Deciphering the Relationship between Cell Growth and Cell Cycle in Individual Escherichia coli Cells by Flow Cytometry.

Accurate coordination of chromosome replication and cell division is essential for cellular processes, yet the regulatory mechanisms governing the bacterial cell cycle remain contentious. The lack of quantitative data connecting key cell cycle players at the single-cell level across large samples hinders consensus. Employing high-throughput flow cytometry, we quantitatively correlated the expression levels of key cell cycle proteins (FtsZ, MreB, and DnaA) with DNA content in individual bacteria. Our findings reveal distinct correlations depending on the chromosome number (CN), specifically whether CN ≤2 or ≥4, unveiling a mixed regulatory scenario in populations where CN of 2 or 4 coexist. We observed function-dependent regulations for these key proteins across nonoverlapping division cycles and various nutrient conditions. Notably, a logarithmic relationship between total protein content and replication origin number across nutrient conditions suggests a unified mechanism governing cell cycle progression, confirming the applicability of Schaechter's growth law to cells with CN ≥4. For the first time, we established a proportional relationship between the synthesis rates of key cell cycle proteins and chromosome dynamics in cells with CN ≥4. Drug experiments highlighted CN 2 and 4 as pivotal turning points influencing cellular resource allocation. This high-throughput, single-cell analysis provides interconnected quantitative insights into key molecular events, facilitating a predictive understanding of the relationship between cell growth and cell cycle.

Nanoleaf-derived carbon materials as a sensitivity coating for solid­phase microextraction of polycyclic aromatic hydrocarbons.

Metal-organic framework-derived carbon materials have shown extensive application in the sensitive extraction of polycyclic aromatic hydrocarbons (PAHs), but more active sites for its adsorption were still a tireless pursuit. In this study, ZIF-nanoleaf-derived carbon (NLCs) was synthesized and developed as a solid-phase microextraction (SPME) fiber (NLCs-F). The extraction performance was compared with ZIF-dodecahedron-derived carbon (DHCs) coated fiber (DHCs-F), which was prepared by only changing the ratio of the reactants. The unique morphology of NLCs provided abundant adsorption active sites for the selected PAHs, while the large average aperture facilitated selective extraction of high molecular weight analytes. Additionally, the high carbon content enhanced the strong enrichment capability for hydrophobic PAHs. Hence, the prepared NLCs-F coupled with GC-MS showed a good correlation coefficient (0.9975) in a wide linear range, low limits of detection (0.3-1.8 ng L-1), satisfactory repeatability, and reproducibility, which made it apply in the enrichment of PAHs in actual tea and coffee samples.

The effects of different extraction methods on the structure and antioxidant properties of Bletilla striata polysaccharide.

Bletilla striata polysaccharide (BSP) is one of the main active ingredients of the traditional Chinese medicine Bletilla striata (Thunb) Richb.f and the extraction method of BSP has a significant impact on its properties. This study investigated the effects of four extraction methods, namely hot water extraction, ultrasonic extraction, enzyme extraction, and microwave extraction, on the structure and antioxidant properties of BSP. Characterization results from FTIR and NMR showed that all four BSP consisted mainly of glucose and mannose, forming α-glycosidic and ß-glycosidic linkages to form glucomannan. Hot water extraction had the lowest extraction rate of BSP at 21.78% ± 0.73%. The polysaccharide BSP-H obtained from hot water extraction had the smallest absolute Zeta potential and Grain size, but the largest molecular weight at 204 kDa. It exhibited the best thermal stability and superior antioxidant activity compared to polysaccharides extracted using the other three methods, as evaluated by three different antioxidant assays. Although the antioxidant activity of BSP-V was slightly weaker, it showed a significant improvement compared to the remaining two polysaccharides. These results suggest that hot water extraction is the most suitable method for large-scale application of BSP, preserving its activity effectively, thus facilitating practical production and product development.

Effects of Different Drying Methods on the Quality of Bletilla striata Scented Tea.

