[Critical influencing factors on quality of traditional Chinese medicine preparations from perspective of process and equipment].

The quality of traditional Chinese medicine preparations is directly related to the safety of patients. Among the various factors, the process and corresponding critical equipment are critical factors influencing the quality of the preparations. To improve the quality of traditional Chinese medicine preparations, this article summarizes and analyzes the problems in the process links and corresponding critical equipment in the manufacturing process of traditional Chinese medicine preparations. Furthermore, a critical quality attribute evaluation system is established based on safety and effectiveness combined with the drug properties, preparation process, and preparation characteristics, providing a basis for the process and equipment improvements aimed at quality enhancement.

[Scientific connotation of temperature control in processing of Chinese medicinal materials based on theory of quality evaluation through morphological identification].

Processing of Chinese medicinal materials is an important part in the Chinese medicine heritage, and the temperature control in the processing has a direct impact on the quality and efficacy of traditional Chinese medicines. However, the processing of Chinese medicinal materials has the problems of subjective temperature judgement, determination of the end point based on experience, unclear processing mechanism, unstable quality of products, and inconsistent processing standards. The temperature control in the processing is reflected in the appearance and internal quality of Chinese medicinal materials. The theory of quality evaluation through morphological identification is developed based on the comprehensive evaluation of the shape, color, taste, and components, which is associated with the temperature control in the processing. To solve the problems above, this paper puts forward the following solutions. The first is literature mining. By review of the ancient medical works and pharmaceutical experience, the temperature control in processing and the evolution of processing methods can be revealed. Second, according to the ancient method, the processing principle can be explored, on the basis of which the processing technology can be innovated. Third, the standard operating procedure(SOP) should be established to quantify the fire temperature, providing a theoretical basis for the formulation of Chinese medicinal material processing standards. Moreover, it provides a basis for improving the quality of processed products and increasing the safety and effectiveness of traditional Chinese medicines.

[Current situation and development trend of digital traditional Chinese medicine pharmacy].

The 21st century is a highly information-driven era, and traditional Chinese medicine(TCM) pharmacy is also moving towards digitization and informatization. New technologies such as artificial intelligence and big data with information technology as the core are being integrated into various aspects of drug research, manufacturing, evaluation, and application, promoting interaction between these stages and improving the quality and efficiency of TCM preparations. This, in turn, provides better healthcare services to the general population. The deep integration of emerging technologies such as artificial intelligence, big data, and cloud computing with the TCM pharmaceutical industry will innovate TCM pharmaceutical technology, accelerate the research and industrialization process of TCM pharmacy, provide cutting-edge technological support to the global scientific community, boost the efficiency of the TCM industry, and promote economic and social development. Drawing from recent developments in TCM pharmacy in China, this paper discussed the current research status and future trends in digital TCM pharmacy, aiming to provide a reference for future research in this field.

Natural product piperlongumine inhibits proliferation of oral squamous carcinoma cells by inducing ferroptosis and inhibiting intracellular antioxidant capacity.

Background: As a new form of cell death, ferroptosis has been shown to have inhibitory effects on a variety of tumor cells except oral squamous cell carcinoma (OSCC). There were few investigations on the effects and molecular mechanisms of piperlongumine (PL, a ferroptosis inducer) and CB-839 (a GLS1 inhibitor which promotes ferroptosis) on OSCC cells. This article assesses the anticancer effect and mechanism of PL as well as combined with CB-839. Methods: OSCC cells were treated with specified concentration of PL alone or with ferroptosis inhibitor Ferrostatin-1 (Fer-1) and antioxidant N-Acetylcysteine (NAC) to assess their effects on biological characteristics such as cell proliferation, cell death and intracellular ferroptosis related pathways. Also, cells were treated with PL combined with CB-839 to evaluate the synergistic effect of CB-839 on PL's anticancer effects. Results: The results showed that the proliferation rate of PL-treated OSCC cells were decreased in a dose- and time-dependent manner. PL can induce OSCC cells apoptosis. Lipid peroxidation (LPO) and intracellular reactive oxygen species (ROS) were accumulated after PL treatment. We found some protein changes significantly such as the expression of DMT1 increased, and the expression of FTH1, SLC7A11 and GPX4 decreased. In addition, the anti-proliferation effect of PL can be reversed by Fer-1 and NAC and the level of LPO and ROS was decreased accordingly. Importantly, we found that PL and CB-839 in combination could decrease the cell viability and the LPO level synergistically, accompanied by a large consumption of glutathione (GSH). These evidences prove that PL can induce ferroptosis of OSCC cells, which can be enhanced by CB-839. Conclusions: Our study suggested that the nature product PL can induce the ferroptotic death of OSCC cells, which is further enhanced when combined with CB-839. The synergistic anticancer effect of these two may prove new strategy for OSCC treatment.

