Why is oral-induced RNAi inefficient in Diatraea saccharalis? A possible role for DsREase and other nucleases.

The efficiency of RNAi technology in insects varies considerably, particularly in lepidopterans. An important limiting factor of RNAi-mediated gene silencing is the degradation of dsRNA by insect nucleases before cellular uptake. To date, few studies have reported effective gene knockdown in the sugarcane borer Diatraea saccharalis. However, yielding contradictory results when using oral delivery. Further, the RNAi efficiency in D. saccharalis and presumed activity of gut nucleases remain poorly understood. Therefore, we investigated whether gene silencing was feasible via dsRNA feeding in D. saccharalis. Two different genes were tested, juvenile hormone esterase (DsJHE) and chitin synthase 1 (DsCHS1). Discrete knockdown was verified only for DsCHS1 with high dsRNA dosages and long exposure times. Neither mortality nor abnormal phenotypes were observed after treatment with any tested dsRNA. It was also verified that dsRNAs were quickly degraded when incubated with gut juice. Furthermore, we identified four possible nucleases that could reduce the knockdown efficiency in D. saccharalis. Three of them had the endonuclease_NS domain (DsNucleases), and one had the PIN domain (DsREase), with REase-like genes being scarcely represented in databanks. We further remark that DsNuclease1 and DsREase are highly expressed in the larval gut, and DsREase was upregulated as insects were fed with artificial diet (without dsRNA), and also when injected with dsRNA. Conversely, no nuclease was triggered when insects were fed with a sucrose droplet containing dsRNA. Thus, our findings suggest that nuclease activity within the gut is one of the possible reasons for the inefficiency of RNAi in D. saccharalis. Our data may shed light on the challenges to overcome when introducing RNAi as a strategy for controlling lepidopteran pests.

Transcriptome Analysis and Knockdown of the Juvenile Hormone Esterase Gene Reveal Abnormal Feeding Behavior in the Sugarcane Giant Borer.

The sugarcane giant borer (SGB), Telchin licus licus, is a pest that has strong economic relevance for sugarcane producers. Due to the endophytic behavior of the larva, current methods of management are inefficient. A promising biotechnological management option has been proposed based on RNA interference (RNAi), a process that uses molecules of double-stranded RNA (dsRNA) to specifically knock down essential genes and reduce insect survival. The selection of suitable target genes is often supported by omic sciences. Studies have shown that genes related to feeding adaptation processes are good candidates to be targeted by RNAi for pest management. Among those genes, esterases are highlighted because of their impact on insect development. In this study, the objective was to evaluate the transcriptome responses of the SGB's gut in order to provide curated data of genes that could be used for pest management by RNAi in future studies. Further, we validated the function of an esterase-coding gene and its potential as a target for RNAi-based control. We sequenced the gut transcriptome of SGB larvae by Illumina HiSeq and evaluated its gene expression profiles in response to different diets (sugarcane stalk and artificial diet). We obtained differentially expressed genes (DEGs) involved in detoxification, digestion, and transport, which suggest a generalist mechanism of adaptation in SGB larvae. Among the DEGs, was identified and characterized a candidate juvenile hormone esterase gene (Tljhe). We knocked down the Tljhe gene by oral delivery of dsRNA molecules and evaluated gene expression in the gut. The survival and nutritional parameters of the larvae were measured along the developmental cycle of treated insects. We found that the gene Tljhe acts as a regulator of feeding behavior. The knockdown of Tljhe triggered a forced starvation state in late larval instars that significantly reduced the fitness of the larvae. However, the mechanism of action of this gene remains unclear, and the correlation between the expression of Tljhe and the levels of juvenile hormone (JH) metabolites in the hemolymph of the SGB must be assessed in future research.

Transcriptional modulation of AREB-1 by CRISPRa improves plant physiological performance under severe water deficit.

