Exploring the Diverse Biological Properties of Cannabidiol: A Focus on Plant Growth Stimulation.

The aim of the current study was to compare some biological activities of edible oils enriched with 10 % of cannabidiol (CBD samples) from the Slovak market. In addition, hemp, coconut, argan, and pumpkin pure oils were also examined. The study evaluated the fatty acids content, as well as antibacterial, antifungal, antioxidant, cytotoxic, and phytotoxic activities. The CBD samples presented antimicrobial activity against the tested bacterial strains at higher concentrations (10000 and 5000 mg/L) and antifungal activity against Alternaria alternata, Penicillium italicum and Aspergillus flavus. DPPH⋅ and FRAP assays showed greater activity in CBD-supplemented samples compared to pure oils and vitamin E. In cell lines (IPEC-J2 and Caco-2), a reduced cell proliferation and viability were observed after 24 hours of incubation with CBD samples. The oils showed pro-germinative effects. The tested activities were linked to the presence of CBD in the oils.

Use of Thermoluminescence Dosimetry for QA in High-Dose-Rate Skin Surface Brachytherapy with Custom-Flap Applicator.

Surface brachytherapy (BT) lacks standard quality assurance (QA) protocols. Commercially available treatment planning systems (TPSs) are based on a dose calculation formalism that assumes the patient is made of water, resulting in potential deviations between planned and delivered doses. Here, a method for treatment plan verification for skin surface BT is reported. Chips of thermoluminescent dosimeters (TLDs) were used for dose point measurements. High-dose-rate treatments were simulated and delivered through a custom-flap applicator provided with four fixed catheters to guide the Iridium-192 (Ir-192) source by way of a remote afterloading system. A flat water-equivalent phantom was used to simulate patient skin. Elekta TPS Oncentra Brachy was used for planning. TLDs were calibrated to Ir-192 through an indirect method of linear interpolation between calibration factors (CFs) measured for 250 kV X-rays, Cesium-137, and Cobalt-60. Subsequently, plans were designed and delivered to test the reproducibility of the irradiation set-up and to make comparisons between planned and delivered dose. The obtained CF for Ir-192 was (4.96 ± 0.25) µC/Gy. Deviations between measured and TPS calculated doses for multi-catheter treatment configuration ranged from -8.4% to 13.3% with an average of 0.6%. TLDs could be included in clinical practice for QA in skin BT with a customized flap applicator.

Chemical Composition, Phytotoxic and Antibiofilm Activity of Seven Eucalyptus Species from Tunisia.

This study was carried out to characterize the chemical composition of the essential oils from seven Eucalyptus species (E. griffithsii, E. hemiphloia, E. lesouefii, E. longicornis, E. pyriformis, E. viminalis, and E. wandoo), as well as their phytotoxic and antibacterial activities. The essential oils were analyzed by GC/MS and the potential in vitro phytotoxicity was evaluated against germination and radical elongation of Raphanus sativus, Lolium multiflorum, and Sinapis arvensis seeds. The antibiofilm activity was studied against both Gram-negative (Pseudomonas aeruginosa, Escherichia coli and Acinetobacter baumannii) and Gram-positive (Staphylococcus aureus and Listeria monocytogenes) bacteria. The inhibition of biofilm formation and its metabolism was determined at different times. Eucalyptol was the most abundant component in all essential oils studied (ranging from 40.8% for E. lesouefii EO to 73.6% for E. wandoo) except for that of E. pyriformis where it was present but at 15.1%. E. pyriformis was the most active against both germination and radical elongation of S. arvensis. The action of all essential oils proved to be highly effective in inhibiting the bacterial adhesion process of the five strains considered. In light of these results, these essential oils could have potential applications both in the agricultural and health fields.

Impact of drying methods on the yield and chemistry of Origanum vulgare L. essential oil.

