Indirect Binding Research Articles

Comprehensive clinical evaluation of indirect and direct bonding techniques in orthodontic treatment: a single-centre, open-label, quasi-randomized controlled clinical trial.

Few prospective investigations have compared direct and indirect techniques through comprehensive and detailed clinical evaluations, considering the impact of all factors. This study aimed to compare and evaluate direct and indirect bonding methods at a single institution and to clarify the selection criteria for the bonding method. This single-centre, quasi-randomized controlled clinical trial included 153 patients who required fixed orthodontic treatment. They were randomly divided into indirect and direct binding groups by the project lead (K.K.), who was blinded to all clinical data, and performed the allocation using medical record numbers. The chair time for bracket bonding, discomfort during bracket bonding, oral hygiene after bonding, number of bracket failures, number of intentional bracket reattachments, post-treatment occlusal index, and total treatment time were assessed. Outcomes were compared using a two-sample t-test or Mann-Whitney U test (P < .05). Fifty-eight patients were included in the indirect bonding group (20 male, 38 female; mean age: 20.63 ± 5.69 years) and 66 (14 male, 52 female; mean age: 23.17 ± 8.83 years) in the direct bonding group. Compared to the direct bonding group, the indirect bonding group had shorter chair time (P < .001), a shorter total treatment period (P < .01), and a better final occlusal relationship (P < .001). The number of bracket detachments was higher (P < .001) in the indirect bonding group, but the number of intentional reattachments was lower (P < .001). Indirect bonding may improve the efficiency of orthodontic treatment. No harm was observed during the study. This trial was approved by the Ethics Review Committee of Okayama University (approval number: d10001), UMIN registration number 000022182.

Mycoplasma synoviae surface-located elongation factor G functions as a cytoadhesin to promote adhesion to synovial sheath cells through binding to vimentin

Mycoplasma synoviae infection has caused serious economic losses to the poultry industry worldwide. The molecular mechanism by which M. synoviae colonizes the synovium and induces synovitis is unclear. In this study, desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry analyses were used to screen M. synoviae membrane proteins that bind the membrane proteins of synovial sheath cells (SSCs). Among the 128 screened proteins, elongation factor G (EF-G) of M. synoviae was identified as a surface-located protein using colony blotting and dual fluorescence analyses. The immunogenicity of EF-G was confirmed by the preparation of a rabbit polyclonal antibody. EF-G was identified as a cytoadhesin that directly binds to SSCs using indirect immunofluorescence assay and ELISA plate binding assay. In addition, antibody adhesion inhibition and protein adhesion inhibition demonstrated that EF-G could significantly promote the adhesion of M. synoviae to SSCs. Co-IP, GST pull-down, bacterial two-hybridization, and ELISA plate binding assays were performed to demonstrate the binding of EF-G and vimentin in vivo and in vitro. Antibody adhesion inhibition, protein adhesion inhibition, and siRNA interference adhesion inhibition assays demonstrated that vimentin significantly affected M. synoviae adhesion to SSCs. These studies indicate that two interacting proteins, EF-G, a novel cytoadhesin, and vimentin, an important cell surface receptor, play important roles in the adhesion of M. synoviae to SSCs, laying a foundation for subsequent studies on the mechanism of M. synoviae-induced synovitis and providing meaningful targets for screening target drugs against M. synoviae infection.

