Aptamer-Based Detection of Disease Biomarkers in Mouse Models for Chagas Drug Discovery

Drug discovery initiatives, aimed at Chagas treatment, have been hampered by the lack of standardized drug screening protocols and the absence of simple pre-clinical assays to evaluate treatment efficacy in animal models. In this study, we used a simple Enzyme Linked Aptamer (ELA) assay to detect T. cruzi biomarker in blood and validate murine drug discovery models of Chagas disease. In two mice models, Apt-29 ELA assay demonstrated that biomarker levels were significantly higher in the infected group compared to the control group, and upon Benznidazole treatment, their levels reduced. However, biomarker levels in the infected treated group did not reduce to those seen in the non-infected treated group, with 100% of the mice above the assay cutoff, suggesting that parasitemia was reduced but cure was not achieved. The ELA assay was capable of detecting circulating biomarkers in mice infected with various strains of T. cruzi parasites. Our results showed that the ELA assay could detect residual parasitemia in treated mice by providing an overall picture of the infection in the host. They suggest that the ELA assay can be used in drug discovery applications to assess treatment efficacy in-vivo.     

Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival

BackgroundsSpontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms.MethodsPCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis.ResultsThe 25.4 kDa native PCMT from C. utilis was found to have a K_m of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro.General significanceIdentification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.     

Model structures of inactive and peptide agonist bound C5aR: Insights into agonist binding,selectivity and activation

C5a receptor (C5aR) is one of the major chemoattractant receptors of the druggable proteome that binds C5a, the proinflammatory polypeptide of complement cascade, triggering inflammation and SEPSIS. Here, we report the model structures of C5aR in both inactive and peptide agonist (YSFKPMPLaR; a=D-Ala) bound meta-active state. Assembled in CYANA and evolved over molecular dynamics (MD) in POPC bilayer, the inactive C5aR demonstrates a topologically unique compact heptahelical bundle topology harboring a β-hairpin in extracellular loop 2 (ECL2), derived from the atomistic folding simulations. The peptide agonist bound meta-active C5aR deciphers the “site2” at an atomistic resolution in the extracellular surface (ECS), in contrast to the previously hypothesized inter-helical crevice. With estimated Ki≈2.75 μM, the meta-active C5aR excellently rationalizes the IC₅₀ (0.1–13 μM) and EC₅₀ (0.01–6 μM) values, displayed by the peptide agonist in several signaling studies. Moreover, with Ki≈5.3×10⁵ μM, the “site2” also illustrates selectivity, by discriminating the stereochemical mutant peptide (YSFkPMPLaR; k=D-Lys), known to be inert toward C5aR, up to 1 mM concentration. Topologically juxtaposed between the structures of rhodopsin and CXCR1, the C5aR models also display excellent structural correlations with the other G-protein coupled receptors (GPCRs). The models elaborated in the current study unravel many important structural insights previously not known for regulating the agonist binding and activation mechanism of C5aR.     

Ion-pairing liquid chromatography–tandem mass spectrometry-based quantification of uridine diphosphate-linked intermediates in the Staphylococcus aureus cell wall biosynthesis pathway

Bacterial cell wall biosynthesis is the target of several antibiotics and is of interest as a target for new inhibitor development. The cytoplasmic steps of this pathway involve a series of uridine diphosphate (UDP)-linked peptidoglycan intermediates. Quantification of these intermediates is essential for studies of current agents targeting this pathway and for the development of new agents targeting this pathway. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for quantification of these intermediates in Staphylococcus aureus. To address the problem of poor retention of UDP-linked intermediates on reverse phase media, an ion-pairing (IP) approach using N,N-dimethylhexylamine was developed. MS/MS detection in negative mode was optimized for UDP-GlcNAc, UDP-MurNAc, UDP-MurNAc-l-Ala, UDP-MurNAc-l-Ala-d-Glu, UDP-MurNAc-l-Ala-d-Glu-l-Lys, and UDP-MurNAc-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala. The lower limits of quantification (LLOQs) for these analytes were 1.8, 1.0, 0.8, 2.2, 0.6, and 0.5 pmol, respectively, which correspond to LLOQs of 6, 3, 3, 7, 2, and 2 nmol/g bacteria, respectively. This method was demonstrated for quantification of in vivo levels of these intermediates from S. aureus (0.3 mg dry weight analyzed) treated with fosfomycin, d-boroAla, d-cycloserine, and vancomycin. Metabolite accumulation is consistent with the known targets of these antibiotics and indicates potential regulatory loops within this pathway.     

