Multiple Lines of Evidence for a Potentially Seismogenic Fault Along the Central-Apennine (Italy) Active Extensional Belt–An Unexpected Outcome of the MW6.5 Norcia 2016 Earthquake

The Apenninic chain, in central Italy, has been recently struck by the Norcia 2016 seismic sequence. Three mainshocks, in 2016, occurred on August 24 (MW6.0), October 26 (MW 5.9) and October 30 (MW6.5) along well-known late Quaternary active WSW-dipping normal faults. Coseismic fractures and hypocentral seismicity distribution are mostly associated with failure along the Mt Vettore-Mt Bove (VBF) fault. Nevertheless, following the October 26 shock, the aftershock spatial distribution suggests the activation of a source not previously mapped beyond the northern tip of the VBF system. In this area, a remarkable seismicity rate was observed also during 2017 and 2018, the most energetic event being the April 10, 2018 (MW4.6) normal fault earthquake. In this paper, we advance the hypothesis that the Norcia seismic sequence activated a previously unknown seismogenic source. We constrain its geometry and seismogenic behavior by exploiting: 1) morphometric analysis of high-resolution topographic data; 2) field geologic- and morphotectonic evidence within the context of long-term deformation constraints; 3) 3D seismological validation of fault activity, and 4) Coulomb stress transfer modeling. Our results support the existence of distributed and subtle deformation along normal fault segments related to an immature structure, the Pievebovigliana fault (PBF). The fault strikes in NNW-SSE direction, dips to SW and is in right-lateral en echelon setting with the VBF system. Its activation has been highlighted by most of the seismicity observed in the sector. The geometry and location are compatible with volumes of enhanced stress identified by Coulomb stress-transfer computations. Its reconstructed length (at least 13 km) is compatible with the occurrence of MW≥6.0 earthquakes in a sector heretofore characterized by low seismic activity. The evidence for PBF is a new observation associated with the Norcia 2016 seismic sequence and is consistent with the overall tectonic setting of the area. Its existence implies a northward extent of the intra-Apennine extensional domain and should be considered to address seismic hazard assessments in central Italy

Rare among rare: phenotypes of uncommon CMT genotypes

(1) Background: Charcot-Marie-Tooth disease (CMT) is the most frequent form of inherited chronic motor and sensory polyneuropathy. Over 100 CMT causative genes have been identified. Previous reports found PMP22, GJB1, MPZ, and MFN2 as the most frequently involved genes. Other genes, such as BSCL2, MORC2, HINT1, LITAF, GARS, and autosomal dominant GDAP1 are responsible for only a minority of CMT cases. (2) Methods: we present here our records of CMT patients harboring a mutation in one of these rare genes (BSCL2, MORC2, HINT1, LITAF, GARS, autosomal dominant GDAP1). We studied 17 patients from 8 unrelated families. All subjects underwent neurologic evaluation and genetic testing by next-generation sequencing on an Ion Torrent PGM (Thermo Fischer) with a 44-gene custom panel. (3) Results: the following variants were found: BSCL2 c.263A > G p.Asn88Ser (eight subjects), MORC2 c.1503A > T p.Gln501His (one subject), HINT1 c.110G > C p.Arg37Pro (one subject), LITAF c.404C > G p.Pro135Arg (two subjects), GARS c.1660G > A p.Asp554Asn (three subjects), GDAP1 c.374G > A p.Arg125Gln (two subjects). (4) Expanding the spectrum of CMT phenotypes is of high relevance, especially for less common variants that have a higher risk of remaining undiagnosed. The necessity of reaching a genetic definition for most patients is great, potentially making them eligible for future experimentations

Minimally invasive aortic valve replacement with a sutureless valve through a right anterior mini-thoracotomy versus transcatheter aortic valve implantation in high-risk patients

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Identification and Functional Studies of Two New Dual-Oxidase 2 (DUOX2) Mutations in a Child with Congenital Hypothyroidism and a Eutopic Normal-Size Thyroid Gland

