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Heparin-like macromolecules are widely used in clinics as anticoagulant, antiviral, and anticancer drugs. However, the search of heparin antidotes based on small synthetic molecules to control blood coagulation still remains a challenging task due to the physicochemical properties of this anionic polysaccharide. Here, we use a dynamic combinatorial chemistry approach to optimize heparin binders with submicromolar affinity. The recognition of heparin by the most amplified members of the dynamic library has been studied with different experimental (SPR, fluorescence, NMR) and theoretical approaches, rendering a detailed interaction model. The enzymatic assays with selected library members confirm the correlation between the dynamic covalent screening and the in vitro heparin inhibition. Moreover, both ex vivo and in vivo blood coagulation assays with mice show that the optimized molecules are potent antidotes with potential use as heparin reversal drugs. Overall, these results underscore the power of dynamic combinatorial chemistry targeting complex and elusive biopolymers., This work was supported by the Spanish Ministry of Science and Innovation, Spanish Research Agency (10.13039/501100011033), and European Social Fund (MCI/AEI/FEDER, RTI2018-096182-B-I00, CSIC13-4E-2076), as well as AGAUR (2017 SGR 208). The authors acknowledge the assistance from the ICTS “NANBIOSIS” in some key experiments: SPR carried out in the Unit U2, Custom Antibody Service (CAbS, CIBER-BBN) and DLS performed by the Nanostructured Liquid Characterization Unit, both located at IQAC-CSIC. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

The use of aminals in dynamic covalent chemistry is slightly underexplored, probably due to their inherent instability. Here we report the spontaneous [2+2] macrocyclization of tetrakis(aminals). Their unexpected stability and structural modularity, the dynamic nature of the connections and their water tolerance make them appealing systems for future applications as stimulus-responsive materials.

A new family of hybrid β,γ-peptidomimetics consisting of a repetitive unit formed by a chiral cyclobutane-containing trans-β-amino acid plus a Nα-functionalized trans-γ-amino-l-proline joined in alternation were synthesized and evaluated as cell penetrating peptides (CPP). They lack toxicity on the human tumoral cell line HeLa, with an almost negligible cell uptake. The dodecapeptide showed a substantial microbicidal activity on Leishmania parasites at 50 µM but with a modest intracellular accumulation. Their previously published γ,γ-homologues, with a cyclobutane γ-amino acid, showed a well-defined secondary structure with an average inter-guanidinium distance of 8-10 Å, a higher leishmanicidal activity as well as a significant intracellular accumulation. The presence of a very rigid cyclobutane β-amino acid in the peptide backbone precludes the acquisition of a defined conformation suitable for their cell uptake ability. Our results unveiled the preorganized charge-display as a relevant parameter, additional to the separation among the charged groups as previously described. The data herein reinforce the relevance of these descriptors in the design of CPPs with improved properties., The authors would like to thank the staff at the Servei de Microscòpia de la Universitat Autònoma de Barcelona and the Peptide Synthesis Unit of the NANBIOSIS ICTS (U3, CIBER BBN-IQAC-CSIC) for technical assistance.

Characterization of the T cell response in individuals who recover from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8(+) T cell recognition in a cross-sectional sample of 30 coronavirus disease 2019 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis and from humoral and inflammatory responses. There were 132 SARS-CoV-2–specific CD8(+) T cell responses detected across 6 different HLAs, corresponding to 52 unique epitope reactivities. CD8(+) T cell responses were detected in almost all convalescent individuals and were directed against several structural and nonstructural target epitopes from the entire SARS-CoV-2 proteome. A unique phenotype for SARS-CoV-2–specific T cells was observed that was distinct from other common virus-specific T cells detected in the same cross-sectional sample and characterized by early differentiation kinetics. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8(+) T cell response. Overall, T cells exhibited distinct differentiation into stem cell and transitional memory states (subsets), which may be key to developing durable protection.

