Differential Binding Patterns Research Articles

Molecular docking based comparative study of antiviral compounds on SARS-CoV-2 spike protein.

The urgent need for effective therapeutic interventions against SARS-CoV-2 has prompted extensive exploration of potential drug candidates. Among the viral proteins, the spike (S) protein presents an attractive target due to its critical role in viral entry and infection. In this study, we employed molecular docking techniques to investigate the binding affinities and interaction profiles of a panel of active compounds against the SARS-CoV-2 spike protein. Utilising computational simulations, we assessed the binding properties of these compounds within the receptor-binding domain (RBD) and other key regions of the spike protein. Our comparative analysis elucidates the differential binding patterns and identifies promising lead compounds with high binding affinity and favourable interaction profiles. Furthermore, we discuss the implications of these findings for the development of potential therapeutics targeting the SARS-CoV-2 spike protein. Using molecular docking and the Lipinski five rule, this study illustrates possible compounds with strong binding affinities, their molecular interactions, for both naturally occurring and man-made drugs. Computational approach is applied, and it is concluded that, drugs like Withanolide, Dihydroergocristine, Fenebrutinib, and Ergotamine shows binding energies between -8.3 and -9.1 kcal/mol, and are possible candidate for anti covid drug.

Abstract LB200: Defining the mechanisms of cancer specific transcription factor, tGLI1

Abstract Glioblastoma (GBM) is the most common malignant central nervous system tumor in adults with a five-year survival rate of less than 7%. First identified in GBM, truncated GLI1 (tGLI1), an alternative splicing isoform of GLI1, has emerged as a cancer specific GLI1 variant as tGLI1 is highly expressed in both GBM cell lines and primary specimens but not in normal tissue. Despite an in-frame loss of 41 amino acids, the tGLI1 protein has retained all known GLI1 functional domains and appears to respond to sonic hedgehog in a similar manner as GLI1. However, tGLI1 operates as a gain of function transcription factor with the ability to bind to and activate genes unique from GLI1 to promote invasion, migration, angiogenesis, and stemness in both GBM and breast cancer. Despite the advancements in our understanding of tGLI1, a genome wide DNA binding pattern of tGLI1 has not been established and the mechanism by which tGLI1 gains access to these genes is unknown. To establish a more robust understanding of the differential DNA binding patterns of GLI1 and tGLI1, we carried out ChIP-sequencing (ChIP-seq) and found only 14% of GLI1 and tGLI1 binding sites are shared. These results suggest GLI1 and tGLI1 have even more unique binding pattens across the entire genome that previously thought. However, the underlying mechanism for why GLI1 and tGLI1 bind different regions of the genome remains elusive. To determine whether variations in protein interactions of GLI1 and tGLI1 contribute to the unique binding pattern of tGLI1, we carried out immunoprecipitation (IP) followed by mass spectrometry (mass spec). Results identified 45 GLI1 associated proteins and 52 tGLI1 associated proteins with only a 29% overlap between the two. Furthermore, gene ontology analysis revealed 43% of the tGLI1-specific associated proteins are involved in processes related to RNA binding, processing, and metabolism. From these RNA-related proteins, we validated an interaction between tGLI1 and the protein NONO. NONO, which can bind both RNA and DNA, is involved in a range of molecular processes including paraspeckle formation and transcriptional regulation. In GBM, high NONO expression correlated with poor patient outcomes and the mesenchymal subtype. Interestingly, we observed that NONO, along with another essential paraspeckle component, SFPQ, had a greater interaction with tGLI1 compared to GLI1. Furthermore, co-expressing NONO with tGLI1 significantly increased clonogenic growth as well as CD44 gene transactivation using a luciferase reporter, suggesting it may play a role in tGLI1-driven stem-like properties. Furthermore, co-expression of NONO and tGLI1 increased the nuclear localization of both proteins. Together, these results show NONO may enhance tGLI1 nuclear localization, transcriptional activity, and exacerbate tGLI1-driven phenotypes. These effects of NONO could also contribute to the differential binding pattern between GLI1 and tGLI1. Citation Format: Haddie DeHart, Nolan Gregg, Richard Carpenter. Defining the mechanisms of cancer specific transcription factor, tGLI1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB200.

Chemokine Binding to Tenascin-C Influences Chemokine-Induced Immune Cell Migration.

