The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity

This is the final version. Available from Life Science Alliance via the DOI in this recordDuring mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in Drosophila and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar–phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in Drosophila when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.This project received funding from the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013), ERC grant agreement no. 310957, and the Deutsche Forschungsgemeinschaft (FOR2333, BO3588/2-1 to F Bono)

Identification of key receptor residues discriminating human chorionic gonadotropin (Hcg)-and luteinizing hormone (lh)-specific signaling

(1) The human luteinizing hormone (LH)/chorionic gonadotropin (hCG) receptor (LHCGR) discriminates its two hormone ligands and differs from the murine receptor (Lhr) in amino acid residues potentially involved in qualitative discerning of LH and hCG. The latter gon-adotropin is absent in rodents. The aim of the study is to identify LHCGR residues involved in hCG/LH discrimination. (2) Eight LHCGR cDNAs were developed, carrying “murinizing” mutations on aminoacidic residues assumed to interact specifically with LH, hCG, or both. HEK293 cells expressing a mutant or the wild type receptor were treated with LH or hCG and the kinetics of cyclic adenosine monophosphate (cAMP) and phosphorylated extracellular signal-regulated ki-nases 1/2 (pERK1/2) activation was analyzed by bioluminescence resonance energy transfer (BRET). (3) Mutations falling within the receptor leucine reach repeat 9 and 10 (LRR9 and LRR10; K225S +T226I and R247T), of the large extracellular binding domain, are linked to loss of hormone-specific induced cAMP increase, as well as hCG-specific pERK1/2 activation, leading to a Lhr-like modulation of the LHCGR-mediated intracellular signaling pattern. These results support the hypothesis that LHCGR LRR domain is the interaction site of the hormone β-L2 loop, which differs between LH and hCG, and might be fundamental for inducing gonadotropin-specific signals. (4) Taken to-gether, these data identify LHCGR key residues likely evolved in the human to discriminate LH/hCG specific binding

The “hitchhiker’s guide to the galaxy” of endothelial dysfunction markers in human fertility

Endothelial dysfunction is an early event in the pathogenesis of atherosclerosis and represents the first step in the pathogenesis of cardiovascular diseases. The evaluation of endothelial health is fundamental in clinical practice and several direct and indirect markers have been suggested so far to identify any alterations in endothelial homeostasis. Alongside the known endothelial role on vascular health, several pieces of evidence have demonstrated that proper endothelial functioning plays a key role in human fertility and reproduction. Therefore, this stateof‐the‐art review updates the endothelial health markers discriminating between those available for clinical practice or for research purposes and their application in human fertility. Moreover, new molecules potentially helpful to clarify the link between endothelial and reproductive health are evaluated herein

Anestesia locoregionale dell'orecchio nel cavallo: descrizione del blocco tramite l'ausilio di un neurostimolatore