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado–Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination., We thank Axel Knebel, Richard Ewan, Clare Johnson, and Daniel Fountaine from the Medical Research Council (MRC) Protein Production and Assay Development team, and MRC Reagents and Services, who all contributed to the generation of protein reagents required for the MALDI-TOF DUB assay platform. We thank Ronald Hay for provision of the plasmid encoding the constitutively active RNF4 E3 ligase. This work was funded by the United Kingdom MRC (MC_UU_12016/8), the Biotechnology and Biological Sciences Research Council (BB/P003982/1), and The Michael J. Fox Foundation (12756). We also acknowledge pharmaceutical companies supporting the Division of Signal Transduction Therapy (Boehringer-Ingelheim, GlaxoSmithKline, and Merck KGaA).

Background Understanding the diversity, breadth, magnitude, and functional profile of the T cell response against SARS-CoV-2 in recovered COVID-19 individuals is critical to evaluate the contribution of T cells to produce a potentially protective immune response. Methods We used a multiplexed peptide-MHC tetramer approach to screen a total of 408 SARS-CoV-2 candidate peptide epitopes for CD8+ T cell recognition in a cohort of 30 individuals recovered from COVID-19. The peptides spanned the whole viral genome and were restricted to six prevalent HLA alleles; T cells were simultaneously characterized by a 28-marker phenotypic panel. The evolution of the SARS-CoV-2 T cell responses was then statistically modeled against time from diagnosis, and in relation to humoral and inflammatory response. Workflow for Study. A multiplexed peptide-MHC tetramer approach was used to screen SARS-CoV-2 candidate peptide epitopes in a cohort of 30 COVID-19 recovered patients across 6 prevalent HLA alleles, and T cells profiled with a 28-marker phenotypic panel. Multiplex tetramer screen. One representative COVID-19 recovered patient and one healthy donor were screened for HLA- relevant SARS-CoV-2 epitopes, as well as epitopes for CMV, EBV, Influenza, Adenovirus and MART-1. Shown are the frequencies of tetramer-positive CD8 T cells from 2 technical replicates per subject. Results Almost all individuals screened showed a T cell response against SARS-CoV-2 (29/30): 132 SARS-CoV-2-specific CD8+ T cells hits were detected, corresponding to 52 unique reactive epitopes. Twelve of the 52 unique SARS-CoV-2-specific epitopes were recognized by more than 40% of the individuals screened, indicating high prevalence in the subjects. Importantly, these CD8+ T cell responses were directed against both structural and non-structural viral proteins, with the highest magnitude against nucleocapsid derived peptides, but without any antigen-driven bias in the phenotype of specific T cells. Overall, SARS-CoV-2 T cells showed specific states of differentiation (stem-cell memory and transitional memory), which differed from those of MART-1, influenza, CMV and EBV-specific T cells. UMAP visualization revealed a phenotypic profile of SARS-CoV-2-specific CD8 T cells in COVID-19 convalescent donors that is distinct from other viral specificities, such as influenza, CMV, EBV and Adenovirus. SARS-CoV-2 epitope screening revealed CD8+ T cell responses directed against both structural and non-structural viral proteins, with the highest magnitude response against nucleocapsid derived peptides Conclusion The kinetics modeling demonstrates a dynamic, evolving immune response characterized by a time-dependent decrease in overall inflammation, increase in neutralizing antibody titer, and progressive differentiation of a broad SARS-CoV-2 CD8 T cell response. It could be desirable to aim at recapitulating the hallmarks of this robust CD8 T cell response in the design of protective COVID-19 vaccines. Disclosures Hassen Kared, PhD, ImmunoScape (Shareholder) Alessandra Nardin, DvM, ImmunoScape (Shareholder) Hermi Sumatoh, BSc, Dip MTech, ImmunoScape (Shareholder) Faris Kairi, BSc, ImmunoScape (Shareholder) Daniel Carbajo, PhD, ImmunoScape (Shareholder) Brian Abel, PhD, MBA, ImmunoScape (Shareholder) Evan Newell, PhD, ImmunoScape (Shareholder)

