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Gene editing technologies have revolutionized plant molecular biology, providing powerful tools for precise gene manipulation for understanding function and enhancing or modifying agronomically relevant traits. Among these technologies, the CRISPR-Cas9 system has emerged as a versatile and widely accepted strategy for targeted gene manipulation. This protocol provides detailed, step-by-step instructions for implementing CRISPR-Cas9 genome editing in tomato plants, with a specific focus in generating knockout lines for a target gene. For that, the guide RNA should preferentially be designed within the first exon downstream and closer to the start codon. The edited plants obtained are free of transgene cassette for expression of the CRISPR-Cas9 machinery.

Abstract Microorganisms present in mosquitoes and their interactions are key factors affecting insect development. Among them, Wolbachia is closely associated with the host and affects several fitness parameters. In this study, the bacterial and fungal microbiota from two laboratory Culex quinquefasciatus isolines (wild type and tetracycline-cured) were characterized by metagenome amplicon sequencing of the ITS2 and 16S rRNA genes at different developmental stages and feeding conditions. We identified 572 bacterial and 61 fungal OTUs. Both isolines presented variable bacterial communities and different trends in the distribution of diversity among the groups. The lowest bacterial richness was detected in sugar-fed adults of the cured isoline, whereas fungal richness was highly reduced in blood-fed mosquitoes. Beta diversity analysis indicated that isolines are an important factor in the differentiation of mosquito bacterial communities. Considering composition, Penicillium was the dominant fungal genus, whereas Wolbachia dominance was inversely related to that of Enterobacteria (mainly Thorsellia and Serratia). This study provides a more complete overview of the mosquito microbiome, emphasizing specific highly abundant components that should be considered in microorganism manipulation approaches to control vector-borne diseases.

Soybean’s protein concentration and amino acid profile vary geographically due to genotype and environment. We used 1817 on-farm grain samples from the United States, Brazil, and the Southern Cone to assess soybean protein variability in the Western Hemisphere, which accounts for 85% of global production. Our analysis found consistent spatial patterns for protein concentration and amino acid composition, with differences in protein concentration of up to 5.4% among regions. While there was a negative correlation between protein concentration and the abundance of critical amino acids (lysine, cysteine, threonine, methionine, and tryptophane), their amount per ton of grain increased with protein concentration. Our findings can guide market and industry in benchmarking soybean protein quality across the Western Hemisphere, which according to our projection could supply close to 120 million metric tons of protein and 17 million metric tons of the most critical amino acids annually by 2030.

Advances in sensors have revolutionized the biomedical engineering field, having an extreme affinity for specific analytes also providing an effective, real-time, point-of-care testing for an accurate diagnosis. Quartz Crystal Microbalance (QCM) is a well-established sensor that has been successfully applied in a broad range of applications to monitor and explore various surface interactions, in situ thin-film formations, and layer properties. This technology has gained interest in biomedical applications since novel QCM systems are able to work in liquid media. QCM with dissipation monitoring (QCM-D) is an expanded version of a QCM that measures changes in damping properties of adsorbed layers thus providing information on its viscoelastic nature. In this article, an open source and low cost QCM-D prototype for biomedical applications was developed. In addition, the system was validated using different Polyethylene Glycol (PEG) concentrations due to its importance for many medical applications. The statistics show a bigger dissipation of the system as the fluid becomes more viscous, also having a very acceptable sensibility when temperature is controlled.

Baculoviruses are viral pathogens that infect different species of Lepidoptera, Diptera, and Hymenoptera, with a global distribution. Due to their biological characteristics and the biotechnological applications derived from these entities, the Baculoviridae family is an important subject of study and manipulation in the natural sciences. With the advent of RNA interference mechanisms, the presence of baculoviral genes that do not code for proteins but instead generate transcripts similar to microRNAs (miRNAs) has been described. These miRNAs are functionally associated with the regulation of gene expression, both in viral and host sequences. This article provides a comprehensive review of miRNA biogenesis, function, and characterization in general, with a specific focus on those identified in baculoviruses. Furthermore, it delves into the specific roles of baculoviral miRNAs in regulating viral and host genes and presents structural and thermodynamic stability studies that are useful for detecting shared characteristics with predictive utility. This review aims to expand our understanding of the baculoviral miRNAome, contributing to improvements in the production of baculovirus-based biopesticides, management of resistance phenomena in pests, enhancement of recombinant protein production systems, and development of diverse and improved BacMam vectors to meet biomedical demands.

