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1.
Artículo en Inglés | MEDLINE | ID: mdl-38940759

RESUMEN

BACKGROUND: Food-induced anaphylaxis (FIA) is a major public health problem resulting in serious clinical complications, emergency department visits, hospitalization, and death. OBJECTIVE: The objectives of this investigation were to assess the epidemiology and the trends in hospitalizations because of FIA in Spain between 2016 and 2021. METHODS: An observational descriptive study was conducted using data from the Spanish National hospital discharge database. Information is coded based on the International Classification of Diseases, Tenth Revision. The study population was analyzed by sex and age group and according to food triggers, clinical characteristics, admission to the intensive care unit (ICU), severity, and in-hospital mortality (IHM). The annual incidence of hospitalizations because of FIA per 100,000 person-years was estimated and analyzed using Poisson regression models. Multivariable logistic regression models were constructed to identify which variables were associated with severe FIA. RESULTS: A total of 2161 hospital admissions for FIA in were recorded in Spain from 2016 to 2021. The overall incidence rate was 0.77 cases per 100,000 person-years. The highest incidence was found in the <15-year age group (3.68), with lower figures among those aged 15-59 years (0.25) and ≥60 years (0.29). Poisson regression showed a significant increase in incidence from 2016 to 2021 only among children (3.78 per 100,000 vs. 5.02 per 100,000 person-years; p=.047). The most frequent food triggers were ""Milk and dairy products" (419/2161, 19.4% of cases) and "Peanuts and tree nuts and seeds" (409/2161, 18.9%). Overall, 256 of 2161 (11.9%) patients hospitalized because of FIA required admission to the ICU, and 11 patients (0.5%) died in hospital. Among children, the most severe cases of FIA appeared in patients aged 0 to 4 years (40/99, 40.4%). Among adults, 69.4% (111/160) of cases occurred in those aged 15 to 59 years. Multivariable logistic regression showed the variables associated with severe FIA to be age 15-59 years (OR, 5.1; 95% CI, 3.11-8.36), age ≥60 years (OR, 3.87; 95% CI, 1.99-7.53), and asthma. CONCLUSIONS: In Spain, the incidence of hospitalization because of FIA increased slightly, although the only significant increase was among children. Even if IHM remains low and stable, the proportion of severe cases is high and has not improved from 2016 to 2021, with older age and asthma being risk factors for severity. Surveillance must be improved, and preventive strategies implemented to reduce the burden of FIA.

2.
J Clin Med ; 13(10)2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38792346

RESUMEN

Background/Objectives: To analyze changes in the prevalence of atrial fibrillation (AF) in patients hospitalized for acute exacerbation of chronic obstructive pulmonary disease (AE-COPD); to evaluate hospital outcomes according to AF status, assessing sex differences; to identify factors associated with AF presence; and to analyze variables associated with in-hospital mortality (IHM) in AE-COPD patients with AF. Methods: We used data from the Registry of Specialized Care Activity-Basic Minimum Data Set (RAE-CMBD) to select patients aged ≥40 years with COPD in Spain (2016-2021). We stratified the study population according to AF presence and sex. The propensity score matching (PSM) methodology was employed to create comparable groups based on age, admission year, and comorbidities at the time of hospitalization. Results: We identified 399,196 hospitalizations that met the inclusion criteria. Among them, 20.58% had AF. The prevalence of AF rose from 2016 to 2021 (18.26% to 20.95%), though the increase was only significant in men. The median length of hospital stay (LOHS) and IHM were significantly higher in patients with AF than in those without AF. After PSM, IHM remained significantly higher for man and women with AF. Older age, male sex, and several comorbidities were factors associated with AF. Additionally, older age, male sex, different comorbidities including COVID-19, hospitalization in the year 2020, mechanical ventilation, and intensive care unit (ICU) admission were associated with higher IHM in patients with AE-COPD and AF. Conclusions: AF prevalence was high in patients hospitalized for AE-COPD, was higher in men than in women, and increased over time. AF presence was associated with worse outcomes. The variables associated with IHM in hospitalized AE-COPD patients with AF were older age, male sex, different comorbidities including COVID-19 presence, hospitalization in the year 2020, need of mechanical ventilation, and ICU admission.

