Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch.

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2ß2) disorders, sickle cell disease, and ß-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to ß- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays.

CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.

Structural Features of Transcription Factors Associating with Nucleosome Binding.

Fate-changing transcription factors (TFs) scan chromatin to initiate new genetic programs during cell differentiation and reprogramming. Yet the protein structure domains that allow TFs to target nucleosomal DNA remain unexplored. We screened diverse TFs for binding to nucleosomes containing motif-enriched sequences targeted by pioneer factors in vivo. FOXA1, OCT4, ASCL1/E12α, PU1, CEBPα, and ZELDA display a range of nucleosome binding affinities that correlate with their cell reprogramming potential. We further screened 593 full-length human TFs on protein microarrays against different nucleosome sequences, followed by confirmation in solution, to distinguish among factors that bound nucleosomes, such as the neuronal AP-2α/ß/γ, versus factors that only bound free DNA. Structural comparisons of DNA binding domains revealed that efficient nucleosome binders use short anchoring α helices to bind DNA, whereas weak nucleosome binders use unstructured regions and/or ß sheets. Thus, specific modes of DNA interaction allow nucleosome scanning that confers pioneer activity to transcription factors.

Identifying parental and cell-division origins of aneuploidy in the human blastocyst.

Preimplantation genetic testing commonly employs simplistic copy-number analyses to screen for aneuploidy in blastocyst trophectoderm biopsies. Interpreting intermediate copy number alone as evidence of mosaicism has led to suboptimal estimation of its prevalence. Because mosaicism originates from mitotic nondisjunction, utilizing SNP microarray technology to identify the cell-division origins of aneuploidy might provide a more accurate estimation of its prevalence. The present study develops and validates a method of determining the cell-division origin of aneuploidy in the human blastocyst by using both genotyping and copy-number data in parallel. The concordance of predicted origins with expected results was demonstrated in a series of truth models (99%-100%). This included determination of X chromosome origins from a subset of normal male embryos, determination of the origins of translocation chromosome-related imbalances via embryos from couples with structural rearrangements, and prediction of either mitotic or meiotic origins via multiple rebiopsies of embryos with aneuploidy. In a cohort of blastocysts with parental DNA (n = 2,277), 71% were euploid, 27% were meiotic aneuploid, and 2% were mitotic aneuploid, indicating a low frequency of bona fide mosaicism in the human blastocyst (mean maternal age: 34.4). Chromosome-specific trisomies in the blastocyst were also consistent with observations previously established in products of conception. The ability to accurately identify mitotic-origin aneuploidy in the blastocyst could benefit and better inform individuals whose IVF cycle results in all aneuploid embryos. Clinical trials with this methodology might also help provide a definitive answer regarding the reproductive potential of bona fide mosaic embryos.

Systematic evaluation of genome sequencing for the diagnostic assessment of autism spectrum disorder and fetal structural anomalies.

Short-read genome sequencing (GS) holds the promise of becoming the primary diagnostic approach for the assessment of autism spectrum disorder (ASD) and fetal structural anomalies (FSAs). However, few studies have comprehensively evaluated its performance against current standard-of-care diagnostic tests: karyotype, chromosomal microarray (CMA), and exome sequencing (ES). To assess the clinical utility of GS, we compared its diagnostic yield against these three tests in 1,612 quartet families including an individual with ASD and in 295 prenatal families. Our GS analytic framework identified a diagnostic variant in 7.8% of ASD probands, almost 2-fold more than CMA (4.3%) and 3-fold more than ES (2.7%). However, when we systematically captured copy-number variants (CNVs) from the exome data, the diagnostic yield of ES (7.4%) was brought much closer to, but did not surpass, GS. Similarly, we estimated that GS could achieve an overall diagnostic yield of 46.1% in unselected FSAs, representing a 17.2% increased yield over karyotype, 14.1% over CMA, and 4.1% over ES with CNV calling or 36.1% increase without CNV discovery. Overall, GS provided an added diagnostic yield of 0.4% and 0.8% beyond the combination of all three standard-of-care tests in ASD and FSAs, respectively. This corresponded to nine GS unique diagnostic variants, including sequence variants in exons not captured by ES, structural variants (SVs) inaccessible to existing standard-of-care tests, and SVs where the resolution of GS changed variant classification. Overall, this large-scale evaluation demonstrated that GS significantly outperforms each individual standard-of-care test while also outperforming the combination of all three tests, thus warranting consideration as the first-tier diagnostic approach for the assessment of ASD and FSAs.