The current TB treatment regimen involves a combination of drugs administered for an extended duration that could last for 6 months to 2 years. This could lead to noncompliance and the emergence of newer drug resistance strains., The worldwide increase in the frequency of multidrug-resistant and extensively drug-resistant cases of tuberculosis is mainly due to therapeutic noncompliance associated with a lengthy treatment regimen. Depending on the drug susceptibility profile, the treatment duration can extend from 6 months to 2 years. This protracted regimen is attributed to a supposedly nonreplicating and metabolically inert subset of the Mycobacterium tuberculosis population, called “persisters.” The mechanism underlying stochastic generation and enrichment of persisters is not fully known. We have previously reported that the utilization of host cholesterol is essential for mycobacterial persistence. In this study, we have demonstrated that cholesterol-induced activation of a RNase toxin (VapC12) inhibits translation by targeting proT tRNA in M. tuberculosis. This results in cholesterol-specific growth modulation that increases the frequency of generation of the persisters in a heterogeneous M. tuberculosis population. Also, a null mutant strain of this toxin (ΔvapC12) demonstrated an enhanced growth phenotype in a guinea pig model of M. tuberculosis infection, depicting its role in disease persistence. Thus, we have identified a novel strategy through which cholesterol-specific activation of a toxin-antitoxin module in M. tuberculosis enhances persister formation during infection. The current findings provide an opportunity to target persisters, a new paradigm facilitating tuberculosis drug development. IMPORTANCE The current TB treatment regimen involves a combination of drugs administered for an extended duration that could last for 6 months to 2 years. This could lead to noncompliance and the emergence of newer drug resistance strains. It is widely perceived that the major culprits are the so-called nonreplicating and metabolically inactive “persister” bacteria. The importance of cholesterol utilization during the persistence stage of M. tuberculosis infection and its potential role in the generation of persisters is very intriguing. We explored the mechanism involved in the cholesterol-mediated generation of persisters in mycobacteria. In this study, we have identified a toxin-antitoxin (TA) system essential for the generation of persisters during M. tuberculosis infection. This study verified that M. tuberculosis strain devoid of the VapBC12 TA system failed to persist and showed a hypervirulent phenotype in a guinea pig infection model. Our studies indicate that the M. tuberculosis VapBC12 TA system acts as a molecular switch regulating persister generation during infection. VapBC12 TA system as a drug target offers opportunities to develop shorter and more effective treatment regimens against tuberculosis.

Characterization of the T cell response in individuals who recover from SARS-CoV-2 infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 COVID-19 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis, humoral and inflammatory responses. 132 distinct SARS-CoV-2-specific CD8+ T cell epitope responses across six different HLAs were detected, corresponding to 52 unique reactivities. T cell responses were directed against several structural and non-structural virus proteins. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem-cell and transitional memory states, subsets, which may be key to developing durable protection.

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome and many have been implicated with pathologies including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct and its physiological importance is emerging. Here we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain (OTU) DUB class demonstrate specific (iso)peptidase activity. Strikingly, we find the Machado Josephin Domain (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity that rivals the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad but proximal hydrophobic residues appear to be general determinants of non-lysine activity. These findings suggest that non-lysine ubiquitination is an integral component of the ubiquitin system, with regulatory sophistication comparable to that of canonical ubiquitination.

