Identification of reference genes for quantitative PCR during C3H10T1/2 chondrogenic differentiation

C3H10T1/2, a mouse mesenchymal stem cell line, is a well-known in vitro model of chondrogenesis that can be easily employed to recapitulate some of the mechanisms intervening in this process. Moreover, these cells can be used to validate the effect of candidate molecules identified by high throughput screening approaches applied to the development of targeted therapy for human disorders in which chondrogenic differentiation may be involved, as in conditions characterized by heterotopic endochondral bone formation. Chondrogenic differentiation of C3H10T1/2 cells can be monitored by applying quantitative polymerase chain reaction (qPCR), one of the most sensitive methods that allows detection of small dynamic changes in gene expression between samples obtained under different experimental conditions. In this work, we have used qPCR to monitor the expression of specific markers during chondrogenic differentiation of C3H10T1/2 cells in micromass cultures. Then we have applied the geNorm approach to identify the most stable reference genes suitable to get a robust normalization of the obtained expression data. Among 12 candidate reference genes (Ap3d1, Csnk2a2, Cdc40, Fbxw2, Fbxo38, Htatsf1, Mon2, Pak1ip1, Zfp91, 18S, ActB, GAPDH) we identified Mon2 and Ap3d1 as the most stable ones during chondrogenesis. ActB, GAPDH and 18S, the most commonly used in the literature, resulted to have an expression level too high compared to the differentiation markers (Sox9, Collagen type 2a1, Collagen type 10a1 and Collagen type 1a1), therefore are actually less recommended for these experimental conditions. In conclusion, we identified nine reference genes that can be equally used to obtain a robust normalization of the gene expression variation during the C3H10T1/2 chondrogenic differentiation

Nitrate sensing by the maize root apex transition zone: A merged transcriptomic and proteomic survey

Nitrate is an essential nutrient for plants, and crops depend on its availability for growth and development, but its presence in agricultural soils is far from stable. In order to overcome nitrate fluctuations in soil, plants have developed adaptive mechanisms allowing them to grow despite changes in external nitrate availability. Nitrate can act as both nutrient and signal, regulating global gene expression in plants, and the root tip has been proposed as the sensory organ. A set of genome-wide studies has demonstrated several nitrate-regulated genes in the roots of many plants, although only a few studies have been carried out on distinct root zones. To unravel new details of the transcriptomic and proteomic responses to nitrate availability in a major food crop, a double untargeted approach was conducted on a transition zone-enriched root portion of maize seedlings subjected to differing nitrate supplies. The results highlighted a complex transcriptomic and proteomic reprogramming that occurs in response to nitrate, emphasizing the role of this root zone in sensing and transducing nitrate signal. Our findings indicated a relationship of nitrate with biosynthesis and signalling of several phytohormones, such as auxin, strigolactones, and brassinosteroids. Moreover, the already hypothesized involvement of nitric oxide in the early response to nitrate was confirmed with the use of nitric oxide inhibitors. Our results also suggested that cytoskeleton activation and cell wall modification occurred in response to nitrate provision in the transition zone

mRNA-Sequencing Analysis Reveals Transcriptional Changes in Root of Maize Seedlings Treated with Two Increasing Concentrations of a New Biostimulant

Biostimulants are a wide range of natural or synthetic products containing substances and/or microorganisms that can stimulate plant processes to improve nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality ( http://www.biostimulants.eu/ , accessed September 27, 2017). The use of biostimulants is proposed as an advanced solution to face the demand for sustainable agriculture by ensuring optimal crop performances and better resilience to environment changes. The proposed approach is to predict and characterize the function of natural compounds as biostimulants. In this research, plant growth assessments and transcriptomic approaches are combined to investigate and understand the specific mode(s) of action of APR, a new product provided by the ILSA group (Arzignano, Vicenza). Maize seedlings (B73) were kept in a climatic chamber and grown in a solid medium to test the effects of two different combinations of the protein hydrolysate APR (A1 and A1/2). Data on root growth evidenced a significant enhancement of the dry weight of both roots and root/shoot ratio in response to APR. Transcriptomic profiles of lateral roots of maize seedlings treated with two increasing concentrations of APR were studied by mRNA-sequencing analysis (RNA-seq). Pairwise comparisons of the RNA-seq data identified a total of 1006 differentially expressed genes between treated and control plants. The two APR concentrations were demonstrated to affect the expression of genes involved in both common and specific pathways. On the basis of the putative function of the isolated differentially expressed genes, APR has been proposed to enhance plant response to adverse environmental conditions