Antibody Profiling of Pan-Cancer Viral Proteome Reveals Biomarkers for Nasopharyngeal Carcinoma Diagnosis and Prognosis.

Diagnosing, predicting disease outcome, and identifying effective treatment targets for virus-related cancers are lacking. Protein biomarkers have the potential to bridge the gap between prevention and treatment for these types of cancers. While it has been shown that certain antibodies against EBV proteins could be used to detect nasopharyngeal carcinoma (NPC), antibodies targeting are solely a tiny part of the about 80 proteins expressed by the EBV genome. Furthermore, it remains unclear what role other viruses play in NPC since many diseases are the result of multiple viral infections. For the first time, this study measured both IgA and IgG antibody responses against 646 viral proteins from 23 viruses in patients with NPC and control subjects using nucleic acid programmable protein arrays. Candidate seromarkers were then validated by ELISA using 1665 serum samples from three clinical cohorts. We demonstrated that the levels of five candidate seromarkers (EBV-BLLF3-IgA, EBV-BLRF2-IgA, EBV-BLRF2-IgG, EBV-BDLF1-IgA, EBV-BDLF1-IgG) in NPC patients were significantly elevated than controls. Additional examination revealed that NPC could be successfully diagnosed by combining the clinical biomarker EBNA1-IgA with the five anti-EBV antibodies. The sensitivity of the six-antibody signature at 95% specificity to diagnose NPC was comparable to the current clinically-approved biomarker combination, VCA-IgA, and EBNA1-IgA. However, the recombinant antigens of the five antibodies are easier to produce and standardize compared to the native viral VCA proteins. This suggests the potential replacement of the traditional VCA-IgA assay with the 5-antibodies combination to screen and diagnose NPC. Additionally, we investigated the prognostic significance of these seromarkers titers in NPC. We showed that NPC patients with elevated BLLF3-IgA and BDLF1-IgA titers in their serum exhibited significantly poorer disease-free survival, suggesting the potential of these two seromarkers as prognostic indicators of NPC. These findings will help develop serological tests to detect and treat NPC in the future.

Galectin-4 Antimicrobial Activity Primarily Occurs Through its C-Terminal Domain.

Although immune tolerance evolved to reduce reactivity with self, it creates a gap in the adaptive immune response against microbes that decorate themselves in self-like antigens. This is particularly apparent with carbohydrate-based blood group antigens, wherein microbes can envelope themselves in blood group structures similar to human cells. In this study, we demonstrate that the innate immune lectin, galectin-4 (Gal-4), exhibits strain-specific binding and killing behavior towards microbes that display blood group-like antigens. Examination of binding preferences using a combination of microarrays populated with ABO(H) glycans and a variety of microbial strains, including those that express blood group-like antigens, demonstrated that Gal-4 binds mammalian and microbial antigens that have features of blood group and mammalian-like structures. Although Gal-4 was thought to exist as a monomer that achieves functional bivalency through its two linked carbohydrate recognition domains, our data demonstrate that Gal-4 forms dimers and that differences in the intrinsic ability of each domain to dimerize likely influences binding affinity. While each Gal-4 domain exhibited blood group-binding activity, the C-terminal domain (Gal-4C) exhibited dimeric properties, while the N-terminal domain (Gal-4N) failed to similarly display dimeric activity. Gal-4C not only exhibited the ability to dimerize but also possessed higher affinity toward ABO(H) blood group antigens and microbes expressing glycans with blood group-like features. Furthermore, when compared to Gal-4N, Gal-4C exhibited more potent antimicrobial activity. Even in the context of the full-length protein, where Gal-4N is functionally bivalent by virtue of Gal-4C dimerization, Gal-4C continued to display higher antimicrobial activity. These results demonstrate that Gal-4 exists as a dimer and exhibits its antimicrobial activity primarily through its C-terminal domain. In doing so, these data provide important insight into key features of Gal-4 responsible for its innate immune activity against molecular mimicry.

