Cost-effectiveness of dapagliflozin plus standard treatment compared to standard therapy for the management of chronic kidney disease in Colombia.

BACKGROUND: The DAPA-CKD study showed that dapagliflozin added to standard treatment reduced the risk of chronic kidney disease progression, and death from renal or cardiovascular causes compared to placebo. OBJECTIVE: Assess the cost-effectiveness of dapagliflozin and standard treatment versus standard treatment alone for chronic kidney disease within the Colombian health system. METHODS: We employed a Markov model based on the DAPA-CKD study, tailored to the Colombian scenario. The model forecasted hospitalizations for heart failure, overall and cardiovascular mortality, and chronic kidney disease progression over a 10-year horizon with a 5% discount rate. RESULTS: Dapagliflozin combined with standard treatment is a cost-effective intervention in treating stage 2-4 CKD. In the base case, the ICER was US $5,366, below 1 GDP (US $6.558) per capita. This was consistent in the sensitivity analyses. CONCLUSION: Our study showed that dapagliflozin, when combined with standard treatment, is cost-effective against standard treatment alone, aligning with Colombia's willingness-to-pay threshold.

Dynamic changes in mRNA nucleocytoplasmic localization in the nitrate response of Arabidopsis roots.

Nitrate is a nutrient and signal that regulates gene expression. The nitrate response has been extensively characterized at the organism, organ, and cell-type-specific levels, but intracellular mRNA dynamics remain unexplored. To characterize nuclear and cytoplasmic transcriptome dynamics in response to nitrate, we performed a time-course expression analysis after nitrate treatment in isolated nuclei, cytoplasm, and whole roots. We identified 402 differentially localized transcripts (DLTs) in response to nitrate treatment. Induced DLT genes showed rapid and transient recruitment of the RNA polymerase II, together with an increase in the mRNA turnover rates. DLTs code for genes involved in metabolic processes, localization, and response to stimulus indicating DLTs include genes with relevant functions for the nitrate response that have not been previously identified. Using single-molecule RNA FISH, we observed early nuclear accumulation of the NITRATE REDUCTASE 1 (NIA1) transcripts in their transcription sites. We found that transcription of NIA1, a gene showing delayed cytoplasmic accumulation, is rapidly and transiently activated; however, its transcripts become unstable when they reach the cytoplasm. Our study reveals the dynamic localization of mRNAs between the nucleus and cytoplasm as an emerging feature in the temporal control of gene expression in response to nitrate treatment in Arabidopsis roots.

The inorganic pyrophosphatases of microorganisms: a structural and functional review.

Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H+/Na+ pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure-function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences.

Persistence of COVID-19 Symptoms and Quality of Life at Three and Twelve Months after Hospital Discharge.

Background and Objectives: Medical and public recognition of "long-COVID or post-COVID syndrome", as well as its impact on the quality of life (QoL), is required to better address the disease burden. Objectives: We aimed to describe the persistence of COVID-19 symptoms and QoL among patients at three and twelve months after their discharge from the hospital. Materials and Methods: We conducted an observational, prospective, and longitudinal analytic study from September 2021 to April 2022. To measure QoL, we used a validated version of the 36-item Short-Form Health Survey (SF-36). Results: We included 68 patients in the study. A total of 54 (79.4%) patients reported at least one persistent symptom at three months vs. 52 (76.4%) at twelve months (p = 0.804). Some persistent symptoms (myalgia, alopecia, and cough) decreased significantly at twelve months (50% vs. 30.9%, 29.4% vs. 13.2%, and 23.5% vs. 7.4%; respectively, p = 0.007); in contrast, other persistent symptoms (sleep-wake and memory disorders) were more frequent (5.9% vs. 32.4% and 4.4% vs. 20.6%; respectively, p = ≤0.001). Regarding QoL, a statistically significant improvement was observed in some scores over time, p = ≤0.037. At twelve months, dyspnea, myalgia, and depression were risk factors associated with a poor physical component summary (PCS), p = ≤0.027, whereas anxiety, depression, and fatigue were associated with a poor mental component summary (MCS), p = ≤0.015. Conclusion: As the proportion of persistent symptoms at twelve months is high, we suggest that patients must continue under long-term follow up to reclassify, diagnose, and treat new onset symptoms/diseases.

Filling the gaps on root hair development under salt stress and phosphate starvation using current evidence coupled with a meta-analysis approach.

