Rapid and signal crowdedness-robust in situ sequencing through hybrid block coding.

Spatial transcriptomics technology has revolutionized our understanding of cell types and tissue organization, opening possibilities for researchers to explore transcript distributions at subcellular levels. However, existing methods have limitations in resolution, sensitivity, or speed. To overcome these challenges, we introduce SPRINTseq (Spatially Resolved and signal-diluted Next-generation Targeted sequencing), an innovative in situ sequencing strategy that combines hybrid block coding and molecular dilution strategies. Our method enables fast and sensitive high-resolution data acquisition, as demonstrated by recovering over 142 million transcripts using a 108-gene panel from 453,843 cells from four mouse brain coronal slices in less than 2 d. Using this advanced technology, we uncover the cellular and subcellular molecular architecture of Alzheimer's disease, providing additional information into abnormal cellular behaviors and their subcellular mRNA distribution. This improved spatial transcriptomics technology holds great promise for exploring complex biological processes and disease mechanisms.

Ultra-high-performance liquid chromatography-mass spectrometry combined with molecular docking and molecular dynamics simulation reveals the source of bitterness in the traditional Chinese medicine formula Runchang-Tongbian.

The Runchang-Tongbian (RCTB) formula is a traditional Chinese medicine (TCM) formula consisting of four herbs, namely Cannabis Fructus (Huomaren), Rehmanniae Radix (Dihuang), Atractylodis Macrocephalae Rhizoma (Baizhu), and Aurantii Fructus (Zhiqiao). It is widely used clinically because of its beneficial effect on constipation. However, its strong bitter taste leads to poor patient compliance. The bitter components of TCM compounds are complex and numerous, and inhibiting the bitter taste of TCM has become a major clinical challenge. Here, we use ultra-high-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) and high-resolution mass spectrometry to identify 59 chemical components in the TCM compound RCTB formula. Next, four bitter taste receptors, TAS2R39, TAS2R14, TAS2R7, and TAS2R5, which are tightly bound to the compounds in RCTB, were screened as molecular docking receptors using the BitterX database. The top-three-scoring receptor-small-molecule complexes for each of the four receptors were selected for molecular dynamics simulation. Finally, seven bitter components were identified, namely six flavonoids (rhoifolin, naringin, poncirin, diosmin, didymin, and narirutin) and one phenylpropanoid (purpureaside C). Thus, we proposed a new method for identifying the bitter components in TCM compounds, which provides a theoretical reference for bitter taste inhibition in TCM compounds.

Three-dimensional digital PCR through light-sheet imaging of optically cleared emulsion.

The realization of the vast potential of digital PCR (dPCR) to provide extremely accurate and sensitive measurements in the clinical setting has thus far been hindered by challenges such as assay robustness and high costs. Here we introduce a lossless and contamination-free dPCR technology, termed CLEAR-dPCR, which addresses these challenges by completing the dPCR sample preparation, PCR, and readout all in one tube. Optical clearing of the droplet dPCR emulsion was combined with emerging light-sheet fluorescence microscopy, to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds. CLEAR-dPCR provides ultrahigh-throughput readout results in situ and fundamentally eliminates the possibility of either sample loss or contamination. This approach exhibits improved accuracy over existing dPCR platforms and enables a greatly increased dynamic range to be comparable to that of real-time quantitative PCR.

Butanol Extract of Acanthopanax senticosus (Rupr. et Maxim.) Harms Alleviates Atherosclerosis in Apolipoprotein E-Deficient Mice Fed a High-Fat Diet.

This study investigated the effect of butanol extract of AS (ASBUE) on atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. The mice were administered ASBUE (390 or 130 mg/kg/day) or rosuvastatin (RSV) via oral gavage for eight weeks. In ApoE-/- mice, ASBUE suppressed the abnormal body weight gain and improved serum and liver biochemical indicators. ASBUE remarkably reduced the aortic plaque area, improved liver pathological conditions, and lipid metabolism abnormalities, and altered the intestinal microbiota structure in ApoE-/- mice. In the vascular tissue of ASBUE-treated mice, P-IKKß, P-NFκB, and P-IκBα levels tended to decrease, while IκB-α increased in high fat-diet-fed atherosclerotic mice. These findings demonstrated the anti-atherosclerotic potential of ASBUE, which is mediated by the interaction between the gut microbiota and lipid metabolism and regulated via the Nuclear Factor-kappa B (NF-κB) pathway. This work paves the groundwork for subsequent studies to develop innovative drugs to treat atherosclerosis.