Altering the competitive environment of B cell epitopes significantly extends the duration of antibody production.

Persistent immunoglobulin G (IgG) production (PIP) provides long-term vaccine protection. While variations in the duration of protection have been observed with vaccines prepared from different pathogens, little is known about the factors that determine PIP. Here, we investigated the impact of three parameters on the duration of anti-peptide IgGs production, namely amino acid sequences, protein carriers, and immunization programs. We show that anti-peptide IgGs production can be transformed from transient IgG production (TIP) to PIP, by placing short peptides (Pi) containing linear B cell epitopes in different competitive environments using bovine serum albumin (BSA) conjugates instead of the original viral particles. When goats were immunized with the peste des petits ruminants (PPR) live-attenuated vaccine (containing Pi as the constitutive component) and BSA-Pi conjugate, anti-Pi IgGs production exhibited TIP (duration <60 days) and PIP (duration >368 days), respectively. Further, this PIP was unaffected by subsequent immunization with the PPR live-attenuated vaccine in the same goat. When goats were co-immunized with PPR live-attenuated vaccine and BSA-Pi, the induced anti-Pi IgGs production showed a slightly extended TIP (from ~60 days to ~100 days). This discovery provides new perspectives for studying the fate of plasma cells in humoral immune responses and developing peptide vaccines related to linear neutralizing epitopes from various viruses.

Functional consequences of familial ALS-associated SOD1L84F in neuronal and muscle cells.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder characterized by progressive skeletal muscle denervation and loss of motor neurons that results in muscle atrophy and eventual death due to respiratory failure. Previously, we identified a novel SOD1L84F variation in a familial ALS case. In this study, we examined the functional consequences of SOD1L84F overexpression in the mouse motor neuron cell line (NSC-34). The cells expressing SOD1L84F showed increased oxidative stress and increased cell death. Interestingly, SOD1L84F destabilized the native dimer and formed high molecular weight SDS-resistant protein aggregates. Furthermore, SOD1L84F also decreased the percentage of differentiated cells and significantly reduced neurite length. A plethora of evidence suggested active involvement of skeletal muscle in disease initiation and progression. We observed differential processing of the mutant SOD1 and perturbations of cellular machinery in NSC-34 and muscle cell line C2C12. Unlike neuronal cells, mutant protein failed to accumulate in muscle cells probably due to the activated autophagy, as evidenced by increased LC3-II and reduced p62. Further, SOD1L84F altered mitochondrial dynamics only in NSC-34. In addition, microarray analysis also revealed huge variations in differentially expressed genes between NSC-34 and C2C12. Interestingly, SOD1L84F hampered the endogenous FUS autoregulatory mechanism in NSC-34 by downregulating retention of introns 6 and 7 resulting in a two-fold upregulation of FUS. No such changes were observed in C2C12. Our findings strongly suggest the differential processing and response towards the mutant SOD1 in neuronal and muscle cell lines.

Profiling Humoral Immunity After Mixing and Matching COVID-19 Vaccines Using SARS-CoV-2 Variant Protein Microarrays.

In November 2022, 68% of the population received at least one dose of COVID-19 vaccines. Owing to the ongoing mutations, especially for the variants of concern (VOCs), it is important to monitor the humoral immune responses after different vaccination strategies. In this study, we developed a SARS-CoV-2 variant protein microarray that contained the spike proteins from the VOCs, e.g., alpha, beta, gamma, delta, and omicron, to quantify the binding antibody and surrogate neutralizing antibody. Plasmas were collected after two doses of matching AZD1222 (AZx2), two doses of matching mRNA-1273 (Mx2), or mixing AZD1222 and mRNA-1273 (AZ+M). The results showed a significant decrease of surrogate neutralizing antibodies against the receptor-binding domain in all VOCs in AZx2 and Mx2 but not AZ+M. A similar but minor reduction pattern of surrogate neutralizing antibodies against the extracellular domain was observed. While Mx2 exhibited a higher surrogate neutralizing level against all VOCs compared with AZx2, AZ+M showed an even higher surrogate neutralizing level in gamma and omicron compared with Mx2. It is worth noting that the binding antibody displayed a low correlation to the surrogate neutralizing antibody (R-square 0.130-0.382). This study delivers insights into humoral immunities, SARS-CoV-2 mutations, and mixing and matching vaccine strategies, which may provide a more effective vaccine strategy especially in preventing omicron.