Introduzione. L\u2019esecuzione di procedure diagnostiche e terapeutiche a carico dell\u2019orecchio del ca- vallo \ue8 spesso complicata e frustrante per il veterinario. Ci\uf2 \ue8 particolarmente vero per procedure dolorose quali biopsie o gestione di ferite che richiedono l\u2019immobilit\ue0 del paziente1. A tale propo- sito nell\u2019ambito chirurgico, di grossi e piccoli animali, si \ue8 assistito negli ultimi anni, ad un cre- scente interesse nei confronti di tecniche di analgesia selettiva dell\u2019orecchio. Il blocco diretto di ner- vi che innervano la pinna a livello della base dell\u2019orecchio costituiscono una tecnica in grado di ri- durre lo stress del paziente e il tempo richiesto per l\u2019esecuzione delle procedure1. Nonostante un\u2019ot- tima conoscenza anatomica locale, la corretta localizzazione nervosa viene meno a causa di un\u2019ele- vata variabilit\ue0 individuale1. La stimolazione nervosa periferica (tramite neurostimolatore) permet- te di applicare in modo mirato ed in stretta vicinanza alla fibra nervosa, una serie di stimoli elettri- ci che, provocando una contrazione muscolare, consentono di identificare le strutture di interesse. L\u2019utilizzo del neurostimolatore consente una veloce e precisa localizzazione nervosa e abbassa i ri- schi di lesioni da inoculazione intraneurale. Descrizione del caso. Un cavallo di 9 anni, di 420 Kg viene riferito per drenaggio chirurgico di un otoematoma a carico dello scafo dell\u2019orecchio sinistro. L\u2019innervazione auricolare si compone di 3 nervi fondamentali: branca mandibolare del nervo trigemino, branca auricolopalpebrale del nervo facciale e secondo nervo cervicale o grande auricolare. Si decide quindi per il blocco di questi 3 ner- vi tramite l\u2019utilizzo di un neurostimolatore. Il soggetto viene sedato e lo strumento (Vygon Italia) viene connesso ad un ago atraumatico (22G, 50 mm, 30\ub0), infisso nel punto di repere specifico per i singoli nervi e viene impostato ad una intensit\ue0 di 1.0 mA con una stimolazione di 2 Hz della du- rata di 0.1 ms, per ottenere una contrazione dei muscoli target2. Per anestetizzare la branca mandi- bolare del nervo trigemino l\u2019ago, dopo accurata tricotomia e preparazione asettica della cute del- l\u2019area, viene infisso a livello dell\u2019articolazione temporo-mandibolare con direzione rostro-caudale e latero-mediale fino ad ottenere una contrazione dei muscoli digastrico, pterigoideo mediale e la- terale e massetere (spostamento rostrale della pinna e masticazione). Vengono quindi inoculati 5 ml di mepivacaina cloridrato 2%. Viene poi individuata ed anestetizzata la branca auricolo-palpebrale del nervo facciale. Il suo punto di repere \ue8 rappresentato dalla cresta occipitale e dalla base della pinna, costituita dai margini mediale e laterale dell\u2019elice. Dopo accurata tricotomia e preparazione asettica della cute dell\u2019area, l\u2019ago atraumatico viene inserito al centro del triangolo idealmente di- segnato dai due vertici tra la base della pinna e la cresta occipitale. Con le stesse procedure utiliz- zate precedentemente viene eseguita una stimolazione dei muscoli della pinna e della palpebra su- periore e, una volta ottenuti movimenti auricolari caudorostrali e ammiccamento, vengono inocula- ti 5 ml di mepivacaina cloridrato 2%. Per effettuare il blocco del II nervo cervicale, l\u2019ago atraumatico viene inserito ventrocaudalmente all\u2019ala dell\u2019atlante in direzione lateromediale e leggermente ventrodorsale sino ad ottenere una con- trazione dei muscoli del collo e movimenti di abduzione dell\u2019orecchio. Vengono quindi inoculati 5 ml di mepivacaina cloridrato 2%. Dopo 10 minuti dall\u2019esecuzione di ogni singolo blocco viene testata la sensibilit\ue0 tattile e dolorifi- ca dell\u2019orecchio e, visto l\u2019esito favorevole, si procede all\u2019intervento chirurgico. Conclusioni. Il blocco della branca mandibolare del trigemino, della branca auricolopalpebrale del nervo facciale e della branca auricolare del II nervo cervicale \ue8 risultato di facile esecuzione ed ef- ficace per la desensibilizzazione dell\u2019orecchio in tutta la sua struttura. La tecnica \ue8 stata ben tolle- rata e ha permesso di eseguire in maniera agevole la chirurgia riducendo il dosaggio dei farmaci anestetici utilizzati per l\u2019anestesia generale. Questo tipo di anestesia locoregionale potrebbe essere efficace per effettuare procedure chirurgiche a carico dell\u2019orecchio con la sola sedazione dell\u2019ani- male (drenaggio otoematoma, escissione masse neoplastiche o criochirurgia), ovviando cos\uec ai ri- schi legati all\u2019anestesia generale3. La buona conoscenza anatomica e l\u2019utilizzo dello stimolatore nervoso periferico ha permesso la cor- retta localizzazione nervosa con conseguente desensibilizzazione del sito di chirurgia. L\u2019impiego di anestetici locali a media/lunga durata d\u2019azione consente una riduzione del dolore nel periodo pe- rioperatorio, condizione indispensabile affinch\ue9 diminuisca il rischio di autotraumatismi. In ultima analisi, il blocco dei nervi sopracitati ha permesso non solo l\u2019esecuzione della procedura chirurgica ma anche una miglior gestione del paziente nel periodo post-operatorio