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Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis

Protection against deadly pathogens requires the production of high-affinity antibodies by B cells, which are generated in germinal centers (GCs). Alteration of the GC developmental program is common in many B cell malignancies. Identification of regulators of the GC response is crucial to develop targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte–induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.Published versio

Racial differences in systemic sclerosis disease presentation: a European Scleroderma Trials and Research group study

Objectives. Racial factors play a significant role in SSc. We evaluated differences in SSc presentations between white patients (WP), Asian patients (AP) and black patients (BP) and analysed the effects of geographical locations.Methods. SSc characteristics of patients from the EUSTAR cohort were cross-sectionally compared across racial groups using survival and multiple logistic regression analyses.Results. The study included 9162 WP, 341 AP and 181 BP. AP developed the first non-RP feature faster than WP but slower than BP. AP were less frequently anti-centromere (ACA; odds ratio (OR) = 0.4, P < 0.001) and more frequently anti-topoisomerase-I autoantibodies (ATA) positive (OR = 1.2, P = 0.068), while BP were less likely to be ACA and ATA positive than were WP [OR(ACA) = 0.3, P < 0.001; OR(ATA) = 0.5, P = 0.020]. AP had less often (OR = 0.7, P = 0.06) and BP more often (OR = 2.7, P < 0.001) diffuse skin involvement than had WP.AP and BP were more likely to have pulmonary hypertension [OR(AP) = 2.6, P < 0.001; OR(BP) = 2.7, P = 0.03 vs WP] and a reduced forced vital capacity [OR(AP) = 2.5, P < 0.001; OR(BP) = 2.4, P < 0.004] than were WP. AP more often had an impaired diffusing capacity of the lung than had BP and WP [OR(AP vs BP) = 1.9, P = 0.038; OR(AP vs WP) = 2.4, P < 0.001]. After RP onset, AP and BP had a higher hazard to die than had WP [hazard ratio (HR) (AP) = 1.6, P = 0.011; HR(BP) = 2.1, P < 0.001].Conclusion. Compared with WP, and mostly independent of geographical location, AP have a faster and earlier disease onset with high prevalences of ATA, pulmonary hypertension and forced vital capacity impairment and higher mortality. BP had the fastest disease onset, a high prevalence of diffuse skin involvement and nominally the highest mortality

