Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca

Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca ( Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested

Metabolic and molecular changes of the phenylpropanoid pathway in tomato (Solanum lycopersicum) lines carrying different Solanum pennellii wild Chromosomal regions

Solanum lycopersicum represents an important dietary source of bioactive compounds including the antioxidants flavonoids and phenolic acids. We previously identified two genotypes (IL7-3 and IL12-4) carrying loci from the wild species Solanum pennellii, which increased antioxidants in the fruit. Successively, these lines were crossed and two genotypes carrying both introgressions at the homozygous condition (DHO88 and DHO88-SL) were selected. The amount of total antioxidant compounds was increased in DHOs compared to both ILs and the control genotype M82. In order to understand the genetic mechanisms underlying the positive interaction between the two wild regions pyramided in DHO genotypes, detailed analyses of the metabolites accumulated in the fruit were carried out by colorimetric methods and LC/MS/MS. These analyses evidenced a lower content of flavonoids in DHOs and in ILs, compared to M82. By contrast, in the DHOs the relative content of phenolic acids increased, particularly the fraction of hexoses, thus evidencing a redirection of the phenylpropanoid flux towards the biosynthesis of phenolic acid glycosides in these genotypes. In addition, the line DHO88 exhibited a lower content of free phenolic acids compared to M82. Interestingly, the two DHOs analyzed differ in the size of the wild region on chromosome 12. Genes mapping in the introgression regions were further investigated. Several genes of the phenylpropanoid biosynthetic pathway were identified, such as one 4-coumarate:CoA ligase and two UDP-glycosyltransferases in the region 12-4 and one chalcone isomerase and one UDP-glycosyltransferase in the region 7-3. Transcriptomic analyses demonstrated a different expression of the detected genes in the ILs and in the DHOs compared to M82.These analyses, combined with biochemical analyses, suggested a central role of the 4-coumarate:CoA ligase in redirecting the phenylpropanoid pathways towards the biosynthesis of phenolic acids in the pyramided lines. Moreover, analyses here carried out suggest the presence in the introgression regions of novel regulatory proteins, such as one Myb4 detected on chromosome 7 and one bHLH detected in chromosome 12. Overall our data indicates that structural and regulatory genes identified in this study might have a key role for the manipulation of the phenylpropanoid metabolic pathway in tomato fruit

The MAP kinase MpkA controls cell wall integrity, oxidative stress response, gliotoxin production and iron adaptation in Aspergillus fumigatus

The saprophytic fungus Aspergillus fumigatus is the most important air-borne fungal pathogen. The cell wall of A. fumigatus has been studied intensively as a potential target for development of effective antifungal agents. A major role in maintaining cell wall integrity is played by the mitogen-activated protein kinase (MAPK) MpkA. To gain a comprehensive insight into this central signal transduction pathway, we performed a transcriptome analysis of the ΔmpkA mutant under standard and cell wall stress conditions. Besides genes involved in cell wall remodelling, protection against ROS and secondary metabolism such as gliotoxin, pyomelanin and pseurotin A, also genes involved in siderophore biosynthesis were regulated by MpkA. Consistently, northern and western blot analyses indicated that iron starvation triggers phosphorylation and thus activation of MpkA. Furthermore, localization studies indicated that MpkA accumulates in the nucleus under iron depletion. Hence, we report the first connection between a MAPK pathway and siderophore biosynthesis. The measurement of amino acid pools and of the pools of polyamines indicated that arginine was continuously converted into ornithine to fuel the siderophore pool in the ΔmpkA mutant strain. Based on our data, we propose that MpkA fine-tunes the balance between stress response and energy consuming cellular processes

Cardiac Hypertrophy: from Pathophysiological Mechanisms to Heart Failure Development

Cardiac hypertrophy develops in response to increased workload to reduce ventricular wall stress and maintain function and efficiency. Pathological hypertrophy can be adaptive at the beginning. However, if the stimulus persists, it may progress to ventricular chamber dilatation, contractile dysfunction, and heart failure, resulting in poorer outcome and increased social burden. The main pathophysiological mechanisms of pathological hypertrophy are cell death, fibrosis, mitochondrial dysfunction, dysregulation of Ca2+-handling proteins, metabolic changes, fetal gene expression reactivation, impaired protein and mitochondrial quality control, altered sarcomere structure, and inadequate angiogenesis. Diabetic cardiomyopathy is a condition in which cardiac pathological hypertrophy mainly develop due to insulin resistance and subsequent hyperglycaemia, associated with altered fatty acid metabolism, altered calcium homeostasis and inflammation. In this review, we summarize the underlying molecular mechanisms of pathological hypertrophy development and progression, which can be applied in the development of future novel therapeutic strategies in both reversal and prevention

Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing

The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon (Cynara cardunculus L. var. altilis) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through Agrobacterium tumefaciens-mediated metabolic engineering. Bioinformatic data mining in the C. cardunculus transcriptome allowed the selection and molecular characterization of SAD (stearic acid desaturase) and FAD2.2 (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent CcSAD overexpressing (OE) and CcFAD2.2 RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, SAD overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the CcSADOE6 line compared with 30.4% in the wild type (WT), whereas FAD2.2 silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the CcFAD2.2KO2 line with respect to 17.7% in the WT. Moreover, CcSADOE6 and CcFAD2.2KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts

Role of Arabidopsis UV RESISTANCE LOCUS 8 in plant growth reduction under osmotic stress and low levels of UV-B

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.This work was supported by the Interuniversity Attraction Poles Programme (IUAP P7/29 'MARS') initiated by the Belgian Science Policy Office, by grants from Ghent University (Bijzonder Onderzoeksfonds Methusalem project no. BOF08/01M00408), and by grants to the MIUR project FIRB Plant-STRESS.Fasano, R.; Gonzalez, N.; Tosco, A.; Dal Piaz, F.; Docimo, T.; Serrano Salom, R.; Grillo, S.... (2014). Role of Arabidopsis UV RESISTANCE LOCUS 8 in plant growth reduction under osmotic stress and low levels of UV-B. Molecular Plant. 7(5):773-791. https://doi.org/10.1093/mp/ssu002S7737917

Breakthrough SARS-CoV-2 infections in MS patients on disease-modifying therapies

Background: Patients with multiple sclerosis (pwMS) treated with anti-CD20 or fingolimod showed a reduced humoral response to SARS-CoV-2 vaccines. Objective: In this study we aimed to monitor the risk of breakthrough SARS-CoV-2 infection in pwMS on different disease-modifying therapies (DMTs). Methods: Data on the number of vaccinated patients and the number of patients with a breakthrough infection were retrospectively collected in 27 Italian MS centers. We estimated the rate of breakthrough infections and of infection requiring hospitalization per DMT. Results: 19,641 vaccinated pwMS were included in the database. After a median follow-up of 8 months, we observed 137 breakthrough infections. Compared with other DMTs, the rate of breakthrough infections was significantly higher on ocrelizumab (0.57% vs 2.00%, risk ratio (RR) = 3.55, 95% CI = 2.74-4.58, p < 0.001) and fingolimod (0.58% vs 1.62%, RR = 2.65, 95% CI = 1.75-4.00, p < 0.001), while there were no significant differences in any other DMT group. In the ocrelizumab group the hospitalization rate was 16.7% versus 19.4% in the pre-vaccination era (RR = 0.86, p = 0.74) and it was 3.9% in all the other DMT groups versus 11.9% in the pre-vaccination period (RR = 0.33, p = 0.02). Conclusions: The risk of breakthrough SARS-CoV-2 infections is higher in patients treated with ocrelizumab and fingolimod, and the rate of severe infections was significantly reduced in all the DMTs excluding ocrelizumab

COVID-19 Severity in Multiple Sclerosis: Putting Data Into Context

Background and objectives: It is unclear how multiple sclerosis (MS) affects the severity of COVID-19. The aim of this study is to compare COVID-19-related outcomes collected in an Italian cohort of patients with MS with the outcomes expected in the age- and sex-matched Italian population. Methods: Hospitalization, intensive care unit (ICU) admission, and death after COVID-19 diagnosis of 1,362 patients with MS were compared with the age- and sex-matched Italian population in a retrospective observational case-cohort study with population-based control. The observed vs the expected events were compared in the whole MS cohort and in different subgroups (higher risk: Expanded Disability Status Scale [EDSS] score > 3 or at least 1 comorbidity, lower risk: EDSS score ≤ 3 and no comorbidities) by the χ2 test, and the risk excess was quantified by risk ratios (RRs). Results: The risk of severe events was about twice the risk in the age- and sex-matched Italian population: RR = 2.12 for hospitalization (p < 0.001), RR = 2.19 for ICU admission (p < 0.001), and RR = 2.43 for death (p < 0.001). The excess of risk was confined to the higher-risk group (n = 553). In lower-risk patients (n = 809), the rate of events was close to that of the Italian age- and sex-matched population (RR = 1.12 for hospitalization, RR = 1.52 for ICU admission, and RR = 1.19 for death). In the lower-risk group, an increased hospitalization risk was detected in patients on anti-CD20 (RR = 3.03, p = 0.005), whereas a decrease was detected in patients on interferon (0 observed vs 4 expected events, p = 0.04). Discussion: Overall, the MS cohort had a risk of severe events that is twice the risk than the age- and sex-matched Italian population. This excess of risk is mainly explained by the EDSS score and comorbidities, whereas a residual increase of hospitalization risk was observed in patients on anti-CD20 therapies and a decrease in people on interferon