Tenascin-C (TNC) is a complex glycoprotein of the extracellular matrix (ECM) involved in a plethora of (patho-)physiological processes, such as oncogenesis and inflammation. Since chemokines play an essential role in both disease processes, we have investigated here the binding of TNC to some of the key chemokines, namely CCL2, CCL26, CXCL8, CXCL10, and CXCL12. Thereby, a differential chemokine-TNC binding pattern was observed, with CCL26 exhibiting the highest and CCL2 the lowest affinity for TNC. Heparan sulfate (HS), another member of the ECM, proved to be a similarly high-affinity ligand of TNC, with a Kd value of 730 nM. Chemokines use glycosa-minoglycans such as HS as co-receptors to induce immune cell migration. Therefore, we assumed an influence of TNC on immune cell chemotaxis due to co-localization within the ECM. CCL26- and CCL2-induced mobilization experiments of eosinophils and monocytes, respectively, were thus performed in the presence and the absence of TNC. Pre-incubation of the immune cells with TNC resulted in a 3.5-fold increase of CCL26-induced eosinophil chemotaxis, whereas a 1.3-fold de-crease in chemotaxis was observed when monocytes were pre-incubated with CCL2. As both chemokines have similar HS binding but different TNC binding affinities, we speculate that TNC acts as an attenuator in monocyte and as an amplifier in eosinophil mobilization by impeding CCL2 from binding to HS on the one hand, and by reinforcing CCL26 to bind to HS on the other hand.

Differential affinity chromatography reveals a link between Porphyromonas gingivalis-induced changes in vascular smooth muscle cell differentiation and the type 9 secretion system.

Porphyromonas gingivalis is implicated in adverse pregnancy outcome. We previously demonstrated that intrauterine infection with various strains of P. gingivalis impairs the physiologic remodeling of the uterine spiral arteries (IRSA) during pregnancy, which underlies the major obstetrical syndromes. Women diagnosed with IRSA also have a greater risk for premature cardiovascular disease in later life. The dysregulated plasticity of vascular smooth muscle cells (VSMCs) is present in both IRSA and premature cardiovascular events. We hypothesized that VSMCs could serve as a bait to identify P. gingivalis proteins associated with dysregulated VSMC plasticity as seen in IRSA. We first confirmed that dams with P. gingivalis A7UF-induced IRSA also show perturbed aortic smooth muscle cell (AoSMC) plasticity along with the P. gingivalis colonization of the tissue. The in vitro infection of AoSMCs with IRSA-inducing strain A7UF also perturbed AoSMC plasticity that did not occur with infection by non-IRSA-inducing strain W83. Far-Western blotting with strain W83 and strain A7UF showed a differential binding pattern to the rat aorta and primary rat AoSMCs. The affinity chromatography/pull-down assay combined with mass spectrometry was used to identify P. gingivalis/AoSMC protein interactions specific to IRSA. Membrane proteins with a high binding affinity to AoSMCs were identified in the A7UF pull-down but not in the W83 pull-down, most of which were the outer membrane components of the Type 9 secretion system (T9SS) and T9SS cargo proteins. Additional T9SS cargo proteins were detected in greater abundance in the A7UF pull-down eluate compared to W83. None of the proteins enriched in the W83 eluate were T9SS components nor T9SS cargo proteins despite their presence in the prey preparations used in the pull-down assay. In summary, differential affinity chromatography established that the components of IRSA-inducing P. gingivalis T9SS as well as its cargo directly interact with AoSMCs, which may play a role in the infection-induced dysregulation of VSMC plasticity. The possibility that the T9SS is involved in the microbial manipulation of host cell events important for cell differentiation and tissue remodeling would constitute a new virulence function for this system.

Comparative Analysis of Genome-Scale Gene Regulatory Networks in Human Hematopoietic Stem and Myeloid Progenitor Fractions