Low Risk of SARS-CoV-2 Reinfection for Fully or Boosted mRNA Vaccinated Subjects in Sicily: A Population-Based Study Using Real-World Data

: Background: Reinfections occur as a response to natural infections wanes and novel strains of SARS-CoV-2 emerge. The present research explored the correlation between sex, age, COVID-19 vaccination, prior infection hospitalization, and SARS-CoV-2 reinfection in Sicily, Italy. Materials and Methods: A population-based retrospective cohort study was articulated using the vaccination flux from a regional registry and the Sicilian COVID-19 monitoring system of the Italian Institute of Health. Only adult Sicilians were included in the study, and hazard ratios were calculated using Cox regression. Results: Partial vaccination provided some protection (adj-HR: 0.92), when compared to unvaccinated individuals; furthermore, reinfection risk was reduced by full vaccination (adj-HR: 0.43), and the booster dose (adj-HR: 0.41). Males had a lower risk than females of reinfection with SARS-CoV-2 (adj-HR: 0.75). Reinfection with SARS-CoV-2 was diminished by hospitalization during the first infection (adj-HR: 0.78). Reinfection risk was higher among those aged 30-39 and 40-49 compared to those aged 18-29, whereas those aged 60-69, 70-79, and 80+ were statistically protected. Reinfection was significantly more frequent during the wild-type-Alpha, Delta, Delta-Omicron, and Omicron dominance/codominance waves compared to the wild type. Conclusions: This study establishes a solid base for comprehending the reinfection phenomenon in Sicily by pinpointing the most urgent policy hurdles and identifying some of the major factors. COVID-19 vaccination, one of the most effective public health tools, protects against reinfection, mostly caused by the Omicron strain. Elderly and hospitalized people's lower risk suggests stricter PPE use

High-throughput screening for modulators of ACVR1 transcription: discovery of potential therapeutics for fibrodysplasia ossificans progressiva.

open12noopenCappato, S; Tonachini, L; Giacopelli, F; Tirone, M; Galietta, Lj; Sormani, M; Giovenzana, A; Spinelli, Antonello E.; Canciani, B; Brunelli, S; Ravazzolo, R; Bocciardi, R.Cappato, S; Tonachini, L; Giacopelli, F; Tirone, M; Galietta, Lj; Sormani, M; Giovenzana, A; Spinelli, Antonello; Canciani, B; Brunelli, S; Ravazzolo, R; Bocciardi, R

Metabolic and molecular rearrangements of Sauvignon Blanc (Vitis vinifera L.) berries in response to foliar applications of specific dry yeast

Dry yeast extracts (DYE) are applied to vineyards to improve aromatic and secondary metabolic compound content and wine quality; however, systematic information on the underpinning molecular mechanisms is lacking. This work aimed to unravel, through a systematic approach, the metabolic and molecular responses of Sauvignon Blanc berries to DYE treatments. To accomplish this, DYE spraying was performed in a commercial vineyard for two consecutive years. Berries were sampled at several time points after the treatment, and grapes were analyzed for sugars, acidity, free and bound aroma precursors, amino acids, and targeted and untargeted RNA-Seq transcriptional profiles. The results obtained indicated that the DYE treatment did not interfere with the technological ripening parameters of sugars and acidity. Some aroma precursors, including cys-3MH and GSH3MH, responsible for the typical aromatic nuances of Sauvignon Blanc, were stimulated by the treatment during both vintages. The levels of amino acids and the global RNA-seq transcriptional profiles indicated that DYE spraying upregulated ROS homeostatic and thermotolerance genes, as well as ethylene and jasmonic acid biosynthetic genes, and activated abiotic and biotic stress responses. Overall, the data suggested that the DYE reduced berry oxidative stress through the regulation of specific subsets of metabolic and hormonal pathway