Population expansion is a global issue, especially for food production. Meanwhile, global climate change is damaging our soils, making it difficult for crops to thrive and lowering both production and quality. Poor nutrition and salinity stress affect plant growth and development. Although the impact of individual plant stresses has been studied for decades, the real stress scenario is more complex due to the exposure to multiple stresses at the same time. Here we investigate using existing evidence and a meta-analysis approach to determine molecular linkages between two contemporaneous abiotic stimuli, phosphate (Pi) deficiency and salinity, on a single plant cell model, the root hairs (RHs), which is the first plant cell exposed to them. Understanding how these two stresses work molecularly in RHs may help us build super-adaptable crops and sustainable agriculture in the face of global climate change.

Silencing of Sporothrix schenckii GP70 Reveals Its Contribution to Fungal Adhesion, Virulence, and the Host-Fungus Interaction.

Sporothrix schenckii is one of the etiological agents of sporotrichosis, a cutaneous and subcutaneous infection distributed worldwide. Like other medically relevant fungi, its cell wall is a molecular scaffold to display virulence factors, such as protective pigments, hydrolytic enzymes, and adhesins. Cell wall proteins with adhesive properties have been previously reported, but only a handful of them have been identified and characterized. One of them is Gp70, an abundant cell wall protein mainly found on the surface of yeast-like cells. Since the protein also has a role in the activity of 3-carboxy-cis,cis-muconate cyclase and its abundance is low in highly virulent strains, its role in the Sporothrix-host interaction remains unclear. Here, a set of GP70-silenced strains was generated, and the molecular and phenotypical characterization was performed. The results showed that mutants with high silencing levels showed a significant reduction in the adhesion to laminin and fibrinogen, enzyme activity, and defects in the cell wall composition, which included reduced mannose, rhamnose, and protein content, accompanied by an increment in ß-1,3-glucans levels. The cell wall N-linked glycan content was significantly reduced. These strains induced poor TNFα and IL-6 levels when interacting with human peripheral blood mononuclear cells in a dectin-1-, TLR2-, and TLR4-dependent stimulation. The IL-1ß and IL-10 levels were significantly higher and were stimulated via dectin-1. Phagocytosis and stimulation of neutrophil extracellular traps by human granulocytes were increased in highly GP70-silenced strains. Furthermore, these mutants showed virulence attenuation in the invertebrate model Galleria mellonella. Our results demonstrate that Gp70 is a versatile protein with adhesin properties, is responsible for the activity of 3-carboxy-cis,cis-muconate cyclase, and is relevant for the S. schenckii-host interaction.

Heart Failure With Reduced Ejection Fraction in a Patient With Erdheim-Chester Disease on Dabrafenib.

A 54-year-old woman with a diagnosis of Erdheim-Chester disease under therapy with dabrafenib presents with clinical signs of heart failure. After discontinuing the offending medication and initiating guideline-directed medical therapy for heart failure with reduced ejection fraction, the clinical picture improved.

Deep convolutional neural network for weld defect classification in radiographic images.

The quality of welds is critical to the safety of structures in construction, so early detection of irregularities is crucial. Advances in machine vision inspection technologies, such as deep learning models, have improved the detection of weld defects. This paper presents a new CNN model based on ResNet50 to classify four types of weld defects in radiographic images: crack, pore, non-penetration, and no defect. Stratified cross-validation, data augmentation, and regularization were used to improve generalization and avoid over-fitting. The model was tested on three datasets, RIAWELC, GDXray, and a private dataset of low image quality, obtaining an accuracy of 98.75 %, 90.255 %, and 75.83 %, respectively. The model proposed in this paper achieves high accuracies on different datasets and constitutes a valuable tool to improve the efficiency and effectiveness of quality control processes in the welding industry. Moreover, experimental tests show that the proposed approach performs well on even low-resolution images.

Biochemical parameters, oxidative stress biomarkers, and anatomopathological changes in Wistar rats treated with 3'-demethoxy-6-O-demethylisoguaiacin and norisoguaiacin.

