TLC-bioautography-guided identification and assessment of the antibacterial compounds from Feijoa sellowiana.

INTRODUCTION: A rapid procedure was developed for the targeted isolation and assessment of antibacterial compounds from plant-based materials. The effectiveness of this method was demonstrated using Feijoa sellowiana fruit peels. OBJECTIVE: The objectives of this study are as follows: develop an efficient procedure utilizing direct thin-layer chromatography (TLC)-bioautography to facilitate the targeting, identification, and purification of antibacterial compounds from plant extracts and delineate a method based on TLC-bioautography to determine the minimum effective dose (MED), alongside a colorimetric broth microdilution aided by high-performance liquid chromatography (HPLC) for evaluating the isolated active compounds. METHODOLOGY: Active compounds were targeted using TLC-bioautography against Staphylococcus aureus, and the identification was achieved through liquid chromatography-mass spectrometry (LC-MS) combined with Compound Discoverer. Purification was carried out using a customized separation method. The structure was confirmed using nuclear magnetic resonance (NMR) spectroscopy. The MED, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were determined by two enhanced antibacterial assays. RESULTS: The main antibacterial compound identified was flavone. A TLC-bioautography-based antibacterial assay and a colorimetric broth microdilution assisted by HPLC were described as the enhanced antibacterial assay protocols. The MED, MIC, and MBC of flavone against S. aureus were found to be 4.2-5.2 µg/cm2, 225-275 µg/mL, and 550-650 µg/mL, respectively. Similarly, the MED, MIC, and MBC against Escherichia coli were determined to be 5.2-6.1 µg/cm2, 325-375 µg/mL, and 375-425 µg/mL, respectively. CONCLUSION: This study proposed an enhanced bioassay-guided separation technique for the isolation of antibacterial compounds from plants, along with two improved methods for assessing the antibacterial efficacy of insoluble or colored compounds.

TLC-Bioautography-Guided Isolation and Assessment of Antibacterial Compounds from Manuka (Leptospermum scoparium) Leaf and Branch Extracts.

A rapid procedure for the targeted isolation of antibacterial compounds from Manuka (Leptospermum scoparium) leaf and branch extracts was described in this paper. Antibacterial compounds from three different Manuka samples collected from New Zealand and China were compared. The active compounds were targeted by TLC-bioautography against S. aureus and were identified by HR-ESI-MS, and -MS/MS analysis in conjunction with Compound Discoverer 3.3. The major antibacterial component, grandiflorone, was identified, along with 20 ß-triketones, flavonoids, and phloroglucinol derivatives. To verify the software identification, grandiflorone underwent purification via column chromatography, and its structure was elucidated through NMR analysis, ultimately confirming its identity as grandiflorone. This study successfully demonstrated that the leaves and branches remaining after Manuka essential oil distillation serve as excellent source for extracting grandiflorone. Additionally, we proposed an improved TLC-bioautography protocol for evaluating the antibacterial efficacy on solid surfaces, which is suitable for both S. aureus and E. coli. The minimum effective dose (MED) of grandiflorone was observed to be 0.29-0.59 µg/cm2 against S. aureus and 2.34-4.68 µg/cm2 against E. coli, respectively. Furthermore, the synthetic plant growth retardant, paclobutrazol, was isolated from the samples obtained in China. It is hypothesized that this compound may disrupt the synthesis pathway of triketones, consequently diminishing the antibacterial efficacy of Chinese Manuka extract in comparison to that of New Zealand.

An improved MTT colorimetric method for rapid viable bacteria counting.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay has been employed in the analysis of bacterial growth. In comparison to experiments conducted on mammalian cells, the MTT bacterial assay encounters a greater number of interfering factors and obstacles that impact the accuracy of results. In this study, we have elucidated an improved MTT assay protocol and put forth an equation that establishes a correlation between colony-forming units (CFU) and the amount of formazan converted by the bacteria, drawing upon the fundamental principle of the MTT assay. This equation is represented as CFU=kF. Furthermore, we have explicated a methodology to determine the scale factor "k" by employing S. aureus and E. coli as illustrative examples. The findings indicate that S. aureus and E. coli reduce MTT by a cyclic process, from which the optimal reduction time at room temperature was determined to be approximately 30 mins. Furthermore, individual E. coli exhibits an MTT reduction capacity approximately four times greater than that of S. aureus. HPLC analysis proves to be the most accurate method for mitigating interferences during the dissolution and quantification of formazan. Additionally, this study has identified a new constraint related to the narrow linear range (0-125 µg/mL) of formazan concentration-absorbance and has presented strategies to circumvent this limitation.

