Many macromolecules, including transmembrane proteins and apolipoproteins, interact with lipids and membranes and play crucial roles in diverse biological processes. Understanding macromolecule-lipid interactions at the structural level is essential for elucidating their functions and mechanisms. However, determining the structure of macromolecules, particularly proteins in their lipid-bound state, has traditionally been challenging for X-ray crystallography due to the conformational and compositional heterogeneity of macromolecule-lipid complexes. Transmission electron microscopy (TEM) offers a unique capability to directly visualizing individual macromolecular particles in the presence of lipid interactions. Among TEM techniques, negative staining (NS) is a fast and widely used approach for imaging macromolecules. However, conventional NS protocols often introduce artifacts in lipid-associated protein samples, such as rouleaux formation in lipoproteins, which can compromise structural interpretation. To address these limitations, Ren and colleagues developed an optimized negative staining (OpNS) protocol by refining earlier methods and validating the results against cryo-electron microscopy (cryo-EM) images of lipoproteins embedded in vitrified ice. This optimized protocol minimizes artifacts and produces "near-native" particle images with high quality. It enables more accurate structural analysis, particularly for three-dimensional (3D) reconstructions of single macromolecular particles without averaging, using individual-particle electron tomography (IPET). This improved protocol provides a robust, efficient, and reliable method for imaging macromolecules in their lipid-binding states, offering significant advances in understanding macromolecule-lipid interactions.

Phytochemical investigation on Chrysosplenium carnosum Hook. f. Thomson, the source plant of Tibetan medicine “Yajima,” resulted in the isolation and characterization of a new hexanor-cucurbitane triterpenoid, namely 3-epi-hexanorcucurbitacin F (1), along with 20 known compounds (2–21). Their structures were determined by comprehensive spectroscopic analysis, including 1D and 2D NMR, UV, IR, and HR-ESI-MS, and comparison of the spectroscopic data with those reported in the literature.

The whole plant of Astragalus rigidulus Benth. ex Bunge (Fabaceae) has been employed in Tibet to clear lung heat, alleviate diarrhea and edema. In the present study, a phytochemical investigation of A. rigidulus led to the isolation of a new alkaloid, n-butyl hematinate (1), and a new benzyl ester, astrigioside A (2), along with twenty known analogs (3-22). Their structures were elucidated through comprehensive spectroscopic analysis, including 1D/2D NMR, UV, IR, HR-ESI-MS, and comparison of the spectroscopic data with those reported in literatures. The UT-B inhibitory activities of all isolates were evaluated by erythrocyte lysis assay model. As a result, compounds 7, 8, 10, and 18 possessed significant UT-B inhibitory effects, with IC50 values of 22.93 ± 0.88, 26.40 ± 0.73, 21.83 ± 0.68, 29.60 ± 0.75 µM, respectively.

Chrysosplenium carnosum, an original plant used in traditional Tibetan medicine known as “Yajima”, was subjected to phytochemical study. This led to the isolation of a new hexanor-cucurbitane triterpenoid, namely chrycarnin A (1), along with three previously known structurally related analogs (2–4). Their structures were determined by spectroscopic methods, including 1D and 2D NMR, UV, IR, ECD, and HR-ESI-MS, and comparison of the spectroscopic data with those reported in the literature.

Intestinal stem cell (ISC) aging diminishes the regenerative capacity of the intestinal epithelia, but effective therapeutic strategies to counteract human ISC aging remain elusive. Here, we find that the synthesis of α-lipoic acid (ALA) is reduced in old human small intestine. Notably, ALA supplementation inhibits ISC aging and decreases the number of atypical Paneth cells in old human intestinal organoids and in old mouse small intestines. Importantly, we discern that the effect of ALA on mitigating ISC aging is contingent upon the presence of Paneth cells. Inhibiting the mTOR pathway in Paneth cells with ALA or rapamycin significantly increases cyclic ADP ribose (cADPR) secretion and decreases Notum secretion, which, in turn, enhances ISC functions. In this work, our findings substantiate the role of ALA in inhibiting human ISC aging and present a potential therapeutic approach for managing age-related human intestinal diseases.