Graphene-based immunoassay for human lipocalin-2

We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human lipocalin-2 (LCN2) in plasma, serum, and whole blood. It has the dynamic range, linear range, limit of detection, and analytical sensitivity of 0.6 to 5120, 80 to 2560, 0.7, and 1 pg/ml, respectively. It is the most sensitive assay for the detection of LCN2, which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit. The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3-aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on MTP and GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody by its carboxyl groups using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as the heterobifunctional crosslinker. The anti-LCN2 antibody-bound MTPs were highly stable given that they did not show any significant decrease in their functional activity when stored at 4 °C in 0.1 M phosphate-buffered saline (PBS) for 8 weeks. The developed immunoassay correlated well with the conventional ELISA, thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings.     

Revising angiotensinogen from phylogenetic and genetic variants perspectives

Angiotensinogen (AGT) belongs to the serpin superfamily. It acts as the unique substrate of all angiotensin peptides, which generates a spectrum of angiotensin peptides in the renin-angiotensin system and regulates hypertension. This serpin belongs to the multiple member group V2 of the intron encoded vertebrate serpin classification. Despite huge advancements in the understanding of angiotensinogen based on biochemical properties and its roles in the RAS, phylogenetic history of AGT remains forgotten. To date, there is no comprehensive study illustrating the phylogenetic history of AGT. Herein, we investigated phylogenetic traits of AGT gene across vertebrates. Gene structures of AGT gene from selected ray-finned fishes varied in exon I and II with insertions of two novel introns in the core domain for ray-finned fishes at the position 77c and 233c. We that found AGT loci is conserved from lampreys to human and estimated to be older than 500 MY. By comparing AGT protein in 57 vertebrate genomes, we illustrated that the reactive center loop (RCL) of AGT protein became from inhibitory (in lampreys, GTEAKAETVVGIMPI†SMPPT) to non-inhibitory (in human, EREPTESTQQLNKPE†VLEVT) during period of 500 MY. We identified 690 AGT variants by analysis of 1092 human genomes with top three variation classes belongs to SNPs (89.7%), somatic SNVs (5.2%) and deletion (2.9%). There are 32 key residues out of 121 missense variants, which are deleterious for AGT protein, computed by combination of SIFT and PolyPhen V2 methods. These results may have clinical implications for understanding hypertension.     

Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys.     

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.     

ILCD Handbook Public Consultation Workshop

Introduction

The European Commission is supporting the development of the International Reference Life Cycle Data System (ILCD). This consists primarily of the ILCD Handbook and the ILCD Data Network. This paper gives an insight into the scientific positions of business, governments, consultants, academics, and others that were expressed at this public consultation workshop.

Workshop focus

The workshop focused on four of the topics of the main guidance documents of the ILCD Handbook: (1) general guidance on life cycle assessment (LCA); (2) guidance for generic and average life cycle inventory (LCI) data sets; (3) requirements for environmental impact assessment methods, models and indicators for LCA; and (4) review schemes for LCA.

Workshop participation

This consultation workshop was attended by more than 120 participants during the 4 days of the workshop. Representatives came from 23 countries, from both within and outside the European Union.

Workshop structure

Approximately half of the participants were from business associations or individual companies. Another 20% were governmental representatives. Others came predominantly from consultancies and academia.

Results

This public consultation workshop provided valuable inputs into the overall ILCD Handbook developments as well as for further development. This paper focuses on some of the main scientific issues that were raised.