Porphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by stimulating the release of neutrophil extracellular traps in the tumor immune microenvironment.

BACKGROUND: The aim of this study was to investigate the impact of Porphyromonas gingivalis (P. gingivalis) on the progression of oral squamous cell carcinoma (OSCC) through neutrophil extracellular traps (NETs) in the tumor immune microenvironment. METHODS: The expression of NETs-related markers was identified through immunohistochemistry, immunofluorescence, and Western blotting in different clinical stages of OSCC samples. The relationship between NETs-related markers and clinicopathological characteristics in 180 samples was analyzed using immunohistochemistry data. Furthermore, the ability to predict the prognosis of OSCC patients was determined by ROC curve analysis and survival analysis. The effect of P. gingivalis on the release of NETs was identified through immunofluorescence and immunohistochemistry, both in vitro and in vivo. CAL27 and SCC25 cell lines were subjected to NETs stimulation to elucidate the influence of NETs on various cellular processes, including cell proliferation, migration, invasion, and metastasis in vitro. Furthermore, the impact of NETs on the growth and metastatic potential of OSCC was assessed using in vivo models involving tumor-bearing mice and tumor metastasis mouse models. RESULTS: Immunochemistry analysis revealed a significant correlation between the NETs-related markers and clinical stage, living status as well as TN stage. P. gingivalis has demonstrated its ability to effectively induce the release of NETs both in vivo and in vitro. NETs have the potential to facilitate cell migration, invasion, and colony formation. Moreover, in vivo experiments have demonstrated that NETs play a pivotal role in promoting tumor metastasis. CONCLUSION: High expression of NETs-related markers demonstrates a strong correlation with the progression of OSCC. Inhibition of the NETs release process stimulated by P. gingivalis and targeted NETs could potentially open up a novel avenue in the field of immunotherapy for patients afflicted with OSCC.

Tetrachloromaxamycins: Divergent Total Synthesis and Initial Assessments.

Divergent total syntheses of binding pocket and peripherally modified tetrachlorovancomycins, a non-native synthetic glycopeptide, and their evaluation are disclosed. Central to the approach is the synthesis of a single late-stage intermediate that bears a residue 4 thioamide ([Ψ[C(═S)NH]Tpg4]tetrachlorovancomycin (3), LLS 15 steps, 14% overall) as a precursor to either of two key pocket modifications and their pairing with any combination of two peripheral modifications conducted without protecting groups. A stereochemical simplification achieved by the addition of two aryl chlorides removes two synthetically challenging atropisomer centers in native glycopeptides and streamlines the synthesis. Key features include in a convergent epimerization-free thioacylation of the AB ring system amine with an N-thioacylbenzotriazolyl DE tetrapeptide (85%) followed by simultaneous room-temperature SNAr macrocyclizations of the CD and DE ring systems (96%). The approach provided 3 from which [Ψ[C(═N)NH]Tpg4]tetrachlorovancomycin (4) and [Ψ(CH2NH)Tpg4]tetrachlorovancomycin (5) were prepared in a single-step and bear binding pocket modifications that convey dual d-Ala-d-Ala/d-Lac ligand binding to overcome vancomycin resistance. The newest maxamycin members are disclosed, bearing two additional peripheral modifications that introduce two independent synergistic MOAs that do not rely on native ligand binding for activity. Ligand binding properties of pocket-modified tetrachlorovancomycins 3-5, antibacterial activity of a key compound series, and PK assessments of two tetrachloromaxamycins are reported.

Blocking Gremlin1 inhibits M1 macrophage polarization through Notch1/Hes1 signaling pathway in apical periodontitis.

