Rapid sequential determination of the explosives 2,4,6-trinitrotoluene and cyclotrimethylenetrinitramine in forensic samples employing a graphite sheet sensor and cyclic square-wave stripping voltammetry.

The development of a portable analytical procedure is described for rapid sequential detection and quantification of the explosives 2,4,6-trinitrotoluene (TNT) and cyclotrimethylenetrinitramine (RDX) in forensic samples using a graphite sheet (GS). A single GS platform works as a collector of explosive residues and detector after its assembly into a 3D-printed cell. The detection strategy is based on cyclic square-wave stripping voltammetry. The cathodic scan from + 0.1 to -1.0 V with accumulation at 0.0 V enables the TNT detection (three reduction peaks), and the anodic scan from + 0.2 to + 1.55 V with accumulation at -0.9 V provides the RDX detection (two oxidation processes). Low detection limit values (0.1 µmol L-1 for TNT and 2.4 µmol L-1 for RDX) and wide linear ranges (from 1 to 150 µmol L-1 for TNT and from 20 to 300 µmol L-1 for RDX) were obtained. The sensor did not respond to pentaerythritol tetranitrate (PETN), which was evaluated as a potential interferent, because plastic explosives contain mixtures of TNT, RDX, and PETN. The GS electrode was also evaluated as a collector of TNT and RDX residues spread on different surfaces to simulate forensic scenarios. After swiping over different surfaces (metal, granite, wood, cloths, hands, money bills, and cellphone), the GS electrode was assembled in the 3D-printed cell ready to measure both explosives by the proposed method. In all cases, the presence of TNT and RDX was confirmed, attesting the reliability of the proposed device to act as collector and sensor.

Polyploid goldback and silverback ferns (Pentagramma) occupy a wider, colder, and wetter bioclimatic niche than diploid counterparts.

PREMISE: The western North American fern genus Pentagramma (Pteridaceae) is characterized by complex patterns of ploidy variation, an understanding of which is critical to comprehending both the evolutionary processes within the genus and its current diversity. METHODS: We undertook a cytogeographic study across the range of the genus, using a combination of chromosome counts and flow cytometry to infer ploidy level. Bioclimatic variables and elevation were used to compare niches. RESULTS: We found that diploids and tetraploids are common and widespread, and triploids are rare and sporadic; in contrast with genome size inferences in earlier studies, no hexaploids were found. Diploids and tetraploids show different geographic ranges: only tetraploids were found in the northernmost portion of the range (Washington, Oregon, and British Columbia) and only diploids were found in the Sierra Nevada of California. Diploid, triploid, and tetraploid cytotypes were found to co-occur in relatively few localities: in the southern (San Diego County, California) and desert Southwest (Arizona) parts of the range, and along the Pacific Coast of California. CONCLUSIONS: Tetraploids occupy a wider bioclimatic niche than diploids both within P. triangularis and at the genus-wide scale. It is unknown whether the wider niche of tetraploids is due to their expansion upon the diploid niche, if diploids have contracted their niche due to competition or changing abiotic conditions, or if this wider niche occupancy is due to multiple origins of tetraploids.

The InBIO Barcoding Initiative Database: DNA barcodes of Iberian Bees.

Background: Bees are important actors in terrestrial ecosystems and are recognised for their prominent role as pollinators. In the Iberian Peninsula, approximately 1,100 bee species are known, with nearly 100 of these species being endemic to the Peninsula. A reference collection of DNA barcodes, based on morphologically identified bee specimens, representing 514 Iberian species, was constructed. The "InBIO Barcoding Initiative Database: DNA Barcodes of Iberian bees" dataset contains records of 1,059 sequenced specimens. The species of this dataset correspond to about 47% of Iberian bee species diversity and 21% of endemic species diversity. For peninsular Portugal only, the corresponding coverage is 71% and 50%. Specimens were collected between 2014 and 2022 and are deposited in the research collection of Thomas Wood (Naturalis Biodiversity Center, The Netherlands), in the FLOWer Lab collection at the University of Coimbra (Portugal), in the Andreia Penado collection at the Natural History and Science Museum of the University of Porto (MHNC-UP) (Portugal) and in the InBIO Barcoding Initiative (IBI) reference collection (Vairão, Portugal). New information: Of the 514 species sequenced, 75 species from five different families are new additions to the Barcode of Life Data System (BOLD) and 112 new BINs were added. Whilst the majority of species were assigned to a single BIN (94.9%), 27 nominal species were assigned to multiple BINs. Although the placement into multiple BINs may simply reflect genetic diversity and variation, it likely also represents currently unrecognised species-level diversity across diverse taxa, such as Amegillaalbigena Lepeletier, 1841, Andrenarussula Lepeletier, 1841, Lasioglossumleucozonium (Schrank, 1781), Nomadafemoralis Morawitz, 1869 and Sphecodesalternatus Smith, 1853. Further species pairs of Colletes, Hylaeus and Nomada were placed into the same BINs, emphasising the need for integrative taxonomy within Iberia and across the Mediterranean Basin more broadly. These data substantially contribute to our understanding of bee genetic diversity and DNA barcodes in Iberia and provide an important baseline for ongoing taxonomic revisions in the West Palaearctic biogeographical region.