Dynamic covalent chemistry combines in a single step the screening and synthesis of ligands for biomolecular recognition. In order to do that, a chemical entity is used as template within a dynamic combinatorial library of interconverting species, so that the stronger binders are amplified due to the efficient interaction with the target. Here we employed whole A549 living cells as template in a dynamic mixture of imines, for which amplification reflects the efficient and selective interaction with the corresponding extracellular matrix. The amplified polyamine showed strong interaction with the A549 extracellular matrix in on‐cell NMR experiments, while combination of NMR, SPR, and molecular dynamics simulations in model systems provided insights on the molecular recognition event. Notably, our work pioneers the use of whole living cells in dynamic combinatorial chemistry, which paves the way towards the discovery of new bioactive molecules in a more biorelevant environment. Dynamic combinatorial chemistry (DCC) proposes the use of dynamic combinatorial libraries (DCL) for the generation of species able to exchange through reversible covalent bonds.1 These molecular systems are responsive to external stimuli by modification of the DCL composition,2 with the stabilized members being amplified at the expense of the other components in the mixture.3 Within the chemical biology field, the DCC approach has led to the discovery of new protein ligands,4 nucleic acids binders,5 and even replicators.6 For biological applications, it should be desirable that the conditions used for the DCC screening resemble those in the place of action.7 Inspired by Sander's comparison of DCC with the immune system,8 we envisioned targeting the extracellular matrix (ECM).9 The external surface of the cells is formed by a complex network of glycoproteins and anionic polysaccharides that is fundamental for processes such as cell communication,10 regeneration,11 metastasis,12 and host‐pathogen infection.13 The ECM is the first barrier for a molecule (i.e. a drug) to enter inside the cell; thus, navigating the ECM is fundamental in biomedicine and in chemical biology.14 However, the chemical and structural complexity of the ECM have hindered its detailed molecular characterization and frustrated the rational design of synthetic ligands.15 Paradoxically, the intrinsic complexity of the ECM offers an ideal playground for the realization of the self‐organizing features of DCC (Figure 1 A), which has demonstrated its power for the discovery of strong binders to challenging biomolecules.1, 2-4, 5-8 Considering our recent results in the identification of a strong heparin binder16 and the chemical similarity between heparin and the glycosaminoglycans (GAGs) of the ECM,14 we designed a library (Figure 1 B) combining spermine as a cationic polyamine scaffold17 with a set of aromatic aldehydes, which would mediate binding through CH–aryl interactions with the saccharide units.18 Thus, the dynamic mixture of imines (2XY ) obtained by the reaction between spermine (1 ) and an equimolecular mixture of four aromatic aldehydes (A , B , K , L ) was incubated with living cells and reduced in situ with NaBH3CN to the corresponding polyamines (3XY ). As an initial model, we used the A549 human lung adenocarcinoma cell line since the ECM of these cells is rich in anionic GAGs.19 The supernatant was analyzed by UPLC‐MS allowing the identification and quantification of each member of the library (Figure 1 C). The normalized area of the UPLC‐MS peaks for the reactions performed in the presence (A T) and in the absence (A 0) of cells was compared by the calculation of the corresponding amplification factors (AF=A T/A 0)., We thank Prof. A. Messeguer for helpful discussion. The ICTS “NANOBIOSIS” (CAbS, IQAC‐CSIC, CIBER‐BBN) is acknowledged for the assistance and support with the use of OpenSPR. This work was supported by the Spanish Ministry of Science and Innovation/ Spanish Research Agency (MCI/AEI/FEDER, RTI2018‐096182‐B‐I00, CSIC13‐4E‐2076) and AGAUR (2017 SGR 208).

Despite heparin being the most widely used macromolecular drug, the design of small-molecule ligands to modulate its effects has been hampered by the structural properties of this polyanionic polysaccharide. Now a dynamic covalent selection approach is used to identify a new ligand for heparin, assembled from extremely simple building blocks. The amplified molecule strongly binds to heparin (K D in the low μm range, ITC) by a combination of electrostatic, hydrogen bonding, and CH–π interactions as shown by NMR and molecular modeling. Moreover, this ligand reverts the inhibitory effect of heparin within an enzymatic cascade reaction related to blood coagulation. This study demonstrates the power of dynamic covalent chemistry for the discovery of new modulators of biologically relevant glycosaminoglycans. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Financial Support from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO/FEDER, CTQ2015-70117-R) and AGAUR (2017 SGR 208) is gratefully acknowledged. The 500 MHz spectrometer and cryoprobe in the IQAC NMR facility were funded by the CSIC13-4E2076 MINECO-FEDER grant.

A worldwide increase in the frequency of multidrug-resistant and extensively drug-resistant cases of tuberculosis is mainly due to therapeutic noncompliance associated with a lengthy treatment regimen. Depending on the drug susceptibility profile, the treatment duration can extend from 6 months to 2 years. This protracted regimen is attributed to a supposedly non-replicating and metabolically inert subset of theMycobacterium tuberculosis(Mtb) population, called ‘persisters’. The mechanism underlying stochastic generation and enrichment of persisters is not fully known. We have previously reported that the utilization of host cholesterol is essential for mycobacterial persistence. In this study, we have demonstrated that cholesterol-induced activation of a ribonuclease toxin (VapC12) inhibits translation by targeting proT tRNA in Mtb. This results in cholesterol-specific growth modulation that increases the frequency of the generation of persisters in a heterogeneous Mtb population. Also, a null mutant strain of this toxin (ΔvapC12) failed to persist and demonstrated an enhanced growth phenotype in a guinea pig model of Mtb infection. Thus, we have identified a novel strategy through which cholesterol-specific activation of a toxin–antitoxin (TA) module in Mtb enhances persister formation during infection. In addition to identifying the mechanism, the study provides opportunity for targeting persisters, a new paradigm facilitating tuberculosis drug development.

