An Open One-Step RT-qPCR for SARS-CoV-2 detection.

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.

Insights into molecular links and transcription networks integrating drought stress and nitrogen signaling.

Drought and the availability of nitrate, the predominant source of nitrogen (N) in agriculture, are major factors limiting plant growth and crop productivity. The dissection of the transcriptional networks' components integrating droght stress and nitrate responses provides valuable insights into how plants effectively balance stress response with growth programs. Recent evidence in Arabidopsis thaliana indicates that transcription factors (TFs) involved in abscisic acid (ABA) signaling affect N metabolism and nitrate responses, and reciprocally, components of nitrate signaling might affect ABA and drought gene responses. Advances in understanding regulatory circuits of nitrate and drought crosstalk in plant tissues empower targeted genetic modifications to enhance plant development and stress resistance, critical traits for optimizing crop yield and promoting sustainable agriculture.

An Open One-Step RT-qPCR for SARS-CoV-2 detection.

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, and the kit showed comparable sensitivity to approved commercial kits. The One-Step RT-qPCR was then tested on clinical samples and demonstrated similar performance to commercial kits in terms of positive and negative calls. 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.

A simple RNA preparation method for SARS-CoV-2 detection by RT-qPCR.

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.

Functional characterisation and in silico modelling of MdPSY2 variants and MdPSY5 phytoene synthases from Malus domestica.

Carotenoids are plastid isoprenoid pigments that play critical roles in light harvesting, photoprotection, and phytohormone biosynthesis. They are also vitamin-A precursors and antioxidant molecules important for human nutrition. Apples (e.g. Malus x domestica Borkh), one of the most widely consumed fruits with high nutrient levels, have a very low carotenoid concentration in flesh, compared with other fruits and vegetables. This could be explained by a deficiency in carotenoid synthesis/accumulation and/or accelerated degradation. We analysed the contribution of M. domestica cv. 'Fuji' phytoene synthase (PSY) in the biosynthesis of carotenoids and determined that among four MdPSY genes present in the organism, MdPSY2 and MdPSY5 are highly expressed in leaves and during fruit ripening in line with an increment in carotenoid content in fruits. Furthermore, two representative polymorphic MdPSY2 variants were found, one with a Tyr358Phe substitution (MdPSY2_F) and the other that additionally has a six-amino-acid deletion in the signal peptide (MdPSY2_CG). MdPSY2, MdPSY5, MdPSY2_F and MdPSY2_CG are all localised in plastids. Interestingly, the polymorphic MdPSY2_F and MdPSY2_CG variants show lower enzymatic activity than the wild-type form in a heterologous complementation assay, which could be attributed to the Tyr358Phe substitution close to the active-site pocket, as was suggested by 3-D modelling analysis. The presence of polymorphic MdPSY2 variants with lower enzymatic activity could be partially responsible for the low carotenoid content in Fuji apple fruits.

Universal loop assembly: open, efficient and cross-kingdom DNA fabrication.

Standardized type IIS DNA assembly methods are becoming essential for biological engineering and research. These methods are becoming widespread and more accessible due to the proposition of a 'common syntax' that enables higher interoperability between DNA libraries. Currently, Golden Gate (GG)-based assembly systems, originally implemented in host-specific vectors, are being made compatible with multiple organisms. We have recently developed the GG-based Loop assembly system for plants, which uses a small library and an intuitive strategy for hierarchical fabrication of large DNA constructs (>30 kb). Here, we describe 'universal Loop' (uLoop) assembly, a system based on Loop assembly for use in potentially any organism of choice. This design permits the use of a compact number of plasmids (two sets of four odd and even vectors), which are utilized repeatedly in alternating steps. The elements required for transformation/maintenance in target organisms are also assembled as standardized parts, enabling customization of host-specific plasmids. Decoupling of the Loop assembly logic from the host-specific propagation elements enables universal DNA assembly that retains high efficiency regardless of the final host. As a proof-of-concept, we show the engineering of multigene expression vectors in diatoms, yeast, plants and bacteria. These resources are available through the OpenMTA for unrestricted sharing and open access.

Loop assembly: a simple and open system for recursive fabrication of DNA circuits.

High-efficiency methods for DNA assembly have enabled the routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customization, elaborate vector sets or serial manipulations for the different stages of assembly. We have developed Loop assembly based on a recursive approach to DNA fabrication. The system makes use of two Type IIS restriction endonucleases and corresponding vector sets for efficient and parallel assembly of large DNA circuits. Standardized level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly enables efficient and versatile DNA fabrication for plant transformation. We show the construction of plasmids up to 16 genes and 38 kb with high efficiency (> 80%). We have characterized Loop assembly on over 200 different DNA constructs and validated the fidelity of the method by high-throughput Illumina plasmid sequencing. Our method provides a simple generalized solution for DNA construction with standardized parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

Carotenoid Biosynthesis in Daucus carota.

Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main source of dietary provitamin A. Contrary to other plants, almost all carrot varieties accumulate massive amounts of carotenoids in the root, resulting in a wide variety of colors, including those with purple, yellow, white, red and orange roots. During the first weeks of development the root, grown in darkness, is thin and pale and devoid of carotenoids. At the second month, the thickening of the root and the accumulation of carotenoids begins, and it reaches its highest level at 3 months of development. This normal root thickening and carotenoid accumulation can be completely altered when roots are grown in light, in which chromoplasts differentiation is redirected to chloroplasts development in accordance with an altered carotenoid profile. Here we discuss the current evidence on the biosynthesis of carotenoid in carrot roots in response to environmental cues that has contributed to our understanding of the mechanism that regulates the accumulation of carotenoids, as well as the carotenogenic gene expression and root development in D. carota.