Spinning Reactors for Process Intensification of Flow Photochemistry.

The design of new and more sustainable synthetic protocols to access new materials or valuable compounds will have a high impact on the broader chemistry community. In this sense, continuous-flow photochemistry has emerged as a powerful technique which has been employed successfully in various areas such as biopharma, organic chemistry, as well as materials science. However, it is important to note that chemical processes must not only advance towards new or improved chemical transformations, but also implement new technologies that enable new process opportunities. For this reason, the design of novel photoreactors is key to advancing photochemical strategies. In this sense, the use of equipment and techniques embracing processes intensification is important in developing more sustainable protocols. Among the most recent applications, spinning continuous flow reactors, such as rotor reactors or vortex reactors, have shown promising performance as new synthetic tools. Nevertheless, there is currently no review in the literature that effectively summarizes and showcases the most recent applications of such type of photoreactors. Herein, we highlight fundamental aspects and applications of two categories of spinning reactors, the Spinning Disc Reactors (SDRs) and Thin Film Vortex reactors, critiquing the scope and limitations of these advanced processing technologies. Further, we take a view on the future of spinning reactors in flow as a synthetic toolbox to explore new photochemical transformations.

Providing an in vitro depiction of microglial cells challenged with immunostimulatory extracellular vesicles of Naegleria fowleri.

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a rapid and acute infection of the central nervous system with a fatal outcome in >97% of cases. Due to the infrequent report of cases and diagnostic gaps that hinder the possibility of recovering clinic isolates, studies related to pathogenesis of the disease are scarce. However, the secretion of cytolytic molecules has been proposed as a factor involved in the progression of the infection. Several of these molecules could be included in extracellular vesicles (EVs), making them potential virulence factors and even modulators of the immune response in this infection. In this work, we evaluated the immunomodulatory effect of EVs secreted by two clinic isolates of Naegleria fowleri using in vitro models. For this purpose, characterization analyses between EVs produced by both isolates were first performed, for subsequent gene transcription analyses post incubation of these vesicles with primary cultures from mouse cell microglia and BV-2 cells. Analyses of morphological changes induced in primary culture microglia cells by the vesicles were also included, as well as the determination of the presence of nucleic acids of N. fowleri in the EV fractions. Results revealed increased expression of NOS, proinflammatory cytokines IL-6, TNF-α, and IL-23, and the regulatory cytokine IL-10 in primary cultures of microglia, as well as increased expression of NOS and IL-13 in BV-2 cells. Morphologic changes from homeostatic microglia, with small cellular body and long processes to a more amoeboid morphology were also observed after the incubation of these cells with EVs. Regarding the presence of nucleic acids, specific Naegleria fowleri DNA that could be amplified using both conventional and qPCR was confirmed in the EV fractions. Altogether, these results confirm the immunomodulatory effects of EVs of Naegleria fowleri over microglial cells and suggest a potential role of these vesicles as biomarkers of primary acute meningoencephalitis.

Bridging the Gap between Field Experiments and Machine Learning: The EC H2020 B-GOOD Project as a Case Study towards Automated Predictive Health Monitoring of Honey Bee Colonies.

