CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors

[EN] The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial CRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.This research was supported by grants BIO2017-83184-R and PID2019-108203RB-I00 from the Ministerio de Ciencia e Innovacion (Spain) through the Agencia Estatal de Investigacion (co-financed by the European Regional Development Fund) and H2020-760331 Newcotiana from the European Commission. M.U. andM.V.-V. are the recipients of fellowships FPU17/05503 from the Ministerio de Ciencia e Innovacion (Spain) and APOSTD/2020/096 from the Generalitat Valenciana (Spain), respectivelyUranga, M.; Vázquez-Vilar, M.; Orzáez Calatayud, DV.; Daròs, J. (2021). CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors. The CRISPR Journal. 4(5):1-9. https://doi.org/10.1089/crispr.2021.0049194

Radiographic and histopathological study of gastrointestinal dysmotility in lipopolysaccharide-induced sepsis in the rat

AbstractSepsis is a highly incident condition in which a cascade of proinflammatory cytokines is involved. One of its most frequent consequences is ileus, which can increase mortality. Animal models such as that induced by systemic administration of lipopolysaccharide (LPS) are useful to deeply evaluate this condition. The effects of sepsis on the gastrointestinal (GI) tract have been explored but, to our knowledge, in vivo studies showing the motor and histopathological consequences of endotoxemia in an integrated way are lacking. Our aim was to study in rats the effects of sepsis on GI motility, using radiographic methods, and to assess histological damage in several organs.MethodsMale rats were intraperitoneally injected with saline or E. coli LPS at 0.1, 1, or 5 mg kg−1. Barium sulfate was intragastrically administered, and X‐rays were performed 0–24 h afterwards. Several organs were collected for organography, histopathology, and immunohistochemistry studies.Key ResultsAll LPS doses caused gastroparesia, whereas changes in intestinal motility were dose‐and time‐dependent, with an initial phase of hypermotility followed by paralytic ileus. Lung, liver, stomach, ileum, and colon (but not spleen or kidneys) were damaged, and density of neutrophils and activated M2 macrophages and expression of cyclooxygenase 2 were increased in the colon 24 h after LPS 5 mg kg−1.Conclusions and InferencesUsing radiographic, noninvasive methods for the first time, we show that systemic LPS causes dose‐, time‐, and organ‐dependent GI motor effects. Sepsis‐induced GI dysmotility is a complex condition whose management needs to take its time‐dependent changes into account

Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector

[EN] Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.This work was supported by grants BIO2017-83184-R and PID2019-108203RB-I00 from Ministerio de Ciencia e Innovacion (Spain) through the Agencia Estatal de Investigacion (co-financed European Regional Development Fund), and H2020-760331 Newcotiana from the European Commission. M.U. and S.S. are the recipients of fellowships FPU17/05503 and BES-2017-0890098, respectively, from Ministerio de Ciencia e Innovacion (Spain)Uranga-Ruiz De Eguino, M.; Aragones, V.; Selma García, S.; Vázquez-Vilar, M.; Orzáez Calatayud, DV.; Daròs, J. (2021). Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector. The Plant Journal. 106(2):555-565. https://doi.org/10.1111/tpj.15164555565106

Allergenicity to worldwide invasive grass Cortaderia selloana as environmental risk to public health

Allergies to grass pollen affects about 20% of the population worldwide. In the last few decades, the South American grass Cortaderia selloana (CS, Pampas grass) has expanded worldwide in a variety of countries including the USA, Australia and Western Europe. In many of these locations, CS has strikingly spread and has now been classified an invasive species. Many pernicious consequences of CS have been reported for local biodiversity, landscape and structures. However, the effect on human health has not been studied. To investigate this issue, we have chosen a European region on the northern cost of Spain where CS spread is overwhelming, Cantabria. We obtained CS pollen extract and analysed the allergenic reaction of 98 patients that were allergic to pollen of local grasses. We determined the skin reaction and the presence of specific IgE antibodies (sIgE) to CS or to a typical autochthonous grass, Phleum pratense. We also compared the seasonal symptoms with reported grass pollen counts in the area. The results strongly suggest that CS can cause respiratory allergies at a similar extent to the local grasses. Given that CS pollinises later than the local grasses, this would extend the period of grass allergies in the region for about three months every year, as stated by most of the patients. This is the first study reported on the effects of the striking expansion of CS on human health. Considering the strong impact that respiratory allergies have on the population, our results suggest that CS can currently constitute a relevant environmental health issue.We thank Daniel Liebana Uranga (Cantabria, Spain) for permission to include authorship and data obtained by Marta Uranga, sadly deceased before preparation of the manuscript. We thank Thalia Burn (England, UK) for manuscript text revision and Life Stop Cortaderia (Cantabria, Spain) for image in Fig. 2B-right. This work received financial support from ALK-Abello S.A (Madrid, Spain). This article is dedicated to the memory of Marta Uranga

Prácticas y abandonos

Este artículo presenta uno de los aspectos trabajados en el Primer Informe de Avance del Proyecto de Investigación «No lo quiero tener más, lo vengo a devolver. Características de los circuitos burocráticos-administrativos en el proceso de las  adopciones tramitadas a través del Consejo Provincial del Menor en el período 1973 a 1983 bajo el régimen de la Ley 19.134». Su propósito es realizar una vinculación analítico-reflexiva entre las prácticas descriptas y registradas en loslegajos durante el período estudiado y la construcción de la ctegoría «abandono», siendo esta uno de los motivos principales para declarar el estado de adoptabilidad del menor. El acoplamiento de lo moral a lo material permite observar la trama por la que los circuitos burocráticos administrativos se advienen normalizantes y renovar las posibles razones que dan origen al título este Proyecto: «No lo quiero tener más, lo vengo a devolver[…]»

Population-based colorectal cancer screening programmes using a faecal immunochemical test:Should faecal haemoglobin cut-offs differ by age and sex?

