and secretion, are performed in a compartmentalized manner, at the site of hormone receptor stimulation, and involving the compartmentalized formation of the steroid molecule-synthesizing complex.

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Objetivo: conhecer a experiência de manejo familiar da criança à espera de um transplante de órgãos sólidos de acordo com o Modelo de estilos de manejo familiar. Método: foi realizado um estudo qualitativo que utilizou o modelo supracitado como referencial teórico. Foram aplicadas entrevistas, de forma remota, a nove familiares de crianças que aguardavam por um transplante de órgãos. Os dados foram analisados através do modelo híbrido de análise temática. Resultados: as famílias definem que essa fase oscila entre o medo do transplante, da rejeição do órgão e possibilidade de morte em fila de espera, e a esperança na melhora do quadro clínico. Considerações finais: identificar as necessidades da família que aguarda por um transplante possibilita ao profissional de saúde implementar intervenções

Objective: to understand the family management experience of children waiting for a solid organ transplant in accordance with the Family Management Styles Model. Method: a qualitative study was carried out using the aforementioned model as a theoretical framework. Interviews were conducted remotely with nine family members of children waiting for an organ transplant. Data were analyzed using the hybrid thematic analysis model. Results: families define that this phase oscillates between fear of transplantation, organ rejection and the possibility of death on the waiting list, and hope for an improvement in the clinical condition. Final considerations: identifying the needs of the family waiting for a transplant allows the health professional to implement interventions

Objetivos:comprender la experiencia de gestión familiar de niños en espera de trasplante de órgano sólido de acuerdo con el Modelo de Estilos de Gestión Familiar. Método: se realizó un estudio cualitativo utilizando como marco teórico el modelo antes mencionado. Las entrevistas se realizaron de forma remota con nueve familiares de niños que esperaban un trasplante de órgano. Los datos se analizaron utilizando el modelo de análisis temático híbrido. Resultados: las familias definen que esta fase oscila entre el miedo al trasplante, el rechazo del órgano y la posibilidad de muerte en lista de espera, y la esperanza de mejora del cuadro clínico. Consideraciones finales: identificar las necesidades de la familia en espera de un trasplante permite al profesional de la salud implementar intervenciones

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RESUMO

Objetivo: conhecer a experiência de manejo familiar da criança à espera de um transplante de órgãos sólidos de acordo com o Modelo de estilos de manejo familiar. Método: foi realizado um estudo qualitativo que utilizou o modelo supracitado como referencial teórico. Foram aplicadas entrevistas, de forma remota, a nove familiares de crianças que aguardavam por um transplante de órgãos. Os dados foram analisados através do modelo híbrido de análise temática. Resultados: as famílias definem que essa fase oscila entre o medo do transplante, da rejeição do órgão e possibilidade de morte em fila de espera, e a esperança na melhora do quadro clínico. Considerações finais: identificar as necessidades da família que aguarda por um transplante possibilita ao profissional de saúde implementar intervenções

Objective: to understand the family management experience of children waiting for a solid organ transplant in accordance with the Family Management Styles Model. Method: a qualitative study was carried out using the aforementioned model as a theoretical framework. Interviews were conducted remotely with nine family members of children waiting for an organ transplant. Data were analyzed using the hybrid thematic analysis model. Results: families define that this phase oscillates between fear of transplantation, organ rejection and the possibility of death on the waiting list, and hope for an improvement in the clinical condition. Final considerations: identifying the needs of the family waiting for a transplant allows the health professional to implement interventions

Objetivos:comprender la experiencia de gestión familiar de niños en espera de trasplante de órgano sólido de acuerdo con el Modelo de Estilos de Gestión Familiar. Método: se realizó un estudio cualitativo utilizando como marco teórico el modelo antes mencionado. Las entrevistas se realizaron de forma remota con nueve familiares de niños que esperaban un trasplante de órgano. Los datos se analizaron utilizando el modelo de análisis temático híbrido. Resultados: las familias definen que esta fase oscila entre el miedo al trasplante, el rechazo del órgano y la posibilidad de muerte en lista de espera, y la esperanza de mejora del cuadro clínico. Consideraciones finales: identificar las necesidades de la familia en espera de un trasplante permite al profesional de la salud implementar intervenciones

