943 research outputs found
Characterization of non-driver mutations and identification of different outcomes and treatment strategies based on NGS results in myelofibrosis patients in different clinical stages
Myelofibrosis (MF), a chronic Ph-negative myeloproliferative neoplasm, is a clinically and genetically heterogeneous disease. Beside driver mutations that represent the hallmark of pathogenesis, the rapid advancements in gene sequencing technology, like Next Generation Sequencing (NGS), have led to discover additional mutations revealing biological insights in MF and possible novel prognostic markers. However, current clinical prognostic risk-stratification models are the most used in clinical practice. The aim of our project is to use and validate NGS technology in transplant-eligible MF patients, prospectively refining a more reliable prognostic risk assessment and risk-adapted treatment strategy, in real-life setting.
We enrolled 68 MF patients, consecutively diagnosed and followed at Sapienza University. Twenty-two out of 68 (32%) subjects had secondary MF (SMF), diagnosed post essential thrombocythemia and polycythaemia vera. As for driver mutations, 52%, 28% and 3% of patients, carried JAK2V617F, CALR and MPL mutation, respectively. One patient had double mutation (JAK2V617F/MPL); 10 (15%) patients were identified as triple negative. We found 72 non-driver mutations; 13 out of 68 (19%) patients had a high molecular risk (HMR) profile. The most frequently mutated genes were TET2 (n=14, 20%), DNMT3A (n=7, 10%) and ASXL1 (n=11, 16%). ASXL1 mutated patients carried distinct high-risk clinical features, including higher value of LDH (p<0.001), monocytes (p<0.001), spleen diameter (p=0.035) and symptoms (p=0.042). Focusing on mutational profile, no significant differences were detected comparing PMF and SMF. According to the IPSS survival risk distribution at diagnosis in PMF, 32 patients were classified as low risk (70%), 9 as intermediate-1 (20%), 3 as intermediate-2 (6%) and 2 as high (4%). In SMF, the MYSEC-PM risk distribution identified 7 patients as low risk (32%), 13 (59%) as intermediate-1 and 2 as intermediate-2 (9%). The real-life application of MIPSS70 model identified 22 patients, who were previously categorized as low risk according to IPSS/MYSEC-PM, in intermediate risk and allocated 3 patients, previously considered as intermediate risk, in high-risk category. Category shift was due to HMR profile in 7 (10%) patients. Allotransplant was recommended in 5 high-risk patients immediately after NGS results. HMR profile was determinant in proposing transplant choice in 3/9 (33%) intermediate MIPSS70 risk patients. High-risk MIPSS70 category showed inferior OS (p=0.017) and EFS (p=0.005) than low/intermediate risks. HMR profile negatively influenced overall outcome, both in terms of OS and EFS (p<0.05). ASXL1 mutated patients had inferior EFS (p=0.012) compared to ASXL1 wt. These findings were confirmed only in PMF. Moreover, RUNX1 mutated patients had significantly shorter OS than RUNX1 wt (p=0.002). We analysed 21 patients who received ruxolitinib confirming its clinical benefit irrespective of biological findings. Overall, 28 (41%) patients were on clinical treatment-free follow-up. Thirty-nine (57%) patients needed a treatment: 21 (54%) ruxolitinib, 3 (8%) interferon, 14 (36%) hydroxyurea, 1 (2%) allotransplant without a bridge therapy. Globally, 7 patients were allografted. Overall, 4 out of 68 (6%) patients died: 2 due to blast crisis, 1 for transplant complication and 1 for SARS-CoV-2 infection in MF progression.
In our monocentric prospective real-life study, NGS analysis allows a better risk stratification and a more accurate risk-adapted therapy of MF patients, contributing to characterize mutational landscape of the disease
A role in pH homeostasis regulation by genes related to Neurodevelopmental Disorders: TBC1D24 and ATP6V1A.
TBC1D24 is a gene mutated in a spectrum of neurodevelopmental disorders, from mild epilepsy to severe epileptic encephalopathy. TBC1D24 is involved in brain development, synaptic vesicle trafficking and synaptic function; yet the molecular mechanisms mediating these roles and their relationship to brain dysfunction are largely unknown. TBC1D24 is unique in containing conserved TBC and TLDc domains; importantly, TLDc proteins have been recently described as interactors of the essential complex of V-ATPase. V-ATPase is a multisubunit proton pump that acidifies intracellular organelles through the hydrolysis of ATP. In this thesis, we aimed to explore the interaction between Tbc1d24 and V-ATPase and its physio-pathological role in neuronal cells. We found that Tbc1d24 interacts with the V-ATPase V1 cytosolic domain subunits ATP6V1A and ATP6V1B2 in the brain. By employing a mouse model of chronic loss of Tbc1d24, we demonstrated that loss of Tbc1d24 led to a cytosolic shift of ATP6V1A and ATP6V1B2 subunits, suggesting an unproper assembly state of the complex. This phenotype was accompanied by the alteration of intracellular organelles acidification with increased pH and impairment of autophagic flux. Given the localization of Tbc1d24 at synaptic sites and the relevance of V-ATPase proton pumping activity in synaptic vesicles, we evaluated synaptic ultrastructure and synaptic vesicles acidification. In lack of Tbc1d24, presynaptic compartments showed fewer synaptic vesicles and the accumulation of aberrant endosomal-like structures that engulfed the pre-synapse. Moreover, we found an increased luminal pH of synaptic vesicles.
