Rapid and reversible knockdown of endogenously tagged endosomal proteins via an optimized HaloPROTAC degrader

Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag-directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC₅₀ of 3–10 nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min) and complete (<i>D</i>_max of ∼95% at 48 h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function

An Italian survey of opioids misuse: Epidemiological and psychopathological aspects

© 2021 Published by Elsevier Ltd on behalf of International Society for the Study of Emerging Drugs. This is an open access article under the CC BY-NC-ND license, http://creativecommons.org/licenses/by-nc-nd/4.0/Objective Opioid abuse is a worrying reality especially in the US. The increase in the prescription of opioids in Europe poses the risk of a possible increase in the number of abusers also in Italy. The aim of the study is to evaluate the abuse of opioids in the youth population and to evaluate possible correlations with some psychopathological aspects. Methods A survey, conducted from July 2019 to March 2020, about the use of opioids was spread to a group of subjects aged between 18 and 40 years. A socio-demographical investigation and psychometric scales evaluating internet game addiction, gaming online, quality of life and general psychometric features were administrated. Results Nine-hundred and thirteen subjects completed the survey. Seventeen-five subjects (8.21%) have used one opioid at least once in their lifetime. Weak correlations were found between codeine and morphine intensity of use and sleep disturbance, cigarette smoked per day, while codeine correlates with the number of coffees taken per day and somatization. Conclusion Although the data of this survey do not show high percentages of use in Italy (8.21% of the sample have used one of the listed opioids at least once in their lifetime), the correlations founded confirm the literature data already present highlighting the need for constant monitoring of this phenomenon.Peer reviewedFinal Published versio

Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

<p>Abstract</p> <p>Background</p> <p>It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.</p> <p>Results</p> <p>A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice.</p> <p>Conclusion</p> <p>Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.</p

Seamounts as hot-spots of large pelagic aggregations

The distribution of four top predators in the Tyrrhenian Sea, a sub-basin of the Mediterranean Sea, was investigated by means of random forest regression considering depth, distance from the coast, seafloor slope, and distance from seamounts as habitat descriptors on a 2x2–nautical mile regular grid. RF results are processed to estimate variable importance and model performance. Random forest architecture reached optimal sensitivity and specificity, thus providing a consistent support tool for identifying suitable habitats. The considered species are characterized as having patched suitable habitats with a number of hot-spot areas where the different species' habitats overlap. These hot-spot areas' locations correspond to those of specific seamounts identifying the attraction effect of these topographic structures. The mean features typifying the most attractive seamounts are investigated and found to be shallow peak and base depths but wide base area and high relative elevation

Surface Functionalization of Grown-on-Tip ZnO Nanopyramids: From Fabrication to Light-Triggered Applications

We report on a combined chemical vapor deposition (CVD)/radio frequency (RF) sputtering synthetic strategy for the controlled surface modification of ZnO nanostructures by Ti-containing species. Specifically, the proposed approach consists in the CVD of grown-on-tip ZnO nanopyramids, followed by titanium RF sputtering under mild conditions. The results obtained by a thorough characterization demonstrate the successful ZnO surface functionalization with dispersed Ti-containing species in low amounts. This phenomenon, in turn, yields a remarkable enhancement of photoactivated superhydrophilic behavior, self-cleaning ability, and photocatalytic performances in comparison to bare ZnO. The reasons accounting for such an improvement are unravelled by a multitechnique analysis, elucidating the interplay between material chemico-physical properties and the corresponding functional behavior. Overall, the proposed strategy stands as an amenable tool for the mastering of semiconductor-based functional nanoarchitectures through <i>ad hoc</i> engineering of the system surface

Enantioselective Cytotoxicity of Chiral Diphosphine Ruthenium(II) Complexes Against Cancer Cells

The chiral cationic complex [Ru(η1-OAc)(CO)((R,R)-Skewphos)(phen)]OAc (2R), isolated from reaction of [Ru(η1-OAc)(η2-OAc)(R,R)-Skewphos)(CO)] (1R) with phen, reacts with NaOPiv and KSAc affording [RuX(CO)((R,R)-Skewphos)(phen)]Y (X=Y=OPiv 3R; X=SAc, Y=OAc 4R). The corresponding enantiomers 2S-4S have been obtained from 1S containing (S,S)-Skewphos. Reaction of 2R and 2S with (S)-cysteine and NaPF6 at pH=9 gives the diastereoisomers [Ru((S)-Cys)(CO)(PP)(phen)]PF6 (PP=(R,R)-Skewphos 2R-Cys; (S,S)-Skewphos 2S-Cys). The DFT energetic profile for 2R with (S)-cysteine in H2O indicates that aquo and hydroxo species are involved in formation of 2R-Cys. The stability of the ruthenium complexes in 0.9 % w/v NaCl solution, PBS and complete DMEM medium, as well as their n-octanol/water partition coefficient (logP), have been evaluated. The chiral complexes show high cytotoxic activity against SW1736, 8505 C, HCT-116 and A549 cell lines with EC50 values of 2.8–0.04 μM. The (R,R)-Skewphos derivatives show higher cytotoxicity compared to their enantiomers, 4R (EC50=0.04 μM) being 14 times more cytotoxic than 4S against the anaplastic thyroid cancer 8505 C cell line