7615 Genomic Consequences of GRHL2 Overexpression in ER+ Breast Cancer Cells

Abstract Disclosure: K.O. Allen: None. C. Zheng: None. N. Solodin: None. M.S. Ozers: Owner/Co-Owner; Self; Proteovista, LLC. C.L. Warren: None. P.E. Messa: None. D.T. Vogt: None. E.T. Alarid: None. Breast cancer progression is characterized by the presence and prevalence of metastatic disease. At a genomic level, metastasis can be facilitated via rearrangement of the chromatin landscape to expose oncogenic genes for transcription. Grainyhead like protein 2 (GRHL2) is a nuclear transcription factor implicated in epithelial cell differentiation. The GRHL2 motif has been found near activated estrogen receptor (ER) binding sites, and elevated levels of GRHL2 have been linked to worse prognosis in hormone receptor positive breast cancer patients. In its capacity to contribute to a more severe disease state when overexpressed, GRHL2 may function as either a transcription or pioneer factor to aid in the expression of metastasis-associated genes. Our lab previously showed that GRHL2 genomic binding precedes ER binding at co-regulated binding sites. Also, overexpression of GRHL2 in MCF7 cells for 24 hours did not appreciably alter gene expression by RNA-Seq. These data taken together indicate that GRHL2 may be exhibiting pioneer factor activity. However, the specific role of GRHL2 overexpression in hormone positive breast cancer progression remains unknown. In order to examine the mechanism of GRHL2 action, GRHL2 genomic binding was investigated using a tetracycline-inducible MCF7 model that allows controlled overexpression of GRHL2. ChIP studies were performed to determine GRHL2 binding intensity at specific and genome wide GRHL2 binding sites. Specific focus was given to established GRHL2 binding sites near oncogenic genes responsible for epithelial to mesenchymal transition (EMT), dormancy, and proto-oncogenic behavior. A similar analysis examined H3K27 acetylation to validate active gene transcription. An orthogonal technology, Specificity and Affinity for Protein (SNAP) microarrays, which characterizes direct GRHL2 binding to tiled genomic regions identified in ChIP-Seq analyses from multiple labs, was utilized to discriminate direct and indirect GRHL2 binding sites. The requirement for GRHL2 transactivation function in regulation of EMT related genes was further interrogated using transactivation GRHL2 dominant negative mutants. This work aims to distinguish the transcriptional and non-transcriptional activities of GRHL2 in breast cancer cells on a genome wide level and thereby, provide a deeper understanding of how overexpressed GRHL2 contributes to hormone positive breast cancer. Presentation: 6/3/2024

Ion specificity on cysteine tripeptides in aqueous environments revealed by molecular simulation

Molecular dynamics simulations were conducted to investigate the interactions of cysteine tripeptides with ions and water molecules in solutions of salts (MA, M = Li+ and Cs+, A = halide ions). The results revealed that as the anion size increases, cations aggregate more easily around the tripeptide, irrespective of capping. Radial distribution function (RDF) analysis showed direct interactions between F− and tricysteine, whereas Cl−, Br−, and I− exhibited indirect binding. Comparison between Li+ and Cs+ systems indicated that the smaller Li+ ions tend to distribute more around the tripeptide, engaging in direct interactions, whereas Cs+ ions interact indirectly, mediated by water molecules. Interaction energy analysis demonstrated that in lithium salt solutions, the total energy of anions with tricysteine decreased with anion size for both capped and uncapped systems. In cesium salt solutions, the anionic I− had the strongest interaction with cysteine tripeptides, and the cationic Cs+ interaction with tripeptides peaked in the CsI solution. The results highlight differences in ionic interactions depending on the anion–cation correlations of different salts.

Specific detection of crustacean allergens in food: Development of indirect competitive and sandwich ELISA targeting sarcoplasmic calcium binding protein

To overcome the problem of cross-reactivity between crustaceans, mollusks, insects, and mites in current crustacean allergen immunodetection, the significant stable crustacean allergen, sarcoplasmic calcium binding protein (SCP) was innovatively selected as the target of ELISA detection. An indirect competitive ELISA (icELISA) and a sandwich ELISA (sELISA) were constructed and evaluated with spiked model foods. The sELISA (LOD 0.001 mg/kg and LOQ 0.003 mg/kg) is more sensitive than icELISA (LOD 0.11 mg/kg and LOQ 0.56 mg/kg). But the icELISA presented a wider standard range (10–10000 ng/mL) and greater recoveries of SCP in matrices (84.04%–118.94%). Sufficient precision (repeatability and reproducibility <15%), excellent specificity distinguishing crustaceans from others, and adequate detectability in diverse commercial processed foods were observed from the SCP-based icELISA. These results demonstrated the feasibility and applicability of SCP-targeted icELISA and provided a new direction to improve the capacity for crustacean allergen detection.

