The tiny giant of the sea, Ostreococcus's unique adaptations.

Ostreococcus spp. are unicellular organisms with one of the simplest cellular organizations. The sequencing of the genomes of different Ostreococcus species has reinforced this status since Ostreococcus tauri has one most compact nuclear genomes among eukaryotic organisms. Despite this, it has retained a number of genes, setting it apart from other organisms with similar small genomes. Ostreococcus spp. feature a substantial number of selenocysteine-containing proteins, which, due to their higher catalytic activity compared to their selenium-lacking counterparts, may require a reduced quantity of proteins. Notably, O. tauri encodes several ammonium transporter genes, that may provide it with a competitive edge for acquiring nitrogen (N). This characteristic makes it an intriguing model for studying the efficient use of N in eukaryotes. Under conditions of low N availability, O. tauri utilizes N from abundant proteins or amino acids, such as L-arginine, similar to higher plants. However, the presence of a nitric oxide synthase (L-arg substrate) sheds light on a new metabolic pathway for L-arg in algae. The metabolic adaptations of O. tauri to day and night cycles offer valuable insights into carbon and iron metabolic configuration. O. tauri has evolved novel strategies to optimize iron uptake, lacking the classic components of the iron absorption mechanism. Overall, the cellular and genetic characteristics of Ostreococcus contribute to its evolutionary success, making it an excellent model for studying the physiological and genetic aspects of how green algae have adapted to the marine environment. Furthermore, given its potential for lipid accumulation and its marine habitat, it may represent a promising avenue for third-generation biofuels.

Presence of SARS-CoV-2 in urban effluents in south-east Buenos Aires, Argentina, May 2020 to March 2022.

Objectives: To implement and evaluate the use of wastewater sampling for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in two coastal districts of Buenos Aires Province, Argentina. Methods: In General Pueyrredon district, 400 mL of wastewater samples were taken with an automatic sampler for 24 hours, while in Pinamar district, 20 L in total (2.2 L at 20-minute intervals) were taken. Samples were collected once a week. The samples were concentrated based on flocculation using polyaluminum chloride. RNA purification and target gene amplification and detection were performed using reverse transcription polymerase chain reaction for clinical diagnosis of human nasopharyngeal swabs. Results: In both districts, the presence of SARS-CoV-2 was detected in wastewater. In General Pueyrredon, SARS-CoV-2 was detected in epidemiological week 28, 2020, which was 20 days before the start of an increase in coronavirus virus disease 2019 (COVID-19) cases in the first wave (epidemiological week 31) and 9 weeks before the maximum number of laboratory-confirmed COVID-19 cases was recorded. In Pinamar district, the virus genome was detected in epidemiological week 51, 2020 but it was not possible to carry out the sampling again until epidemiological week 4, 2022, when viral circulation was again detected. Conclusions: It was possible to detect SARS-CoV-2 virus genome in wastewater, demonstrating the usefulness of the application of wastewater epidemiology for long-term SARS-CoV-2 detection and monitoring.

Presence of SARS-CoV-2 in urban effluents in south-east Buenos Aires, Argentina, May 2020 to March 2022

[ABSTRACT]. Objectives. To implement and evaluate the use of wastewater sampling for detection of severe acute respira- tory syndrome coronavirus 2 (SARS-CoV-2) in two coastal districts of Buenos Aires Province, Argentina. Methods. In General Pueyrredon district, 400 mL of wastewater samples were taken with an automatic sam- pler for 24 hours, while in Pinamar district, 20 L in total (2.2 L at 20-minute intervals) were taken. Samples were collected once a week. The samples were concentrated based on flocculation using polyaluminum chloride. RNA purification and target gene amplification and detection were performed using reverse transcription poly- merase chain reaction for clinical diagnosis of human nasopharyngeal swabs. Results. In both districts, the presence of SARS-CoV-2 was detected in wastewater. In General Pueyrre- don, SARS-CoV-2 was detected in epidemiological week 28, 2020, which was 20 days before the start of an increase in coronavirus virus disease 2019 (COVID-19) cases in the first wave (epidemiological week 31) and 9 weeks before the maximum number of laboratory-confirmed COVID-19 cases was recorded. In Pinamar district, the virus genome was detected in epidemiological week 51, 2020 but it was not possible to carry out the sampling again until epidemiological week 4, 2022, when viral circulation was again detected. Conclusions. It was possible to detect SARS-CoV-2 virus genome in wastewater, demonstrating the useful- ness of the application of wastewater epidemiology for long-term SARS-CoV-2 detection and monitoring.