AMS-VegBank: a new database of vegetation plots for the Italian territory

The importance of collection, storage and exchange of georeferenced vegetation plot-based data has significantly grown in the recent decades, because of the new potentialities offered by ecoinformatics. In this article we introduce the Alma Mater Studiorum – University of Bologna vegetation database (AMS-VegBank; GIVD code EU-IT-021) compiling 17,505 georeferenced vegetation-plot observations within a time span of 90 years. This database includes 337,799 occurrence data of vascular plant species, belonging to many different habitat types. The historical relevance of the presented database is highlighted by the presence of some of the most ancient vegetation-plot observations in Europe (years 1930–1938). The geographic coverage of the database is mostly for Italian territory but it includes also data from other countries. The thematic focuses represented in the database are various, such as small Mediterranean islands, the Dolomite Mountains and the Italian National Parks. The large amount of historical plots available for the country not previously included in existing databases, combined with the constant action to improve the georeferencing of existing data and the addition of new data, highlight the uniqueness of this database. AMS-VegBank represents thus an important tool for studying plant biodiversity within the context of continental and global vegetation plot databases

Characterizing the building blocks of Problematic Use of the Internet (PUI): The role of obsessional impulses and impulsivity traits among Italian young adults

BACKGROUND: Problematic Use of the Internet (PUI) is a considerable issue of the modern era, but its risk factors are still poorly understood. Impulsivity and obsessive-compulsive symptoms have been associated with PUI, but this relationship is still debated. In this article we focus on the relationships of PUI with obsessive-compulsive and impulsive symptoms in a cohort of Italian young adults, in order to identify possible vulnerability factors for PUI.METHODS: A sample of 772 Italian individuals aged 18-30 (mean age 23.3±3.3years old; 38% males and 62% females) was assessed via online survey using the Internet Addiction Test (IAT), the Mini International Neuropsychiatric Interview (MINI) Screen, the Padua Inventory-Washington State University Revision (PI-WSUR) and the Barratt Impulsiveness Scale (BIS-11).RESULTS: Ninety-seven subjects (12.6% of the sample) reported IAT scores at risk for PUI. PUI participants reported higher levels of impulsivity, obsessive-compulsive symptoms and a higher burden of co-occurrent psychiatric symptoms. In a logistic regression model, obsessional impulses to harm (OR =1.108, p&lt;0.001), attentional impulsivity (OR=1.155, p&lt;0.001) and depressive symptomatology (OR=1.246, p=0.012) had significant association with PUI. Finally, higher severity of PUI has been associated with manic/psychotic symptoms and with attentional impulsivity.CONCLUSIONS: Our findings confirmed the role of impulsivity in PUI, while also underling the association of obsessional impulses with this pathological behavior. We could hypothesize a trigger role of obsessive impulses for the engagement in PUI, together with factors as negative affective states. Further research is needed with respect to more severe forms of PUI, also for establishing tailored interventions

Autobiographical memory in contact tracing: evidence from the COVID-19 pandemic

IntroductionThe recent COVID-19 pandemic has compelled various governments to trace all contacts of a confirmed case, as well as to identify the locations visited by infected individuals. This task, that requires the activation of our autobiographical memories, can make a difference in the spread of the contagion and was based primarily on telephone interviews with infected people. In this study, we examined whether participants were able to provide contact tracing information and whether their memories were influenced by salient events occurring during the initial phases of the pandemic.MethodsParticipants were asked to fill in an online standardized form in which they recounted every day of the 2 weeks before, reporting as much information as possible. The time period selected included, among other things, the day on which the Italian government issued the decree initiating the COVID-19 lockdown. The task was completed twice, the first time relying solely on their memory, and the second time using external aids (diaries, mobile phones etc.). Reports were then coded using a scheme that segmented accounts into informational details, divided into two broad categories, internal and external.ResultsOur findings showed that (i) the use of external aids was effective only when participants had to recall the day furthest away or if to-be-recalled events have low distinctiveness, and (ii) memories of internal details were recalled better than memories of external details. Participants were overall accurate and reported a large amount of information about people and places. However, because of the connection with key pandemic-related events, the effect was somewhat stronger on specific days (e.g., the day in which the lockdown was announced).DiscussionThe results of this work could provide a useful tool for improving the design of contact tracing procedures in the event of an unwanted future public health crisis caused by a highly infectious agent