Hydrogels for Nucleic Acid Drugs Delivery.

Nucleic acid drugs are one of the hot spots in the field of biomedicine in recent years, and play a crucial role in the treatment of many diseases. However, its low stability and difficulty in target drug delivery are the bottlenecks restricting its application. Hydrogels are proven to be promising for improving the stability of nucleic acid drugs, reducing the adverse effects of rapid degradation, sudden release, and unnecessary diffusion of nucleic acid drugs. In this review, the strategies of loading nucleic acid drugs in hydrogels are summarized for various biomedical research, and classify the mechanism principles of these strategies, including electrostatic binding, hydrogen bond based binding, hydrophobic binding, covalent bond based binding and indirect binding using various carriers. In addition, this review also describes the release strategies of nucleic acid drugs, including photostimulation-based release, enzyme-responsive release, pH-responsive release, and temperature-responsive release. Finally, the applications and future research directions of hydrogels for delivering nucleic acid drugs in the field of medicine are discussed.

Xenoestrogen and Its Interaction with Human Genes and Cellular Proteins: An In-Silico Study.

Breast cancer represents one of the leading causes of death worldwide. Apart from genetic factors, the sex hormone estrogen plays a pivotal role in breast cancer development. We are exposed to a plethora of estrogen mimics on a daily basis via various routes. Nevertheless, how xenoestrogens, the exogenous estrogen mimics, modulate cancer-associated signaling pathways and interact with specific genes is still underexplored. Hence, this study aims to explore the direct or indirect binding partners of xenoestrogens and their expression upon exposure to these estrogenic compounds. The collection of genes linked to the xenoestrogens Octylphenol, Nonylphenol, Bisphenol-A, and 2,2-bis(4-hydroxyphenyl)-1,1,1-trichloroethane were gathered from the Comparative Toxicogenomics Database. Venny 2.1 was utilized to pinpoint the genes shared by these xenoestrogens. Subsequently, the shared genes underwent Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis using the Database for Annotation, Visualization, and Integrated Discovery bioinformatics resource. A xenoestrogen-protein interaction network was constructed using Search Tool for Interactions of Chemicals. The expressions of common genes were studied with the microarray dataset GSE5200 from the Gene Expression Omnibus database. Also, the expression of a common gene set within different breast cancer subtypes was identified using the University of California, Santa Cruz Xena. The genes linked to xenoestrogens were identified, and 13 genes were found to interact with all four xenoestrogens. Through DAVID analysis, the genes chosen are found to be enriched for various functions and pathways, including pathways in cancer, chemical carcinogenesis-receptor activation, and estrogen signaling pathways. The results of the Comparative Toxicogenomics Database and the chemical-protein interaction network derived from STITCH were similar. Microarray data analysis showed significantly high expression of all 13 genes in another study, with Bisphenol-A and Nonylphenol treated MCF-7 cells, most of the genes are expressed in luminal A or basal breast cancer subtype. In summary, the genes associated with the four xenoestrogens were mostly linked to pathways related to tumorigenesis, and the expression of these genes was found to be higher in breast cancer.

The activation of Chlamydomonas reinhardtii alpha amylase 2 by glutamine requires its N-terminal aspartate kinase-chorismate mutase-tyrA (ACT) domain.

The coordination of assimilation pathways for all the elements that make up cellular components is a vital task for every organism. Integrating the assimilation and use of carbon (C) and nitrogen (N) is of particular importance because of the high cellular abundance of these elements. Starch is one of the most important storage polymers of photosynthetic organisms, and a complex regulatory network ensures that biosynthesis and degradation of starch are coordinated with photosynthetic activity and growth. Here, we analyzed three starch metabolism enzymes of Chlamydomonas reinhardtii that we captured by a cyclic guanosine monophosphate (cGMP) affinity chromatography approach, namely, soluble starch synthase STA3, starch-branching enzyme SBE1, and α-amylase AMA2. While none of the recombinant enzymes was directly affected by the presence of cGMP or other nucleotides, suggesting an indirect binding to cGMP, AMA2 activity was stimulated in the presence of L-glutamine (Gln). This activating effect required the enzyme's N-terminal aspartate kinase-chorismate mutase-tyrA domain. Gln is the first N assimilation product and not only a central compound for the biosynthesis of N-containing molecules but also a recognized signaling molecule for the N status. Our observation suggests that AMA2 might be a means to coordinate N and C metabolism at the enzymatic level, increasing the liberation of C skeletons from starch when high Gln levels signal an abundance of assimilated N.