Proteomics Identifies Circulating TIMP-1 as a Prognostic Biomarker for Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, although disease stratification using in-depth plasma proteomics has not been performed to date. By measuring more than 1000 proteins in the plasma of 147 DLBCL patients using data-independent acquisition mass spectrometry and antibody array, DLBCL patients were classified into four proteomic subtypes (PS-I-IV). Patients with the PS-IV subtype and worst prognosis had increased levels of proteins involved in inflammation, including a high expression of metalloproteinase inhibitor-1 (TIMP-1) that was associated with poor survival across two validation cohorts (n = 180). Notably, the combination of TIMP-1 with the international prognostic index (IPI) identified 64.00% to 88.24% of relapsed and 65.00% to 80.49% of deceased patients in the discovery and two validation cohorts, which represents a 24.00% to 41.67% and 20.00% to 31.70% improvement compared to the IPI score alone, respectively. Taken together, we demonstrate that DLBCL heterogeneity is reflected in the plasma proteome and that TIMP-1, together with the IPI, could improve the prognostic stratification of patients.

Computationally guided conversion of the specificity of E-selectin to mimic that of Siglec-8.

Sulfated glycans have been found to be associated with various diseases and therefore have significant potential in molecular pathology as biomarkers. Although lectins are useful reagents for detecting glycans, there is a paucity of sulfate-recognizing lectins, and those that exist, such as from Maackia amurensis, display mixed specificities. Recombinant lectin engineering offers an emerging tool for creating novel glycan recognition by altering and/or enhancing endogenous specificities. The present study demonstrated the use of computational approaches in the engineering of a mutated form of E-selectin that displayed highly specific recognition of 6'-sulfo-sialyl Lewis X (6'-sulfo-sLex), with negligible binding to its endogenous nonsulfated ligand, sLex. This new specificity mimics that of the unrelated protein Siglec-8, for which 6'-sulfo-sLex is its preferred ligand. Molecular dynamics simulations and energy calculations predicted that two point mutations (E92A/E107A) would be required to stabilize binding to the sulfated oligosaccharide with E-selectin. In addition to eliminating putative repulsions between the negatively charged side chains and the sulfate moiety, the mutations also abolished favorable interactions with the endogenous ligand. Glycan microarray screening of the recombinantly expressed proteins confirmed the predicted specificity change but also identified the introduction of unexpected affinity for the unfucosylated form of 6'-sulfo-sLex (6'-sulfo-sLacNAc). Three key requirements were demonstrated in this case for engineering specificity for sulfated oligosaccharide: 1) removal of unfavorable interactions with the 6'-sulfate, 2) introduction of favorable interactions for the sulfate, and 3) removal of favorable interactions with the endogenous ligand.

Neutrophil-associated Proteins as Novel Biomarkers Elevated in Cerebrospinal Fluid of Neurosyphilis Patients.

