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This study was supported by a UK Biotechnology and Biological Sciences Research Council (BBSRC) Grant (BB/I019405/1) to DJS, grant 727.011.001 from the Netherlands Organization for Scientific Research (NWO) Forensic Science Program to MK and by Erasmus MC University Medical Centre Rotterdam. DJS is a Royal Society Wolfson Research Merit Award holder. RAH and IH were funded by the Dutch applied research foundation (STW Perspectief Program ‘OnTime’ project 12185). Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor’s alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose. Publisher PDF

Acne vulgaris is one of the most common chronic dermatological diseases among adolescents. Recent data indicated that a specific diet may affect the course and appearance of acne. The aim of the study was to analyze the relationship between body mass index (BMI) and acne prevalence and severity. This cross-sectional study was conducted among 143 consecutively recruited adolescents aged between 12 and 18. All participants were physically examined, including measurement of weight and height and type and severity of acne. Acne lesions were evaluated as not present (0), mild (1), moderate (2), and severe (3). The predominant type of acne lesions was used to classify the acne into one of the following subtypes: comedonic acne, papulo-pustular acne, and nodulo-cystic acne. Acne was present in 123 adolescents (86.0%) being more prevalent in teenagers ≥15 years old than in those between 12 and 14 years old (97.1% vs. 76.0%, respectively, P

ImmTAC (Immune mobilizing monoclonal TCRs against cancer) molecules are a new class of bi-specific biologic that combine a T-cell receptor (TCR)-targeting system with an anti-CD3 effector function to activate highly potent and specific T-cell responses to cancer cells. ImmTAC molecules have the potential to overcome many of the limitations of other immuno-oncology agents; using TCRs to target intracellularly processed peptides presented by HLA allows access to a vast landscape of cancer-specific antigens that antibody-based therapies cannot currently target. Soluble agents also have a number of benefits over cellular therapies in terms of ease of manufacture and supply. At Immunocore we are constantly striving for innovation and looking for new opportunities to advance the ImmTAC platform. One limitation of TCR-targeting is HLA-restriction, which means that a single ImmTAC molecule is highly specific towards a single HLA-subtype. Lead ImmTAC molecules target HLA-A2, which has the highest coverage in the Caucasian population of approximately 55%. Our desire is to cover all patients in need; therefore, we are developing ImmTAC molecules that target peptides on other HLA alleles with an aim to increase patient access across a range of ethnic groups. In addition, the ability to target peptides presented in multiple HLA alleles would significantly increase the number of patients eligible for this treatment. HLA-A3 and HLA-A11 are part of the same superfamily and share largely overlapping peptide repertoires. We have identified peptides from gp100, a melanoma associated-antigen, which are presented by both HLA-A3 and HLA-A11. Identifying a TCR that is able to recognize a peptide in both these HLA alleles is the first step in generating our first dual-HLA-specific ImmTAC. Here we describe the generation of new TCRs suitable for ImmTAC generation, using our integrated in-house TCR discovery process. TCRs were isolated using multiple methods from HLA-A3 or HLA-A11 donors. We were able to identify antigen-specific T cells from multiple donors that recognized the gp100-peptide-HLA complex. The majority of these T cells were specific to the HLA-allele of the donor and failed to recognize the same peptide in the alternative HLA allele. However, we were able to identify TCRs that had a measurable binding affinity to the gp100 peptide presented in both HLA-A3 and HLA-A11. These data illustrate the successful isolation of wild-type TCRs suitable for affinity maturation and the first step in generating our first dual-HLA-restricted ImmTAC molecule. Citation Format: Vanessa L. Clark, Linda Hibbert, Debbie Wright, Sarah Bailey, Alice Barkell, Kathy Hale, Elizabeth Evans, Alasdair Gunn, Maria Busz, Karolina Lech, Michelle Teng, Luke Williams, Joseph Dukes, Annelise Vuidepot, Bent Jakobsen. ImmTAC molecules: Beyond HLA-A*02:01—the identification and isolation of dual-HLA-specific T cell receptors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B44.

