Dimerization of GPCRs:Novel insight into the role of FLNA and SSAs regulating SST₂ and SST₅ homo- and hetero-dimer formation

The process of GPCR dimerization can have profound effects on GPCR activation, signaling, and intracellular trafficking. Somatostatin receptors (SSTs) are class A GPCRs abundantly expressed in pituitary tumors where they represent the main pharmacological targets of somatostatin analogs (SSAs), thanks to their antisecretory and antiproliferative actions. The cytoskeletal protein filamin A (FLNA) directly interacts with both somatostatin receptor type 2 (SST2) and 5 (SST5) and regulates their expression and signaling in pituitary tumoral cells. So far, the existence and physiological relevance of SSTs homo- and hetero-dimerization in the pituitary have not been explored. Moreover, whether octreotide or pasireotide may play modulatory effects and whether FLNA may participate to this level of receptor organization have remained elusive. Here, we used a proximity ligation assay (PLA)-based approach for the in situ visualization and quantification of SST2/SST5 dimerization in rat GH3 as well as in human melanoma cells either expressing (A7) or lacking (M2) FLNA. First, we observed the formation of endogenous SST5 homo-dimers in GH3, A7, and M2 cells. Using the PLA approach combined with epitope tagging, we detected homo-dimers of human SST2 in GH3, A7, and M2 cells transiently co-expressing HA- and SNAP-tagged SST2. SST2 and SST5 can also form endogenous hetero-dimers in these cells. Interestingly, FLNA absence reduced the basal number of hetero-dimers (-36.8 ± 6.3% reduction of PLA events in M2, P &lt; 0.05 vs. A7), and octreotide but not pasireotide promoted hetero-dimerization in both A7 and M2 (+20.0 ± 11.8% and +44.1 ± 16.3% increase of PLA events in A7 and M2, respectively, P &lt; 0.05 vs. basal). Finally, immunofluorescence data showed that SST2 and SST5 recruitment at the plasma membrane and internalization are similarly induced by octreotide and pasireotide in GH3 and A7 cells. On the contrary, in M2 cells, octreotide failed to internalize both receptors whereas pasireotide promoted robust receptor internalization at shorter times than in A7 cells. In conclusion, we demonstrated that in GH3 cells SST2 and SST5 can form both homo- and hetero-dimers and that FLNA plays a role in the formation of SST2/SST5 hetero-dimers. Moreover, we showed that FLNA regulatesSST2 and SST5 intracellular trafficking induced by octreotide and pasireotide. </p

Strongly structured populations and reproductive habitat fragmentation increase the vulnerability of the Mediterranean starry ray Raja asterias (Elasmobranchii, Rajidae)

1. The Mediterranean starry ray (Raja asterias) populations within the Mediterranean Sea are susceptible to high rates of bycatch in the multispecies trawl fisheries. Understanding its population structure and identifying critical habitats are crucial for assessing species vulnerability and setting the groundwork for specific management measures to prevent population decline. 2. To assess the population structure of R. asterias in the Mediterranean, the genetic variation in nine population samples at one mitochondrial marker and eight nuclear microsatellite loci was analysed. Moreover, 172 egg cases collected in the Strait of Sicily were identified at species level using integrated molecular and morphological approaches. 3. Genetic analyses revealed that the Mediterranean starry ray comprises three distinct units inhabiting the western, the central-western, and the central-eastern areas of the Mediterranean. An admixture zone occurs in the Strait of Sicily and the Ionian Sea, where individuals of the central-western and central-eastern population units intermingle. 4. The joint morphometric–genetic analyses of rajid egg cases confirmed the presence of more than one species in the admixture area, with a predominance of egg cases laid by R. asterias. DNA barcoding revealed that egg cases and embryos of R. asterias shared several haplotypes with adult individuals from the centralwestern and central-eastern Mediterranean Sea, revealing that females of both populations laid numerous eggs in this area. 5. According to these findings, detailed taxonomic determination of egg cases, when combined with seasonal migration studies, could improve the capability to identify important spawning or nursery areas for the Mediterranean starry ray, particularly in those admixture zones relevant to maintaining genetic diversity. 6. Finally, these new insights should be considered to update the Action Plan for the Conservation of Cartilaginous Fishes in the Mediterranean Sea with effective measures to reduce the impact of skate bycatch in trawling and safeguard egg cases in nursery areas

Evaluating the Effectiveness of Serious Games for Cultural Awareness: the Icura User Study

Abstract. There is an increasing awareness about the potential of serious games for education and training in many disciplines. However, research still witnesses a lack of methodologies, guidelines and best practices on how to develop effective serious games and how to integrate them in the actual learning and training processes. This process of integration heavily depends on providing and spreading evidence of the effectiveness of serious games This paper reports a user study to evaluate the effectiveness of Icura, a serious game about Japanese culture and etiquette. The evaluation methodology extends the set of instruments used in previous studies by evaluating the effects of the game on raising awareness, by avoiding the selective attention bias and by assessing the medium-term retention. . With this research we aim to provide a handy toolkit for evaluating the effectiveness a serious games for cultural awareness and heritage

