July 1, 2024
Participants: Yijia Huang, Jinming Cui, Zikuan Bai, Yuesi Xu, Jingchen Shi, Xiaoman Li, Yanzhi Xiao, Ziqing Jin, Yufan Dong, Andi Hu, Huier Peng, Jianing Wu, Hang Su, Chengjun Jiang, Hamo Wan, Yihao Tao, Jeffrey (Jiarui) Shen, Qiyun Wang, Jiayang Wang, Zhuxu Zhang, Haonan Deng, Junqiao Chen, Yanrong Li, Chenming Jin, Chang Wang, Yueyan Ma
A.Understand the phenomenon of aging and the mechanisms of action of antioxidants and anti-aging substances.
B.Learn the background related to Alzheimer’s disease.
C.Study basic knowledge and common command-line operations about Linux.
July 2, 2024
Participants: Xu Yuesi, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junqiao, Li Yanrong, Wu Jianing, Jin Chenming, Peng Huier, Ma Yueyan
A.Learn how to install common programs on Linux and understand virtual machines.
B.Study the Notepad++ text editor and learn how to use it for coding and text editing.
July 3, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Chen Junqiao,Wu Jianing Su Hang, Jiang, Chengjun Jiang
A.Know about the background related to wgx-50 molecule screening and discovery.
B.Learn statistics and related knowledge of hypothesis testing.
C.Learn common statistical analyses such as t-tests, ANOVA, non-parametric tests, survival
analysis (Kaplan-Meier survival curve), correlation analysis, and regression analysis.
July 4, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Chen Junqiao
A.Further study the functions and skills of text editors, such as syntax highlighting, code folding, multi-tab editing, document comparison, etc.
B.Learn how to study the survival of nematodes under antioxidant treatment and understand the steps of survival analysis.
July 5, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Chen Junqiao
A.Learn about popular text editors in scientific research, including Visual Studio Code (VS Code), Sublime Text, and Vim.
B.Understand how to set up experimental and control groups for survival analysis experiments: Experimental group: Nematodes are exposed to antioxidants or other oxidative stress conditions (e.g., hydrogen peroxide, para-hydroxybenzaldehyde). Control Group: No antioxidant treatment is administered. For example, the subjects are only exposed to the same oxidative stress conditions as the experimental group but without the addition of antioxidants.
July 6, 2024
Participants: Yijia Huang, Jinming Cui, Zikuan Bai, Yuesi Xu, Jingchen Shi, Xiaoman Li, Yanzhi Xiao, Ziqing Jin, Yufan Dong, Andi Hu, Huier Peng, Jianing Wu, Hang Su, Chengjun Jiang, Hamo Wan, Yihao Tao, Jeffrey (Jiarui) Shen, Haonan Deng, Junqiao Chen, Yanrong Li, Chenming Jin
A.Learn to use the Windows terminal emulator Xshell and master how to remotely log in and
manage servers via SSH, Telnet, Rlogin, SFTP, and Serial protocols.
B.Understand how to prepare nematodes for survival analysis experiments, and select a commonly used nematode species among model organisms: Caenorhabditis elegans (C. elegans), to perform nematode synchronization (to make them at the same developmental stage). A common method is to use eggs floating in egg solution for synchronization.
July 7, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Junqiao Chen
A.Advanced learning of XShell scripting and automation, text editing and searching, batch operations and automation.
B.Understand how to handle antioxidants in survival analysis experiments.
C.Preparation of medium: Dissolve antioxidants into the standard Nematode Growth Medium (NGM) with concentrations set according to the experimental design. Different concentration gradients of antioxidants can be used.
D.Addition of nematodes: Distribute synchronized nematodes into different petri dishes, with each dish containing nematodes from the same treatment group.
July 8, 2024
Participants: Xu Yuesi, Jin Ziqing, Xiao Yanzhi, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junqiao, Li Yanrong, Wu Jianing, Jin Chenming, Peng Huier,
A.Learn about and understand the PDB database(https://www.rcsb.org/) , its introduction and usage, and study how to download the crystal structure of proteins.
B.Learn about how to conduct temperature control in survival analysis experiments: typically, worms are cultured at a specific temperature (e.g., 20℃) to ensure their normal growth. Learn how to regularly observe the survival status of worms. Learn to use a microscope to confirm whether worms are alive. Living worms will exhibit obvious movement, while dead worms will show no response and have stiff bodies.
C.The experiment on prolonging the lifespan of nematodes (C. elegans, N2 strain) with WGX-50 (a type of xanthoxylin) at 20℃ involves treating wild-type nematodes with different concentration gradients of WGX-50 to determine the optimal concentration, recording the data, and plotting a lifespan curve.
July 9, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Chen Junqiao
A.Learn to use the biological macromolecule visualization software Pymol （https://pymolwiki.org/index.php/Main_Page）
B.Observe the state of nematodes regularly: Check the survival status of nematodes daily. Use a microscope to confirm whether the nematodes are alive. Living nematodes will show obvious movement, while dead nematodes will be unresponsive and have stiff bodies.
