Strategies for the adult haemoglobin (HbA) production in β0-thalassemia patients

Background: Nonsense mutations, giving rise to UAA, UGA, UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of about 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy, thalassemia. Currently, there are two approaches to directly overcome diseases caused by nonsense mutations: gene therapy, meaning introduction of an exogenous gene, and translational read-through induced by aminoglycosides, which decrease the accuracy of translation elongation and reduce the efficacy of the translation termination machinery. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. On the other hand, in recent years, many investigators have been studying the development of safe lentiviral vectors to avoid the side effects as activation of oncogenes and transcription silencing. Aim: Our purpose was to verify the effects and clinical utility in b0- thalassemia of translational read-through and gene therapy. Methods: First, we started the development of a cellular model of the b039- thalassemia mutation that could be used for the screening of high numbers of aminoglycosides and analogous molecules. We produced two lentiviral vectors containing the bwt- or b039-thalassemia globin gene under a minimal LCR control region and used such constructs for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different copynumber of the integrated constructs. These clones were then treated with geneticin (G418) and other aminoglycosides and the production of β-globin was analysed by FACS analysis. Secondly, we screened several thalassemia patients and isolated erythroid progenitors from such individuals resulted homozygous for the b039 mutation. Then, we treated the cells with geneticin and analysed the production of b-globin and adult haemoglobin (HbA) by FACS and HPLC analysis, respectively. Finally, we subjected the b039 erythroid precursor cells to gene therapy, through the use of the lentiviral vector expressing the βwt-globin mRNA, with the aim to verify the increase of HbA through HPLC analysis. Moreover, by treating the transduced cells with mithramycin we wanted to verify the combined effects of gene therapy and fetal haemoglobin (HbF) induction. Main results: We obtained and characterized six clones carrying the b039- globin gene and seven clones carrying the bwt-globin gene, to be used as a control. We then chose two clones, a bwt and a b039, showing similar content of mRNA and used them to test various aminoglycosides to verify their read-through activity. Geneticin (G418) and gentamicin showed the highest capacity to readthrough of nonsense mutation, although the effect of G418 was found to be higher than that displayed by gentamicin. A significant increase in β-globin containing cells was also detected when b039 progenitor cells were treated with G418. This last result was confirmed by HPLC analysis, which showed an increase in the relative concentration of HbA compared to total Hbs. Finally, we obtained a high increase in the proportion of HbA when the same progenitor cells were subjected to gene therapy, through transduction with the lentiviral vector containing the bwt-globin gene, and a clear increment of HbF was detected after treatment with mithramycin. Conclusion: Both the therapeutic approaches for the cure of β0-thalassemias analysed in this work, namely translational read-through and gene therapy, while still presenting many disadvantages, including toxicity of aminoglycosides and the uncertainty of the effects of lentiviral vectors on endogenous genes, can be considered techniques with a high curative potential. Naturally, in the case of translational read-through is hoped the identification of new compounds with a therapeutic effect similar to but greater than that of aminoglycosides, with less toxicity and oral bioavailability, while in the case of gene therapy, further investigations should be made to eliminate its well known disadvantages, primarily including the possible activation of oncogenes

Therapy for Cystic Fibrosis Caused by Nonsense Mutations

Nonsense mutations cover about 10% of cystic fibrosis (CF) patients and generate premature termination codons (PTCs) leading to premature translational termination and causing the synthesis of truncated non-functional or partially functional CFTR (cystic fibrosis transmembrane conductance regulator) protein. The read-through approach is the suppression of translation terminations at PTCs and it has been developed as a therapeutic strategy to restore full-length protein using aminoglycoside antibiotics or PTC124. Phenotypic consequences of PTCs can be exacerbated by the nonsense-mediated mRNA decay (NMD) pathway, which detects and degrades mRNA containing PTC. Therefore, modulation of NMD is also of interest as a potential target for suppression therapy. Not all PTCs are susceptible to the read-through treatment alone, especially where the nonsense mutations are combined with other CFTR mutations. For example, many CF patients present the highly frequent F508del CF mutation, causing an alteration of the cell membrane positioning of the CFTR channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect. A combined administration of correctors/potentiators, read-through molecules, and/or NMD inhibitors, depending on the genotype of the CF patients, could be the basis for the design of a personalized therapeutic approach

Radiometric investigation of different snow covers in Svalbard

This paper examines the relationship between reflectance and physical characteristics of the snow cover in the Arctic. Field data were acquired for different snow and ice surfaces during a survey carried out at Ny-Ålesund, Svalbard, in spring 1998. In each measurement reflectance in the spectral range 350 - 2500 nm, snow data (including temperature, grain size and shape, density and water content), surface layer morphology, and vertical profile of the snow pack were recorded detailed analysis of reflectance based on the physical was performed. Field reflectance data were also re-sampled at the spectral intervals of Landsat TM to compare the ability of identifying different snow targets at discrete wavelength intervals. This analysis shows that reliable data on snow structure and thickness are necessary to understand albedo changes of the snow surfaces

