Antibody Profiling and In Silico Functional Analysis of Differentially Reactive Antibody Signatures of Glioblastomas and Meningiomas.

Studies on tumor-associated antigens in brain tumors are sparse. There is scope for enhancing our understanding of molecular pathology, in order to improve on existing forms, and discover new forms, of treatment, which could be particularly relevant to immuno-oncological strategies. To elucidate immunological differences, and to provide another level of biological information, we performed antibody profiling, based on a high-density protein array (containing 8173 human transcripts), using IgG isolated from the sera of n = 12 preoperative and n = 16 postoperative glioblastomas, n = 26 preoperative and n = 29 postoperative meningiomas, and n = 27 healthy, cancer-free controls. Differentially reactive antigens were compared to gene expression data from an alternate public GBM data set from OncoDB, and were analyzed using the Reactome pathway browser. Protein array analysis identified approximately 350-800 differentially reactive antigens, and revealed different antigen profiles in the glioblastomas and meningiomas, with approximately 20-30%-similar and 10-15%-similar antigens in preoperative and postoperative sera, respectively. Seroreactivity did not correlate with OncoDB-derived gene expression. Antigens in the preoperative glioblastoma sera were enriched for signaling pathways, such as signaling by Rho-GTPases, COPI-mediated anterograde transport and vesicle-mediated transport, while the infectious disease, SRP-dependent membrane targeting cotranslational proteins were enriched in the meningiomas. The pre-vs. postoperative seroreactivity in the glioblastomas was enriched for antigens, e.g., platelet degranulation and metabolism of lipid pathways; in the meningiomas, the antigens were enriched in infectious diseases, metabolism of amino acids and derivatives, and cell cycle. Antibody profiling in both tumor entities elucidated several hundred antigens and characteristic signaling pathways that may provide new insights into molecular pathology and may be of interest for the development of new treatment strategies.

Functional Analysis of Autoantibody Signatures in Rheumatoid Arthritis.

For the identification of antigenic protein biomarkers for rheumatoid arthritis (RA), we conducted IgG profiling on high density protein microarrays. Plasma IgG of 96 human samples (healthy controls, osteoarthritis, seropositive and seronegative RA, n = 24 each) and time-series plasma of a pristane-induced arthritis (PIA) rat model (n = 24 total) were probed on AIT's 16k protein microarray. To investigate the analogy of underlying disease pathways, differential reactivity analysis was conducted. A total of n = 602 differentially reactive antigens (DIRAGs) at a significance cutoff of p < 0.05 were identified between seropositive and seronegative RA for the human samples. Correlation with the clinical disease activity index revealed an inverse correlation of antibodies against self-proteins found in pathways relevant for antigen presentation and immune regulation. The PIA model showed n = 1291 significant DIRAGs within acute disease. Significant DIRAGs for (I) seropositive, (II) seronegative and (III) PIA were subjected to the Reactome pathway browser which also revealed pathways relevant for antigen presentation and immune regulation; of these, seven overlapping pathways had high significance. We therefore conclude that the PIA model reflects the biological similarities of the disease pathogenesis. Our data show that protein array analysis can elucidate biological differences and pathways relevant in disease as well be a useful additional layer of omics information.

Multiplexed DNA Methylation Analysis in Colorectal Cancer Using Liquid Biopsy and Its Diagnostic and Predictive Value.

Early diagnosis of colorectal cancer (CRC) is of high importance as prognosis depends on tumour stage at the time of diagnosis. Detection of tumour-specific DNA methylation marks in cfDNA has several advantages over other approaches and has great potential for solving diagnostic needs. We report here the identification of DNA methylation biomarkers for CRC and give insights in our methylation-sensitive restriction enzyme coupled qPCR (MSRE-qPCR) system. Targeted microarrays were used to investigate the DNA methylation status of 360 cancer-associated genes. Validation was done by qPCR-based approaches. A focus was on investigating marker performance in cfDNA from 88 patients (44 CRC, 44 controls). Finally, the workflow was scaled-up to perform 180plex analysis on 110 cfDNA samples, to identify a DNA methylation signature for advanced colonic adenomas (AA). A DNA methylation signature (n = 44) was deduced from microarray experiments and confirmed by quantitative methylation-specific PCR (qMSP) and by MSRE-qPCR, providing for six genes' single areas under the curve (AUC) values of >0.85 (WT1, PENK, SPARC, GDNF, TMEFF2, DCC). A subset of the signatures can be used for patient stratification and therapy monitoring for progressed CRC with liver metastasis using cfDNA. Furthermore, we identified a 35-plex classifier for the identification of AAs with an AUC of 0.80.

