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Lipidation, a common strategy to improve half-life of therapeutic peptides, affects their tendency to oligomerize, their interaction with plasmatic proteins, and their catabolism. In this work, we have leveraged the use of NMR and SPR spectroscopy to elucidate oligomerization propensity and albumin interaction of different analogs of the two marketed lipidated GLP-1 agonists liraglutide and semaglutide. As most lipidated therapeutic peptides are administered by subcutaneous injection, we have also assessed in vitro their catabolism in the SC tissue using the LC-HRMS-based SCiMetPep assay. We observed that oligomerization had a shielding effect against catabolism. At the same time, binding to albumin may provide only limited protection from proteolysis due to the higher unbound peptide fraction present in the subcutaneous compartment with respect to the plasma. Finally, identification of catabolites in rat plasma after SC dosing of semaglutide showed a good correlation with the in vitro data, with Tyr

Zika virus (ZIKV) is a member of the Flaviviridae family that can cause neurological disorders and congenital malformations. The NS2B-NS3 viral serine protease is an attractive target for the development of new antiviral agents against ZIKV. We report here a SAR study on a series of substrate-like linear tripeptides that inhibit in a non-covalent manner the NS2B-NS3 protease. Optimization of the residues at positions P1, P2, P3 and of the N-terminal and C-terminal portions of the tripeptide allowed the identification of inhibitors with sub-micromolar potency with phenylglycine as arginine-mimicking group and benzylamide as C-terminal fragment. Further SAR exploration and application of these structural changes to a series of peptides having a 4-substituted phenylglycine residue at the P1 position led to potent compounds showing double digit nanomolar inhibition of the Zika protease (IC

Aim To compare nasal glucagon (NG) with intramuscular glucagon (IMG) for the treatment of insulin‐induced hypoglycaemia in Japanese patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM). Materials and methods This phase 3, randomized, open‐label, two‐treatment, two‐period crossover non‐inferiority study enrolled Japanese adults with T1DM or T2DM on insulin therapy, with glycated haemoglobin levels ≤86 mmol/mol (≤10%). After ≥8 hours of fasting, hypoglycaemia was induced with human regular insulin (intravenous infusion). Patients received NG 3 mg or IMG 1 mg approximately 5 minutes after insulin termination. The primary endpoint was the proportion of patients achieving treatment success [plasma glucose (PG) increase to ≥3.9 mmol/L (≥70 mg/dL) or ≥1.1 mmol/L (≥20 mg/dL) increase from the PG nadir within 30 minutes of receiving glucagon]. Non‐inferiority was declared if the upper limit of the two‐sided 95% confidence interval (CI) of the mean difference in the percentage of patients achieving treatment success (IMG minus NG) was 2 patients were rhinalgia, increased blood pressure, nausea, ear pain and vomiting in the NG group, and nausea and vomiting in the IMG group. Conclusion Nasal glucagon was non‐inferior to IMG for successful treatment of insulin‐induced hypoglycaemia in Japanese patients with T1DM/T2DM, supporting use of NG as a rescue treatment for severe hypoglycaemia.

We present Terminus, a voting system based on blockchain technology. Terminus relies on technology solutions pioneered by Monero, a privacy-focused Blockchain, and on specifically designed operational procedures: this guarantees full anonymity of the vote and addresses several concerns of digital voting systems. Terminus was tested at an event of an Italian political movement, and will be used to carry out polls to drive some of the political decisions of this movement. We also introduce an evaluation framework for DLT voting systems, and use it to compare existing systems.

The main aim of this work is to study the behaviour of users in social media analysis. In particular, the work involves Twitter users about the percepetion of the Italian guaranteed minimum income (”Reddito di cittadinanza”) on the basis of different categories of users. The main distinction about users is made between verified Twitter users and not verified users. The first category is related to politician, institutional authorities and other official stakeholders. The second one is represented by citizens and other subjects not directly involved in the process of realization of this measure. A classification method based on tweets, retweets and quotes posted by users with hashtag #redditodicittadinanza will be able to discern between verified and not verified users. Moreover, an analysis of the KPI (Key Performance Indicators) will be conducted using their presence and absence through the use of the complementary values. This tool is very useful to give a meaning to an absence of behaviour distinguishing between no interest and a negative opinion. Data have been collected in Italy in April 2019 using all tweets containing #redditodicittadinanza using the official Twitter API. Data will be analysed using R and Python. The use of this classification method allowed also to propose a model to recognize the presence of a Bot, a simulated Twitter user that login using same channels of the humans. Bot are often used to spread the effect of messages and topic in order to influence positive or negative effects.