Concurrently, in collaboration with the group of Professor Guerrini (Meyer Hospital, Florence) we identified de novo mutations in ATP6V1A in patients affected by epileptic encephalopathy with different severity. We characterized patients’ fibroblasts finding that pathogenic mutations can alter protein stability and the overall functionality of V-ATPase complex, leading to alterations in the endo-lysosomal pH. Ultrastructural analysis of patients’ fibroblasts and patients-derived iNeurons revealed the presence of aberrant lysosomes engulfed with different non-degraded materials, a typical hallmark of lysosomal diseases.
All together these findings uncover a novel function for Tbc1d24 as regulator of V-ATPase activity and suggest pH dysregulation as key cellular mechanism that possibly underpin the pathogenesis in TBC1D24- and ATP6V1A-associated disorders
Physical retrieval of sea surface temperature with SEVIRI infrared measurements: Application to the Mediterranean in the period 2013-2019
In this work, we show the results of one of the very few physical-based approaches for the estimation of surface parameters from infrared instruments on board geostationary platforms. The approach has been developed for the infrared channels of the Spinning Enhanced Visible and Infrared Imager (SEVIRI) on board the Meteosat Second Generation (MSG) geostationary platform and here has been applied to the region encompassing the Mediterranean, observed by more than 170000 SEVIRI pixels every 15 minutes, for the physical retrieval of the Sea Surface Temperature (SST). The methodology is based on a Kalman filter and enables simultaneous retrieval of surface emissivity and temperature from SEVIRI infrared radiance measurements using channels at 8.7, 10.8, and 12 μm. When run on a PC with a CPU clocked at 2.7GHz and 8GB of RAM, the processor needs about 0.002 s for each pixel to retrieve SST. So for the Mediterranean region, it takes about 7 minutes with a single CPU, i.e. this processor is ready for real-time computing for this region. We tested the processor by comparing its results with SST retrieved from the Advanced Very High Resolution Radiometer (AVHRR) satellite measurements. AVHRR and SEVIRI L2 SST show an excellent agreement with correlation coefficients larger than 0.99, with no bias and a root mean squared difference of less than 0.2 °C. Finally, this methodology shows that the Mediterranean Sea has warmed by four cents of Celsius per year in the last decade
Predictive Multi Experiment Approach for the Determination of Conjugated Phenolic Compounds in Vegetal Matrices by Means of LC-MS/MS
Polyphenols (PCs) are a numerous class of bioactive molecules and are known for their antioxidant activity. In this work, the potential of the quadrupole/linear ion trap hybrid mass spectrometer (LIT-QqQ) was exploited to develop a semi-untargeted method for the identification of polyphenols in different food matrices: green coffee, Crocus sativus L. (saffron) and Humulus lupulus L. (hop). Several conjugate forms of flavonoids and hydroxycinnamic acid were detected using neutral loss (NL) as a survey scan coupled with dependent scans with enhanced product ion (EPI) based on information-dependent acquisition (IDA) criteria. The presented approach is focused on a specific class of molecules and provides comprehensive information on the different conjugation models that are related to specific base molecules, thus allowing a quick and effective identification of all possible combinations, such as mono-, di-, or tri-glycosylation or another type of conjugation such as quinic acid esters
Human-Derived Cortical Neurospheroids Coupled to Passive, High-Density and 3D MEAs:A Valid Platform for Functional Tests
: With the advent of human-induced pluripotent stem cells (hiPSCs) and differentiation protocols, methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality (3D) would make them more representative of an in-vivo environment. Thus, human-derived 3D structures are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, methodologies to create 3D structures with controlled cellular density and composition and platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapidly generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by using micro-electrode arrays (MEAs) with different types (i.e., passive, C-MOS, and 3D) and number of electrodes. Neurospheroids grown in free culture and transferred on MEAs exhibited functional activity that can be chemically and electrically modulated. Our results indicate that this model holds great potential for an in-depth study of signal transmission to drug screening and disease modeling and offers a platform for in-vitro functional testing
Complication of Gastric Cancer Surgery: A Single Centre Experience
Background/aim: Gastric cancer surgery is still characterised by high morbidity and mortality. However, in 2018 an online platform, GASTRODATA has been proposed in Europe to standardize the recording of gastric surgery complications. The aim of the study was to present a single center experience regarding incidence and grading of acute postoperative complications in a population of patients treated surgically for gastric cancer on the basis of the gastrodata online platform.
Patients and methods: The present study was a single center, observational, retrospective trial held in the General Surgery Unit of the Sant'Andrea Hospital of Rome. The study included 181 consecutive patients who underwent gastric surgical resection for cancer from May 2004 to December 2020 with curative R0 purpose.
Results: Thirty-three percent of patients reported at least one complication, while seventeen percent of the whole population reported a complication classified as at least grade 3 on the Clavien Dindo Classification. The most frequent complications were disorders of the respiratory system (13.3%), followed by bleeding (7.6%) and wound infections (6.2%). Deaths accounted for 3.7% of the population.
Conclusion: A list of defined complications of gastrectomy, if systematically adopted in the Literature, could lead to a reduction in the wide variation of proposals for treatment and assessment. Objectively evaluating the impact of complications on outcomes can lead to quality improvement project proposals
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