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The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

Nitrogen (N) is an essential macronutrient that impacts many aspects of plant physiology, growth, and development. Besides its nutritional role, N nutrient and metabolites act as signaling molecules that regulate the expression of a wide range of genes and biological processes. In this review, we describe recent advances in the understanding of components of the nitrate signaling pathway. Recent evidence posits that in one nitrate signaling pathway, nitrate sensed by NRT1.1 activates a phospholipase C activity that is necessary for increased cytosolic calcium levels. The nitrate-elicited calcium increase presumably activates calcium sensors, kinases, or phosphatases, resulting in changes in expression of primary nitrate response genes. Consistent with this model, nitrate treatments elicit proteome-wide changes in phosphorylation patterns in a wide range of proteins, including transporters, metabolic enzymes, kinases, phosphatases, and other regulatory proteins. Identifying and characterizing the function of the different players involved in this and other nitrate signaling pathways and their functional relationships is the next step to understand N responses in plants.

The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages.

The COVID-19 pandemic has resulted in millions of deaths globally, and while several diagnostic systems were proposed, real-time reverse transcription polymerase chain reaction (RT-PCR) remains the gold standard. However, diagnostic reagents, including enzymes used in RT-PCR, are subject to centralized production models and intellectual property restrictions, which present a challenge for less developed countries. With the aim of generating a standardized One-Step open RT-qPCR protocol to detect SARS-CoV-2 RNA in clinical samples, we purified and tested recombinant enzymes and a non-proprietary buffer. The protocol utilized M-MLV RT and Taq DNA pol enzymes to perform a Taqman probe-based assay. Synthetic RNA samples were used to validate the One-Step RT-qPCR components, demonstrating sensitivity comparable to a commercial kit routinely employed in clinical settings for patient diagnosis. Further evaluation on 40 clinical samples (20 positive and 20 negative) confirmed its comparable diagnostic accuracy. This study represents a proof of concept for an open approach to developing diagnostic kits for viral infections and diseases, which could provide a cost-effective and accessible solution for less developed countries.

Nitrógeno (N) es un macronutriente esencial para el crecimiento y desarrollo de lasplantas. Diferentes nutrientes o metabolitos de N también pueden actuar comomoléculas de señalización que regulan la expresión génica y numerosos procesosbiológicos en las plantas. En un trabajo previo realizado en nuestro laboratorio, sedemostró que la vía de señalización del nitrato dependiente de NRT1.1 induce laactividad de las proteínas fosfolipasa C (PLC) requerida para aumentar laconcentración de calcio (Ca2+) citoplasmático. Ca2+ actúa como segundo mensajeroen la señalización de nitratos en Arabidopsis thaliana.En este trabajo, demostramos que la vía NRT1.1-PLCs afecta la expresión génicade genes de respuesta a nitrato en raíces de A. thaliana. En un análisistranscriptomico y de expresión génica se identificaron genes que requieren de laactividad de PLCs para responder a los tratamientos con nitrato. Además, en estacaracterización, destaca el papel de las diferentes isoformas de las proteínas PLCsespecíficas de la raíz A. thaliana en la respuesta a nitrato. Seis de las nueveproteínas PLCs identificadas, se expresan en raíces de A. thaliana. Las distintasisoformas de las PLCs son reguladas en tratamientos con nitrato en diferentestiempos: solo PLC1, PLC4 y PLC5 fueron reguladas significativamente entratamientos con nitrato. Además, evaluamos la ubicación de las PLCs involucradasen la respuesta de nitrato, encontramos que PLC1, PLC2, PLC4 y PLC5 co-localizancon NRT1.1 en hojas de tabaco transformadas transientemente. PLC4 tiene unancestro divergente de las otras PLCs reguladas por nitrato e interesantemente,PLC4 es la única PLC que interactúa físicamente con los transportadores de nitrato:NRT1.1 y NRT2.1. Además, identificamos que la región C-terminal de NRT1.1 esesencial para la interacción física con PLC4. También, plantas sobre-expresoras y“knock-down” de PLC4 mostraron expresión diferencial en genes canónicos de lavía de asimilación de nitrato. Nuestros hallazgos sugieren un modelo en el queNRT1.1 detecta nitrato y se inicia una cascada de señalización a travez de lainteracción física con la proteína PLC4. De alguna manera, esta interacción podríamediar el aumento de Ca2+ en el citosol, que a su vez es necesario para cambiosen la expresión de genes sensibles a nitrato.

Abstract The technique RT-qPCR for viral RNA detection is the current worldwide strategy used for early detection of the novel coronavirus SARS-CoV-2. RNA extraction is a key pre-analytical step in RT-qPCR, often achieved using commercial kits. However, the magnitude of the COVID-19 pandemic is causing disruptions to the global supply chains used by many diagnostic laboratories to procure the commercial kits required for RNA extraction. Shortage in these essential reagents is even more acute in developing countries with no means to produce kits locally. We sought to find an alternative procedure to replace commercial kits using common reagents found in molecular biology laboratories. Here we report a method for RNA extraction that takes about 40 min to complete ten samples, and is not more laborious than current commercial RNA extraction kits. We demonstrate that this method can be used to process nasopharyngeal swab samples and yields RT-qPCR results comparable to those obtained with commercial kits. Most importantly, this procedure can be easily implemented in any molecular diagnostic laboratory. Frequent testing is crucial for individual patient management as well as for public health decision making in this pandemic. Implementation of this method could maintain crucial testing going despite commercial kit shortages. Competing Interest Statement The authors have declared no competing interest. Footnotes * ↵$ co-first authors