Oxidative stress has been associated with pathogenesis in several diseases including Huntington's disease (HD), a neurodegenerative disorder caused by a mutation in the huntingtin gene. Oxidative stress induced reactive oxygen species (ROS) are normally controlled at the cellular level by the nuclear factor (erythroid-derived 2)-like 2 (NRF2) a transcription factor that regulates the expression of various antioxidants and detoxifying proteins. Normally NRF2 is largely inactivated in the cytoplasm by the Kelch-like ECH-associated protein 1 (KEAP1)/Cullin-3 (CUL3) mediated ubiquitination and subsequent proteosomal degradation. In the presence of ROS, KEAP1 sensor cysteines are directly or indirectly engaged resulting in NRF2 release, nuclear translocation, and activation of its target genes. Consequently the activation of NRF2 by a small-molecule drug may have the therapeutic potential to control oxidative stress by upregulation of the endogenous antioxidant responses. Here we attempted to validate the use of a reversible non-acidic KEAP1 binder (Compound 2) to activate NRF2 with better cellular activity than similar acidic compounds. When tested head to head with sulforaphane, a covalent KEAP1 binder, Compound 2 had a similar ability to induce the expression of genes known to be modulated by NRF2 in neurons and astrocytes isolated from wild-type rat, wild type mouse and zQ175 (an HD mouse model) embryos. However, while sulforaphane also negatively affected genes involved in neurotoxicity in these cells, Compound 2 showed a clean profile suggesting its mode of action has lower off-target activity. We show that Compound 2 was able to protect cells from an oxidative insult by preserving the ATP content and the mitochondrial potential of primary astrocytes, consistent with the hypothesis that neurotoxicity induced by oxidative stress can be limited by upregulation of innate antioxidant response.

Glycosylation is a key posttranslational modification that tags protein to membranes, organelles, secretory pathways, and degradation. Aberrant protein glycosylation is present both in acquired diseases, such as cancer and neurodegeneration, and in congenital disorders of glycosylation (CDGs). Consequently, the ability to interrogate the activity of enzymes that can modify protein glycan moieties is key for drug discovery projects aimed at finding modulators of these enzymes. To date, low-throughput technologies such as SDS-PAGE and mass spectrometry have been used, which are not suitable for compound screening in drug discovery. In the present work, a broadly applicable time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed that can determine the activity of endoglycosidase enzymes in high-throughput formats. The assay was validated using PNGaseF and EndoH as tool deglycosylases. Even though the current setup is based on the recognition of glycans that bind concanavalin A (ConA), the assay concept can be adapted to glycans that bind other lectins.

Rationale Several phthalates and bisphenol A are endocrine-disrupting chemicals (EDCs). Recently, their use has been partially restricted and less toxic compounds, such as di-2-ethylhexyl terephthalate (DEHTP), have been placed on the market. The aim of this work was to develop and validate a method for the simultaneous quantitation of bisphenol A and urinary metabolites of phthalates, including DEHTP. Methods An isotopic dilution high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the determination of bisphenol A (BPA), monobenzyl phthalate (MBzP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP), mono-2-ethyl-5-hydroxyhexyl terephthalate (MEHHTP), monoethyl phthalate (MEP), and mono-n/i-butyl phthalates (MnBP/MiBP) in human urine was developed. A complete validation was carried out and the method was applied to 36 non-occupationally exposed adults. Results Limits of quantitation ranged from 0.02 (MECPP) to 1 μg/L (MnBP and MiBP). Relative standard deviations below 10% indicated a suitable precision; accuracy, evaluated using a standard reference material, ranged from 74.3% to 117.5%; isotopically labelled internal standards were suitable for correcting the matrix effect. The accuracy was confirmed by the successful participation in an external verification exercise. However, for terephthalates, the validation was incomplete due to the lack of reference materials and external verification. Levels of the investigated chemicals in subjects were in line with those previously reported. Conclusions An LC/MS/MS assay for the simultaneous measurement of BPA and phthalate metabolites in human urine was developed and validated; it is useful to investigate exposure in epidemiological studies involving the general population.

Alterations in the ferrochelatase gene (FECH) are the basis of the phenotypic expressions in erythropoietic protoporphyria. The phenotype is due to the presence of a mutation in the FECH gene associated in trans to the c.315–48 T > C variant in the intron 3. The latter is able to increase the physiological quota of alternative splicing events in the intron 3. Other two variants in the FECH gene (c.1–252A > G and c.68–23C > T) have been found to be associated to the intron 3 variant in some populations and together, they constitute a haplotype (ACT/GTC), but eventually, their role in the alternative splicing event has never been elucidated. The absolute number of the aberrantly spliced FECH mRNA molecules and the absolute expression of the FECH gene were evaluated by digital PCR technique in a comprehensive cohort. The number of splicing events that rose in the presence of the c.315–48 T > C variant, both in the heterozygous and homozygous condition was reported for the first time. Also, the percentage of the inserted FECH mRNA increased, even doubled in the T/C cases, compared to T/T cases. The constant presence of variants in the promoter and intron 2 did not influence or modulate the aberrant splicing. The results of FECH gene expression suggested that the homozygosity for the c.315–48 T > C variant could be considered pathological. Thus, this study identified the homozygotes for the c.315–48 T > C variant as pathological. By extension, when the samples were categorised according to the haplotypes, the GTC haplotype in homozygosis was pathological.