[RESUMEN]. Objetivos. Aplicar y evaluar la utilización de muestreos de aguas residuales como método para la detección del coronavirus del síndrome respiratorio agudo severo de tipo 2 (SARS-CoV-2) en dos distritos costeros de la Provincia de Buenos Aires, Argentina. Métodos. Se utilizó un dispositivo de muestreo automático para tomar muestras de 400 mL de las aguas residuales de 24 horas en el distrito de General Pueyrredon, mientras que en el distrito de Pinamar se tomaron muestras de 2,2 L a intervalos de 20 minutos hasta un volumen total de 20 L. Los muestreos se realizaron una vez por semana. Las muestras se concentraron mediante floculación con policloruro de aluminio. La purificación del ARN y la amplificación y detección del gen diana se llevaron a cabo mediante la prueba de reacción en cadena de la polimerasa con retrotranscripción para el diagnóstico clínico a partir de hisopados nasofaríngeos. Resultados. Se observó la presencia de SARS-CoV-2 en las aguas residuales de ambos distritos. En General Pueyrredon, el SARS-CoV-2 se halló en la semana epidemiológica 28 del 2020, es decir, 20 días antes del inicio del aumento de casos de enfermedad por coronavirus 2019 (COVID-19) registrado en la primera ola (semana epidemiológica 31) y nueve semanas antes de que se alcanzara el número máximo de casos de COVID-19 con confirmación de laboratorio. En el distrito de Pinamar se detectó el genoma viral en la semana epidemiológica 51 del 2020, pero solo se pudo volver a realizar el muestreo en la semana epidemiológica 4 del 2022, en la que se volvió a detectar la circulación del virus. Conclusiones. Se pudo detectar el genoma del virus SARS-CoV-2 en aguas residuales, lo que muestra la utilidad de la aplicación de la epidemiología de aguas residuales como método para la detección y el segui- miento del SARS-CoV-2 a largo plazo.

[RESUMO]. Objetivos. Implementar e avaliar o uso de amostragem de águas residuais na detecção do coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2) em dois distritos costeiros da Província de Buenos Aires, Argentina. Métodos. No distrito de General Pueyrredon, amostras de 400 mL de águas residuais foram coletadas ao longo de 24 horas com um amostrador automático; já no distrito de Pinamar, foram coletados 20 L no total (2,2 L a intervalos de 20 minutos). As amostras foram coletadas uma vez por semana e concentradas por flocu- lação com cloreto de polialumínio. A purificação do RNA e a amplificação e detecção de genes-alvo foram realizadas por meio de reação em cadeia da polimerase com transcrição reversa para diagnóstico clínico de esfregaços nasofaríngeos humanos. Resultados. Detectou-se presença de SARS-CoV-2 em águas residuais dos dois distritos. Em General Puey- rredon, o SARS-CoV-2 foi detectado na semana epidemiológica 28 de 2020, ou seja, 20 dias antes do início de um aumento no número de casos da doença provocada pelo coronavírus de 2019 (COVID-19) na primeira onda (semana epidemiológica 31) e 9 semanas antes de se registrar o número máximo de casos de COVID-19 confirmados em laboratório. No distrito de Pinamar, o genoma viral foi detectado na semana epidemiológica 51 de 2020, mas não foi possível realizar a amostragem novamente até a semana epidemiológica 4 de 2022, quando a circulação do vírus foi novamente constatada. Conclusões. Foi possível detectar o genoma do vírus SARS-CoV-2 em águas residuais, demonstrando a uti- lidade da aplicação da epidemiologia baseada em águas residuais para detectar e monitorar o SARS-CoV-2 em longo prazo.

Presence of SARS-CoV-2 in urban effluents in south-east Buenos Aires, Argentina, May 2020 to March 2022 / Presencia del SARS-CoV-2 en aguas residuales urbanas del sudeste de Buenos Aires, Argentina, de mayo del 2020 a marzo del 2022 / Presença de SARS-CoV-2 em águas residuais urbanas no sudeste de Buenos Aires, Argentina, de maio de 2020 a março de 2022