Flavonoids serve as bioactive components and contribute to medicinal and nutritional profile of Lycii fructus. However, there is limited information regarding the influence of ecological environments on the flavonoid biosynthesis pathway. In this study, we integrated transcriptome sequencing and metabonomic techniques across three distinct cultivation regions to elucidate the processes of flavonoids biosynthesis and the associated gene expression levels in L. fructus. LC-MS/MS based metabolomics revealed significant variations in metabolite profiles including 43 differential flavonoid metabolites, predominantly consisting of flavanol compounds across diverse regions. Additionally, 154 significantly differentially expressed genes (DEGs) were categorized in the flavonoid biosynthesis identified by de novo transcriptome assembly. Transcription factors C2C2 MYB, NAC, WRKY, AP2/ERF and B3 superfamily were the mainly hub genes regulating the flavonoids biosynthesis. The flavonoid pathway was built through integrated analysis of DEGs and DAMs to illustrate the molecular mechanism of flavonoid biosynthesis. Precipitation and temperature may serve as the primary environmental factors that affected the flavonoids variations. This study proposed a schematic of flavonoid biosynthesis in L. fructus, and further provided evidence for environmental response of L. fructus.

Chrysosplenium axillare Maxim. is used in traditional Tibetan medicine for the treatment of various human diseases, such as fever, headache, cholecystitis, acute icterohepatitis and acute liver necrosis. In this study, five new cucurbitane triterpenoid derivatives, chrysosaxillins A-E (1-5), along with three known structurally related compounds (6-8) have been isolated from whole herb of C. axillare. Their structures were elucidated by spectroscopic methods, including 1D and 2D NMR, HRESIMS, UV, IR, ECD and single-crystal X-ray diffraction. All isolates were evaluated for cytotoxic activities against four tumor cell lines including PC-3, A549, MCF-7, and HepG2. The results discovered that compound 1 possessed the most potent cytotoxicity against A549 cells with IC50 value of 0.05 mu M, while compounds 2 and 4 have mild cytotoxicities against cells tested with IC50 values ranging from 8.78 to 41.72 mu M. Our study suggests that C. axillare might serve as a valuable source of cucurbitane triterpenoids potentially useful for the development of new anti-tumor agents and support its use as a crop benefits to local economic.

Large-scale and continuous conformational changes in the RNA self-folding process present significant challenges for structural studies, often requiring trade-offs between resolution and observational scope. Here, we utilize individual-particle cryo-electron tomography (IPET) to examine the post-transcriptional self-folding process of designed RNA origami 6-helix bundle with a clasp helix (6HBC). By avoiding selection, classification, averaging, or chemical fixation and optimizing cryo-ET data acquisition parameters, we reconstruct 120 three-dimensional (3D) density maps from 120 individual particles at an electron dose of no more than 168 e-& Aring;-2, achieving averaged resolutions ranging from 23 to 35 & Aring;, as estimated by Fourier shell correlation (FSC) at 0.5. Each map allows us to identify distinct RNA helices and determine a unique tertiary structure. Statistical analysis of these 120 structures confirms two reported conformations and reveals a range of kinetically trapped, intermediate, and highly compacted states, demonstrating a maturation folding landscape likely driven by helix-helix compaction interactions. Using cryogenic electron tomography (cryo-ET) and individual-particle cryo-electron tomography (IPET), non-averaged ternary structures of individual RNA origami 6HBC particles were determined, allowing observation of structural diversity and self-folding dynamics during the maturation process after transcription.

Contactin 2 (CNTN2) is a cell adhesion molecule involved in axon guidance, neuronal migration, and fascic-ulation. The ectodomains of CNTN1-CNTN6 are composed of six Ig domains (Ig1-Ig6) and four FN domains.Here, we show that CNTN2 forms transient homophilic interactions (KD 200 nM). Cryo-EM structures of full-length CNTN2 and CNTN2_Ig1-Ig6 reveal a T-shaped homodimer formed by intertwined, parallel monomers.Unexpectedly, the horseshoe-shaped Ig1-Ig4 headpieces extend their Ig2-Ig3 tips outwards on either side ofthe homodimer, while Ig4, Ig5, Ig6, and the FN domains form a central stalk. Cross-linking mass spectrometryand cell-based binding assays confirm the 3D assembly of the CNTN2 homodimer. The interface mediatinghomodimer formation differs between CNTNs, as do the homophilic versus heterophilic interaction mecha-nisms. The CNTN family thus encodes a versatile molecular platform that supports a very diverse portfolio ofprotein interactions and that can be leveraged to strategically guide neural circuit development.