C.Learn about the settings of the survival experiment, which is divided into 10 experimental groups (2 mM, 1 mM, 0.5 mM, 50 μM, 125 μM, 62.5 μM, 31.25 μM, 15 μM, 7.5 μM, 3.75 μM) and 1 control group (0.1% ethanol). On the nematode solid growth medium containing OP50 (nematode food), wild-type nematodes are first subjected to synchronized culture.
July 10, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Junqiao Chen
A.Learn about the Pymol interface, how to set the working path, how to import proteins, and how to select objects.
B.Record the data for nematode survival analysis: Record the number of surviving nematodes in each group daily. Dead nematodes should be removed promptly to prevent affecting the health of other nematodes.
C.Understand the basic principles and concepts of RNA-seq (RNA Sequencing).
D.Conduct the survival experiment: When the synchronized nematodes have developed to the L4 stage, use a nematode picker under a stereomicroscope (Motic Company) to select well-grown nematodes of uniform size and transfer 30 nematodes each to the NGM solid medium containing 5-fluoro-2'-deoxyuridine (FUDR, Beijing Puxitang Biotechnology) for each experimental group. This process ensures that the nematodes are of the same age and developmental stage to avoid experimental errors. Set up 5 culture dishes for each group, meaning approximately 150 nematodes are included in each group as observation subjects.
July 11, 2024
Participants: Xu Yuesi, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junqiao, Li Yanrong, Wu Jianing, Jin Chenming
A.Learn about the measurement tools in Pymol software, how to set conformation parameters, and how to save files and export images.
B.Master the RNA-seq experimental process, including RNA extraction and RNA quality control.
C.Learn Adobe Illustrator, a vector-based graphic design software from Adobe Systems, and learn to use the “Pen Tool” to set “Anchor Points” and “Direction Lines” for graphic drawing in the later stage, and learn about advanced drawing tools and controls.
D.After the experiment, data was organized, survival curves were created, experimental results were compared to draw conclusions, and the optimal drug concentration of WGX-50 for extending C. elegans lifespan was determined.
July 12, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Junqiao Chen
A.Learn about Pymol software, including how to modify label properties, set custom colors, and perform breakthrough rendering.
B.Continue learning to use Adobe Illustrator.
C.Perform RNA-seq library construction, including fragmentation, end modification, and PCR amplification.
D.Control group: 50 µM of WGX-50; Treatment group: 0.1% ethanol. Three biological replicates per group, treated for 5 days at 25℃. C. elegans are washed from NGM plates using M9 buffer.
July 13, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Junqiao Chen
A.Gain initial understanding of quantum chemistry software Gaussian and its development history, and grasp how to simulate the quantum mechanical behavior of molecular systems through calculations.
B.Learn about how to analyze sequencing data through bioinformatics to obtain quantitative and qualitative gene expression data.
C.Conduct quality control for RNA-seq: The constructed library needs to undergo quality inspection to confirm whether the fragment size distribution and concentration meet the sequencing requirements. Agilent Bioanalyzer or qPCR is selected for library quantification and fragment analysis.
July 14, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Junqiao Chen
A.Learn how to install and run Gaussian software on both Windows and Linux versions, and understand the input and output files for Gaussian.
B.Master raw data processing for RNA-Seq: Including removing adapters and low-quality sequences, and aligning to the reference genome.
C.Continue with the RNA-Seq experiment. To avoid phenotypic changes related to reproduction inhibition, reproduction-inhibiting drugs are not used when handling the nematodes. Therefore, throughout the research process, we transferred the adult worms to newly prepared plates every day.
July 15, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Junqiao Chen
A.Learn the visualization and modeling tool GaussView accompanying Gaussian software. Study how to use Gaussian for geometric optimization of structures, understand commonly used methods in quantum chemistry calculations to aid in the convergence of geometric optimization, master how to use different coordinate systems and coordinate definition methods, how to adjust the maximum step size, and how to adjust symmetry.
B.Continue to transfer the adult worms to newly prepared plates.
C.Learn to conduct differential expression analysis: Use differential expression analysis tools (such as DESeq2, edgeR) to compare gene expression levels between different experimental groups and identify significantly up-regulated or down-regulated genes.
D.Continue learning and getting familiar with the use of Adobe Illustrator software.
July 16, 2024
Participants: Xu Yuesi, Jin Ziqing, Xiao Yanzhi, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junqiao, Li Yanrong, Wu Jianing, Jin Chenming, Peng Huier, Ma Yueyan
A.Learn the chemical structure visualization software tool CYLView and master the main techniques for displaying and analyzing molecular structures and reaction mechanisms. Further deepen the understanding of commonly used methods in quantum chemistry calculations to aid in the convergence of geometric optimization, and grasp how to try different initial structures, as well as various theoretical methods and basis sets.
B.Study the molecular biology method of RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction), understanding its applications in quantitative gene expression, viral load detection, miRNA and lncRNA detection, and gene mutation analysis.
C.Continue learning and getting familiar with the use of Adobe Illustrator software.
July 17, 2024
Participants: Xu Yuesi, Jin Ziqing, Xiao Yanzhi
A.Further study the data analysis and output features of the CYLView software, as well as the analysis of molecular simulation and computational chemistry results.