Artemisia cina (Ac) is a plant with anthelmintic compounds such as 3'-demethoxy-6-O-demethylisoguaiacin (D) and norisoguaiacin (N). Three major objectives were proposed: (1) To evaluate biochemical parameters in blood (2) to determine the tissue oxidative stress by biomarkers as TBARS and glutathione peroxidase activity, and (3) to evaluate anatomopathological changes in organs such as the brain, liver, kidney, and lung after oral administration of n-hexane extract of Ac and D and N. D and N were administrated following the OECD guides for acute oral toxicity evaluation (Guide 420). Fifty Wistar rats were distributed into ten groups as follows: Group 1 (G1): 4 mg/Kg; G2: 40 mg/Kg; G3: 240 mg/Kg; G4: 1600 mg/Kg of n-hexane extract of Ac. G5: 2 mg/Kg; G6: 20 mg/Kg; G7: 120 mg/Kg; G8: 800 mg/Kg of D and N, G9: water and G10: polyvinylpyrrolidone at 2000 mg/Kg. At 14 days, the rats were euthanized, and the blood, liver, brain, kidney, and lung were taken for biochemical analysis, anatomopathological changes, and TBARS and GSH evaluation. Glucose, cholesterol, and phosphorus were altered. Histopathological analysis showed multifocal neuronal degeneration in the brain (G2). The kidney and lungs had changes in G7. The GSH and TBARS increased in G6 and G7. The TBARS activity was higher in G1 and G2. In conclusion, extract and D and N of Ac did not have damage at therapeutic doses. D, N, and n-hexane extract of A. cina do not cause histopathological damage at pharmaceutical doses. Still, the brain, kidney, and liver are related to biochemical parameters at higher doses. However, compounds are proposed as antioxidant agents.

High-Throughput Screening Method Using Escherichia coli Keio Mutants for Assessing Primary Damage Mechanism of Antimicrobials.

The Escherichia coli Keio mutant collection has been a tool for assessing the role of specific genes and determining their role in E. coli physiology and uncovering novel functions. In this work, specific mutants in the DNA repair pathways and oxidative stress response were evaluated to identify the primary targets of silver nanoparticles (NPs) and their mechanism of action. The results presented in this work suggest that NPs mainly target DNA via double-strand breaks and base modifications since the recA, uvrC, mutL, and nfo mutants rendered the most susceptible phenotype, rather than involving the oxidative stress response. Concomitantly, during the establishment of the control conditions for each mutant, the katG and sodA mutants showed a hypersensitive phenotype to mitomycin C, an alkylating agent. Thus, we propose that KatG catalase plays a key role as a cellular chaperone, as reported previously for the filamentous fungus Neurospora crassa, a large subunit catalase. The Keio collection mutants may also be a key tool for assessing the resistance mechanism to metallic NPs by using their potential to identify novel pathways involved in the resistance to NPs.

Machine learning regression algorithms to predict emissions from steam boilers.

Currently, the modeling of complex chemical-physical processes is drastically influencing industrial development. Therefore, the analysis and study of the combustion process of the boilers using machine learning (ML) techniques are vital to increase the efficiency with which this equipment operates and reduce the pollution load they contribute to the environment. This work aims to predict the emissions of CO, CO2, NOx, and the temperature of the exhaust gases of industrial boilers from real data. Different ML algorithms for regression analysis are discussed. The following are input variables: ambient temperature, working pressure, steam production, and the type of fuel used in around 20 industrial boilers. Each boiler's emission data was collected using a TESTO 350 Combustion Gas Analyzer. The modeling, with a machine learning approach using the Gradient Boosting Regression algorithm, showed better performance in the predictions made on the test data, outperforming all other models studied. It was achieved with predicted values showing a mean absolute error of 0.51 and a coefficient of determination of 99.80%. Different regression models (DNN, MLR, RFR, GBR) were compared to select the most optimal. Compared to models based on Linear Regression, the DNN model has better prediction performance. The proposed model provides a new method to predict CO2, CO, NOx emissions, and exhaust gas outlet temperature.

An ELISA-based method for Galleria mellonella apolipophorin-III quantification.

Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune response with humans. However, non-vertebrate animal models have emerged as alternative models to study the host-pathogen interaction with minimal ethical concerns. Galleria mellonella is an alternative model that has proved useful in studying the interaction of the host with either bacteria or fungi, performing drug testing, and assessing the immunological response to different microorganisms. The G. mellonella immune response includes cellular and humoral components with structural and functional similarities to the immune effectors found in higher vertebrates, such as humans. An important humoral effector stimulated during infections is apolipophorin III (apoLp-III), an opsonin characterized by its lipid and carbohydrate-binding properties that participate in lipid transport, as well as immunomodulatory activity. Despite some parameters, such as the measurement of phenoloxidase activity, melanin production, hemocytes counting, and expression of antimicrobial peptides genes are already used to assess the G. mellonella immune response to pathogens with different virulence degrees, the apoLp-III quantification remains to be a parameter to assess the immune response in this invertebrate. Here, we propose an immunological tool based on an enzyme-linked immunosorbent assay that allows apoLp-III quantification in the hemolymph of larvae challenged with pathogenic agents. We tested the system with hemolymph coming from larvae infected with Escherichia coli, Candida albicans, Sporothrix schenckii, Sporothrix globosa, and Sporothrix brasiliensis. The results revealed significantly higher concentrations of apoLp-III when each microbial species was inoculated, in comparison with untouched larvae, or inoculated with phosphate-buffered saline. We also demonstrated that the apoLp-III levels correlated with the strains' virulence, which was already reported. To our knowledge, this is one of the first attempts to quantify apoLp-III, using a quick and easy-to-use serological technique.

