Phage immunoprecipitation sequencing (PhIP-Seq) is a high-throughput and low-cost method for analyzing the specific binding of target proteins to peptide libraries. The method uses oligonucleotide library synthesis (OLS) to encode proteome-scale peptide libraries for display on phages, and then immunoprecipitates these library phages with target proteins (such as antibodies) for subsequent analysis by high-throughput DNA sequencing. PhIP-Seq enables the screening of peptide targets that react specifically with hundreds of proteins or pathogens. PhIP-Seq has been successfully applied in various fields such as disease detection, screening of autoimmune disease biomarkers, vaccine development, and allergen detection, becoming a high-throughput diagnostic technology. This article systematically describes the development, applications, and result evaluation of PhIP-Seq, in order to gain a more comprehensive understanding of the application and future development prospects of this technology in various fields.
Peptide Library ; Humans ; Immunoprecipitation/methods* ; High-Throughput Nucleotide Sequencing/methods* ; Bacteriophages/genetics*

Objective To investigate the effects of folate deficiency on changes in histone H3 lysine 4 (H3K4) mono-methylation (me1)-marked enhancers and the molecular mechanism underpinning the folate deficiency-induced neural tube defects (NTD). Methods Mouse embryonic stem cells (mESCs) were cultured in the folate-free DMEM medium (folate-deficient group) and the DMEM medium containing 4 mg/L folate (normal control group),respectively.Chromatin immunoprecipitation sequencing (ChIP-seq) was performed for H3K4me1. The mouse model of folate-induced NTD was established,and transcriptome sequencing (RNA-seq) was performed for the brain tissue of fetal mice to reveal the differential expression profiles.The results were validated through real-time quantitative polymerase chain reaction (RT-qPCR).The activity of the differential peak regions of H3K4me1 was verified through the luciferase reporter assay. Results The folate content in the mESCs cultured in the folate-free medium reduced compared with that in the normal control group (P=0.008).The H3K4me1-maked enhancers in the mESCs cultured in the folate-free medium induced significant changes in intronic regions,and these changes were concentrated in metabolic and energy metabolism processes (q=9.56×10^-48,P=1.28×10^-47).The differentially expressed genes harboring H3K4me1-marked enhancers in mESCs were mainly enriched in the Wnt signaling pathway (q=0.004,P=0.004 7).ChIP-qPCR results confirmed that H3K4me1 binding decreased in the differential peak regions of the Ldlrap1 gene (P=0.008),Camta1 gene (P=0.002),and Apc2 gene (P=0.012).The H3K4 demethylase inhibitor T-448 effectively reversed the H3K4me1 binding in the differential peak regions of the aforementioned genes (P=0.01).The results of RNA-seq for the brain tissue of NTD fetal mice showed significant enrichment of the differentially expressed genes in the Wnt signaling pathway (P=1.52×10^-5).The enrichment of differential peak regions of H3K4me1-marked enhancers in Apc2,Ldlrap1,and Camta1 genes in the brain tissue also showed significant changes.The differential peak region in Apc2 exhibited transcription factor activity (P=0.020). Conclusion Folate deficiency may affect changes in H3K4me1-marked enhancers to participate in the regulation of neural tube closure genes,thereby inducing the occurrence of NTD.
Neural Tube Defects/genetics* ; Animals ; Mice ; Folic Acid Deficiency/complications* ; Histones/metabolism* ; Folic Acid/metabolism* ; Methylation ; Mouse Embryonic Stem Cells/metabolism* ; Wnt Signaling Pathway ; Lysine/metabolism* ; Chromatin Immunoprecipitation Sequencing

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.
Cricetinae ; Animals ; Antibodies, Monoclonal/metabolism* ; Caprylates/chemistry* ; Cell Culture Techniques ; Chromatography, Affinity ; CHO Cells ; Cricetulus ; Chemical Precipitation