BACKGROUND: The immunopathological mechanisms underlying neurosyphilis remain incompletely elucidated, and the diagnosis of neurosyphilis presents challenges. METHODS: We used an antibody microarray to detect 640 proteins in cerebrospinal fluid (CSF) samples collected from 6 non-neurosyphilis and 10 neurosyphilis patients. The levels of CSF CXCL1, CXCL8, G-CSF, LCN2, MMP8, and MMP9 in 46 non-neurosyphilis, 51 untreated neurosyphilis, and 31 post-treatment neurosyphilis patients were quantified using enzyme-linked immunosorbent assay. The associations between the levels of these proteins and clinical parameters in neurosyphilis were evaluated using Spearman's analysis, and the diagnostic performance of these proteins in neurosyphilis was assessed using receiver operating characteristic curve. RESULTS: A total of 102 differentially expressed proteins between neurosyphilis and non-neurosyphilis were identified. The levels of significantly elevated neutrophil-associated proteins (CXCL1, CXCL8, G-CSF, LCN2, MMP8, and MMP9) in neurosyphilis were positive correlations with WBC counts, RPR titer, and protein concentration in CSF. The combination of CSF CXCL8, MMP9, and LCN2 yielded an AUC of 0.92 for diagnosing neurosyphilis, surpassing that of CSF RPR. CONCLUSIONS: CXCL1, CXCL8, G-CSF, LCN2, MMP8, and MMP9 could be associated with central nervous system damage of neurosyphilis. The combination of CSF CXCL8, MMP9, and LCN2 is a promising biomarker for diagnosing neurosyphilis.

Feature-specific quantile normalization and feature-specific mean-variance normalization deliver robust bi-directional classification and feature selection performance between microarray and RNAseq data.

BACKGROUND: Cross-platform normalization seeks to minimize technological bias between microarray and RNAseq whole-transcriptome data. Incorporating multiple gene expression platforms permits external validation of experimental findings, and augments training sets for machine learning models. Here, we compare the performance of Feature Specific Quantile Normalization (FSQN) to a previously used but unvalidated and uncharacterized method we label as Feature Specific Mean Variance Normalization (FSMVN). We evaluate the performance of these methods for bidirectional normalization in the context of nested feature selection. RESULTS: FSQN and FSMVN provided clinically equivalent bidirectional model performance with and without feature selection for colon CMS and breast PAM50 classification. Using principal component analysis, we determine that these methods eliminate batch effects related to technological platforms. Without feature selection, no statistical difference was identified between the performance of FSQN and FSMVN of cross-platform data compared to within-platform distributions. Under optimal feature selection conditions, balanced accuracy was FSQN and FSMVN were statistically equivalent to the within-platform distribution performance in multivariable linear regression analysis. FSQN and FSMVN also provided similar performance to within-platform distributions as the number of selected genes used to create models decreases. CONCLUSIONS: In the context of generating supervised machine learning classifiers for molecular subtypes, FSQN and FSMVN are equally effective. Under optimal modeling conditions, FSQN and FSMVN provide equivalent model accuracy performance on cross-platform normalization data compared to within-platform data. Using cross-platform data should still be approached with caution as subtle performance differences may exist depending on the classification problem, training, and testing distributions.

Proteomics Platform Reveals Broad-Spectrum Nanobodies for SARS-CoV-2 Variant Neutralization.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the emergence of different variants of concerns with immune evasion that have been prevalent over the past three years. Nanobodies, the functional variable regions of camelid heavy-chain-only antibodies, have garnered interest in developing neutralizing antibodies due to their smaller size, structural stability, ease of production, high affinity, and low immunogenicity, among other characteristics. In this work, we describe an integrated proteomics platform for the high-throughput screening of nanobodies against different SARS-CoV-2 spike variants. To demonstrate this platform, we immunized a camel with subunit 1 (S1) of the wild-type spike protein and constructed a nanobody phage library. The binding and neutralizing activities of the nanobodies against 72 spike variants were then measured, resulting in the identification of two nanobodies (C-282 and C-39) with broad neutralizing activity against six non-Omicron variants (D614G, Alpha, Beta, Gamma, Delta, Kappa) and five Omicron variants (BA.1-5). Their neutralizing capability was validated using in vitro pseudovirus-based neutralization assays. All these results demonstrate the utility of our proteomics platform to identify new nanobodies with broad neutralizing capability and to develop a treatment for patients with SARS-CoV-2 variant infection in the future.

Combinatorial Approach with Mass Spectrometry and Lectin Microarray Dissected Site-Specific Glycostem and Glycoleaf Features of the Virion-Derived Spike Protein of Ancestral and γ Variant SARS-CoV-2 Strains.

The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (γ-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the γ-variant S protein; however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.

Modeling interactions between Heparan sulfate and proteins based on the Heparan sulfate microarray analysis.