B.Understand the process of RT-qPCR, including how to perform reverse transcription of RNA to convert it into complementary DNA (cDNA); then use quantitative real-time PCR (qPCR) to amplify the cDNA while monitoring the amount of amplification products in real-time.
C.Aspirate the supernatant from the worm samples, freeze the nematodes in liquid nitrogen, store the frozen samples using dry ice, and then proceed with the specific sequencing work.
July 18, 2024
Participants: Xu Yuesi, Jin Ziqing, Xiao Yanzhi, Cui Jinming, Li Xiaoman, Huang Yijia, Wang Chang, Chen Junqiao
A.Understand the concepts in computational chemistry and computational physics, comprehend the basic principles of molecular dynamics simulations, and master simulating the behavior of molecular systems as they evolve over time. Master how to numerically solve the classical mechanical equations between molecules.
B.Understand the main steps of RT-qPCR reactions, including Denaturation, Annealing, and Extension.
C.Sequencing: Worms are lysed in TRIzol (Thermo Fisher Scientific, USA) reagent using a tissue homogenizer to extract total RNA. The concentration and purity of RNA samples are measured at 260nm using a Nanodrop 2000 (Thermo Fisher Scientific, USA). The integrity of RNA is assessed using an Agilent 2100 Bioanalyzer and RNA nano 6000 Assay Kit (Agilent Technologies, USA). Poly-A mRNA is enriched using Oligo-(dT) magnetic beads provided by the TIANSeq mRNA Capture Kit (TIANGEN, China).
July 19, 2024
Participants: Xu Yuesi, Jin Ziqing, Xiao Yanzhi
A.Learn about the concept and basic principles of molecular docking, and understand the
characteristics of common molecular docking software such as AutoDock, MOE, Glide, DOCK, etc.
B.Understand the functions and main features of AlphaFold3, and the AlphaFold 3 model based on diffusion-based architecture, which is capable of jointly predicting the structures of complexes including proteins, nucleic acids, small molecules, ions, and modified residues.
C.Continue learning and getting familiar with the use of Adobe Illustrator software.
D.Continue learning and getting familiar with the use of Pymol software.
July 20, 2024
Participants: Xu Yuesi, Jin Ziqing, Xiao Yanzhi
A.Understand and master RT-expression level changes: Demonstrate the relative or absolute expression levels of target genes under different treatment conditions based on calculated ΔΔCt values or standard curves.
B.Continue with the sequencing steps by generating a transcriptome RNA-seq library using the TIANSeq Fast RNA Library Prep Kit (Illumina).
C.Subsequently, the enriched poly-A mRNA samples undergo random fragmentation, complementary cDNA strand synthesis, end repair, and the addition of fluorescently labeled tags.
D.Measure the prepared mRNA-cDNA samples (with activity > 2 nM) using a Qubit 2.0 Fluorometer (Life Technologies, USA) and dilute them to 1 ng/µL.
E.Use an Agilent 2100 Bioanalyzer to assess the size of the mRNA-cDNA sample fragments mentioned above.
F.Following the manufacturer’s instructions, perform bridge amplification on the cBot Cluster Generation System (Illumina, USA) using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, USA).
July 21, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Chen Junqiao
A.Learn about the concept, applications, fundamental principles, and processes of virtual screening, including how to rapidly and effectively identify potential candidate compounds with high binding affinity to specific targets (such as proteins, enzymes, receptors, etc.) from large compound libraries. Understand how computer models and algorithms can be used to predict the interactions between compounds and targets, screening out molecules with higher drug activity.
B.Further explore and comprehend the contents of virtual screening, including Ligand-Based Virtual Screening (LBVS) and Structure-Based Virtual Screening (SBVS).
C.Continue with the sequencing steps by completing paired-end sequencing of the mRNA-cDNA on the NovaSeq 6000 Sequencer (Illumina, USA).
D.Continue learning and gaining familiarity with the use of Adobe Illustrator software.
July 22, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Chen Junqiao
A.Further understand the concepts of false positives and false negatives, and comprehend the reasons why virtual screening generates false positives (inactive compounds mistakenly identified as active) and false negatives (active compounds excluded from the selection).
B.Learn about the fundamental principles of gene silencing mechanism RNA interference (RNAi), its application in bacteria, and study CRISPR interference (CRISPRi).
C.For the obtained raw sequencing data in fastq format, use Trimmomatic (v0.39) software for analysis, whcih involves reading the sequencing sequences, removing the artificially added fluorescent tag sequences at the ends of the sequences, and low-confidence sequencing sequences, ultimately obtaining accurate sequencing DNA sequence data (clean data).
D.Utilize FastQC (v0.12.0) to evaluate sequence quality and sequencing depth, thereby conducting quality control over the entire sequencing process.
July 23, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Chen Junqiao,Wu Jianing Su Hang, Jiang, Chengjun Jiang
A.Learn about molecular docking and its general process. Understand how to search for and predict the structures of receptors and ligands. Learn how to evaluate the scoring function (Binding Score) and assess the quality and results after docking, such as whether the ligand’s geometry is reasonable, including whether the binding pose of the ligand to the receptor aligns with the ligand’s physicochemical properties. The ligand conformations in docking results should avoid excessive deformation or unreasonable distortions.