EPIC-TABSAT: analysis tool for targeted bisulfite sequencing experiments and array-based methylation studies.

DNA methylation is one of the major epigenetic modifications and has frequently demonstrated its suitability as diagnostic and prognostic biomarker. In addition to chip and sequencing based epigenome wide methylation profiling methods, targeted bisulfite sequencing (TBS) has been established as a cost-effective approach for routine diagnostics and target validation applications. Yet, an easy-to-use tool for the analysis of TBS data in combination with array-based methylation results has been missing. Consequently, we have developed EPIC-TABSAT, a user-friendly web-based application for the analysis of targeted sequencing data that additionally allows the integration of array-based methylation results. The tool can handle multiple targets as well as multiple sequencing files in parallel and covers the complete data analysis workflow from calculation of quality metrics to methylation calling and interactive result presentation. The graphical user interface offers an unprecedented way to interpret TBS data alone or in combination with array-based methylation studies. Together with the computation of target-specific epialleles it is useful in validation, research, and routine diagnostic environments. EPIC-TABSAT is freely accessible to all users at https://tabsat.ait.ac.at/.

Systemic autoinflammatory diseases.

Systemic autoinflammatory diseases (SAIDs) are a growing group of disorders caused by a dysregulation of the innate immune system leading to episodes of systemic inflammation. In 1997, MEFV was the first gene identified as disease causing for Familial Mediterranean Fever, the most common hereditary SAID. In most cases, autoinflammatory diseases have a strong genetic background with mutations in single genes. Since 1997 more than 30 new genes associated with autoinflammatory diseases have been identified, affecting different parts of the innate immune system. Nevertheless, for at least 40-60% of patients with phenotypes typical for SAIDs, a distinct diagnosis cannot be met, leading to undefined SAIDs (uSAIDs). However, SAIDs can also be of polygenic or multifactorial origin, with environmental influence modulating the phenotype. The implementation of a disease continuum model combining the adaptive and the innate immune system with autoinflammatory and autoimmune diseases shows the complexity of SAIDs and the importance of new methods to elucidate molecular changes and causative factors in SAIDs. Diagnosis is often based on clinical presentation and genetic testing. The timeline from onset to diagnosis takes up to 7.3 years, highlighting the indisputable need to identify new treatment and diagnostic targets. Recently, other factors are under investigation as additional contributors to the pathogenesis of SAIDs. This review gives an overview of pathogenesis and etiology of SAIDs, and summarizes recent diagnosis and treatment options.

Preventive sublingual immunotherapy with House Dust Mite extract modulates epitope diversity in pre-school children.

BACKGROUND: The preventive effect of allergen immunotherapy (AIT) on allergy and asthma development is currently assessed using primary and secondary AIT approaches. Knowledge of the immunological effects of these interventions is limited and the impact on epitope diversity remains to be defined. METHODS: We used high-density peptide arrays that included all known Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) allergens and the whole proteome of Der f to study changes in House Dust Mite (HDM) linear peptide recognition during a 2-year preventive double-blind placebo-controlled sublingual HDM AIT pilot study in 2-5-year-old children with sensitization to HDM but without symptoms. RESULTS: Preventive AIT-treated patients showed significantly higher IgG epitope diversity to HDM allergens compared to placebo-treated individuals at 24 months of treatment (P < 0.05), while no increase in IgE diversity was seen. At 24 months of treatment, IgG4 diversity for HDM allergens was significantly higher in the pAIT-treated patients compared to placebo group (P < 0.05). Potentially beneficial changes in epitope recognition throughout the treatment are also seen in peptides derived from Der f proteome. CONCLUSION: These data suggest a beneficial immunomodulation of preventive sublingual immunotherapy at a molecular level by favoring a broader blocking repertoire and inhibiting epitope spreading.

Circulating Free Methylated Tumor DNA Markers for Sensitive Assessment of Tumor Burden and Early Response Monitoring in Patients Receiving Systemic Chemotherapy for Colorectal Cancer Liver Metastasis.

