Chemical Synthesis of the Thymocyte Differentiation Antigen 1 (Thy-1) N-glycoprotein

The glycosylation of the proteins is a common post-translational modification found in eukaryotic cells. Glycosylated proteins play a crucial role in cell adhesion, cell-matrix interaction, and immune response. Several studies suggest that the overexpression of these glycoconjugates is linked to many diseases, inflammation, viral infections, cancer metastasis, and cellular apoptosis among other pathologies. Over the years, the scientific community has tried to answer questions about the influence of the oligosaccharides on the protein structure or the relationship between the oligosaccharide structure and glycoproteins function. The main limitation to study these important aspects is the difficulty to obtain homogeneous isoforms of glycoproteins due to the lack of genetic control in the biosynthesis. The crescent interest in the understanding of structure-activity relationship of glycoproteins has led to the development of new technologies to obtain complex glycans and proteins. In this work, different methodologies were investigated to synthesize defined isoforms of the ubiquitous glycoprotein called Thy-1 or CD90. This heavily glycosylated protein was first discovered in 1964 in mouse T lymphocytes and later detected in human fibroblast neurons, blood stem cells, and endothelial cells. The 25kDa glycoprotein is translated as a 161 and 160 amino acids length protein in humans and mouse, respectively. The protein is transferred from ribosomes into the ER and receives several post-translational modifications including N-glycosylation at three different asparagine residues and a glypiation at the C-terminal cysteine residue. Several studies showed the association between the expression of Thy-1 and T cell activation, neurite outgrowth, apoptosis, leukocyte and melanoma cell adhesion and migration, tumor suppression, and fibroblast proliferation. The exploration of the structure-activity relationship to determine the role of the glycosylation on this protein required defined glycoprotein and motivated this work to develop a de novo synthesis of the Thy-1 pure glycoforms. The retrosynthesis of the mature glycoprotein was designed considering three key steps: 1) the assembly of the primary sequence of the protein; 2) the introduction of glycans into the protein; 3) the differentiation of the oligosaccharides at the glycosylation sites. The primary sequence of the glycoprotein was obtained using a sequential native chemical ligation (NCL) of three segments: the peptide fragment (1-18), the glycopeptide (19-84) having two N glycosylations, and the glycopeptide (85-110) containing one N glycosylation. The three peptide fragments were synthesized by solid-phase peptide synthesis (SPPS) and were obtained having a hydrazide moiety on the C-terminus that was converted into a thioester prior to the NCL. For this purpose, a Wang and a trityl resin were functionalized with hydrazine and modified manually with the amino acid on the C-termini. The protecting acetamidomethyl group was used to mask the thiol of the N-terminal cysteine residues in the fragment (19-84) and (85-110) to allow the stepwise ligation of the fragments. The elongation of the polypeptide chains was carried out in the microwave-assisted synthesizer and careful optimization of deprotection and coupling cycles was executed for the three fragments. The incorporation of amino acids was performed considering the common problems that affect the peptide synthesis including the cyclization reaction on asparagine and aspartic acid, and the racemization of cysteine and histidine at temperatures higher than 50°C. These precautions were not enough to obtain the Thy-1 glycopeptide fragments in high yield and with desired quality. The synthesis of the designed peptides required multiple optimizations to avoid side reactions. Some general requirements were established. Hindered amino acids (glutamic acid, isoleucine, and phenylalanine) required double coupling to assure complete attachment to the growing chain. Vicinal identical amino acids (e.g. serine 25-serine 26) were introduced using single coupling for the first residue in the sequence and a double coupling for the following residue. One of the most crucial point for the optimization of the synthesis was the minimization of the aspartimide formation involving the cyclization of the aspartic acid. Among different cocktails for the removal of the Fmoc group promising the suppression of this side reaction, the best results were obtained by using 20% piperidine and 0.7% of formic acid in dimethylformamide, which reduced the aspartimide formation up to 70%. The glycosylations were introduced following two approaches to allow differentiation between the glycan at each glycosylation site. In the cassette-method, a glycosylated asparagine was synthesized and incorporated in the corresponding glycosylation site during the assembly of the peptides. Different procedures were evaluated and optimized for the synthesis of the N-acetyl-O-per-acetylated glucosaminyl asparagine 2 and its introduction into SPPS to get the fragments 6a-c (19-84) and 37a-b (85-110) with a peracetylated glucosamine on each glycosylation site. The convergent approach was used to synthesize