PTBP1 regulates autism-associated FOXP2 gene by Alternative Splicing

Il "gene del linguaggio" Forkhead Box P2 (FOXP2) codifica per un fattore di trascrizione e diverse mutazioni nella regione codificante sono state correlate con il disturbo della parola e del linguaggio di tipo 1 (SPCH1). Esperimenti di linkage disequilibrium e genome wide association hanno associato FOXP2 anche a disturbi dello sviluppo neuronale, tra i quali i disturbi dello spettro autistico (ASDs). Tuttavia non sono note mutazioni nella regione codificante di FOXP2 in pazienti ASD, portando all'ipotesi di possibili difetti a livello post-trascrizionale. A tal proposito, mentre sono disponibili informazioni sulla regolazione da parte di miRNA, poco \ue8 noto sulla regolazione dello splicing alternativo di FOXP2 e sulle ribonucleoproteine coinvolte. La proteina FOXP2 \ue8 codificata dal trascritto full-length (FOXP2-FL), formato da 17 esoni canonici pi\uf9 due piccoli esoni alternativi, 3b e 4a, in grado di mantenere la cornice di lettura. L'analisi bioinformatica della sequenza genomica di FOXP2 ha rivelato la presenza di molteplici ipotetici siti di legame per la ribonucleoproteina Polypyrimidine Binding Tract Protein 1 (PTBP1) nelle regioni introniche attorno agli esoni 11 e 3b. PTBP1 \ue8 una ribonucleoproteina ubiquitaria in grado di regolare l\u2019esclusione esonica. Inoltre, PTBP1 ed il suo paralogo PTBP2, che spesso riconosce gli stessi siti di legame, svolgono un ruolo chiave durante il differenziamento neuronale. Mediante immunoprecipitazione di PTBP1 e degli mRNA ad esso legati (RIP) e sovraespressione di PTBP1 in cellule HEK293 abbiamo dimostrato che PTBP1 \ue8 in grado di legare i trascritti di FOXP2 e promuovere l\u2019esclusione dell'esone 11, generando il trascritto FOXP2-\u25b311. L'aumentato livello del trascritto FOXP2-\u25b311 \ue8 stato accompagnato da una significativa diminuzione del trascritto FOXP2-FL e della proteina da esso codificata. Abbiamo ulteriormente confermato il ruolo di PTBP1 nel promuovere l\u2019esclusione dell'esone 11 di FOXP2 tramite un saggio con minigene. Similmente, abbiamo dimostrato che anche la proteina PTBP2 marcata con Myc \ue8 in grado di promuovere la generazione del trascritto FOXP2-\u25b311. Successivamente, abbiamo analizzato l'abilit\ue0 di PTBP1 di regolare l'esone 3b. Malgrado la presenza di numerosi siti putativi di legame per PTBP1 attorno all'esone 3b, PTBP1 non \ue8 stato in grado di promuoverne l\u2019esclusione, suggerendo la specificit\ue0 di PTBP1 nella regolazione dell'esone 11. L'esclusione dell'esone 11 introduce un codone di terminazione prematuro (PTC) all'interno dell'esone 12 nel trascritto di FOXP2 che, se tradotto, codificherebbe per una proteina tronca mancante del dominio FOX per il legame al DNA, tuttavia un simile peptide non \ue8 stato rilevato nel nostro sistema cellulare in vitro. L'analisi della sequenza di FOXP2-\u25b311 ci ha fatto ipotizzare una sua degradazione mediante Nonsense Mediated Decay (NMD), che elimina gli mRNA contenenti dei PTC. Usando inibitori specifici abbiamo dimostrato che FOXP2-\u25b311 \ue8 un target di NMD. Infine, il silenziamento genico di PTBP1 non ha modificato l'espressione proteica di FOXP2, ma poich\ue9 il silenziamento di PTBP1 promuove l'espressione di PTBP2, abbiamo co-silenziato PTBP1 e PTBP2, ed osservato un aumento della proteina FOXP2 di 2.5 volte. L'analisi dei trascritti ha rilevato un incremento di FOXP2-FL di circa il 26%, ma nessuna variazione nell'espressione di FOXP2-\u25b311, suggerendo una regolazione operata da PTBP1 e PTBP2 indipendente dallo splicing alternativo. Poich\ue8 PTBP1 \ue8 in grado anche di controllare la trascrizione dei trascritti, abbiamo analizzato bioinformaticamente le regioni 5' dei trascritti e trovato un cluster di siti putativi di legame di PTBP1 in uno dei 5' alternativi di FOXP2, tuttavia ulteriori studi sono necessari. In conclusione, proponiamo un modello di controllo dei