Background: Dynamic remodelling of gene regulatory networks (GRN) guides cell state changes during differentiation to establish and maintain cell identity and gene expression. We previously described combinatorial binding of 7 transcription factors (TF (heptad: FLI1, ERG, GATA2, RUNX1, TAL1, LYL1 and LMO2)) which form an autoregulatory network in human hematopoietic stem and progenitor cells (HSPC: Beck et al Blood 2013, Thoms et al Blood 2021). Here we mapped genome-wide binding of TFs and chromatin regulators and explored their roles in establishing and maintaining cell type specific GRNs across human hematopoietic stem cell (HSC) differentiation. Methods: We flow sorted cryopreserved CD34+ LIN- cells collected by apheresis into HSCs (CD38loCD45RA-), common myeloid progenitors (CMP: CD38hiCD45RA-CD123+), granulocyte monocyte progenitors (GMP: CD38hiCD45RA+CD123+) and megakaryocyte erythroid progenitors (MEP: CD38hiCD45RA-CD123-). Active regulatory regions (Fig A) were identified by a) chromatin immunoprecipitation (ChIP)/ChIPmentation for genome wide identification of active (H3K27ac, H3K4me3) and inactive (H3K27me3) histone marks, and TF and co-factor (heptad, PU.1, STAG2, CTCF) binding, b) HiC to broadly classify high order 3D genome structures and c) H3K27ac HiChIP at 5kb. Results: Combinatorial binding of heptad TFs in all cell types was confirmed, with the most overrepresented pattern being co-binding of all 7 factors. However, certain multifactor combinations were identified in restricted cell types or associated with lineage specific gene promoters. For example, the master erythroid TF GATA1 showed combinatorial binding in CMPs and MEPs, while the myeloid TF MPO showed combinatorial binding in CMPs and GMPs. Chromatin looping was highly variable across the four cell types, although high order genome architecture was conserved. We first focussed on HiChIP interactions involving promoters of the heptad genes and observed chromatin looping patterns consistent with an interconnected circuit. Promoter looping patterns were dynamic across cell types, and we found multiple distal regions, each with specific patterns of TF binding, connected to each heptad promoter (Fig B). Many of the distal regions were previously undescribed and may constitute novel regulators of heptad genes. To further understand the role of heptad TFs in regulating cell type specific gene expression, we used our HiChIP data to link promoters to candidate distal regulatory regions and focussed on promoter-regulator pairs with differential heptad binding across the cell types. In general, differential binding was accompanied by differential gene expression. Genes with high heptad binding in MEPs were enriched for pathways related to erythroid cells (e.g., ferroptosis signalling) while genes with high heptad binding in GMPs were enriched for pathways related to myeloid cells (e.g., TREM1 signalling). We then focussed on promoter-regulator pairs corresponding to genes expressed in mature erythroid and myeloid cells and observed cell specific patterns of TF binding. At erythroid genes we saw increasing GATA2/TAL1/LYL1/LMO2 binding and decreasing ERG binding along the HSC to MEP trajectory, while at myeloid genes we saw high GATA2/LYL1/LMO2 binding and low TAL1 binding along the HSC to GMP trajectory. To conduct an unbiased genome wide analysis of HSPC GRNs we used our data to annotate and cluster the 85,100 accessible chromatin regions present in HSPCs (Corces et al Nature Genetics 2016). Accessible regions clustered broadly as promoters, regulatory regions, or CTCF-bound. Regions with high heptad binding in either MEPs or GMPs were located in different subclusters, suggesting dynamic reorganisation of heptad TFs across the genome as HSCs proceed along differentiation trajectories. Taken together our data strongly supports a mechanistic link between differential heptad TF binding and cell type specific expression patterns and provides insight into how expression of multiple genes is coordinated during cell state transitions. Conclusion: This study provides a comprehensive characterisation of the gene regulatory landscape in rare subpopulations of human HSPCs and will be a key resource for understanding adult hematopoiesis. Our work provides a framework for analysing aberrant regulatory networks in leukemic cells and leveraging these to devise novel therapeutic strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cross-reactive neutralizing human monoclonal antibodies mapping to variable antigenic sites on the norovirus major capsid protein.

Human noroviruses are the major viral cause of acute gastroenteritis around the world. Although norovirus symptoms are in most cases mild and self-limited, severe and prolonged symptoms can occur in the elderly and in immunocompromised individuals. Thus, there is a great need for the development of specific therapeutics that can help mitigate infection. In this study, we sought to characterize a panel of human monoclonal antibodies (mAbs; NORO-123, -115, -273A, -263, -315B, and -250B) that showed carbohydrate blocking activity against the current pandemic variant, GII.4 Sydney 2012. All antibodies tested showed potent neutralization against GII.4 Sydney virus in human intestinal enteroid culture. While all mAbs recognized only GII.4 viruses, they exhibited differential binding patterns against a panel of virus-like particles (VLPs) representing major and minor GII.4 variants spanning twenty-five years. Using mutant VLPs, we mapped five of the mAbs to variable antigenic sites A (NORO-123, -263, -315B, and -250B) or C (NORO-115) on the major capsid protein. Those mapping to the antigenic site A showed blocking activity against multiple variants dating back to 1987, with one mAb (NORO-123) showing reactivity to all variants tested. NORO-115, which maps to antigenic site C, showed reactivity against multiple variants due to the low susceptibility for mutations presented by naturally-occurring variants at the proposed binding site. Notably, we show that cross-blocking and neutralizing antibodies can be elicited against variable antigenic sites. These data provide new insights into norovirus immunity and suggest potential for the development of cross-protective vaccines and therapeutics.