BACKGROUND: Gremlin1 is a multifunctional protein whose expression is demonstrated to be involved in a series of physiology and pathological processes. The association between Gremlin1 and apcial periodontitis (AP) has been established. M1-polarized macrophages are crucial immune cells that exacerbate the progression of apical periodontal inflammatory response, but the function of Gremlin1 during macrophages activation in periapical lesions is still unclear. This study attempts to explore the regulatory effects of Gremlin1 on macrophage polarization on apical periodontitis microenviroment. METHODS: Clinical specimens were used to determine the expression of Gremlin1 in periapical tissues by immunohistochemical (IHC) staining. Then, the disease models of periapical inflammation in rats were established, and adenovirus- associated virus (AAVs) was used to blockade Gremlin1 expression. Lentivirus carrying sh-Gremlin1 particles were used to transfect THP-1 induced M1-subtype macrophages. To assess the expression of associated molecules, Western blot, immunofluorescence staining were performed. RESULTS: Gremlin1 was significantly up-regulated in the periapical tissues of subjects with AP as identified by IHC staining, and positively correlated with levels of M1 macrophage-associated genes. Rats AP model with inhibition of Gremlin1 in periapical lesions exhibited limited infiltration of macrophages and decreased expression of M1 macrophage-related genes in periapical lesions. Furthermore, Gremlin1 blockade substantially decreased the Notch1/Hes1 signaling pathway activation level. The in vitro experiments confirmed the above results. CONCLUSION: Taken together, current study illustrated that the Gremlin1 suppression in periapical lesions inhibited M1 macrophage polarization through Notch1/Hes1 axis. Moreover, Gremlin1 may act as a potential candidate in the treatment of AP.

Host-Guest Metal-Organic Frameworks-Based Long-Afterglow Luminescence Materials.

Long-afterglow materials have a broad of applications in optoelectronic devices, sensors, medicine and other fields due to their excellent luminescent properties. The host-guest long-afterglow MOFs material combines the advantages of multi-component characteristics and the stability of MOFs, which improves its luminous performance and expands its other properties. This review introduces the classification, synthesis and application of host-guest MOFs materials with long afterglow. Due to their rigid frames and multi-channel characteristics, MOFs can load common guest materials including rare earth metals, organic dyes, carbon dots, etc. The synthesis methods of loading guest materials into MOFs include solvothermal synthesis, post-encapsulation, post-modification, etc. Those long-afterglow host-guest MOFs have a wide range of applications in the fields of sensors, information security and biological imaging.

A randomized controlled clinical trial of concentrated growth factor combined with sodium hyaluronate in the treatment of temporomandibular joint osteoarthritis.

OBJECTIVE: To investigate the effect of concentrated growth factor (CGF) combined with sodium hyaluronate (SH) on temporomandibular joint osteoarthritis (TMJOA). METHODS: Sixty patients with TMJOA who were diagnosed by cone-beam computed tomography (CBCT) between March 2020 and March 2023 at the Stomatological Hospital of Xi'an Jiaotong University were randomly divided into a control group (n = 30) and an experimental group (n = 30). The patients in the experimental group were treated with CGF + SH, and those in the control group were treated with SH only. The visual analogue scale (VAS) score indicating pain in the temporomandibular joint (TMJ) area; the Helkimo Clinical Dysfunction Index (Di); and changes in condylar CBCT at the first visit and 2 weeks, 3 months and 6 months after treatment were recorded. The CBCT data of the patients in the experimental and control groups were collected, and the three-dimensional CBCT image sequences were imported into Mimics Medical 19.0 software in DICOM format for condylar reconstruction. RESULTS: The VAS scores at 2 weeks, 3 months and 6 months after treatment were significantly lower in the experimental group than in the control group (P < 0.05), and the pain in the experimental group was significantly relieved. The Di was significantly lower in the experimental group than in the control group (P < 0.05), and the clinical function of the TMJ improved. After treatment, the CBCT score was significantly lower in the experimental group than in the control group (P < 0.05), and the condylar bone cortex was obviously repaired. Observation of the condylar bone cortex by three-dimensional reconstruction showed the same results as those obtained by CBCT. CONCLUSION: CGF combined with SH is effective in the treatment of TMJOA and can improve muscle pain, TMJ pain, Impaired TMJ function, Impaired range of movement, Pain on movement of the mandible and promote bone repair. THE REGISTRATION NUMBER (TRN): ChiCTR2400082712. THE DATE OF REGISTRATION: April 5, 2024.