Transcription factor OsWRKY11 induces rice heading at low concentrations but inhibits rice heading at high concentrations.

The heading date of rice is a crucial agronomic characteristic that influences its adaptability to different regions and its productivity potential. Despite the involvement of WRKY transcription factors in various biological processes related to development, the precise mechanisms through which these transcription factors regulate the heading date in rice have not been well elucidated. The present study identified OsWRKY11 as a WRKY transcription factor which exhibits a pivotal function in the regulation of the heading date in rice through a comprehensive screening of a clustered regularly interspaced palindromic repeats (CRISPR) ‒ CRISPR-associated nuclease 9 mutant library that specifically targets the WRKY genes in rice. The heading date of oswrky11 mutant plants and OsWRKY11-overexpressing plants was delayed compared with that of the wild-type plants under short-day and long-day conditions. Mechanistic investigation revealed that OsWRKY11 exerts dual effects on transcriptional promotion and suppression through direct and indirect DNA binding, respectively. Under normal conditions, OsWRKY11 facilitates flowering by directly inducing the expression of OsMADS14 and OsMADS15. The presence of elevated levels of OsWRKY11 protein promote formation of a ternary protein complex involving OsWRKY11, Heading date 1 (Hd1), and Days to heading date 8 (DTH8), and this complex then suppresses the expression of Ehd1, which leads to a delay in the heading date. Subsequent investigation revealed that a mild drought condition resulted in a modest increase in OsWRKY11 expression, promoting heading. Conversely, under severe drought conditions, a significant upregulation of OsWRKY11 led to the suppression of Ehd1 expression, ultimately causing a delay in heading date. Our findings uncover a previously unacknowledged mechanism through which the transcription factor OsWRKY11 exerts a dual impact on the heading date by directly and indirectly binding to the promoters of target genes.

Comparison of Hedging Strategies between Vietnam and the Philippines amid the U.S.-China Strategic Competition

This paper examines and compares the hedging strategies of Vietnam and the Philippines towards both the United States and China. The study is grounded in the context of heightened geopolitical and economic rivalry, where smaller states confront the challenge of navigating the complex dynamics between these two major powers. Hedging, as a strategy to manage uncertainties and risks, is analyzed through a comparative case study approach, focusing on five key components: indirect balancing, dominance denial, economic pragmatism, binding engagement, and limited bandwagoning. The findings indicate that both Vietnam and the Philippines utilize hedging, but with differences. Vietnam's approach is more systematic and comprehensive, involving all five components, whereas the Philippines' strategy is more reactive and less planned, with a stronger tilt towards the U.S. due to shared democratic values and historical ties. The research underscores the nuanced nature of hedging and its implications for middle powers navigating great power rivalries in the Indo-Pacific region.

Protein complexes from mouse and chick brain that interact with phospho-KXGS motif tau/microtubule associated protein antibody.