Taking Pudilan Xiaoyan Oral Liquid as a demonstration, the effective delivery of quality markers in alcohol precipitation of Chinese medicine oral liquid preparations was studied. With the transfer rates of adenosine, corynoline, cichoric acid, baicalin, and wogonin as evaluation indexes, the effect of the density of concentrate before alcoholic precipitation, volume fraction of ethanol, stirring speed, temperature of concentrated solution, stirring time, alcohol concentration, alcohol precipitation time, alcoholic precipitation temperature, alcohol addition rate, and the pH of concentrate on the alcohol precipitation process was investigated by Plackett-Burman trial design, thus obtaining the key factors that influenced the alcohol precipitation process. The key factors were further optimized by Box-Behnken design to determine the optimal alcohol precipitation conditions. When the density of concentrate before alcoholic precipitation was 1.12 g·mL~(-1), the pH of concentrate was 6.86, and the alcohol concentration was 50.00%, the transfer rates of baicalin and wogonin were 91.86% and 87.78%, respectively. When the density of concentrate before alcoholic precipitation was 1.13 g·mL~(-1), the concentration of alcohol was 74.50%, and the alcoholic precipitation temperature was 17.0 ℃, the transfer rates of adenosine, corynoline, and cichoric acid were 85.95%, 71.62% and 83.19%, respectively. The method of optimizing alcohol precipitation techniques and determining the parameters of Pudilan Xiaoyan Oral Liquid by response surface methodology is reasonable and feasible, which provides guidance and experience for the effective delivery of quality markers in Chinese medicine oral liquid preparations.
Drugs, Chinese Herbal ; Ethanol ; Adenosine ; Chemical Precipitation

Microbial induced calcium carbonate precipitation (MICP) refers to the natural biological process of calcium carbonate precipitation induced by microbial metabolism in its surrounding environment. Based on the principles of MICP, microbial cement has been developed and has received widespread attention in the field of biology, civil engineering, and environment owing to the merits of environmental friendliness and economic competence. Urease and carbonic anhydrase are the key enzymes closely related to microbial cement. This review summarizes the genes, protein structures, regulatory mechanisms, engineering strains and mutual synergistic relationship of these two enzymes. The application of bioinformatics and synthetic biology is expected to develop biocement with a wide range of environmental adaptability and high performance, and will bring the MICP research to a new height.
Calcium Carbonate/metabolism* ; Chemical Precipitation ; Urease/metabolism*

Intense utilization and mining of fossil fuels for energy production have resulted in environmental pollution and climate change. Compared to fossil fuels, microalgae is considered as a promising candidate for biodiesel production due to its fast growth rate, high lipid content and no occupying arable land. However, monocultural microalgae bear high cost of harvesting, and are prone to contamination, making them incompetent compared with traditional renewable energy sources. Co-culture system induces self-flocculation, which may reduce the cost of microalgae harvesting and the possibility of contamination. In addition, the productivity of lipid and high-value by-products are higher in co-culture system. Therefore, co-culture system represents an economic, energy saving, and efficient technology. This review aims to highlight the advances in the co-culture system, including the mechanisms of interactions between microalgae and other microorganisms, the factors affecting the lipid production of co-culture, and the potential applications of co-culture system. Finally, the prospects and challenges to algal co-culture systems were also discussed.
Biofuels ; Biomass ; Coculture Techniques ; Flocculation ; Microalgae

Lager yeast is the most popular yeast strain used for beer production in China. The flocculation of yeast plays an important role in cell separation at the end of fermentation. Therefore, appropriately enhancing the flocculation capability of the lager yeast without affecting its fermentation performance would be desirable for beer industry. Our previous study showed that the defect of gene RIM21 might contribute to the enhanced flocculation capability of a lager yeast G03. To further investigate the role of the RIM21 gene in flocculation of strain G03, this study constructed a RIM21-deleted mutant strain G03-RIM21Δ through homologous recombination. Deletion of RIM21 improved the flocculation capability of strain G03 during wort fermentation at 11 °C without changing its fermentation performance significantly. The expression of FLO5, Lg-FLO1 and some other genes involved in cell wall integrity pathway were up-regulated in strain G03-RIM21Δ. In addition, the disruption of RIM21 enhanced resistance of yeast cells to cell wall inhibitors. These results provide a basis for elucidating the flocculation mechanism of lager yeast under low-temperature fermentation conditions.
Beer ; Fermentation ; Flocculation ; Receptors, Cell Surface ; Saccharomyces/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

