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Large biomolecular systems are at the heart of many essential cellular processes. The dynamics and energetics of an increasing number of these systems are being studied by computer simulations. Pushing the limits of length- and timescales that can be accessed by current hard- and software has expanded the ability to describe biomolecules at different levels of detail. We focus in this review on the ribosome, which exemplifies the close interplay between experiment and various simulation approaches, as a particularly challenging and prototypic nanomachine that is pivotal to cellular biology due to its central role in translation. We sketch widely used simulation methods and demonstrate how the combination of simulations and experiments advances our understanding of the function of the translation apparatus based on fundamental physics.

Clonal haematopoiesis of indeterminant potential (CHIP) increases in frequency with age. The effect of CHIP on the mobilization of autologous CD34+ peripheral blood stem cells (PBSC) has not been reported. This study uses a DNA-based targeted candidate gene approach to identify the presence of somatic mutations in ASXL1, DNMT3A, JAK2, SF3B1, TET2 and TP53 in CD34+ haematopoietic progenitor cell-apheresis products of 96 patients who undergo PBSC mobilization for autologous stem cell transplantation (ASCT). Variants were identified in a significantly greater proportion of patients who experience poor CD34+ PBSC mobilization. A DNA-based targeted candidate gene array is able to predict poor CD34+ PBSC mobilization and may be deployed pre-emptively to minimize mobilization and graft failures.

This study evaluated the impact of a school-based program designed to reduce implicit prejudice towards migrants in fifth-grade school children. The program used empathy and perspective taking and direct and indirect contact as strategies to reduce ethnic prejudice. Multiple activities were used, including drawings by migrant children as instruments to promote inclusive behaviors. One hundred and five students were divided into two groups (control, experimental), and the children in each completed the Child-IAT (Implicit Association Test) before and after the program, to measure their implicit prejudice. Only the experimental group participated in the program. The results showed a significant reduction in the implicit prejudice in the experimental group after the educational program, but not in the control group. The results are discussed considering the practical implications of such a school program.

La Società Italiana di Ricerca Didattica (SIRD) ha promosso una ricerca nazionale per avviare una riflessione sulla didattica a distanza (DaD) adottata durante l’emergenza Covid-19. La ricerca è stata condotta mediante l’utilizzo di un questionario a cui hanno risposto oltre 16.000 insegnanti di ogni ordine e grado, dislocati in tutto il territorio nazionale. Il contributo descrive la metodologia utilizzata per l’analisi dei dati qualitativi e presenta la struttura categoriale emersa. Le domande aperte erano volte a far emergere i punti di forza e di debolezza della DaD, nonché le difficoltà riscontrate dagli studenti. Uno spazio dedicato a commenti e riflessioni, inoltre, ha consentito di cogliere in modo maggiormente circostanziato il punto di vista degli insegnanti interpellati.

This chapter addresses building and enhancing resilience in children experiencing a crisis or facing adversity in the twenty-first century. It reflects on an experimental research project for developing an educational and didactical path in order to promote resilience which could be introduced into teaching practices and to verify the effectiveness of this formative path. The chapter examines the possibility of influencing the connection between learning and the affective, cognitive, and psychological variables, especially when promoting resilience. The teaching contents for the project were chosen in a way to directly connect theory and practice. Resilience was connected to scientific thinking, history, geography, sport and storytelling, and the didactical path was structured into learning units in which different methods and instruments were used. The project focused on enhancing social competences, problem-solving, creativity and humour to recognise resources at three levels: personal, classroom and environmental. The result makes a case for the need to realise the full potential of school teachers as resilience tutors.

Fatty acid synthesis is crucial in supporting the survival and proliferation of multiple forms of cancer. The high metabolic demands of fatty acid synthesis are regulated by the AMP-activated kinase and activity of the fatty acid synthase enzyme. In this study, the roles of these enzymes in diffuse large B-cell lymphoma (DLBCL) were investigated by genetic knock-down and pharmacological activation of AMP-activated kinase by metformin, and selective inhibition of fatty acid synthase using the novel drug Fasnall. We observed distinct heterogeneity and adaptive plasticity of lipid metabolism in a panel of DLBCL cell lines and demonstrate the therapeutic potential of inhibiting fatty acid synthesis in a subset of DLBCL cells. The translational relevance of these

Il contesto scolastico rappresenta un luogo privilegiato per rispondere alle sfide della complessità del reale, promuovendo una metamorfosi del rapporto apprendimento-insegnamento (Morin, 2001). La gestione dei contesti educativi, caratterizzati per natura da molteplici differenze di cultura, abilità, genere e sensibilità (Dovigo, 2008), richiede lo sviluppo di un corpo di competenze che consentano all'insegnante di operare in una direzione interculturale e inclusiva. Il contributo presenta l'articolazione e gli esiti di un percorso di formazione sulla riduzione del pregiudizio etnico rivolto a docenti pre-servizio iscritti al Corso di Laurea in Scienze della formazione primaria della Sapienza, Università di Roma. Tra gli obiettivi principali dell'intervento formativo, quello di ridurre il pregiudizio implicito ed esplicito degli studenti in una prospettiva più ampia di promozione delle loro competenze interculturali.