Tetrachlorovancomycin: Total Synthesis of a Designed Glycopeptide Antibiotic of Reduced Synthetic Complexity.

A technically straightforward total synthesis of a new class of vancomycin analogues of reduced synthetic complexity was developed that provided tetrachlorovancomycin (1, LLS = 15 steps, 15% overall yield) and its precursor aglycon 29 (nearly 20% overall yield). The class retains all the intricate vancomycin structural features that contribute to its target binding affinity and selectivity, maintains the antimicrobial activity of vancomycin, and achieves the simplification by an unusual addition, not removal, of benign substituents to the core structure. The modification, accomplished by addition of two aryl chloride substituents to provide 1, permitted a streamlined total synthesis of the new glycopeptide antibiotic class by removing the challenges associated with CD and DE ring system atropisomer stereochemical control. This also enabled their simultaneous and further-activated SNAr macrocyclizations that establish the tricyclic skeleton of 1. Key elements of the approach include catalyst-controlled diastereoselective formation of the AB biaryl axis of chirality (>30:1 dr), an essentially instantaneous macrolactamization of the AB ring system free of competitive epimerization (>30:1 dr), racemization free coupling of the E ring tetrapeptide, room temperature simultaneous CD and DE ring system cyclizations, a highly refined 4-step conversion of the cyclization product to the aglycon, and a protecting-group-free one-pot enzymatic glycosylation for disaccharide introduction. In addition to the antimicrobial evaluation of tetrachlorovancomycin (1), the preparation of key peripherally modified derivatives, which introduce independent and synergistic mechanisms of action, revealed their exceptional antimicrobial potency and provide the foundation for future use of this new class of synthetic glycopeptide analogues.

Divergent Total Synthesis and Characterization of Maxamycins.

A new streamlined and scaled divergent total synthesis of pocket-modified vancomycin analogs is detailed that provides a common late-stage intermediate [Ψ[C(═S)NH]Tpg4]vancomycin (LLS = 18 steps, 12% overall yield, >5 g prepared) to access both existing and future pocket modifications. Highlights of the approach include an atroposelective synthesis of [Ψ[C(═S)NH]Tpg4]vancomycin aglycon (11), a one-pot enzymatic glycosylation for direct conversion to [Ψ[C(═S)NH]Tpg4]vancomycin (12), and new powerful methods for the late-stage conversion of the embedded thioamide to amidine/aminomethylene pocket modifications. Incorporation of two peripheral modifications provides a scalable total synthesis of the maxamycins, all prepared from aglycon 11 without use of protecting groups. Thus, both existing and presently unexplored pocket-modified analogues paired with a range of peripheral modifications are accessible from this common thioamide intermediate. In addition to providing an improved synthesis of the initial member of the maxamycins, this is illustrated herein with the first synthesis and examination of maxamycins that contain the most effective of the pocket modifications (amidine) described to date combined with two additional peripheral modifications. These new amidine-based maxamycins proved to be potent, durable, and efficacious antimicrobial agents that display equipotent activity against vancomycin-sensitive and vancomycin-resistant Gram-positive organisms and act by three independent synergistic mechanisms of action. In the first such study conducted to date, one new maxamycin (21, MX-4) exhibited efficacious in vivo activity against a feared and especially challenging multidrug-resistant (MRSA) and vancomycin-resistant (VRSA) S. aureus bacterial strain (VanA VRS-2) for which vancomycin is inactive.

Porphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by activating the neutrophil chemotaxis in the tumour microenvironment.