Background Although thousands of different chemicals have been identified in cigarette smoke, the characterization of their urinary metabolites still requires significant research. The aim of this work was to perform an untargeted metabolomic approach to a pilot cross-sectional study conducted on subjects with different smoking habits and to compare the results with those of the targeted measurement of mercapturic acids. Methods Urine samples from 67 adults, including 38 non-smokers, 7 electronic cigarette users, and 22 traditional tobacco smokers were collected. Samples were analysed by liquid chromatography/time-of flight mass spectrometry. Data were processed using the R-packages IPO and XCMS to perform feature detection, retention time correction and alignment. One-way ANOVA test was used to identify different features among groups. Quantitative determination of 17 mercapturic acids was available from a previous study. Results One hundred and seventeen features, out of 3613, were different among groups. They corresponded to 91 potential metabolites, 5 of which were identified vs authentic standards, 43 were putatively annotated and 13 were attributed to chemical classes. Among identified compounds there were the mercapturic acids of acrolein, 1,3-butadiene, and crotonaldehyde; among putatively annotated compounds there were the glucuronide conjugated of 3-hydroxycotinine and the sulfate conjugate of methoxyphenol; with the lowest degree of confidence several sulfate conjugates of small molecules were annotated. Considering mercapturic acids, the coherence between the targeted and untargeted approach was found for a limited number of chemicals, typically the most abundant. Conclusions Differences in the urinary levels of several compounds were associated to the different smoking habits, suggesting that the proposed approach is useful for the investigation of the metabolite patterns related to the exposure to toxicants. However, limitations were highlighted, in particular regarding the identification of low concentration compounds.

Background While tobacco smoke contains thousands of chemicals, some of which are carcinogenic to humans, the content of electronic cigarette smoke is less known. This work aimed to assess and compare the exposure associated with different smoking habits by profiling urinary mercapturic acids as biomarkers of toxic compounds. Methods In this pilot study, sixty-seven healthy adults with different smoking habits were investigated: 38 non-smokers (NS), 7 electronic cigarette users (ECU), and 22 traditional tobacco smokers (TTS). Seventeen urinary mercapturic acids, metabolites of 1,3-butadiene (DHBMA, MHBMA), 4-chloronitrobenze (NANPC), acrolein (3-HPMA), acrylamide (AAMA, GAMA), acrylonitrile (CEMA), benzene (SPMA), crotonaldehyde (CMEMA, HMPMA), ethylating agents (EMA), methylating agents (MMA), ethylene oxide (HEMA), N,N-dimethylformamide (AMCC), propylene oxide (2-HPMA), styrene (PHEMA), and toluene (SBMA), were quantified, along with urinary nicotine and cotinine. Results Median urinary cotinine was 0.4, 1530 and 1772 μg/L in NS, ECU and TTS, respectively. Most mercapturic acids were 2–165 fold-higher in TTS compared to NS, with CEMA, MHBMA, 3-HPMA and SPMA showing the most relevant increases. Furthermore, some mercapturic acids were higher in ECU than NS; CEMA and 3-HPMA, in particular, showed significant increases and were 1.8 and 4.9 fold-higher, respectively. Conclusions This study confirms that tobacco smoking is a major source of carcinogenic chemicals such as benzene and 1,3-butadiene; electronic cigarette use is a minor source, mostly associated with exposure to chemicals with less carcinogenic potential such as acrylonitrile and acrolein.

Phototoxic reaction is a known feature of EPP at least in part triggered by the oxidative status, complement system activation, and mast cell response. The aim of this study was to verify some aspects involved in phototoxic reaction during a season. The complement system was evaluated by C3 assay, alternative pathway by factor-B, and classical pathway by C1q; oxidative status was tested with malondialdehyde (MDA) and mast cell by IL-10 assay. The serum samples were collected in winter and summer from 19 EPP patients and 13 controls. The reaction to sun exposure within each group was monitored without any invasive treatment. In summer, C3 and factor B were higher in patients than in controls (p = 0.002 and

Inhibition of KEAP1-NRF2 protein-protein interaction is considered a promising strategy to selectively and effectively activate NRF2, a transcription factor which is involved in several pathologies such as Huntington's disease (HD). A library of linear peptides based on the NRF2-binding motifs was generated on the nonapeptide lead Ac-LDEETGEFL-NH2 spanning residues 76–84 of the Neh2 domain of NRF2 with the aim to replace E78, E79 and E82 with non-acidic amino acids. A deeper understanding of the features and accessibility of the T80 subpocket was also targeted by structure-based design. Approaches to improve cell permeability were investigated using both different classes of cyclic peptides and conjugation to cell-penetrating peptides. This insight will guide future design of macrocycles, peptido-mimetics and, most importantly, small neutral brain-penetrating molecules to evaluate whether NRF2 activators have utility in HD.