Multi-omics analyses of MEN1 missense mutations identify disruption of menin–MLL and menin–JunD interactions as critical requirements for molecular pathogenicity

BackgroundLoss-of-function mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are causal to the MEN1 tumor syndrome, but they are also commonly found in sporadic pancreatic neuroendocrine tumors and other types of cancers. The MEN1 gene product, menin, is involved in transcriptional and chromatin regulation, most prominently as an integral component of KMT2A/MLL1 and KMT2B/MLL2 containing COMPASS-like histone H3K4 methyltransferase complexes. In a mutually exclusive fashion, menin also interacts with the JunD subunit of the AP-1 and ATF/CREB transcription factors.ResultsHere, we applied and in silico screening approach for 253 disease-related MEN1 missense mutations in order to select a set of nine menin mutations in surface-exposed residues. The protein interactomes of these mutants were assessed by quantitative mass spectrometry, which indicated that seven of the nine mutants disrupt interactions with both MLL1/MLL2 and JunD complexes. Interestingly, we identified three missense mutations, R52G, E255K and E359K, which predominantly reduce the MLL1 and MLL2 interactions when compared with JunD. This observation was supported by a pronounced loss of binding of the R52G, E255K and E359K mutant proteins at unique MLL1 genomic binding sites with less effect on unique JunD sites.ConclusionsOur results underline the effects of MEN1 gene mutations in both familial and sporadic tumors of endocrine origin on the interactions of menin with the MLL1 and MLL2 histone H3K4 methyltransferase complexes and with JunD-containing transcription factors. Menin binding pocket mutants R52G, E255K and E359K have differential effects on MLL1/MLL2 and JunD interactions, which translate into differential genomic binding patterns. Our findings encourage future studies addressing the pathophysiological relevance of the separate MLL1/MLL2- and JunD-dependent functions of menin mutants in MEN1 disease model systems.

P989: DYNAMIC CHANGES IN THE HISTONE H3 LYSINE 27 TRIMETHYLATION EPIGENETIC LANDSCAPE FOLLOWING RUXOLITINIB ADMINISTRATION

Background: Dysregulation of normal epigenetic processes is increasingly recognised as a feature of myeloid malignancy generally and implicated in the pathogenesis of MPN. Epigenetic changes may directly result from the mutation of key epigenetic regulators including TET2, ASXL1 and EZH2 or indirectly from the activation of intracellular signalling cascades. We have previously observed that ruxolitinib, a JAK1/2 inhibitor, may alter the epigenetic landscape of histone modifications in MPN. CUT&RUN is a novel experimental approach providing high resolution profiling of epigenetic modifications and transcription factor binding. Aims: To comprehensively profile the H3K27me3 epigenetic landscape of JAK2 V617F positive cells resulting from JAK inhibition and determine the dynamic changes occurring between short term and persistent ruxolitinib therapy to identify potential genetic contributors to pathogenesis and options for future therapeutics. Methods:JAK2 V617F positive UKE1 cells were cultured in duplicate in the presence of 1000nM ruxolitinib or DMSO 0.1% vehicle control continuously for approximately five weeks. Samples were harvested at 37 days for CUT&RUN analysis using H3K27me3, EZH2 and IgG (negative control) antibodies. CUT&RUN performed using Epicypher CUTANA Kit according to manufacturer instructions. Libraries prepared using NEBNext® Ultra™ II DNA Library Prep kit with 50 base pair, paired end sequencing undertaken on Illumina NovaSeq 6000. Resulting FASTQ files were trimmed using Trim Galore! and aligned to reference genome hg38 using Bowtie2. Duplicate reads were removed using SAMtools rmdup and peaks called with MACS2 callpeak on the usegalaxy.eu platform. Differential peak analysis undertaken with Diffbind package in R. Samples were normalised to library size prior to differential analysis with DESeq2 methodology. Bigwig files generated using deepTools bamcoverage for visualisation. Results: UKE1 cells remain sensitive to ruxolitinib after a period of treatment with high dose ruxolitinib lasting over 5 weeks. CUT&RUN libraries were sequenced to a minimum depth of 3.6 million unique reads per sample. Initial comparisons between Day 37 high dose treated cells and vehicle control revealed 1004 differentially enriched H3K27me3 peaks. Of these, 545 (54.2%) peaks were increased in the ruxolitinib treated cells and 459 (45.7%) peaks were increased in the control cells. Analysis of the difference between day 37 and day 1 high dose ruxolitinib treated cells revealed 290 peaks differentially enriched. Of these peaks, 279 (96.2%) were increased and 11 (3.8%) were decreased at day 37. Analysis of the overlap between these results identified 33 peaks increased at D37 in comparison to DMSO control and day 1 treatment samples. Analysis of the differential binding patterns and direct visualisation of the results in a genome browser revealed particular genetic locations of interest including the promoter region of SPRY1, a potential regulator of erythropoiesis and JAK signalling, UGCG and BTBD11. Figure 1 shows an example. Differential H3K27me3 was also noted at regions associated with PLXNA1 and NRP2 genes which act as co-receptors for class III semaphorin signalling. Image:Summary/Conclusion: The dynamic changes in the H3K27me3 epigenetic landscape of JAK2 V617F positive cells following persistent high dose ruxolitinib administration has potential therapeutic implications and may sensitise cells to epigenetic therapies. Specific gene loci identified demonstrating differential H3K27me3 may offer some insight into JAK2 V617F mediated pathology and require further investigation.