Germination, root elongation, and photosynthetic performance of plants exposed to sodium lauryl ether sulfate (SLES). An emerging contaminant

The anionic surfactant SLES (sodium lauryl ether sulfate) is an emerging contaminant, being the main component of foaming agents that are increasingly used by the tunnel construction industry. To fill the gap of knowledge about the potential SLES toxicity on plants, acute and chronic effects were assessed under controlled conditions. The acute ecotoxicological test was performed on Lepidum sativum L. (cress) and Zea mays L. (maize). Germination of both species was not affected by SLES in soil, even at concentrations (1200 mg kg−1) more than twice higher than the maximum realistic values found in contaminated debris, thus confirming the low acute SLES toxicity on terrestrial plants. The root elongation of the more sensitive species (cress) was instead reduced at the highest SLES concentration. In the chronic phytotoxicity experiment, photosynthesis of maize was downregulated, and the photosynthetic performance (PITOT) significantly reduced already under realistic exposures (360 mg kg−1), owing to the SLES ability to interfere with water and/or nutrients uptake by roots. However, such reduction was transient, likely due to the rapid biodegradation of the surfactant by the soil microbial community. Indeed, SLES amount decreased in soil more than 90% of the initial concentration in only 11 days. A significant reduction of the maximum photosynthetic capacity (Pnmax) was still evident at the end of the experiment, suggesting the persistence of negative SLES effects on plant growth and productivity. Overall results, although confirming the low phytotoxicity and high biodegradability of SLES in natural soils, highlight the importance of considering both acute and nonlethal stress effects to evaluate the environmental compatibility of soil containing SLES residues

Confocal Laser Endomicroscopy in the Study of Colonic Mucosa in IBD Patients: A Review

Confocal laser endomicroscopy (CLE) is one of several novel methods that provide real-time, high-resolution imaging at a micronscale via endoscopes. CLE and related technologies are often termed “virtual biopsy” as they simulate the images seen in traditional histology. Recently, the use of CLE was reported in the study of colonic mucosa in patients with inflammatory bowel diseases and in particular in patients affected by ulcerative colitis. CLE has the potential to have an important role in management of IBD patients as it can be used to assess the grading of colitis and in detection of microscopic colitis in endoscopically silent segments. Moreover, CLE can be used in surveillance programs especially in high-risk patients. This report aims to evaluate the current data on the application of confocal endomicroscopy in clinical gastroenterology and particularly in the study of colonic mucosa in UC patients

Capsule impaction presenting as acute small bowel perforation: a case series

INTRODUCTION: Perforation caused by capsule endoscopy impaction is extremely rare and, at present, only five cases of perforation from capsule endoscopy impaction are reported in the literature. CASE PRESENTATION: We report here two cases of patients with undiagnosed small bowel stenosis presenting with acute perforation after capsule endoscopy. Strictures in the small bowel were likely the inciting mechanism leading to acute small bowel obstruction and subsequent distension and perforation above the capsule in the area of maximal serosal tension. Case 1 was a 55-year-old Italian woman who underwent capsule endoscopy because of recurrent postprandial cramping pain and iron deficiency anemia, in the setting of negative imaging studies including an abdominal ultrasound, upper endoscopy, colonoscopy and small bowel follow-through radiograph. She developed a symptomatic bowel obstruction approximately 36 hours after ingestion of the capsule. Emergent surgery was performed to remove the capsule, which was impacted at a stenosis due to a previously undiagnosed ileal adenocarcinoma, leading to perforation. Case 2 was a 60-year-old Italian man with recurrent episodes of abdominal pain and diarrhea who underwent capsule endoscopy after conventional modalities, including comprehensive blood and stool studies, computed tomography, an abdominal ultrasound, upper endoscopy, colonoscopy, barium enema and small bowel follow-through, were not diagnostic. Our patient developed abdominal distension, acute periumbilical pain, fever and leukocytosis 20 hours after capsule ingestion. Emergent surgery was performed to remove the capsule, which was impacted at a previously undiagnosed ileal Crohn’s stricture, leading to perforation. CONCLUSIONS: The present report shows that, although the risk of acute complication is very low, the patient should be informed of the risks involved in capsule endoscopy, including the need for emergency surgical exploration

Oxidative stress and gut-derived lipopolysaccharides in children affected by paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections