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BACKGROUND: Type 2 Diabetes (T2DM) prevalence is increasing in low- and middle-income countries along with high levels of obesity which vary according to socioeconomic and contextual characteristics. We aim to estimate the prevalence of T2DM and obesity in men and women in a hard-to-reach rural area in northern Ecuador considering socio-demographic characteristics. METHODS: Cross-sectional descriptive study based on a population-based survey in the Eloy Alfaro health district of Esmeraldas between October 2020 and January 2022. We collected sociodemographic information and risk factors for non-communicable diseases with an adapted version of the STEPS survey, performed oral glucose tolerance tests, biochemistry and took physical measurements. We estimated the prevalence of T2DM, obesity, and calculated Odds Ratios (OR) with confidence intervals by logistic regression in Stata v.15. RESULTS: The overall prevalence of T2DM was 6.8% (CI95%: 4.9-8.7), markedly higher among women compared to men (10.4%, CI95% 7.3-13.4%, compared 2.0%, CI95% 0.4-3.7% respectively). The risk of having T2DM in women was 5 times higher than in men adjusting for age, ethnicity, employment, household earnings and obesity (OR: 5.03; 95%CI: 1.68-15.01). Regarding age, the risk of T2DM increased by 6% per year (adjusted OR: 1.06; 95%CI: 1.03-1.08). Obesity prevalence was 30.8% (CI95%: 27.3-34.3), in women was nearly three times higher than in men (43.2% CI95%: 38.2-48.2, compared to 14.7% prevalence, CI95%: 10.6-18.8). Indigenous women had a lower prevalence of obesity compared with the Afro-Ecuadorian women (OR: 0.05; 95%CI: 0.02-0.18) after adjusting for age, employment status, household earnings and setting. CONCLUSION: We found alarming differences between the prevalence of T2DM and obesity in women and men that may be explained by gender roles, exacerbated in the rural context. Health promotion measures with a gender perspective should be adapted according to the characteristics of isolated rural contexts.

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An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far.

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This qualitative study aims to explore how the COVID-19 pandemic impacted healthcare access for patients with chronic conditions in Ecuador from the patient's perspective. We interviewed 19 patients diagnosed with arterial hypertension or type 2 diabetes in rural and urban areas of Ecuador during August and September 2020. We used the Framework Method to analyse the interview transcripts with ATLAS.Ti 8.4 and organised the ideas discussed using categories from the World Health Organization Commission on the Social Determinants of Health conceptual framework. Reorganization of health services during the pandemic meant that patients with arterial hypertension or diabetes could no longer attend face-to-face appointments for disease follow-up. System failures related to medication supply led to increased out-of-pocket payments, which, together with reduced or absent earnings, and in a context with limited social protection policies, meant that patients frequently went for prolonged periods without medication. Rural health initiatives, support from family and use of traditional medicine were reported as ways to manage their chronic condition during this time. Barriers to disease management disproportionately affected individuals with low socioeconomic positions. Stock shortages, lack of protective labour policies and limited reach of anticipatory policies for health emergencies likely worsened pre-existing health inequities in Ecuador.

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COVID-19 lockdowns greatly affected the mental health of populations and collectives. This study compares the mental health and self-perceived health in five countries of Latin America and Spain, during the first wave of COVID 19 lockdown, according to social axes of inequality. This was a cross-sectional study using an online, self-managed survey in Brazil, Chile, Ecuador, Mexico, Peru, and Spain. Self-perceived health (SPH), anxiety (measured through GAD-7) and depression (measured through PHQ-9) were measured along with lockdown, COVID-19, and social variables. The prevalence of poor SPH, anxiety, and depression was calculated. The analyses were stratified by gender (men = M; women = W) and country. The data from 39,006 people were analyzed (W = 71.9%). There was a higher prevalence of poor SPH and bad mental health in women in all countries studied. Peru had the worst SPH results, while Chile and Ecuador had the worst mental health indicators. Spain had the lowest prevalence of poor SPH and mental health. The prevalence of anxiety and depression decreased as age increased. Unemployment, poor working conditions, inadequate housing, and the highest unpaid workload were associated with worse mental health and poor SPH, especially in women. In future policies, worldwide public measures should consider the great social inequalities in health present between and within countries in order to tackle health emergencies while reducing the health breach between populations.

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