[Current status, trends, and challenges of continuous manufacturing technology for oral traditional Chinese medicine solid preparations].

In recent years, continuous manufacturing technology has attracted considerable attention in the pharmaceutical industry. This technology is highly sought after for its significant advantages in cost reduction, increased efficiency, and improved productivity, making it a growing trend in the future of the pharmaceutical industry. Compared to traditional batch production methods, continuous manufacturing technology features real-time control and environmentally friendly intelligence, enabling pharmaceutical companies to produce drugs more efficiently. However, the adoption of continuous manufacturing technology has been slow in the field of traditional Chinese medicine(TCM) pharmaceuticals. On the one hand, there is insufficient research on continuous manufacturing equipment and technology that align with the characteristics of TCM preparations. On the other hand, the scarcity of talent with diverse expertise hampers its development. Therefore, in order to promote the modernization and upgrading of the TCM pharmaceutical industry, this article combined the current development status of the TCM industry to outline the development status and regulatory requirements of continuous manufacturing technology. At the same time, it analyzed the problems with existing TCM manufacturing models and explored the prospects and challenges of applying continuous manufacturing technology in the field of TCM pharmaceuticals. The analysis focused on continuous manufacturing control strategies, technical tools, and pharmaceutical equipment, aiming to provide targeted recommendations to drive the development of the TCM pharmaceutical industry.

Global translational control by the transcriptional repressor TrcR in the filamentous cyanobacterium Anabaena sp. PCC 7120.

Transcriptional and translational regulations are important mechanisms for cell adaptation to environmental conditions. In addition to house-keeping tRNAs, the genome of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena) has a long tRNA operon (trn operon) consisting of 26 genes present on a megaplasmid. The trn operon is repressed under standard culture conditions, but is activated under translational stress in the presence of antibiotics targeting translation. Using the toxic amino acid analog ß-N-methylamino-L-alanine (BMAA) as a tool, we isolated and characterized several BMAA-resistance mutants from Anabaena, and identified one gene of unknown function, all0854, named as trcR, encoding a transcription factor belonging to the ribbon-helix-helix (RHH) family. We provide evidence that TrcR represses the expression of the trn operon and is thus the missing link between the trn operon and translational stress response. TrcR represses the expression of several other genes involved in translational control, and is required for maintaining translational fidelity. TrcR, as well as its binding sites, are highly conserved in cyanobacteria, and its functions represent an important mechanism for the coupling of the transcriptional and translational regulations in cyanobacteria.

A tRNA t6A modification system contributes to the sensitivity towards the toxin ß-N-methylamino-L-alanine (BMAA) in the cyanobacterium Anabaena sp. PCC 7120.