Hyperoside Nanomicelles Alleviate Atherosclerosis by Modulating the Lipid Profile and Intestinal Flora Structure in High-Fat-Diet-Fed Apolipoprotein-E-Deficient Mice.

Atherosclerosis (AS) is a serious threat to human health and the main pathological basis of cardiovascular disease. Hyperoside (Hyp), a flavonoid found mainly in traditional Chinese herbs, can exert antitumor, anti-inflammatory, antioxidant, and cardiovascular-protective effects. Herein, we prepared hybrid nanomicelles (HFT) comprising Hyp loaded into pluronic F-127 and polyethylene glycol 1000 vitamin E succinate and assessed their effects on AS. To establish an AS model, apolipoprotein-E-deficient (ApoE-/-) mice were fed a high-fat diet. We then analyzed the effects of HFT on AS-induced changes in aortic tissues and metabolic markers, simultaneously assessing changes in gut flora community structure. In mice with AS, HFT significantly reduced the aortic plaque area; decreased levels of total cholesterol, triglyceride, low-density lipoprotein cholesterol, inflammatory factors, and inducible nitric oxide synthase (NOS); increased high-density lipoprotein cholesterol, endothelial NOS, superoxide dismutase, catalase, and glutathione levels; and promoted the proliferation of beneficial gut bacteria. HFT could regulate intestinal flora structure and lipid metabolism and inhibit inflammatory responses. These beneficial effects may be mediated by inhibiting nuclear factor kappa B signal activation, reducing inflammatory factor expression and improving gut microflora structure and dyslipidemia. The present study provides an empirical basis for the development and clinical application of new dosage forms of Hyp.

Optimization of the extraction process for Shenshou Taiyi powder based on Box-Behnken experimental design, standard relation, and FAHP-CRITIC methods.

BACKGROUND: Ancient classic prescription play a crucial role in the preservation and advancement of traditional Chinese medicine (TCM) theories. They represent a significant milestone in the ongoing development and transmission of TCM knowledge and practices and are considered one of the breakthroughs in the development of TCM inheritance. In the process of developing ancient classic prescriptions, many problems may still arise in ensuring quality consistency between traditional methods and modern production processes, among which the extraction process poses major challenges. This paper introduces a practical approach extracting an ancient classic prescription using a modern extraction process. The technique is demonstrated through the study of the extraction process of Shenshou Taiyi powder (STP). METHODS: This study focuses on optimising the STP extraction process to ensure consistency in the quality of the product obtained through ancient and modern processes using the standard relation and fuzzy analytic hierarchical process (FAHP) and criteria importance through intercriteria correlation (CRITIC) method integrated weights combined with the Box-Behnken response surface test. Using the contents of rosmarinic acid, isoimperatorin, puerarin, as well as the extract yield and fingerprint similarity as evaluation indexes of STP, the Box-Behnken response surface method was employed to examine the varying extraction parameters, including water addition ratio, extraction duration, and number of extractions. The weighted coefficients for each parameter were calculated by combining the benchmark correlation and FAHP-CRITIC method, deriving a comprehensive score. RESULTS: The optimal extraction process for STP consisted of a two extractions, each using at a tenfold quantity of water, performed for one hour. Process verification across three separate batches yielded a comprehensive score of 94.7, with a relative standard deviation of 0.76%. CONCLUSIONS: The application of the Box-Behnken response surface method combined with standard relation and FAHP-CRITIC approach proved to be stable and feasible for optimising the extraction process of STP.

Design and High-Resolution Analysis of an Efficient Periodic Split-and-Recombination Microfluidic Mixer.

We developed a highly efficient passive mixing device based on a split-and-recombine (SAR) configuration. This micromixer was constructed by simply bonding two identical microfluidic periodical open-trench patterns face to face. The structure parameters of periodical units were optimized through numerical simulation to facilitate the mixing efficiency. Despite the simplicity in design and fabrication, it provided rapid mixing performance in both experiment and simulation conditions. To better illustrate the mixing mechanism, we developed a novel scheme to achieve high-resolution confocal imaging of serial channel cross-sections to accurately characterize the mixing details and performance after each SAR cycle. Using fluorescent IgG as an indicator, nearly complete mixing was achieved using only four SAR cycles in an aqueous solution within a device's length of less than 10 mm for fluids with a Péclet number up to 8.7 × 104. Trajectory analysis revealed that each SAR cycle transforms the input fluids using three synergetic effects: rotation, combination, and stretching to increase the interfaces exponentially. Furthermore, we identified that the pressure gradients in the parallel plane of the curved channel induced vertical convection, which is believed to be the driving force underlying these effects to accelerate the mixing process.