The Role of Polyphenols in the Treatment of Alzheimer's Disease: Narrative Review

Many epidemiological studies have suggested that consuming a diet rich in polyphenols can help prevent Alzheimer's disease (AD). Based on well-known in vitro and in vivo models of cerebral Aβ amyloidosis, we examined the data on the effects of various natural polyphenols on the aggregation of amyloid-protein (Aβ). These polyphenols effectively prevent oligomerization and fibril formation of Aβ through differential binding patterns, lowering Aβ oligomer-induced synaptic and neuronal toxicity, according to in vitro investigations. Furthermore, in a transgenic mouse model fed orally with such polyphenolic compounds, soluble Aβ oligomers as well as insoluble Aβ deposits in the brain were significantly reduced. Natural polyphenols exhibit anti-amyloidogenic effects on Aβ, in addition to well-known anti-oxidative and anti-inflammatory activities, according to an updated assessment of the literature, implying their potential as therapeutic and/or preventive agents for AD treatment. To prove polyphenols' efficacy as disease-modifying agents, well-designed clinical trials or preventive treatments using various polyphenols are required.

Evolution of Thyroglobulin Loop Kinetics in EpCAM

Epithelial cell-activating molecule (EpCAM) is an important cancer biomarker and therapeutic target given its elevated expression in epithelial cancers. EpCAM is a type I transmembrane protein that forms cis-dimers along the thyroglobulin type-1A-like domain (TYD) in the extracellular region. The thyroglobulin loop (TY loop) within the TYD is structurally dynamic in the monomer state of human EpCAM, binding reversibly to a TYD site. However, it is not known if this flexibility is prevalent across different species. Here, we conduct over 17 μs of all-atom molecular dynamics simulations to study EpCAM TY loop kinetics of five different species, including human, mouse, chicken, frog, and fish. We find that the TY loop remains dynamic across evolution. In addition to the TYD binding site, we discover a second binding site for the TY loop in the C-terminal domain (CTD). Calculations of the dissociation rate constants from the simulation trajectories suggest a differential binding pattern of fish EpCAM and other organisms. Whereas fish TY loop has comparable binding for both TYD and CTD sites, the TY loops of other species preferably bind the TYD site. A hybrid construct of fish EpCAM with human TY loop restores the TYD binding preference, suggesting robust effects of the TY loop sequence on its dynamic behavior. Our findings provide insights into the structural dynamics of EpCAM and its implication in physiological functions.

Occupational Allergic Sensitization Among Workers Processing King Crab (Paralithodes camtschaticus) and Edible Crab (Cancer pagurus) in Norway and Identification of Novel Putative Allergenic Proteins.

Introduction: Asthma and allergy occur frequently among seafood processing workers, with the highest prevalence seen in the crustacean processing industry. In this study we established for the first time the prevalence of allergic sensitization in the Norwegian king- and edible crab processing industry and characterized the IgE-reactive proteins.Materials and Methods: Two populations of crab processing workers participated; 119 king crab and 65 edible crab workers. The investigation included information on work tasks and health through a detailed questionnaire. Allergic sensitization was investigated by crab-specific IgE quantification and skin prick tests (SPT) to four in-house prepared crab extracts; raw meat, cooked meat, raw intestines and raw shell. Allergen-specific IgE binding patterns were analyzed by IgE immunoblotting to the four allergen extracts using worker serum samples. Total proteins in crab SPT extracts and immunoblot-based IgE binding proteins were identified by mass spectrometric analysis.Results: Positive SPTs were established in 17.5% of king- and 18.1% of edible crab workers, while elevated IgE to crab were demonstrated in 8.9% of king- and 12.2% of edible crab processing workers. There was no significant difference between the king and edible crab workers with respect to self-reported respiratory symptoms, elevated specific IgE to crab or SPT results. Individual workers exhibited differential IgE binding patterns to different crab extracts, with most frequent binding to tropomyosin and arginine kinase and two novel IgE binding proteins, hemocyanin and enolase, identified as king- and edible crab allergens.Conclusions: Occupational exposure to king- and edible crabs may frequently cause IgE mediated allergic sensitization. Future investigations addressing the diagnostic value of crab allergens including tropomyosin and arginine kinase and the less well-known IgE-binding proteins hemocyanin and enolase in a component-resolved diagnostic approach to crab allergy should be encouraged.