Plants are sessile organisms, which are vulnerable to environmental stresses. As such, plants have developed multiple molecular, physiological, and cellular mechanisms to cope with natural stressors. However, these environmental adversities, including drought, are sources of the main agribusiness problems since they interfere with plant growth and productivity. Particularly under water deprivation conditions, the abscisic acid-responsive element-binding protein AREB1/ABF2 plays an important role in drought stress response and physiological adaptation. In this investigation, we provide substantial confirmation for the role of AREB1/ABF2 in plant survival under severe water deficit using the CRISPR activation (CRISPRa) technique to enhance the AREB1 gene expression. In our strategy, the inactive nuclease dCas9 was fused with an Arabidopsis histone acetyltransferase 1, which improves gene expression by remodeling chromatin. The AREB1 overexpression promotes an improvement in the physiological performance of the transgenic homozygous plants under drought, which was associated with an increase in chlorophyll content, antioxidant enzyme activity, and soluble sugar accumulation, leading to lower reactive oxygen species accumulation. Finally, we found that the CRISPR-mediated up-regulation of AREB1 changes the abundance of several downstream ABA-inducible genes, allowing us to report that CRISPRa dCas9-HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.

Comparative gut transcriptome analysis of Diatraea saccharalis in response to the dietary source.

The sugarcane borer (Diatraea saccharalis, Fabricius, 1794) is a devastating pest that causes millions of dollars of losses each year to sugarcane producers by reducing sugar and ethanol yields. The control of this pest is difficult due to its endophytic behavior and rapid development. Pest management through biotechnological approaches has emerged in recent years as an alternative to currently applied methods. Genetic information about the target pests is often required to perform biotechnology-based management. The genomic and transcriptomic data for D. saccharalis are very limited. Herein, we report a tissue-specific transcriptome of D. saccharalis larvae and a differential expression analysis highlighting the physiological characteristics of this pest in response to two different diets: sugarcane and an artificial diet. Sequencing was performed on the Illumina HiSeq 2000 platform, and a de novo assembly was generated. A total of 27,626 protein-coding unigenes were identified, among which 1,934 sequences were differentially expressed between treatments. Processes such as defence, digestion, detoxification, signaling, and transport were highly represented among the differentially expressed genes (DEGs). Furthermore, seven aminopeptidase genes were identified as candidates to encode receptors of Cry proteins, which are toxins of Bacillus thuringiensis used to control lepidopteran pests. Since plant-insect interactions have produced a considerable number of adaptive responses in hosts and herbivorous insects, the success of phytophagous insects relies on their ability to overcome challenges such as the response to plant defences and the intake of nutrients. In this study, we identified metabolic pathways and specific genes involved in these processes. Thus, our data strongly contribute to the knowledge advancement of insect transcripts, which can be a source of target genes for pest management.

Vitamin D and other environmental risk factors in Colombian patients with multiple sclerosis.

OBJECTIVE: The aim of this study was to explore the association between MS and vitamin D levels, as well as Epstein-Barr virus (EBV) seropositivity and smoking history in a Colombian population. METHODS: We conducted a cross-sectional study between 2017 and 2018. We measured vitamin D levels and EBV antibody titers and administered a questionnaire to assess dietary habits, smoking, second-hand smoking and duration of smoking, sunlight exposure, physical activity, and personal and family history in individuals with and without multiple sclerosis during adolescence. A multivariable logistic regression model was then performed to explore the association between vitamin D status and MS. RESULTS: A total of 87 individuals with MS (mean age 40.9 years; 65.52% females) and 87 without MS (mean age 55 years; 65.52% females) were included in the analysis. In the multivariable analysis, after controlling for supplementation vitamin D levels did not differ between both groups and no difference was found regarding tobacco smoke exposure. The proportion of individuals who tested positive for anti-EBV nuclear antigen was significantly higher in individuals with MS (95.4% vs 82.76%, p = 0.028) CONCLUSION: : We did not find a statistically significant association between MS and vitamin D levels while anti-EBV nuclear antigen titers behaved as previously described in the literature. This study provides new evidence of the association between MS and different risk factors in our country, reinforcing the hypothesis that the pathogenesis of MS is multifactorial. Further studies are needed to better define the association between environmental factors and the development of MS in low prevalence areas.

Transcriptome and gene expression analysis of three developmental stages of the coffee berry borer, Hypothenemus hampei.

