The role of prolyl-isomerase PIN1 in GABAergic and glutamatergic synaptic transmission

The correct functioning of the central nervous system relies on the rapid and efficient communication between neurons. This occurs at highly specialized functional points of contact called synapses. Synapses are extremely plastic in structure and function, strongly influenced by their own histories of impulse traffic and by signals from nearby cells. Synaptic contacts are fundamental to the development, homeostasis and remodeling of neural circuits. All these events are achieved through different mechanisms operating at both pre- and postsynaptic sites. At the level of the post synaptic density (PSD) compartment, scaffolding molecules and trans-membrane proteins are known to orchestrate proper synapses formation, maturation and rearrangement required to sustain plasticity processes. Protein phosphorylation represents one of the most important mechanisms engaged in affecting the molecular composition of the post-synaptic device. Most studies have focused on the impact of phosphorylation on the gating properties, surface mobility and trafficking of neurotransmitter receptors while much less is known about the effect of post-translational modifications on scaffolding and cell adhesion molecules functionally linked to neurotransmitter receptors. At GABAergic synapses specific phosphorylation events of the scaffolding molecule gephyrin were shown to alter its multimerization properties, thus producing parallel changes in the number of receptors trapped by the scaffold leading to alterations of synaptic strength. Most of these phosphorylation events occur at serine or threonine residues preceding a proline, underlying a potential role of proline-directed phosphorylation as modulator of synaptic strength. The key player of such signalling cascade is represented by a small enzyme called peptidyl-prolyl isomerase Pin1 (protein interacting with NIMA 1). Pin1, upon recruitment by its substrates in a phosphorylation-dependent manner, catalyzes the cis/tran isomerization of phospho\u2010Ser/Thr\u2010Pro motifs leading to changes in target protein conformation and biological activity. Pin1 is highly expressed in neurons suggesting that it can exert a crucial role in synaptic transmission and plasticity processes at both inhibitory and excitatory synapses. In the first part of my PhD thesis I focused on the impact of Pin1-dependent signalling on GABAergic transmission. I found that the cell adhesion molecule of the neuroligin family enriched at GABAergic synapses, Neuroligin 2 (NL2), undergoes post-phosphorylation prolyl-isomerization modulation of its activity. Using biochemical approaches I found that the unique Pin1 consensus motif present within the cytoplasmic tail of NL2, Serine 714-proline, is indeed phosphorylated in vivo. Proline-directed phosphorylation at Serine 714 of NL2 strongly impacts on NL2 ability to complex with gephyrin. In particular, at this site, post-phosphorylation prolyl-isomerization negatively regulates the ability of NL2 to interact with gephyrin. In line with biochemical results, immunocytochemical analysis reveal that, in the absence of Pin1 expression, NL2/gephyrin complexes are enriched at GABAergic post-synaptic sites and this enrichment is accompanied by an enhanced synaptic recruitment of GABAA receptors (GABAAR). This effect was associated with a concomitant increase in the amplitude, but not in frequency, of spontaneous inhibitory post-synaptic currents (IPSCs). These findings unveil the existence of a new signalling pathway operating at inhibitory GABAergic synapses able to alter the efficacy of GABAergic transmission by modulating NL2/gephyrin interaction. Given the high abundance of Pin1 at excitatory synaptic contacts, in the second part of my PhD thesis I focused on the impact of Pin1-dependent signalling on excitatory glutamatergic transmission. In particular, I started to investigate whether the scaffolding molecule PSD-95, a member of the Disc-Large (DGL)-Membrane-associated guanylate kinase, could be a target of Pin1-dependent signalling cascade. I observed that Pin1, known to reside in post-synaptic structures, is recruited by PSD-95 at specific Serine-Threonine/Proline consensus motifs localized in the linker region connecting PDZ2 to PDZ3 domains. These sites are represented by Treonine287-Proline, Serine290-Proline and Serine295-Proline, and deletion of all of them almost completely abolished Pin1 interaction with PSD-95. Pin1 exerts a negative control on PSD-95 ability to complex with N-Methyl-D-Aspartate receptors (NMDARs). Indeed an enhanced PSD-95/NMDAR complex formation was detected in brain extracts derived from Pin1-/- mice. In electrophysiological experiments, larger NMDA-mediated synaptic currents were detected in CA1 principal cells in hippocampal slices obtained from Pin1-/- mice as compared to controls, an effect that was associated with an enhancement in spine density and size. These data indicate that Pin1 controls the synaptic content of NMDARs via PSD-95 prolyl-isomerization and the expression of dendritic spines, both required for the maintenance of long-term potentiation. Overall, this study highlights the crucial role of Pin1-dependent signalling in the functional organization of both inhibitory and excitatory synapses

Fast determination of biogenic amines in beverages by a core-shell particle column