B.Understand the functions and technical features of the deep learning-based structure prediction framework AlphaFold2, such as the incorporation of the attention mechanism, which allows the model to effectively communicate information between amino acid residues that interact over long distances in the protein sequence.
C.The Evoformer module is a transformer network that incorporates Multiple Sequence Alignment (MSA), responsible for processing MSA: Evoformer extracts co-evolutionary information from a set of aligned sequences of homologous proteins, helping the model understand how different proteins retain specific structural features during evolution. Sequence-structure interaction: Evoformer simultaneously processes protein sequences and related distance matrices (representing spatial relationships between amino acid residues), effectively integrating both sequence and spatial information.
D.Use HISAT2 (v2.1.0) software to align the clean data to the reference genome of the nematode (GCA_000002985.3, Ensembl). The purpose of the alignment is to assign gene names to the high-quality sequences from the sequencing.
E.Use HTSeq (v0.6.0) to calculate the read counts mapped to a specific gene on the nematode genome.
F.Through gene reads, calculate the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for each gene, using the FPKM value as a measure of a gene’s abundance at the mRNA level.
July 24, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Chen Junqiao,Wu Jianing, Chengjun Jiang
A.Learn about the format of PDB files, including information such as the coordinate data of each atom in a protein, protein secondary structure information, and the interactions between atoms.
B.Understand the construction of an expression vector during RNA interference: inserting a designed RNA sequence into a bacterial expression vector, such as a plasmid.
C.Conduct differential expression gene (DEG) analysis between two groups of nematodes (control group and xanthoxylin-treated group) using the DESeq2 R package (v1.30.0) for screening. The criteria for screening differentially expressed genes are that the expression level of a gene in the xanthoxylin group must be more than 1.5 times higher than that in the control group, and the significance test for such changes should achieve a p-value (corrected p-value or p-adj, or q-value) < 0.05.
July 25, 2024
Participants: Xu Yuesi, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junqiao, Li Yanrong, Wu Jianing, Jin Chenming
A.Understand the popular molecular dynamics simulation software Amber (Assisted Model Building with Energy Refinement) in scientific research, and learn the basic concepts of molecular dynamics, applicable problems, conventional simulation processes, GPU acceleration, etc.
B.Familiarize with the modules in Amber software for molecular dynamics simulations, free energy calculations, data analysis, and more.
C.Understand the concept of Gene Set Enrichment Analysis (GSEA) used in genomic enrichment analysis. Annotate differentially expressed genes with their validated biological functions, identifying gene sets that collectively participate in processes such as cell mitosis, cell proliferation, or cell death. This approach can capture subtle changes in gene expression levels while integrating multiple functionally related genes, thereby enabling more accurate identification of biological signals.
D.Continue learning and engaging with Pymol software.
July 26, 2024
Participants: Shi Jingchen, Li Xiaoman, Xiao Yanzhi
A.Master the process of molecular dynamics simulations and learn the relevant knowledge and simulation techniques involved in molecular dynamics, including phase space, ensembles, calculation of statistical thermodynamic quantities, energy minimization algorithms, integration algorithms for equations of motion, periodic boundary conditions, and the calculation of non-bonded interactions.
B.Learn how to prepare the initial structure of a target molecular system (such as proteins, nucleic acids, small molecules, solvents, etc.), and how to select an appropriate force field.
C.Utilize the Gene Set Enrichment Analysis (GSEA) method for genomic enrichment analysis. The initial steps involve preparing the input data, specifically: gene expression data, typically from microarray or RNA-seq, requiring expression values for genes in each sample; phenotype data, representing different groups in the experimental design, such as control and treatment groups; and a gene set database, which are predefined gene sets used for enrichment analysis.
July 27, 2024
Participants: Shi Jingchen, Li Xiaoman, Xiao Yanzhi
A.Continue learning the process of molecular dynamics simulations.
B.Learn how to generate molecular force fields for small molecules.
C.Continue utilizing the Gene Set Enrichment Analysis (GSEA) method for genomic enrichment analysis. The gene ranking step involves sorting genes based on their expression differences according to a specific phenotype. Common ranking statistics include t-values, signal-to-noise ratios, or other metrics derived from the expression data. The result of this ranking is a list of genes ordered from highest to lowest significance of their association with the phenotype.
July 28, 2024
Participants: Shi Jingchen, Li Xiaoman, Xiao Yanzhi
A.Understand and master the common wavefunction analysis software Multiwfn. Learn about the basic features of Multiwfn and how to get started with it, including downloading, installing, and executing the program. Download the program manual and begin reading it.
B.Calculate the Enrichment Score (ES). For each predefined gene set, GSEA examines whether these genes tend to appear at the top or bottom of the ranked list. If a gene set shows clustering at the high or low ranks of the sorted list (i.e., most genes appear in regions of significant expression), the gene set is considered “enriched”.
C.Continue learning and engaging with Pymol software.
July 29, 2024
Participants: Shi Jingchen, Li Xiaoman, Xiao Yanzhi
A.Continue studying the knowledge of MD (Molecular Dynamics) simulation, while learning about various amino acid types in protein structural biology, protein structural hierarchies, helices, folds, turns, coils, disulfide bonds, salt bridges, and the time scales of protein structure formation.
