Identification and expression analysis of two steamer-like retrotransposons in the Chilean blue mussel (Mytilus chilensis).

BACKGROUND: Disseminated neoplasia (DN) is a proliferative cell disorder of the circulatory system of bivalve mollusks. The disease is transmitted between individuals and can also be induced by external chemical agents such as bromodeoxyuridine. In Mya arenaria, we have cloned and characterized an LTR-retrotransposon named Steamer. Steamer mRNA levels and gene copy number correlates with DN and can be used as a marker of the disease. So far, the only mollusk where a retrotransposon expression relates to DN is Mya arenaria. On the other hand, it has been reported that the Chilean blue mussel Mytilus chilensis can also suffers DN. Our aim was to identify retrotransposons in Mytilus chilensis and to study their expression levels in the context of disseminated neoplasia. RESULTS: Here we show that 7.1% of individuals collected in August 2018, from two farming areas, presents morphological characteristics described in DN. Using Steamer sequence to interrogate the transcriptome of M. chilensis we found two putative retrotransposons, named Steamer-like elements (MchSLEs). MchSLEs are present in the genome of M. chilensis and MchSLE1 is indeed an LTR-retrotransposon. Neither expression, nor copy number of the reported MchSLEs correlate with DN status but both are expressed at different levels among individual animals. We also report that in cultured M. chilensis haemocytes MchSLEs1 expression can be induced by bromodeoxyuridine. CONCLUSIONS: We conclude that SLEs present in Mytilus chilensis are differentially expressed among individuals and do not correlate with disseminated neoplasia. Treatment of haemocytes with a stressor like bromodeoxyuridine induces expression of MchSLE1 suggesting that in Mytilus chilensis environmental stressors can induce activation of LTR-retrotransposon.

Safety of a controlled human infection model of tuberculosis with aerosolised, live-attenuated Mycobacterium bovis BCG versus intradermal BCG in BCG-naive adults in the UK: a dose-escalation, randomised, controlled, phase 1 trial.

BACKGROUND: Mycobacterium tuberculosis is the main causative agent of tuberculosis. BCG, the only licensed vaccine, provides inadequate protection against pulmonary tuberculosis. Controlled human infection models are useful tools for vaccine development. We aimed to determine a safe dose of aerosol-inhaled live-attenuated Mycobacterium bovis BCG as a surrogate for M tuberculosis infection, then compare the safety and tolerability of infection models established using aerosol-inhaled and intradermally administered BCG. METHODS: This phase 1 controlled human infection trial was conducted at two clinical research facilities in the UK. Healthy, immunocompetent adults aged 18-50 years, who were both M tuberculosis-naive and BCG-naive and had no history of asthma or other respiratory diseases, were eligible for the trial. Participants were initially enrolled into group 1 (receiving the BCG Danish strain); the trial was subsequently paused because of a worldwide shortage of BCG Danish and, after protocol amendment, was restarted using the BCG Bulgaria strain (group 2). After a dose-escalation study, during which participants were sequentially allocated to receive either 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 colony-forming units (CFU) of aerosol BCG, the maximum tolerated dose was selected for the randomised controlled trial. Participants in this trial were randomly assigned (9:12), by variable block randomisation and using sequentially numbered sealed envelopes, to receive aerosol BCG (1 × 107 CFU) and intradermal saline or intradermal BCG (1 × 106 CFU) and aerosol saline. Participants were masked to treatment allocation until day 14. The primary outcome was to compare the safety of a controlled human infection model based on aerosol-inhaled BCG versus one based on intradermally administered BCG, and the secondary outcome was to evaluate BCG recovery in the airways of participants who received aerosol BCG or skin biopsies of participants who received intradermal BCG. BCG was detected by culture and by PCR. The trial is registered at ClinicalTrials.gov, NCT02709278, and is complete. FINDINGS: Participants were assessed for eligibility between April 7, 2016, and Sept 29, 2018. For group 1, 15 participants were screened, of whom 13 were enrolled and ten completed the study; for group 2, 60 were screened and 33 enrolled, all of whom completed the study. Doses up to 1 × 107 CFU aerosol-inhaled BCG were sufficiently well tolerated. No significant difference was observed in the frequency of adverse events between aerosol and intradermal groups (median percentage of solicited adverse events per participant, post-aerosol vs post-intradermal BCG: systemic 7% [IQR 2-11] vs 4% [1-13], p=0·62; respiratory 7% [1-19] vs 4% [1-9], p=0·56). More severe systemic adverse events occurred in the 2 weeks after aerosol BCG (15 [12%] of 122 reported systemic adverse events) than after intradermal BCG (one [1%] of 94; difference 11% [95% CI 5-17]; p=0·0013), but no difference was observed in the severity of respiratory adverse events (two [1%] of 144 vs zero [0%] of 97; 1% [-1 to 3]; p=0·52). All adverse events after aerosol BCG resolved spontaneously. One serious adverse event was reported-a participant in group 2 was admitted to hospital to receive analgesia for a pre-existing ovarian cyst, which was deemed unrelated to BCG infection. On day 14, BCG was cultured from bronchoalveolar lavage samples after aerosol infection and from skin biopsy samples after intradermal infection. INTERPRETATION: This first-in-human aerosol BCG controlled human infection model was sufficiently well tolerated. Further work will evaluate the utility of this model in assessing vaccine efficacy and identifying potential correlates of protection. FUNDING: Bill & Melinda Gates Foundation, Wellcome Trust, National Institute for Health Research Oxford Biomedical Research Centre, Thames Valley Clinical Research Network, and TBVAC2020.