Differential Binding of Autoantibodies to MOG Isoforms in Inflammatory Demyelinating Diseases.

ObjectiveTo analyze serum immunoglobulin G (IgG) antibodies to major isoforms of myelin oligodendrocyte glycoprotein (MOG-alpha 1-3 and beta 1-3) in patients with inflammatory demyelinating diseases.MethodsRetrospective case-control study using 378 serum samples from patients with multiple sclerosis (MS), patients with non-MS demyelinating disease, and healthy controls with MOG alpha-1-IgG positive (n = 202) or negative serostatus (n = 176). Samples were analyzed for their reactivity to human, mouse, and rat MOG isoforms with and without mutations in the extracellular MOG Ig domain (MOG-ecIgD), soluble MOG-ecIgD, and myelin from multiple species using live cell-based, tissue immunofluorescence assays and ELISA.ResultsThe strongest IgG reactivities were directed against the longest MOG isoforms alpha-1 (the currently used standard test for MOG-IgG) and beta-1, whereas the other isoforms were less frequently recognized. Using principal component analysis, we identified 3 different binding patterns associated with non-MS disease: (1) isolated reactivity to MOG-alpha-1/beta-1 (n = 73), (2) binding to MOG-alpha-1/beta-1 and at least one other alpha, but no beta isoform (n = 64), and (3) reactivity to all 6 MOG isoforms (n = 65). The remaining samples were negative (n = 176) for MOG-IgG. These MOG isoform binding patterns were associated with a non-MS demyelinating disease, but there were no differences in clinical phenotypes or disease course. The 3 MOG isoform patterns had distinct immunologic characteristics such as differential binding to soluble MOG-ecIgD, sensitivity to MOG mutations, and binding to human MOG in ELISA.ConclusionsThe novel finding of differential MOG isoform binding patterns could inform future studies on the refinement of MOG-IgG assays and the pathophysiologic role of MOG-IgG.

Generation of single stranded DNA with selective affinity to bovine spermatozoa.

ObjectiveThis study was conducted to generate single stranded DNA oligonucleotides with selective affinity to bovine spermatozoa, assess its binding potential and explore its potential utility in trapping spermatozoa from suspensions.MethodsA combinatorial library of 94 mer long oligonucleotide was used for systematic evolution of ligands by exponential enrichment (SELEX) with bovine spermatozoa. The amplicons from sixth and seventh rounds of SELEX were sequenced, and the reads were clustered employing cluster database at high identity with tolerance (CD-HIT) and FASTAptamer. The enriched nucleotides were predicted for secondary structures by Mfold, motifs by Multiple Em for Motif Elicitation and 5′ labelled with biotin/6-FAM to determine the binding potential and binding pattern.ResultsWe generated 14.1 and 17.7 million reads from sixth and seventh rounds of SELEX respectively to bovine spermatozoa. The CD-HIT clustered 78,098 and 21,196 reads in the top ten clusters and FASTAptamer identified 2,195 and 4,405 unique sequences in the top three clusters from the sixth and seventh rounds, respectively. The identified oligonucleotides formed secondary structures with delta G values between −1.17 to −26.18 kcal/mol indicating varied stability. Confocal imaging with the oligonucleotides from the seventh round revealed different patterns of binding to bovine spermatozoa (fluorescence of the whole head, spot of fluorescence in head and mid- piece and tail). Use of a 5′-biotin tagged oligonucleotide from the sixth round at 100 pmol with 4×106 spermatozoa could trap almost 80% from the suspension.ConclusionThe binding patterns and ability of the identified oligonucleotides confirms successful optimization of the SELEX process and generation of aptamers to bovine spermatozoa. These oligonucleotides provide a quick approach for selective capture of spermatozoa from complex samples. Future SELEX rounds with X- or Y- enriched sperm suspension will be used to generate oligonucleotides that bind to spermatozoa of a specific sex type.