B.Continue reading and studying the Multiwfn manual to gain a deeper understanding of the significance, functions, and applications of the Multiwfn wave function analysis program.
C.Study MD simulation to master how to perform energy minimization on the system, eliminating unreasonable parts in the structure (such as excessively short interatomic distances). Grasp how to gradually increase the temperature to reach the target temperature (e.g., 300K) and achieve thermodynamic equilibrium by balancing the system at that temperature.
D.Continue to conduct statistical significance evaluation of the GSEA (Gene Set Enrichment Analysis) analysis process. To assess the significance of the enrichment scores, GSEA uses a permutation-based test. By randomly permuting the labels of the experimental and control groups multiple times, a distribution of random enrichment scores is generated. The p-value of the actual gene set enrichment score is calculated by comparing it to the enrichment scores in the random distribution to determine its significance.
July 30, 2024
Participants: Shi Jingchen, Li Xiaoman, Xiao Yanzhi, Xu Yuesi, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junqiao, Li Yanrong, Wu Jianing, Jin Chenming, Peng Huier, Ma Yueyan
A.Continue learning about MD (Molecular Dynamics) simulation knowledge, while also studying the definitions of backbone dihedral angles parameters, Ramachandran plots, and standard naming conventions for residue atoms.
B.Learn the basic features and usage methods of Multiwfn, mastering the supported formats of wavefunction files; understand the real-space functions supported by Multiwfn, and learn how to inspect and modify wavefunctions through the Multiwfn software.
C.Continue to conduct genome enrichment analysis, focusing on the normalization of enrichment scores (NES).
July 31, 2024
Participants: Shi Jingchen, Li Xiaoman, Xiao Yanzhi
A.Understand and learn about the introduction of relevant force fields involved in MD simulation.
B.Conduct RNA interference analysis by constructing HT115 bacteria from the Escherichia coli strain, which should contain the L4440 plasmid within its cells. This plasmid harbors the cDNA sequence of the exon region (CDS) of the gene targeted for interference (silencing, reduction of expression level).
C.Construct the L4440-target interference gene plasmid.
August 1, 2024
Participants: Xu Yuesi, Shi Jingchen, Li Xiaoman, Xiao Yanzhi, Cui Jinming, Jin Ziqing, Huang Yijia, Wang Chang
A.Learn about the Cpptraj module in Amber software. Master how to obtain trajectory files and structure files from MD simulations in Amber. Understand how to set up analysis tasks: write analysis scripts using Cpptraj to define the analysis tasks to be executed, such as calculating RMSD (Root Mean Square Deviation), hydrogen bonds, distances, etc. Grasp how to run Cpptraj analysis: execute Cpptraj via the command line, read trajectory files, and perform analysis commands in the script. Additionally, learn how to view the output results.
B.Continue to conduct genome enrichment analysis. To control the false positive rate, GSEA applies multiple testing correction to the analysis results of multiple gene sets using the FDR (False Discovery Rate) correction method.
C.According to the methods described in the sequencing protocol, extract total RNA from nematodes and obtain complementary DNA (cDNA) sequences of the total RNA using a reverse transcription kit. Design specific primers based on the gene to be interfered with, and use PCR to amplify the target gene, facilitating its cloning.
August 2, 2024
Participants: Xu Yuesi, Shi Jingchen, Li Xiaoman, Xiao Yanzhi
A.Learn about the scientific graphing software Origin, and understand common 2D and 3D graph types used in scientific plotting, such as line charts, bar charts, scatter plots, contour plots, surface plots, etc., which are suitable for presenting experimental data results.
B.Perform double enzyme digestion on the L4440 plasmid using XhoI and HindIII enzymes to obtain a linear plasmid with sticky ends. The plasmid is originally a closed circular DNA.
C.Also perform double enzyme digestion on the cloned gene obtained above, and then use the Hieff clone®plus one-step cloning kit to insert it into the double-digested L4440 plasmid, ultimately obtaining a gene interference plasmid containing the cDNA sequence of the gene to be interfered with.
August 3, 2024
Participants: Xu Yuesi, Shi Jingchen, Li Xiaoman, Xiao Yanzhi
A.Continue learning about the scientific graphing software Origin, and understand common data analysis tools, including basic statistical analysis (such as mean, variance), regression analysis, curve fitting, signal processing (such as Fourier transform, filtering), etc.
B.Continue with the RNAi experiment by amplifying the above gene interference plasmid and sending it for sequencing to confirm the correctness of the inserted target gene fragment sequence.
C.Transform the sequencing-confirmed gene interference plasmid into HT115 bacteria.
D.Concentrate the bacteria transformed with the plasmid on an ampicillin-resistant solid culture plate. After overnight incubation at 37℃, pick single clones and use the aforementioned PCR method to confirm again that the gene interference plasmid has been successfully transformed into HT115 bacteria.
August 4, 2024
Participants: Xu Yuesi, Li Xiaoman, Xiao Yanzhi, Jin Ziqing
A.Learn about conformational search and related algorithms, such as potential energy surface scanning, molecular dynamics, and Monte Carlo algorithms.