Honey bee colonies have great societal and economic importance. The main challenge that beekeepers face is keeping bee colonies healthy under ever-changing environmental conditions. In the past two decades, beekeepers that manage colonies of Western honey bees (Apis mellifera) have become increasingly concerned by the presence of parasites and pathogens affecting the bees, the reduction in pollen and nectar availability, and the colonies' exposure to pesticides, among others. Hence, beekeepers need to know the health condition of their colonies and how to keep them alive and thriving, which creates a need for a new holistic data collection method to harmonize the flow of information from various sources that can be linked at the colony level for different health determinants, such as bee colony, environmental, socioeconomic, and genetic statuses. For this purpose, we have developed and implemented the B-GOOD (Giving Beekeeping Guidance by computational-assisted Decision Making) project as a case study to categorize the colony's health condition and find a Health Status Index (HSI). Using a 3-tier setup guided by work plans and standardized protocols, we have collected data from inside the colonies (amount of brood, disease load, honey harvest, etc.) and from their environment (floral resource availability). Most of the project's data was automatically collected by the BEEP Base Sensor System. This continuous stream of data served as the basis to determine and validate an algorithm to calculate the HSI using machine learning. In this article, we share our insights on this holistic methodology and also highlight the importance of using a standardized data language to increase the compatibility between different current and future studies. We argue that the combined management of big data will be an essential building block in the development of targeted guidance for beekeepers and for the future of sustainable beekeeping.

PolLimCrop, a global dataset of pollen limitation in crops.

Pollination is a crucial ecosystem service for maintaining plant communities and food production. 75% of the main crops depend on or benefit from pollination services provided by animal pollinators. However, when these services are insufficient and/or inefficient, crops experience pollen limitation with, often, lower associated yield, which may translate into economic losses. We constructed a global dataset that gathers studies with pollination experiments, aiming to provide pollen limitation values of animal-pollinated crops worldwide. Pollination experiments included hand pollen supplementation treatments, where plants were subjected to pollen supplementation of outcross pollen, and natural pollination treatments. The PolLimCrop dataset comprises 294 studies and 1169 unique pollen supplementation experiments with values of pollen limitation for 108 crops, spanning 50 years and 62 countries.

Descrição das características clínicas e epidemiológicas de pacientes pediátricos internados e diagnosticados com COVID-19 / Description of clinical and epidemiological features of hospitalized pediatric patients diagnosed with COVID-19

INTRODUÇÃO: Apesar da disseminação mundial da COVID-19, os padrões epidemiológicos e clínicos da doença ainda permanecem incertos na faixa etária pediátrica. OBJETIVO: Descrever as características epidemiológicas, clínicas, laboratoriais e radiológicas dos pacientes pediátricos diagnosticados com doença causada pelo SARS-CoV-2, hospitalizados na enfermaria do Hospital Regional João Penido, situado em Juiz de Fora/MG. MÉTODOS: Foi realizado um estudo do tipo coorte, com coleta de dados do período da internação referente a critérios epidemiológicos, clínicos, laboratoriais e radiológicos através de formulário próprio desenvolvido para a pesquisa de pacientes pediátricos hospitalizados no período de 11 de março de 2020 até 11 de março de 2021, que receberam diagnóstico de COVID-19, confirmado por RT-PCR ou por sorologia IgM/IgG. RESULTADOS: Dos pacientes com COVID-19, 49,2% tinham idade até 3 anos, 66,2% eram do sexo masculino e, dentre as comorbidades, doença respiratória crônica foi a de maior prevalência (26,9%). A maioria dos pacientes apresentou quadro clínico respiratório (72,7%). Com relação à gravidade da doença, 38,5% apresentaram doença leve, 23,1% moderada, 23,1% grave e 12,3% crítica. No desfecho, 98,5% tiveram alta hospitalar e apenas 1 paciente, que possuía diversas comorbidades, evoluiu a óbito. CONCLUSÃO: A COVID-19 possui apresentações clínicas, radiológicas e laboratoriais variadas e, não existindo tratamento único ou específico, os casos devem ser analisados e conduzidos de forma individual.

INTRODUCTION: Despite the worldwide dissemination of COVID-19, the epidemiological and clinical patterns of the disease remain uncertain in the pediatric age group. OBJECTIVE: To describe the epidemiological, clinical, laboratory and radiological characteristics of pediatric patients diagnosed with disease caused by SARS-CoV-2, hospitalized in the ward of Hospital Regional João Penido, located in Juiz de Fora, MG. METHODS: A cohort study was carried out, with data collection from the period of hospitalization regarding epidemiological, clinical, laboratory and radiological criteria through a specific form developed for the research of pediatric patients, hospitalized from March 11, 2020 to March 11, 2021, who received a diagnosis of COVID-19, confirmed by RT-PCR or by IgM/IgG serology. RESULTS: Of the patients with COVID-19, 49.2% were aged up to 3 years, 66.2% were male and, among the comorbidities, chronic respiratory disease was the most prevalent (26.9%). Most patients had a clinical respiratory condition (72.7%). Regarding disease severity, 38.5% had mild disease, 23.1% moderate, 23.1% severe and 12.3% critical. In the outcome, 98.5% were discharged from the hospital and only 1 patient, who had several comorbidities, died. CONCLUSION: COVID-19 has varied clinical, radiological and laboratory presentations and, since there is no single or specific treatment, cases must be analyzed and managed individually.