A fast and reliable HPLC method for the determination of 11 biogenic amines in beverages has been performed. After pre-column derivatization with dansyl-chloride a Kinetex C18 core–shell particle column (100 mm × 4.6 mm, 2.6 μm particle size) has been employed and the biogenic amines were identified and quantified in a total run time of 13 min with ultraviolet (UV) or fluorescence detection (FLD). Chromatographic conditions such as column temperature (kept at 50 °C), gradient elution and flow rate have been optimized and the method has been tested on red wine and fruit nectar. The proposed method is enhanced in terms of reduced analysis time and eluent consumption with respect of classical HPLC method as to be comparable to UHPLC methods. Green and cost-effective, this method can be used as a quality-control tool for routine quantitative analysis of biogenic amines in beverages for the average laboratory

Archaeometric Classification of Scattered Marble Fragments to Help the Reconstruction of Statues

A multi-technique approach combining petrographic, cathodoluminescence, and stable isotope analyses is commonly used in provenance studies of archaeological marbles. In the present paper, this characterization approach transcends provenance, and it is applied to the reconstruction of fragmented sculptures. The potential of this novel application of archaeometric measurements is illustrated with a case study consisting in 16 scattered marble fragments retrieved from a Roman villa (Els Munts) near Tarraco (presently Northeastern Spain). The samples were grouped taking into account their similarity in quantified parameters such as the cathodoluminescence color clusters and the stable carbon and oxygen isotopic ratios. The results permitted classification of the fragments into three groups corresponding to three different statues depicting Antinous (7 fragments), Minerva goddess (4 fragments), and an undetermined character (3 fragments). Two other fragments could not be ascribed to any particular statue. The archaeometric grouping provides arguments that can be used to confirm or refute archaeological hypotheses of statue reconstructions. © 2022 by the authors

Pin1 Modulates the Synaptic Content of NMDA Receptors via Prolyl-Isomerization of PSD-95

Phosphorylation of serine/threonine residues preceding a proline regulates the fate of its targets through postphosphorylation conformational changes catalyzed by the peptidyl-prolyl cis-/trans isomerase Pin1. By flipping the substrate between two different functional conformations, this enzyme exerts a fine-tuning of phosphorylation signals. Pin1 has been detected in dendritic spines and shafts where it regulates protein synthesis required to sustain the late phase of long-term potentiation (LTP). Here, we demonstrate that Pin1 residing in postsynaptic structures can interact with postsynaptic density protein-95 (PSD-95), a key scaffold protein that anchors NMDA receptors (NMDARs) in PSD via GluN2-type receptor subunits. Pin1 recruitment by PSD-95 occurs at specific serine-threonine/proline consensus motifs localized in the linker region connecting PDZ2 to PDZ3 domains. Upon binding, Pin1 triggers structural changes in PSD-95, thus negatively affecting its ability to interact with NMDARs. In electrophysiological experiments, larger NMDA-mediated synaptic currents, evoked in CA1 principal cells by Schaffer collateral stimulation, were detected in hippocampal slices obtained from Pin1(-/-) mice compared with controls. Similar results were obtained in cultured hippocampal cells expressing a PSD-95 mutant unable to undergo prolyl-isomerization, thus indicating that the action of Pin1 on PSD-95 is critical for this effect. In addition, an enhancement in spine density and size was detected in CA1 principal cells of Pin1(-/-) or in Thy-1GFP mice treated with the pharmacological inhibitor of Pin1 catalytic activity PiB.Our data indicate that Pin1 controls synaptic content of NMDARs via PSD-95 prolyl-isomerization and the expression of dendritic spines, both required for LTP maintenance

Family-Centered Care to Improve Family Consent for Organ Donation

The need for organ donation has increased over time, but the shortage of available donors is the major limiting factor in transplantation. Organ donation refusal from relatives of potential donors with brain death significantly reduces organ availability. We report a brief analysis about family conflicts in decision-making and causes for refusing donation; moreover, we describe new family-centered strategies in the intensive care unit (ICU) and our systematic communication approach between medical staff and patients’ relatives. In 2016 we conducted a single-center, non-randomized, controlled and before and after study in our ICU, an 18-bed intensive care unit (ICU) of a university hospital. We compared the rate of consent for organ donation before and after the introduction of the new communication approach. The application of a new communication approach between medical staff and relatives of brain-dead patients was associated with a significant increase in the rate of consent to donation. The positive results of the 3-year period 2013–2015 have been confirmed in the 2-year period 2016–2017. Our results highlight the importance of empathy and counselor support of relatives in the ICU