Mouse monoclonal 12E8 antibody, which recognises conserved serine phosphorylated KXGS motifs in the microtubule binding domains of tau/tau-like microtubule associated proteins (MAPs), shows elevated binding in brain during normal embryonic development (mammals and birds) and at the early stages of human Alzheimer's disease (AD). It also labels ADF/cofilin-actin rods that form in neurites during exposure to stressors. We aimed to identify direct and indirect 12E8 binding proteins in postnatal mouse brain and embryonic chick brain by immunoprecipitation (IP), mass spectrometry and immunofluorescence. Tau and/or MAP2 were major direct 12E8-binding proteins detected in all IPs, and actin and/or tubulin were co-immunoprecipitated in most samples. Additional proteins were different in mouse versus chick brain IP. In mouse brain IPs, FSD1l and intermediate filament proteins - vimentin, α-internexin, neurofilament polypeptides - were prominent. Immunofluorescence and immunoblot using recombinant intermediate filament subunits, suggests an indirect interaction of these proteins with the 12E8 antibody. In chick brain IPs, subunits of eukaryotic translation initiation factor 3 (EIF3) were found, but no direct interaction between 12E8 and recombinant Eif3e protein was detected. Fluorescence microscopy in primary cultured chick neurons showed evidence of co-localisation of Eif3e and tubulin labelling, consistent with previous data demonstrating cytoskeletal organisation of the translation apparatus. Neither total tau or MAP2 immunolabelling accumulated at ADF/cofilin-actin rods generated in primary cultured chick neurons, and we were unable to narrow down the major antigen recognised by 12E8 antibody on ADF/cofilin-actin rods.

Adaptor Anti-P329G CAR T Cells for Modular Targeting of AML

Introduction Chimeric antigen receptor (CAR) T cell therapy has achieved promising results for treatment of B cell and plasma cell malignancies, but success has been limited in the treatment of acute myeloid leukemia (AML). Numerous antigens including CD33, CD123 or CSF1R have been proposed for AML therapy. However, clinical success of CAR T cell therapy in AML is impaired by antigen heterogeneity, therapy-associated toxicities, and antigen escape. Adaptor CAR T cells which allow sequential, transient or simultaneous targeting of multiple antigens have the potential to overcome these limitations. These adaptor CAR T cells were combined with tumor antigen-directed CAR-adaptor molecules for indirect tumor binding. We developed a modular P329G-directed CAR targeting the P329G mutation in the Fc part of tumor-targeting human IgG1 antibodies containing P329G L234A/L235A (PGLALA) mutations. These PGLALA mutations render the Fc part inactive for binding to Fc gamma receptors (FcgRs) and to the complement system, and up to now, more than 20 such Fc-silenced therapeutic antibodies including an approved T cell recruiting bispecific antibody have been clinically validated. Compared to other modular CAR T cell platforms this approach does not rely on haptens or artificial tags fused to the targeting antibody and may ease development and testing of the platform. Methods A scFv-based second generation CAR vector system was used in primary human T cells. P329G-targeting CAR T cells were combined with PGLALA-Fc-mutated antibodies targeting CD33, CD123 or CSF1R. Classical, directly binding anti-CD33 and anti-CSF1R CAR T cells have been used as positive controls, while anti-CD19 CAR T cells and untransduced T cells have been used as negative controls. Different AML (MOLM-13, OCI-AML-3, PL-21, Mv4-11, THP-1 and U-937) and an ALL cell line (NALM-6) were used as target cell lines expressing the target antigens in different intensity. Effector function of CAR T cells was also tested against primary human AML blasts derived from patients. Luciferase-based killing assays, ELISA-based and flow-based analysis were used to evaluate CAR effector functions. Results Unlike CD16-CAR T cells, which bind human IgG in a non-selective manner, anti-P329G CAR T cells revealed specific effector functions only when combined with antibodies carrying PGLALA-Fc-mutations. Anti-P329G CAR T cells alone revealed no activation. Anti-P329G CAR T cells could be specifically activated by recombinant protein when combined with antibodies targeting CD33, CD123 and CSF1R leading to upregulation of T cell activation markers. Anti-P329G CAR T cells combined with PGLALA-mutated antibodies targeting CD33, CD123 und CSF1R achieved efficient in vitro cytotoxicity against tested AML cell lines and primary human AML blasts, while antigen-negative ALL cell line NALM-6 was not killed. Remarkably, effector functions of anti-P329G CAR T cells were similar to classical CAR T cells particularly when targeting CD33. This led to specific cytokine production when anti-P329G CAR T cells were co-cultured with respective antibodies and tumor cells. P329G-CAR T cells activated by recombinant CD33, CD123 or CSF1R showed no cytokine production in the presence of an antibody which was not recognizing the respective antigen. However, by switching the antibody after 24 h of stimulation towards the tumor antigen-targeting antibody, CAR T cell activation could be again achieved by the recombinant protein. Depleting the tumor antigen-targeting antibody after 24 h of stimulation decreased the cytokine production after 48 h and 72 h despite ongoing presence of the recombinant protein. This confirms the modularity and reversibility of this adaptor CAR T cell platform. Conclusions Taken together, anti-P329G CAR T cells combined with Fc-silenced tumor antigen-targeting IgG1 antibodies carrying the clinically validated PGLALA-mutations in the Fc part achieved profound effector functions against various human AML cell lines and primary AML blasts. The modular platform has the potential to overcome recent limitations of CAR T cell therapy in AML. These results support the rational for clinical translation of this novel and modular CAR T cell platform.