Oregano (Origanum vulgare L.) is mainly cultivated, both as fresh and dried herb, for several purposes, such as ailments, drugs, and spices. To evaluate the influence of some drying methods on the chemical composition of the essential oil of oregano, its aerial parts were dehydrated by convective drying techniques (shade, static oven), microwave-assisted heating (three different treatments) and osmotic treatment. The oils were analyzed by GC-FID and GC-MS. The highest essential oil yield was achieved from microwave and shade drying methods. In total, 39 components were found, with carvacrol (ranging from 56.2 to 81.4%) being the main constituent; other compounds present in lower amounts were p-cymene (1.6-17.7%), γ-terpinene (0.8-14.2%), α-pinene (0.1-2.1%), thymol methyl ether (0.4-1.8%) and thimoquinone (0.5-3.5%). The essential oil yields varied among the different treatments as well as the relative compositions. The percentages of p-cymene, γ-terpinene and α-pinene decreased significantly in the dried sample compared with the fresh sample; on the other hand, carvacrol, isoborneol and linalool increased significantly in the dried materials. The choice of the drying method for obtaining the essential oil therefore appears crucial not only in relation to the higher yield but also and above all in reference to the percentage presence of components that can direct the essential oil toward an appropriate use.

Corrigendum: Characterization of Two Cases of Congenital Dyserythropoietic Anemia Type I Shed Light on the Uncharacterized C15orf41 Protein.

[This corrects the article DOI: 10.3389/fphys.2019.00621.].

RAP-011 Rescues the Disease Phenotype in a Cellular Model of Congenital Dyserythropoietic Anemia Type II by Inhibiting the SMAD2-3 Pathway.

Congenital dyserythropoietic anemia type II (CDA II) is a hypo-productive anemia defined by ineffective erythropoiesis through maturation arrest of erythroid precursors. CDA II is an autosomal recessive disorder due to loss-of-function mutations in SEC23B. Currently, management of patients with CDA II is based on transfusions, splenectomy, or hematopoietic stem-cell transplantation. Several studies have highlighted benefits of ACE-011 (sotatercept) treatment of ineffective erythropoiesis, which acts as a ligand trap against growth differentiation factor (GDF)11. Herein, we show that GDF11 levels are increased in CDA II, which suggests sotatercept as a targeted therapy for treatment of these patients. Treatment of stable clones of SEC23B-silenced erythroleukemia K562 cells with the iron-containing porphyrin hemin plus GDF11 increased expression of pSMAD2 and reduced nuclear localization of the transcription factor GATA1, with subsequent reduced gene expression of erythroid differentiation markers. We demonstrate that treatment of these SEC23B-silenced K562 cells with RAP-011, a "murinized" ortholog of sotatercept, rescues the disease phenotype by restoring gene expression of erythroid markers through inhibition of the phosphorylated SMAD2 pathway. Our data also demonstrate the effect of RAP-011 treatment in reducing the expression of erythroferrone in vitro, thus suggesting a possible beneficial role of the use of sotatercept in the management of iron overload in patients with CDA II.

Kinome multigenic panel identified novel druggable EPHB4-V871I somatic variant in high-risk neuroblastoma.

Neuroblastoma (NB) is the most common extracranial neoplasm in children. The overall outcome for high-risk NB patients is still unacceptable, therefore, it is critical to deeply understand molecular mechanisms associated with NB, which in turn can be utilized for developing drugs towards the treatment of NB. Protein kinases (TKs) play an essential role in the regulation of cell survival and proliferation. Different kinases, such as anaplastic lymphoma kinase (ALK), Aurora kinase, RET receptor tyrosine kinase, are potential therapeutic targets in various cancers, including NB. We analysed a cohort of 45 high-risk NB patients and 9 NB cell lines by a targeted-(t)NGS custom gene panel (genes codifying for the kinase domains of 90 TKs). We identified somatic variants in four TK genes (ALK, EPHB4, LMTK3 and EPHB6) in NB patients and we functionally characterized an interesting somatic variant, V871I, in EPHB4 gene. EPHB4 plays a crucial role in cardiovascular development and regulates vascularization in cancer-promoting angiogenesis, tumour growth and metastasis. Several EPHB4 mutations have previously been identified in solid and haematological tumour specimens but EPHB4 mutations were not described until now in NB. Interestingly, a re-analysis of public CGH-array showed that the EPHB4 gain is associated with advanced diseases in NB. We further demonstrated that higher EPHB4 expression is correlated to stage 4 of NB and with poor overall survival. Additionally, we also revealed that the EPHB4-V871I accounts for increased proliferation, migration and invasion properties in two NB cell lines by acting on VEGF, c-RAF and CDK4 target genes and by increasing the phosphorylation of ERK1-2 pathway. The use of two EPHB4 inhibitors, JI-101 and NVP-BHG712, was able to rescue the phenotype driven by the variant. Our study suggested that EPHB4 is a promising therapeutic target in high-risk NB.