A handle for the protection of the C-terminus of peptides after cleaving with low concentration of trifluoroacetic acid (2-4%) is reported. The handle prevents polymerization reactions in the convergent condensation of peptidic fragments. Moreover, it is traceless, being removed during the final deprotection step of the peptide synthesis. This cheap and convenient handle is easily attached to the solid support, causing no disturbance to peptide elongation and thus proving to be useful in the convergent synthesis of long peptides. © 2019 American Chemical Society., This work was funded in part by the following: Lonza AG (Wisp, Switzerland), the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO) (CTQ2015-67870-P & RTC-2014-2207-1), CIBER-BBN, the Generalitat de Catalunya (2017 SGR 1439) (Spain), and the International Scientific Partnership Program ISPP at King Saud University (ISPP# 0061) (Saudi Arabia).

INTRODUCTION Resident tissue macrophages (RTMs) are a heterogeneous population of immune cells occupying multiple tissue niches and exhibiting microenvironment-specific phenotypes and functions. In certain tissues such as the brain, lung, and liver, embryonically derived RTMs maintain themselves by self-renewal, whereas others, including those in the gut, dermis, and pancreas, are replaced by monocytes, at levels that are tissue specific. Once they arrive in their tissue of residence, monocytes undergo extensive differentiation according to molecular cues provided by their distinct tissue-specific niches, enabling their development into specialized RTMs that support local tissue function. RATIONALE As a result of this ontogenetic and tissue niche heterogeneity, each tissue contains multiple populations of macrophages. For example, in the murine lung, alveolar macrophages are the major embryonically derived population in the alveolar spaces, whereas a minor population named interstitial macrophages (IMs) resides within the lung parenchyma. Previous results reported several phenotypically distinct IM subpopulations, whose relationship remained unknown. Do they represent independent populations or, rather, different points on the spectrum of maturation and activation states? How do these differences relate to their localization in tissue or roles in tissue function in health and disease? Does such macrophage heterogeneity also exist in other tissues? RESULTS Here, using single-cell mRNA sequencing, we unbiasedly identified two independent populations exhibiting distinct gene expression profiles and phenotypes: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) RTMs. We uncovered evidence of parallel populations in multiple others tissues, including the heart, fat, and dermis, as well as in human lung and omental and subcutaneous fat tissues, suggesting that a similar dichotomy is observed in human tissues. We further demonstrated that both populations are slowly replaced by Ly6Chi monocytes. Importantly, using complementary fate-mapping models, we showed that monocyte-derived RTMs (MRTMs) are two separate lineages, rather than representing points along a developmental or maturation continuum. Notably, these distinct MRTM populations preferentially reside within different, but conserved, subtissular niches, located either adjacent to nerve bundles and fibers (Lyve1loMHCIIhi) or blood vessels (Lyve1hiMHCIIlo) across tissues. Finally, by acutely depleting Lyve1hiMHCIIlo MRTMs using a mouse model of inducible macrophage depletion during the induction of fibrosis, we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung and heart fibrosis, demonstrating their critical role in tissue inflammation. CONCLUSION Two independent MRTMs populations exist across tissues with specific niche-dependent phenotype and functional programming. Their different roles in homeostasis, immune regulation, and fibrosis renders them attractive and separate cellular targets for the therapeutic exploitation of RTM subsets.

The incorporation of flexible aliphatic units into otherwise rigid aromatic foldamer sequences may result in different outcomes. The flexible units may have conformational preferences of their own that can be expressed orthogonally to those of the aromatic units. Alternatively, the latter may dictate their folding behavior onto the former. Hybrid aliphatic–aromatic peptidic oligomers combining oxazole-based (O) and quinoline-based (Q) amino acids have been synthesized, and their folding behavior has been investigated in solution by NMR spectroscopy and CD spectroscopy, and in the solid state by X-ray crystallography. Sequences based on the OQQ repeat motif were shown to fold into a canonical aromatic helix motif dominated by the preferences of the quinoline, whereas sequences based on OQ repetitions preferred to fold into a herringbone helix in which the conformational preference of the oxazole units is also expressed.