BACKGROUND: We aimed to determine the significance of Porphyromonas gingivalis (P. gingivalis) in promoting tumour progression in the tumour microenvironment (TME) of oral squamous cell carcinoma (OSCC). METHODS: The Gene Expression Omnibus (GEO) was used to screen out the differentially expressed genes from the two datasets of GEO138206 and GSE87539. Immunohistochemistry and immunofluorescence analysis of samples, cell biological behaviour experiments, and tumour-bearing animal experiments were used to verify the results in vivo and in vitro. The mechanism was revealed at the molecular level, and rescue experiments were carried out by using inhibitors and lentiviruses. RESULTS: CXCL2 was selected by bioinformatics analysis and was found to be related to a poor prognosis in OSCC patients. Samples with P. gingivalis infection in the TME of OSCC had the strongest cell invasion and proliferation and the largest tumour volume in tumour-bearing animal experiments and exhibited JAK1/STAT3 signalling pathway activation and epithelial-mesenchymal transition (EMT). The expression of P. gingivalis, CXCL2 and TANs were independent risk factors for poor prognosis in OSCC patients. A CXCL2/CXCR2 signalling axis inhibitor significantly decreased the invasion and proliferation ability of cells and the tumour volume in mice. When lentivirus was used to block the CXCL2/CXCR2 signalling axis, the activity of the JAK1/STAT3 signalling pathway was decreased, and the phenotype of EMT was reversed. CONCLUSION: Porphyromonas gingivalis promotes OSCC progression by recruiting TANs via activation of the CXCL2/CXCR2 axis in the TME of OSCC.

Discovery of two biotin-PEG4­diarylidenyl piperidone prodrugs as potent antitumor agents with good efficacy, limited toxicity, and low resistance.

Two biotin-polyethylene glycol (PEG)4­diarylidenyl piperidone (DAP) prodrugs, compounds 3a and 3b, were designed as antineoplastic agents and synthesized by coupling biotin to bifluoro- and binitro-substituted DAP derivatives (DAP-F and DAP-NO2) through a PEG4 linker, respectively. The results of the MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide) assay and a SW480 xenograft model identified compounds 3a and 3b as candidate antitumor agents with good efficacy, limited toxicity, and low resistance, as compared to the original drugs (DAP-F and DAP-NO2), cisplatin, and doxorubicin (dox). The results of a preliminary pharmacokinetic study showed that compounds 3a and 3b slowly released their original drug DAP-F and DAP-NO2 within 12 h after intraperitoneal injection, respectively. Western blot analysis and computer docking simulations indicated that DAP-F, DAP-NO2, and compounds 3a and 3b were indeed inhibitors of signal transducer and activator of transcription 3 (STAT3) and the antitumor effects of compounds 3a and 3b were exerted by sequentially interacting with the SH2-binding domain followed by the DNA-binding domain after releasing the original drugs DAP-F and DAP-NO2, respectively. These results suggest that the targeted prodrug model led to good antitumor efficacy with reduced toxicity, while a dual STAT3-binding model may promote antitumor efficacy and resistance.

Programmable DNA hydrogel provides suitable microenvironment for enhancing autophagy-based therapies in intervertebral disc degeneration treatment.

The pathogenesis of intervertebral disc degeneration (IVDD) is attributed to metabolic dysregulation within the extracellular matrix and heightened apoptosis of nucleus pulposus cells (NPC). Therefore, a potential therapeutic strategy for managing IVDD involves the reestablishment of metabolic equilibrium within the extracellular matrix and the suppression of excessive myeloid cell apoptosis. The microRNA, miR-5590, displays marked differential expression in degenerative nucleus pulposus (NP) tissues and exerts a direct influence on the regulation of DDX5 expression. This, in turn, modulates mammalian target of rapamycin (mTOR) phosphorylation, thereby impacting autophagy and apoptosis. However, ensuring the smooth delivery of miRNA to a specific injury site poses a significant challenge. To address this issue, a multifunctional DNA hydrogel was developed and subsequently loaded with miR-5590 via spherical nucleic acids (SNAs) for the treatment of IVDD. The hydrogel, which exhibits versatility, has the potential to be administered through injection at the site of injury, resulting in a consistent and prolonged release of miR-5590. This leads to the creation of a genetic microenvironment within the NP, which triggers the onset of autophagy in NPCs and subsequently suppresses apoptosis. As a result, this process regulates the metabolic equilibrium within the extracellular matrix, thereby impeding the in vitro and in vivo progression of IVDD. The amalgamation of miRNAs and biomaterials offers a promising therapeutic strategy for the management of IVDD in clinical settings.

Advances in the application of induced pluripotent stem cells in pediatric diseases.