Assessing the dynamic resilience of Urban Rail Transit Networks during their evolution using a ridership-weighted network.

The assessment of the resilience of Urban Rail Transit Networks (URTNs) and the analysis of their evolutionary characteristics during network growth can help in the design of efficient, safe, and sustainable networks. However, there have been few studies regarding the change of resilience in long-term network development. As for the existing resilience studies, they rarely consider the entire cycle of accident occurrence and repair; furthermore, they ignore the changes in network transportation performance during emergencies. Moreover, the measurement metrics of the important nodes have not been comprehensively considered. Therefore, to remedy these deficiencies, this paper proposes a URTN dynamic resilience assessment model that integrates the entire cycle of incident occurrence and repair, and introduces the network transport effectiveness index E(Gw) to quantitatively assess the network resilience. In addition, a weighted comprehensive identification method of the important nodes (the WH method) is proposed. The application considers the Xi'an urban rail transit network (XURTN) during 2011-2021. The obtained results identify the resilience evolutionary characteristics during network growth. And longer peripheral lines negatively affect the resilience of XURTN during both the attack and the repair processes. The central city network improves the damage index Rdam and the recovery index Rrec by up to 123.46% and 11.65%, respectively, over the overall network. In addition, the WH method can comprehensively and accurately identify the important nodes in the network and their evolutionary characteristics. Compared to the single-factor and topological strategies, the Rdam is 1.17%~178.89% smaller and the Rrec is 1.68%~84.81% larger under the WH strategy. Therefore, this method improves the accuracy of the important node identification. Overall, the insights from this study can provide practical and scientific references for the synergistic development of URTN and urban space, the enhancement of network resilience, and the protection and restoration of important nodes.

Vulnerability assessment of freeway network considering the probabilities and consequences from a perspective based on network cascade failure.

Freeway networks are vulnerable to natural disasters and man-made disruptions. The closure of one or more toll stations of the network often causes a sharp decrease in freeway performance. Therefore, measuring the probability and consequences of vulnerability to identify critical parts in the network is crucial for road emergency management. Most existing techniques only measure the consequences of node closure and rarely consider the probability of node closure owing to the lack of an extensive historical database; moreover, they ignore highways outside the study area, which can lead to errors in topological analysis and traffic distribution. Furthermore, the negative effects produced by the operation of freeway tunnels in vulnerability assessment have been neglected. In this study, a framework for freeway vulnerability assessment that considers both the probability and consequences of vulnerability is proposed, based on the perspective of network cascade failure analysis. The cascade failure analysis is conducted using an improved coupled map lattice model, developed by considering the negative effects of tunnels and optimizing the rules of local traffic redistribution. The perturbation threshold and propagation time step of network cascade failure are captured to reflect the probabilities and consequences of vulnerability. A nodal vulnerability index is established based on risk assessment, and a hierarchical clustering method is used to identify the vulnerability classification of critical nodes. The freeway network of Fuzhou in China is utilized to demonstrate the effectiveness of the proposed approach. Specifically, the toll stations in the study area are classified into five clusters of vulnerability: extremely high, high, medium, low, and extremely low. Approximately 31% of the toll stations were classified as the high or extremely high cluster, and three extremely vulnerable freeway sections requiring different precautions were identified. The proposed network vulnerability analysis method provides a new perspective to examine the vulnerability of freeway networks.

Codelivery of DOX and siRNA by folate-biotin-quaternized starch nanoparticles for promoting synergistic suppression of human lung cancer cells.