Microorganisms associated with mosquitoes have fundamental roles, not only in their nutrition, but also in physiological and immunological processes, and in their adaptation to the environment as well. Studies on mosquito hologenomes have increased significantly during the last years, achieving important advances in the characterization of the “core bacteriome” of some species of health importance. However, the fungal mycobiome has not been exhaustively researched, especially throughout the life cycle of some hematophagous mosquito species. In this work, the diversity and composition of fungal communities in different developmental stages, sexes, and adult nutrition of Culex quinquefasciatus reared on laboratory conditions were characterized, using internal transcribed spacer high throughput amplicon sequencing. Larvae presented a higher fungal richness, while sucrose-fed males and females showed a similar diversity between them. Blood-fed females presented few operational taxonomic units with an even distribution. Results are consistent with the reduction of larval microbiota after molting, observed for the bacterial microbiome in other mosquito species. The filamentous Ascomycota Penicillium polonicum and Cladosporium sp. were present in all stages of the mosquitoes; in addition, the presence of yeasts in the insects or their subsequent colonization associated with their diet is also discussed. These results suggest that some species of fungi could be essential for the nutrition and development of mosquitoes throughout their life cycle.

Abstract Sunflower Verticillium Wilt and Leaf Mottle (SVW), caused by Verticillium dahliae (Kleb.; Vd), is a soil-borne disease affecting sunflower worldwide. A single dominant locus, known as V1, was formerly effective in controlling North-American Vd races, whereas races from Argentina, Europe and an emerging race from USA overcome its resistance. This emphasizes the need for identifying broad-spectrum genetic resistance (BSR) sources. Here we characterize two sunflower mapping populations (MPs) for SVW resistance: a biparental MP and the association MP from the National Institute of Agricultural Technology (INTA), under field growing conditions. Nine field-trials (FTs) were conducted in highly infested fields in the most SVW-affected region of Argentina. Several disease descriptors (DDs), including incidence and severity, were scored across four phenological stages. Generalized linear models were fitted according to the nature of each variable, adjusting mean phenotypes for inbred lines across and within FTs. Comparison of these responses allowed the identification of novel BSR sources. Furthermore, we present the first report of SVW resistance heritability, with estimates ranging from 35 to 45% for DDs related to disease incidence and severity, respectively. This study constitutes the largest SVW resistance characterization reported to date in sunflower, identifying valuable genetic resources for BSR-breeding to cope with a pathogen of increasing importance worldwide.

Baculoviruses are entomopathogens that carry large, double-stranded circular DNA genomes and infect insect larvae of Lepidoptera, Hymenoptera and Diptera, with applications in the biological control of agricultural pests, in the production of recombinant proteins and as viral vectors for various purposes in mammals. These viruses have a variable genetic composition that differs between species, with some sequences shared by all known members, and others that are lineage-specific or unique to isolates. Based on the analysis of nearly 300 sequenced genomes, a thorough bioinformatic investigation was conducted on all the baculoviral protein coding sequences, characterizing their orthology and phylogeny. This analysis confirmed the 38 protein coding sequences currently considered as core genes, while also identifying novel coding sequences as candidates to join this set. Accordingly, homology was found among all the major occlusion body proteins, thus proposing that the polyhedrin, granulin and CUN085 genes be considered as the 39th core gene of Baculoviridae.

Tear viscosity is a critical property affecting tear distribution and ocular surface stability. While not widely established as a primary diagnostic marker, deviations from normal viscosity can impact ocular health, potentially contributing to conditions such as dry eye syndrome. Despite their importance, traditional viscometers require sample volumes that are not feasible to use with tear volume. This research introduces a novel Quartz Crystal Microbalance (QCM)-based method for tear viscosity measurement, offering a viscometer prototype that operates with minimal sample volumes. Human tear samples, solutions used in artificial eye drops, and various commercial eye drop brands were evaluated. Results show that the QCM method aligns with established viscosity ranges. The average viscosity of healthy human tears was found to be 1.73 ± 0.61 cP, aligning with the typical range of 1–10 cP. Variability in the viscosities of eye drop can be attributed to differences in their chemical compositions. The QCM method offers benefits such as reduced sample consumption and rapid results, enhancing understanding of tear dynamics for ocular health. Further research with larger sample sizes is needed to establish normative viscosity values in healthy individuals and those with dry eye syndrome, which is crucial for validating the device's clinical efficacy. [ABSTRACT FROM AUTHOR]