Gain-of-function mutations in PIEZO1 directly impair hepatic iron metabolism via the inhibition of the BMP/SMADs pathway.

Dehydrated hereditary stomatocytosis (DHS), or xerocytosis, is an autosomal dominant hemolytic anemia. Most patients with DHS carry mutations in the PIEZO1 gene encoding a mechanosensitive cation channel. We here demonstrate that patients with DHS have low levels of hepcidin and only a slight increase of ERFE, the erythroid negative regulator of hepcidin. We demonstrated that at the physiological level, PIEZO1 activation induced Ca2+ influx and suppression of HAMP expression in primary hepatocytes. In two hepatic cellular models expressing PIEZO1 WT and two PIEZO1 gain-of-function mutants (R2456H and R2488Q), we highlight altered expression of a few genes/proteins involved in iron metabolism. Mutant cells showed increased intracellular Ca2+ compared to WT, which was correlated to increased phosphorylation of ERK1/2, inhibition of the BMP-SMADs pathway, and suppression of HAMP transcription. Moreover, the HuH7 cells, treated with PD0325901, a potent inhibitor of ERK1/2 phosphorylation, reduced the phosphorylation of ERK1/2 with the consequent increased phosphorylation of SMAD1/5/8, confirming the link between the two pathways. Another "proof of concept" for the mechanism that links PIEZO1 to HAMP regulation was obtained by mimicking PIEZO1 activation by cell Ca2+ overload, by the Ca2+ ionophore A23187. There was strong down-regulation of HAMP gene expression after this Ca2+ overload. Finally, the inhibition of PIEZO1 by GsMTx4 leads to phenotype rescue. This is the first demonstration of a direct link between PIEZO1 and iron metabolism, which defines the channel as a new hepatic iron metabolism regulator and as a possible therapeutic target of iron overload in DHS and other iron-loading anemias.

The BMP-SMAD pathway mediates the impaired hepatic iron metabolism associated with the ERFE-A260S variant.

The erythroferrone (ERFE) is the erythroid regulator of hepatic iron metabolism by suppressing the expression of hepcidin. Congenital dyserythropoietic anemia type II (CDAII) is an inherited hyporegenerative anemia due to biallelic mutations in the SEC23B gene. Patients with CDAII exhibit marked clinical variability, even among individuals sharing the same pathogenic variants. The ERFE expression in CDAII is increased and related to abnormal erythropoiesis. We identified a recurrent low-frequency variant, A260S, in the ERFE gene in 12.5% of CDAII patients with a severe phenotype. We demonstrated that the ERFE-A260S variant leads to increased levels of ERFE, with subsequently marked impairment of iron regulation pathways at the hepatic level. Functional characterization of ERFE-A260S in the hepatic cell system demonstrated its modifier role in iron overload by impairing the BMP/SMAD pathway. We herein described for the first time an ERFE polymorphism as a genetic modifier variant. This was with a mild effect on disease expression, under a multifactorial-like model, in a condition of iron-loading anemia due to ineffective erythropoiesis.

Characterization of Two Cases of Congenital Dyserythropoietic Anemia Type I Shed Light on the Uncharacterized C15orf41 Protein.

CDA type I is a rare hereditary anemia, characterized by relative reticulocytopenia, and congenital anomalies. It is caused by biallelic mutations in one of the two genes: (i) CDAN1, encoding Codanin-1, which is implicated in nucleosome assembly and disassembly; (ii) C15orf41, which is predicted to encode a divalent metal ion-dependent restriction endonuclease with a yet unknown function. We described two cases of CDA type I, identifying the novel variant, Y94S, in the DNA binding domain of C15orf41, and the H230P mutation in the nuclease domain of the protein. We first analyzed the gene expression and the localization of C15orf41. We demonstrated that C15orf41 and CDAN1 gene expression is tightly correlated, suggesting a shared mechanism of regulation between the two genes. Moreover, we functionally characterized the two variants, establishing that the H230P leads to reduced gene expression and protein level, while Y94S induces a slight decrease of expression. We demonstrated that C15orf41 endogenous protein exhibits nuclear and cytosolic localization, being mostly in the nucleus. However, no altered nuclear-cytosolic compartmentalization of mutated C15orf41 was observed. Both mutants accounted for impaired erythroid differentiation in K562 cells, and H230P mutant also exhibits an increased S-phase of the cell cycle in these cells. Our functional characterization demonstrated that the two variants have different effects on the stability of the mutated mRNA, but both resulted in impaired erythroid maturation, suggesting the block of cell cycle dynamics as a putative pathogenic mechanism for C15orf41-related CDA I.