The optimal diagnosis and treatment of pediatric diseases depend on more adequate understanding of pathophysiology. The advent of induced pluripotent stem cells (iPSCs) has provided new strategies for the research and therapy of pediatric diseases. iPSCs are pluripotent stem cells induced by reprogramming of mature cells. Now they can be induced from many types of somatic cells (such as fibroblasts, peripheral blood mononuclear cells and urine cells).With the improvement of various reprogramming methods, its generation procedure is more and more optimized, and the use of small molecules to induce iPSCs is one of the research focus now. Due to their ability to differentiate into a variety of cells, combined with the development of gene editing technology, iPSCs have been increasingly favored in the modeling of diseases and cell therapy, especially hereditary diseases, and have achieved some success in clinical treatment. But before they can be widely used in clinical treatment, there are still some problems to be solved, such as tumorigenicity, immunogenicity and heterogeneity. This article reviewed the source of iPSCs, reprogramming technology, applications of iPSCs in common childhood diseases, current problems and prospects, in order to deepen the understanding of iPSCs and provide reference for in-depth research in field of exploring mechanisms of diseases and therapy of diseases.

Identification of DELLA gene family in head cabbage and analysis of mRNA transport in the heterograft.

DELLA gene family is involved in the regulation of signal transduction of plant hormones. mRNAs of GA insensitive (GAI), the member of DELLA gene family, are also signaling molecules of long-distance transport in plants. Genome-wide identification and mRNA transport analysis of the members of DELLA gene family in head cabbage (Brassica oleracea var. capitata) can provide basic data for their application in head cabbage. In this study, five members of DELLA gene family (BoRGA1, BoRGA2, BoRGL1, BoRGL2, and BoRGL3) were identified in head cabbage using genome and transcriptome data. However, head cabbage lacked a GAI gene in its genome. The scion (head cabbage, inbred line G27) and the rootstock Chinese flowering cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) (sijiucaixin) were cleft-grafted together to produce the heterograft. Inflorescence stem of the rootstock and the corresponding inflorescence stem in Chinese flowering cabbage seedlings (as controls) were purified and analyzed with transcriptome sequencing. The total of 8, 9, 3, 5, and 1 exogenous read(s), derived respectively from BoRGA1, BoRGA2, BoRGL1, BoRGL2, and BoRGL3, were identified in the transcriptomes of the rootstocks. Nevertheless, mRNA transport of DELLA family genes from scion to rootstock did not increase the transcriptional level of the members of DELLA gene family in the rootstocks. Correlation analysis suggested that mRNA transport efficiency of the DELLA family genes was correlated with the sequence and the transcriptional level of the respective DELLA gene in the scion (head cabbage). This study lays the foundation for further investigation on the molecular mechanism of mRNA transport of the members of DELLA gene family in head cabbage.

[Quality evaluation of Lysimachiae Herba from different habitats based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis].

A method based on ultra-high performance liquid chromatography coupled with triple quadrupole linear ion trap-tandem mass spectrometry(UHPLC-QTRAP-MS/MS) was developed for the simultaneous determination of 41 bioactive constituents of flavonoids, organic acids, nucleosides, and amino acids in Lysimachiae Herba. The content of multiple bioactive constituents was compared among the samples from different habitats. The chromatographic separation was performed in a Waters XBridge®C_(18) column(4.6 mm×100 mm, 3.5 µm) at 30 ℃. The gradient elution was performed with 0.4% methanol(A)-formic acid water(B) as the mobile phase at a flow rate of 0.8 mL·min~(-1), and the multiple-reaction monitoring(MRM) mode was adopted. According to the content of 41 constituents, hierarchical cluster analysis(HCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and gray relational analysis(GRA) were perfomed to comprehensively evaluate the samples from different habitats. The results showed that the 41 constituents exhibited good linear relationship within the tested concentration ranges, with the correlation coefficients(r) greater than 0.999 4. The method featured good precision, repeatability, and stability with the relative standard deviations(RSDs) less than 5.0%. The average recoveries of the 41 constituents ranged from 98.06% to 101.9%, with the RSDs of 0.62%-4.6%. HCA and OPLS-DA separated 48 batches of Lysimachiae Herba samples from different habitats into three categories: the producing areas in Sichuan and Chongqing, the producing areas in Jiangsu, Zhejiang, and Jiangxi, and the producing areas in Guizhou. The content of 41 constituents varied among the Lysimachiae Herba samples from different habitats. The GRA results revealed that the Lysimachiae Herba sample from Nanchong City, Sichuan Province had the best comprehensive quality. The method developed in this study was accurate and reliable and thus can be used for comprehensive evaluation of Lysimachiae Herba quality and provide basic information for the selection of habitats.