4.
J Fungi (Basel) ; 10(3)2024 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-38535190

RESUMEN

Sweet chestnut, an Asiatic tree introduced in many parts of Europe including the United Kingdom, is planted for nut production, timber, and amenity. Its major threat is the disease called blight, caused by the fungus Cryphonectria parasitica, which infects through wounds by airborne spores. Field trapping using sticky rods rotating traps was performed in an infected area in Devon (between May 2021 and April 2023). An improved dual hydrolysis Taqman probes real-time PCR was used. The number of spores was calculated by comparing the cycle threshold to the Ct of standards with known amounts of conidia or known target fragment copies cloned into a plasmid. Weekly spore counts were in the range of around 60 to approximately 8.5 × 103, with fluctuations of peaks (mainly in late summer-autumn 2021) and troughs. The effects of weather parameters were modelled, finding correlations between spore numbers and temperature, humidity, dewpoint, rainfall, wind speed, and wind duration. Additionally, an insect trapping was performed to confirm the presence/absence and quantity of C. parasitica conidia potentially phoretic on some insects by using the same molecular approach. None of the ten collected insect species harboured spores of this fungus.

5.
J Fungi (Basel) ; 9(10)2023 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-37888292

RESUMEN

In England, Cryphonectria parasitica was detected for the first time in 2011 in a nursery and in 2016 in the wider environment. Surveys between 2017 and 2020 identified the disease at different sites in Berkshire, Buckinghamshire, Cornwall, Derbyshire, Devon, Dorset, London, West Sussex, and the island of Jersey, while the present study comprises the results of the 2020-2023 survey with findings in Derbyshire, Devon, Kent, Nottinghamshire, Herefordshire, Leicestershire, London, West Sussex, and the islands of Jersey and Guernsey. A total of 226 suspected samples were collected from 72 surveyed sites, as far north as Edinburgh and as far west as Plymouth (both of which were negative), and 112 samples tested positive by real-time PCR and isolation from 35 sites. The 112 isolates were tested for the vegetative compatibility group (VCG), mating type, and Cryphonectria hypovirus 1 (CHV1). Twelve VCGs were identified, with two of them (EU-5 and EU-22) being the first records in the UK. Both mating types were present (37% MAT-1 and 63% MAT-2), but only one mating type was present per site and VCG, and perithecia were never observed. Cryphonectria hypovirus 1 (CHV1), consistently subtype-I haplotype E-5, was detected in three isolates at a low concentration (5.9, 21.1, and 33.0 ng/µL) from locations in London, Nottinghamshire, and Devon.

6.
J Pediatr Nurs ; 73: 67-71, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37647790

RESUMEN

OBJECTIVES: A multimedia medication training program for parents and legal guardians of children with chronic kidney disease (CKD) aimed to improve comprehension CKD and general information about medications used in pediatric patients attending The Foundation for Children with Kidney Disease (FUNDANIER, Guatemala City). METHODS: A quasi-experimental study was carried out to measure the impact of the educational intervention on medication knowledge, at FUNDANIER from September to October 2019. Means and standard deviations was used to described test results. A Wilcoxon test was performed, to compare scores of pre and post-tests. Odds Ratio (OR) was used to determine if there was an improvement in the knowledge score before and after the intervention. Results There was significant improvement in knowledge scores knowledge before and after the intervention(27/35 versus 33/35, P < 0.005). Mestizo participants had higher odds of improvement before and after the intervention (OR 7, CI: 0.6-78). Parent-guardians with prior education, and who spoke Spanish had higher odds of improved knowledge scores (OR 3.2, CI:0.3-35; OR 1.1 CI: 0.1-14 respectively). CONCLUSION: Caregivers who participated in the educational workshop improved and retained information related to CKD comprehension and medications used. This study provides a model for educational modules that can be used, tested, and applied in other chronic disease settings in low to middle income countries. PRACTICE IMPLICATIONS: A culturally relevant multimedia CKD educational platform was effective in improving medication knowledge among parent/guardians of children with CKD in a low literacy setting.