Cyanobacteria are oxygen-evolving photosynthetic autotrophs essential for nutrient cycling in the environment. They possess multiple control mechanisms for their cellular activities in order to adapt to the environment. While protein translation is essential for cell survival and adaptation, the regulation and the flexibility of this process are poorly understood in cyanobacteria. ß-N-methylamino-L-alanine (BMAA), an amino acid analog proposed as an environmental neurotoxin, is highly toxic to the filamentous diazotrophic cyanobacterium Anabaena PCC 7120. In this study, through genetic analysis of BMAA-resistant mutants, we demonstrate that the system responsible for modification of ANN-decoding tRNAs with N(6)-threonylcarbamoyl adenosine (t6A) is involved in BMAA sensitivity through the control of translation. Both BMAA and inactivation of the t6A biosynthesis pathway affect translational fidelity and ribosome assembly. However, the two factors display either additive effects on translational elongation, or attenuate each other over translational fidelity or the resistance/sensitivity to antibiotics that inhibit different steps of the translational process. BMAA has a broad effect on translation and transcription, and once BMAA enters the cells, the presence of the t6A biosynthesis pathway increases the sensitivity of the cells towards this toxin. BMAA-resistant mutants screening is an effective method for getting insight into the toxic mechanisms of BMAA. In addition, BMAA is a useful tool for probing translational flexibility of cyanobacteria, and the characterization of the corresponding resistant mutants should help us to reveal translational mechanism allowing cyanobacteria to adapt to changing environments.

Quantitative Determination of 15 Active Components in Lepidium meyenii with UHPLC-PDA and GC-MS.

In this study, a method using ultrahigh-performance liquid chromatography with photodiode array (UHPLC-PDA) was established and validated for the simultaneous quantification of 10 active components, including eight macamides and two glucosinolates, in Lepidium meyenii (maca). A gas chromatographic mass spectroscopy (GC-MS) method was used to determine the levels of three benzyl isothiocyanates and two sterols in maca. Liquid chromatographic separation was achieved on a Waters Acquity UHPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm) with gradient elution over 15 min. The mobile phase was (B) acetonitrile-(A) 10 mM aqueous ammonium phosphate, and the detection wavelength was 210 nm. The gas chromatographic separation was performed on an SH-Rxi-1 MS column, and the ionization mode was electron ionization (EI). Two methods were confirmed to have desirable precision (RSD < 1.58%), repeatability (RSD < 1.97%), stability (RSD < 1.76%), and good linearity (R 2 ≥ 0.999) within the test range. The recoveries were in the range of 96.79-109.99%, with an RSD below 2.39%. We applied the established methods and successfully analyzed 15 compounds in maca processed under different drying conditions, providing a comprehensive reference for maca processing method of development. In summary, this study provided two rapid and effective methods for the quantification of 15 active components, which contributed to the in-depth maca quality control and provided a reference for the development of maca products.

HetF Protein Is a New Divisome Component in a Filamentous and Developmental Cyanobacterium.

Bacterial cell division, with a few exceptions, is driven by FtsZ through a treadmilling mechanism to remodel and constrict the rigid peptidoglycan (PG) layer. Yet different organisms may differ in the composition of the cell division complex (divisome). In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, hetF is required for the initiation of the differentiation of heterocysts, cells specialized in N2 fixation under combined-nitrogen deprivation. In this study, we demonstrate that hetF is expressed in vegetative cells and necessary for cell division under certain conditions. Under nonpermissive conditions, cells of a ΔhetF mutant stop dividing, consistent with increased levels of HetF under similar conditions in the wild type. Furthermore, HetF is a membrane protein located at midcell and cell-cell junctions. In the absence of HetF, FtsZ rings are still present in the elongated cells; however, PG remodeling is abolished. This phenotype is similar to that observed with the inhibition of the septal PG synthase FtsI. We further reveal that HetF is recruited to or stabilized at the divisome by interacting with FtsI and that this interaction is necessary for HetF function in cell division. Our results indicate that HetF is a member of the divisome depending mainly on light intensity and reveal distinct features of the cell division machinery in cyanobacteria that are of high ecological and environmental importance. IMPORTANCE Cyanobacteria shaped the Earth's evolutionary history and are still playing important roles for elementary cycles in different environments. They consist of highly diverse species with different cell shapes, sizes, and morphologies. Although these properties are strongly affected by the process of cytokinesis, the mechanism remains largely unexplored. Using different approaches, we demonstrate that HetF is a new component of the cell division machinery under certain environmental conditions in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. The common and diverged characteristics of cell division in prokaryotes reflect the evolutionary history of different bacteria as an adaptive measure to proliferate under certain environmental conditions. As a protein for cell differentiation, the recruitment of HetF to the septum illustrates such an adaptive mechanism in cyanobacteria.