Fibrodysplasia Ossificans Progressiva: what have we achieved and where are we now? follow-up to the 2015 Lorentz Workshop

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics

The maize root response to nitrogen fluctuations: signalling crosstalk with strigolactones, auxin and transcriptional regulation

Nitrogen (N) plays a vital role in plant life, with nitrate (NO3-) and ammonium (NH4+) as the most common inorganic N compounds. Strigolactones (SLs) are carotenoid-derived phytohormones, acting as both endogenous and exogenous signalling molecules to play multiple roles in regulating plant development in response to various environmental stimuli and in concert with many other regulators. Starting from the hypothesis that nitric oxide (NO), auxin and SLs could take part to complex pathway governing the maize root adaptation to different N availabilities, this PhD work mainly investigated the involvement of SLs in the maize root developmental response to both nitrate and ammonium, thanks to a combined physiological and molecular approach. The research was initially focused on studying the effect of the two different N source on SL exudation and biosynthesis. A LC-MS/MS method was applied to identify and quantify the already known SLs in maize root exudates obtained by seedlings grown with different N availabilities. The results indicated a clear inhibitory effect of nitrate on SL production. The expression of genes encoding key SL biosynthesis and transport components was then measured in roots in response to nitrate, ammonium and N-starvation, and a germination bioassay was also performed. The results further confirmed the presence of SLs in the exudates harvested from N-deprived plants, while a massive inhibition of SL exudation resulted as a specific response to nitrate provision. In situ hybridization (ISH) experiments were then performed both in roots and shoot. The effects of 24 h of N-deficiency, nitrate and ammonium supply in the presence of a SL biosynthesis inhibitor and of a synthetic SL analogue on lateral root (LR) density and primary root (PR) length were then evaluated. The results suggested that the stimulation of LR development might be linked to the complete or partial inhibition of SL production observed in response to nitrate and ammonium, respectively. Since our previous results suggest that SLs and auxin might cooperate to regulate the response of maize primary root to nitrate, the hypothesis that the negative effect of nitrate on SL biosynthesis/exudation could depend on auxin was further studied with a SL quantification in planta, gene expression assessment and lateral root density evaluation. Results obtained are in accordance with our previous results on exudates, thus confirming the role of zealactones production as a clear response to N-deprivation. The expression of the auxin-related genes evidenced peculiar trends, thus allowing to select few of them as good candidates to better characterize and deepen the auxinic action involved in the nitrate signalling. LR density was also assessed in seedlings treated as for gene expression analysis. Our preliminary results suggest that SLs and auxin share overlapping and divergent pathways to regulate maize lateral root development in response to nitrate availability. The maize root response to different N provision was then assessed, trying to outline the different signature between nitrate and ammonium. Accordingly, a root transcriptome analysis was assessed to compare gene expression profiles in maize root apex of seedlings exposed to N-depleted solution or supplied with nitrate or ammonium for 24 h. In addition, physiological evaluation of plant development in response to nitrate and ammonium was also performed. The results provided new insight to better characterize how the early sensing of N-deficiency or nitrate/ammonium provision by root could impact on the overall plant growth and physiology

The maize root response to nitrogen fluctuations: signalling crosstalk with strigolactones, auxin and transcriptional regulation