The "StemDif Sensor Test": A Straightforward, Non-Invasive Assay to Characterize the Secreted Stemness and/or Differentiation Activities of Tumor-Derived Cancer Cell Lines.

Cancer stem cells are a subpopulation of tumor cells characterized by their ability to self-renew, induce tumors upon engraftment in animals and exhibit strong resistance to chemotherapy and radiotherapy. These cells exhibit numerous characteristics in common with embryonic stem cells, expressing some of their markers, typically absent in non-pathological adult differentiated cells. The aim of this study was to investigate the potential of conditioned media from cancer stem cells to modulate the fate of Leukemia Inhibitory Factor (LIF)-dependent murine embryonic stem cells (mESCs) as a way to obtain a direct readout of the secretome of cancer cells. A functional assay, "the StemDif sensor test", was developed with two types of cancer stem cells derived from grade IV glioblastoma (adult and pediatric) or from gastric adenocarcinoma. We show that conditioned media from the selection of adult but not pediatric Glioma-Inducing Cells (GICs) maintain mESCs' pluripotency in correlation with LIF secretion and activation of STAT3 protein. In contrast, conditioned media from gastric adenocarcinoma cells display LIF-independent stemness and differentiation activities on mESC. Our test stands out for its user-friendly procedures, affordability and straightforward output, positioning it as a pioneering tool for in-depth exploration of cancer stem cell secretome characteristics.

Population size as a major determinant of mating system and population genetic differentiation in a narrow endemic chasmophyte.

BACKGROUND: Mating system is one of the major determinants of intra- and interspecific genetic structure, but may vary within and between plant populations. Our study model included all known populations of Moehringia tommasinii (Caryophyllaceae), a narrow endemic plant inhabiting rock crevices in the northwestern Adriatic, and some populations of co-occurring and widespread M. muscosa, an ecologically divergent relative with an overlapping flowering period. We performed reciprocal crosses within and between taxa and used molecular markers to assess the extent of gene flow within and between populations and taxa. Using coefficient of inbreeding, population size, seed weight, pollen-to-ovule ratio, and flower display size, we also looked for evidence of a selfing syndrome. RESULTS: A surprisingly high variation in mating systems was observed among populations of M. tommasinii. These populations exhibited genetic structuring, with their size positively correlated with both seed weight and pollen production. Although a selfing syndrome could not be confirmed as the majority of selfing resulted from allogamous treatments, the occurrence of selfing was notable. In the presence of M. muscosa, at a site where both species coexist closely, a distinct pattern of fruit production was observed in M. tommasinii following various pollination treatments. Molecular and morphometric data provided evidence of hybridization followed by local extinction at this site. CONCLUSIONS: Population size proved to be the most important factor affecting the mating system in genetically structured populations of M. tommasinii. Lighter seeds and lower pollen production observed in populations with pronounced selfing do not provide enough evidence for the selfing syndrome. Detected gene flow between M. tommasinii and the sympatric M. muscosa suggested weak reproductive barriers between the taxa, which could pose a conservation problems for the former species. Hybridization leading to local extinction may also resulted in floral polymorphism and disruption of mating patterns of M. tommasinii.

Ploidy effects on the relationship between floral phenotype, reproductive investment, and fitness in an autogamous species complex.