Establishing a Multi-Vial Design Space for the Freeze-Drying Process by Means of Mathematical Modeling of the Primary Drying Stage.

Primary drying is the most critical stage of the freeze-drying process. This work aimed to establish a design space for this process by means of mathematical modeling of the primary drying stage, capable of addressing the thermal characteristics of distinct vial suppliers. Modeling of primary drying was implemented on Microsoft Excel using steady-state heat and mass transfer equations at two extreme conditions as assessed by risk analysis, to predict product temperature and primary-drying time. The heat transfer coefficients (Kv) of four different vial suppliers were experimentally determined, both, at the center and edge of the freeze-dryer's shelf. Statistically significant differences (ANOVA p<0.05) were observed between suppliers throughout the assessed pressure range. Overall, the average Kve/Kvc (edge/center) ratio was higher than 1.6 for all suppliers due to the radiation effect. A design space for the drying process was established using mathematical modeling taking into account the Kv of the worst-case supplier, in the shelf edge. A primary drying cycle was carried out at a shelf temperature of -25 °C and a chamber pressure of 45 mTorr for 8 % sucrose and at -10 °C and 75 mTorr for 5 % NaCl. Freeze-dried products with good cosmetic appearance were obtained for the four vial suppliers both, in the shelf center and edge. The results show that it is possible to predict and establish the critical parameters for the primary drying stage, under a design space concept, considering the differences in the Kv of vial suppliers without adverse consequences on the quality of the finished freeze-dried product.

Dominance of ST131, B2, blaCTX-M-15, and papA-papC-kpsMII-uitA among ESBL Escherichia coli isolated from bloodstream infections in Quito, Ecuador: a 10-year surveillance study (2009-2019).

AIMS: This study aimed to examine antibiotic resistance and the epidemiology of extended-spectrum ß-lactamases (ESBL)-producing Escherichia coli associated with bloodstream infections over a period of 10 years. METHODS AND RESULTS: Isolates were collected from January 2009 to December 2019 and those testing for E. coli were included. Antibiotic susceptibility was tested using the VITEK® system. Selected isolates were further characterized by amplification of marker genes (virulence traits, phylogroups, and sequence types). A total of 166 ESBL-producing E. coli were recovered. The blaCTX-M-15 allele was the most abundant. Most of the isolates were resistant to ceftriaxone, cefepime, ceftazidime, ampicillin/sulbactam, piperacillin/tazobactam, and ciprofloxacin. No resistance to carbapenems was registered. More than 80% of bacteria were classified as extraintestinal pathogenic E. coli (ExPEC), and the combination of virulence traits:papA-papC-kpsMII-uitA was the most common. Phylogroup B2 was the most prevalent, and bacteria predominantly belonged to ST131. CONCLUSIONS: There was an increase in the ExPEC ESBL-E coli in bloodstream infections and the relationship between the isolates found in these infections during these 10 years.