Open-top light-sheet imaging of CLEAR emulsion for high-throughput loss-free analysis of massive fluorescent droplets.

Digital droplet PCR (ddPCR) is classified as the third-generation PCR technology that enables absolute quantitative detection of nucleic acid molecules and has become an increasingly powerful tool for clinic diagnosis. We previously established a CLEAR-dPCR technique based on the combination of CLEAR droplets generated by micro-centrifuge-based microtubule arrays (MiCA) andinsitu3D readout by light-sheet fluorescence imaging. This CLEAR-dPCR technique attains very high readout speed and dynamic range. Meanwhile, it is free from sample loss and contamination, showing its advantages over commercial d-PCR technologies. However, a conventional orthogonal light-sheet imaging setup in CLEAR d-PCR cannot image multiple centrifuge tubes, thereby limiting its widespread application to large-scale, high-speed dd-PCR assays. Herein, we propose an in-parallel 3D dd-PCR readout technique based on an open-top light-sheet microscopy setup. This approach can continuously scan multiple centrifuge tubes which contain CLEAR emulsions with highly diverse concentrations, and thus further boost the scale and throughput of our 3D dd-PCR technique.

Rotational scan digital LAMP for accurate quantitation of nucleic acids.

Digital quantitation of nucleic acids is precise and sensitive because of its molecular-level resolution. However, only several quantitation formats are common, especially pertaining to how one obtains digital signals from multiple droplets. Here we present rotational scan digital loop-mediated amplification, termed RS-dLAMP. Droplets generated by centrifugation undergo isothermal loop-mediated amplification (LAMP), and self-tile by gravitation into a tubular space between two coaxial cylinders, which are then rotated and scanned to acquire droplet fluorescence signals. RS-dLAMP is quantitatively comparable to commercial digital PCR, yet has higher throughput. Moreover, by sealing the sample throughout analysis, RS-dLAMP eliminates contamination, facilitating point-of-care diagnosis and other applications.

Reconstruction of Dynamic and Reversible Color Change using Reflectin Protein.

Cephalopods have remarkable ability to change their body color across a wide range of wavelengths, yet the structural basis remains largely unknown. Reflectin, a protein family assumed to be responsible for structural color in cephalopods, has unique features of higher-order assembly that are tightly regulated by aromatic molecules. Here, we reconstructed the dynamic and reversible color change using purified reflectin protein and demonstrated how the conformational change and the status of assembly led to the change in optical properties. In addition, optical spectral and structural analyses indicated that the "cephalopod-blue" primarily resulted from wavelength-dependent light scattering rather than reflection. Our results suggest a possible role of reflectin in color dynamics. The in vitro reconstruction system we present here may serve as an initial step for designing bio-inspired optical materials based on reflectin protein.

High-throughput single-cell whole-genome amplification through centrifugal emulsification and eMDA.

Single-cell whole-genome sequencing (scWGS) is mainly used to probe intercellular genomic variations, focusing on the copy number variations or alterations and the single-nucleotide variations (SNVs) occurring within single cells. Single-cell whole-genome amplification (scWGA) needs to be applied before scWGS but is challenging due to the low copy number of DNA. Besides, many genomic variations are rare within a population of cells, so the throughput of currently available scWGA methods is far from satisfactory. Here, we integrate a one-step micro-capillary array (MiCA)-based centrifugal droplet generation technique with emulsion multiple displacement amplification (eMDA) and demonstrate a high-throughput scWGA method, MiCA-eMDA. MiCA-eMDA increases the single-run throughput of scWGA to a few dozen, and enables the assessment of copy number variations and alterations at 50-kb resolution. Downstream target enrichment further enables the detection of SNVs with 20% allele drop-out.

Centrifugal micro-channel array droplet generation for highly parallel digital PCR.

Stable water-in-oil emulsion is essential to digital PCR and many other bioanalytical reactions that employ droplets as microreactors. We developed a novel technology to produce monodisperse emulsion droplets with high efficiency and high throughput using a bench-top centrifuge. Upon centrifugal spinning, the continuous aqueous phase is dispersed into monodisperse droplet jets in air through a micro-channel array (MiCA) and then submerged into oil as a stable emulsion. We performed dPCR reactions with a high dynamic range through the MiCA approach, and demonstrated that this cost-effective method not only eliminates the usage of complex microfluidic devices and control systems, but also greatly suppresses the loss of materials and cross-contamination. MiCA-enabled highly parallel emulsion generation combines both easiness and robustness of picoliter droplet production, and breaks the technical challenges by using conventional lab equipment and supplies.