The Impact of High-mobility Group Box Mutation of T-cell Factor 4 on Its Genomic Binding Pattern in Non–small Cell Lung Cancer

T-cell factor 4 (TCF-4) is determined to play a crucial role in Wnt/β-catenin signaling pathway activation. The mutations and alternative splice isoforms of TCF-4 can cause cancers and other diseases. The high-mobility group (HMG) box domain of TCF-4 contributes to interacting with DNA motif for transcriptional regulation. However, the impact of the mutations within HMG box of TCF-4 on the genomic binding pattern is poorly investigated. Herein, we generated non–small cell lung cancer (NSCLC) cell line A549 with stably overexpressed TCF-4 with HMG box hot spot mutation (10th exon partial deletion), and conducted TCF-4 and β-catenin chromatin immunoprecipitation sequence to explore the differential genomic binding patterns. Our results revealed that TCF-4 lost 19365 but gained 1724 peaks, and β-catenin lost 4035 but gained 5287 peaks upon mutant TCF-4 compared with the wild type (log2FC > 1 or < -1, FDR<0.01). The transcriptional levels of the genes associated with these differential peaks such as H3F3C, KRT1, KRT14, MMp1, and MMP15 were all found to strongly change responding to TCF-4 binding (P < 0.05). Furthermore, A549 cells with TCF-4 mutation displayed a more compromising tumor characterization on cell proliferation and invasion. Our data determined the important role of TCF-4 in gene transcription controlling and provided the gain function evidence of TCF-4 caused by the TCF-4 mutation in NSCLC.

Differential binding patterns of anti-sulfatide antibodies to glial membranes

Sulfatide is a major glycosphingolipid in myelin and a target for autoantibodies in autoimmune neuropathies. However neuropathy disease models have not been widely established, in part because currently available monoclonal antibodies to sulfatide may not represent the diversity of anti-sulfatide antibody binding patterns found in neuropathy patients. We sought to address this issue by generating and characterising a panel of new anti-sulfatide monoclonal antibodies. These antibodies have sulfatide reactivity distinct from existing antibodies in assays and in binding to peripheral nerve tissues and can be used to provide insights into the pathophysiological roles of anti-sulfatide antibodies in demyelinating neuropathies.

Gut immunoglobulin alpha anti-glycan binding profiles as a research tool for local disease detection.

A promising approach capitalizing on the specific and highly sensitive characteristics of the body's own immune system is demonstrated in the context of revealing pancreatic ductal adenocarcinoma cancer (PDAC). IgA from a local biofluid called gastrointestinal lavage fluid (GLF) is used to investigate glycan reactivity to show the potential of this approach. IgA antibody responses, just as with IgG, result in amplification of a small signal which aids in detecting changes from a healthy state. IgA from GLF was screened against glycan arrays containing 609 glycan structures to investigate differential binding patterns associated with the disease. Samples included PDAC (n = 14) and non-PDAC (n = 6). Non-PDAC conditions included samples from healthy patients and the potentially confounding conditions of colon cancer and its precancerous lesion, colon adenoma. Results demonstrated characteristic reactivity in the PDAC sample group to a glycan structure. Also, IgA non-reactive motifs arose showing remarkable consistency within and between sample groups. While sample sizes are too small to identify putative biomarkers, these data show the use of IgA from GLF to be a promising avenue of research for local disease biomarker discovery.

Molecular modeling and docking of recombinant HAP-phytase of a thermophilic mould Sporotrichum thermophile reveals insights into molecular catalysis and biochemical properties

A thermostable and protease-resistant HAP-phytase of Sporotrichum thermophile was over-expressed in Pichia pastoris X-33. Purified recombinant phytase displayed all its biochemical properties similar to wild type. Molecular modeling and docking of phytase with various substrates showed differential binding patterns with GoldScore values ranging from 40.61 to 79.78. Docking with different substrates revealed strong binding affinity with ATP and phytic acid, while the lowest with AMP and phosphoenol pyruvate. This was further confirmed using biochemical assays, as the recombinant enzyme displayed broad substrate specificity. Docking with inhibitors also showed differential binding with GoldScore values ranging from 22.94 (2,3-butanedione) to 85.72 (myo-inositol hexasulphate). Validation using biochemical analysis revealed that both 2,3-butanedione and phenyl glyoxal inhibited the phytase activity significantly. Furthermore, presence of inorganic phosphate in the reaction mixture also inhibited the phytase activity, as there was no activity at and beyond 0.8 mM. Docking of phytase with metavanadate showed binding at the same atom in the active-site where the substrate i.e. phytic acid binds. Vanadium incorporation resulted in the catalytic conversion of phytase into a peroxidase with concomitant inhibition of phytase activity. Peroxidase activity was high in acidic range and the product formation showed correlation with reaction time. Furthermore, molecular modeling and docking of recombinant HAP-phytase of a thermophilic mould S. thermophile reveals insights into molecular catalysis that is validated by the biochemical properties.