To study the chemical constituents of n-butanol extract from Astragalus rigidulus, HP-20 macroporous adsorption resin, Sephadex LH-20 gel, ODS gel column chromatography and semi-preparative high performance liquid chromatography were used to separate the chemical constituents. The structures of all isolates were identified by spectroscopic methods, including NMR and HR-ESI-MS. The results showed that twenty compounds including nineteen flavonoid derivatives and one sesquiterpene glycoside were isolated and purified from n-butanol extract of A. rigidulus. Their structures were identified as 7-O-methylorobol-4′-O-β-D-glucopyranoside (1), mildiside A (2), naringenin (3), purine 4′-O-β-D-glucoside (4), orobot (5), kaempferol-3-O-β-D-(6′-acetyl) glucopyranoside (6), 5,7-dihydroxy-4′-methoxyisoflavone-2′-O-β-D-glucopyranoside (7), amarantholidoside IV (8), kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (9), kaempferol (10), 5,7,4′-trihydroxyisoflavone (11), kaempferol-3-O-β-D-glucopyranoside (12), (S)-mucronulatol (13), calycosin (14), quercetin (15), pratensein-7-O-β-D-glucoside (16), 2′-hydroxy-3′,4′-dimethoxyisoflavan-7-O-β-D-glucoside (17), kaempferol-3-O-rutinoside (18), 5,7,4′-trihydroxy-3′-methoxyflavonol-3-O-rutinoside (19), quercetin-3-O-β-D-glucoside (20). It is the first report for the compounds 1-9 found in the genus Astragalus. The other compounds are isolated from the title plant for the first time. The results of this study provide basic data for the pharmacodynamic material study of A. rigidulus, and provide a theoretical reference for the rational development and utilization of the plant resources in the future.

Macromolecules, such as proteins and nucleic acids, play essential roles in cellular functions through dynamic structural changes. Understanding these functions requires detailed characterization of structural dynamics. While techniques like X-ray crystallography and cryo-EM resolve high-resolution static structures, they struggle to capture low-resolution, flexible structures and the full distribution of conformations during chemical reactions. These limitations arise from averaging processes that enhance signal-to-noise ratio (SNR) but exclude flexible regions, distort resolution, and miss rare high-energy states. To address this, we developed individual-particle electron tomography (IPET), a method for determining 3D structures of single particles at low-to-intermediate resolution (up to 2 nm) without averaging. IPET reconstructs a detailed 3D density map by capturing images at multiple tilt angles, facilitating flexible model fitting and revealing unique particle structures. This method reveals unbiased structural distributions, enhancing the study of molecular dynamics, phase transitions, and structural alterations during chemical reactions and self-folding.

The decline in intestinal stem cell (ISC) function is a hallmark of aging, contributing to compromised intestinal regeneration and increased incidence of age-associated diseases. Novel therapeutic agents that can rejuvenate aged ISCs are of paramount importance for extending healthspan. Here, we report on the discovery of Chrysosplenosides I and A (CAs 1 & 2), flavonol glycosides from the Xizang medicinal plant Chrysosplenium axillare Maxim., which exhibit potent anti-aging effects on ISCs. Our research, using Drosophila models, reveals that CAs 1 & 2 treatments not only restrain excessive ISC proliferation, thereby preserving intestinal homeostasis, but also extend the lifespan of aging Drosophila. In aged mouse intestinal organoids, CAs 1 & 2 enhance the growth and budding of intestinal organoids, indicating improved regenerative capacity. Mechanistic investigations show that CAs 1 & 2 exert their effects by activating the peroxisome proliferator-activated receptor-gamma (PPARγ) and concurrently inhibiting the epidermal growth factor receptor (EGFR) signaling pathways. Our findings position CAs 1 & 2 as promising candidates for ameliorating ISC aging and suggest that targeting PPARγ, in particular, may offer a therapeutic strategy to counteract age-related intestinal dysfunction.

Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1-184) and a hydrophobic C-terminal domain (residues 185-243). When a recombinant fusion protein construct [bacterial pelB leader sequence - human apoA-I (1-243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1-184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1-184) was secreted from the bacteria. When the pelB-apoA-I (1-184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1-184) was the sole major protein present. Incubation of apoA-I (1-184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.

As a part of systematic research, an ongoing phytochemical investigation of the sesquiterpenoid-containing fraction led to the isolation of five new sesquiterpenoids from the peeled stems of Syringa pinnatifolia, including two pairs of enantiomeric humulane-type (±)-alashanoids A1 and B1 (1 and 2) and one eremophilane-type alashanoid C1 (3). These structures were elucidated by the analysis of extensive spectroscopic data, including ESI-MS and 1D and 2D NMR, and the absolute configuration was determined by comparing its experimental and calculated electronic circular dichroism and calculated NMR. These isolates exhibited moderate in vitro cardioprotective effects against oxidative injuries in H9c2 cells.