Introduction Mean platelet volume (MPV) assists the differential diagnosis of inherited thrombocytopenia (IT) but lacks standardisation and varies between automated analysers. Classification of IT based on mean platelet diameter (MPD) has been proposed by an international collaborative study but has not been validated. Methods To assess the applicability of MPD to classify forms of IT, digital images of blood films from patients with established genetic causes for IT were generated, and the MPD measured (ZEISS Axio-scanner and Image J software) by a blinded reviewer. Comparison was made to the proposed classification system. Results Mean platelet volume was measured in thrombocytopenia with different genetic aetiologies, bilallelic BSS (bBSS) (n = 1), monoallelic BSS (mBSS) (n = 2), MYH9-related disorders (MYH9-RD) (n = 11), GFI1B-related thrombocytopenia (RT) (n = 15), FLI1-RT (n = 2), TUBB1-RT (n = 3), ITGA2B/ITGB3-RT (n = 1), RUNX1-RT (n = 2) and controls (n = 54). bBSS and 82% of MYH9-RD samples had MPD >4 μm which correlated with “IT with giant platelets.” Only 55% of samples expected in the “large platelet group” had MPD meeting the classification cut-off (MPD >3.2 μm). FLI1-RT MPD were significantly larger than expected whilst ITGA2B/ITGB3-RT MPD were smaller than proposed. MPD in FPD/AML were “normal.” Conclusion Platelet MPD measurements are a useful guide to classify IT, but the time taken to record measurements may limit clinical applicability.

MYH9-related disorders (MYH9-RDs) caused by mutation of the MYH9 gene which encodes non-muscle myosin heavy-chain-IIA (NMMHC-IIA), an important motor protein in hemopoietic cells, are the most commonly encountered cause of inherited macrothrombocytopenia. Despite distinguishing features including an autosomal dominant mode of inheritance, giant platelets on the peripheral blood film accompanied by leucocytes with cytoplasmic inclusion bodies (dohle-like bodies), these disorders remain generally under-recognized and often misdiagnosed as immune thrombocytopenia (ITP). This may result in inappropriate treatment with corticosteroids, immunosupressants and in some cases, splenectomy. We explored the efficacy of next generation sequencing (NGS) with a candidate gene panel to establish the aetiology of thrombocytopenia for individuals who had been referred to our center from hematologists in the Australasian region in whom the cause of thrombocytopenia was suspected to be secondary to an inherited condition but which remained uncharacterized despite phenotypic investigations. Pathogenic MYH9 variants were detected in 15 (15/121, 12.4%) individuals and the pathogenecity of a novel variant of uncertain significance was confirmed in a further two related individuals following immunofluorescence (IF) staining performed in our laboratory. Concerningly, only one (1/17) individual diagnosed with MYH9-RD had been referred with this as a presumptive diagnosis, in all other cases (16/17, 94.1%), a diagnosis was not suspected by referring clinicians, indicating a lack of awareness or a failing of our diagnostic approach to these conditions. We examined the mean platelet diameter (MPD) measurements as a means to better identify and quantify platelet size. MPDs in cases with MYH9-RDs were significantly larger than controls (p < 0.001) and in 91% were greater than a previously suggested threshold for platelets in cases of ITP. In addition, we undertook IF staining in a proportion of cases and confirm that this test and/or NGS are satisfactory diagnostic tests. We propose that fewer cases of MYH9-RDs would be missed if diagnostic algorithms prioritized IF and/or NGS in cases of thrombocytopenia associated with giant platelets, even if dohle-like bodies are not appreciated on the peripheral blood film. Finally, our report describes the long-term use of a thrombopoietin agonist in a case of MYH9-RD that had previously been diagnosed as ITP, and demonstrates that treatment with these agents may be possible, and is well tolerated, in this group of patients.