Pharmacokinetic-Pharmacodynamic Modeling of Midazolam in Pediatric Surgery.

Midazolam (MDZ) is used for sedation in surgical procedures; its clinical effect is related to its receptor affinity and the dose administered. Therefore, a pharmacokinetic-pharmacodynamic (PK-PD) population model of MDZ in pediatric patients undergoing minor surgery is proposed. A descriptive, observational, prospective, and longitudinal, study that included patients of both sexes, aged 2-17 years, ASA I/II, who received MDZ in IV doses (0.05 mg/kg) before surgery. Three blood samples were randomly taken between 5-120 min; both sedation by the Bispectral Index Scale (BIS) and its adverse effects were recorded. The PK-PD relationship was determined using a nonlinear mixed-effects, bicompartmental first-order elimination model using Monolix Suite™. Concentrations and the BIS were fitted to the sigmoid Emax PK-PD population and sigmoid Emax PK/PD indirect binding models, obtaining drug concentrations at the effect site (biophase). The relationship of concentrations and BIS showed a clockwise hysteresis loop, probably indicating time-dependent protein binding. Of note, at half the dose used in pediatric patients, adequate sedation without adverse effects was demonstrated. Further PK-PD studies are needed to optimize dosing schedules and avoid overdosing or possible adverse effects.

The natural product, echinatin, protects mice from methicillin-resistant Staphylococcus aureus pneumonia by inhibition of alpha-hemolysin expression.

Antimicrobial resistance (AMR) is a global, multifaceted crisis that poses significant challenges to the successful eradication of devastating pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA), a persistent superbug that causes devastating infections. The scarcity of new antibacterial drugs is obvious, and antivirulence strategies that reduce the pathogenicity of bacteria by weakening their virulence have become the subject of intense investigation. Alpha-hemolysin (Hla), a cytolytic pore-forming toxin, has a pivotal role in S. aureus pathogenesis. Here, we demonstrated that echinatin, a natural compound isolated from licorice, effectively inhibited the hemolytic activity of MRSA at 32 μg/mL. In addition, echinatin did not interfere with bacterial growth and had no significant cytotoxicity at the inhibitory concentration of S. aureus hemolysis. Heptamer formation tightly correlated with Hla-mediated cell invasion, whereas echinatin did not affect deoxycholic acid-induced oligomerization of Hla. Echinatin affected hemolytic activity through indirect binding to Hla as confirmed by the neutralization assay and cellular thermal shift assay (CETSA). Furthermore, qRT-PCR and western blot analyses revealed that echinatin suppressed Hla expression at both the mRNA and protein levels as well as the transcript levels of Agr quorum-sensing system-related genes. Additionally, when echinatin was added to a coculture system of A549 cells and S. aureus, it significantly reduced cell damage. Importantly, echinatin exhibited a significant therapeutic effect in an MRSA-induced mouse pneumonia model. In conclusion, the present findings demonstrated that echinatin significantly inhibits the hemolysin effect and may be a potential candidate compound for combating drug-resistant MRSA infections.