PIEZO1 Hypomorphic Variants in Congenital Lymphatic Dysplasia Cause Shape and Hydration Alterations of Red Blood Cells.

PIEZO1 is a cation channel activated by mechanical force. It plays an important physiological role in several biological processes such as cardiovascular, renal, endothelial and hematopoietic systems. Two different diseases are associated with alteration in the DNA sequence of PIEZO1: (i) dehydrated hereditary stomatocytosis (DHS1, #194380), an autosomal dominant hemolytic anemia caused by gain-of-function mutations; (ii) lymphatic dysplasia with non-immune fetal hydrops (LMPH3, #616843), an autosomal recessive condition caused by biallelic loss-of-function mutations. We analyzed a 14-year-old boy affected by severe lymphatic dysplasia already present prenatally, with peripheral edema, hydrocele, and chylothoraces. By whole exome sequencing, we identified compound heterozygosity for PIEZO1, with one splicing and one deletion mutation, the latter causing the formation of a premature stop codon that leads to mRNA decay. The functional analysis of the erythrocytes of the patient highlighted altered hydration with the intracellular loss of the potassium content and structural abnormalities with anisopoikolocytosis and presence of both spherocytes and stomatocytes. This novel erythrocyte trait, sharing features with both hereditary spherocytosis and overhydrated hereditary stomatocytosis, complements the clinical features associated with loss-of-function mutations of PIEZO1 in the context of the generalized lymphatic dysplasia of LMPH3 type.

Genotype-phenotype correlation and risk stratification in a cohort of 123 hereditary stomatocytosis patients.

Hereditary stomatocytoses (HSts) are a wide spectrum of hemolytic anemias in which the erythrocyte membrane cation permeability is increased. Dehydrated hereditary stomatocytosis is the most frequent among HSts. It is caused by missense mutations in PIEZO1 and KCNN4 genes. We described 123 patients enrolled in our Genetic Unit from 2013 to 2017. Overall HSt subjects exhibit macrocytic mild anemia. We found that PIEZO1 is the most frequent mutated gene within our families (47% of pedigrees). In 59.1% of cases the mutations localized in the nonpore protein domain, while in 40.9% of patients they localized in the central pore region. The genotype-phenotype correlation analysis on 29 PIEZO1-patients demonstrated that most of severely affected patients carried mutations in the pore domain, suggesting that the severity of this condition is related to the pore properties and intracellular domain that could be responsible of interactions with intracellular components. This is the first cohort study on a large set of hereditary stomatocytosis patients, stratified according to their causative gene useful for diagnosis, prognosis, and management of these patients.

Multi-gene panel testing improves diagnosis and management of patients with hereditary anemias.

Mutations in more than 70 genes cause hereditary anemias (HA), a highly heterogeneous group of rare/low frequency disorders in which we included: hyporegenerative anemias, as congenital dyserythropoietic anemia (CDA) and Diamond-Blackfan anemia; hemolytic anemias due to erythrocyte membrane defects, as hereditary spherocytosis and stomatocytosis; hemolytic anemias due to enzymatic defects. The study describes the diagnostic workflow for HA, based on the development of two consecutive versions of a targeted-NGS panel, including 34 and 71 genes, respectively. Seventy-four probands from 62 unrelated families were investigated. Our study includes the most comprehensive gene set for these anemias and the largest cohort of patients described so far. We obtained an overall diagnostic yield of 64.9%. Despite 54.2% of cases showed conclusive diagnosis fitting well to the clinical suspicion, the multi-gene analysis modified the original clinical diagnosis in 45.8% of patients (nonmatched phenotype-genotype). Of note, 81.8% of nonmatched patients were clinically suspected to suffer from CDA. Particularly, 45.5% of the probands originally classified as CDA exhibited a conclusive diagnosis of chronic anemia due to enzymatic defects, mainly due to mutations in PKLR gene. Interestingly, we also identified a syndromic CDA patient with mild anemia and epilepsy, showing a homozygous mutation in CAD gene, recently associated to early infantile epileptic encephalopathy-50 and CDA-like anemia. Finally, we described a patient showing marked iron overload due to the coinheritance of PIEZO1 and SEC23B mutations, demonstrating that the multi-gene approach is valuable not only for achieving a correct and definitive diagnosis, but also for guiding treatment.