ABSTRACT Objectives. To implement and evaluate the use of wastewater sampling for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in two coastal districts of Buenos Aires Province, Argentina. Methods. In General Pueyrredon district, 400 mL of wastewater samples were taken with an automatic sampler for 24 hours, while in Pinamar district, 20 L in total (2.2 L at 20-minute intervals) were taken. Samples were collected once a week. The samples were concentrated based on flocculation using polyaluminum chloride. RNA purification and target gene amplification and detection were performed using reverse transcription polymerase chain reaction for clinical diagnosis of human nasopharyngeal swabs. Results. In both districts, the presence of SARS-CoV-2 was detected in wastewater. In General Pueyrredon, SARS-CoV-2 was detected in epidemiological week 28, 2020, which was 20 days before the start of an increase in coronavirus virus disease 2019 (COVID-19) cases in the first wave (epidemiological week 31) and 9 weeks before the maximum number of laboratory-confirmed COVID-19 cases was recorded. In Pinamar district, the virus genome was detected in epidemiological week 51, 2020 but it was not possible to carry out the sampling again until epidemiological week 4, 2022, when viral circulation was again detected. Conclusions. It was possible to detect SARS-CoV-2 virus genome in wastewater, demonstrating the usefulness of the application of wastewater epidemiology for long-term SARS-CoV-2 detection and monitoring.

RESUMEN Objetivos. Aplicar y evaluar la utilización de muestreos de aguas residuales como método para la detección del coronavirus del síndrome respiratorio agudo severo de tipo 2 (SARS-CoV-2) en dos distritos costeros de la Provincia de Buenos Aires, Argentina. Métodos. Se utilizó un dispositivo de muestreo automático para tomar muestras de 400 mL de las aguas residuales de 24 horas en el distrito de General Pueyrredon, mientras que en el distrito de Pinamar se tomaron muestras de 2,2 L a intervalos de 20 minutos hasta un volumen total de 20 L. Los muestreos se realizaron una vez por semana. Las muestras se concentraron mediante floculación con policloruro de aluminio. La purificación del ARN y la amplificación y detección del gen diana se llevaron a cabo mediante la prueba de reacción en cadena de la polimerasa con retrotranscripción para el diagnóstico clínico a partir de hisopados nasofaríngeos. Resultados. Se observó la presencia de SARS-CoV-2 en las aguas residuales de ambos distritos. En General Pueyrredon, el SARS-CoV-2 se halló en la semana epidemiológica 28 del 2020, es decir, 20 días antes del inicio del aumento de casos de enfermedad por coronavirus 2019 (COVID-19) registrado en la primera ola (semana epidemiológica 31) y nueve semanas antes de que se alcanzara el número máximo de casos de COVID-19 con confirmación de laboratorio. En el distrito de Pinamar se detectó el genoma viral en la semana epidemiológica 51 del 2020, pero solo se pudo volver a realizar el muestreo en la semana epidemiológica 4 del 2022, en la que se volvió a detectar la circulación del virus. Conclusiones. Se pudo detectar el genoma del virus SARS-CoV-2 en aguas residuales, lo que muestra la utilidad de la aplicación de la epidemiología de aguas residuales como método para la detección y el seguimiento del SARS-CoV-2 a largo plazo.

RESUMO Objetivos. Implementar e avaliar o uso de amostragem de águas residuais na detecção do coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2) em dois distritos costeiros da Província de Buenos Aires, Argentina. Métodos. No distrito de General Pueyrredon, amostras de 400 mL de águas residuais foram coletadas ao longo de 24 horas com um amostrador automático; já no distrito de Pinamar, foram coletados 20 L no total (2,2 L a intervalos de 20 minutos). As amostras foram coletadas uma vez por semana e concentradas por floculação com cloreto de polialumínio. A purificação do RNA e a amplificação e detecção de genes-alvo foram realizadas por meio de reação em cadeia da polimerase com transcrição reversa para diagnóstico clínico de esfregaços nasofaríngeos humanos. Resultados. Detectou-se presença de SARS-CoV-2 em águas residuais dos dois distritos. Em General Pueyrredon, o SARS-CoV-2 foi detectado na semana epidemiológica 28 de 2020, ou seja, 20 dias antes do início de um aumento no número de casos da doença provocada pelo coronavírus de 2019 (COVID-19) na primeira onda (semana epidemiológica 31) e 9 semanas antes de se registrar o número máximo de casos de COVID-19 confirmados em laboratório. No distrito de Pinamar, o genoma viral foi detectado na semana epidemiológica 51 de 2020, mas não foi possível realizar a amostragem novamente até a semana epidemiológica 4 de 2022, quando a circulação do vírus foi novamente constatada. Conclusões. Foi possível detectar o genoma do vírus SARS-CoV-2 em águas residuais, demonstrando a utilidade da aplicação da epidemiologia baseada em águas residuais para detectar e monitorar o SARS-CoV-2 em longo prazo.

TOR signaling in the green picoalga Ostreococcus tauri.