[Analysis and evaluation of bioactive constituents from different parts of Epimedium brevicornum].

A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 45 bioactive constituents including flavonoids, alkaloids, amino acids, phenolic acids, and nucleosides in Epimedium brevicornum. The multiple bioactive constituents in leaves, petioles, stems and rhizomes of E. brevicornum were analyzed. The gradient elution was performed at 30 ℃ in an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 µm) with 0.4% formic acid aqueous solution-acetonitrile as the mobile phase at a flow rate of 0.8 mL·min~(-1). Single factor experiment and response surface methodology were employed to optimize the extraction conditions. Multivariate statistical analyses including systematic cluster analysis(SCA), principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and one-way analysis of variance(One-way ANOVA) were carried out to classify the samples from different parts and identify different constituents. Grey relation analysis(GRA) and entropy weight-TOPSIS analysis were performed to build a multi-index comprehensive evaluation model for different parts of E. brevicornum. The results showed that there was a good relationship between the mass concentrations of 45 constituents and the corresponding peak areas, with the correlation coefficients(r) not less than 0.999 0. The precision, repeatability, and stability of the established method were good for all the target constituents in this study, with the relative standard deviations(RSDs) less than 5.0%(0.62%-4.9%) and the average recovery of 94.51%-105.7%. The above results indicated that the bioactive constituents varied in different parts of E. brevicornum, and the overall quality followed the trend of leaves > petioles > rhizomes > stems. This study verified the rationality of the Chinese Pharmacopoeia(2020 edition) stipulating that the medicinal part of E. brevicornum is the leaf. Moreover, our study indicated that the rhizome had the potential for medicinal development. The established method was accurate and reliable, which can be used to comprehensive evaluate and control the quality of E. brevicornum. This study provides data reference for clarifying the medicinal parts and rationally utilizing the resources of E. brevicornum.

Mechanistic Insights into the Reaction of Amidines with 1,2,3-Triazines and 1,2,3,5-Tetrazines.

1,2,3-Triazines and 1,2,3,5-tetrazines react rapidly, efficiently, and selectively with amidines to form pyrimidines/1,3,5-triazines, exhibiting an orthogonal reactivity with 1,2,4,5-tetrazine-based conjugation chemistry. Whereas the mechanism of the reaction of the isomeric 1,2,4-triazines and 1,2,4,5-tetrazines with alkenes is well understood, the mechanism of the 1,2,3-triazine/1,2,3,5-tetrazine-amidine reaction as well as its intrinsic reactivity remains underexplored. By using 15N-labeling, kinetic investigations, and kinetic isotope effect studies, complemented by extensive computational investigations, we show that this reaction proceeds through an addition/N2 elimination/cyclization pathway, rather than the generally expected concerted or stepwise Diels-Alder/retro Diels-Alder sequence. The rate-limiting step in this transformation is the initial nucleophilic attack of an amidine on azine C4, with a subsequent energetically favored N2 elimination step compared with a disfavored stepwise formation of a Diels-Alder cycloadduct. The proposed reaction mechanism is in agreement with experimental and computational results, which explains the observed reactivity of 1,2,3-triazines and 1,2,3,5-tetrazines with amidines.

1,2,3,5-Tetrazines: A General Synthesis, Cycloaddition Scope, and Fundamental Reactivity Patterns.

