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Background and Aim: Dialysis patients are a high-risk population and have a reduced immune response to vaccination against SARS-CoV-2. The aim of this study was to assess the humoral response to homologous Gam-COVID-Vac (Sputnik V) and heterologous Sputnik V/mRNA-1273 (Moderna) vaccination in dialysis patients. The vaccination scheme depended on dose availability and the prioritization of risk populations as established by the Argentine Ministry of Health. Methods: Previous COVID-19 infection was determined in symptomatic patients. Binding IgG antibodies against the spike (S) receptor-binding domain (RBD) of SARS-CoV-2 (anti-S-RBD) concentration was assessed between 3 and 16 weeks after the boost dose. Anti-S-RBD antibodies were quantified using the Abbott Diagnostics SARS-CoV-2 IgG II Quant chemiluminescent microparticle immunoassay (CMIA) on an Architect i2000 SR and an Alinity I analyzer (Abbott Diagnostics, Abbott Park, Illinois, USA). To standardize the results to WHO binding antibody units (BAU), a correction factor for Abbott arbitrary units (AU) was applied where 1 BAU/mL equals 0.142 AU, as previously established by Abbott with the WHO international standard NIBSC 20–136. Following the manufacturer’s recommendations, samples were considered reactive for anti-S-RBD when titers were above 50 AU/mL (7.2 BAU/mL). An 80% protective effect (PROT-80) against symptomatic SARS-CoV-2 infection was assumed when anti-S-RBD titers were 506 BAU/ml or higher. Charlson Comorbidity Index (CCI) score was classified as mild = 1–2, moderate = 3–4, and severe ≥ 5. Side effects were evaluated until day 7 by patients´ self-reported questionnaire. Results: One hundred seven participants were enrolled [n= 84 homologous (SpV/SpV), nn 23 heterologous (SpV/Mod)]. Median (IQR) age was 64 (50–75) years old and 79 (73.8%) were male. Additionally, 19 (22.6%) of the SpV/SpV and 4 (17.4%) of the SpV/Mod group had a prior confirmed SARS-CoV-2 infection (p= 0.589). In the overall population, 103 patients reached seroconversion (96.3%). Anti-S-RBD IgG median titers (IQR) were higher in the heterologous [1222 (288–5680) BAU/mL] than in the homologous scheme [447 (100–1551) BAU/mL], p= 0.022. In a linear model adjusted for age, gender, days from first vaccination to boost dose and days from the boost dose to the anti-S-RBD IgG determination, previous SARS-COV-2 infection (B: 2062.2; CI95: 1231.8–2892.6; p< 0.001), and SpV/Mod vaccination scheme (B: 1294.6; CI95: 435.58–2147.6; p= 0.003) were independently associated with anti-S-RBD levels. Finally, a higher frequency of adverse effects was associated with the heterologous scheme, although they were well tolerated by all individuals. Conclusions: The present study provides evidence that the homologous SpV/SpV and heterologous SpV/Mod schemes showed good efficacy and safety in patients on chronic dialysis. These results could be useful for designing future vaccination strategies, especially aimed at this risk group. Graphical abstract:

Nuclear magnetic resonance (NMR) data from NOESY (nuclear Overhauser enhancement spectroscopy) and ROESY (rotating frame Overhauser enhancement spectroscopy) experiments can easily be combined with distance geometry (DG) based conformer generators by modifying the molecular distance bounds matrix. In this work, we extend the modern DG based conformer generator ETKDG, which has been shown to reproduce experimental crystal structures from small molecules to large macrocycles well, to include NOE-derived interproton distances. In noeETKDG, the experimentally derived interproton distances are incorporated into the distance bounds matrix as loose upper (or lower) bounds to generate large conformer sets. Various subselection techniques can subsequently be applied to yield a conformer bundle that best reproduces the NOE data. The approach is benchmarked using a set of 24 (mostly) cyclic peptides for which NOE-derived distances as well as reference solution structures obtained by other software are available. With respect to other packages currently available, the advantages of noeETKDG are its speed and that no prior force-field parametrization is required, which is especially useful for peptides with unnatural amino acids. The resulting conformer bundles can be further processed with the use of structural refinement techniques to improve the modeling of the intramolecular nonbonded interactions. The noeETKDG code is released as a fully open-source software package available at www.github.com/rinikerlab/customETKDG.