Circulating thyrotropin is upregulated by estradiol

After encountering two women with serum thyrotropin (TSH) levels greater in periovulatory phase than in other days of the menstrual cycle, we hypothesized that TSH levels could be sensitive to changes in circulating estrogens in women. The objective of this study was to evaluate whether serum TSH increases after an induced acute increase of serum estradiol, and compare serum TSH increase with that of prolactin (PRL) which is a classic estradiol-upregulated pituitary hormone. In this retrospective study, we resorted to stored frozen sera from 55 women who had undergone the GnRH agonist (buserelin)-acute stimulation test of ovarian steroidogenesis. This test, that is preceded by dexamethasone administration to suppress adrenal steroidogenesis, had been performed to show an increased buserelin-stimulated response of 17-hydroxyprogesterone, a response that is frequent in polycystic ovary syndrome. Fifty-five women had enough serum volume at pertinent times (first observation early in the follicular phase and all times of the test) to permit assay of serum estradiol, TSH and PRL. Before dexamethasone administration, estradiol averaged 26.4 ± 15.5 pg/ml (reference range 23–139, follicular phase), TSH 1.78 ± 0.86 mU/L (reference range 0.3–4.2) and PRL 409.4 ± 356 mU/L (reference range 70.8–556) (mean ± SD). Serum estradiol, TSH and PRL averaged 47.2 ± 27 pg/ml, 0.77 ± 0.48 mU/L and 246.4 ± 206.8 mU/L just prior to the buserelin injection, but they peaked at 253.4 ± 113.5 pg/ml (nv 83–495, midcycle), 3.30 ± 1.65 mU/L and 540.3 ± 695.2 mU/L after injection. The responses to buserelin of estradiol, TSH and PRL were of wide magnitude. There was a significant correlation between TSH peak and serum estradiol peak, betweeen AUC0-24 h-TSH and AUC0-24 h-estradiol, or between PRL peak and estradiol peak and AUC0-24 h -PRL and AUC0-24 h-estradiol in only a subgroup of women. Therefore, women with estradiol-dependent increase in serum TSH do exist. Reference bands of serum TSH dependent on the phases of the menstrual cycle should be available

Validation of Two Commercial Multiplex Real-Time PCR Assays for Detection of SARS-CoV-2 in Stool Donors for Fecal Microbiota Transplantation

Recurrent infection by Clostridioides difficile has recently been treated by fecal microbiota transplantation (FMT). As viable SARS-CoV-2 was recovered from stool of asymptomatic individuals, the FMT procedure could be a potential risk of SARS-CoV-2 transmission, thus underlying the need to reliably detect SARS-CoV-2 in stool. Here, we performed a multicentric study to explore performances of two commercially available assays for detection of SARS-CoV-2 RNA in stool of potential FMT donors. In three hospitals, 180 stool samples were spiked with serial 10-fold dilutions of a SARS-CoV-2 inactivated lysate to evaluate the Seegene Allplex ™ SARS-CoV-2 (SC2) and SARS-CoV- 2/FluA/FluB/RSV (SC2FABR) Assays for the detection of viral RNA in stool of FMT donors. The results revealed that both assays detected down to 2 TCID50/mL with comparable limit of detection values, SC2 showing more consistent target positivity rate than SC2FABR. Beyond high amplification efficiency, correlation between CT values and log concentrations of inactivated viral lysates showed R2 values ranging from 0.88 to 0.90 and from 0.87 to 0.91 for the SC2 and SC2FABR assay, respectively. The present results demonstrate that both methods are highly reproducible, sensitive, and accurate for SARS-CoV-2 RNA detection in stool, suggesting a potential use in FMT-donor screening

Validation of the Italian version of wisconsin stone quality of life (WISQOL): a prospective Italian multicenter study

Urolithiasis is a worldwide spread condition that affects patients' Health-Related Quality of Life (HRQOL), which measurement is an important tool for routine clinical and research practice. Disease-specific HRQOL measures demonstrated to perform better in assessing the effects of specific conditions. A disease-specific questionnaire for kidney stones, the WISQOL, has been validated in different languages, but an Italian version is still missing. Our aim is to produce and validate the Italian version of WISQOL (IT-WISQOL). Patients undergoing any elective treatment for upper urinary tract stones were enrolled. A multi-step process with forward- and back-translation was used to translate WISQOL into Italian. Patients were evaluated within 15 days pre-operatively and then at 30-, 90 days post-operatively and administered both IT-WISQOL and SF-36v2. Post-operative data such as 30 days postoperative complications, late stone-related events, successful status, and stone complexity were collected. Cronbach's α was used to evaluate the internal consistency of IT-WISQOL, while Spearman's rho was used for item and inter-domain correlations and IT-WISQOL with SF-36v2 correlation. We found excellent internal consistency across all domains (α ≥ 0.88), particularly when the total score is considered (α = 0.960). Test-retest reliability showed excellent results for the total questionnaire (Pearson correlation value: 0.85). The Inter-domain association ranged from 0.497 to 0.786. Convergent validity was confirmed by a good correlation with subdomains of the SF-36v2 measures. IT-WISQOL is a reliable tool to measure HRQOL in stone patients. It shows analog characteristics if compared to English WISQOL