Asunto(s)
Multimedia , Insuficiencia Renal Crónica , Humanos , Niño , Guatemala , Cuidadores , Insuficiencia Renal Crónica/terapia , Enfermedad Crónica
7.
Sci Total Environ ; 902: 166084, 2023 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-37549704

RESUMEN

This study presents an analysis of the impacts of the changes in bottom depth along the Guadalquivir Estuary on tidal dynamics. A realistic non-linear 1D numerical model, incorporating changes in both breadth and bottom depth, was employed to investigate the involved effects. The findings reveal a significant amplification of the M2 tidal wave towards the upper region of the Estuary, resulting from the gradual deepening caused by multiple dredging operations. The Estuary exhibits a pronounced tendency towards resonance, which is further enhanced by its deepening, resulting in reduced bottom friction and a smaller decrease in tidal wave amplitude as it propagates through the Estuary. The alterations in depth, particularly in breadth, along the Estuary play a crucial role in determining the magnitude of the resonant response of the M2 tidal wave.

8.
Viruses ; 15(6)2023 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-37376560

RESUMEN

Biological control of Cryphonectria parasitica fungus, the causal agent of chestnut blight, by virus infection (hypovirulence) is an effective control strategy against chestnut blight in Europe and some parts of North America. The most studied mycovirus is the Cryphonectria hypovirus 1 (CHV1) type species of the Hypoviridae family. In this study, the CHV1 virus was studied within some highly infected British isolates of Cryphonectria parasitica, gained in the past through co-culture transmissions. The effects of six temperatures (5-30 °C, in 5 °C steps) on six infected isolates (three with viral strain E-5, and other three with viral strain L-18) and their respective negative non-infected controls, three isogenic virulent fungal isolates, were examined. Experiments were performed with the nine isolate types with three replicates on potato dextrose agar (PDA) with cellophane sheets per isolate and temperature. A recently developed rapid, specific, quantitative reverse transcription PCR (RT-qPCR) screening method was used. This enabled quantifying the concentration (nanograms per microliter or copy numbers) of the virus within each isolate repetition. The presence of the virus had a significant negative effect between 20 and 25 °C on the C. parasitica growth rate, which was anyway highly influenced by and positively correlated with the temperature. The temperature clearly determined the virus accumulation and its recovery from cold or heat, and the virus optimum temperature was estimated at 15-25 °C.


Asunto(s)
Virus Fúngicos , Virus ARN , Temperatura , Enfermedades de las Plantas/microbiología
9.
ACS Synth Biol ; 12(7): 2015-2028, 2023 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-37155828

RESUMEN

The reconstitution of basic cellular functions in micrometer-sized liposomes has led to a surge of interest in the construction of synthetic cells. Microscopy and flow cytometry are powerful tools for characterizing biological processes in liposomes with fluorescence readouts. However, applying each method separately leads to a compromise between information-rich imaging by microscopy and statistical population analysis by flow cytometry. To address this shortcoming, we here introduce imaging flow cytometry (IFC) for high-throughput, microscopy-based screening of gene-expressing liposomes in laminar flow. We developed a comprehensive pipeline and analysis toolset based on a commercial IFC instrument and software. About 60 thousands of liposome events were collected per run starting from one microliter of the stock liposome solution. Robust population statistics from individual liposome images was performed based on fluorescence and morphological parameters. This allowed us to quantify complex phenotypes covering a wide range of liposomal states that are relevant for building a synthetic cell. The general applicability, current workflow limitations, and future prospects of IFC in synthetic cell research are finally discussed.