PREMISE: The relationships between reproductive investment, phenotype, and fitness have been broadly studied in cross-pollinated plants in contrast to selfing species, which are considered less interesting in this area because they are supposed to be a dead end in any evolutionary pathway. Still, selfing plants are unique systems to study these questions since the position of reproductive structures and traits related to flower size play an important role in female and male pollination success. METHODS: Erysimum incanum s.l. is a selfing species complex that has three levels of ploidy (diploids, tetraploids, and hexaploids) and traits that are typically associated with the selfing syndrome. Here, we used 1609 plants belonging to these three ploidies to characterize the floral phenotype and spatial configuration of reproductive structures, reproductive investment (pollen and ovule production), and plant fitness. Then, we used structural equation modelling to analyze the relationship between all these variables across ploidy levels. RESULTS: An increase in ploidy level leads to bigger flowers with anthers exserted farther and more pollen and ovules. In addition, hexaploid plants had higher absolute values for herkogamy, which is positively correlated with fitness. Ovule production significantly mediated the natural selection acting on different phenotypic traits and pollen production, a pattern that is maintained across ploidies. CONCLUSIONS: The changes in floral phenotypes, reproductive investment, and fitness with ploidy level suggest that genome duplication can be a driver for transitions in reproductive strategy by modifying the investment in pollen and ovules and linking them with plant phenotype and fitness.

The Use of Flow Cytometry for Estimating Genome Sizes and DNA Ploidy Levels in Plants.

Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided.

Ecological niches in the polyploid complex Linum suffruticosum s.l.

Introduction: The high frequency of polyploidy in the evolutionary history of many plant groups occurring in the Mediterranean region is likely a consequence of its dynamic paleogeographic and climatic history. Polyploids frequently have distinct characteristics that allow them to overcome the minority cytotype exclusion. Such traits may enable polyploid individuals to grow in habitats different from their parentals and/or expand to new areas, leading to spatial segregation. Therefore, the successful establishment of polyploid lineages has long been associated with niche divergence or niche partitioning and the ability of polyploids to cope with different, often more stressful, conditions. In this study, we aimed to explore the role of environmental variables associated with the current distribution patterns of cytotypes within the polyploid complex Linum suffruticosum s.l.. Methods: The distribution and environmental niches of the five main cytotypes of Linum suffruticosum s.l. (diploids, tetraploids, hexaploids, octoploids and decaploids) were studied across its distribution range. Realized environmental niche of each cytotype was determined using niche modelling tools, such as maximum entropy modelling and niche equivalency and similarity tests. Results: Differences in the environmental conditions of L. suffruticosum s.l. cytotypes were observed, with polyploids being associated with habitats of increased drought and soil pH, narrower temperature ranges and decreased soil water and cation exchange capacities. Diploids present the widest environmental niche, and polyploids occupy part of the diploid niche. Although some polyploids have equivalent potential ecological niches, cytotypes do not co-occur in nature. Additionally, the ecological niche of this polyploid complex is different between continents, with North African habitats being characterised by differences in soil texture, higher pH, and low cation exchange capacity, precipitation and soil water capacity and higher temperatures than habitats in southwest Europe. Discussion: The different ecological conditions played a role in the distribution of cytotypes, but the mosaic distribution could not be entirely explained by the environmental variables included in this study. Other factors, such as reproductive isolation and competitive interactions among cytotypes, could further explain the current diversity and distribution patterns in white flax. This study provides relevant data on the niche requirements of each cytotype for further competition and reciprocal transplant experiments. further competition and reciprocal transplant experiments.

Helicobacter pylori infection induces gastric precancerous lesions and persistent expression of Angpt2, Vegf-A and Tnf-A in a mouse model.

Introduction: Helicobacter pylori colonizes the gastric mucosa and induces chronic inflammation. Methods: Using a mouse model of H. pylori-induced gastritis, we evaluated the mRNA and protein expression levels of proinflammatory and proangiogenic factors, as well as the histopathological changes in gastric mucosa in response to infection. Five- to six-week-old female C57BL/6N mice were challenged with H. pylori SS1 strain. Animals were euthanized after 5-, 10-, 20-, 30-, 40- and 50-weeks post infection. mRNA and protein expression of Angpt1, Angpt2, VegfA, Tnf-α, bacterial colonization, inflammatory response and gastric lesions were evaluated. Results: A robust bacterial colonization was observed in 30 to 50 weeks-infected mice, which was accompanied by immune cell infiltration in the gastric mucosa. Compared to non-infected animals, H. pylori-colonized animals showed an upregulation in the expression of Tnf-A, Angpt2 and VegfA at the mRNA and protein levels. In contrast, Angpt1 mRNA and protein expression was downregulated in H. pylori-colonized mice. Conclusion: Our data show that H. pylori infection induces the expression of Angpt2, Tnf-A and Vegf-A in murine gastric epithelium. This may contribute to the pathogenesis of H. pylori-associated gastritis, however the significance of this should be further addressed.

