Plasma and dried blood spot lysosphingolipids for the diagnosis of different sphingolipidoses: a comparative study.

Abstract Background Lysosphingolipids, the N-deacylated forms of sphingolipids, have been identified as potential biomarkers of several sphingolipidoses, such as Gaucher, Fabry, Krabbe and Niemann-Pick diseases and in GM1 and GM2 gangliosidoses. To date, different methods have been developed to measure various lysosphingolipids (LysoSLs) in plasma. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a simultaneous quantification of LysoSLs (HexSph, LysoGb3, LysoGM1, LysoGM2, LysoSM and LysoSM509) in dried blood spot (DBS). This LC-MS/MS method was used to compare the levels of LysoSLs in DBS and plasma in both affected patients and healthy controls. Methods Lysosphingolipids were extracted from a 3.2 mm diameter DBS with a mixture of methanol:acetonitrile:water (80:15:5, v/v) containing internal stable isotope standards. Chromatographic separation was performed using a C18 column with a gradient of water and acetonitrile both with 0.1% formic acid in a total run time of 4 min. The compounds were detected in the positive ion mode electrospray ionization (ESI)-MS/MS by multiple reaction monitoring (MRM). Results The method was validated on DBS to demonstrate specificity, linearity, lowest limit of quantification, accuracy and precision. The reference ranges were determined in pediatric and adult populations. The elevated levels of LysoSLs were identified in Gaucher disease (HexSph), Fabry disease (LysoGb3), prosaposin deficiency (HexSph and LysoGb3) and Niemann-Pick disease types A/B and C (LysoSM and LysoSM509). The correlation in the levels between DBS and plasma was excellent for LysoGb3 and HexSph but poor for LysoSM and LysoSM509. Conclusions Despite the fact that plasma LysoSLs determination remains the gold standard, our LC-MS/MS method allows a rapid and reliable quantification of lysosphingolipids in DBS. The method is a useful tool for the diagnosis of different sphingolipidoses except for Niemann-Pick type C

Agronomic performance of 21 new disease resistant winegrape varieties grown in northeast Italy

The goal of the field trial was to evaluate the agronomic performance of 21 (10 red and 11 white) winegrape varieties obtained from recent breeding programmes for disease resistance developed in Hungary, Germany, and Italy. The tested red varieties were as follows: ‘Cabernet Carbon’, ‘Cabernet Eidos’, ‘Cabernet Volos’, ‘Julius’, ‘Merlot Khorus’, ‘Merlot Kanthus’, ‘Monarch’, ‘Prior’, UD. 31.103, ‘Vinera’. The tested white varieties were as follows: ‘Aromera’, ‘Bronner’, ‘Fleurtai’, ‘Johanniter’, ‘Muscaris’, ‘Souvignier Gris’, ‘Sauvignon Kretos’, ‘Sauvignon Nepis’, ‘Sauvignon Rytos’, ‘Solaris’, ‘Soreli’. ‘Merlot’ (red) and ‘Glera’ (white) were included as control. The experimental vineyard was established in Castelfranco Veneto on the plain, in 2014. Spray treatments were applied against downy and powdery mildew, by using only copper and sulphur. Grape production, grape quality, and phenology were recorded over a six-year-period, while disease resistance (downy mildew, powdery mildew, black rot and anthracnose) was detected only during a few years. The most significant findings were: a) all varieties showed a good level of downy mildew resistance, especially ‘Cabernet Carbon’, ‘Monarch’, ‘Prior’, UD 31.103, ‘Muscaris’, ‘Solaris’, ‘Souvignier Gris’, ‘Bronner’, ‘Fleurtai’, ‘Aromera’; b) no powdery mildew attacks were detected in any variety; c) ‘Monarch’, ‘Muscaris’, ‘Solaris’ and ‘Souvignier Gris’ also showed a high level of resistance towards black rot and anthracnose; d) red grape varieties had an earlier bud burst as compared to ‘Merlot’, and, concerning ripening, some varieties were earlier than ‘Merlot’, other ones were later; e) white varieties had a later bud burst but an earlier ripening time as compared to ‘Glera’; f) grape production and quality changed significantly depending on the varieties, being titratable acidity higher than 6.4 g L-1 tartaric acid and pH lower than 3.5; also the year affected in a significant way those parameters as well as the interaction between the genotype and the year. In conclusion, the tested varieties behaved positively in terms of environmental sustainability

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules

BACKGROUND: The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson's disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown. RESULTS: By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca2+ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains. CONCLUSIONS: Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease

Is it really advantageous to operate proximal femoral fractures within 48 h from diagnosis? – A multicentric retrospective study exploiting COVID pandemic-related delays in time to surgery