Ibuprofen: a weak inhibitor of carbonic anhydrase II.

Carbonic anhydrases (CAs) are drug targets for a variety of diseases. While many clinically relevant CA inhibitors are sulfonamide-based, novel CA inhibitors are being developed that incorporate alternative zinc-binding groups, such as carboxylic acid moieties, to develop CA isoform-specific inhibitors. Here, the X-ray crystal structure of human CA II (hCA II) in complex with the carboxylic acid ibuprofen [2-(4-isobutylphenyl)propanoic acid, a common over-the-counter nonsteroidal anti-inflammatory drug] is reported to 1.54 Å resolution. The binding of ibuprofen is overlaid with the structures of other carboxylic acids in complex with hCA II to compare their inhibition mechanisms by direct or indirect (via a water) binding to the active-site zinc. Additionally, enzyme-inhibition assays using ibuprofen, nicotinic acid and ferulic acid were performed with hCA II to determine their IC50 values and were compared with those of other carboxylic acid binders. This study discusses the potential development of CA inhibitors utilizing the carboxylic acid moiety.

Genome-wide binding analysis of transcription factor Rice Indeterminate 1 reveals a complex network controlling rice floral transition.

RICE INDETERMINATE 1 (RID1) plays a critical role in controlling floral transition in rice (Oryza sativa). However, the molecular basis for this effect, particularly the target genes and regulatory specificity, remains largely unclear. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) in young leaves at the pre-floral-transition stage to identify the target genes of RID1, identifying 2,680 genes associated with RID1 binding sites genome-wide. RID1 binding peaks were highly enriched for TTTGTC, the direct binding motif of the INDETERMINATE DOMAIN protein family that includes RID1. Interestingly, CACGTG and GTGGGCCC, two previously uncharacterized indirect binding motifs, were enriched through the interactions of RID1 with the novel flowering-promoting proteins OsPIL12 and OsTCP11, respectively. Moreover, the ChIP-seq data demonstrated that RID1 bound to numerous rice heading-date genes, such as HEADING DATE 1 (HD1) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1). Notably, transcriptome sequencing (RNA-seq) analysis revealed roles of RID1 in diverse developmental pathways. Genetic analysis combined with genome-wide ChIP-seq and RNA-seq results showed that RID1 directly binds to the promoter of OsERF#136 (a repressor of rice flowering) and negatively regulates its expression. Overall, our findings provide new insights into the molecular and genetic mechanisms underlying rice floral transition and characterize OsERF#136 as a previously unrecognized direct target of RID1.

2,3-Dehydrokievitone combats methicillin-resistant Staphylococcus aureus infection by reducing alpha-hemolysin expression

Due to powerful drug resistance and fatal toxicity of methicillin-resistant Staphylococcus aureus (MRSA), therapeutic strategies against virulence factors present obvious advantages since no evolutionary pressure will induce bacterial resistance. Alpha-hemolysin (Hla) is an extracellular toxin secreted by Staphylococcus aureus and contributes to bacterial pathogenicity. Herein, we identified a natural product 2,3-dehydrokievitone (2,3-DHKV) for inhibiting Hla activity of MRSA strain USA300 but not affecting bacteria growth. 2,3-DHKV significantly decreased hemolysin expression in a dose-dependent manner, but it did not potently neutralize hemolysin activity. Subsequently, cellular thermal shift and heptamer formation assays confirmed that 2,3-DHK affects hemolytic activity through indirect binding to Hla. RT-qPCR and western blot revealed that 2,3-DHKV suppressed Hla expression at the mRNA and protein levels, and further decreased accessory gene regulator A (agrA) transcription levels. We also observed that 2,3-DHK significantly attenuated the damage of A549 cells by S. aureus and reduced the release of lactate dehydrogenase (LDH). Moreover, in the MRSA-induced pneumonia mouse model, 2,3-DHK treatment prolonged the life span of mice and reduced the bacterial load in the lungs, which significantly alleviated the damage to the lungs. In summary, this study proved that 2,3-DHK as a Hla inhibitor is a potential antivirulence agent against MRSA infection.