The leafcutter ants (LCA) are considered plague in a great part of the American continent, causing great damage in production fields. Knowing the locomotion and foraging rhythm in LCA on a continuous basis would imply a significant advance for ecological studies, fundamentally of animal behavior. However, studying the forage rhythm of LCA in the field involves a significant human effort. This also adds a risk of subjective results due to the operator fatigue. In this work a new development named ‘AntVideoRecord’ is proposed to address this issue. This device is a low-cost autonomous system that records videos of the LCA path in a fixed position. The device can be easily reproduced using the freely accessible source code provided. The evaluation of this novel device was successful because it has exceeded all the basic requirements in the field: record continuously for at least seven days, withstand high and low temperatures, capture acceptable videos during the day and night, and have a simple configuration protocol by mobile devices and laptops. It was possible to confirm the correct operation of the device, being able to record more than 1900 h in the field at different climate conditions and times of the day.

Muster of men and equipment sent as reinforcements to Juan de Oñate.

The involvement of natriuretic peptides was studied during the hypertrophic remodeling transition mediated by sequential exposure to chronic hemodynamic overload. We induced hypertension in rats by pressure (renovascular) or volume overload (DOCA-salt) during 6 and 12 weeks of treatment. We also studied the consecutive combination of both models in inverse sequences: RV 6 weeks/DS 6 weeks and DS 6 weeks/RV 6 weeks. All treated groups developed hypertension. Cardiac hypertrophy and left ventricular ANP gene expression were more pronounced in single DS than in single RV groups. BNP gene expression was positively correlated with left ventricular hypertrophy only in RV groups, while ANP gene expression was positively correlated with left ventricular hypertrophy only in DS groups. Combined models exhibited intermediate values between those of single groups at 6 and 12 weeks. The latter stimulus associated to the second applied overload is less effective than the former to trigger cardiac hypertrophy and to increase ANP and BNP gene expression. In addition, we suggest a correlation of ANP synthesis with volume overload and of BNP synthesis with pressure overload-induced hypertrophy after a prolonged treatment. Volume and pressure overload may be two mechanisms, among others, involved in the differential regulation of ANP and BNP gene expression in hypertrophied left ventricles. Plasma ANP levels reflect a response to plasma volume increase and volume overload, while circulating BNP levels seem to be regulated by cardiac BNP synthesis and ventricular hypertrophy.

Background and Objectives. The aim of this study is to compare clinical and epidemiological characteristics and outcomes in patients with versus without nosocomial COVID-19 after exposure to SARS-CoV-2 and to analyze the risk factors for severe outcomes of COVID-19 in a long-term hospital in Spain. Materials and methods. This retrospective, single-center observational study included all inpatients in a long-term hospital during a COVID-19 outbreak from 21 January to 15 March 2021. Results. Of 108 admitted patients, 65 (60.2%) were diagnosed with nosocomial COVID-19 disease (n = 34 women (52.3%), median age 77 years). In the univariable analysis, risk factors associated with nosocomial COVID-19 were dementia (OR 4.98 95% CI 1.58–15.75), dyspnea (OR 5.34 95% CI 1.69–16.82), asthenia (OR 5.10, 95% CI 1.40–18.60) and NECesidades PALiativas (NECPAL) (OR 1.28 95% CI 1.10–1.48). In the multivariable analysis, risk factors independently associated with nosocomial COVID-19 infection were dyspnea (aOR 7.39; 95% CI 1.27–43.11) and NECPAL (aOR 1.25; 95% CI 1.03–1.52). Of the 65 patients diagnosed with nosocomial COVID-19, 29 (44.6%) died, compared to 7/43 (16.2%) non-infected patients (OR 4.14, 95% CI 1.61–10.67). Factors associated with mortality in nosocomial COVID-19 were confusion (aOR 3.83; 95% CI 1.03–14.27) and dyspnea (aOR 7.47; 95% CI 1.87–29.82). The NECPAL tool played an important predictive role in both nosocomial COVID-19 infection and mortality (aOR 1.19, 95% CI: 1.00–1.41). Conclusions. In a long-term hospital, nosocomial COVID-19 main clinical characteristics associated with infection were dyspnea and NECPAL. Mortality was higher in the group with nosocomial COVID-19; risk factors were confusion and dyspnea. The NECPAL tool may help to predict progression and death in COVID-19.