Group 1 innate lymphoid cells include natural killer (NK) cells and ILC1s, which mediate the response to intracellular pathogens. Thymic NK (tNK) cells were described with hybrid features of immature NK cells and ILC1 but whether these cells are related to NK cells or ILC1 has not been fully investigated. We report that murine tNK cells expressed the NK-cell associated transcription factor EOMES and developed independent of the essential ILC1 factor TBET, confirming their placement within the NK lineage. Moreover, tNK cells resemble NK cells rather than ILC1 in their requirements for the E protein transcription factor inhibitor ID2. We provide further insight into the mechanisms governing tNK-cell development by showing that the transcription factor ETS1 prevented tNK cell acquisition of the conventional NK-cell maturation markers CD11b and KLRG1. Our data reveal few ILC1 in the thymus and clarify the identity and developmental requirements of tNK cells.

The NK cell population is characterized by distinct NK cell subsets that respond differently to the various activating stimuli. For this reason, the determination of the optimal cytotoxic activation of the different NK cell subsets can be a crucial aspect to be exploited to counter cancer cells in oncologic patients. To evaluate how the triggering of different combination of activating receptors can affect the cytotoxic responses of different NK cell subsets, we developed a microbead-based degranulation assay. By using this new assay, we were able to detect CD107a+ degranulating NK cells even within the less cytotoxic subsets (i.e., resting CD56bright and unlicensed CD56dim NK cells), thus demonstrating its high sensitivity. Interestingly, signals delivered by the co-engagement of NKp46 with 2B4, but not with CD2 or DNAM-1, strongly cooperate to enhance degranulation on both licensed and unlicensed CD56dim NK cells. Of note, 2B4 is known to bind CD48 hematopoietic antigen, therefore this observation may provide the rationale why CD56dim subset expansion correlates with successful hematopoietic stem cell transplantation mediated by alloreactive NK cells against host T, DC and leukemic cells, while sparing host non-hematopoietic tissues and graft versus host disease. The assay further confirms that activation of LFA-1 on NK cells leads to their granule polarization, even if, in some cases, this also takes to an inhibition of NK cell degranulation, suggesting that LFA-1 engagement by ICAMs on target cells may differently affect NK cell response. Finally, we observed that NK cells undergo a time-dependent spontaneous (cytokine-independent) activation after blood withdrawal, an aspect that may strongly bias the evaluation of the resting NK cell response. Altogether our data may pave the way to develop new NK cell activation and expansion strategies that target the highly cytotoxic CD56dim NK cells and can be feasible and useful for cancer and viral infection treatment.

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. SummaryBackground Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.

Hemizygous deletion of a variable region on chromosome 11q containing FLI1 causes an inherited platelet-related bleeding disorder in Paris-Trousseau thrombocytopenia and Jacobsen syndrome. These multisystem disorders are also characterized by heart anomalies, changes in facial structure, and intellectual disability. We have identified a consanguineous family with autosomal recessive inheritance of a bleeding disorder that mimics Paris-Trousseau thrombocytopenia but has no other features of the 11q23 deletion syndrome. Affected individuals in this family have moderate thrombocytopenia; absent collagen-induced platelet aggregation; and large, fused α-granules in 1% to 5% of circulating platelets. This phenotype was caused by a FLI1 homozygous c.970C>T-point mutation that predicts an arginine-to-tryptophan substitution in the conserved ETS DNA-binding domain of FLI1. This mutation caused a transcription defect at the promoter of known FLI1 target genes GP6, GP9, and ITGA2B, as measured by luciferase assay in HEK293 cells, and decreased the expression of these target proteins in affected members of the family as measured by Western blotting of platelet lysates. This kindred suggests abnormalities in FLI1 as causative of Paris-Trousseau thrombocytopenia and confirms the important role of FLI1 in normal platelet development.

Experimental models of diabetic retinopathy (DR) have had a crucial role in the comprehension of the pathophysiology of the disease and the identification of new therapeutic strategies. Most of these studies have been conductedin vivo, in animal models. However, a significant contribution has also been provided by studies on retinal cultures, especially regarding the effects of the potentially toxic components of the diabeticmilieuon retinal cell homeostasis, the characterization of the mechanisms on the basis of retinal damage, and the identification of potentially protective molecules. In this review, we highlight the contribution given by primary retinal cultures to the study of DR, focusing on early neuroglial impairment. We also speculate on possible themes into which studies based on retinal cell cultures could provide deeper insight.