Kinome expression profiling of human neuroblastoma tumors identifies potential drug targets for ultra high-risk patients.

Neuroblastoma (NBL) accounts for >7% of malignancies in patients younger than 15 years. Low- and intermediate-risk patients exhibit excellent or good prognosis after treatment, whereas for high-risk (HR) patients, the estimated 5-year survival rates is still <40%. The ability to stratify HR patients that will not respond to standard treatment strategies is critical for informed treatment decisions. In this study, we have generated a specific kinome gene signature, named Kinome-27, which is able to identify a subset of HR-NBL tumors, named ultra-HR NBL, with highly aggressive clinical behavior that not adequately respond to standard treatments. We have demonstrated that NBL cell lines expressing the same kinome signature of ultra-HR tumors (ultra-HR-like cell lines) may be selectively targeted by the use of two drugs [suberoylanilide hydroxamic acid (SAHA) and Radicicol], and that the synergic combination of these drugs is able to block the ultra-HR-like cells in G2/M phase of cell cycle. The use of our signature in clinical practice will allow identifying patients with negative outcome, which would benefit from new and more personalized treatments. Preclinical in vivo studies are needed to consolidate the SAHA and Radicicol treatment in ultra-HR NBL patients.

Data demonstrating the role of peroxiredoxin 2 as important anti-oxidant system in lung homeostasis.

The data presented in this article are related to the research paper entitled "peroxiredoxin-2 plays a pivotal role as multimodal cytoprotector in the early phase of pulmonary hypertension" (Federti et al., 2017) [1]. Data show that the absence of peroxiredoxin-2 (Prx2) is associated with increased lung oxidation and pulmonary vascular endothelial dysfunction. Prx2-/- mice displayed activation of the redox-sensitive transcriptional factors, NF-kB and Nrf2, and increased expression of cytoprotective system such as heme-oxygenase-1 (HO-1). We also noted increased expression of both markers of vascular activation and extracellular matrix remodeling. The administration of the recombinant fusion protein PEP Prx2 reduced the activation of NF-kB and Nrf2 and was paralleled by a decrease in HO-1 and in vascular endothelial abnormal activation. Prolonged hypoxia was used to trigger pulmonary artery hypertension (PAH). Prx2-/- precociously developed PAH compared to wildtype animals.

Peroxiredoxin-2 plays a pivotal role as multimodal cytoprotector in the early phase of pulmonary hypertension.

Pulmonary-artery-hypertension (PAH) is a life-threatening and highly invalidating chronic disorder. Chronic oxidation contributes to lung damage and disease progression. Peroxiredoxin-2 (Prx2) is a typical 2-cysteine (Cys) peroxiredoxin but its role on lung homeostasis is yet to be fully defined. Here, we showed that Prx2-/- mice displayed chronic lung inflammatory disease associated with (i) abnormal pulmonary vascular dysfunction; and (ii) increased markers of extracellular-matrix remodeling. Hypoxia was used to induce PAH. We focused on the early phase PAH to dissect the role of Prx2 in generation of PAH. Hypoxic Prx2-/-mice showed (i) amplified inflammatory response combined with cytokine storm; (ii) vascular activation and dysfunction; (iii) increased PDGF-B lung levels, as marker of extracellular-matrix deposition and remodeling; and (iv) ER stress with activation of UPR system and autophagy. Rescue experiments with in vivo the administration of fused-recombinant-PEP-Prx2 show a reduction in pulmonary inflammatory vasculopathy and in ER stress with down-regulation of autophagy. Thus, we propose Prx2 plays a pivotal role in the early stage of PAH as multimodal cytoprotector, targeting oxidation, inflammatory vasculopathy and ER stress with inhibition of autophagy. Collectively, our data indicate that Prx2 is able to interrupt the hypoxia induced vicious cycle involving oxidation-inflammation-autophagy in the pathogenesis of PAH.