Objectives: Hip fractures in the elderly are common injuries that need timely surgical management. Since the beginning of the pandemic, patients with a proximal femoral fracture (PFF) experienced a delay in time to surgery. The primary aim of this study was to evaluate a possible variation in mortality in patients with PFF when comparing COVID-19 negative versus positive. Methods: This is a multicentric and retrospective study including 3232 patients with PFF who underwent surgical management. The variables taken into account were age, gender, the time elapsed between arrival at the emergency room and intervention, pre-operative American Society of Anesthesiology score, pre-operative cardiovascular and respiratory disease, and 10-day/1-month/6-month mortality. For 2020, we had an additional column, “COVID-19 swab positivity.” Results: COVID-19 infection represents an independent mortality risk factor in patients with PFFs. Despite the delay in time-to-surgery occurring in 2020, no statistically significant variation in terms of mortality was detected. Within our sample, a statistically significant difference was not detected in terms of mortality at 6 months, in patients operated within and beyond 48 h, as well as no difference between those operated within or after 12/24/72 h. The mortality rate among subjects with PFF who tested positive for COVID-19 was statistically significantly higher than in patients with PFF who tested. COVID-19 positivity resulted in an independent factor for mortality after PFF. Conclusion: Despite the most recent literature recommending operating PFF patients as soon as possible, no significant difference in mortality was found among patients operated before or after 48 h from diagnosis

RADICL-seq identifies general and cell type–specific principles of genome-wide RNA-chromatin interactions

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Targeting the complexity of mouse olfactory system

In recent years the research on the olfactory system has entered a phase of deep innovation, regardless of the animal model taken as a reference. While the advancements achieved in different fields have provided answer to old questions, the striking evidences that have emerged in this new olfactory landscape have brought new ideas, new hypothesis and new scientific problems that necessarily need to be approached with adequate tools and strategies. The work presented in this thesis has targeted three different issues among the more intriguing ones concerning the murine olfactory system. The project described in the first section has conf ronted with the molecular identity of the Calcium-activated chloride channel responsible for the amplification of cationic currents in olfactory sensory neurons, a key mechanism for the triggering of action potentials after binding of odour molecules with their specific receptors. Olfactory microvillar cells constitute a cell population largely represented in the main olfactory epithelium, but their role is still poorly understood mostly because a precise genomic characterization of this cell-type has never been undertaken; the project presented in the second section has tried to reveal the genomic fingerprint of microvillar cells through a custom gene expression profiling. The data presented in the third section of this thesis are the result of a deep genomic investigation that has targeted the entire transcriptome of the olfactory sensory epithelium exploiting a newly developed high-throughput tagging approach derived from the Cap-Analysis of Gene Expression (CAGE) technology. The potential of this workflow has allowed revealing new details about the expression of pheromone vomeronasal receptors in the main olfactory epithelium

Capture-seq protocol and TE-reX pipeline guidelines for detection of recombination of repeat elements in short- and long-DNA reads libraries

Summary: Recombination of repeat elements is an important source of genomic variation in human tissues. Here, we describe steps to prepare libraries enriched for repeat elements starting from the genomic DNA of any species. We also detail the computational post-processing of the TE-reX pipeline output to generate datasets of putative somatic and polymorphic recombination events.For complete details on the use and execution of this protocol, please refer to Pascarella et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

ReUse: REgressive Unet for Carbon Storage and Above-Ground Biomass Estimation

The United Nations Framework Convention on Climate Change (UNFCCC) has recently established the Reducing Emissions from Deforestation and forest Degradation (REDD+) program, which requires countries to report their carbon emissions and sink estimates through national greenhouse gas inventories (NGHGI). Thus, developing automatic systems capable of estimating the carbon absorbed by forests without in situ observation becomes essential. To support this critical need, in this work, we introduce ReUse, a simple but effective deep learning approach to estimate the carbon absorbed by forest areas based on remote sensing. The proposed method’s novelty is in using the public above-ground biomass (AGB) data from the European Space Agency’s Climate Change Initiative Biomass project as ground truth to estimate the carbon sequestration capacity of any portion of land on Earth using Sentinel-2 images and a pixel-wise regressive UNet. The approach has been compared with two literature proposals using a private dataset and human-engineered features. The results show a more remarkable generalization ability of the proposed approach, with a decrease in Mean Absolute Error and Root Mean Square Error over the runner-up of 16.9 and 14.3 in the area of Vietnam, 4.7 and 5.1 in the area of Myanmar, 8.0 and 1.4 in the area of Central Europe, respectively. As a case study, we also report an analysis made for the Astroni area, a World Wildlife Fund (WWF) natural reserve struck by a large fire, producing predictions consistent with values found by experts in the field after in situ investigations. These results further support the use of such an approach for the early detection of AGB variations in urban and rural areas