B.Study the common conformational search software CREST (Conformer–Rotamer Ensemble Sampling Tool) and its accompanying XTB algorithm and program.
C.Further learn about Origin software, including how to batch process data, use templates and themes, and export images.
D.Continue with the RNAi experiment by recording the numbers of positive clones, inoculating them into liquid LB medium, and incubating them overnight at 37℃ in medium containing 50 μg/mL ampicillin.
August 5, 2024
Participants: Xu Yuesi, Li Xiaoman, Xiao Yanzhi, Jin Ziqing
A.Delve deeper into using CREST software and its accompanying XTB algorithm and program.
B.Learn about the concepts and technical points of visualization analysis in non-covalent interactions.
C.Continue with the RNAi experiment by centrifuging the above RNAi cultures to concentrate them to 20 mg/mL. Inoculate 250 μL of the concentrated culture onto each 6 cm nematode growth medium (NGM) plate. After air-drying the NGM plates in a clean bench, incubate them overnight at 25℃ for later use.
D.Add 10 mM isopropyl β-D-thiogalactopyranoside (IPTG, from Bioteke Life Sciences Co., Ltd. in China) to the NGM plates. IPTG can induce the expression of dsRNA synthesis of the target gene in the L4440 plasmid within the gene interference bacteria.
E.When nematodes consume the HT115 bacteria, the L4440 plasmid within their bodies can be induced to express double-stranded DNA of the target interference gene. After degradation by the dicer enzyme in the nematodes, 20-nucleotide double-stranded DNA fragments are formed, which then inhibit the expression of the target gene at the transcriptional level. Feed the interference bacteria to nematodes starting from the L1 stage, while also administering nematodes (control group, xanthohumol treatment group).
August 6, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Junqiao Chen
A.Use CREST software to perform conformational search for the low-energy structures of wgx-50, and optimize the structure of wgx-50 using Gaussian software.
B.Learn about the principles of Bader's Atoms-in-Molecules (AIM) topology analysis in non-covalent interactions.
C.Conduct an RT-qPCR experiment by washing the nematodes three times with M9 buffer to collect them from the plates.
D.Extract total RNA from the nematodes using the RNAiso plus reagent kit (Takara Bio, Japan) as a reference method. Perform quantitative reverse transcription polymerase chain reaction (RT-qPCR) using the Hieff® qPCR SYBR Green Master Mix kit (Yeasen Biotech) to measure the expression levels of multiple genes at the mRNA level in nematode and mouse tissues.
August 7, 2024
Participants: Xu Yuesi, Li Xiaoman, Xiao Yanzhi, Jin Ziqing
A.Dock the optimized wgx-50 structure with the protein and perform MD (Molecular Dynamics) simulations using Amber software.
B.Learn about the method for graphical representation of custom inter- and intra-fragment interactions called the independent gradient model based on Hirshfeld partition (IGMH). Study and compare the differences between IGMH and IGM methods for interaction analysis.
C.Continue with the RT-qPCR experiment, collecting PCR data on the CFX Connect system. Use the 2-ΔΔCT method to calculate the relative expression of genes, with gpd-1 and gapdh serving as internal reference controls.
August 8, 2024
Participants: Xu Yuesi, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junjiong, Li Yanrong, Wu Jianing, Jin Chenming, Peng Huier, Ma Yueyan
A.Learn how to determine the protonation states of residue side chains and apply this to determine the protonation states of residues in the studied wgx-50 protein system.
B.Conduct multiple parallel MD simulations and monitor the progress of the MD simulations.
C.Perform fluorescence microscopy analysis. All nematodes are cultured at 25°C. Control group: L4 nematodes with 0.1% ethanol. Treatment group: L4 nematodes with 50 μM WGX-50. Harvest nematodes after treating them with WGX-50 (50 μM) for 5 days according to the protocol.
August 9, 2024
Participants: Xu Yuesi, Li Xiaoman, Xiao Yanzhi, Jin Ziqing
A.Understand the concept of binding free energy.
B.Learn about popular free energy calculation methods in MD (Molecular Dynamics) simulations, including the Molecular Mechanics combined with Poisson-Boltzmann (PB) or Generalized Born (GB) Surface Area (SA) methods, known as MM/PBSA and MM/GBSA respectively.
C.Study the nonpolar solvation free energy, which is typically calculated through the solvent-accessible surface area (SA) and is used to describe molecular surface tension and hydrophobic effects.
August 10, 2024
Participants: Xu Yuesi, Li Xiaoman, Xiao Yanzhi, Jin Ziqing
A.Learn how to add solvent, box, and counter ions, as well as perform restrained dynamics.
B.Learn how to download MD (Molecular Dynamics) simulation trajectories, specifically the simulation trajectories of the wgx-50 protein complex.
C.Learn how to calculate binding free energy in Amber.
D.Understand how to read and interpret the output files for binding free energy, including the meaning of each energy term, such as ELE (electrostatic energy calculated by the molecular mechanics (MM) force field), VDW (van der Waals contribution from MM), and INT (internal energy arising from bond, angle, and dihedral terms in the MM force field).
August 11, 2024
Participants: Xu Yuesi, Huang Yijia, Wan Hamo
A.Understand the concepts of trajectory analysis, including Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF).