Impact of genome duplications in drought tolerance and distribution of the diploid-tetraploid Jasione maritima.

Polyploidy has important ecological effects, including ploidy-mediated effects on morphology, breeding system and ecological tolerances. However, there is still little comprehensive research available to test its adaptive significance and its role in driving distributional patterns. This work aimed to assess the contribution of genome duplications to ecological divergence using an experimental approach with the diploid-tetraploid Jasione maritima polyploid complex. We explored if individuals with different ploidy differ in their tolerance to water deficit and if this may contribute to explaining the distribution patterns along a latitudinal gradient in the northwest Iberian Peninsula. For that, we used three cytogenetic entities: diploids and established tetraploids collected in natural populations along a latitudinal gradient, and neotetraploids synthesized from diploid populations after treatments with colchicine. Thirty plants from each of the nine populations were grown under controlled conditions with half randomly assigned to the water deficit treatment, and half used as control. We determined experimental plants' response by measuring fitness-related parameters, such as above and belowground biomass, plant water status, photosynthetic efficiency and pigments, membrane stability, antioxidant capacity and sugars content. Our data shows that biomass, chlorophyll content, photochemical quenching (qP) and non-photochemical quenching (NPQ) in neotetraploids and established tetraploids were significantly higher than in diploids and that these differences could be attributed to genome duplications. In response to the water deficit, diploids seem to use a strategy of avoidance, whereas tetraploids seem to employ the strategy of tolerance to overcome water deficit stress, which appears equally efficient. Additionally, we did not observe a response pattern along the latitudinal gradient of the distributional range of the J. maritima complex. The results indicate that the response to water deficit is population dependent. Further studies are necessary to understand the role of ploidy in explaining the distribution patterns of the J. maritima complex.

Stable production of recombinant SARS-CoV-2 receptor-binding domain in mammalian cells with co-expression of a fluorescent reporter and its validation as antigenic target for COVID-19 serology testing.

SARS-CoV-2 receptor binding domain (RBD) recognizes the angiotensin converting enzyme 2 (ACE2) receptor in host cells that enables infection. Due to its antigenic specificity, RBD production is important for development of serological assays. Here we have established a system for stable RBD production in HEK 293T mammalian cells that simultaneously express the recombinant fluorescent protein dTomato, which enables kinetic monitoring of RBD expression by fluorescence microscopy. In addition, we have validated the use of this recombinant RBD in an ELISA assay for the detection of anti-RBD antibodies in serum samples of COVID-19 convalescent patients. Recombinant RBD generated using this approach can be useful for generation of antibody-based therapeutics against SARS-CoV-2, as well serological assays aimed to test antibody responses to this relevant virus.

Hybridization and introgression are prevalent in Southern European Erysimum (Brassicaceae) species.

BACKGROUND AND AIMS: Hybridization is a common and important force in plant evolution. One of its outcomes is introgression - the transfer of small genomic regions from one taxon to another by hybridization and repeated backcrossing. This process is believed to be common in glacial refugia, where range expansions and contractions can lead to cycles of sympatry and isolation, creating conditions for extensive hybridization and introgression. Polyploidization is another genome-wide process with a major influence on plant evolution. Both hybridization and polyploidization can have complex effects on plant evolution. However, these effects are often difficult to understand in recently evolved species complexes. METHODS: We combined flow cytometry, analyses of transcriptomic sequences and pollen tube growth assays to investigate the consequences of polyploidization, hybridization and introgression on the recent evolution of several Erysimum (Brassicaceae) species from the South of the Iberian Peninsula, a well-known glacial refugium. This species complex differentiated in the last 2 million years, and its evolution has been hypothesized to be determined mainly by polyploidization, interspecific hybridization and introgression. KEY RESULTS: Our results support a scenario of widespread hybridization involving both extant and 'ghost' taxa. Several taxa studied here, most notably those with purple corollas, are polyploids, probably of allopolyploid origin. Moreover, hybridization in this group might be an ongoing phenomenon, as pre-zygotic barriers appeared weak in many cases. CONCLUSIONS: The evolution of Erysimum spp. has been determined by hybridization to a large extent. Species with purple (polyploids) and yellow flowers (mostly diploid) exhibit a strong signature of introgression in their genomes, indicating that hybridization occurred regardless of colour and across ploidy levels. Although the adaptive value of such genomic exchanges remains unclear, our results demonstrate the significance of hybridization for plant diversification, which should be taken into account when studying plant evolution.