Despite the explosion of interest in heterocyclic azadienes, 1,2,3,5-tetrazines remain unexplored. Herein, the first general synthesis of this new class of heterocycles is disclosed. Its use in the preparation of a series of derivatives, and the first study of substituent effects on their cycloaddition reactivity, mode, and regioselectivity provide the foundation for future use. Their reactions with amidine, electron-rich, and strained dienophiles reveal unique fundamental reactivity patterns (4,6-dialkyl-1,2,3,5-tetrazines > 4,6-diaryl-1,2,3,5-tetrazines for amidines but slower with strained dienophiles), an exclusive C4/N1 mode of cycloaddition, and dominant alkyl versus aryl control on regioselectivity. An orthogonal reactivity of 1,2,3,5-tetrazines and the well-known isomeric 1,2,4,5-tetrazines is characterized, and detailed kinetic and mechanistic investigations of the remarkably fast reaction of 1,2,3,5-tetrazines with amidines, especially 4,6-dialkyl-1,2,3,5-tetrazines, established the mechanistic origins underlying the reactivity patterns and key features needed for future applications.

[Analysis and evaluation of dynamic accumulation of multiple active constituents in Taxilli Herba in Guangxi].

A method based on ultra-high performance liquid chromatography with triple quadrupole/linear ion trap mass spectrometry(UHPLC-QTRAP-MS/MS) was developed for the simultaneous determination of 33 active constituents, including flavonoids, organic acids, nucleosides, and amino acids in Taxilli Herba to analyze and evaluate the dynamic accumulation of their multiple active constituents. The separation was performed at 30 ℃ on an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 µm) with gradient elution using 0.1% formic acid aqueous solution-methanol as the mobile phase at a flow rate of 0.5 mL·min~(-1), and the injection volume was 2 µL. The constituents were ionized in the electrospray ionization source(ESI) and quantitated by the multiple reaction monitoring(MRM) mode. The entropy weight TOPSIS method was used to objectively assign weights to the target constituents and rank them according to their relative closeness coefficient(C_i) to construct a multi-index comprehensive evaluation model of Taxilli Herba. The results showed that the concentrations and peak areas of 33 target constituents had good linearity in their respective linear ranges, and the correlation coefficients(r) were not less than 0.999 0. The RSD of precision, reproducibility, and stability were not higher than 4.7%. The average recoveries were 98.03%-101.3% with RSD less than 4.0%. There were differences in the content of 33 active constituents in Taxilli Herba at different harvest periods. The overall quality of Taxilli Herba harvested from mid-February to early March was better, which was consistent with the traditional harvest period. This study provides basic information for revealing the rule of dynamic accumulation of multiple active constituents in Taxilli Herba and determining the suitable harvesting period. Meanwhile, it also provides a new methodological reference for the comprehensive evaluation of the intrinsic quality of Taxilli Herba.

Maxamycins: Durable Antibiotics Derived by Rational Redesign of Vancomycin.