Nutritional, Fatty Acids, (Poly)phenols and Technological Properties of Flower Powders from Fuchsia hybrida and Alcea rosea.

Fuchsia hybrida (pena pena) and Alcea rosea L. (malvagoma) are predominant flowers in the "Horchata" infusion, a traditional beverage in southern Ecuador, to which some medicinal properties are attributed. However, there is very little published information about these two flower species. The current study aimed to obtain two dehydrated powders of these flowers and to determine their chemical composition, physicochemical and technological properties, polyphenols, and fatty acids profile. In both powdered flowers, carbohydrates predominated, with a significant content of dietary fiber and fructose. The fat content was low, mainly comprising polyunsaturated fats (62% pena pena and 52% malvagoma), with a significant presence of omega-3 (C18:3n-3,6,9) and omega-6 (C18:2n-6,9) fatty acids, showing a better n-6/n-3 balance in the malvagoma flowers. Pena pena flowers are highlighted by high anthocyanin and ellagic acid amounts, whereas malvagoma contains a high content of flavanones. In conclusion, the studied powder flowers, could be used in the formulation of new foods or as source of anthocyanins as food colorants.

Bone samples' behavior in sunlight, IR light, and temperature increase with FEM simulation.

Biological and environmental factors produce biochemical processes that modify the bone structure. A few studies have attempted to show the adverse biological effects of sun radiation. The bone tissue exposures to infrared and sunlight radiation are analyzed by using focused sound, characterization spectroscopy techniques, and image processing. The study is complemented with a finite element method simulation on temperature behaviors. The crystal morphology on the bone hydroxyapatite and functional groups was characterized by X-ray diffraction and infrared spectroscopy. The infrared spectra confirmed the hydroxyl group of bovine hydroxyapatite, amines, and lipids are also correlated with modifications of the hydroxyapatite. The diffractograms showed the characteristic peaks of hydroxyapatite, with the main intensity at 2θ = 32.02°. Bone samples exposed to sun radiation presented a peak at 2θ = 27.5°, evidencing the possible formation of ß-TCP y α-TCP. The analysis with the spectroscopy techniques about the structural changes in the samples suggests interpreting an increase of sound obtained by expanding the exposure time. It is possible to verify that there are some structural changes in the bone samples due to exposure to non-ionizing radiation. These results show an increase in the registered intensity sound correlated with the interpretation of the structural changes of bone. Thanks to the different novel analysis techniques established in the present study, it could establish the changes that experienced the bone structure under different sources of radiation, which will help to better detect scenarios of bone deficiency.

Insights into molecular links and transcription networks integrating drought stress and nitrogen signaling.

Drought and the availability of nitrate, the predominant source of nitrogen (N) in agriculture, are major factors limiting plant growth and crop productivity. The dissection of the transcriptional networks' components integrating droght stress and nitrate responses provides valuable insights into how plants effectively balance stress response with growth programs. Recent evidence in Arabidopsis thaliana indicates that transcription factors (TFs) involved in abscisic acid (ABA) signaling affect N metabolism and nitrate responses, and reciprocally, components of nitrate signaling might affect ABA and drought gene responses. Advances in understanding regulatory circuits of nitrate and drought crosstalk in plant tissues empower targeted genetic modifications to enhance plant development and stress resistance, critical traits for optimizing crop yield and promoting sustainable agriculture.