An elevated HScore is associated with poor clinical outcomes in COVID-19.

BACKGROUND: Patients with Coronavirus Disease 2019 (COVID-19) frequently experience a hyperinflammatory syndrome leading to unfavorable outcomes. This condition resembles Secondary Hemophagocytic Lymphohistiocytosis (sHLH) described in neoplastic, rheumatic and other infectious diseases. A scoring system (HScore) that evaluates underlying immunosuppression, temperature, organomegaly, cytopenias, ferritin, triglycerides, fibrinogen and AST was validated for sHLH, and recently proposed to evaluate hyperinflammation in COVID-19. AIM: To assess the presence of sHLH among patients with COVID-19 admitted for hospitalization and to evaluate Hscore as a prognostic tool for poor outcomes. MATERIAL AND METHODS: One hundred forty-three patients aged 21-100 years (64% males) admitted because of COVID-19 were enrolled in a prospective study. HScore was calculated within 72 hours admission. The incidence of sHLH during hospitalization was evaluated. Additionally, the relationship between a HScore ≥ 130 points and either the requirement of mechanical ventilation or 60-days mortality was explored. RESULTS: The median HScore was 96 (33-169). A SHLH was diagnosed in one patient (incidence 0.7%), whose HScore was 169. After adjusting for age, sex, comorbidities and obesity, HScore ≥ 130 was independently associated with the composite clinical outcome (Hazard rartio 2.13, p = 0.022). CONCLUSIONS: sHLH is not frequent among COVID-19 patients. HScore can be useful to predict the risk for poor outcomes.

An Overview of the Use and Applications of Limosilactobacillus fermentum in Broiler Chickens.

The implementation of government regulations on antibiotic use, along with the public's concern for drug resistance, has strengthened interest in developing alternatives not only aimed at preserving animal production but also at reducing the effects of pathogenic infections. Probiotics, in particular, are considered microorganisms that induce health benefits in the host after consumption of adequate amounts; they have been established as a potential strategy for improving growth, especially by stimulating intestinal homeostasis. Probiotics are commonly associated with lactic acid bacteria, and Limosilactobacillus fermentum is a well-studied species recognized for its favorable characteristics, including adhesion to epithelial cells, production of antimicrobial compounds, and activation of receptors that prompt the transcription of immune-associated genes. Recently, this species has been used in animal production. Different studies have shown that the application of L. fermentum strains not only improves the intestinal ecosystem but also reduces the effects caused by potentially pathogenic microorganisms. These studies have also revealed key insights into the mechanisms behind the actions exerted by this probiotic. In this manuscript, we aim to provide a concise overview of the effects of L. fermentum administration on broiler chicken health and performance.

Effects of Lactobacillus fermentum Administration on Intestinal Morphometry and Antibody Serum Levels in Salmonella-Infantis-Challenged Chickens.

There are no studies reporting the effects of Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) on intestinal architecture and immunoglobulin serum levels in chickens. Here, we measured these parameters and hypothesized whether probiotic administration could modulate the observed outcomes. Two-hundred 1-day-old COBB 500 male chicks were allocated into four groups: (I) the control, (II) the group treated with L. fermentum, (III) the group exposed to S. Infantis, and (IV) the group inoculated with both bacteria. At 11 days post infection, blood was gathered from animals which were then euthanized, and samples from the small intestine were collected. Intestinal conditions, as well as IgA and IgM serum levels, were assessed. S. Infantis reduced villus-height-to-crypt-depth (VH:CD) ratios in duodenal, jejunal, and ileal sections compared to control conditions, although no differences were found regarding the number of goblet cells, muc-2 expression, and immunoglobulin concentration. L. fermentum improved intestinal measurements compared to the control; this effect was also evidenced in birds infected with S. Infantis. IgM serum levels augmented in response to the probiotic in infected animals. Certainly, the application of L. fermentum elicited positive outcomes in S. Infantis-challenged chickens and thus must be considered for developing novel treatments designed to reduce unwanted infections.