livelli di espressione di FOXP2 durante lo sviluppo neuronale mediante uno splicing alternativo regolato da PTBP1, che genera il trascritto non codificante FOXP2-\u25b311 poi degradato attraverso NMD.The language gene Forkhead Box P2 (FOXP2) encodes a transcription factor and mutations in gene coding region have been associated with human speech and language disorder type 1 (SPCH1). Linkage disequilibrium and genome wide association studies (GWAS) linked FOXP2 also to neurodevelopmental diseases, among others autism spectrum disorders (ASDs), but since no mutations in the coding region were found, post-transcriptional defects were hypothesized. While some data on miRNA regulation are available, little is known on alternative splicing regulation of FOXP2 and the ribonucleoproteins (RBPs) involved. FOXP2 protein is encoded by full-length (FOXP2-FL) transcript, composed of 17 canonical exons and two alternative small in frame alternatively spliced exons, 3b and 4a. Bioinformatic analysis of FOXP2 genomic sequence revealed several putative binding sites in the intronic regions around FOXP2 exon 11 and exon 3b for the Polypyrimidine Tract Binding Protein 1 (PTBP1). PTBP1 is a ubiquitous ribonucleoprotein often regulating exon skipping. Moreover, PTBP1 and its paralog PTBP2, that recognizes the same binding sequences in most cases, play key roles during neuronal differentiation. By RNA ImmunoPrecipitation (RIP) and PTBP1 overexpression in HEK293 cells we demonstrated that PTBP1 binds FOXP2 transcripts and promotes the exclusion of exon 11 generating a FOXP2-\u25b311 transcript. Interestingly, up-regulation of FOXP2-\u25b311 transcript was accompanied by down-regulation of FOXP2-FL transcript and subsequent FOXP2 protein decrement of about 50%. These findings were further confirmed by in vitro minigene assays performed on FOXP2 gene region from exon 9 to exon 13 subcloned in a suitable expression vector. Interestingly, Myc-tagged PTBP2 was also able to up-regulate FOXP2-\u25b311. The minigene approach allowed us also to restrict the intronic regions required for PTBP1 binding sufficient to promote the exclusion of exon 11. Similarly, we analyzed the ability of PTBP1 to regulate the alternatively spliced 3b exon. Despite the presence of numerous predicted binding sites for PTBP1 around exon 3b, PTBP1 appeared unable to promote its skipping, suggesting specificity towards exon 11. The exclusion of exon 11 creates a Premature Termination Codon (PTC) within exon 12, which will generate a truncated FOXP2 protein lacking the DNA binding domain, if translated. Nevertheless, we could not find FOXP2-\u25b311-derived peptides in our cell system. Feature analysis of FOXP2-\u25b311 nucleotide sequence led us to hypothesize this transcript could be degraded by Nonsense-Mediated Decay (NMD), a conserved degradation pathway for aberrant or PTC-bearing mRNAs. By using selective inhibitors, we showed that FOXP2-\u25b311 transcript is indeed a target of NMD. Finally, siRNA-mediated PTBP1 silencing did not show a significative variation of FOXP2 protein or transcript in HEK293 cells, but since the silencing of PTBP1 will promote PTBP2 expression, we co-silenced PTBP1 and PTBP2 and observed a FOXP2 protein overexpression of about 2.5 fold. Transcripts analysis of the same samples revealed a FOXP2-FL increment of about 26%, but no variation of FOXP2-\u25b311 transcript expression, thus pointing to an alternative splicing-indipendent regulation performed by PTBP1 and PTBP2. Since PTBP1 may also control transcript translation, we performed a bioinformatic analysis of FOXP2 5' UTR regions and found a cluster of putative binding sites for PTBP1 in one of the alternatively spliced 5\u2019 UTR of FOXP2 gene, whose function is yet to be elucidated. In conclusion, we propose a model for the control of FOXP2 expression mediated by PTBP1 via upregulation of the noncoding FOPX2-\u25b311 transcript by alternative splicing and degraded by NMD