Contribución de la asignatura Historia de Cuba al proceso de comprensión-expresión en estudiantes de Medicina

Fundamento: el desarrollo exitoso de la comprensión-expresión posibilita la efectividad del proceso comunicativo, y consecuentemente, el objetivo de la atención médica en el cumplimiento de sus deberes funcionales, expresados en el perfil del profesional. Objetivo: valorar la funcionalidad de un modelo didáctico desde la asignatura Historia de Cuba para favorecer la comprensión-expresión de los estudiantes de Medicina. Métodos: se desarrolló una investigación cualitativa, con enfoque dialéctico materialista, en la Facultad "Manuel Fajardo" de la Universidad de Ciencias Médicas de la Habana, en el período 2019-2021. Se emplearon métodos teóricos: histórico- lógico, inductivo-deductivo y análisis-síntesis; empíricos: revisión documental y observación; las técnicas: taller y tormenta de ideas; e instrumentos: diario de observación y autorreporte de estudiantes. Se realizó la consulta a especialistas y triangulación metodológica. Resultados: en el diagnóstico inicial: 37 estudiantes iniciaron en el nivel bajo, 19 en el nivel medio y 11 en el nivel alto de comprensión-expresión. Durante la aplicación del modelo, se modificaron los resultados: 8 en nivel bajo; 35 en nivel medio y 24 en nivel alto. Tras culminar la asignatura: solo 2 quedaron en el nivel bajo, 4 en el nivel medio y 61 en el nivel alto. Conclusiones: el modelo didáctico es funcional, flexible, participativo, abierto y colaborativo, con sustentos filosóficos, psicológicos, pedagógicos y didácticos, dialécticamente relacionados. Con su aplicación, se estrechó la brecha entre las exigencias del perfil del profesional y el déficit de comprensión-expresión de los estudiantes.

Background: the successful development of understanding-expression enables the effectiveness of the communicative process, and consequently, the objective of medical care in the fulfillment of its functional duties, expressed in the professional's profile. Objective: assess the functionality of a didactic model from the subject History of Cuba to promote the understanding-expression of medical students. Methods: a qualitative investigation was developed, with a materialist dialectical approach, at the "Manuel Fajardo" Faculty of Havana University of Medical Sciences, from 2019 to 2021. Theoretical methods were used: historical-logical, inductive-deductive and analysis-synthesis; empirical ones: documentary review and observation; the techniques: workshop and brainstorming; and instruments: observation diary and student self-report. Consultation with specialists and methodological triangulation were carried out. Results: in the initial diagnosis: 37 students started at the low level, 19 at average level and 11 at the high level of comprehension-expression. During the application of the model, the results were modified: 8 at a low level; 35 at average level and 24 at high level. After completing the subject: only 2 remained at the low level, 4 at the average level and 61 at the high level. Conclusions: the didactic model is functional, flexible, participatory, open and collaborative, with philosophical, psychological, pedagogical and didactic foundations, dialectically related. With its application, the gap between the demands of the professional profile and the students' comprehension-expression deficit was shortened.

Medicina regenerativa de cartílago: efecto del tratamiento con ácido hialurónico reticulado bifásico en lesiones osteocondrales / Regenerative medicine: effect of treatment with biphasic cross-linked hyaluronic acid in osteochondral lesions

Objetivo: Evaluar si el tratamiento con ácido hialurónico reticulado bifásico de lesiones osteocondrales promovería la regeneración del tejido cartilaginoso, favoreciendo así la reparación de la lesión. Materiales y métodos: Quince conejos hembra adultos fueron divididos aleatoriamente en tres grupos: grupo 1, de control; grupo 2 y grupo 3, sometidos a una estrategia quirúrgica de lesión osteocondral en la rodilla derecha (4 mm de diámetro, 5 mm de profundidad), el grupo 3 recibió tratamiento con 0,2 ml de ácido hialurónico por vía intrarticular después de la cirugía. Se realizaron controles clínicos, bioquímicos, histopatológicos y estudios por imágenes. Resultados: Se detectaron menos casos de dolor a la palpación en el grupo 3 que en el grupo 2 a partir de los 45 días. En la resonancia magnética, casi todas las muestras del grupo 3 tenían signos de regeneración del tejido cartilaginoso en el sitio de la lesión, sin edema óseo, ni derrame articular significativo. Los estudios histopatológicos de las muestras del grupo 3 indicaron un aumento de la matriz extracelular propia de tejido cartilaginoso, comparada con la del grupo 2, con hipercelularidad, dada por condrocitos, los que formaban grupos isogénicos axiales y coronales. Conclusiones: Este estudio brinda evidencias de que el tratamiento con ácido hialurónico reticulado bifásico en unidades experimentales de conejos con lesión osteocondral no tuvieron dolor en etapas tempranas después de la lesión, a diferencia de las unidades intervenidas y sin dicho tratamiento. A su vez, los estudios por imágenes e histopatológico mostraron la reparación del tejido dañado.