Nitrogen (N) plays a vital role in plant life, with nitrate (NO3-) and ammonium (NH4+) as the most common inorganic N compounds. Strigolactones (SLs) are carotenoid-derived phytohormones, acting as both endogenous and exogenous signalling molecules to play multiple roles in regulating plant development in response to various environmental stimuli and in concert with many other regulators. Starting from the hypothesis that nitric oxide (NO), auxin and SLs could take part to complex pathway governing the maize root adaptation to different N availabilities, this PhD work mainly investigated the involvement of SLs in the maize root developmental response to both nitrate and ammonium, thanks to a combined physiological and molecular approach. The research was initially focused on studying the effect of the two different N source on SL exudation and biosynthesis. A LC-MS/MS method was applied to identify and quantify the already known SLs in maize root exudates obtained by seedlings grown with different N availabilities. The results indicated a clear inhibitory effect of nitrate on SL production. The expression of genes encoding key SL biosynthesis and transport components was then measured in roots in response to nitrate, ammonium and N-starvation, and a germination bioassay was also performed. The results further confirmed the presence of SLs in the exudates harvested from N-deprived plants, while a massive inhibition of SL exudation resulted as a specific response to nitrate provision. In situ hybridization (ISH) experiments were then performed both in roots and shoot. The effects of 24 h of N-deficiency, nitrate and ammonium supply in the presence of a SL biosynthesis inhibitor and of a synthetic SL analogue on lateral root (LR) density and primary root (PR) length were then evaluated. The results suggested that the stimulation of LR development might be linked to the complete or partial inhibition of SL production observed in response to nitrate and ammonium, respectively. Since our previous results suggest that SLs and auxin might cooperate to regulate the response of maize primary root to nitrate, the hypothesis that the negative effect of nitrate on SL biosynthesis/exudation could depend on auxin was further studied with a SL quantification in planta, gene expression assessment and lateral root density evaluation. Results obtained are in accordance with our previous results on exudates, thus confirming the role of zealactones production as a clear response to N-deprivation. The expression of the auxin-related genes evidenced peculiar trends, thus allowing to select few of them as good candidates to better characterize and deepen the auxinic action involved in the nitrate signalling. LR density was also assessed in seedlings treated as for gene expression analysis. Our preliminary results suggest that SLs and auxin share overlapping and divergent pathways to regulate maize lateral root development in response to nitrate availability. The maize root response to different N provision was then assessed, trying to outline the different signature between nitrate and ammonium. Accordingly, a root transcriptome analysis was assessed to compare gene expression profiles in maize root apex of seedlings exposed to N-depleted solution or supplied with nitrate or ammonium for 24 h. In addition, physiological evaluation of plant development in response to nitrate and ammonium was also performed. The results provided new insight to better characterize how the early sensing of N-deficiency or nitrate/ammonium provision by root could impact on the overall plant growth and physiology

Nitrate Regulates Maize Root Transcriptome through Nitric Oxide Dependent and Independent Mechanisms

Maize root responds to nitrate by modulating its development through the coordinated action of many interacting players. Nitric oxide is produced in primary root early after the nitrate provision, thus inducing root elongation. In this study, RNA sequencing was applied to discover the main molecular signatures distinguishing the response of maize root to nitrate according to their dependency on, or independency of, nitric oxide, thus discriminating the signaling pathways regulated by nitrate through nitric oxide from those regulated by nitrate itself of by further downstream factors. A set of subsequent detailed functional annotation tools (Gene Ontology enrichment, MapMan, KEGG reconstruction pathway, transcription factors detection) were used to gain further information and the lateral root density was measured both in the presence of nitrate and in the presence of nitrate plus cPTIO, a specific NO scavenger, and compared to that observed for N-depleted roots. Our results led us to identify six clusters of transcripts according to their responsiveness to nitric oxide and to their regulation by nitrate provision. In general, shared and specific features for the six clusters were identified, allowing us to determine the overall root response to nitrate according to its dependency on nitric oxide