Coffee production is a global industry valued at approximately 173 billion US dollars. One of the main challenges facing coffee production is the management of the coffee berry borer (CBB), Hypothenemus hampei, which is considered the primary arthropod pest of coffee worldwide. Current control strategies are inefficient for CBB management. Although biotechnological alternatives, including RNA interference (RNAi), have been proposed in recent years to control insect pests, characterizing the genetics of the target pest is essential for the successful application of these emerging technologies. In this study, we employed RNA-seq to obtain the transcriptome of three developmental stages of the CBB (larva, female and male) to increase our understanding of the CBB life cycle in relation to molecular features. The CBB transcriptome was sequenced using Illumina Hiseq and assembled de novo. Differential gene expression analysis was performed across the developmental stages. The final assembly produced 29,434 unigenes, of which 4,664 transcripts were differentially expressed. Genes linked to crucial physiological functions, such as digestion and detoxification, were determined to be tightly regulated between the reproductive and nonreproductive stages of CBB. The data obtained in this study help to elucidate the critical roles that several genes play as regulatory elements in CBB development.

Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a Histone AcetylTransferase.

Drought episodes decrease plant growth and productivity, which in turn cause high economic losses. Plants naturally sense and respond to water stress by activating specific signalling pathways leading to physiological and developmental adaptations. Genetically engineering genes that belong to these pathways might improve the drought tolerance of plants. The abscisic acid (ABA)-responsive element binding protein 1/ABRE binding factor (AREB1/ABF2) is a key positive regulator of the drought stress response. We investigated whether the CRISPR activation (CRISPRa) system that targets AREB1 might contribute to improve drought stress tolerance in Arabidopsis. Arabidopsis histone acetyltransferase 1 (AtHAT1) promotes gene expression activation by switching chromatin to a relaxed state. Stable transgenic plants expressing chimeric dCas9HAT were first generated. Then, we showed that the CRISPRa dCas9HAT mechanism increased the promoter activity controlling the ß-glucuronidase (GUS) reporter gene. To activate the endogenous promoter of AREB1, the CRISPRa dCas9HAT system was set up, and resultant plants showed a dwarf phenotype. Our qRT-PCR experiments indicated that both AREB1 and RD29A, a gene positively regulated by AREB1, exhibited higher gene expression than the control plants. The plants generated here showed higher chlorophyll content and faster stomatal aperture under water deficit, in addition to a better survival rate after drought stress. Altogether, we report that CRISPRa dCas9HAT is a valuable biotechnological tool to improve drought stress tolerance through the positive regulation of AREB1.

Qué se debe saber en esclerosis múltiple: 16 preguntas prácticas / What one needs to know in multiple sclerosis: 16 practical questions

RESUMEN La esclerosis múltiple (EM) es la más frecuente de las enfermedades desmielinizantes del sistema nervioso central. Se trata de una patología compleja, con mecanismos fisiopatológicos aún no elucidados completamente y una presentación clínica variada. Es una entidad crónica, de alto costo para el sistema de salud, y usualmente se asocia a una importante discapacidad en los pacientes afectados. No existe una cura para la EM, sin embargo, se cuenta con más de 13 terapias modificadoras de la enfermedad que actúan en distintas dianas terapéuticas, enlentecen la progresión y mejoran el pronóstico. El objetivo de este escrito es presentar una actualización de EM, a través de 16 preguntas y temas controversiales. Estos 16 puntos responden a dudas puntuales y actuales, son presentados, analizados y, en los casos en los que la evidencia lo permite, se plantean recomendaciones. Las preguntas abordadas incluyen temas como diagnóstico, tratamiento, uso de nuevas tecnologías y manejo de los efectos adversos. Este escrito está destinado a neurólogos, médicos generales, residentes de neurología y cualquier profesional interesado en las enfermedades desmielinizantes.

SUMMARY Multiple sclerosis (MS) is the most common demyelinating disorder of the central nervous system. It is a complex disease, with a pathogenesis not fully understood and multiple clinical presentations. MS is a chronic disease, represents a great cost to healthcare systems and is often associated with a high burden of disability in patients. Despite the absence of a cure, there are at least13 disease-modifying therapies that act on different targets of the pathogenic process. The main purpose of this review is to solve 16 controversial and current topics in MS. These 16 topics are analysed, and when there is enough evidence, we issue recommendations. The topics include diagnosis, treatment, use of new technologies in MS, and medication side effects. This paper is meant for neurologists, neurology residents, general physicians and any other healthcare personnel interested in demyelinating diseases.

Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts.

The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.