BACKGROUND: Neoadjuvant chemotherapy (neoCTx) followed by hepatic resection is the treatment of choice for patients with colorectal cancer liver metastasis (CLM). Treatment response is generally assessed using radiologic imaging after several cycles of chemotherapy. However, earlier assessment of response would be desirable since nonresponders could be switched early to an alternative chemotherapy regimen. Recent evidence suggests that circulating free methylated tumor DNA is a highly sensitive biomarker and may more accurately reflect tumor burden and treatment response than conventional markers for CRC. PATIENTS AND METHODS: Thirty-four patients with CLM who received neoCTx prior to intended hepatic resection were included in this prospective nonrandomized study. Peripheral blood plasma was collected at baseline and before each cycle of neoCTx and was then analyzed for aberrant methylation of 48 CRC-associated genes. Methylation marker levels were correlated with baseline tumor volume and treatment response and compared with the standard tumor markers CEA and CA 19-9. RESULTS: The methylation markers SEPT9, DCC, BOLL, and SFRP2 were present in all patients at baseline and displayed a stronger correlation with tumor volume than CEA and CA 19-9. Serial measurement of these methylation markers allowed for discrimination between operated and nonoperated patients already after 1 cycle of neoCTx with high sensitivity and specificity. The early dynamic changes of SEPT9 and DCC also seemed to correlate with pathohistological response. CONCLUSION: Our data suggest that serial measurements of CRC-associated methylation markers could be a particularly valuable tool for early response assessment in patients receiving neoCTx for CLM.

DaMold: A data-mining platform for variant annotation and visualization in molecular diagnostics research.

Next-generation sequencing (NGS) has become a powerful and efficient tool for routine mutation screening in clinical research. As each NGS test yields hundreds of variants, the current challenge is to meaningfully interpret the data and select potential candidates. Analyzing each variant while manually investigating several relevant databases to collect specific information is a cumbersome and time-consuming process, and it requires expertise and familiarity with these databases. Thus, a tool that can seamlessly annotate variants with clinically relevant databases under one common interface would be of great help for variant annotation, cross-referencing, and visualization. This tool would allow variants to be processed in an automated and high-throughput manner and facilitate the investigation of variants in several genome browsers. Several analysis tools are available for raw sequencing-read processing and variant identification, but an automated variant filtering, annotation, cross-referencing, and visualization tool is still lacking. To fulfill these requirements, we developed DaMold, a Web-based, user-friendly tool that can filter and annotate variants and can access and compile information from 37 resources. It is easy to use, provides flexible input options, and accepts variants from NGS and Sanger sequencing as well as hotspots in VCF and BED formats. DaMold is available as an online application at http://damold.platomics.com/index.html, and as a Docker container and virtual machine at https://sourceforge.net/projects/damold/.

The prostate cancer immunome: In silico functional analysis of antigenic proteins from microarray profiling with IgG.

The study of the immunome of prostate cancer (PCa) and characterization of autoantibody signature from differentially reactive antigens can uncover disease stage proteins, reveal enriched networks and even expose aberrant cellular mechanisms during the disease process. By conducting plasma IgG profiling on protein microarrays presenting 5449 unique human proteins expressed in 15 417 E. coli human cDNA expression clones, we elucidated 471 (21 higher reactive in PCa) differentially reactive antigens in 50 PCa versus 49 patients with benign prostate hyperplasia (BPH) at initial diagnosis. Functional analyzes show that the immune-profile of PCa compared to BPH control samples is significantly enriched in features targeting Cellular assembly, Cell death and pathways involved in Cell cycle, translation, and assembly of proteins as EIF2 signaling, PCa related genes as AXIN1 and TP53, and ribosomal proteins (e.g. RPS10). An overlap of 61 (out of 471) DIRAGs with the published 1545 antigens from the SEREX database has been found, however those were higher reactive in BPH. Clinical relevance is shown when antibody-reactivities against eight proteins were significantly (p < 0.001) correlated with Gleason-score. Herewith we provide a biological and pathophysiological characterization of the immunological layer of cancerous (PCa) versus benign (BPH) disease, derived from antibody profiling on protein microarrays.

ClinQC: a tool for quality control and cleaning of Sanger and NGS data in clinical research.