Flower tea is widely loved as a drink, especially for the beautiful and rich flowers of the orchid family, and the drying method for different flowers is also unique. GC-MS is widely used to study volatile substances to determine the quality of flower teas. The findings show that the freeze-drying method can retain the original aroma and flavor of Bletilla striata has the highest sensory evaluation score, with the key flavor substances ethyl caproate and N-heptanal containing 1.14% and 6.28%, respectively, and their ROAV values reaching 54.46 and 100.00. Additionally, the freeze-drying method can well retain flavonoids, polysaccharides, and phenolic components, while providing better antioxidant and antibacterial properties. The stove-drying method would make Bletilla striata slightly burnt and less flavorful and efficacious than freeze-drying; the air-drying method is difficult to retain the special odor and fragrance of Bletilla striata flowers and has the lowest sensory evaluation score, with the presence of volatile components with irritating and unpleasant odors such as pyrazine and 2-pentylfuran, while not showing better efficacy. In addition, steam fixation would destroy the morphology and flavor of Bletilla striata, lose polysaccharide and phenolic components, and reduce the efficacy of Bletilla striata scented tea, but could retain the flavonoid components well. In summary, direct freeze-drying without steam fixation is the best process for drying Bletilla striata scented tea, stove-drying without steam fixation is more economical and convenient in actual production and application, steam fixation and air-drying are not suitable as drying processes for Bletilla striata scented tea. This study analyzed the quality of Bletilla striata scented tea under different drying methods, promoted the further processing of Bletilla striata scented tea, and provided a reference for the comprehensive utilization of Bletilla striata scented tea.

[Correlation research of photosynthetic characteristics and medicinal materials production with 4 Uncariae Cum Uncis].

Using four Uncariae Cum Uncis materials including Uncaria sinensis (HGT), U. hirsutea (MGT), Jianhe U. rhynchophylla (JHGT) and U. rhynchophylla(GT) as the research objects, the correlations between medicinal materials' yield and photosynthetic ecophysiology-factors in the plant exuberant growth period were studied. Results showed that the Uncaria plants net photosynthetic rate (Pn) changed by unimodal curve. There was not "midday depression" phenomenon. There was a different relationship among the photosynthetic ecophysiology-factors and between photosynthetic ecophysiology-factors and medicinal materials' yield. Pn,Tl,Gs had a significant correlation with medicinal materials' yield（M）and were the most important factors of growth.

Discovery of an EP300 Inhibitor using Structure-based Virtual Screening and Bioactivity Evaluation.

BACKGROUND: EP300 (E1A binding protein p300) played a significant role in serial diseases such as cancer, neurodegenerative disease. Therefore, it became a significant target. METHODS: Targeting EP300 discovery of a novel drug to alleviate these diseases. In this paper, 17 candidate compounds were obtained using a structure-based virtual screening approach, 4449-0460, with an IC50 of 5.89 ± 2.08 uM, which was identified by the EP300 bioactivity test. 4449-0460 consisted of three rings. The middle benzene ring connected the 5-ethylideneimidazolidine-2,4-dione group and the 3-F-Phenylmethoxy group. RESULTS: Furthermore, the interaction mechanism between 4449-0460 and EP300 was explored by combining molecular dynamics (MD) simulations and binding free energy calculation methods. CONCLUSION: The binding free energy of EP300 with 4449-0460 was -10.93 kcal/mol, and mainly came from the nonpolar energy term (ΔGnonpolar). Pro1074, Phe1075, Val1079, Leu1084, and Val1138 were the key residues in EP300/4449-0460 binding with more -1 kcal/mol energy contribution. 4449-0460 was a promising inhibitor targeting EP300, which had implications for the development of drugs for EP300-related diseases.

Discovery of Two GSK3ß Inhibitors from Sophora flavescens Ait. using Structure-based Virtual Screening and Bioactivity Evaluation.

OBJECTIVE: Kushen (Sophora flavescens Ait.) has a long history of medicinal use in China due to its medicinal values, such as antibacterial, antiviral, and anti-inflammatory. Rapid discovery of the components and the medicinal effects exerted by Kushen will help elucidate the science of Kushen in curing diseases. GSK3ß (glycogen synthase kinase-3 beta) is a protein kinase with a wide range of physiological functions, such as antibacterial, antiviral, and anti-inflammatory. The discovery of inhibitors targeting GSK3ß from Kushen was not only helpful for the rapid discovery of the components responsible for the efficacy of Kushen but also important for the development of novel drugs. METHODS: In this study, the chemical composition of Kushen was extracted from the TMSCP database. Molecular docking, GSK3ß enzyme assay, and molecular dynamics simulations were used to discover the GSK3ß inhibitors from the chemical composition of Kushen. RESULTS: A total of 113 chemical compositions of Kushen were extracted from the TMSCP database. Molecular docking indicated that 15 chemical compositions of Kushen scored better than -8 kcal/mol against GSK3ß. GSK3ß enzyme assay demonstrated several inhibitory activities of kushenol I and kushenol F with IC50 values of 7.53 ± 2.55 µM and 4.96 ± 1.29 µM, respectively. Molecular dynamics simulations were used to reveal the interactions of kushenol I and kushenol F with GSK3ß from structural and energetic perspectives. CONCLUSION: Kushenol I and kushenol F could be the material basis for the antibacterial, antiviral, and anti-inflammatory properties of Kushen.