Asunto(s)
Células Artificiales , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Liposomas/química , Transcripción Genética , Genes Reporteros , Biosíntesis de Proteínas , Replicación del ADN , Microtúbulos , Células Artificiales/química
10.
ACS Synth Biol ; 12(4): 1187-1203, 2023 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-37014369

RESUMEN

In cell-free gene expression, low input DNA concentration severely limits the phenotypic output, which may impair in vitro protein evolution efforts. We address this challenge by developing CADGE, a strategy that is based on clonal isothermal amplification of a linear gene-encoding dsDNA template by the minimal Φ29 replication machinery and in situ transcription-translation. We demonstrate the utility of CADGE in bulk and in clonal liposome microcompartments to boost up the phenotypic output of soluble and membrane-associated proteins, as well as to facilitate the recovery of encapsulated DNA. Moreover, we report that CADGE enables the enrichment of a DNA variant from a mock gene library via either a positive feedback loop-based selection or high-throughput screening. This new biological tool can be implemented for cell-free protein engineering and the construction of a synthetic cell.


Asunto(s)
ADN Polimerasa Dirigida por ADN , ADN , ADN/genética , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Proteínas de la Membrana/genética , Biblioteca de Genes , Expresión Génica
11.
Gene ; 863: 147302, 2023 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-36813062

RESUMEN

INTRODUCTION: Genetic-association studies have shown that some polymorphisms are associated with different aspects of athletic performance, including very specific features, such as players' position in team sports, like soccer, rugby, and Australian football. However, this type of association has not been investigated in Basketball yet. The present study analyzed the association of ACTN3 R577X, AGT M268T, ACE I/D and BDKRB2+9/-9 polymorphisms with the position of basketball players. METHODS: One hundred fifty-two male athletes from 11 teams of the first division of Brazilian Basketball League and 154 male Brazilian controls were genotyped. The analyses of the ACTN3 R577X and AGT M268T were performed by the allelic discrimination method, while ACE I/D and BDKRB2+9/-9 by conventional PCR followed by electrophorese in agarose gel. RESULTS: The results showed a significant effect of height on all positions and an association between the genetic polymorphisms analyzed and basketball positions. In addition, a significantly higher frequency of ACTN3 577XX genotype was observed in Point Guards. Also, compared to Point Guard, ACTN3 RR and RX were more prevalent in the Shooting Guard and Small Forward group and RR genotype was also more prevalent in the Power Forward and Center group. CONCLUSION: The main finding of our study was the positive association of ACTN3 R577X polymorphism and basketball playing position, and a suggestion of genotypes related to strength/power performance with post players and genotypes related to endurance performance with point guard players.


Asunto(s)
Baloncesto , Humanos , Masculino , Brasil , Actinina/genética , Australia , Polimorfismo Genético , Atletas , Genotipo
12.
Qual Health Res ; 33(4): 297-307, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36715082

RESUMEN

This study explores adolescents' subjective experiences when facing body measurements at school, performed to evaluate the effects of a school-based intervention to promote physical activity. Three semi-structured interviews (n = 3) and two focus groups (n = 7 and 3) were conducted with adolescents after measuring some variables related to their weight and adiposity. Observational data (n = 88) collected by the two researchers in charge of the measurements were also obtained. Findings derived from our thematic analysis question the suitability of body measurements as social contexts where the core ideas of healthism emerge. Some adolescents lived body measurements as negative social experiences, promoting body dissatisfaction, social comparison, embarrassment, and simplistic associations between health and body shape, which could hinder their adherence to healthy habits. We emphasize the role of qualitative research when evaluating pre/posttests of school-based intervention studies, not just the interventions themselves, to generate knowledge to improve research protocols and prevent unwanted psychological outcomes in participants.


Asunto(s)
Ejercicio Físico , Obesidad , Adolescente , Humanos , Ejercicio Físico/psicología , Medio Social , Instituciones Académicas , Estudiantes/psicología , Servicios de Salud Escolar
13.
Viruses ; 14(12)2022 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-36560682

RESUMEN

Biological control of Cryphonectria parasitica fungus, causal agent of chestnut blight, by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe and some parts of North America. The most studied mycovirus is the Cryphonectria hypovirus 1 (CHV-1) type species of the Hypoviridae family. To efficiently provide biocontrol, the virus must be able to induce hypovirulence in its fungal host in chestnut trees. Here, two different CHV-1 subtype I virus strains (E-5 and L-18), gained by transmissions, were tested for their hypovirulence induction, biocontrol potential, and transmission between vegetatively compatible (VCG) and incompatible fungal isolate groups in sweet chestnut seedlings and branches. Both strains of CHV-1 showed great biocontrol potential and could protect trees by efficiently transmitting CHV-1 by hyphal anastomosis between fungal isolates of the same VCG and converting virulent to hypovirulent cankers. The hypovirulent effect was positively correlated with the virus concentration, tested by four different reverse-transcription PCRs, two end-point and two real-time methods, one of which represents a newly developed real-time PCR for the detection and quantification of CHV-1.