A time-series analysis of altered histone H3 acetylation and gene expression during the course of MMAIII-induced malignant transformation of urinary bladder cells.

Our previous studies have shown that chronic exposure to low doses of monomethylarsonous acid (MMAIII) causes global histone acetylation dysregulation in urothelial cells (UROtsa cells) during the course of malignant transformation. To reveal the relationship between altered histone acetylation patterns and aberrant gene expression, more specifically, the carcinogenic relevance of these alterations, we performed a time-course analysis of the binding patterns of histone 3 lysine 18 acetylation (H3K18ac) across the genome and generated global gene-expression profiles from this UROtsa cell malignant transformation model. We showed that H3K18ac, one of the most significantly upregulated histone acetylation sites following MMAIII exposure, was enriched at gene promoter-specific regions across the genome and that MMAIII-induced upregulation of H3K18ac led to an altered binding pattern in a large number of genes that was most significant during the critical window for MMAIII-induced UROtsa cells' malignant transformation. Some genes identified as having a differential binding pattern with H3K18ac, acted as upstream regulators of critical gene networks with known functions in tumor development and progression. The altered H3K18ac binding patterns not only led to changes in expression of these directly affected upstream regulators but also resulted in gene-expression changes in their regulated networks. Collectively, our data suggest that MMAIII-induced alteration of histone acetylation patterns in UROtsa cells led to a time- and malignant stage-dependent aberrant gene-expression pattern, and that some gene regulatory networks were altered in accordance with their roles in carcinogenesis, probably contributing to MMAIII-induced urothelial cell malignant transformation and carcinogenesis.

C0 Domain of Cardiac Myosin Binding Protein-C Modulates Interaction of the Neighboring C1 Domain with Tropomyosin Through the Allosteric Interaction with F-Actin

Mutations in cardiac myosin binding protein C (cMyBP-C) are the most common cause of hypertrophic cardiomyopathy affecting millions of people worldwide. Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown. Recently, we demonstrated that despite being structural homologs, C0 and C1 NTDs exhibit different patterns of binding on the surface of F-actin. Moreover, we showed that C1 but not C0 can activate the TF by means of shifting the tropomyosin (Tm) cable from the “closed” to the “open” structural state via direct interaction with Tm. Here we used cryo electron microscopy and image analysis to reveal how C0 and C1 Ig-domains connected by the Proline/Alanine-linker communicate with each other when interacting with the TF in tandem. Our results show that C0 binds to the front of actin subdomain-1 in two distinct modes, and that in these two modes C0 has different effect on the interaction of C1 domain with both actin filament and Tm cable. In one mode C0 enhances the activating effect of C1 domain so that the Tm cable is shifted towards the subdomain-4 of actin to an even larger extent than found in the “open” state of Tm. In the second mode C0 reverses the activation effect of C1 by means of disrupting the interaction of C1 with Tm. Importantly, we demonstrate that the two domains communicate with each other allosterically through the actin filament. Our data suggest that the internal dynamics of the actin molecule is essential for the regulation of the actomyosin interaction in cardiac muscle. We propose a mechanism by which cMyBP-C may modulate actomyosin interactions in cardiac muscle.

Characterization of the Essential Residues of Cyclooxygenase-1 and −2 Responsible for their Inter-Subunit Communications Upon their Binding to the Corresponding Substrates and Inhibitors

Various conformational changes of a protein upon interaction with its endogenous partner molecules or artificial synthetic compounds imply often an essential role structurally or functionally. Induction triggered by its binding to small ligands can contribute a long-range communication between intra- or inter-subunits, which has been exemplified in several model studies. Among these, cyclooxygenases (COX-1 and −2), also known as prostaglandin endoperoxide synthases, display a differential binding pattern between two sequentially identical monomers. COXs catalyze the first committed step in the conversion of arachidonic acid into prostaglandins and thromboxanes. Potential drug-like compounds against COXs have been enormously developed and characterized up to date, which are exploited in this study with an aim to find core regions or residues responsible for their inter-subunit and domain-domain communications. In addition to a careful examination of the COX crystallographic data, extensive analysis of docking experiments with categorized NSAIDs and prediction of hot-spot(s) for protein-protein interaction were performed, which suggest the theoretical basis for the functionally heterodimeric nature and half-of-the-sites behavior of COXs.