fragment 6b (19-84) having the peracetylated N acetylglucosamine 2 in one glycosylation site and an unprotected N-acetylglucosamine, installed via Lansbury aspartylation on the second site. The Lansbury strategy required the formation of the amide bond between the free carboxylate at an aspartic acid and the N-acetylglucosamine amine. The reaction was performed on the fully protected peptide fragment and required the synthesis of two building blocks, an orthogonal protected aspartic acid 3, and a pseudoproline dipeptide 4. The aspartic acid was synthesized with a photolabile protecting group on the side chain, which was selectively removed on resin to give a free carboxylic acid. To avoid the undesired rearrangement of the amino acid, the protected pseudoproline dipeptide Thr-Ser(Me,MePro) was incorporated before the aspartic acid. The three (glyco)peptides were efficiently synthesized by a combination of manual and automated processes and were characterized by their challenging purifications. The difficulties in obtaining the isoforms from the Thy-1 fragment (19-84) glycopeptides 6a-b, were related to the low solubility of the generated glycopeptides and the loss of acetyl groups on the peracetylated glucosamine. The analysis of these problems led to the design of the optimal strategy to synthesize in high yield the fragment 6c having a protected and non-protected N‑acetylglucosamine. The synthesis of glycoform 6c involved the coupling of a per-acetylated N-acetylglucosaminyl asparagine building block in the peptide sequence that was de-acetylated before the introduction of the second glycosylated asparagine residue. The assembly of the Thy-1 glycoprotein involved the ligation of the synthesized fragments by the chemoselective reaction between the C-terminal peptide thioester in one peptide fragment and the free N-terminal cysteine residue of another fragment. The process required the conversion of the synthesized peptide hydrazides into the corresponding thioesters prior to each ligation and the removal of the Acm-group of the fragments having the N-terminal cysteine. Two combinations were studied to ligate the three fragments: from C- to N-terminus and from N- to the C-terminus. The first approach consisted of the initial ligation of the glycopeptides 6a-c (19-84) and 37a-b (85-110) and the subsequent ligation with the peptide 1 (1-18). The second strategy involved the reaction of peptide 1 (1-18) with the glycopeptide 6a-c (19-84) and the following ligation with the fragment 37a-b (85-110). The ligation conditions were investigated, and the strategy was selected considering factors such as the availability and solubility of the fragments, number of steps and the efficiency of the processes. The N-to C-strategy was the first approach adopted. The hydrazide precursor 6a was converted into the thioester fragment 38 by formation of the peptide azide at low pH and thiolysis with the corresponding thiol. The Acm group from the cysteine in 37b was removed with mercury (II) acetate and reduction of the sulfur-mercury complex with mercaptoethanol. The ligation of the fragments delivered the glycoprotein 40 and the hydrolysed fragment 38 as by-product. The low solubility of the glycoprotein 40 hampered the isolation of the product and suggested the need of more polar fragment. For this purpose, glycoform 6b was converted into the thioester 41 and reacted with fragment 37a having a free N-terminal cysteine. Surprisingly, this ligation did not proceed suggesting that the treatment with mercury (II) acetate could be beneficial for the ligation. A N- to C- ligation of fragment 6c treated with the mercury (II) acetate with the thioester of fragment 1 proceeded successfully and the isolation of the ligation product yielded the glycoprotein 45, confirming the better behaviour of polar fragments and reactivity of the cysteine-containing fragments treated with mercury. A ligation of the MPAA thioester of Thy-1 fragment (1-84) with 37b to obtain the full glycoprotein Thy-1 (1-120) was hindered by the poor solubility of thioester 46 in the ligation media. A new C- to N- ligation involving the polar glycoform 6c with the fragment 37b delivered the glycoprotein 49 that was treated in one-pot with PdCl2 to release the N-terminal cysteine and get 50. Finally, a methyl 3-mercaptopropionate MMP thioester of fragment 1-18 (51) was ligated with glycoprotein fragment 50 to obtain the desired Thy-1 glycoprotein (1-120) having three glycosylations. In this work was presented the design and evaluation of different strategies for the synthesis of the glycoprotein Thy-1. The assembly of the 13kDa glycoprotein required different steps and the optimized synthesis of amino acid building blocks for the solid-phase assembly of glycopeptides. Various methods were applied for the generation of glycopeptides, the exploration of the chemical properties of the obtained fragments, and the modulation of the chemical conditions for the ligation of the peptide fragments to get the target glycoprotein. This work focused on the production of homogeneous glycoforms of Thy-1. However, the synthetic methods and protocols established in this work are applicable for the synthesis of any peptides and glycopeptides and contribute to the chemical synthesis of other important glycoproteins