USP7 is a deubiquitinase implicated in destabilizing the tumor suppressor p53, and for this reason it has gained increasing attention as a potential oncology target for small molecule inhibitors. Herein we describe the biophysical, biochemical, and computational approaches that led to the identification of 4-(2-aminopyridin-3-yl)phenol compounds described by Kategaya(Nature2017, 550, 534–538)as specific inhibitors of USP7. Fragment based lead discovery (FBLD) by NMR combined with virtual screening and re-mining of biochemical high-throughput screening (HTS) hits led to the discovery of a series of ligands that bind in the “palm” region of the catalytic domain of USP7 and inhibit its catalytic activity. These ligands were then optimized by structure-based design to yield cell-active molecules with reasonable physical properties. This discovery process not only involved multiple techniques working in concert but also illustrated a unique way in which hits from orthogonal screening approaches complemented each other for lead identification.

Disulfide-constrained peptides (DCPs) have gained increased attention as a drug modality due to their exceptional stability and combined advantages of large biologics and small molecules. Chemical synthesis, although widely used to produce DCPs, is associated with high cost, both economically and environmentally. To reduce the dependence on solid phase peptide synthesis and the negative environmental footprint associated with it, we present a highly versatile, low-cost, and environmentally friendly bioproduction platform to generate DCPs and their conjugates as well as chemically modified or isotope-labeled DCPs. Using the DCP against the E3 ubiquitin ligase Zinc and Ring Finger 3, MK1-3.6.10, as a model peptide, we have demonstrated the use of bacterial expression, combined with Ser ligation or transglutaminase-mediated XTEN ligation, to produce multivalent MK1-3.6.10 and MK1-3.6.10 with N-terminal functional groups. We have also developed a bioproduction method for the site-specific incorporation of unnatural amino acids into recombinant DCPs by the amber codon suppression system. Lastly, we produced 15N/13C-labeled MK1-3.6.10 with high yield and assessed the performance of a semiautomated resonance assignment workflow that could be used to accelerate binding studies and structural characterization of DCPs. This study provides a proof of concept to generate functionalized DCPs using bioproduction, providing a potential solution to alleviate the reliance on hazardous chemicals, reduce the cost, and expedite the timeline for DCP discovery.

The development of selective ubiquitin-specific protease-7 (USP7) inhibitors GNE-6640 and GNE-6776, which induce tumour cell death and reveal differential kinetics of Lys-48 and Lys-63-linked ubiquitin chain depolymerization by USP7.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma LDL cholesterol (LDL-c) levels by promoting the degradation of liver LDL receptors (LDLRs). Antibodies that inhibit PCSK9 binding to the EGF(A) domain of the LDLR are effective in lowering LDL-c. However, the discovery of small-molecule therapeutics is hampered by difficulty in targeting the relatively flat EGF(A)-binding site on PCSK9. Here we demonstrate that it is possible to target this site, based on the finding that the PCSK9 P′ helix displays conformational flexibility. As a consequence, the vacated N-terminal groove of PCSK9, which is adjacent to the EGF(A)-binding site, is in fact accessible to small peptides. In phage-display experiments, the EGF(A)-mimicking peptide Pep2-8 was used as an anchor peptide for the attachment of an extension peptide library directed toward the groove site. Guided by structural information, we further engineered the identified groove-binding peptides into antagonists, which encroach on the EGF(A)-binding site and inhibit LDLR binding.

Bacterial richness in maritime Antarctica has been poorly described to date. Phylogenetic affiliation of seawater free-living microbial assemblages was studied from three locations near the Argentinean Jubany Station during two Antarctic summers. Sixty 16S RNA cloned sequences were phylogenetically affiliated to Alphaproteobacteria(30/60 clones), Gammaproteobacteria(19/60 clones), Betaproteobacteriaand Cytophaga–Flavobacteriia–Bacteroides(CFB), which were (2/60) and (3/60) respectively. Furthermore, six out of 60 clones could not be classified. Both, Alphaproteobacteriaand Gammaproteobacteria, showed several endemic and previously undescribed sequences. Moreover, the absence of Cyanobacteriasequences in our samples is remarkable. In conclusion, we are reporting a rich sequence assemblage composed of widely divergent isolates among themselves and distant from the most closely related sequences currently deposited in data banks.

There are two main trends in the area of home and service robotics. The classical one aims at the development of a single skilled servant robot, able to perform complex tasks in a passive environment. The second, more recent trend aims at the achievement of complex tasks through the cooperation of a network of simpler robotic devices pervasively embedded in the domestic environment. This paper contributes to the latter trend by describing the PEIS Table, an autonomous robotic table that can be embedded in a smart environment. The robotic table can operate alone, performing simple point-to-point navigation, or it can collaborate with other devices in the environment to perform more complex tasks. Collaboration follows the PEIS Ecology model. The hardware and software design of the PEIS Table are guided by a set of requirements for robotic domestic furniture that differ, to some extent, from the requirements usually considered for service robots.