BACKGROUND: Traditional Sanger sequencing has been used as a gold standard method for genetic testing in clinic to perform single gene test, which has been a cumbersome and expensive method to test several genes in heterogeneous disease such as cancer. With the advent of Next Generation Sequencing technologies, which produce data on unprecedented speed in a cost effective manner have overcome the limitation of Sanger sequencing. Therefore, for the efficient and affordable genetic testing, Next Generation Sequencing has been used as a complementary method with Sanger sequencing for disease causing mutation identification and confirmation in clinical research. However, in order to identify the potential disease causing mutations with great sensitivity and specificity it is essential to ensure high quality sequencing data. Therefore, integrated software tools are lacking which can analyze Sanger and NGS data together and eliminate platform specific sequencing errors, low quality reads and support the analysis of several sample/patients data set in a single run. RESULTS: We have developed ClinQC, a flexible and user-friendly pipeline for format conversion, quality control, trimming and filtering of raw sequencing data generated from Sanger sequencing and three NGS sequencing platforms including Illumina, 454 and Ion Torrent. First, ClinQC convert input read files from their native formats to a common FASTQ format and remove adapters, and PCR primers. Next, it split bar-coded samples, filter duplicates, contamination and low quality sequences and generates a QC report. ClinQC output high quality reads in FASTQ format with Sanger quality encoding, which can be directly used in down-stream analysis. It can analyze hundreds of sample/patients data in a single run and generate unified output files for both Sanger and NGS sequencing data. Our tool is expected to be very useful for quality control and format conversion of Sanger and NGS data to facilitate improved downstream analysis and mutation screening. CONCLUSIONS: ClinQC is a powerful and easy to handle pipeline for quality control and trimming in clinical research. ClinQC is written in Python with multiprocessing capability, run on all major operating systems and is available at https://sourceforge.net/projects/clinqc.

Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers.

BACKGROUND: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. METHODS: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma. RESULTS: We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients' age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients. CONCLUSIONS: The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer.

Clinical Neuropathology practice news 1-2014: pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma.

Testing of the MGMT promoter methylation status in glioblastoma is relevant for clinical decision making and research applications. Two recent and independent phase III therapy trials confirmed a prognostic and predictive value of the MGMT promoter methylation status in elderly glioblastoma patients. Several methods for MGMT promoter methylation testing have been proposed, but seem to be of limited test reliability. Therefore, and also due to feasibility reasons, translation of MGMT methylation testing into routine use has been protracted so far. Pyrosequencing after prior DNA bisulfite modification has emerged as a reliable, accurate, fast and easy-to-use method for MGMT promoter methylation testing in tumor tissues (including formalin fixed and paraffin-embedded samples). We performed an intra- and inter-laboratory ring trial which demonstrates a high analytical performance of this technique. Thus, pyrosequencing- based assessment of MGMT promoter methylation status in glioblastoma meets the criteria of high analytical test performance and can be recommended for clinical application, provided that strict quality control is performed. Our article summarizes clinical indications, practical instructions and open issues for MGMT promoter methylation testing in glioblastoma using pyrosequencing.

Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoassays.

Antibody (AB) testing or serotesting for reactive ABs against antigenic proteins is broadly used. Parallel examination of many antigens is of high interest to identify autoantibodies (AAB) or differential antigenic reactivities in many biological settings like allergy and infectious autoimmune, cancerous, or systemic disease. The resulting AAB profiles can be used for diagnosis, prognosis, and monitoring of such conditions. Protein microarrays have been used for AB profiling over the past decade but show some significant limitations which make them unsuitable for clinical applications. Alternative multiplexing platforms such as bead arrays were shown to provide a versatile tool for the confirmation and efficient analysis of high numbers of biological samples. Luminex' bead-based xMAP technology combines advantages such as multiplexing and lower demand for sample volume and at the same time overcomes the challenges of microarrays. It works faster, shows better antigen stability, is more reproducible, and allows the analysis of up to 500 analytes in one sample well. In this chapter we introduce our established workflow for the use of the xMAP technology for AB profiling including an overview of the method principle and protocols for the covalent immobilization of proteins to the MagPlex beads, confirmation of protein coupling, the execution of a multiplexed bead-based protein immunoassay, and subsequent data handling.

Multiplexed Bead-Based Peptide Immunoassays for the Detection of Antibody Reactivities.

Antigenic peptides are commonly used in serological test settings such as enzyme-linked immunosorbent assays (ELISA) to determine reactive antibodies (ABs) from serum or plasma samples. The use of synthetic peptides provides advantages like lower production effort and easier incorporation of specific chemical modifications compared to full-length antigenic proteins. Multiplexed antibody (AB) profiling methods such as microarray technologies enable the simultaneous identification of multiple novel biomarkers for the use in early disease diagnostics, vaccine development, or monitoring of immune responses. Despite various benefits they still show major limitations which can be overcome with bead-based assay technologies like the multi-analyte profiling (xMAP) technology developed by Luminex. In this chapter we introduce our established workflow for AB profiling with a multiplexed bead-based peptide immunoassay. The workflow is based on copper-catalyzed click chemistry to immobilize designed synthetic peptides onto uniquely color-coded paramagnetic beads in an orientation-specific manner. The individual peptide-coupled beads can be distinguished by their unique emission spectra during readout in the xMAP instrument and therefore allow testing of up to 500 different antigenic peptides in one multiplexed reaction. The multistep process described in this chapter is divided into separate sections for peptide design, coupling of functionalized peptides to MagPlex beads via click chemistry, confirmation of successful peptide immobilization, processing of serum or plasma samples, or preferably purified IgG thereof, with the multiplexed bead-based peptide immunoassay and subsequent data export and analysis.