CD38 and bone marrow microenvironment.

This review summarizes the events ruled by CD38 shaping the bone marrow environment, recapitulating old and new aspects derived from the body of knowledge on the molecule. The disease models considered were myeloma and chronic lymphocytic leukemia (CLL). CD38 has been analyzed considering its twin function as receptor and enzyme, roles usually not considered in clinics, where it is used as a routine marker. Another aspect pertaining basic science concerns the role of the molecule as a member of an ectoenzyme network, potentially metabolizing soluble factors not yet analyzed (e.g., NAD+, ATP, NAM) or influencing hormone secretion (e.g., oxytocin). The last point is focused on the use of CD38 as a target of an antibody-mediated therapeutic approach in myeloma and CLL. A recent observation is that CD38 may run an escape circuit leading to the production of adenosine. The generation of local anergy may be blocked by using anti-CD38 antibodies. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes

Polygenic Susceptibility to Papillary Thyroid Cancer in Italian Subjects.

olygenic Susceptibility to Papillary Thyroid Cancer in Italian Subjects INTRODUCTION AND AIM. Thyroid cancer is the most common endocrine neoplasia, with an estimated age- standardized incidence rate of 6.7 per 100000 worldwide in 2018 [1]. This rate is rapidly increasing and papillary thy- roid carcinoma (PTC) is the main histotype. PTC suscepti- bility is the result of genetic predisposition, environmental factors and lifestyle. We studied the genetic combination that characterizes PTC affected subjects, differentiating them from healthy controls. METHODS AND RESULTS. We considered the genetic variants (SNPs) significantly associated with PTC on the PubMed database. 184 informative SNPs were selected, considering linkage disequilibrium. Then, SNPs data were extracted from the online 1000 Genomes database,comprising genome of 2504 unselected individuals col- lected worldwide. The combination of 184 SNPs associ- ated with PTC was used to group individuals in different risk-clusters according to their genetic structure, calcu- lated by Bayesian statistics, as previously performed for polycystic ovary syndrome [2]. Individuals were distrib- uted among 7 groups worldwide, indicating different de- gree of genetic predisposition to PTC. We then considered genetic data from about 1200 individuals (697 PTC versus 497 healthy controls) of Central/South Italian origin reg- istered in a GWAS, specific for PTC [3]. This first analysis was refined using the 33 SNPs reasonably most causa- tive of genetic clustering (26 with p<0.05 at trend test in GWAS and 7 with p<0.05 in the model of recessive inher- itance). At multivariate logistic regression analysis, PTC and healthy controls resulted genetically different (ODDS RATIO 188.6, 95%CI 64.35-552.8), revealing diverse pre- disposition to develop cancer. Afterwards, these results have been confirmed in an independent cohort of Italian subjects (234 PTC and 100 controls). Then, the genetic structure of each subject was indicated as a percentage of affinity to each risk-cluster and re-analyzed together with other risk factors: sex, body-mass index, area of origin and familiarity (quantified in a growing score as the degree of kinship increases). These data were analyzed together by principal component analysis and clustering of the two groups was even more pronounced. The most contributive factors to the diversity between PTC and healthy controls were genetics and familiarity. CONCLUSION. We demonstrated that PTC affected subjects are genetically different from healthy controls, and that the difference is identifiable in a peculiar combi- nation of genetic variants