In addition to orchestrating the expression of all erythroid-specific genes, GATA-1 controls the growth, differentiation, and survival of the erythroid lineage through the regulation of genes that manipulate the cell cycle and apoptosis. The stages of mammalian erythropoiesis include global gene inactivation, nuclear condensation, and enucleation to yield circulating erythrocytes, and some of the genes whose expression are altered by GATA-1 during this process are members of the p53 pathway. In this study, we demonstrate a specific in vitro interaction between the transactivation domain of p53 (p53TAD) and a segment of the GATA-1 DNA-binding domain that includes the carboxyl-terminal zinc-finger domain. We also show by immunoprecipitation that the native GATA-1 and p53 interact in erythroid cells and that activation of p53-responsive promoters in an erythroid cell line can be inhibited by the overexpression of GATA-1. Mutational analysis reveals that GATA-1 inhibition of p53 minimally requires the segment of the GATA-1 DNA-binding domain that interacts with p53TAD. This inhibition is reciprocal, as the activation of a GATA-1–responsive promoter can be inhibited by p53. Based on these findings, we conclude that inhibition of the p53 pathway by GATA-1 may be essential for erythroid cell development and survival.

In addition to orchestrating the expression of all erythroid-specific genes, GATA-1 controls the growth, differentiation, and survival of the erythroid lineage through the regulation of genes that manipulate the cell cycle and apoptosis. The stages of mammalian erythropoiesis include global gene inactivation, nuclear condensation, and enucleation to yield circulating erythrocytes, and some of the genes whose expression are altered by GATA-1 during this process are members of the p53 pathway. In this study, we demonstrate a specific in vitro interaction between the transactivation domain of p53 (p53TAD) and a segment of the GATA-1 DNA-binding domain that includes the carboxyl-terminal zinc-finger domain. We also show by immunoprecipitation that the native GATA-1 and p53 interact in erythroid cells and that activation of p53-responsive promoters in an erythroid cell line can be inhibited by the overexpression of GATA-1. Mutational analysis reveals that GATA-1 inhibition of p53 minimally requires the segment of the GATA-1 DNA-binding domain that interacts with p53TAD. This inhibition is reciprocal, as the activation of a GATA-1–responsive promoter can be inhibited by p53. Based on these findings, we conclude that inhibition of the p53 pathway by GATA-1 may be essential for erythroid cell development and survival.

Digital droplet PCR (ddPCR) is an implementation of conventional PCR, with the potential of overcoming some limitations of real-time quantitative PCR (RQ-PCR). To evaluate if ddPCR may improve the quantification of disease levels and refine patients' risk stratification, 116 samples at four time points from 44 (35 B-lineage and 9 T-lineage) adult Philadelphia-negative acute lymphoblastic leukemia patients enrolled in the GIMEMA LAL1913 protocol were analyzed by RQ-PCR and ddPCR. A concordance rate between RQ-PCR and ddPCR of 79% (P < 0.0001) was observed; discordances were identified in 21% of samples, with the majority being RQ-PCR-negative (NEG) or positive not quantifiable (PNQ). ddPCR significantly reduced the proportion of PNQ samples—2.6% versus 14% (P = 0.003)—and allowed disease quantifiability in 6.6% of RQ-PCR-NEG, increasing minimal residual disease quantification in 14% of samples. Forty-seven samples were also investigated by next-generation sequencing, which confirmed the ddPCR results in samples classified as RQ-PCR-PNQ or NEG. By reclassifying samples on the basis of the ddPCR results, a better event-free survival stratification of patients was observed compared with RQ-PCR: indeed, ddPCR captured more true-quantifiable samples, with five relapses occurring in three patients who resulted RQ-PCR-PNQ/NEG but proved ddPCR positive quantifiable. At variance, no relapses were recorded in patients whose follow-up samples were RQ-PCR-PNQ but reclassified as ddPCR-NEG. A broader application of ddPCR in acute lymphoblastic leukemia clinical trials will help to improve patients' stratification.

The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well-known helix-turn-helix motif. The recognition helix penetrates the DNA major groove, gives specific protein–DNA contacts and forms direct, or water-mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42–53, where residues W48 and F49, not involved in the protein–DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well-defined secondary structure in TFE and in TFE/water (90/10, v/v) with an α-helix encompassing residues 4–9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted α-helical conformation involving residues 4–8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the α-helical conformation even if separated from the rest of the molecule.