The Proposed Neurotoxin ß-N-Methylamino-l-Alanine (BMAA) Is Taken up through Amino-Acid Transport Systems in the Cyanobacterium Anabaena PCC 7120.

Produced by cyanobacteria and some plants, BMAA is considered as an important environmental factor in the occurrence of some neurodegenerative diseases. Neither the underlying mechanism of its toxicity, nor its biosynthetic or metabolic pathway in cyanobacteria is understood. Interestingly, BMAA is found to be toxic to some cyanobacteria, making it possible to dissect the mechanism of BMAA metabolism by genetic approaches using these organisms. In this study, we used the cyanobacterium Anabaena PCC 7120 to isolate BMAA-resistant mutants. Following genomic sequencing, several mutations were mapped to two genes involved in amino acids transport, suggesting that BMAA was taken up through amino acid transporters. This conclusion was supported by the protective effect of several amino acids against BMAA toxicity. Furthermore, targeted inactivation of genes encoding different amino acid transport pathways conferred various levels of resistance to BMAA. One mutant inactivating all three major amino acid transport systems could no longer take up BMAA and gained full resistance to BMAA toxicity. Therefore, BMAA is a substrate of amino acid transporters, and cyanobacteria are interesting models for genetic analysis of BMAA transport and metabolism.

Synthesis of methionine methyl ester-modified coumarin as the fluorescent-colorimetric chemosensor for selective detection Cu2+ with application in molecular logic gate.

A methionine methyl ester-modified coumarin derivative was designed and synthesized, which could discriminate Cu2+ from other metal ions in HEPES buffer (10 mM, pH 7.4)/CH3CN (40:60, V/V). The detection limit of WM toward Cu2+ was 1.84 × 10-7 M, which was lower than the concentration of Cu2+ in drinking water suggested by WHO and EPA. And the proposed coordination mode exhibiting the interaction between WM and Cu2+ was studied by UV-Vis, fluorescence spectrum, ESI-MS and FT-IR. Based on the fluorescent reversibility of WM, WM was considered as a molecular logic gate and molecular keypad lock. In addition, the test strips and the silica gel plates prepared from the solution of WM also demonstrate the favorable selectivity toward Cu2+.

Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120.

CRISPR systems, such as CRISPR-Cas9 and CRISPR-Cpf1, have been successfully used for genome editing in a variety of organisms. Although the technique of CRISPR-Cpf1 has been applied in cyanobacteria recently, its use was limited without exploiting the full potential of such a powerful genetic system. Using the cyanobacterium Anabaena PCC 7120 as a model strain, we improved the tools and designed genetic strategies based on CRISPR-Cpf1, which enabled us to realize genetic experiments that have been so far difficult to do in cyanobacteria. The development includes: (1) a "two-spacers" strategy for single genomic modification, with a success rate close to 100%; (2) rapid multiple genome editing using editing plasmids with different resistance markers; (3) using sacB, a counter-selection marker conferring sucrose sensitivity, to enable the active loss of the editing plasmids and facilitate multiple rounds of genetic modification or phenotypic analysis; (4) manipulation of essential genes by the creation of conditional mutants, using as example, polA encoding the DNA polymerase I essential for DNA replication and repair; (5) large DNA fragment deletion, up to 118 kb, from the Anabaena chromosome, corresponding to the largest bacterial chromosomal region removed with CRISPR systems so far. The genome editing vectors and the strategies developed here will expand our ability to study and engineer cyanobacteria, which are extensively used for fundamental studies, biotechnological applications including biofuel production, and synthetic biology research. The vectors developed here have a broad host range, and could be readily used for genetic modification in other microorganisms.