Heparan sulfate (HS), a sulfated polysaccharide abundant in the extracellular matrix, plays pivotal roles in various physiological and pathological processes by interacting with proteins. Investigating the binding selectivity of HS oligosaccharides to target proteins is essential, but the exhaustive inclusion of all possible oligosaccharides in microarray experiments is impractical. To address this challenge, we present a hybrid pipeline that integrates microarray and in silico techniques to design oligosaccharides with desired protein affinity. Using fibroblast growth factor 2 (FGF2) as a model protein, we assembled an in-house dataset of HS oligosaccharides on microarrays and developed two structural representations: a standard representation with all atoms explicit and a simplified representation with disaccharide units as "quasi-atoms." Predictive Quantitative Structure-Activity Relationship (QSAR) models for FGF2 affinity were developed using the Random Forest (RF) algorithm. The resulting models, considering the applicability domain, demonstrated high predictivity, with a correct classification rate of 0.81-0.80 and improved positive predictive values (PPV) up to 0.95. Virtual screening of 40 new oligosaccharides using the simplified model identified 15 computational hits, 11 of which were experimentally validated for high FGF2 affinity. This hybrid approach marks a significant step toward the targeted design of oligosaccharides with desired protein interactions, providing a foundation for broader applications in glycobiology.

Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNA methylation profiling.

BACKGROUND: The Illumina family of Infinium Methylation BeadChip microarrays has been widely used over the last 15 years for genome-wide DNA methylation profiling, including large-scale and population-based studies, due to their ease of use and cost effectiveness. Succeeding the popular HumanMethylationEPIC BeadChip (EPICv1), the recently released Infinium MethylationEPIC v2.0 BeadChip (EPICv2) claims to extend genomic coverage to more than 935,000 CpG sites. Here, we comprehensively characterise the reproducibility, reliability and annotation of the EPICv2 array, based on bioinformatic analysis of both manifest data and new EPICv2 data from diverse biological samples. RESULTS: We find a high degree of reproducibility with EPICv1, evidenced by comparable sensitivity and precision from empirical cross-platform comparison incorporating whole genome bisulphite sequencing (WGBS), and high correlation between technical sample replicates, including between samples with DNA input levels below the manufacturer's recommendation. We provide a full assessment of probe content, evaluating genomic distribution and changes from previous array versions. We characterise EPICv2's new feature of replicated probes and provide recommendations as to the superior probes. In silico analysis of probe sequences demonstrates that probe cross-hybridisation remains a significant problem in EPICv2. By mapping the off-target sites at single nucleotide resolution and comparing with WGBS we show empirical evidence for preferential off-target binding. CONCLUSIONS: Overall, we find EPICv2 a worthy successor to the previous Infinium methylation microarrays, however some technical issues remain. To support optimal EPICv2 data analysis we provide an expanded version of the EPICv2 manifest to aid researchers in understanding probe design, data processing, choosing appropriate probes for analysis and for integration with methylation datasets from previous versions of the Infinium Methylation BeadChip.

A comparison of the TempO-Seq and Affymetrix microarray platform using RTqPCR validation.

Next-generation risk assessment relies on mechanistic data from new approach methods, including transcriptome data. Various technologies, such as high-throughput targeted sequencing methods and microarray technologies based on hybridization with complementary probes, are used to determine differentially expressed genes (DEGs). The integration of data from different technologies requires a good understanding of the differences arising from the use of various technologies.To better understand the differences between the TempO-Seq platform and Affymetrix chip technology, whole-genome data for the volatile compound dimethylamine were compared. Selected DEGs were also confirmed using RTqPCR validation. Although the overlap of DEGs between TempO-Seq and Affymetrix was no higher than 37%, a comparison of the gene regulation in terms of log2fold changes revealed a very high concordance. RTqPCR confirmed the majority of DEGs from either platform in the examined dataset. Only a few conflicts were found (11%), while 22% were not confirmed, and 3% were not detected.Despite the observed differences between the two platforms, both can be validated using RTqPCR. Here we highlight some of the differences between the two platforms and discuss their applications in toxicology.