Essentials The phenotypes of different growth factor-independent 1B (GFI1B) variants are not established. GFI1B variants produce heterogeneous clinical phenotypes dependent on the site of mutation. Mutation of the first non-DNA-binding zinc-finger causes a mild platelet and clinical phenotype. GFI1B regulates the CD34 promoter; platelet CD34 expression is an indicator of GFI1B mutation.Background Mutation of the growth factor-independent 1B (GFI1B) fifth DNA-binding zinc-finger domain causes macrothrombocytopenia and α-granule deficiency leading to clinical bleeding. The phenotypes associated with GFI1B variants disrupting non-DNA-binding zinc-fingers remain uncharacterized. Objectives To determine the functional and phenotypic consequences of GFI1B variants disrupting non-DNA-binding zinc-finger domains. Methods The GFI1B C168F variant and a novel GFI1B c.2520 + 1_2520 + 8delGTGGGCAC splice variant were identified in four unrelated families. Phenotypic features, DNA-binding properties and transcriptional effects were determined and compared with those in individuals with a GFI1B H294 fs mutation of the fifth DNA-binding zinc-finger. Patient-specific induced pluripotent stem cell (iPSC)-derived megakaryocytes were generated to facilitate disease modeling. Results The DNA-binding GFI1B variant C168F, which is predicted to disrupt the first non-DNA-binding zinc-finger domain, is associated with macrothrombocytopenia without α-granule deficiency or bleeding symptoms. A GFI1B splice variant, c.2520 + 1_2520 + 8delGTGGGCAC, which generates a short GFI1B isoform that lacks non-DNA-binding zinc-fingers 1 and 2, is associated with increased platelet CD34 expression only, without quantitative or morphologic platelet abnormalities. GFI1B represses the CD34 promoter, and this repression is attenuated by different GFI1B zinc-finger mutations, suggesting that deregulation of CD34 expression occurs at a direct transcriptional level. Patient-specific iPSC-derived megakaryocytes phenocopy these observations. Conclusions Disruption of GFI1B non-DNA-binding zinc-finger 1 is associated with mild to moderate thrombocytopenia without α-granule deficiency or bleeding symptomatology, indicating that the site of GFI1B mutation has important phenotypic implications. Platelet CD34 expression appears to be a common feature of perturbed GFI1B function, and may have diagnostic utility.

Group 1 innate lymphoid cells include natural killer (NK) cells and ILC1s, which mediate the response to intracellular pathogens. Thymic NK (tNK) cells were described with hybrid features of immature NK cells and ILC1 but whether these cells are related to NK cells or ILC1 has not been fully investigated. We report that murine tNK cells expressed the NK-cell associated transcription factor EOMES and developed independent of the essential ILC1 factor TBET, confirming their placement within the NK lineage. Moreover, tNK cells resemble NK cells rather than ILC1 in their requirements for the E protein transcription factor inhibitor ID2. We provide further insight into the mechanisms governing tNK-cell development by showing that the transcription factor ETS1 prevented tNK cell acquisition of the conventional NK-cell maturation markers CD11b and KLRG1. Our data reveal few ILC1 in the thymus and clarify the identity and developmental requirements of tNK cells.

Although NK cells are considered part of the innate immune system, a series of evidences has demonstrated that they possess characteristics typical of the adaptive immune system. These NK adaptive features, in particular their memory-like functions, are discussed from an ontogenetic and evolutionary point of view.

Trisomy 12, the third most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), confers an intermediate prognosis. In our cohort of 104 untreated patients carrying +12, NOTCH1 mutations occurred in 24% of cases and were associated to unmutated IGHV genes (P=0.003) and +12 as a sole cytogenetic abnormality (P=0.008). NOTCH1 mutations in +12 CLL associated with an approximately 2.4 fold increase in the risk of death, a significant shortening of survival (P

The present work reports on the investigation of oxidation states, local symmetry, and optical properties of manganese and iron species in natural cordierite (Mg, Fe) 2 Al 4 Si 5 O 18 , by means of energy-dispersive X-ray fluorescence, electron paramagnetic resonance (EPR), photoluminescence (PL) and optical absorption techniques. In particular, specific EPR characteristics show the presence of distinct Mn 2+ octahedral sites in the crystal lattice, and a small amount of Mn 4+ ions in tetrahedral sites. The absence of EPR signal ascribable to Mn 4+ in octahedral sites turns out to be consistent with the lack of narrow PL lines from d 3 ions in octahedral crystal field (CF). Evidence of Mn 3+ in octahedral sites is instead found in steady-state and time-resolved PL measurements. EPR spectra give further details on iron site occupancy, showing the occurrence of Fe 3+ ions in octahedral sites. Characteristics ascribable to all the identified metal species have been found in the optical absorption spectrum or in the PL excitation spectrum, as arising from the expected CF transitions of Fe 2+ , Fe 3+ , Mn 2+ , Mn 3+ , and Mn 4+ ions. Interestingly, the results show a polarization-dependent correlation between the manganese luminescence and the pleochroic absorption band at about 17,000 cm −1 . This result reveals a pleochroic contribution from the Mn 3+ spin-allowed 5 E→ 5 T 2 transition under the visible pleochroic band attributed to Fe 2+ (oct)→Fe 3+ (tet) transitions.