Asunto(s)
Ascomicetos , Virus Fúngicos , Virus ARN , Virus Fúngicos/genética , Enfermedades de las Plantas , Virus ARN/genética
14.
Annu Int Conf IEEE Eng Med Biol Soc ; 2022: 3051-3054, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-36085792

RESUMEN

Meningioma is the most common intracranial tumor in adulthood. With a clear female predominance and a recurrence rate that reaches 20%, it is, despite being considered a benign tumor, a pathology that greatly compromises post-diagnosis quality of life. Its prone to recur or progress to a higher degree is difficult to predict in the absence of obvious histological criteria. This project aims to develop an automatic methodology to aid in the diagnosis of meningiomas that is objective and easily reproducible. The methodology is based on histopathological image analysis using artificial intelligence and machine learning algorithms. It includes a semi-automatic process of identification and cleaning of the scanned samples, an automatic detection of the nuclei of each image and, finally, the parameterization of the samples. The obtained data together with the clinical information will be analyzed using statistical methods in order to provide a methodology to support clinical diagnosis and decision-making in patient management. The result is the development of an effective methodology that generates a set of data associated with morphological parameters with different trends according to the pathological groups studied. A tool has been developed that allows an effective semiautomatic analysis of the images to evaluate these parameters in an objective and reproducible way, helping in clinical decision-making and facilitating to undertake projects with large sample series. Clinical Relevance- The main contribution of this project is in the field of neuropathology, for the diagnosis of meningiomas, the most common brain tumor. The present project provides an objective and quantifiable prognosis methodology for the meningiomas, offering a more precise monitoring of the treatment applied to the patient, resulting in a better prognosis and better quality of life.


Asunto(s)
Neoplasias Encefálicas , Neoplasias Meníngeas , Meningioma , Adulto , Inteligencia Artificial , Femenino , Humanos , Masculino , Neoplasias Meníngeas/diagnóstico por imagen , Neoplasias Meníngeas/patología , Meningioma/diagnóstico por imagen , Meningioma/patología , Calidad de Vida
15.
Front Physiol ; 13: 919544, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36117688

RESUMEN

Renin-angiotensin system (RAS) and kallikrein-kinin system (KKS) have a different site of interaction and modulate vascular tone and inflammatory response as well on exercise adaptation, which is modulated by exercise-induced cytokines. The aim of the study was to evaluate the role of ACE I/D and BDKRB2 +9/-9 polymorphism on exercise-induced cytokine response. Seventy-four male marathon finishers, aged 30 to 55 years, participated in this study. Plasma levels of exercise-induced cytokines were determined 24 h before, immediately after, and 24 h and 72 h after the São Paulo International Marathon. Plasma concentrations of MCP-1, IL-6 and FGF-21 increased after marathon in all genotypes of BDKRB2. IL-10, FSTL and BDNF increased significantly after marathon in the genotypes with the presence of the -9 allele. FSTL and BDNF concentrations were higher in the -9/-9 genotype compared to the +9/+9 genotype before (p = 0.006) and after the race (p = 0.023), respectively. Apelin, IL-15, musclin and myostatin concentrations were significantly reduced after the race only in the presence of -9 allele. Marathon increased plasma concentrations of MCP1, IL-6, BDNF and FGF-21 in all genotypes of ACE I/D polymorphism. Plasma concentrations of IL-8 and MIP-1alpha before the race (p = 0.015 and p = 0.031, respectively), of MIP-1alpha and IL-10 after the race (p = 0.033 and p = 0.047, respectively) and VEGF 72 h after the race (p = 0.018) were lower in II homozygotes compared to runners with the presence of D allele. One day after the race we also observed lower levels of MIP-1alpha in runners with II homozygotes compared to DD homozygotes (p = 0.026). Before the marathon race myostatin concentrations were higher in DD compared to II genotypes (p = 0.009). Myostatin, musclin, IL-15, IL-6 and apelin levels decreased after race in genotypes with the presence of D allele. After the race ACE activity was negatively correlated with MCP1 (r = -56, p < 0.016) and positively correlated with IL-8, IL-10 and MIP1-alpha (r = 0.72, p < 0.0007, r = 0.72, p < 0.0007, r = 0.47, p < 0.048, respectively). The runners with the -9/-9 genotype have greater response in exercise-induced cytokines related to muscle repair and cardioprotection indicating that BDKRB2 participate on exercise adaptations and runners with DD genotype have greater inflammatory response as well as ACE activity was positively correlated with inflammatory mediators. DD homozygotes also had higher myostatin levels which modulates protein homeostasis.