Linear and cyclic cyclolinopeptide A (CLA) analogues containing α‐hydroxymethylleucine (HmL) in positions 1, 4, and 1&4, and α‐hydroxymethylvaline (HmV) in position 5, were synthesized by the solid‐phase peptide strategy and cyclized with the 1‐Ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide/1‐hydroxy‐7‐azabenzotriazole (EDC/HOAt) reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation assays (LPA). Only HmL‐containing peptides demonstrated at about 25% lower immunosuppressive activity, but they are four times more soluble in water solutions than the native CLA. It seems from the LPA results that peptide [(HmL4)CLA] is the most promising for further studies. This peptide was characterized in solution, at room temperature in CDCl3, and the conformation compared with that observed for CLA in the solid state. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2005

In this paper we report the synthesis and a detailed NMR solution characterization of a new CCK8 analogue and its indium(III) complex, PK‐CCK8 and In‐PK‐CCK8. The new compounds contain a porphyrin moiety covalently bound through an amide bond to the side chain of a Lys residue introduced at the N‐terminus of CCK8. A molecular dynamics simulation, based on the NMR structure of the complex between CCK8 and the N‐terminal extracellular arm of the CCKAreceptor, is also reported. Both the NMR study and the molecular dynamics simulation indicate that the porphyrin–peptide conjugate might be able to bind to the CCKAreceptor model. The results of the molecular dynamics calculations show that the conformational features of the CCK8/CCKAreceptor model complex and of the PK‐CCK8/CCKAreceptor‐model complex are similar. This evidence supports the view that the introduction of the porphyrin‐Lys moiety does not influence the mode of ligand binding to the CCKAreceptor model. The NMR structure of PK‐CCK8 in DMSO consists of a well defined pseudo‐helical N‐terminal region, while the C‐terminal region is flexible. Moreover, the absence of NOE contacts between the porphyrin and the peptide indicates that the macrocyclic ring is directed away from the peptide region involved in the binding with the receptor. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.

Secondary structure formation and stability are essential features in the knowledge of complex folding topology of biomolecules. To better understand the relationships between preferred conformations and functional properties of β‐homo‐amino acids, the synthesis and conformational characterization by X‐ray diffraction analysis of peptides containing conformationally constrained Cα,α‐dialkylated amino acid residues, such as α‐aminoisobutyric acid or 1‐aminocyclohexane‐1‐carboxylic acid and a single β‐homo‐amino acid, differently displaced along the peptide sequence have been carried out. The peptides investigated are: Boc‐βHLeu‐(Ac6c)2‐OMe, Boc‐Ac6c‐βHLeu‐(Ac6c)2‐OMe and Boc‐βHVal‐(Aib)5‐OtBu, together with the C‐protected β‐homo‐residue HCl·H‐βHVal‐OMe. The results indicate that the insertion of a βH‐residue at position 1 or 2 of peptides containing strong helix‐inducing, bulky Cα,α‐disubstituted amino acid residues does not induce any specific conformational preferences. In the crystal state, most of the NH groups of β‐homoresidues of tri‐ and tetrapeptides are not involved in intramolecular hydrogen bonds, thus failing to achieve helical structures similar to those of peptides exclusively constituted of Cα,α‐disubstituted amino acid residues. However, by repeating the structural motifs observed in the molecules investigated, a β‐pleated sheet secondary structure, and a new helical structure, named (14/15)‐helix, were generated, corresponding to calculated minimum‐energy conformations. Our findings, as well as literature data, strongly indicate that conformations of βH‐residues, with the µtorsion angle equal to −60°, are very unlikely. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.

ABSTRACTThe accuracy of LiPA 2.0 for hepatitis C virus 1 (HCV-1) subtype classification was analyzed. LiPA 2.0 genotype results from 101 HCV-1-infected patients were compared to genotype findings determined by direct core sequencing. Eleven (11%) samples were misclassified. Given the influence of the HCV-1-subtype in the anti-HCV therapy response, an alternative classification method is warranted.