A Survey of AI-Based Anomaly Detection in IoT and Sensor Networks.

Machine learning (ML) and deep learning (DL), in particular, are common tools for anomaly detection (AD). With the rapid increase in the number of Internet-connected devices, the growing desire for Internet of Things (IoT) devices in the home, on our person, and in our vehicles, and the transition to smart infrastructure and the Industrial IoT (IIoT), anomaly detection in these devices is critical. This paper is a survey of anomaly detection in sensor networks/the IoT. This paper defines what an anomaly is and surveys multiple sources based on those definitions. The goal of this survey was to highlight how anomaly detection is being performed on the Internet of Things and sensor networks, identify anomaly detection approaches, and outlines gaps in the research in this domain.

An elevated HScore is associated with poor clinical outcomes in COVID-19 / Un HScore elevado se asocia a mal pronóstico en pacientes con COVID-19

BACKGROUND: Patients with Coronavirus Disease 2019 (COVID-19) frequently experience a hyperinflammatory syndrome leading to unfavorable outcomes. This condition resembles Secondary Hemophagocytic Lymphohistiocytosis (sHLH) described in neoplastic, rheumatic and other infectious diseases. A scoring system (HScore) that evaluates underlying immunosuppression, temperature, organomegaly, cytopenias, ferritin, triglycerides, fibrinogen and AST was validated for sHLH, and recently proposed to evaluate hyperinflammation in COVID-19. AIM: To assess the presence of sHLH among patients with COVID-19 admitted for hospitalization and to evaluate Hscore as a prognostic tool for poor outcomes. MATERIAL AND METHODS: One hundred forty-three patients aged 21-100 years (64% males) admitted because of COVID-19 were enrolled in a prospective study. HScore was calculated within 72 hours admission. The incidence of sHLH during hospitalization was evaluated. Additionally, the relationship between a HScore ≥ 130 points and either the requirement of mechanical ventilation or 60-days mortality was explored. RESULTS: The median HScore was 96 (33-169). A SHLH was diagnosed in one patient (incidence 0.7%), whose HScore was 169. After adjusting for age, sex, comorbidities and obesity, HScore ≥ 130 was independently associated with the composite clinical outcome (Hazard rartio 2.13, p = 0.022). CONCLUSIONS: sHLH is not frequent among COVID-19 patients. HScore can be useful to predict the risk for poor outcomes.

ANTECEDENTES: Los pacientes con Enfermedad por Coronavirus 2019 (COVID-19), experimentan frecuentemente un síndrome hiperinflamatorio que lleva a resultados desfavorables. Esta situación se asemeja al Síndrome Hemofagocítico Secundario (sHLH) descrito en enfermedades neoplásicas, reumatológicas y por otros agentes infecciosos. Un sistema simple de puntaje (HScore) que evalúa inmunosupresión, temperatura organomegalia, citopenias, ferritina, triglicéridos, fibrinógeno y AST ha sido validado para el diagnóstico de sHLH y ha sido propuesto recientemente para evaluar la hiperinflamación en COVID-19. OBJETIVO: Medir la frecuencia de sHLH entre pacientes con COVID-19 hospitalizados, y evaluar a HScore como una herramienta pronóstica. MATERIAL Y MÉTODOS: Ciento cuarenta y tres pacientes de 21 a 100 años (64% hombres) fueron ingresados en este estudio de cohorte prospectivo, unicéntrico. Se calculó HScore dentro de las primeras 72 horas desde el ingreso, y se midió la incidencia de sHLH durante la hospitalización. Adicionalmente, se evaluó la relación entre HScore ≥ 130 puntos y un desenlace compuesto de ventilación mecánica o muerte a los 60 días. RESULTADOS: La mediana de HScore fue 96 (33-169) puntos. Un paciente fue diagnosticado con sHLH (incidencia 0,7%). Luego de ajustar por edad, sexo, comorbilidades y obesidad, un HScore ≥ 130 se asoció de manera independiente con el desenlace compuesto. CONCLUSIONES: El sHLH no es frecuente en los pacientes con COVID-19. El uso de HScore puede ser útil para predecir el riesgo de desenlaces clínicos desfavorables.