Abstract 1784 Introduction: The introduction of whole exome sequencing has allowed to unravel novel molecular lesions in CLL. NOTCH1, SF3B1 and BIRC3 mutations are detected, according to the phases of disease, in 4–12%, 5–17% and 4–24% of patients, respectively. In retrospective studies, their presence has been shown to correlate with overall survival (OS) and treatment-free interval shortening. Aims: To define the incidence, correlation with known prognostic factors and clinical impact of NOTCH1, SF3B1 and BIRC3 mutations in CLL patients undergoing first-line treatment. Methods: We evaluated 162 CLL patients enrolled in the GIMEMA LLC0405 protocol (n=80) for patients aged 65 yrs or 60–65 if not eligible for fludarabine-based programs). In the GIMEMA LLC0405 protocol, patients were stratified into low and high-risk: patients with del17p or with del11q plus an unmutated IGHV status and/or CD38 positivity and/or ZAP70 positivity were considered as high-risk (HR) and underwent Fludarabine plus Campath, followed by stem cell transplantation procedures, whereas low-risk patients received Fludarabine and Cyclophosphamide. The MLL21445 protocol consisted of 8 cycles of Chlorambucil and 6 of Rituximab induction treatment. NOTCH1 (exon 34), SF3B1 (exons 14 and 15) and BIRC3 (exons 2–9, including splicing sites) were screened by Sanger sequencing on either genomic DNA (gDNA) or whole genome amplified DNA (WGA) collected at the time of treatment. These studies were not part of the clinical protocols. Results: NOTCH1 mutations were detected at the time of treatment in 18 cases (22%) enrolled in the LLC0405 study. There was a significant association with high-risk stratification (p=0.036), namely with an IGHV unmutated status (p=0.0035), CD38 (p=0.03), +12 (p=0.034) and, partly, ZAP-70 expression (p=0.059). While the overall response rate (ORR) did not differ between NOTCH1 mutated vs wild-type (WT) cases (82% vs 77%, respectively), the complete response (CR) rate was significantly lower in NOTCH1 mutated patients (43% for WT vs 17% for NOTCH1 mutated cases; p=0.05). So far, no significant difference between mutated and WT patients has emerged in terms of OS and progression-free survival (PFS); this may be contributed by the fact that most NOTCH1 mutated cases were HR and were therefore treated more aggressively. SF3B1 mutations were recorded in 9 cases (11%); no significant associations were found with known biological parameters and, so far, with the ORR and CR rate. A single case harbored a BIRC3 mutation; this patient had an IGHV unmutated status, no FISH abnormalities and a concomitant SF3B1 mutation. In the ML21445 cohort, NOTCH1 mutations were found in 12 cases (15%), were associated with an unmutated IGHV status (p=0.047) and ZAP-70 expression (p=0.007), and did not impact on the ORR and CR rate. SF3B1 mutations were found in 11 cases (13%); no significant associations were found with known biological parameters and the ORR rate. Of interest, only 1/11 SF3B1 mutated patients achieved a CR. BIRC3 mutations were recorded in 3 patients (3.6%); of these, 2 were IGHV mutated, 1 had no cytogenetic abnormalities and 1 carried a del11q, while the third patient was IGHV unmutated status and had no cytogenetic abnormalities. No NOTCH1 and/or SF3B1 mutations were detected. Overall, NOTCH1, SF3B1 and BIRC3 mutations were largely mutually exclusive among each other and with TP53 lesions in the whole cohort. Conclusions: This study confirms the association of NOTCH1 mutations with unfavorable biologic markers and +12, while the presence of SF3B1 mutations was not coupled to poor prognostic markers in CLL patients requiring first-line treatment. Furthermore, it suggests that NOTCH1 mutations impact on the CR rate of young patients receiving Fluda-based regimens, while SF3B1 appears to impact on the CR rate of elderly patients treated with Chlorambucil and Rituximab. Given the small numbers of patients harboring BIRC3, it is at present difficult to draw any conclusion on the clinical impact of this mutation in the cohort of patients hereby analyzed. Disclosures: No relevant conflicts of interest to declare.