Identification of antigen-specific T cell receptors (TCRs) is an essential component of the ImmTAC (Immune mobilizing monoclonal TCRs Against Cancer) platform at Immunocore. ImmTAC molecules are formed of a soluble TCR fused to an anti-CD3 effector function that engages, redirects and activates T cells to kill cancerous cells. TCRs recognize peptide antigens presented on the surface of the cancer cell by human leukocyte antigen (HLA). Thus, a fundamental step in our pipeline is to isolate T-cell receptors specific for validated cancer antigens for downstream development. T cells are isolated from whole blood of healthy donors to identify antigen-specific T cell clones using an in vitro model that consists of several stimulation steps. Activated CD8+ T cells are sorted into single cells for subsequent analysis and characterization. Despite the relative success of this approach, often analysis of T-cell clones is limited by low or absent mRNA expression for either or both TCR alpha (TRAV) and beta (TRBV) chains. We predict that TCR downregulation may result from overstimulation of T cells, leading to exhaustion. Here, we aimed to establish an in vitro model of T-cell exhaustion that would enable us to better understand the molecular mechanisms behind this process and thus facilitate method development. We induced extreme conditions of stimulation using CD3/CD28 beads and recombinant human IL-2 (rhIL-2) for 2-3 weeks. Whole-transcriptome analysis of the resulting activated T cells was compared to rested cells using a 5′ single-cell RNA-seq method from 10X Genomics (California, USA). Our preliminary analysis indicates that the activated T cells express several checkpoint inhibitors at higher levels. Our findings will be further investigated by performing full-length RNA sequencing of previously isolated single T-cell clones with low TRAV and/or TRBV detection rates. By identifying a molecular signature that represents the phenotype of exhausted T cells, we can potentially assess the state of T cells in TCR discovery outputs. Most importantly, determination of the expression levels of inhibitory receptors that are responsible for the decrease or loss of TCR expression is expected to enhance our understanding of the potential impact of T-cell exhaustion. Citation Format: Paraskevi Mallini, Filipa Bravo-Lopes, Karolina Lech, Sunir Malla, Maria Busz, Nathaniel Davies, Jacob Hurst, Luke Williams, Sterenn Davis, Michelle Teng. Assessing the T-cell exhaustion status of TCR discovery outputs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A92.

The quality of life of hemodialysis (HD) patients is hampered by reduced nocturnal sleep quality and excessive daytime sleepiness. In addition to the sleep/wake cycle, levels of circadian biomarkers (e.g. melatonin) are disturbed in end-stage renal disease (ESRD). This suggests impaired circadian clock performance in HD patients, but the underlying mechanism is unknown. In this observational study, diurnal rhythms of sleep, serum melatonin and cortisol concentrations and clock gene mRNA expression are compared between HD patients (n = 9) and healthy control subjects (n = 9). In addition, the presence of circulating factors that might affect circadian rhythmicity is tested in vitro with cell culture experiments. Reduced sleep quality (median sleep onset latency [interquartile range] of 23.9 [17.3] min for patients versus 5.0 [10] minutes for controls, p

Numerous studies have shown that platinum compounds stimulate the expression of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase resulting in anti-proliferative activity and apoptosis. As many cancer cell types including pancreatic cancer cells express high levels of polyamines, the possibility to develop anti-tumor strategies to deplete polyamine pools has drawn considerable attention in recent years. This has been effectively accomplished by treating cells with platinum drugs in combination with polyamine analogs such as N(1),N(11)-diethylnorspermine (DENSPM). The present study, examined the cytotoxic effects of oxaliplatin in combination with stimulators of polyamine catabolism in human pancreatic cancer cells, that are notoriously resistant to chemotherapeutic treatment, and colorectal cancer cells. Additionally, as protein kinase CK2 has been shown to be an anti-apoptotic and pro-survival enzyme regulated by the intracellular polyamine pools, we aimed to investigate the effect of combined DENSPM and oxaliplatin treatment on CK2-mRNA and -protein levels. Results reported here show that treatment with oxaliplatin and DENSPM in combination impairs cell viability particularly in the case of colorectal cancer cells. The analysis of CK2 expression and activity indicates that the response to a specific treatment may depend on the impact that individual compounds exert on pro-survival and pro-death proteins at the transcription and translation levels that should be carefully evaluated in view of subsequent clinical studies.

Immunocore’s ImmTAC™ (Immune Mobilising Monoclonal TCR Against Cancer) platform combines affinity-enhanced T-cell receptor (TCR)-based targeting with an anti-CD3 scFv effector function to activate a cytotoxic T-cell response against cancer cells. A key part to this process is the identification of tumour epitope specific TCRs from tumor antigen-reactive T-cells. Here, we describe an integrated in-house process leading to the isolation of TCRs specific for validated cancer epitopes, coupled with rapid identification of TCR chains from individual clones using single cell sequencing. The process involves first strand cDNA generation and universal amplification using SmartSeq2 chemistry, followed by targeted sequencing of the TCR alpha and beta chains using next-generation sequencing (NGS). We have also leveraged the 10x Genomics VDJ/5’ counting platform to label and pool multiple experimental clones for repertoire sequencing within a single run. Together with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-Seq), we can reliably assign each T-cell clone to its sample of origin paired with transcriptomic information of epitope specific T-cell populations, linking TCR sequences to their functional phenotype. Citation Format: Karolina Lech, Lucia Correia, Max Beckmann, Maria Busz, Sean Collison, Sterenn Davis, Paraskevi Mallini, Sarah Scaife, Joseph Dukes, Bent K. Jakobsen, Luke Williams, Michelle Teng. Using single-cell paired sequencing to isolate cancer-specific T-cell receptors for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B024.