Sustainability of water transfer projects: A systematic review.

Inter-basin water transfer projects (IBTs) have significantly increased in number in recent decades due to the unremitting need to solve the problem of global water imbalance. However, given the complex challenges inherent in implementing and maintaining IBTs, there is a need to characterize the multi-faceted aspects of sustainability (or unsustainability) that result from these megaprojects. Through a systematic review of the literature, we sought to identify and characterize the positive and negative impacts that most often influence the sustainability of IBTs, focusing on impacts within the environmental, social, and economic pillars of sustainability. Based on an eligibility criterion, the systematic review selected 68 documents out of an initial total of 1567 for information quality analysis and content evaluation. The qualitative coding of the documents allowed us to characterize the landscape of impacts that result from IBTs across the three pillars of sustainability. The study findings revealed that the most frequently coded positive impacts related to the environmental pillar of sustainability, while the most frequently coded negative impacts related to both social and environmental pillars. In addition, the most frequently coded positive impact overall related to the economic benefits generated by the IBTs. Through a critical analysis of the study findings, we provide an assessment of future IBTs with a focus on the UN sustainable development goals.

A PMAxxTM qPCR Assay Reveals That Dietary Administration of the Microalgae Tetraselmis chuii Does Not Affect Salmonella Infantis Caecal Content in Early-Treated Broiler Chickens.

Salmonella enterica serovars cause infections in humans. S. enterica subsp. enterica serovar Infantis is considered relevant and is commonly reported in poultry products. Evaluating innovative approaches for resisting colonization in animals could contribute to the goal of reducing potential human infections. Microalgae represent a source of molecules associated with performance and health improvement in chickens. Tetraselmis chuii synthesizes fermentable polysaccharides as part of their cell wall content; these sugars are known for influencing caecal bacterial diversity. We hypothesized if its dietary administration could exert a positive effect on caecal microbiota in favor of a reduced S. Infantis load. A total of 72 one-day-old broiler chickens (COBB 500) were randomly allocated into three groups: a control, a group infected with bacteria (day 4), and a group challenged with S. Infantis but fed a microalgae-based diet. Caecal samples (n = 8) were collected two days post-infection. A PMAxxTM-based qPCR approach was developed to assess differences regarding bacterial viable load between groups. The inclusion of the microalga did not modify S. Infantis content, although the assay proved to be efficient, sensitive, and repeatable. The utilized scheme could serve as a foundation for developing novel PCR-based methodologies for estimating Salmonella colonization.

Preventing Postendoscopic Mucosal Resection Bleeding of Large Nonpedunculated Colorectal Lesions.

The most common major adverse event of endoscopic mucosal resection (EMR) is clinically significant post-EMR bleeding (CSPEB), with an incidence of 6%-7% in large lesions. Repeat colonoscopy, blood transfusions, or other interventions are often needed. The associated direct costs are much higher than those of an uncomplicated EMR. In this review, we discuss the aspects related to CSPEB of large nonpedunculated polyps, such as risk factors, predictive models, and prophylactic measures, and we highlight evidence for preventive treatment options and explore new methods for bleeding prophylaxis. We also provide recommendations for steps that can be taken before, during, and after EMR to minimize bleeding risk. Finally, this review proposes future directions to reduce CSPEB incidence.

Antibiotic Resistance Awareness among Undergraduate Students in Quito, Ecuador.