A new freezing box for the managing of semen cryopreservation process

Sperm freezing is of interest not only for animal breeding. The ability to use sperm in frozen form for AI is a key factor in ensuring the long-term preservation of genetic diversity through the creation of a sperm cryobank. The most widely used freezing method involves using straws as packaging sperm, then freezing them on liquid nitrogen (N2) vapor and finally immersing them in N2. The cryosurvival of sperm cells also varies among different animal species and has been correlated with the freezing rate, i.e., the distance of the straws above the N2 level. Identifying a freezing procedure that will report a fertilization rate as close as possible to that of fresh semen appears to be a challenge. Thus, there is a clear need to standardize the entire freezing process. In this research, a new device for semen cryopreservation is presented. The cryopreservation prototype allows management of the freezing rate by varying the heights of the support straws above the N2 level. A stainless steel box with a suitable floating frame equipped with a support for the straws was designed and fabricated. The holder is connected to the floating frame by means of two threaded rods that allow for height adjustment by screwing in appropriate guides. Two K-type thermocouples were used, one to measure the temperature of the nitrogen vapor at the height of the support and the other to measure the temperature of the semen inside the straw. In this way, different freezing protocols can be compared. By managing the freezing rate, the sperm freezing procedure, which is specific to animal species, could be standardized and thus variability in results could be minimized. In addition, improved cryosurvival and post-freezing sperm fertilization rates are expected

Exosomes and Signaling Nanovesicles from the Nanofiltration of Preconditioned Adipose Tissue with Skin-B® in Tissue Regeneration and Antiaging: A Clinical Study and Case Report

: Background and Objectives: This three-year clinical trial aimed to demonstrate that only the signaling vesicles produced by ADSCa, containing mRNA, microRNA, growth factors (GFs), and bioactive peptides, provide an advantage over classical therapy with adipose disaggregate to make the tissue regeneration technique safer due to the absence of interfering materials and cells, while being extremely minimally invasive. The infiltration of disaggregated adipose nanofat, defined by the Tonnard method, for the regeneration of the dermis and epidermis during physiological or pathological aging continues to be successfully used for the presence of numerous adult stem cells in suspension (ADSCa). An improvement in this method is the exclusion of fibrous shots and cellular debris from the nanofat to avoid inflammatory phenomena by microfiltration. Materials and Methods: A small amount of adipose tissue was extracted after surface anesthesia and disaggregated according to the Tonnard method. An initial microfiltration at 20/40 microns was performed to remove fibrous shots and cellular debris. The microfiltration was stabilized with a sterile solution containing hyaluronic acid and immediately ultrafiltered to a final size of 0.20 microns to exclude the cellular component and hyaluronic acid chains of different molecular weights. The suspension was then injected into the dermis using a mesotherapy technique with microinjections. Results: This study found that it is possible to extract signaling microvesicles using a simple ultrafiltration system. The Berardesca Scale, Numeric Rating Scale (NRS), and Modified Vancouver Scale (MVS) showed that it is possible to obtain excellent results with this technique. The ultrafiltrate can validly be used in a therapy involving injection into target tissues affected by chronic and photoaging with excellent results. Conclusions: This retrospective clinical evaluation study allowed us to consider the results obtained with this method for the treatment of dermal wrinkles and facial tissue furrows as excellent. The method is safe and an innovative regenerative therapy as a powerful and viable alternative to skin regeneration therapies, antiaging therapies, and chronic inflammatory diseases because it lacks the inflammatory component produced by cellular debris and fibrous sprouts and because it can exclude the mesenchymal cellular component by reducing multiple inflammatory cytokine levels

A β-Arrestin 2-Biased Dopamine Receptor Type 2 (DRD2) Agonist Is More Efficacious Than Cabergoline in Reducing Cell Proliferation in PRL-Secreting but Not in Non-Functioning Pituitary Tumor Cells

The molecular events underlying the variable effectiveness of dopamine receptor type 2 (DRD2) agonists in pituitary neuroendocrine tumors (PitNETs) are not known. Besides the canonical pathway induced by DRD2 coupling with Gi proteins, the β-arrestin 2 pathway contributes to DRD2′s antimitotic effects in PRL- and NF-PitNETs. A promising pharmacological strategy is the use of DRD2-biased agonists that selectively activate only one of these two pathways. The aim of the present study was to compare the effects of two biased DRD2 ligands, selectively activating the G protein (MLS1547) or β-arrestin 2 (UNC9994) pathway, with unbiased DRD2 agonist cabergoline in PRL- and NF-PitNET cells. In rat tumoral pituitary PRL-secreting MMQ cells, UNC9994 reduced cell proliferation with a greater efficacy compared to cabergoline (−40.2 ± 20.4% vs. −21 ± 10.9%, p n = 23), biased agonists did not show better antiproliferative effects than cabergoline. In conclusion, the preferential activation of the β-arrestin 2 pathway by UNC9994 improves DRD2-mediated antiproliferative effects in PRL-PitNETs, suggesting a new pharmacological approach for resistant or poorly responsive tumors

Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant

Cushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases USP8 and USP48, and in BRAF genes, have been reported in a subset of patients affected by CD. The aim of this study was to characterize the genetic profile of a cohort of 60 patients with ACTH-secreting tumors, searching for somatic mutations in USP8, USP48, and BRAF hotspot regions. Seven patients were found to carry USP8 somatic mutations in the well-characterized 14-3-3 protein binding motif (n = 5 P720R, n = 1 P720Q, n = 1 S718del); 2 patients were mutated in USP48 (M415I); no mutation was identified in BRAF. In addition, a novel USP8 variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of USP8 G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the USP8 G664R variant resulted in a significant increase of ACTH release and cell proliferation (+114.5 ± 53.6% and +28.3 ± 2.6% vs. empty vector transfected cells, p &lt; 0.05, respectively). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58–fold increase of N-terminal USP8 fragment, vs. WT USP8, p &lt; 0.05). Surprisingly, in situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8/14-3-3 proteins colocalization, in G664R USP8 transfected cells with respect to WT USP8 transfected cells (−47.9 ± 6.6%, vs. WT USP8, p &lt; 0.001). No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage, and 14-3-3 proteins interaction was observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT USP8 and other USP8 mutants, G664R USP8 displays an exclusive cytoplasmic localization. In conclusion, somatic mutations were found in USP8 (13.3% vs. 36.5% incidence of all published mutations) and USP48 (3.3% vs. 13.3% incidence) hotspot regions. A novel USP8 variant was identified in a CD patient, and in vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis, expanding the characterization of USP8 functional domains

P720R USP8 Mutation Is Associated with a Better Responsiveness to Pasireotide in ACTH-Secreting PitNETs

Somatic mutations in the ubiquitin specific peptidase 8 (USP8) gene have been associated with higher levels of somatostatin (SS) receptor subtype 5 (SSTR5) in adrenocorticotroph hormone (ACTH)-secreting pituitary neuroendocrine tumors (PitNETs). However, a correlation between the USP8 mutational status and favourable responses to pasireotide, the somatostatin multi-receptor ligand acting especially on SSTR5, has not been investigated yet. Here, we studied the impact of USP8 mutations on pasireotide responsiveness in human and murine corticotroph tumor cells. SSTR5 upregulation was observed in USP8 wild-type primary tumor cells transfected with S718del USP8 mutant. However, cell transfection with S718del USP8 and C40-USP8 mutants in in vitro sensitive cultures from USP8 wild-type tumors abolished their ability to respond to pasireotide and did not confer pasireotide responsiveness to the in vitro resistant culture. Pasireotide failed to reduce ACTH secretion in primary cells from one S718P USP8-mutated tumor but exerted a strong antisecretory effect in primary cells from one P720R USP8-mutated tumor. In agreement, AtT-20 cells transfection with USP8 mutants led to SSTR5 expression increase but pasireotide could reduce ACTH production and cyclin E expression in P720R USP8 overexpressing cells, only. In situ Proximity Ligation Assay and immunoflurescence experiments revealed that P720R USP8 mutant is still able to bind 14-3-3 proteins in AtT-20 cells, without affecting SSTR5 localization. In conclusion, P720R USP8 mutation might be considered as a molecular predictor of favourable response to pasireotide in corticotroph tumor cells

Crustal structure and seismotectonics of central Sicily (southern Italy): new constraints from instrumental seismicity

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Clinical nutrition in surgical oncology: Young AIOM-AIRO-SICO multidisciplinary national survey on behalf of NutriOnc research group

Malnutrition is a common condition in cancer patients which is usually associated with functional limitations, as well as increased morbidity and mortality. Based on the support of the young sections of Italian Association of Medical Oncology (AIOM), Italian Association of Radiotherapy and Clinical Oncology (AIRO) and Italian Society of Surgical Oncology (SICO) merged into the NutriOnc Research Group, we performed a multidisciplinary national survey with the aim to define the awareness of nutritional issues among healthcare professionals delivering anticancer care. The questionnaire was organized in four sections, as follows: Knowledge and practices regarding Nutritional Management of cancer patients; Timing of screening and assessment of Nutritional Status; Nutritional Treatment and prescription criteria; Immunonutrition and educational topics. The modules focused on esophagogastric, hepato-bilio-pancreatic and colorectal malignancies. Overall, 215 physicians completed the survey. As regards the management of Nutritional Status of cancer patients, many responders adopted the ERAS program (49.3%), while a consistent number of professionals did not follow a specific validated nutritional care protocol (41.8%), mainly due to lack of educational courses (14.5%) and financial support (15.3%). Nearly all the included institutions had a multidisciplinary team (92%) to finalize the treatment decision-making. Cancer patients routinely underwent nutritional screening according to 57.2% of interviewed physicians. The timing of nutritional assessment was at diagnosis (37.8%), before surgery (25.9%), after surgery (16.7%), before radiochemotherapy (13.5%) and after radiochemotherapy (7%). Most of the responders reported that nutritional status was assessed throughout the duration of cancer treatments (55.6%). An important gap between current delivery and need of nutritional assessment persists. The development of specific and defined care protocols and the adherence to these tools may be the key to improving nutritional support management in clinical practice