In this paper, the self-assembled folate-biotin-quaternized starch nanoparticles (FBqS NPs) were used as carrier system of doxorubicin (DOX) and siRNAIGF1R for the codelivery of both into human lung adenocarcinoma cell lines (A549 cells) in vitro. The cytotoxicity, targeted ligand competition, cell proliferation inhibition, cellular uptake, endocytosis mechanism and target protein suppression of drug-loaded FBqS NPs were evaluated in detail. Compared with several other drug formulations under same condition, siRNAIGF1R/DOX/FBqS NPs exhibited the greatest cytotoxicity to A549 cells and the cytotoxicity was competitively inhibited by free folate in dose-dependent manner. The A549 cells treated by siRNAIGF1R/DOX/FBqS NPs showed the lowest cell proliferation capacity. The energy-dependent clathrin- and caveolae-mediated endocytosis might be the primary cellular uptake mechanism of drug-loaded FBqS NPs. The expression of IGF1R protein in A549 cells treated by siRNAIGF1R/FBqS NPs declined dramatically. So the FBqS NPs were expected as the co-carrier system of chemotherapeutants and siRNAs for future clinical application.

Oral insulin delivery by carboxymethyl-ß-cyclodextrin-grafted chitosan nanoparticles for improving diabetic treatment.

In this paper, a new oral insulin formulation, insulin-loaded carboxymethyl-ß-cyclodextrin-grafted chitosan nanoparticles (insulin/CMCD-g-CS NPs), was fabricated by ionic crosslinking technique. The therapeutic efficacy of new formulation was investigated in detail. Firstly, the CMCD-g-CS was synthesized by EDC-mediated esterification reaction. The prepared CMCD-g-CS exhibited favourable loading capacity and encapsulation efficiency of drug. The release experiment in vitro showed that the nanocarrier could efficiently protect encapsulated insulin at simulated gastric environment and release drug in the simulated colonic fluid. The insulin/CMCD-g-CS NPs effectively promoted drug internalization into Caco-2 cells and could reversibly open the tight junction between cells. The oral administration of insulin/CMCD-g-CS NPs could lastingly decrease blood sugar level in diabetic mice. The liver function study verified that the insulin/CMCD-g-CS NPs had not obvious toxicity to experimental mice. Therefore, the CMCD-g-CS could be an effective and safe oral insulin delivery carrier for future clinical application. A new biocompatible polysaccharide nanoparticle was fabricated as oral insulin delivery carrier for improving diabetic treatment.

Fabrication of self-assembled folate-biotin-quaternized starch nanoparticles as co-carrier of doxorubicin and siRNA.

In this paper, the starch was firstly modified by quaternary reagent to obtain cationic starch. Then self-assembled folate-biotin-quaternized starch nanoparticles were prepared by a one-pot synthesis via N,N'-dicyclohexylcarbodiimide/N-hydroxysuccinimide/4-dimethylaminopyridine-mediated esterification reaction. The physicochemical properties of the prepared folate-biotin-quaternized starch nanoparticles were characterized. The average diameter of folate-biotin-quaternized starch nanoparticles was 109 nm with polydispersity index of 0.183 and zeta potential of 28.59 mV. The folate-biotin-quaternized starch nanoparticles were used as co-carrier of siRNA and doxorubicin with satisfactory drug loading capacity (6.98%) and encapsulation efficiency (69.66 %), and siRNA could be efficiently encapsulated at 40/1 weight ratio of doxorubicin/folate-biotin-quaternized starch nanoparticles to siRNA. The folate-biotin-quaternized starch nanoparticles could effectively protect siRNA from degradation of serum RNAase for up to 48 h. The release characteristics of doxorubicin and siRNA from folate-biotin-quaternized starch nanoparticles were studied in different pH environment and the release behaviors of two drugs were all pH sensitive. The folate-biotin-quaternized starch nanoparticles as a potential co-carrier of anticancer agents and gene drugs was expected to achieve future practical application in vitro and in vivo.

Optimization of the design of pre-signal system using improved cellular automaton.

The pre-signal system can improve the efficiency of intersection approach under rational design. One of the main obstacles in optimizing the design of pre-signal system is that driving behaviors in the sorting area cannot be well evaluated. The NaSch model was modified by considering slow probability, turning-deceleration rules, and lane changing rules. It was calibrated with field observed data to explore the interactions among design parameters. The simulation results of the proposed model indicate that the length of sorting area, traffic demand, signal timing, and lane allocation are the most important influence factors. The recommendations of these design parameters are demonstrated. The findings of this paper can be foundations for the design of pre-signal system and show promising improvement in traffic mobility.