Inspired by DNA mimic proteins, we have introduced aromatic foldamers bearing phosphonate groups as synthetic mimics of the charge surface of B-DNA and competitive inhibitors of some therapeutically relevant DNA-binding enzymes: the human DNA Topoisomerase 1 (Top1) and the human HIV-1 integrase (HIV-1 IN). We now report on variants of these anionic foldamers bearing carboxylates instead of phosphonates. Several new monomers have been synthesized with protecting groups suitable for solid phase synthesis (SPS). Six hexadecaamides have been prepared using SPS. Proof of their resemblance to B-DNA was brought by the first crystal structure of one of these DNA-mimic foldamers in its polyanionic form. While some of the foldamers were found to be as active as, or even more active than, the original phosphonate oligomers, others had no activity at all or could even stimulate enzyme activity in vitro. Some foldamers were found to have differential inhibitory effects on the two enzymes. These results demonstrate a strong dependence of inhibitory activity on foldamer structure and charge distribution. They open broad avenues for the development of new classes of derivatives that could inhibit the interaction of specific proteins with their DNA target thereby influencing the cellular pathways in which they are involved. © The Author(s) 2019., Agence Nationale de la Recherche [ANR-11-BS07-013-01]; European Research Council under the European Union’s Seventh Framework Programme [ERC-2012-AdG-320892 and PEOPLE-2011-IEF-300948]; Ligue Contre Le Cancer (Comité Languedoc-Roussillon); SIRIC Montpellier Cancer [INCa Inserm DGOS 12553]; SIDACTION (AO2016, VIH2016721002). Funding for open access charge: University of Munich fund.

Studies in human aging mainly focus on αβ T cells while γδ T cells have been neglected despite their role in immunosurveillance. In this study, we investigated γδ T cells and its subsets (i.e. Vδ1 , Vδ2 and DN γδ (γδ Vδ1-Vδ2-)) by deep immunophenotyping. Our data shows that peripheral Vδ2 are differentially susceptible to life-long stresses against all other αβ and γδ T cells. Using aging and cytomegalovirus history (the main confounding factor in immune-aging studies) as immunological models and via functional assays, telomere length determination, epigenetic methylome profiling and DNA damage response capacity, we show that Vδ2 T cells are resistant from cellular senescence. Our results highlight the differential impact of stress on γδ T cells subsets, reveal surface markers that are functionally relevant for γδ T cells subsets and highlight possible mechanisms that enable Vδ2 to be resistant to cellular aging. This new finding reinforces the concept that Vδ2 have an “innate-like” behavior and are more resilient to the environment as compared to “adaptive-like” Vδ1 T cells. Funding Statement: Weili Xu is funded by A*STAR Graduate Academy (AGA). The work was funded by the Singapore Immunology Network, the Agency for Science Technology and Research (JCO grant #1434m00115) and the Skin Research Institute of Singapore (SRG grant #14018). Declaration of Interests: The authors have no conflict of interests. Ethics Approval Statement: Participants of the study were recruited under National University of Singapore Institutional Review Board (IRB code NUS-IRB 01-256). Cone blood was obtained from Health Science Authority (HSA) Singapore, approved under NUS-IRB 10-250.

In this study, we combined a cell-penetrating γ-peptide, PEG-1, with antimicrobial undecapeptides in order to provide compounds with anticancer properties against MDA-MB-231 human breast cancer cells. We demonstrated that the conjugates were more cytotoxic than Ac-PEG-1 and the parent undecapeptides. We also evaluated the toxicity of the conjugates against non-malignant cells. The peptide conjugate with the best biological profile was BP77-PEG-1, which, at 10 μM, showed a 71% growth inhibition in MDA-MB-231 cells and only a 17% inhibition in non-malignant cells. Therefore, this study suggests that PEG-1 mediated the undecapeptide delivery into cancer cells and that these conjugates are the proof-of-concept of this strategy to generate improved anticancer drugs based on peptides.