Nitric oxide (NO) is a second messenger that regulates a broad range of physiological processes in plants. NO-derived molecules called reactive nitrogen species (RNS) can react with unsaturated fatty acids generating nitrated fatty acids (NO2-FA). NO2-FA work as signaling molecules in mammals where production and targets have been described under different stress conditions. Recently, NO2-FAs were detected in plants, however their role(s) on plant physiological processes is still poorly known. Although in this work NO2-OA has not been detected in any Arabidopsis seedling tissue, here we show that exogenous application of nitro-oleic acid (NO2-OA) inhibits Arabidopsis primary root growth; this inhibition is not likely due to nitric oxide (NO) production or impaired auxin or cytokinin root responses. Deep analyses showed that roots incubated with NO2-OA had a lower cell number in the division area. Although this NO2-FA did not affect the hormonal signaling mechanisms maintaining the stem cell niche, plants incubated with NO2-OA showed a reduction of cell division in the meristematic area. Therefore, this work shows that the exogenous application of NO2-OA inhibits mitotic processes subsequently reducing primary root growth.

This paper examines the two lines of analysis that are generally pursued when dealing with discourse phenomena in the generative tradition: syntactico-centric and interface-based approaches. Syntactico-centric analyses are criticized because they need construction-specific mechanisms, while interface-based analyses sometimes challenge standard assumptions about the architecture of grammar. The discussion is mainly theoretical, but three case studies serve as exemplification: focalization, ellipsis and parentheticals. The second part of the paper is focused on parentheticals; a brief proposal is presented regarding the distinction between free and anchored parentheticals from a syntax-phonology interface perspective. The general conclusion is that following an interface-based perspective to approach discourse phenomena can help us gain new insights about the nature of the interfaces and their role in grammar.

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.

Spodoptera ornithogalli (Guenée) (Lepidoptera: Noctuidae) is an important pest in different crops of economic relevance in America. For its control, strategies that include chemicals are usually used; so, the description of entomopathogens would be very useful for the formulation of biopesticides. In this regard, two different baculoviruses affecting S. ornithogalli were isolated in Colombia, with one of them being an NPV and the other a GV. Ultrastructural, molecular, and biological characterization showed that both isolates possess the 38 core genes and are novel species in Baculoviridae, named as Spodoptera ornithogalli nucleopolyhedrovirus (SporNPV) and Spodoptera ornithogalli granulovirus (SporGV). The bioassays carried out in larvae of S. ornithogalli and S. frugiperda showed infectivity in both hosts but being higher in the first. In addition, it was observed that SporGV potentiates the insecticidal action of SporNPV (maximum value in ratio 2.5:97.5). Both viruses are individually infective but coexist in nature, producing mixed infections with a synergistic effect that improves the performance of the NPV and enables the transmission of the GV, which presents a slowly killing phenotype.

Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numerous biotechnological applications, such as the biological control of agricultural pests, the expression of recombinant proteins or the gene delivery of therapeutic sequences in mammals, among others. Their genomes encode between 80 and 200 proteins, of which 38 are shared by all reported species. Thanks to multi-omic studies, there is remarkable information about the baculoviral proteome and the temporality in the virus gene expression. This allows some functional elements of the genome to be very well described, such as promoters and open reading frames. However, less information is available about the transcription termination signals and, consequently, there are still imprecisions about what are the limits of the transcriptional units present in the baculovirus genomes and how is the processing of the 3′ end of viral mRNA. Regarding to this, in this review we provide an update about the characteristics of DNA signals involved in this process and we contribute to their correct prediction through an exhaustive analysis that involves bibliography information, data mining, RNA structure and a comprehensive study of the core gene 3′ ends from 180 baculovirus genomes.