Epigenetic and transcriptional dysregulation in CD4+ T cells in patients with atopic dermatitis.

Atopic dermatitis (AD) is one of the most common skin disorders among children. Disease etiology involves genetic and environmental factors, with 29 independent AD risk loci enriched for risk allele-dependent gene expression in the skin and CD4+ T cell compartments. We investigated the potential epigenetic mechanisms responsible for the genetic susceptibility of CD4+ T cells. To understand the differences in gene regulatory activity in peripheral blood T cells in AD, we measured chromatin accessibility (an assay based on transposase-accessible chromatin sequencing, ATAC-seq), nuclear factor kappa B subunit 1 (NFKB1) binding (chromatin immunoprecipitation with sequencing, ChIP-seq), and gene expression levels (RNA-seq) in stimulated CD4+ T cells from subjects with active moderate-to-severe AD, as well as in age-matched non-allergic controls. Open chromatin regions in stimulated CD4+ T cells were highly enriched for AD genetic risk variants, with almost half of the AD risk loci overlapping AD-dependent ATAC-seq peaks. AD-specific open chromatin regions were strongly enriched for NF-κB DNA-binding motifs. ChIP-seq identified hundreds of NFKB1-occupied genomic loci that were AD- or control-specific. As expected, the AD-specific ChIP-seq peaks were strongly enriched for NF-κB DNA-binding motifs. Surprisingly, control-specific NFKB1 ChIP-seq peaks were not enriched for NFKB1 motifs, but instead contained motifs for other classes of human transcription factors, suggesting a mechanism involving altered indirect NFKB1 binding. Using DNA sequencing data, we identified 63 instances of altered genotype-dependent chromatin accessibility at 36 AD risk variant loci (30% of AD risk loci) that might lead to genotype-dependent gene expression. Based on these findings, we propose that CD4+ T cells respond to stimulation in an AD-specific manner, resulting in disease- and genotype-dependent chromatin accessibility alterations involving NFKB1 binding.

Gangliosides play important roles in the nervous system by regulating ion concentrations.

Gangliosides are important components of the neuronal cell membrane and play a vital role in the development of neurons and the brain. They participate in neurotransmission and are considered as the structural basis of learning and memory. Gangliosides participate in several and important physiological processes, such as cell differentiation, cell signaling, neuroprotection, nerve regeneration and apoptosis. The stability of ion concentration in excitable cells is particularly important in the maintenance of a steady state of cells and in the regulation of physiological functions. Ion concentration has been found to be related to the ganglioside's regulation in many neurological diseases, and several studies have found that they can stabilize intracellular ion concentration by regulating ion channels, which highlights their important regulatory role in neuronal excitability and synaptic transmission. Gangliosides can influence some forms of ion transport, by directly binding to ion transporters or through indirect binding and activation of transport proteins via appropriate signaling pathways. Therefore, the important and special role of gangliosides in the homeostasis of ion concentration is becoming a hot topic in the field and a theoretical basis in promoting help gangliosides use as key drugs for the treatment of nervous system diseases.

Thermodynamics-based modeling reveals regulatory effects of indirect transcription factor-DNA binding

SummaryTranscription factors (TFs) influence gene expression by binding to DNA, yet experimental data suggests that they also frequently bind regulatory DNA indirectly by interacting with other DNA-bound proteins. Here, we used a data modeling approach to test if such indirect binding by TFs plays a significant role in gene regulation. We first incorporated regulatory function of indirectly bound TFs into a thermodynamics-based model for predicting enhancer-driven expression from its sequence. We then fit the new model to a rich data set comprising hundreds of enhancers and their regulatory activities during mesoderm specification in Drosophila embryogenesis and showed that the newly incorporated mechanism results in significantly better agreement with data. In the process, we derived the first sequence-level model of this extensively characterized regulatory program. We further showed that allowing indirect binding of a TF explains its localization at enhancers more accurately than with direct binding only. Our model also provided a simple explanation of how a TF may switch between activating and repressive roles depending on context.