B.Analyze the trajectories after performing MD (Molecular Dynamics) simulations on the wgx-50 protein complex system.
C.Learn to use the Log-rank (Mantel-Cox) method for statistical hypothesis testing. For significance analysis between any two groups, a two-tailed unpaired t-test is used. For significance analysis among multiple groups, a one-way ANOVA analysis is employed.
D.Learn data analysis and plotting using GraphPad Prism software. Data graphs are plotted using GraphPad Prism v9.5 (GraphPad Software, USA) and then arranged and exported using Adobe Illustrator CC (Adobe Systems Inc., USA).
August 12, 2024
Participants: Xu Yuesi, Huang Yijia, Wan Hamo
A.Calculate the RMSD (Root Mean Square Deviation) values for the wgx-50 protein complex system and create corresponding graphs.
B.Learn and consider methods to eliminate translational and rotational motions of the system, remove water from the trajectories, and check the distance between the protein and its image.
C.Continue with the fluorescence microscopy analysis experiment. After treatment, wash the nematodes twice with M9 buffer. Randomly select 10-30 nematodes, transfer them onto a 2% agar pad, anesthetize them with an appropriate amount of 10 mM levamisole solution, and observe the green fluorescence intensity of nematodes in each group under an Inverted and Upright Integrated Fluorescence Microscope (Revolve, ECHO Company).
D.Use ImageJ software to quantitatively analyze the fluorescence intensity.
August 13, 2024
Participants: Xu Yuesi, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junqiao, Li Yanrong, Wu Jianing, Jin Chenming, Peng Huier, Ma Yueyan
A.Calculate the RMSF (Root Mean Square Fluctuation) values for the wgx-50 protein complex system and create corresponding graphs.
B.Learn and understand popular free energy calculation methods in MD simulations, including the MM/GBSA method. This includes preparing topology files (e.g., complex.prmtop) for the protein-ligand complex, understanding the factors that influence binding energy, and learning about residue-based energy decomposition methods.
C.Calculate the binding free energy of the wgx-50 protein complex.
August 14, 2024
Participants: Xu Yuesi, Huang Yijia, Wan Hamo
A.Learn cluster analysis and PCA (Principal Component Analysis), and apply these methods to the wgx-50 protein complex system to identify representative structures during MD simulations. Utilize software such as Pymol for structure visualization.
B.Calculate the radius of gyration values for the wgx-50 protein complex system and create corresponding graphs.
C.Continue learning and familiarizing with Pymol software.
August 15, 2024
Participants: Xu Yuesi, Huang Yijia, Wan Hamo, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Wang Chang, Chen Junqiao
A.Learn how to use Multiwfn for IGMH (Independent Gradient Model based on Hirshfeld partitioning) analysis to clearly and intuitively visualize interactions in chemical systems. Study quantitative analysis of the contributions of atoms and atom pairs to these interactions.
B.Analyze which residues of wgx-50 and the protein form interactions, and use the IGMH method to analyze the formation of non-covalent interactions.
C.Continue learning data analysis and plotting using GraphPad Prism software for creating figures and tables for research papers.
August 16, 2024
Participants: Xu Yuesi, Huang Yijia, Wan Hamo
A.Learn the RDG (Reduced Density Gradient) method for visualizing and quantifying non-covalent interactions. Study how to use RDG isosurfaces to display weak interaction regions and distinguish the strength and type of weak interactions.
B.Apply the RDG method to the wgx-50 protein system.
August 17, 2024
Participants: Xu Yuesi, Huang Yijia, Wan Hamo
A.Learn to create filled RDG (Reduced Density Gradient) scatter plots for weak interaction analysis. Study the gnuplot software required for plotting and understand its basic operation procedures.
B.Continue learning data analysis and plotting using GraphPad Prism software for creating figures and tables for research papers.
C.Continue learning the use of the plotting software Origin.
August 18, 2024
Participants: Yijia Huang, Jinming Cui, Yuesi Xu, Jingchen Shi, Ziqing Jin, Yufan Dong, Andi Hu, Hamo Wan, Haonan Deng, Yihao Tao, Jeffrey (Jiarui) Shen, Haonan Deng
A.Further study how to analyze the state of ligands within proteins and investigate the role and function of water molecules in molecular recognition. Certain water molecules at binding sites may play crucial roles in the binding between ligands and receptors, so it is necessary to consider whether the ligand interacts appropriately with these water molecules.
B.Apply this type of analysis to the wgx-50 system.
August 19, 2024
Participants: Jinming Cui, Yuesi Xu, Ziqing Jin, Yufan Dong, Andi Hu, Hamo Wan, Haonan Deng, Yihao Tao, Jeffrey (Jiarui) Shen, Chenming Jin
A.Learn the key points of writing an introduction, study how to read and summarize previous literature, and clarify the research background and its significance.
B.Learn to write a brief literature review: summarize the main research progress in the related field, study how to cite several representative or recent research achievements, and present the development process and current knowledge level in the field.
August 20, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Chen Junqiao
A.Investigate the skeletal motion characteristics during the simulation process, the stability of conformations in different regions, and the hydrogen bonding situation in the system.