Process optimization and character evaluation of Bletilla striata polysaccharide (BSP) and chitosan (CS) composite hemostatic sponge (BSP-CS).

Bletilla striata polysaccharide (BSP) and chitosan (CS) were chemically cross-linked using oxalyl chloride to prepare a composite hemostatic sponge (BSP-CS), and the process parameters were optimized using the Box-Behnken design (BBD) with response surface methodology. To optimize the performance of the hemostatic sponge, we adjusted the ratio of independent variables, the amount of oxalyl chloride added, and the freeze-dried volume. A series of evaluations were conducted on the hemostatic applicability of BSP-CS. The characterization results revealed that BSP-CS had a stable bacteriostatic effect on Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa within 72 h, and the bacteriostatic rate was above 30%. The CCK-8 cytotoxicity test demonstrated that BSP-CS had a certain effect on promoting cell proliferation of L929 cells. In the mouse tail-cutting experiment, the hemostasis time of BSP-CS was 463.0±38.16 s, shortened by 91.3 s on average compared with 554.3±34.67 s of the gauze group. The blood loss of the BSP-CS group was 28.47±3.74 mg, which was 34.7% lower than that of the control gauze group (43.6±3.83 mg). In the in vitro coagulation experiment, the in vitro coagulation index of the BSP-CS group was 97.29%±1.8%, which was reduced to 8.6% of the control group. The CT value of the BSP-CS group was 240±15 s, which was 155 s lower than that of the gauze group (355±31.22 s). All characterization results indicate that BSP-CS is an excellent hemostatic material.

A scalable field study using leaves as a novel passive air sampler to evaluate the potential source of organophosphate esters in street dust.

Organophosphate esters (OPEs) are widely used as flame retardants and plasticizers in industrial and commercial products. It is generally believed that OPEs in street dust mainly originate from road traffic and anthropogenic activities. The influence of atmospheric deposition is still unknown. In this study, leaves were employed as a novel passive air sampler to collect particle matters (PM) in 12 cities in the central province of Henan, China. Similar compositional profiles of OPEs were found in street dust and PM samples. The concentrations of individual OPEs in PM were 1-4 times higher than in street dust. Chlorinated OPEs concentration in PM shows a moderate correlation (r2 = 0.538, p < 0.01) with that in street dust. The concentration of alkyl OPEs in PM has a high correlation (r2 = 0.843, p < 0.01) with that in street dust. No significant correlation (r2 = 0.133, p = 0.132) was found on the aryl OPEs concentrations between street dust and PM. Spearman correlation reveals that the emission sources of tricresyl phosphate (TCrP) and triethyl phosphate (TEP) may be different from other OPEs in dust and PM samples. Principle component analysis (PCA) provides an appropriate explanation that tris (2-chloroethyl) phosphate (TCEP), triphenyl phosphate (TPhP), tris (chloropropyl) phosphate (TCPP), tributyl phosphate (TnBP), and TEP in street dust and PM may be emitted from the same sources, suggesting that PM has a significant influence on the occurrence of OPEs in street dust. The estimated dry deposition fluxes of particle-bound OPEs show a significant correlation (R2 = 0.969, p < 0.01) with OPEs concentrations in street dust, revealing that the input of atmospheric deposition could be a major source of OPEs in street dust.

Effects of biochar on the degradation of organophosphate esters in sewage sludge aerobic composting.

In this study, the impact of biochar on the degradation of organophosphate esters (OPEs) during the aerobic composting of sewage sludge was investigated. Three treatments were conducted with different percentages of biochar in the compost, including 5 %, 10 %, and 20 %. The treatment with 10 % of biochar showed the longest thermophilic phase compared to that of 5 % and 20 % of biochar, which greatly promoted the decomposition of organic matter. In addition, the degradation rate of the hard-to-degrade chlorinated-OPEs was significantly increased by 10 % biochar, reaching to 57.2 %. Correspondingly, approximately 43.6 % of the total concentration of OPEs (Σ6OPEs) was eliminated in the presence of 10 % of biochar, which was higher than the treatments with 5 % and 20 % of biochar. Biochar significantly influenced the microbial community structure of compost, but the previously reported organophosphorus-degrading bacteria did not play a major role in the degradation of OPEs. The redox ability of the increased oxygen-containing functional groups such as quinone on the surface of biochar and the biochar-mediated electron transfer ability may play an essential role in the degradation of OPEs during the composting process.

Dual-fluorescent bacterial two-hybrid system for quantitative Protein-Protein interaction measurement via flow cytometry.