Target of rapamycin (TOR) is a master regulator that controls growth and metabolism by integrating external and internal signals. Although there was a great progress in the study of TOR in plants and in the model alga Chlamydomonas, scarce data are available in other green algae. Thus, in this work we studied TOR signaling in Ostreococcus tauri, the smallest free-living eukaryote described to date. This picoalga is particularly important because it has a key site at the base of the green lineage and is part of the marine phytoplankton, contributing to global photosynthesis. We investigated OtTOR complex in silico and experimentally, by using first- and second-generation TOR inhibitors, such as rapamycin and PP242. We analyzed the effect of TOR down-regulation on cell growth and on the accumulation of carbon reserves. The results showed that O. tauri responds to TOR inhibitors more similarly to plants than to Chlamydomonas, being PP242 a valuable tool to study this pathway. Besides, Ottor expression analysis revealed that the kinase is dynamically regulated under nutritional stress. Our data indicate that TOR signaling is conserved in O. tauri and we propose this alga as a good and simple model for studying TOR kinase and its regulation.

Ancient signal for nitrogen status sensing in the green lineage: Functional evidence of CDPK repertoire in Ostreococcus tauri.

Calcium-dependent protein kinases (CDPKs) regulate plant development and many stress signalling pathways through the complex cytosolic [Ca2+] signalling. The genome of Ostreococcus tauri (Ot), a model prasinophyte organism that is on the base of the green lineage, harbours three sequences homologous to those encoding plant CDPKs with the three characteristic conserved domains (protein kinase, autoregulatory/autoinhibitory, and regulatory domain). Phylogenetic and structural analyses revealed that putative OtCDPK proteins are closely related to CDPKs from other Chlorophytes. We functionally characterised the first marine picophytoeukaryote CDPK gene (OtCDPK1) and showed that the expression of the three OtCDPK genes is up-regulated by nitrogen depletion. We conclude that CDPK signalling pathway might have originated early in the green lineage and may play a key role in prasinophytes by sensing macronutrient changes in the marine environment.

Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent.

The search for a nitric oxide synthase (NOS) sequence in the plant kingdom yielded two sequences from the recently published genomes of two green algae species of the Ostreococcus genus, O. tauri and O. lucimarinus. In this study, we characterized the sequence, protein structure, phylogeny, biochemistry, and expression of NOS from O. tauri. The amino acid sequence of O. tauri NOS was found to be 45% similar to that of human NOS. Folding assignment methods showed that O. tauri NOS can fold as the human endothelial NOS isoform. Phylogenetic analysis revealed that O. tauri NOS clusters together with putative NOS sequences of a Synechoccocus sp strain and Physarum polycephalum. This cluster appears as an outgroup of NOS representatives from metazoa. Purified recombinant O. tauri NOS has a K(m) for the substrate l-Arg of 12 ± 5 µM. Escherichia coli cells expressing recombinant O. tauri NOS have increased levels of NO and cell viability. O. tauri cultures in the exponential growth phase produce 3-fold more NOS-dependent NO than do those in the stationary phase. In O. tauri, NO production increases in high intensity light irradiation and upon addition of l-Arg, suggesting a link between NOS activity and microalgal physiology.

Sucrose regulated expression of a Ca2+-dependent protein kinase (TaCDPK1) gene in excised leaves of wheat.

Sucrose (Suc) can influence the expression of a large number of genes and thereby regulates many metabolic and developmental processes. However, the Suc sensing and the components of the ensuing signaling transduction pathway leading to the regulation of gene expression are not fully understood. We have shown that protein kinases and phosphatases are involved in the Suc induced expression of fructosyltransferase (FT) genes and fructan accumulation by an hexokinase independent pathway in wheat (Triticum aestivum). In the present study, using an RT-PCR based strategy, we have cloned a calcium-dependent protein kinase (TaCDPK1) cDNA that is upregulated during Suc treatment of excised wheat leaves. The deduced amino-acid sequence of CDPK1 has high sequence similarity (>70%) to known CDPKs from both monocots and dicots. Based on sequence homology, TaCDPK1 sequence shows a variable domain preceding a catalytic domain, an autoinhibitory function domain, and a C-terminal calmodulin-domain containing 4 EF-hand calcium-binding motifs, along with a N-myristoylation motif in the N-terminal variable domain. The recombinant Escherichia coli expressed TaCDPK1 was able to phosphorylate histone III-S in a calcium dependent manner in in vitro assays. The TaCDPK1 gene expression, as determined by quantitative RT-PCR, is induced by Suc and this effect is repressed by the inhibitors of the putative components of the Suc signal transduction pathway (calcium, Ser/Thr protein kinases and protein phosphatases). We propose that TaCDPK1 is involved in the Suc induced signaling pathway in wheat leaves.