Cultivo primario de Helicobacter pylori a partir de biopsias gástricas obtenidas por endoscopia / Primary culture of Helicobacter pylori from endoscopic gastric biopsies

Objetivo: Determinar la viabilidad del cultivo de la bacteria Helicobacter pylori en Costa Rica por medio de la documentación de toma de muestras, la comparación del diagnóstico histopatológico y la descripción de los diagnósticos asociados a los aislamientos obtenidos con los resultados de la ureasa rápida. Métodos: Investigación descriptiva que involucró a pacientes de entre los 35 y 70 años, de ambos sexos, que asistieron al Servicio de Endoscopia Digestiva del Hospital Clínica Bíblica entre febrero y junio del 2019 para estudio gastroscópico. Se obtuvieron biopsias gástricas para diagnóstico histopatológico, prueba de ureasa rápida y cultivo de Helicobacter pylori. Para este último, se transportaron las biopsias en un medio de transporte semisólido, se maceró el tejido y se cultivó enagar Skirrow y agar selectivo para Helicobacter; una placa de cada medio se incubó a 37 °C en microaerofilia entre 48 horas y 10 días. La positividad del cultivo se realizó por observación de la morfología colonial y la bacteria se identificó por análisis microscópico al fresco, tinción de Gram y pruebas bioquímicas (catalasa, ureasa y oxidasa). Resultados: Se incluyó a 44 pacientes (edad: 50.6 ± 10.0, 54.5% masculinos). Se recuperó Helicobacter pylori en biopsias de 27 pacientes (61.4% de éxito). La recuperación de la bacteria fue similar en el medio Skirrow y en el selectivo para Helicobacter. El porcentaje de éxito de recuperación semanal aumentó durante el estudio hasta alcanzar un éxito del 100% en la semana 11. Se comparó el cultivo con la ureasa rápida en 27 pacientes y la concordancia entre ambos métodos fue de un coeficiente kappa de Cohen de 0.48. El cultivo detectó la bacteria en un 56% de los pacientes, la ureasa rápida en un 37% y la combinación de ambas técnicas permitió la detección en un 60%. El diagnóstico endoscópico más frecuente en los pacientes con cultivo positivo fue la gastritis eritematosa y gastritis crónica superficial y el diagnóstico histopatológico predominante fue gastritis crónica con atrofia gástrica. El diagnóstico por cultivo coincidió con la detección por azul de toluidina en un 80.4% de los casos. Conclusiones: Se puede implementar el cultivo de Helicobacter pylori en Costa Rica. Este estudio tuvo un porcentaje de recuperación de la bacteria de 61.4%. La combinación del método de cultivo con la prueba de ureasa rápida y la detección histológica contribuye a un diagnóstico certero y oportuno. Recomendamos que, con base en protocolos descritos en esta investigación, cada laboratorio estandarice las condiciones que le permitan un buen porcentaje de recuperación y una implementación adaptada a sus actividades de rutina.

Aim: To document the recent experiences on the implementation of sampling and culturing Helicobacter pylori in Costa Rica, to compare it with other diagnostic methods: rapid urease test and histopathology and to describe the diagnoses associated with the obtained isolates. Methods: Descriptive research involving patients who visited the digestive endoscopy department of the Clínica Bíblica hospital in San José, Costa Rica between February and July of 2019 for gastroscopy. Gastric biopsies were obtained and histopathological analysis, rapid urease test, and bacteriological culture for Helicobacter pylori were performed. For culture techniques, the sample was transported in an in-house semi-solid medium. Biopsy fragments were macerated and plated on Skirrow agar and Helicobacterselective in-house agar, and incubated in microaerophilic atmosphere for 48 hours to 10 days. Culture positivity was determined by observation of the colonial morphology and microscopic observation; Gram staining and biochemical tests (urease, catalase, and oxidase) were used for bacterial identification. Results: 44 patients (mean aged 50.6 ± 10.0 years old, 54.5% male) were included in the study. Helicobacter pylori was recovered in biopsies from 27 patients (61.4% success rate). Bacterial growth was similar regardless the culture medium, but the physiological state of the bacteria was better in the Helicobacter-selective agar than in Skirrow. The weekly recovery rate increased to reach a 100% recovery plateau on week 11. Culture was compared with the rapid urease test in 27 patients, and the concordance between both methods using Cohen's kappa coefficient was 0.48. Whilst the culture detected Helicobacter pylori in 56% of the patients, and the rapid urease test in 37%, the combination of both allowed a 60% rate. The most frequent endoscopic diagnosis in patients with positive cultures were erythematous gastritis and chronic superficial gastritis, and the predominant histopathological diagnosis was chronic atrophic gastritis. Culture-based diagnosis was consistent with the histopathology detection of Helicobacter pylori in 80.4% of the cases. Conclusions: The implementation of H. pylori culture in Costa Rica is possible. This study had a 61.4% recovery rate. The combination of culture with rapid urease test and histopathology increases the probability of an accurate and timely diagnosis. We recommend that, based on previously described protocols such as ours, each laboratory adjusts the conditions to allow a good recovery rate and implement H. pylori diagnostic methods most suitable to their routine activities.