Parametric and Non-Parametric Analyses for Pedestrian Crash Severity Prediction in Great Britain

The study aims to investigate the factors that are associated with fatal and severe vehicle– pedestrian crashes in Great Britain by developing four parametric models and five non-parametric tools to predict the crash severity. Even though the models have already been applied to model the pedestrian injury severity, a comparative analysis to assess the predictive power of such modeling techniques is limited. Hence, this study contributes to the road safety literature by comparing the models by their capabilities of identifying the significant explanatory variables, and by their performances in terms of the F-measure, the G-mean, and the area under curve. The analyses were carried out using data that refer to the vehicle–pedestrian crashes that occurred in the period of 2016–2018. The parametric models confirm their advantages in offering easy-to-interpret outputs and understandable relations between the dependent and independent variables, whereas the non-parametric tools exhibited higher classification accuracies, identified more explanatory variables, and provided insights into the interdependencies among the factors. The study results suggest that the combined use of parametric and non-parametric methods may effectively overcome the limits of each group of methods, with satisfactory prediction accuracies and the interpretation of the factors contributing to fatal and serious crashes. In the conclusion, several engineering, social, and management pedestrian safety countermeasures are recommended

Role of Sterylglucosidase 1 (Sgl1) on the pathogenicity of Cryptococcus neoformans: Potential applications for vaccine development

Cryptococcosis caused by C. neoformans and C. gattii affects a large population and is a cause of significant morbidity and mortality. Despite its public health burden, there are currently no vaccines against cryptococcosis and new strategies against such infections are needed. In this study, we demonstrate that C. neoformans has the biochemical ability to metabolize sterylglucosides (SGs), a class of immunomodulatory glycolipids. Genetic manipulations that eliminate cryptococccal sterylglucosidase lead to the accumulation of SGs and generate a mutant strain (Îsgl1) that is non-pathogenic in the mouse models of cryptococcosis. Interestingly, this mutant strain acts as a vaccine strain and protects mice against cryptococcosis following infection with C. neoformans or C. gattii. The immunity induced by the Îsgl1 strain is not CD4+ T-cells dependent. Immunocompromised mice, which lack CD4+ T-cells, are able to control the infection by Îsgl1 and acquire immunity against the challenge by wild-type C. neoformans following vaccination with the Îsgl1 strain. These findings are particularly important in the context of HIV/AIDS immune deficiency and suggest that the Îsgl1 strain might provide a potential vaccination strategy against cryptococcosis

Econometric and Machine Learning Methods to Identify Pedestrian Crash Patterns

Walking plays an important role in overcoming many challenges nowadays, and governments and local authorities are encouraging healthy and environmentally sustainable lifestyles. Nevertheless, pedestrians are the most vulnerable road users and crashes with pedestrian involvement are a serious concern. Thus, the identification of pedestrian crash patterns is crucial to identify appropriate safety countermeasures. The aims of the study are (1) to identify the road infrastructure, environmental, vehicle, and driver-related patterns that are associated with an overrepresentation of pedestrian crashes, and (2) to identify safety countermeasures to mitigate the detected pedestrian crash patterns. The analysis carried out an econometric model, namely the mixed logit model, and the association rules and the classification tree algorithm, as machine learning tools, to analyse the patterns contributing to the overrepresentation of pedestrian crashes in Italy. The dataset consists of 874,847 crashes—including 101,032 pedestrian crashes—that occurred in Italy from 2014 to 2018. The methodological approach adopted in the study was effective in uncovering relations among road infrastructure, environmental, vehicle, and driver-related patterns, and the overrepresentation of pedestrian crashes. The mixed logit provided a clue on the impact of each pattern on the pedestrian crash occurrence, whereas the association rules and the classification tree detected the associations among the patterns with insights on how the co-occurrence of more factors could be detrimental to pedestrian safety. Drivers’ behaviour and psychophysical state turned out to be crucial patterns related to pedestrian crashes’ overrepresentation. Based on the identified crash patterns, safety countermeasures have been proposed