The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages-which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues-expressed the hyaluronan (HA) receptor LYVE-l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP-9-dependent proteolysis through engagement of LYVE-1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE-1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE-1 in leukocytes. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore)

et al., The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes., Susana P. Egusquiaguirre thanks the Basque Government (Gobierno Vasco, Departamento de Educación, Universidades e Investigación) for the fellowship research grant. C. Manguan-García is supported by CIBER de Enfermedades Raras. This project was partially supported by the Basque Government (Consolidated Groups, IT-407-07), MINECO from the Spanish Government (INNPACTO, IPT-2012-0674-090000). The authors gratefully acknowledge the support of University of the Basque Country UPV/EHU (UFI11/32). Besides, this work was also supported by grant PI11-00949 (supported by FEDER funds) from Instituto Carlos III.

Two generations of hybrid γ,γ-peptides containing cyclobutane amino acids and cis-γ-amino-L-proline joined in alternation have been synthesized and their capacity to cross the eukaryotic cell membrane has been evaluated. The first generation consists of di-, tetra- and hexapeptides, and their properties have been analyzed as well as the influence of peptide length and chirality of the cyclobutane residues. Results have shown that the absolute configuration of the cyclobutane amino acid does not have a relevant influence. The second generation consists of hybrid γ,γ-hexapeptides with a common backbone and distinct side chains introduced with different linkage types through the α-amino group (N(α)) of the proline monomers. These peptides have been shown to be non-toxic towards HeLa cells and to internalize them effectively, the best results being obtained for the peptides with a spacer of five carbons between the N(α) atom and the guanidinium group. The introduction of cyclobutane residues inside the sequence affords a good balance between charge and hydrophobicity, reducing the number of positive charges. This results in lower toxicity and similar cell-uptake properties when compared to previously described peptide agents.

Two diastereomeric series of hybrid γ,γ-peptides derived from conveniently protected derivatives of (1R,2S)- and (1S,2R)-3-amino-2,2-dimethylcyclobutane-1-carboxylic acid and cis-4-amino-L: -proline joined in alternation have efficiently been prepared through convergent synthesis. High-resolution NMR experiments show that these compounds present defined conformations in solution affording very compact structures as the result of intra and inter residue hydrogen-bonded ring formation. (R,S)-cyclobutane containing peptides adopt more twisted conformations than (S,R) diastereomers. In addition, all these γ-peptides have high tendency to aggregation providing vesicles of nanometric size, which were stable when allowed to stand for several days, as verified by transmission electron microscopy.

Background Increasingly, biologists and biochemists use computational tools to design experiments to probe the function of proteins and/or to engineer them for a variety of different purposes. The most effective strategies rely on the knowledge of the three-dimensional structure of the protein of interest. However it is often the case that an experimental structure is not available and that models of different quality are used instead. On the other hand, the relationship between the quality of a model and its appropriate use is not easy to derive in general, and so far it has been analyzed in detail only for specific application. Results This paper describes a database and related software tools that allow testing of a given structure based method on models of a protein representing different levels of accuracy. The comparison of the results of a computational experiment on the experimental structure and on a set of its decoy models will allow developers and users to assess which is the specific threshold of accuracy required to perform the task effectively. Conclusions The ModelDB server automatically builds decoy models of different accuracy for a given protein of known structure and provides a set of useful tools for their analysis. Pre-computed data for a non-redundant set of deposited protein structures are available for analysis and download in the ModelDB database. Implementation, availability and requirements Project name: A resource for benchmarking the usefulness of protein structure models. Project home page: http://bl210.caspur.it/MODEL-DB/MODEL-DB_web/MODindex.php. Operating system(s): Platform independent. Programming language: Perl-BioPerl (program); mySQL, Perl DBI and DBD modules (database); php, JavaScript, Jmol scripting (web server). Other requirements: Java Runtime Environment v1.4 or later, Perl, BioPerl, CPAN modules, HHsearch, Modeller, LGA, NCBI Blast package, DSSP, Speedfill (Surfnet) and PSAIA. License: Free. Any restrictions to use by non-academics: No.

Aniline-catalyzed oxime chemistry was employed to conjugate a γ-amino-proline-derived cell penetrating peptide to superparamagnetic iron oxide nanoparticles (SPIONs). Internalization of the novel nanoconjugate into HeLa cells was found to be remarkably higher compared to the analogous TAT-SPION conjugate.