Project description:Helicobacter pylori (H. pylori) is currently recognized as the primary carcinogenic pathogen associated with gastric tumorigenesis, and its high prevalence and resistance make it difficult to tackle. A graph neural network-based deep learning model, employing different training sets of 13,638 molecules for pre-training and fine-tuning through rigorous iterative learning processes, was aided in predicting and exploring novel molecules against H. pylori. A positively predicted novel berberine (BBR) derivative 8 with 3,13-disubstituted alkene exhibited a potency against all tested drug-susceptible and resistant H. pylori strains with minimum inhibitory concentrations (MICs) of 0.25–0.5 μg/mL. Strikingly, pharmacokinetic studies demonstrated an ideal gastric retention of 8, with the stomach concentration significantly higher than its MIC value at 24 h post dose. Oral administration of 8 and proton pump inhibitor omeprazole (OPZ) showed a 2.2-log reduction in the gastric bacterial burden compared with the control group, which is comparable to the triple-therapy, namely OPZ + amoxicillin (AMX) + clarithromycin (CLA), and partially restored the diversity of the intestinal flora as well as the abundance of probiotics. A combination of OPZ + AMX + CLA + 8 could further decrease the bacteria load (2.8-log reduction). More importantly, the mono-therapy of 8 showed comparable eradicative efficacies compared with both triple-therapy (OPZ + AMX + CLA) and the quadruple-therapy (OPZ + AMX + CLA + bismuth citrate) groups. SecA and BamD, playing a major role in outer membrane protein (OMP) transport and assembling, were identified and verified as the direct targets of 8 by employing the chemoproteomics technique. The treatment of 8 induced the death of H. pylori caused by OMP deficiency, and the subsequent reduced adhesion to gastric epithelial cells. In summary, by targeting the relatively conserved OMPs transport and assembling system, 8 has the potential to be developed as a novel anti-H. pylori candidate, especially for the eradication of drug-resistant strains. The deep learning model established in this study might provide a reliable prediction tool for future anti-H. pylori candidate discovery.

Project description:Post expression from the host cell, a bio-therapeutic will be subjected to several downstream processing steps prior to final formulation. Analysis of the product under various buffer conditions at each stage is desirable to show that the integrity of the sample is maintained and to monitor any potential product loss. In this study we examine how varying buffer conditions during downstream processing impact the structural integrity of a biopharmaceutical product, with a specific focus on the model IgG1 antibody, mAb4, provided by FUJIFILM Diosynth Biotechnologies. Flexibility, stability, aggregation propensity, and bulk properties were evaluated across the buffers of four sample points, including perfusion media, purification stages, and formulation buffer. Comparisons with Herceptin, an extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible buffer. Although mAb4 and Herceptin present with similar charge state distributions (CSD) in native mass spectrometry (MS) experiments, they exhibit distinct unfolding patterns during the activated ion mobility-mass spectrometry (aIM-MS) and differential scanning fluorimetry (DSF). The greater structural stability and aggregation onset temperature (Tagg) observed in Herceptin, are primarily attributed to the heavier glycosylation and kappa-class light chains present in Herceptin compared to the lambda-class light chains in mAb4. Hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that while mAb4 experiences substantial structural changes during purification, characterized by high flexibility, a low melting temperature (Tm), and prevalent repulsive protein-protein interactions, it eventually transitions to a highly compact and stable structure in buffers with higher salt concentrations and during formulation. Notably, in the formulated environment, the third constant domain (CH3) of the heavy chain retains flexibility and is identified as a region of interest for aggregation formation. Here we showcase the integration of MS and orthogonal techniques to attain comprehensive information which can be utilized early in the development stage to aid in decision-making regarding targeted mutations or to guide the design space of bioprocesses and formulation choices.

Project description:Background: Similar to replicating myoblasts, many rhabdomyosarcoma cells express the myogenic determination gene MyoD. In contrast to myoblasts, rhabdomyosarcoma cells do not make the transition from a regulative growth phase to terminal differentiation. Previously we demonstrated that the forced expression of MyoD with its E-protein dimerization partner was sufficient to induce differentiation and suppress multiple growth-promoting genes, suggesting that the dimer was targeting a switch that regulated the transition from growth to differentiation. Our data also suggested that a balance existed between various inhibitory transcription factors and MyoD activity that kept rhabdomyosarcomas trapped in a proliferative state. Methods: Potential myogenic co-factors identified by analysis of high-throughput sequencing of chromatin immunoprecipitation experiments in normal myogenic cells were tested for their ability to drive differentiation in rhabdomyosarcoma cell culture models, and their relation to MyoD activity determined through molecular biological experiments. Results: Modulation of the transcription factors RUNX1 and ZNF238, factors with poorly delineated roles in myogenic development, can induce differentiation in rhabdomyosarcoma cells and their activity is integrated, at least in part, through the activation of miR-206, which acts as a genetic switch to transition the cell from a proliferative growth phase to differentiation. The inhibitory transcription factor MSC also plays a role in controlling miR-206, appearing to function by occluding a binding site for MyoD in the miR-206 promoter. Conclusions: These findings suggest that nested feed-forward circuits that proceed from MyoD, to RUNX1, to ZNF238, and finally to miR-206 function in both rhabdomyosarcomas as well as normal myogenesis to control the decision point of proliferation versus differentiation. Total RNA samples were collected from human RD cells transduced with lentivirus carrying RUNX1, RP58 (ZNF238), miR-206 or GFP (three biological replicates each) and allowed to differentiate for 72 hours.