Systematic literature review of 10 years of cyclist safety research

Cyclist safety is a research field that is gaining increasing interest and attention, but still offers questions and challenges open to the scientific community. The aim of this study was to provide an exhaustive review of scientific publications in the cyclist safety field. For this purpose, Bibliometrix-R tool was used to analyse 1066 documents retrieved from Web of Science (WoS) between 2012 and 2021. The study examined published sources and productive scholars by exposing their most influential contributions, presented institutions and countries most contributing to cyclist safety and explored countries open towards international collaborations. A keywords analysis provided the most frequent author keywords in cyclist safety shown in a word cloud with E-bike, behaviour, and crash severity representing the primary keywords. Furthermore, a thematic map of cyclist safety field drafted from the author’s keywords was identified. The strategic diagram is divided in four quadrants and, according to both density and centrality, the themes can be classified as follows: 1) motor themes, characterized by high value of both centrality and density; 2) niche themes, defined by high density and low centrality; 3) emerging or declining themes, featured by low value of both centrality and density; and 4) basic themes, distinguished by high centrality and low density. The motor themes (i.e., the main topics in cyclist safety field) crash severity and bike network were further explored. The research findings will be useful to develop strategies for making bike a safer and more confident form of transport as well as to guide researchers towards the future scientific knowledge

Parametric and Non-Parametric Analyses for Pedestrian Crash Severity Prediction in Great Britain

The study aims to investigate the factors that are associated with fatal and severe vehicle–pedestrian crashes in Great Britain by developing four parametric models and five non-parametric tools to predict the crash severity. Even though the models have already been applied to model the pedestrian injury severity, a comparative analysis to assess the predictive power of such modeling techniques is limited. Hence, this study contributes to the road safety literature by comparing the models by their capabilities of identifying the significant explanatory variables, and by their performances in terms of the F-measure, the G-mean, and the area under curve. The analyses were carried out using data that refer to the vehicle–pedestrian crashes that occurred in the period of 2016–2018. The parametric models confirm their advantages in offering easy-to-interpret outputs and understandable relations between the dependent and independent variables, whereas the non-parametric tools exhibited higher classification accuracies, identified more explanatory variables, and provided insights into the interdependencies among the factors. The study results suggest that the combined use of parametric and non-parametric methods may effectively overcome the limits of each group of methods, with satisfactory prediction accuracies and the interpretation of the factors contributing to fatal and serious crashes. In the conclusion, several engineering, social, and management pedestrian safety countermeasures are recommended

Age Associated Decrease of MT-1 Melatonin Receptor in Human Dermal Skin Fibroblasts Impairs Protection Against UV-Induced DNA Damage

The human body follows a physiological rhythm in response to the day/night cycle which is synchronized with the circadian rhythm through internal clocks. Most cells in the human body, including skin cells, express autonomous clocks and the genes responsible for running those clocks. Melatonin, a ubiquitous small molecular weight hormone, is critical in regulating the sleep cycle and other functions in the body. Melatonin is present in the skin and, in this study, we showed that it has the ability to dose-dependently stimulate PER1 clock gene expression in normal human dermal fibroblasts and normal human epidermal keratinocytes. Then we further evaluated the role of MT-1 melatonin receptor in mediating melatonin actions on human skin using fibroblasts derived from young and old subjects. Using immunocytochemistry, Western blotting and RT-PCR, we confirmed the expression of MT-1 receptor in human skin fibroblasts and demonstrated a dramatic age-dependent decrease in its level in mature fibroblasts. We used siRNA technology to transiently knockdown MT-1 receptor in fibroblasts. In these MT-1 knockdown cells, UV-dependent oxidative stress (H2O2 production) was enhanced and DNA damage was also increased, suggesting a critical role of MT-1 receptor in protecting skin cells from UV-induced DNA damage. These studies demonstrate that the melatonin pathway plays a pivotal role in skin aging and damage. Moreover, its correlation with skin circadian rhythm may offer new approaches for decelerating skin aging by modulating the expression of melatonin receptors in human skin

Injury severity prediction of cyclist crashes using random forests and random parameters logit models