Objective: To demonstrate whether treatment with biphasic cross-linked hyaluronic acid in osteochondral lesions promotes the regeneration of cartilage tissue. Materials and methods: Fifteen adult female rabbits were randomly assigned to three groups. G1 was the control group, whereas G2 and G3 underwent surgery to treat an osteochondral injury in the right knee (4mm diameter, 5mm depth). G3 received treatment with 0.2 ml of hyaluronic acid intrarticularly after surgery. Clinical, biochemical, histopathological controls and imaging studies were performed. Results:Clinically, G3 exhibited less pain on palpation than G2 after 45 days. In G3, almost all samples showed evidence of cartilage tissue regeneration at the injury site, with neither bone edema or considerable joint effusion detected on MRI. The histological tests of G3 samples revealed an increase in the extracellular matrix of cartilaginous tissue when compared to G2, with hypercellularity caused by chondrocytes that formed axial and coronal isogenic groups. Conclusions: This study provides evidence that treatment with biphasic cross-linked hyaluronic acid in experimental units of rabbits with osteochondral injuries did not cause pain in the early stages of the injury. In turn, imaging and histopathological studies revealed that the injured tissue had been repaired.

Differential Recognition of Clinically Relevant Sporothrix Species by Human Granulocytes.

Sporotrichosis is a cutaneous mycosis that affects humans and animals and has a worldwide distribution. This infection is mainly caused by Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa. Current research about anti-Sporothrix immunity has been mainly focused on S. schenckii and S. brasiliensis, using different types of human or animal immune cells. Granulocytes are a group of cells relevant for cytokine production, with the capacity for phagocytosis and the generation of neutrophil extracellular traps (NETs). Considering their importance, this study aimed to compare the capacity of human granulocytes to stimulate cytokines, uptake, and form NETs when interacting with different Sporothrix species. We found that conidia, germlings, and yeast-like cells from S. schenckii, S. brasiliensis, and S. globosa play an important role in the interaction with these immune cells, establishing morphology- and species-specific cytokine profiles. S. brasil-iensis tended to stimulate an anti-inflammatory cytokine profile, whilst the other two species had a proinflammatory one. S. globosa cells were the most phagocytosed cells, which occurred through a dectin-1-dependent mechanism, while the uptake of S. brasiliensis mainly occurred via TLR4 and CR3. Cell wall N-linked and O-linked glycans, along with ß-1,3-glucan, played a significant role in the interaction of these Sporothrix species with human granulocytes. Finally, this study indicates that conidia and yeast-like cells are capable of inducing NETs, with the latter being a better stimulant. To the best of our knowledge, this is the first study that reports the cytokine profiles produced by human granulocytes interacting with Sporothrix cells.

DamID-seq: A Genome-Wide DNA Methylation Method that Captures Both Transient and Stable TF-DNA Interactions in Plant Cells.

Capturing the dynamic and transient interactions of a transcription factor (TF) with its genome-wide targets whose regulation leads to plants' adaptation to their changing environment is a major technical challenge. This is a widespread problem with biochemical methods such as chromatin immunoprecipitation-sequencing (ChIP-seq) which are biased towards capturing stable TF-target gene interactions. Herein, we describe how DNA adenine methyltransferase identification and sequencing (DamID-seq) can be used to capture both transient and stable TF-target interactions by DNA methylation. The DamID technique uses a TF protein fused to a DNA adenine methyltransferase (Dam) from E. coli. When expressed in a plant cell, the Dam-TF fusion protein will methylate adenine (A) bases near the sites of TF-DNA interactions. In this way, DamID results in a permanent, stable DNA methylation mark on TF-target gene promoters, even if the target gene is only transiently "touched" by the Dam-TF fusion protein. Here we provide a step-by-step protocol to perform DamID-seq experiments in isolated plant cells for any Dam-TF fusion protein of interest. We also provide information that will enable researchers to analyze DamID-seq data to identify TF-binding sites in the genome. Our protocol includes instructions for vector cloning of the Dam-TF fusion proteins, plant cell protoplast transfections, DamID preps, library preparation, and sequencing data analysis. The protocol outlined in this chapter is performed in Arabidopsis thaliana, however, the DamID-seq workflow developed in this guide is broadly applicable to other plants and organisms.