The inappropriate use of antibiotics leads to antibiotic resistance, which reduces their efficacy. The education of undergraduates is likely to influence their practices. Assessing awareness is critical in the general effort to confront the spread of antibiotic resistance. This cross-sectional investigation was carried out using the questionnaire "Antibiotic resistance: Multi-country public awareness" developed by the World Health Organization. Students from different backgrounds at the Central University participated in the study (n = 733). The survey comprised five sections: demographics, knowledge, usage, sources of information, and attitudes. The rate of correct answers was 64.88%; differences were detected between programs of study (p < 0.001); effect size analysis showed that these differences cannot be considered large. Individuals from applied sciences scored higher than their counterparts from social studies. Mostly, interviewees were knowledgeable about usage, but mistakenly associated antibiotics with conditions such as cold/flu or viral illnesses; also, they associated antibiotic resistance with the patient and not with bacteria. Despite these misconceptions, positive attitudes were registered overall, and students generally adhered to common practices. They cited doctors/nurses and teachers as sources of information. As a consequence, it is recommended to develop courses that address deficient knowledge regarding antibiotic resistance, especially for individuals affiliated to social disciplines.

Neurotoxoplasmosis con presentación imagenológica atípica / Atypical imaging presentation of neurotoxoplasmosis

La toxoplasmosis cerebral o neurotoxoplasmosis es una de las infecciones por agentes oportunistas más frecuentes en pacientes infectados por virus de inmunodeficiencia humana (VIH) y se relaciona con inmunodeficiencia severa. En estos pacientes causa lesiones neurológicas de alto grado e incluso la muerte. Suele aparecer como múltiples focos de lesión de predominio gangliobasal y con realce anular. Se presenta un caso clínico de neurotoxoplasmosis en paciente VIH con un patrón radiológico poco frecuente.

Cerebral toxoplasmosis or neurotoxoplasmosis is one of the most frequent infections by opportunistic agents in HIV-infected patients and is associated with severe immunodeficiency. In these patients, it causes high-grade neurological lesions and even death. It usually appears as multiple foci of lesions with gangliobasal predominance and annular enhancement. We present a clinical case of neurotoxoplasmosis in a HIV patient with an unusual radiological pattern.

Administration of Dietary Microalgae Ameliorates Intestinal Parameters, Improves Body Weight, and Reduces Thawing Loss of Fillets in Broiler Chickens: A Pilot Study.

This pilot investigation aimed at studying the feasibility of using a low dose (0.2%) of dietary microalgae as a means of improving intestinal morphometry, body weight, and selected meat quality parameters in broilers. A total of 72 one-day-old ROSS 308 male chicks were randomly separated into four groups; three experimental pens in which the birds were fed with biomass from Tysochrysis lutea, Tetraselmis chuii, and Porphyridium cruentum over 30 days and a control group. T. chuii and P. cruentum had a positive effect with regard to body weight. In treated animals, duodenal and ileal sections showed characteristic tall and thin villi, with serrated surfaces and goblet cell differentiation. In both sections, values of the villus-height-to-crypt-depth ratio were increased by microalgae ingestion. The thawing weight loss of fillets was reduced in T. chuii-fed animals. The positive effects exerted by T. chuii and P. cruentum on intestinal architecture were associated with the improved body weight. Arguably, these outcomes exhibit the potential of using these species to enhance growth performance in broiler chickens by promoting gut homeostasis and thus nutrient absorption.

In Silico Detection of Antimicrobial Resistance Integrons in Salmonella enterica Isolates from Countries of the Andean Community.

Antimicrobial resistance genes are often associated with integrons, which promote their movement between and within DNA molecules. IntFinder 1.0 and I-VIP v1.2 were used for the detection of integrons and their associated resistance genes in assembled sequences and raw reads. A dataset comprising 1688 sequenced Salmonella enterica isolates from countries of the Andean Community was developed. A total of 749 and 680 integrons were identified by IntFinder 1.0 and I-VIP v1.2, respectively; class 2 integrons were the most abundant followed by class 1, whereas no class 3 integrons were detected. These elements were mainly associated with isolates from animal sources. S. Infantis ST32 contained the majority of integrons. Trimethoprim resistance genes (dfrA) were found in greater numbers than others, including aadA and bla genes. The presence of these resistance integrons may come as a response to antibiotic misuse, especially of co-trimoxazole. This represents a public health risk as novel resistant strains might appear due to gene dissemination. The information gathered from in silico studies not only contributes to our understanding of integron dynamics in pathogenic Salmonella, but also helps identify potential emergent patterns of resistance in the region, which is fundamental for developing pertinent antibiotic surveillance programs.