Since its discovery, vancomycin has been used in the clinic for >60 years. Because of their durability, vancomycin and related glycopeptides serve as the antibiotics of last resort for the treatment of protracted bacterial infections of resistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant (MDR) Streptococcus pneumoniae. After 30 years of use, vancomycin resistance was first observed and is now widespread in enterococci and more recently in S. aureus. The widespread prevalence of vancomycin-resistant enterococci (VRE) and the emergence of vancomycin-resistant S. aureus (VRSA) represent a call to focus on the challenge of resistance, highlight the need for new therapeutics, and provide the inspiration for the design of more durable antibiotics less prone to bacterial resistance than even vancomycin.Herein we summarize progress on efforts to overcome vancomycin resistance, first addressing recovery of its original durable mechanism of action and then introducing additional independent mechanisms of action intended to increase the potency and durability beyond that of vancomycin itself. The knowledge of the origin of vancomycin resistance and an understanding of the molecular basis of the loss of binding affinity between vancomycin and the altered target ligand d-Ala-d-Lac provided the basis for the subtle and rational redesign of the vancomycin binding pocket to remove the destabilizing lone-pair repulsion or reintroduce a lost H-bond while not impeding binding to the unaltered ligand d-Ala-d-Ala. Preparation of the modified glycopeptide core structure was conducted by total synthesis, providing binding pocket-modified vancomycin aglycons with dual d-Ala-d-Ala/d-Lac binding properties that directly address the intrinsic mechanism of resistance to vancomycin. Fully glycosylated pocket-modified vancomycin analogues were generated through a subsequent two-step enzymatic glycosylation, providing a starting point for peripheral modifications used to introduce additional mechanisms of action. A well-established vancosamine N-(4-chlorobiphenyl)methyl (CBP) modification as well as newly discovered C-terminal trimethylammonium cation (C1) or guanidine modifications were introduced, providing two additional synergistic mechanisms of action independent of d-Ala-d-Ala/d-Lac binding. The CBP modification provides an additional stage for inhibition of cell wall synthesis that results from direct competitive inhibition of transglycosylase, whereas the C1/guanidine modification induces bacteria cell permeablization. The synergistic behavior of the three independent mechanisms of action combined in a single molecule provides ultrapotent antibiotics (MIC = 0.01-0.005 µg/mL against VanA VRE). Beyond the remarkable antimicrobial activity, the multiple mechanisms of action suppress the rate at which resistance may be selected, where any single mechanism of action is protected by the action of others. The results detailed herein show that rational targeting of durable vancomycin-derived antibiotics has generated compounds with a "resistance against resistance", provided new candidate antibiotics, and may serve as a generalizable strategy to combat antibacterial resistance.

Reaction Scope of Methyl 1,2,3-Triazine-5-carboxylate with Amidines and the Impact of C4/C6 Substitution.

A comprehensive study of the reaction scope of methyl 1,2,3-triazine-5-carboxylate (3a) with alkyl and aryl amidines is disclosed, reacting at room temperature at remarkable rates (<5 min, 0.1 M in CH3CN) nearly 10000-fold faster than that of unsubstituted 1,2,3-triazine and providing the product pyrimidines in high yields. C4 Methyl substitution of the 1,2,3-triazine (3b) had little effect on the rate of the reaction, whereas C4/C6 dimethyl substitution (3c) slowed the room-temperature reaction (<24 h, 0.25 M) but displayed an unaltered scope, providing the product pyrimidines in similarly high yields. Measured second-order rate constants of the reaction of 3a-c, the corresponding nitriles 3e and 3f, and 1,2,3-triazine itself (3d) with benzamidine and substituted derivatives quantitated the remarkable reactivity of 3a and 3e, verified the inverse electron demand nature of the reaction (Hammett ρ = -1.50 for substituted amidines, ρ = +7.9 for 5-substituted 1,2,3-triazine), and provided a quantitative measure of the impact of 4-methyl and 4,6-dimethyl substitution on the reactivity of the methyl 1,2,3-triazine-5-carboxylate and 5-cyano-1,2,3-triazine core heterocycles.

Quality Evaluation of Taxilli Herba from Different Hosts Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Taxilli Herba (TAXH) is an important traditional Chinese medicine with a long history, dating from the Eastern Han Dynasty to the present times. However, the active constituents in it that parasitize different hosts vary, affecting its clinical efficacy. Given the complexity of the host origins, evaluating the quality of TAXH is critical to ensure the safety and effectiveness of clinical medication. In the present study, a quantitative method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established, which simultaneously determined the content of 33 active constituents, including 12 flavonoids, 4 organic acids, 12 amino acids, and 5 nucleosides in 45 samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to classify and distinguish between TAXH and its adulterants, Tolypanthi Herba (TOLH). A hierarchical clustering analysis (HCA) was conducted combined with a heatmap to visually observe the distribution regularity of 33 constituents in each sample. Furthermore, gray relational analysis (GRA) was applied to evaluate the quality of samples to get the optimal host. The results demonstrated that TAXH excelled TOLH in quality as a whole. The quality of TAXH parasitizing Morus alba was also better, while those that were parasitic on Cinnamomum camphora and Glyptostrobus pensilis had relatively poor quality. This study may provide comprehensive information that is necessary for quality control and supply a scientific basis for further exploring the quality formation mechanism of TAXH.