Patients with Systemic Juvenile Idiopathic Arthritis (SJIA) Show Differences in Autoantibody Signatures Based on Disease Activity.

Systemic juvenile idiopathic arthritis (SJIA) is a severe rheumatic disease in children. It is a subgroup of juvenile idiopathic arthritis (JIA; MIM #604302), which is the most common rheumatic disease in children. The diagnosis of SJIA often comes with a significant delay, and the classification between autoinflammatory and autoimmune disease is still discussed. In this study, we analyzed the immunological responses of patients with SJIA, using human proteome arrays presenting immobilized recombinantly expressed human proteins, to analyze the involvement of autoantibodies in SJIA. Results from group comparisons show several differentially reactive antigens involved in inflammatory processes. Intriguingly, many of the identified antigens had a high reactivity against proteins involved in the NF-κB pathway, and it is also notable that many of the detected DIRAGs are described as dysregulated in rheumatoid arthritis. Our data highlight novel proteins and pathways potentially dysregulated in SJIA and offer a unique approach to unraveling the underlying disease pathogenesis in this chronic arthropathy.

Aberrant Naive CD4-Positive T Cell Differentiation in Systemic Juvenile Idiopathic Arthritis Committed to B Cell Help.

OBJECTIVE: Systemic juvenile idiopathic arthritis (JIA) features characteristics of autoinflammation and autoimmunity, culminating in chronic arthritis. In this study, we hypothesized that aberrant or incomplete polarization of T helper cells contributes to disease pathology. METHODS: Cells or serum samples were obtained from healthy controls (n = 72) and systemic JIA patients (n = 171). Isolated naive T helper cells were cultured under Th1, Th17, and T follicular helper (Tfh) or T peripheral helper (Tph)-polarizing conditions and were partly cocultured with allogenic memory B cells. Cell samples were then analyzed for surface marker, transcription factor, and cytokine expression, as well as plasmablast generation. Serum samples were subjected to multiplexed bead and self-antigen arrays and enzyme-linked immunosorbent assays, and all data were compared to retrospective RNA profiling analyses. RESULTS: Differentiation of systemic JIA-naive T helper cells toward Th1 cells resulted in low expression levels of interferon-γ (IFNγ) and eomesodermin, which was associated in part with disease duration. In contrast, developing Th1 cells in patients with systemic JIA were found to produce elevated levels of interleukin-21 (IL-21), which negatively correlated with cellular expression of IFNγ and eomesodermin. In both in vitro and ex vivo analyses, IL-21 together with programmed cell death 1 (PD-1), inducible T cell costimulator (ICOS), and CXCR5 expression induced naive T helper cells from systemic JIA patients to polarize toward a Tfh/Tph cell phenotype. Retrospective analysis of whole-blood RNA-sequencing data demonstrated that Bcl-6, a master transcription factor in Tfh/Tph cell differentiation, was overexpressed specifically in patients with systemic JIA. Naive T helper cells from systemic JIA patients which were stimulated in vitro promoted B cellular plasmablast generation, and self-antigen array data indicated that IgG reactivity profiles of patients with systemic JIA differed from those of healthy controls. CONCLUSION: In the pathogenesis of systemic JIA, skewing of naive T helper cell differentiation toward a Tfh/Tph cell phenotype may represent an echo of autoimmunity, which may indicate the mechanisms driving progression toward chronic destructive arthritis.

Plasma cells are not restricted to the CD27+ phenotype: characterization of CD27-CD43+ antibody-secreting cells.