A Cross-Sectional Study to Assess Knowledge of COVID-19 among Undergraduate Students in North-Central Ecuador.

Adherence to preventive measures is influenced by people's knowledge, attitudes and practices towards a disease; therefore, assessing knowledge of COVID-19 is critical in the overall effort to contain the outbreak. This cross-sectional study was conducted among undergraduates (n = 3621) of different programs and different levels of education associated with universities in north-central Ecuador. The form consisted of 32 questions covering demographics, symptoms, detection, treatment, transmission, prevention and knowledge of the virus. The rate of correct answers was 75.5% (21.1 ± 5 out of 28), with differences observed regarding program of study, educational level and location of institution (α = 0.05), although effect size analyses showed that these differences could not be considered large. Multiple linear regression analyses showed that lower scores were associated with initial stages of education, careers related to social sciences and location of institution. Participants possessed sufficient knowledge about detection, transmission and prevention, although they overestimated fatality rate and were less confident about the characteristics of the virus and the effectiveness of traditional medicine. Consequently, future educational programs must place emphasis on addressing deficient knowledge. Certainly, improving COVID-19 literacy will promote the appropriate application of protective measures aimed at preventing the virus' spread.

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants.

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme's sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle's genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an 'Fe-only' nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.

A Simple Technology for Generating Marker-Free Chloroplast Transformants of the Green Alga Chlamydomonas reinhardtii.

The availability of routine methods for the genetic engineering of the chloroplast genome of Chlamydomonas reinhardtii is allowing researchers to explore the use of this microalga as a phototrophic cell platform for synthesis of high value recombinant proteins and metabolites. However, the established method for delivering transforming DNA into the algal chloroplast involves microparticle bombardment using an expensive "gene gun". Furthermore, selection of transformant lines most commonly involves the use of a bacterial antibiotic resistance gene. In this chapter, we describe a simple and cheap delivery method in which cell-DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. We also describe the use of plasmid expression vectors that target transgenes to a neutral site within the chloroplast genome between psbH and trnE2, and employ psbH as the selectable marker-thereby avoiding issues of unwanted antibiotic resistance genes in the resulting transgenic lines. Finally, we highlight a feature in our latest vectors in which the presence of a novel tRNA gene on the plasmid results in recognition within the chloroplast of UGA stop codons in transgenes as tryptophan codons. This feature simplifies the cloning of transgenes that are normally toxic to E. coli, serves as a biocontainment strategy restricting the functional escape of transgenes from the algal chloroplast to environmental microorganisms, and offers a simple system of temperature-regulated translation of transgenes.

An On-Line Cross-Sectional Questionnaire to Assess Knowledge of COVID-19 Pandemic among Citizens Tested for the SARS-CoV-2 Virus in Quito and Ibarra, Ecuador.

Several public health measures have been implemented to contain the SARS-CoV-2 outbreak. The adherence to control measures is known to be influenced by people's knowledge, attitudes and practices with regard to the disease. This study aimed at assessing COVID-19 knowledge in individuals who were tested for the virus. An online cross-sectional survey of 32 items, adapted to the national context, was conducted among 1656 Ecuadorians. The mean knowledge score was 22.5 ± 3 out of 28, with significant differences being observed with regard to educational attainment. People with postgraduate training scored higher than those with college, secondary and elementary instruction. Indeed, multiple linear regression revealed that lower scores were associated significantly with the latter three levels of education. Interviewees were knowledgeable about the symptoms, detection, transmission and prevention of the disease. However, they were less assertive regarding the characteristics of the virus as well as the usefulness of traditional and unproven treatments. These outcomes indicated a lack of knowledge in fundamental aspects of virus biology, which may limit the effectiveness of further prevention campaigns. Conclusively, educational and communicational programs must place emphasis on explaining the basic molecular characteristics of SARS-CoV-2; such information will certainly contribute to improve the public's adherence to control measures.