Characterization of protein-protein interactions (PPIs) is essential for understanding cellular signal transduction pathways. However, quantitative measurement of the binding strength remains challenging. Building upon the classical bacterial adenylate cyclase two-hybrid (BACTH) system, we previously demonstrated that the relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, is an intrinsic characteristic associated with the binding strength between the two interacting proteins. In this study, we inserted fluorescent protein tdTomato in the chromosome as the reporter protein by CRISPR/Cas9 technology and employed a 12-amino acid tetracysteine (TC) to tag one of the interacting proteins, which can be further labeled by a membrane-permeable biarsenical dye. The combined use of tdTomato and TC-tag offers rapid and high-throughput analysis of the expression levels of both the reporter protein and one of the interacting proteins at the single-cell level by multicolor flow cytometry, which simplifies the quantitative measurement of PPI. The use of the as-developed RRPE-tdTomato-TC-BACTH approach was demonstrated in three demanding applications. First, binding affinities could be correctly ranked for discriminating interaction strengths with a tenfold difference or of the same order of magnitude. We demonstrate that the method is sensitive enough to discriminate affinities with a small difference of 1.4-fold. Moreover, residues involved in PPI can be easily mapped and ranked. Lastly, protein interaction inhibitors can be rapidly screened.

Full Transcriptome Analysis of Callus Suspension Culture System of Bletilla striata.

BACKGROUND: Bletilla striata has been widely used in the pharmacology industry. To effectively produce the secondary metabolites through suspension cultured cells of B. striata, it is important to exploring the full-length transcriptome data and the genes related to cell growth and chemical producing of all culture stages. We applied a combination of Real-Time Sequencing of Single Molecule (SMRT) and second-generation sequencing (SGS) to generate the complete and full-length transcriptome of B. striata suspension cultured cells. METHODS: The B. striata transcriptome was formed in de novo way by using PacBio isoform sequencing (Iso-Seq) on a pooled RNA sample derived from 23 samples of 10 culture stages, to explore the potential for capturing full-length transcript isoforms. All unigenes were obtained after splicing, assembling, and clustering, and corrected by the SGS results. The obtained unigenes were compared with the databases, and the functions were annotated and classified. RESULTS AND CONCLUSIONS: A total of 100,276 high-quality full-length transcripts were obtained, with an average length of 2530 bp and an N50 of 3302 bp. About 52% of total sequences were annotated against the Gene Ontology, 53,316 unigenes were hit by KOG annotations and divided into 26 functional categories, 80,020 unigenes were mapped by KEGG annotations and clustered into 363 pathways. Furthermore, 15,133 long-chain non-coding RNAs (lncRNAs) were detected. And 68,996 coding sequences were identified based on SSR analysis, among which 31 pairs of primers selected at random were amplified and obtained stable bands. In conclusion, our results provide new full-length transcriptome data and genetic resources for identifying growth and metabolism-related genes, which provide a solid foundation for further research on its growth regulation mechanisms and genetic engineering breeding mechanisms of B. striata.

Antibacterial bibenzyl derivatives from the tubers of Bletilla striata.

Ten previously undescribed bibenzyl derivatives (bletistrins A-J), including 5 that have hydroxyl-substituted chiral centres on the aliphatic bibenzyl bridge, along with twelve known bibenzyl derivatives, were isolated from the rhizomes of Bletilla striata. The structures of bletistrins A-J were primarily elucidated on the basis of their 1D and 2D NMR spectroscopic data. The absolute configurations of bletistrins A, D, F, H and I were determined by electronic circular dichroism (ECD) spectroscopic analysis and optical rotation value. Most of the isolated compounds were evaluated for their antibacterial activities against 3 g-positive bacterial strains and 1 g-negative bacterial strain. Bletistrins F, G, and J, bulbocol, shanciguol and shancigusin B showed inhibitory activities, with MICs of (3-28 µg/mL) against S. aureus ATCC 6538.

Antibacterial stilbenes from the tubers of Bletilla striata.

Three new bibenzyl derivatives (bletstrins A-C, 1-3), including two bibenzyls that have hydroxyl-substituted chiral centers on the aliphatic bibenzyl bridge, along with eighteen known stilbenoids (4-21) were isolated from the tubers of Bletilla striata. The structures of new compounds were elucidated by the use of 1D/2D NMR spectroscopic data. The absolute configurations of bletitrins A and B were determined by optical rotation value. Compounds 13-16 were isolated from the Orchidaceae for the first time. Most of the isolated compounds were evaluated for their antibacterial activities against three gram-positive bacterial strains and one gram-negative bacterial strain. Compounds 4, 10, 12, 14, 15, 16 and 18 showed potent inhibitory activities, with MICs of (6-52 µg/mL) against S. aureus ATCC 6538.