BACKGROUND: Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections syndrome (PANDAS) identifies patients with acute onset of obsessive-compulsive and tic disorders. The objective of this study was to evaluate serum NOX2 levels, as well as 8-iso-prostaglandin F2α (8-iso-PGF2α) and lipopolysaccharide (LPS) of PANDAS patients. METHODS: In this study we wanted to compare serum levels of soluble NOX2-dp (sNOX-2-dp), iso-PGF2α and LPS in 60 consecutive subjects, including 30 children affected by PANDAS and 30 controls (CT) matched for age and gender. Serum zonulin was used as intestinal permeability assay. RESULTS: Compared with CT, PANDAS children had increased serum levels of sNOX-2-dp, 8-iso-PGF2α and LPS. Bivariate analysis showed that serum sNOX2-dp was significantly correlated with LPS (Rs = 0.359; p = 0.005), zonulin (Rs = 0.444; p < 0.001) and 8-iso-PGF2α (Rs = 0.704; p < 0.001). Serum LPS significantly correlated with zonulin (Rs = 0.610; p < 0.001), and 8-iso-PGF2α (Rs = 0.591; p = 0.001). Finally, a multiple linear regression analysis showed that serum 8-iso-PGF2α and zonulin were the only independent variables associated with sNOX2-dp (R2 = 68%). CONCLUSION: This study shows that children affected by PANDAS have high circulating levels of sNOX2-dp, isoprostanes and of LPS that could be involved in the process of neuroinflammation

Quality of life and emotional distress in sarcoma patients diagnosed during COVID-19 pandemic: a supplementary analysis from the SarCorD study

BackgroundThe COVID-19 outbreak had a negative psychological impact on cancer patients. In this study, we analyzed emotional distress and quality of life in patients diagnosed with sarcoma during the first year of the pandemic compared to the previous year.MethodsWe retrospectively enrolled patients with soft tissue, bone sarcoma, and aggressive benign musculoskeletal diseases diagnosed during the pandemic (COVID group) or the year before (control group) at the IRCCS Regina Elena National Cancer Institute in Rome. Patients who had undergone a psychological assessment with the EORTC QLQ-C30 and the Distress Thermometer at diagnosis were included in the final analysis. We analyzed whether there is a difference in the various domains of quality of life between the two groups and whether there are changes over time in each group.ResultsWe enrolled 114 patients (72 control group; 42 COVID group), affected by soft tissue (64%), bone sarcoma (29%), and aggressive benign musculoskeletal diseases (7%). We did not observe significant differences in the health-related quality of life domains in the control and COVID groups, except for the financial domain (p = 0.039), with 9.7% vs. 23.8% of patients with a score > 0 in the control and COVID groups, respectively. We observed emotional distress at diagnosis in 48.6% of patients in the control group vs. 69.0% in the COVID group (p = 0.034). In the control group, we observed an improvement in physical function (p = 0.043) and in QoL (p = 0.022), while in the COVID group, we observed a deterioration in role function (p = 0.044) during follow-up. In the COVID group, 22.2% of patients were concerned about COVID-19, 61.1% by tumor, 91.1% stated that the pandemic worsened their subjective perception of cancer, and 19.4% perceived that their quality of care had worsened.ConclusionWe observed a higher level of distress among patients diagnosed during the pandemic compared to the year before, probably due to the increased concern for both infection and cancer, the worsened perception of health status, and the perception of a poorer quality of health care

Impacts of air pollution on human and ecosystem health, and implications for the National Emission Ceilings Directive. Insights from Italy

Across the 28 EU member states there were nearly half a million premature deaths in 2015 as a result of exposure to PM2.5, O3 and NO2. To set the target for air quality levels and avoid negative impacts for human and ecosystems health, the National Emission Ceilings Directive (NECD, 2016/2284/EU) sets objectives for emission reduction for SO2, NOx, NMVOCs, NH3 and PM2.5 for each Member State as percentages of reduction to be reached in 2020 and 2030 compared to the emission levels into 2005. One of the innovations of NECD is Article 9, that mentions the issue of “monitoring air pollution impacts” on ecosystems. We provide a clear picture of what is available in term of monitoring network for air pollution impacts on Italian ecosystems, summarizing what has been done to control air pollution and its effects on different ecosystems in Italy. We provide an overview of the impacts of air pollution on health of the Italian population and evaluate opportunities and implementation of Article 9 in the Italian context, as a case study beneficial for all Member States. The results showed that SO42− deposition strongly decreased in all monitoring sites in Italy over the period 1999–2017, while NO3− and NH4+ decreased more slightly. As a consequence, most of the acid-sensitive sites which underwent acidification in the 1980s partially recovered. The O3 concentration at forest sites showed a decreasing trend. Consequently, AOT40 (the metric identified to protect vegetation from ozone pollution) showed a decrease, even if values were still above the limit for forest protection (5000 ppb h−1), while PODy (flux-based metric under discussion as new European legislative standard for forest protection) showed an increase. National scale studies pointed out that PM10 and NO2 induced about 58,000 premature deaths (year 2005), due to cardiovascular and respiratory diseases. The network identified for Italy contains a good number of monitoring sites (6 for terrestrial ecosystem monitoring, 4 for water bodies monitoring and 11 for ozone impact monitoring) distributed over the territory and will produce a high number of monitored parameters for the implementation of the NECD