B.Continue learning data analysis and plotting using GraphPad Prism software for creating figures and tables for research papers.
C.Continue learning the use of the plotting software Origin.
August 21, 2024
Participants: Yijia Huang, Jinming Cui, Yuesi Xu, Ziqing Jin, Yufan Dong, Andi Hu, Hamo Wan, Haonan Deng, Yihao Tao, Jeffrey (Jiarui) Shen, Chenming Jin
A.Read typical literature on MD simulations to understand the key points of writing about MD simulation and interaction analysis techniques, especially the generation of topology files, selection of force fields, and the process of establishing simulation systems.
B.Continue learning and practicing the use of Adobe Illustrator software for creating figures and tables for research papers.
August 22, 2024
Participants: Xu Yuesi, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junjiao, Li Yanrong, Wu Jianing, Jin Chenming, Peng Huier, Ma Yueyan
A.Learn scientific paper writing, specifically how to propose research innovations and elaborate on the innovative aspects of your research compared to existing work.
B.Reflect on the research methods, theoretical models, additions to existing theories, or new solutions to practical problems used in this project.
August 23, 2024
Participants: Yijia Huang, Jinming Cui,Yuesi Xu, Ziqing Jin, Yufan Dong, Andi Hu, J Hamo Wan, Haonan Deng
Summarize and organize experimental results, contemplate the potential contributions or application values of this study, and emphasize its importance and potential impact on the field.
August 24, 2024
Participants: Zikuan Bai, Yijia Huang, Yuesi Xu, Huier Peng, Jianing Wu, Hang Su, Chengjun Jiang, Hamo Wan, Jeffrey (Jiarui) Shen, Zhuxu Zhang, Junqiao Chen, Yanrong Li
Write the abstract of the paper: Briefly summarize the research purpose, methods, results, and conclusions. It should be clear and concise, typically 150-250 words, highlighting the paper's innovation and main findings.
August 25, 2024
Participants: Zikuan Bai, Yijia Huang, Yuesi Xu, Huier Peng, Jianing Wu, Hang Su, Chengjun Jiang, Hamo Wan, Jeffrey (Jiarui) Shen, Zhuxu Zhang, Junqiao Chen, Yanrong Li,
Continue summarizing and organizing experimental results, contemplating the potential contributions or application values of this study, and emphasizing its importance and potential impact on the field.
August 26, 2024
Participants: Zikuan Bai, Yijia Huang, Yuesi Xu, Huier Peng, Jianing Wu, Hang Su, Chengjun Jiang, Hamo Wan, Jeffrey (Jiarui) Shen, Zhuxu Zhang, Junqiao Chen, Yanrong Li, Chang Wang, Yueyan Ma
Write the methods section of the paper: Describe the experimental or research methods in detail to ensure the reproducibility of the study. Include research design, experimental materials, data collection, and analysis methods.
August 27, 2024
Participants: Xu Yuesi, Bai Zikuan, Huang Yijia, Wan Hamo, Wang Chang, Chen Junjiao, Li Yanrong, Wu Jianing, Jin Chenming, Peng Huier, Ma Yueyan
A.Refine the scientific paper writing.
B.Write the results section: Objectively present the main findings of the study, including data, charts, and statistical analysis.
August 28, 2024
Participants: Zikuan Bai, Yijia Huang, Yuesi Xu, Huier Peng, Jianing Wu, Hang Su, Chengjun Jiang, Hamo Wan, Jeffrey (Jiarui) Shen, Zhuxu Zhang, Junqiao Chen, Yanrong Li, Chang Wang, Yueyan Ma
A.Refine the scientific paper writing.
B.Finalize the results section: Objectively present the main findings of the study, including data, charts, and statistical analysis.
C.Conduct analysis of MD simulation results.
August 29, 2024
Participants: Xu Yuesi, Cui Jinming, Li Xiaoman, Xiao Yanzhi, Jin Ziqing, Huang Yijia, Wang Chang, Chen Junqiao
A.Refine the scientific paper writing.
B.Write the discussion section: Interpret the research results, compare similarities and differences with other studies, discuss the limitations of the study, and propose directions for future research.
August 30, 2024
Participants: Zikuan Bai, Yijia Huang, Yuesi Xu, Huier Peng, Jianing Wu, Hang Su, Chengjun Jiang, Hamo Wan, Jeffrey (Jiarui) Shen, Zhuxu Zhang, Junqiao Chen, Yanrong Li
A.Refine the scientific paper writing.
B.Supplement and complete the references section: List all cited literature, ensuring the format meets the requirements of the target journal.
C.Polish the language of the paper.
August 31, 2024
Participants: Zikuan Bai, Yijia Huang, Yuesi Xu, Huier Peng, Jianing Wu, Hang Su, Chengjun Jiang, Hamo Wan, Jeffrey (Jiarui) Shen, Zhuxu Zhang, Junqiao Chen, Yanrong Li, Chang Wang, Yueyan Ma
A.Refine the scientific paper writing, such as avoiding excessive citations: While it is necessary to cite relevant literature, the introduction is not a literature review. Citations should revolve around the research question, avoiding the accumulation of references.
B.Polish the language of the paper.