Exploratory multigroup structural equation modeling approach to analyzing H. pylori infection, serological pepsinogen levels, and life style factors in an elderly population in Costa Rica / Enfoque exploratorio del modelo de ecuaciones estructurales multigrupo para el análisis de la infección con H. pylori, niveles de pepsinógenos séricos y factores asociados a estilo de vida en una población de adultos mayores en Costa Rica

Abstract The aim of this work is to describe and analyze the association of PGI/PGII ratio (indicator of gastric atrophy) with H. pylori-CagA and life style factors such as caloric intake, obesity, and harmful habits amongst H. pylori-positive elderly people infected in Costa Rica using an exploratory multigroup structural equations model (SEM). Using a sample of 1748 H. pylori-positive elderly people from CRELES first wave study, a SEM was employed analyze if the relationships between PGI/PGII ratio with levels of H. pylori-CagA, caloric intake, obesity, and harmful habits, differs by sex, age and risk areas subgroups. The proposed SEMs exhibited a good fit in males (RMSEA = 0.039), females (RMSEA = 0.000), low-risk area (RMSEA = 0.038), middle-risk area (RMSEA = 0.042), individuals under 80 years (RMSEA = 0.038) and individuals aged 80 and over (RMSEA = 0.042), while an acceptable fit was observed for the high-risk area (RMSEA = 0.061). Fitted SEMs showed that CagA predicted PG-ratio as expected, with effects increasing with the risk area, but similar between sex and age groups. All indicators measuring obesity (BMI, arms, and waist) showed significant standardized coefficients, with similar effects between sex, age and risk area groups. No other significant effects or differences between groups were identified. We propose a good-fitted SEM model for the possible relationships between CagA and PG ratio and the geographical risk area level for elderly people. No differences were observed on measured parameters between male and female population, or between under 80 years and older individuals.

Resumen El objetivo de este trabajo es describir y analizar la asociación entre PGI/PGII (indicador de atrofia gástrica con H. pylori-CagA y factores asociados a estilo de vida como ingesta calórica, obesidad y hábitos nocivos entre adultos mayores positivos por H. pylori en Costa Rica utilizando modelos de ecuaciones estructurales multigrupo (SEM). Con una muestra de 1748 adultos mayores del estudio CRELES, se utilizó un SEM para analizar las relaciones entre PGI/PGII, CagA, ingesta calórica, obesidad y hábitos nocivos difieren por sexo, edad y áreas de riesgo. Los SEMs propuestos exhibieron un buen ajuste en hombres (RMSEA = 0.039), mujeres (RMSEA = 0.000), área de bajo riesgo (RMSEA = 0.038), áreas de riesgo medio (RMSEA = 0.042), individuos menores de 80 años (RMSEA = 0.038) e individuos de 80 años o más (RMSEA = 0.042), mientras que hubo un ajuste aceptable en áreas de alto riesgo (RMSEA = 0.061). Los SEMs ajustados mostraron que CagA predice la relación PGI/II en la dirección esperada con efectos proporcionales al área de riesgo, pero no por sexo y edad. Todos los indicadores medibles de obesidad (IMC, brazos y cintura) mostraron coeficientes estandarizados significativos con efectos similares entre los grupos por sexo, edad y área de riesgo. No se encontraron otros efectos o diferencias significativas. Proponemos un modelo SEM bien ajustado para las posibles relaciones entre CagA y PGI/II y el nivel de riesgo del área geográfica en adultos mayores. No se encontraron diferencias en las variables analizadas entre hombres y mujeres ni entre los grupos de edad.