A new flavonoid derivative, namely astriginoside A (1), was isolated from the whole plant of Astragalus rigidulus Bunge, together with 19 known ones (2–20). Their structures were determined by spectroscopic methods, including 1D and 2D NMR, UV, IR, and HR-ESI-MS, and comparison of the spectroscopic data with those reported in the literature.

Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a major challenge. Here, we apply a novel but simple liquid-cell technique, developed previously for real-time imaging of the dynamics at a liquid-gas interface, to image wet biological samples. With extra scattering from the liquid phase, the transmission electron micrographs show amplitude contrast comparable to that in negatively stained samples. Single-molecule domains are resolved in the protein complex GroEL imaged in buffer solution at room temperature. Moreover, various stages of virus cell entry, which are transient events with very few structural information to date, are also captured. Morphological details are reconstructed using the technique of individual particle electron tomography. These results demonstrate that this approach can be a valuable yet cost-effective technique complementary to other microscopy techniques for addressing important biological questions at the molecular level.

Sintered agglomerate of synthetic mesoporous silica nanoparticles (MSNs) is an architected geomaterial that provides confinement-mediated flow and transport properties of fluids needed for environmental research such as geological subsurface energy storage or carbon capture. The design of those properties can be guided by numerical simulations but is hindered by the lack of method to characterize the permeable pores within MSNs due to pore size. This work uses the advances of an Individual Particle cryogenic transmission Electron Tomography (IPET) technique to obtain detailed 3D morphology of monodispersed MSNs with diameters below 50 nm. The 3D reconstructed density-maps show the diameters of those MSNs vary from 35-46 nm, containing connected intraparticle pores in diameter of 2-20 nm with a mean of 9.2 ± 3 nm, which is comparable to the mean interparticle pore diameters in sintered agglomerate. The characterization of the pore shape and dimensions provides key information for estimating the flow and transport properties of fluids within the sintered agglomerate of those MSNs and for modeling the atomic MSN structures needed for pore-fluid simulations.

Inflammatory bowel diseases (IBDs) are characterized by chronic relapsing intestinal inflammation that causes digestive system dysfunction. For years, researchers have been working to find more effective and safer therapeutic strategies to treat these diseases. Silibinin (SIL), a flavonoid compound extracted from the seeds of milk thistle plants, possesses multiple biological activities and is traditionally applied to treat liver diseases. SIL is also widely used in the treatment of a variety of inflammatory diseases attributed to its excellent antioxidant and anti-inflammatory effects. However, the efficacy of SIL against IBDs and its mechanisms remain unclear. In this study, using Drosophila melanogaster as a model organism, we found that SIL can effectively relieve intestinal inflammation caused by dextran sulfate sodium (DSS). Our results suggested that SIL supplementation can inhibit the overproliferation of intestinal stem cells (ISCs) induced by DSS, protect intestinal barrier function, acid-base balance, and intestinal excretion function, reduce intestinal reactive oxygen species (ROS) levels and inflammatory stress, and extend the lifespan of Drosophila. Furthermore, our study demonstrated that SIL ameliorates intestinal inflammation via modulating the c-Jun N-terminal kinase (JNK) signaling pathway in Drosophila. Our research aims to provide new insight into the treatment of IBDs.

目的 建立藏药灰蒿药材的质量标准,为灰蒿质量控制提供科学的评价依据.方法 对藏药灰蒿进行本草考证,明确其来源,并对21批不同产地的药材进行性状、显微及薄层鉴别、水分、灰分及浸出物进行测定.其中本标准中以东莨菪内酯作为灰蒿药材薄层鉴别的对照品.结果 灰蒿药材显微特征明显;检测21批药材样品,薄层色谱图与东莨菪内酯对照品一致,显相同颜色的荧光斑点;灰蒿药材水分、总灰分、醇溶性浸出物范围分别为7.15%～11.26%、6.03%～14.79%、7.44%～12.01%.结论 该研究首次全面考察灰蒿的各项质量指标,能有效评价灰蒿的质量.

Two new isoprenylated flavonoids, namely artoheteroids F and G ( 1 and 2 ), were isolated from the roots of Artocarpus heterophyllus . Their structures were determined by spectroscopic methods including 1D and 2D NMR, UV, IR, and HR-ESI-MS.