Abstract Background The Basque Colorectal Cancer Screening Programme has both high participation rate and high compliance rate of colonoscopy after a positive faecal occult blood test (FIT). Although, colorectal cancer (CRC) screening with biannual (FIT) has shown to reduce CRC mortality, the ultimate effectiveness of the screening programmes depends on the accuracy of FIT and post-FIT colonoscopy, and thus, harms related to false results might not be underestimated. Current CRC screening programmes use a single faecal haemoglobin concentration (f-Hb) cut-off for colonoscopy referral for both sexes and all ages. We aimed to determine optimum f-Hb cut-offs by sex and age without compromising neoplasia detection and interval cancer proportion. Methods Prospective cohort study using a single-sample faecal immunochemical test (FIT) on 444,582 invited average-risk subjects aged 50–69 years. A result was considered positive at ≥20 μg Hb/g faeces. Outcome measures were analysed by sex and age for a wide range of f-Hb cut-offs. Results We analysed 17,387 positive participants in the programme who underwent colonoscopy. Participation rate was 66.5%. Men had a positivity rate for f-Hb of 8.3% and women 4.8% (p < 0.0001). The detection rate for advanced neoplasia (cancer plus advanced adenoma) was 44.0‰ for men and 15.9‰ for women (p < 0.0001). The number of colonoscopies required decreased in both sexes and all age groups through increasing the f-Hb cut-off. However, the loss in CRC detection increased by up to 28.1% in men and 22.9% in women. CRC missed were generally at early stages (Stage I-II: from 70.2% in men to 66.3% in women). Conclusions This study provides detailed outcomes in men and women of different ages at a range of f-Hb cut-offs. We found differences in positivity rates, neoplasia detection rate, number needed to screen, and interval cancers in men and women and in younger and older groups. However, there are factors other than sex and age to consider when consideration is given to setting the f-Hb cut-off

Aprendizajes y prácticas educativas en las actuales condiciones de época: COVID-19

“Esta obra colectiva es el resultado de una convocatoria a docentes, investigadores y profesionales del campo pedagógico a visibilizar procesos investigativos y prácticas educativas situadas en el marco de COVI-19. La misma se inscribe en el trabajo llevado a cabo por el equipo de Investigación responsable del Proyecto “Sentidos y significados acerca de aprender en las actuales condiciones de época: un estudio con docentes y estudiantes de la educación secundarias en la ciudad de Córdoba” de la Facultad de Filosofía y Humanidades. Universidad Nacional de Córdoba. El momento excepcional que estamos atravesando, pero que también nos atraviesa, ha modificado la percepción temporal a punto tal que habitamos un tiempo acelerado y angustiante que nos exige la producción de conocimiento provisorio. La presente publicación surge como un espacio para detenernos a documentar lo que nos acontece y, a su vez, como oportunidad para atesorar y resguardar las experiencias educativas que hemos construido, inventado y reinventando en este contexto. En ella encontrarán pluralidad de voces acerca de enseñar y aprender durante la pandemia. Este texto es una pausa para reflexionar sobre el hacer y las prácticas educativas por venir”.Fil: Beltramino, Lucia (comp.). Universidad Nacional de Córdoba. Facultad de Filosofía y Humanidades. Escuela de Archivología; Argentina

Ameliorative effects of egg white hydrolysate on recognition memory impairments associated with chronic exposure to low mercury concentration

et al.The study aimed to investigate whether the Egg White Hydrolysate (EWH) is able to prevent the recognition memory disorders associated with long-term Hg exposure in rats. For this, male Wistar rats were treated for 60 days with: a) Untreated: saline solution (i.m.); b) Hydrolysate: EWH (1 g/kg/day, gavage); c) Mercury: HgCl (1st dose 4.6 μg/kg, subsequent doses 0.07 μg/kg/day, i.m.); d) Hydrolysate-Mercury. Object recognition memory test was performed to verify Short (STM) and Long-Term Memory (LTM) and Open Field, Plus Maze and Tail Flick tests were performed as control for behavioural experiments. Reactive Oxygen Species (ROS) in the hippocampus were determined by the dichlorofluorescein diacetate (DCFH-DA) method, malondialdehyde (MDA) levels by TBARS, antioxidant power by FRAP assay and total Hg concentration by atomic fluorescence spectrometry. We confirm that the STM and LTM were impaired in adult rats exposed to Hg at low concentrations, which may be related to the increased metal deposition, ROS production and subsequent oxidative damage in the hippocampus. In addition, we demonstrated for the first time that EWH treatment is able to prevent memory impairment induced by Hg exposure, reducing Hg content and ROS production in the hippocampus. In conclusion, EWH prevents memory impairments induced by chronic exposure to low doses of Hg. These findings may represent a good public health strategy since they indicate that EWH is a promising candidate as a new natural therapy for heavy metal intoxication.This research was supported by the Brazilian Government (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq – 203440/2014-5) and the Spanish Government (MINECO – AGL2012-32387; CSIC – Intramural 201570I028).Peer Reviewe