Development, optimization and validation of a stability indicating HPLC method for sertraline hydrochloride

Sertraline HCl is an antidepressant compound for oral administration, belonging to the group of selective serotonin reuptake inhibitors (SSRI) in the brain. Chemically, sertraline is a secondary amine with a pKa value of 9.47.The analytical method employed for the determination of sertraline and its impurities (A, Band C) throughout the stability assays is generally based on reversed phase with ion-paring chromatography at acidic pH, employing 1-octane sulphonate and acetonitrile as organic modifier. The drawbacks of ion-pairing chromatography are generally known. One of them, the poor retention time reproducibility, is a source of problems during stability indicating assays. The availability of new stationary phases with new selectivities in reverse phase HPLC gave us the possibility of new separations avoiding these drawbacks. During the development also the replacement of acetonitrile with methanol was tested. Moreover, to our knowledge, there is no validated HPLC method including sertraline and these impurities described in the literature.A screening on RP stationary phases was conducted to explore different selectivities, including a wide range of polarities: C8, C18, cyano, PEG and amide. The best result was obtained with a Zorbax Bonus-RP column, which contains a polar amide group embedded in a C14 alkyl chain and has a triple end-capping. These characteristics yield in a reduction of the interactions between basic compounds and the silica support, improving peak shape and reducing the analysis time. Once the best stationary phase was chosen, a HPLC method was developed by means of an experimental design with four quantitative factors (organic proportion in the mobile phase, temperature, pH and buffer concentration) in order to find the best conditions which maximize the resolution between impurities A and B (positional isomers) and minimize the total run time.Optimal conditions for simultaneously determining sertraline and its impurities, being baseline separated in less than 10 min, were found with Zorbax Bonus-RP column (150 x 4.6 mm, 5 mm, Agilent Technologies), under isocratic conditions with 10 mM phosphate buffer (pH=2.8) and MeOH (63:37, v/v) at 50 ºC.This method has been successfully validated following ICH guidelines and it has proved to be reliable for the determination of sertraline and related impurities in tablets as pharmaceutical forms

Development and validation of a HPLC method for the determination of sertraline and three non-chiral related impurities

In this study, a screening on reversed-phase stationary phases (including C(8), C(18), CN, PEG and amide) was carried out in order to obtain an efficient HPLC method for the determination of sertraline and three of its more closely related synthetical and non-chiral impurities, without using ion-pair reagents. The best results in terms of both retention time and resolution of the target analytes were obtained with a Zorbax Bonus-RP column, which contains a polar amide group embedded in a C(14) alkyl chain. Once the most suitable stationary phase was chosen, the HPLC method was optimized by using a factorial design, evaluating three quantitative factors (column temperature, buffer pH and buffer concentration) in order to find the best conditions which maximize the resolution between impurities A and B (positional isomers) and minimize the total run time. The final HPLC conditions were set by means of a second experimental design, which allowed optimizing the effects of the buffer pH and the proportion of methanol in the mobile phase. The optimal conditions for simultaneously determining sertraline and its impurities, being baseline separated in less than 10min, were finally obtained with Zorbax Bonus-RP column (150mmx4.6mm, 5 um), under isocratic conditions with phosphate buffer (pH 2.8; 10mM)-methanol (63:37, v/v) at 50 degrees C, at the flow-rate of 1.0mL/min. UV detection was set at 220nm. This method was successfully validated following ICH guidelines and it proved to be reliable for the determination of sertraline and related impurities in tablets as pharmaceutical forms

3D Models as a Tool to Assess the Anti-Tumor Efficacy of Therapeutic Antibodies: Advantages and Limitations

Therapeutic monoclonal antibodies (mAbs) are an emerging and very active frontier in clinical oncology, with hundred molecules currently in use or being tested. These treatments have already revolutionized clinical outcomes in both solid and hematological malignancies. However, identifying patients who are most likely to benefit from mAbs treatment is currently challenging and limiting the impact of such therapies. To overcome this issue, and to fulfill the expectations of mAbs therapies, it is urgently required to develop proper culture models capable of faithfully reproducing the interactions between tumor and its surrounding native microenvironment (TME). Three-dimensional (3D) models which allow the assessment of the impact of drugs on tumors within its TME in a patient-specific context are promising avenues to progressively fill the gap between conventional 2D cultures and animal models, substantially contributing to the achievement of personalized medicine. This review aims to give a brief overview of the currently available 3D models, together with their specific exploitation for therapeutic mAbs testing, underlying advantages and current limitations to a broader use in preclinical oncology