immunoprecipitation assay were employed to confirm the direct target of miR-21. MTT assay, flow cytometric analysis, and transwell assay were used to evaluate cell proliferation, apoptosis, and migration and invasion capacities, respectively.RESULTS: Curcumin repressed the proliferation, migration, and invasion abilities and promoted apoptosis in SU-DHL-8 cells. Curcumin inhibited miR-21 expression and curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis effects by miR-21 in SU-DHL-8 cells. VHL was a direct target of miR-21. Moreover, curcumin exerted its regulatory effects on SU-DHL-8 cells by VHL.CONCLUSION: Curcumin exerted its anti-proliferation, anti-migration, anti-invasion, and pro-apoptosis functions, at least partly, by repressing miR-21 and regulating VHL expression in DLBCL cell line. Our findings provided a possible molecular mechanism of curcumin-mediated anti-cancer effect.]]>
Apoptosis ; B-Lymphocytes ; Blotting, Western ; Carcinogenesis ; Caspase 3 ; Cell Line ; Cell Proliferation ; Curcumin ; Humans ; Immunoprecipitation ; In Situ Hybridization ; Lymphoma ; Lymphoma, B-Cell ; MicroRNAs ; Real-Time Polymerase Chain Reaction ; RNA ; RNA, Messenger

PURPOSE: Reduction-oxidation reaction homeostasis is vital for regulating inflammatory conditions and its dysregulation may affect the pathogenesis of chronic airway inflammatory diseases such as asthma. Peroxiredoxin-6, an important intracellular anti-oxidant molecule, is reported to be highly expressed in the airways and lungs. The aim of this study was to analyze the expression pattern of peroxiredoxin-6 in the peripheral blood mononuclear cells (PBMCs) of asthmatic patients and in bronchial epithelial cells (BECs).METHODS: The expression levels and modifications of peroxiredoxin-6 were evaluated in PBMCs from 22 asthmatic patients. Phosphorylated and acetylated peroxiredoxin-6 in hydrogen peroxide-treated human BECs was detected using immunoprecipitation analysis. The expression level of peroxiredoxin-6 was also investigated in BECs treated with hydrogen peroxide. Cycloheximide and proteasome inhibitors were used to determine whether peroxiredoxin-6 is degraded by proteasomes.RESULTS: Peroxiredoxin-6 expression was significantly reduced in the PBMCs of asthmatic patients compared to control subjects. Distinct modification patterns for peroxiredoxin-6 were observed in the PBMCs of asthmatic patients using 2-dimensional-electrophoresis. The levels of phosphorylated serine and acetylated lysine in peroxiredoxin-6 were significantly increased in the BECs following hydrogen peroxide treatment. The level of peroxiredoxin-6 expression was reduced in hydrogen peroxide-stimulated BECs, presumably due to proteasomes.CONCLUSIONS: The expression of peroxiredoxin-6, which is down-regulated in the immune cells of asthmatic patients and BECs, can be modified by oxidative stress. This phenomenon may have an effect on asthmatic airway inflammation.
Asthma ; Cycloheximide ; Epithelial Cells ; Homeostasis ; Humans ; Hydrogen ; Hydrogen Peroxide ; Immunoprecipitation ; Inflammation ; Lung ; Lysine ; Oxidative Stress ; Proteasome Inhibitors ; Protein Processing, Post-Translational ; Serine

OBJECTIVE: To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data. METHODS: Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis. RESULTS: Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%. CONCLUSIONS Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.
Chromatin Immunoprecipitation ; DNA ; Gene Library ; High-Throughput Nucleotide Sequencing ; Humans ; Reproducibility of Results ; Sequence Analysis, DNA