Circulating antibody-secreting cells are present in the peripheral blood of healthy individuals reflecting the continued activity of the humoral immune system. Antibody-secreting cells typically express CD27. Here we describe and characterize a small population of antibody-secreting class switched CD19+CD43+ B cells that lack expression of CD27 in the peripheral blood of healthy subjects. In this study, we characterized CD27-CD43+ cells. We demonstrate that class-switched CD27-CD43+ B cells possess characteristics of conventional plasmablasts as they spontaneously secrete antibodies, are morphologically similar to antibody-secreting cells, show downregulation of B cell differentiation markers, and have a gene expression profile related to conventional plasmablasts. Despite these similarities, we observed differences in IgA and IgG subclass distribution, expression of homing markers, replication history, frequency of somatic hypermutation, immunoglobulin repertoire, gene expression related to Toll-like receptors, cytokines, and cytokine receptors, and antibody response to vaccination. Their frequency is altered in immune-mediated disorders. Conclusion: we characterized CD27-CD43+ cells as antibody-secreting cells with differences in function and homing potential as compared to conventional CD27+ antibody-secreting cells.

A novel click chemistry-based peptide ELISA protocol: development and technical evaluation.

ELISA is the current standard for (auto)antibody diagnostics. Once established, ELISA protocols can be easily adapted for novel antigens; however, peptide-based protocols are rarely available. Herein the authors describe the results of a technical investigation of an indirect ELISA protocol using peptides conjugated onto a protein carrier based on click chemistry and immobilized in standard plastics. The authors compared this approach with the common biotin-avidin system and obtained a slightly improved limit of detection for purified IgG of 25-100 ng/well compared with 25-1000 ng/well. Reproducibility and stability of the methodological approach were conducted for further technical characterization. Indirect ELISA using immunoreactive peptides conjugated to bovine serum albumin offers a reliable method that is complementary to standard plastics and plate readers.

Designed SARS-CoV-2 receptor binding domain variants form stable monomers.

The receptor binding domain (RBD) of the SARS-CoV-2 spike (S)-protein is a prime target of virus-neutralizing antibodies present in convalescent sera of COVID-19 patients and thus is considered a key antigen for immunosurveillance studies and vaccine development. Although recombinant expression of RBD has been achieved in several eukaryotic systems, mammalian cells have proven particularly useful. The authors aimed to optimize RBD produced in HEK293-6E cells towards a stable homogeneous preparation and addressed its O-glycosylation as well as the unpaired cysteine residue 538 in the widely used RBD (319-541) sequence. The authors found that an intact O-glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, it was shown that deletion or substitution of the unpaired cysteine residue C538 reduces the intrinsic propensity of RBD to form oligomeric aggregates, concomitant with an increased yield of the monomeric form of the protein. Bead-based and enzyme-linked immunosorbent assays utilizing these optimized RBD variants displayed excellent performance with respect to the specific detection of even low levels of SARS-CoV-2 antibodies in convalescent sera. Hence, these RBD variants could be instrumental for the further development of serological SARS-CoV-2 tests and inform the design of RBD-based vaccine candidates.

EFS shows biallelic methylation in uveal melanoma with poor prognosis as well as tissue-specific methylation.

BACKGROUND: Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed EFS (embryonal Fyn-associated substrate) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated EFS promoter region CpG island in tumor classification and metastatic progression. METHODS: EFS methylation was determined by direct sequencing of PCR products from bisulfite-treated DNA or by sequence analysis of individual cloned PCR products. The results were associated with clinical features of tumors and tumor-related death of patients. RESULTS: Analysis of 16 UM showed full methylation of the EFS CpG island in 8 (50%), no methylation in 5 (31%) and partial methylation in 3 (19%) tumors. Kaplan-Meier analysis revealed a higher risk of metastatic progression for tumors with EFS methylation (p = 0.02). This correlation was confirmed in an independent set of 24 randomly chosen tumors. Notably, only UM with EFS methylation gave rise to metastases. Real-time quantitative RT-PCR expression analysis revealed a significant inverse correlation of EFS mRNA expression with EFS methylation in UM. We further found that EFS methylation is tissue-specific with full methylation in peripheral blood cells, and no methylation in sperm, cultured primary fibroblasts and fetal muscle, kidney and brain. Adult brain samples, cultured melanocytes from the uveal tract, fetal liver and 3 of 4 buccal swab samples showed partial methylation. EFS methylation always affects both alleles in normal and tumor samples. CONCLUSIONS: Biallelic EFS methylation is likely to be the result of a site-directed methylation mechanism. Based on partial methylation as observed in cultured melanocytes we hypothesize that there might be methylated and unmethylated precursor cells located in the uveal tract. The EFS methylation of a UM may depend on which type of precursor cell the tumor originated from.