Project description:Background: Similar to replicating myoblasts, many rhabdomyosarcoma cells express the myogenic determination gene MyoD. In contrast to myoblasts, rhabdomyosarcoma cells do not make the transition from a regulative growth phase to terminal differentiation. Previously we demonstrated that the forced expression of MyoD with its E-protein dimerization partner was sufficient to induce differentiation and suppress multiple growth-promoting genes, suggesting that the dimer was targeting a switch that regulated the transition from growth to differentiation. Our data also suggested that a balance existed between various inhibitory transcription factors and MyoD activity that kept rhabdomyosarcomas trapped in a proliferative state. Methods: Potential myogenic co-factors identified by analysis of high-throughput sequencing of chromatin immunoprecipitation experiments in normal myogenic cells were tested for their ability to drive differentiation in rhabdomyosarcoma cell culture models, and their relation to MyoD activity determined through molecular biological experiments. Results: Modulation of the transcription factors RUNX1 and ZNF238, factors with poorly delineated roles in myogenic development, can induce differentiation in rhabdomyosarcoma cells and their activity is integrated, at least in part, through the activation of miR-206, which acts as a genetic switch to transition the cell from a proliferative growth phase to differentiation. The inhibitory transcription factor MSC also plays a role in controlling miR-206, appearing to function by occluding a binding site for MyoD in the miR-206 promoter. Conclusions: These findings suggest that nested feed-forward circuits that proceed from MyoD, to RUNX1, to ZNF238, and finally to miR-206 function in both rhabdomyosarcomas as well as normal myogenesis to control the decision point of proliferation versus differentiation. Two biologically independent sets of RD cells were transduced with either MD~E or empty vector pCLBabe retroviruses. After RNA extraction pairs of samples (MD~E versus pCLBabe) were hybridized to arrays, incorporating both dye-swap and technical replicates (i.e. 1 comparison x 2 dye-swaps x 2 biological replicates x 2 technical replicates = 8 arrays)

Project description:Background: Canmei formula (CMF) is a traditional Chinese medicine compound with definite effect on the prevention and treatment of colorectal adenoma (CRA). It can prevent the transformation of intestinal inflammation to cancer. This study explored the mechanism of action of CMF in anti-CRA using multi-omics techniques. Method: Mice were randomly divided into 4 groups: blank group (Control), high-fat diet (HFD) + AOM/DSS colorectal adenoma model (ADH) groups, Canmei formula treatment group (ADH-CMF) and sulfasalazine treatment group (Sul). Except for the blank group, ADH model was established in the other 3 groups by intraperitoneal injection with AOM reagent, and then mice was given 2.5%DSS in free drinking water and high-fat diet. The mice in the blank group and ADH group were intragastrically perfused with normal saline, and the mice in the other 2 groups were treated with corresponding drugs for 20 weeks. During this period, the changes of physical signs of mice in each group were observed. The differentially expressed genes and proteins in the Control group, ADH group and ADH-CMF group were detected by RNA-seq transcriptome sequencing and Tandem Mass Tags (TMT) quantitative proteomics. After the combined analysis and verification. The key targets were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results : A total of 2548 differential genes were obtained by transcriptomics analysis, and 45 differential proteins were obtained by proteomics analysis. The results of proteomics data and experimental verification showed that CMF mainly acted on the Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase (LHPP) target. GO analysis showed that the targets of CMF were involved in the biological processes such as cellular process, metabolic process and biological regulation. KEGG analysis showed that those genes were involved in oxidative phosphorylation, cell senescence, and Metabolic pathways. Studies have shown that Lhpp overexpression of Lhpp impeded Colorectal cancer cell growth and proliferation in vitro, and was associated with a change in PI3K/AKT activity.

			Action	DRS
	EV71.msf	Msf
	EV71.raw	Raw
	EV71_PZH.msf	Msf
	EV71_PZH.raw	Raw
	blank.msf	Msf

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Human rhabdomyosarcoma cells LC-MSMS

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