The synthesis and conformational properties of a series of dipeptide taste ligands, differing from the commercial sweetener aspartame by the presence of a methylene group between the Cαand the C′ carbon atoms (as in homo‐β‐residues) in either the L‐Asp or the L‐Phe residues, are described. Homo‐β‐residues such as homo‐β‐aspartic acid, homo‐β‐phenylglycine and homo‐β‐phenylalanine, obtained by homologation of the corresponding proteinogenic α‐amino acids, have been used in the solution peptide synthesis of the following aspartame analogues in protected and unprotected forms: NH2‐homo‐β‐(Lor D)‐Asp‐L‐Phe‐OMe, NH2‐L‐Asp‐homo‐β‐L‐Phg‐OMe and NH2‐L‐Asp‐homo‐β‐L‐Phe‐OMe. Lengthening of the peptide skeleton at the L‐Asp site results in a drastic loss of sweetness with the production of tasteless compounds; on the other hand, lengthening of the skeleton at the C‐terminal L‐Phe site partially mantains the sweet taste in both NH2‐L‐Asp‐homo‐β‐L‐Phe‐OMe and NH2‐L‐Asp‐homo‐β‐L‐Phg‐OMe.The solution conformation of the synthesized dipeptide taste ligands was investigated by NMR and circular dichroism techniques. The analysis of NMR data combined with restrained molecular dynamics calculations shows that all peptides are fairly flexible and they do not assume a preferred conformation in DMSO and methanol. The peptides containing homo‐β‐L‐Phe and homo‐β‐L‐Phg do adopt a discrete number of conformations among which mainly extended and ‘L‐shaped’ conformation are represented. The circular dichroism spectra are consistent with the NMR results, indicating a significant flexibility for these compounds. Copyright © 1999 John Wiley & Sons, Ltd.

To define the skeletal muscle abnormalities in patients undergoing exercise deconditioning and evaluate the metabolic effect of propionyl‐L‐carnitine (PLC), muscle biopsies were obtained from 28 patients with effort angina and 31 control subjects. Coronary artery disease patients received either placebo (n= 12), PLC (1.5 g IV followed by infusion of 1 mg/kg/min for 30 min, n= 10), or L‐carnitine (1 g IV followed by infusion of 0.65 mg/kg/min for 30 min, n= 6) for 2 days. Exercise deconditioned patients treated with placebo showed normal muscle content of total carnitine and glycogen, and decrease in percentage of type 1 fibers (P< 0.01) and in the activity of citrate synthase (P< 0.05), succinate dehydrogenase (P< 0.05), and cytochrome oxidase (P< 0.05), as compared to controls. Both PLC and L‐carnitine did not modify muscle fiber composition or enzyme activities, but significantly increased muscle levels of total carnitine by 42% and 31%, respectively (P< 0.05). Moreover, PLC significantly increased glycogen muscle content (P< 0.01), while the equimolar dose of L‐carnitine did not. This effect, probably due to the anaplerotic activity of the propionic group of PLC, suggests that this drug may be effective in improving energy metabolism of muscles with impaired oxidative capacity. © John Wiley & Sons, Inc. Muscle Nerve20:1115–1120, 1997

The aim of this study was to investigate the hygiene performance of a camel (Camelus dromedarius) slaughtering process as carried out with the traditional method in the Sahrawi refugee camps located in southwestern Algeria. The camel slaughtering process in this region differs significantly from that carried out in commercial abattoirs. Slaughtering is performed outdoors in desert areas, and dehiding of the carcass is approached via the dorsoventral route rather than the classic ventrodorsal route. Samples were taken from 10 camel carcasses from three different areas: the hide, the carcass meat immediately after dehiding, and the meat after final cutting. Enterobacteriaceaecounts (EC) were enumerated employing conventional laboratory techniques. Carcass meat samples resulted in EC below the detection limit more frequently if the hide samples from the same carcass had also EC counts below the detection limit. Because of the low number of trials, the calculation of statistical significance of the results was not possible. Further experimental research is needed in order to validate the results presented in this study. The comparison of the microbiological hygiene performance between dorsal dehiding and traditional ventral dehiding of slaughtered animals could serve to validate the hypothesis of the potential positive impact of the dorsal dehiding method in carcass meat hygiene.

Aluminum Nitride (AIN) has arised as a promising synthetic material, valued for its high thermal conductivity coupled with high electrical resistivity. These properties, associated with the fact that AIN is nontoxic and has thermal expansion characteristics which closely match those of silicon, makes it an excellent material for electronic packaging. Currently two commercial processes are primaly used for the manufacture of AIN powder, the direct nitridation of the metal, and the carbothermic reduction of the alumina in the presence of nitrogem.In this work, nanosized aluminum nitride particles were synthesized from the phase using ammonia and aluminum chloride vapor as reactants. This system consists of a vaporization system, ammonia (NH3) distributor, tubular synthesis reactor and powder collection system. The aluminum chloride (AlCl3) vapor starts to react with ammonia injected in the synthesis reactor inlet position (mixing zone). The aluminum nitride nucleates and grows along the reactor length. The aluminum nitride powder produced was collected and chemically and physically characterized.