This study was supported in part by the Netherlands Organization for Scientific Research (NWO) Forensic Science Program Grant 27.011.001, the European Union 6th Framework project EUCLOCK (018741), the UK Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/I019405/1, and Erasmus MC University Medical Center Rotterdam. D.J.S. is a Royal Society Wolfson Research Merit Award holder. The identification and investigation of novel clock-controlled genes (CCGs) has been conducted thus far mainly in model organisms such as nocturnal rodents, with limited information in humans. Here, we aimed to characterize daily and circadian expression rhythms of CCGs in human peripheral blood during a sleep/sleep deprivation (S/SD) study and a constant routine (CR) study. Blood expression levels of 9 candidate CCGs (SREBF1, TRIB1, USF1, THRA1, SIRT1, STAT3, CAPRIN1, MKNK2, and ROCK2), were measured across 48 h in 12 participants in the S/SD study and across 33 h in 12 participants in the CR study. Statistically significant rhythms in expression were observed for STAT3, SREBF1, TRIB1, and THRA1 in samples from both the S/SD and the CR studies, indicating that their rhythmicity is driven by the endogenous clock. The MKNK2 gene was significantly rhythmic in the S/SD but not the CR study, which implies its exogenously driven rhythmic expression. In addition, we confirmed the circadian expression of PER1, PER3, and REV-ERBα in the CR study samples, while BMAL1 and HSPA1B were not significantly rhythmic in the CR samples; all 5 genes previously showed significant expression in the S/SD study samples. Overall, our results demonstrate that rhythmic expression patterns of clock and selected clock-controlled genes in human blood cells are in part determined by exogenous factors (sleep and fasting state) and in part by the endogenous circadian timing system. Knowledge of the exogenous and endogenous regulation of gene expression rhythms is needed prior to the selection of potential candidate marker genes for future applications in medical and forensic settings. Postprint

Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor's alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose.

In yeast and mammals, prohibitins (PHBs) are considered as structural proteins that form a scaffold-like structure for interacting with a set of proteins involved in various processes occurring in the mitochondria. The role of PHB in plant mitochondria is poorly understood. In the study, the model organism Arabidopsis thaliana was used to identify the possible roles of type-II PHBs (homologs of yeast Phb2p) in plant mitochondria. The obtained results suggest that the plant PHB complex participates in the assembly of multisubunit complexes; namely, respiratory complex I and enzymatic complexes carrying lipoic acid as a cofactor (pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glycine decarboxylase). PHBs physically interact with subunits of these complexes. Knockout of two Arabidopsis type-II prohibitins (AtPHB2 and AtPHB6) results in a decreased abundance of these complexes along with a reduction in mitochondrial acyl carrier proteins. Also, the absence of AtPHB2 and AtPHB6 influences the expression of the mitochondrial genome and leads to the activation of alternative respiratory pathways, namely alternative oxidase and external NADH-dependent alternative dehydrogenases.

A recent proof-of-concept pilot study proposed using microRNA (miRNA) markers for time of death determination. The markers - miRNA-142-5p and miRNA-541, were reported to show considerable expression differences in vitreous humor between individuals who died during the day or night. Here, we investigated whether these miRNA markers show the same diurnal expression pattern in blood, which would make them useful for estimating bloodstain deposition time to allow molecular alibi testing for forensic casework. We analyzed venous blood samples collected from 12 healthy individuals every 4 h during the 24 h day/night period under controlled sleep-laboratory conditions. MiRNA-142-5p normalized against miRNA-222 showed no statistically significant expression differences between blood samples collected during daytime and nighttime (one-way ANOVA p = 0.81), and also no statistically significant rhythmicity during the 24 h day/night period (cosine fit for all individuals p > 0.05, averaged data p = 0.932). MicroRNA-541 amplification in blood was above the 34-cycle threshold applied in the study, indicating too low quantities for obtaining reliable data. Overall, we conclude that the two miRNA markers previously suggested for time of death determination in vitreous humor are not suitable for estimating the deposition time of forensic bloodstains. Future studies may find out if miRNA markers with significant diurnal expression patterns can be identified and how useful they would be for forensic trace deposition timing. (C) 2014 Elsevier Ireland Ltd. All rights reserved.