16.
J Fungi (Basel) ; 8(8)2022 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-35893146

RESUMEN

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters-diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility-were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes.

17.
Pathogens ; 11(8)2022 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-35894049

RESUMEN

The plant nursery industry has become an ideal reservoir for Phytophthora species and other soilborne pathogens. In this context, isolation from tissues and soil of ornamental and forest plants from nurseries in four regions of Spain was carried out. A high diversity of Phytophthora species was confirmed. Fourteen Phytophthora phylotypes (P. cactorum, P. cambivora, P. cinnamomi, P. citrophthora, P. crassamura, P. gonapodyides, P. hedraiandra, P. nicotianae, P. niederhauserii, P. palmivora, P. plurivora, P. pseudocryptogea, P. sansomeana, and Phytophthora sp. tropicalis-like 2) were isolated from over 500 plant samples of 22 species in 19 plant genera. Nine species were detected in water sources, two of them (P. bilorbang and P. lacustris) exclusively from water samples. P. crassamura was detected for the first time in Spain. This is the first time P. pseudocryptogea is isolated from Chamaecyparis lawsoniana and Yucca rostrata in Spain.

19.
Methods Mol Biol ; 2536: 13-49, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35819596

RESUMEN

Phytophthora species can be found in multiple substrates. Due to dormancy of resting structures and presence of faster-growing antagonists, direct isolation of Phytophthora can be difficult to achieve, and indirect baiting methods often reach higher isolation frequencies. In this chapter, different methodologies are described for sampling and for the successful isolation of Phytophthora species from natural ecosystems. Sampling methods for soil, roots, bark cankers, and waterbodies are described. Agar recipes and guidance on the selection of suitable tissue to perform direct isolations are provided. A range of different baiting techniques are described for isolation of Phytophthora from different substrates. Purification methods to obtain clean and non-mixed cultures and conservation methods for pure cultures are shown. Two pathogenicity test methods for soil infestation and for under-bark inoculation, respectively, are described in detail.


Asunto(s)
Phytophthora , Ecosistema , Suelo , Microbiología del Suelo , Virulencia
20.
PLoS One ; 17(3): e0260420, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35312702

RESUMEN

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of proteins, biological circuits and metabolic pathways. Their encapsulation on microfluidic platforms can generate millions of cell-free reactions in picoliter volume droplets. However, current methods struggle to create DNA diversity between droplets while also reaching sufficient protein expression levels. In particular, efficient multi-gene expression has remained elusive. We here demonstrate that co-encapsulation of DNA-coated beads with a defined cell-free system allows high protein expression while also supporting genetic diversity between individual droplets. We optimize DNA loading on commercially available microbeads through direct binding as well as through the sequential coupling of up to three genes via a solid-phase Golden Gate assembly or BxB1 integrase-based recombineering. Encapsulation with an off-the-shelf microfluidics device allows for single or multiple protein expression from a single DNA-coated bead per 14 pL droplet. We envision that this approach will help to scale up and parallelize the rapid prototyping of more complex biological systems.


Asunto(s)
Técnicas Analíticas Microfluídicas , ADN/genética , Expresión Génica , Dispositivos Laboratorio en un Chip , Microfluídica
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