Cycling provides numerous benefits to individuals and to society but the burden of road traffic injuries and fatalities is disproportionately sustained by cyclists. Without awareness of the contributory factors of cyclist death and injury, the capability to implement context-specific and appropriate measures is severely limited. In this paper, we investigated the effects of the characteristics related to the road, the environment, the vehicle involved, the driver, and the cyclist on severity of crashes involving cyclists analysing 72,363 crashes that occurred in Great Britain in the period 2016–2018. Both a machine learning method, as the Random Forest (RF), and an econometric model, as the Random Parameters Logit Model (RPLM), were implemented. Three different RF algorithms were performed, namely the traditional RF, the Weighted Subspace RF, and the Random Survival Forest. The latter demonstrated superior predictive performances both in terms of F-measure and G-mean. The main result of the Random Survival Forest is the variable importance that provides a ranked list of the predictors associated with the fatal and severe cyclist crashes. For fatal classification, 19 variables showed a normalized importance higher than 5% with the second involved vehicle manoeuvring and the gender of the driver of the second vehicle having the greatest predictive ability. For serious injury classification, 13 variables showed a normalized importance higher than 5% with the bike leaving the carriageway having the greatest normalized importance. Furthermore, each path from the root node to the leaf nodes has been retraced the way back generating 361 if-then rules with fatal crash as consequent and 349 if-then rules with serious injury crash as consequent. The RPLM showed significant unobserved heterogeneity in the data finding four normal distributed indicator variables with random parameters: cyclist age ≥ 75 (fatal prediction), cyclist gender male (fatal and serious prediction), and driver aged 55–64 (serious prediction). The model's McFadden Pseudo R2 is equal to 0.21, indicating a very good fit. Furthermore, to understand the magnitude of the effects and the contribution of each variable to injury severity probabilities the pseudo-elasticity was assessed, gaining valuable insights into the relative importance and influence of the variables. The RF and the RPLM resulted complementary in identifying several roadways, environmental, vehicle, driver, and cyclist-related factors associated with higher crash severity. Based on the identified contributory factors, safety countermeasures useful to develop strategies for making bike a safer and more friendly form of transport were recommended

Parametric and Non-Parametric Analyses for Pedestrian Crash Severity Prediction in Great Britain

The study aims to investigate the factors that are associated with fatal and severe vehicle–pedestrian crashes in Great Britain by developing four parametric models and five non-parametric tools to predict the crash severity. Even though the models have already been applied to model the pedestrian injury severity, a comparative analysis to assess the predictive power of such modeling techniques is limited. Hence, this study contributes to the road safety literature by comparing the models by their capabilities of identifying the significant explanatory variables, and by their performances in terms of the F-measure, the G-mean, and the area under curve. The analyses were carried out using data that refer to the vehicle–pedestrian crashes that occurred in the period of 2016–2018. The parametric models confirm their advantages in offering easy-to-interpret outputs and understandable relations between the dependent and independent variables, whereas the non-parametric tools exhibited higher classification accuracies, identified more explanatory variables, and provided insights into the interdependencies among the factors. The study results suggest that the combined use of parametric and non-parametric methods may effectively overcome the limits of each group of methods, with satisfactory prediction accuracies and the interpretation of the factors contributing to fatal and serious crashes. In the conclusion, several engineering, social, and management pedestrian safety countermeasures are recommended

A combined analytical approach applied to Medieval wall paintings from Puglia (Italy): The study of painting techniques and its conservation state

Journal of Raman Spectroscopy Volume 47, Issue 3, 1 March 2016, Pages 321-328 A combined analytical approach applied to Medieval wall paintings from Puglia (Italy): The study of painting techniques and its conservation state (Article) Fico, D. , Pennetta, A., Rella, G., Savino, A., Terlizzi, V., De Benedetto, G.E. Laboratorio di Spettrometria di Massa Analitica Ed Isotopica, Dipartimento di Beni Culturali, Università Del Salento, Edificio M, campus Ecotekne, s.p. Lecce-Monteroni, Lecce, Italy View references (46) Abstract A combined analytical approach has been applied to the wall paintings, dated from 10th to 14th centuries, of the Santi Stefani crypt at Vaste (Lecce, Southern Italy). These paintings are a precious testimony of Medieval art in Southern Italy. However, the church shows problems of damp as well as clear evidences of flora, fungi and mold presence, and there is little knowledge of the pictorial methodologies used. Raman spectroscopy allowed to determine the palette and to reconstruct the worksite and the chronological sequence of the various paint layers. Kaolinite, calcite, carbon black, hematite, massicot, goethite, indigo and azurite were identified as pigments along with synthetic pigments, like phthalocyanine blue and chrome yellow. Attenuated total reflectance-Fourier transform infrared spectroscopy suggested the presence of egg as a binder in some pictorial layers. The conservation state of the crypt is poor, and detachments of pigmented layers are frequent because of the presence of subflorescence and efflorescence: nitrate, sulfate and chloride salts have been identified spectroscopically and quantified by ion chromatography. The extensive use of kaolinite in Santi Stefani, actually not uncommon in Medieval art, is observed for the first time in a crypt of Puglia: its use to stabilize some pigments and to improve their adhesion on substrate is proposed. Copyright © 2015 John Wiley & Sons, Ltd