Monitoring and quantifying ATP levels in vivo is essential to understanding its role as a signaling molecule in tumor progression and therapy. Nevertheless, the real-time monitoring and quantitative assessment of lysosomal ATP remains challenging due to the lack of accurate tools in deep tissues. In this study, based on the crosslinking enhanced emission (CEE) effect, we successfully synthesized red carbon dots (R-CDs) with dual emission properties for efficient quantification of intracellular ATP. The R-CDs emit in the near-infrared range and target lysosomes with rapid detection capabilities, rendering them exceptionally well-suited for directly observing and analyzing the dynamics of lysosomal ATP through live cell imaging techniques. Importantly, R-CDs have proven their efficacy in real-time monitoring of drug stimulus-induced fluctuations in endogenous lysosomal ATP concentration and have also been employed for quantifying and distinguishing lysosomal ATP levels among normal and cancer cell lines. These noteworthy findings emphasize the versatility of the R-CD as a valuable imaging tool for elucidating the functional role of lysosomal ATP in drug screening and cancer diagnostics and hold the promise of becoming a reference tool for deepening our understanding of drug mechanisms of action.

The spontaneous generation of hydrogen peroxide (H2O2) within atmospheric microdroplets, such as raindrops and aerosols, plays a crucial role in various environmental processes including pollutant degradation and oxidative stress. However, quantifying hydroxyl radicals (•OH), essential for H2O2 formation, remains challenging due to their short lifespan and low concentration. This study addresses this gap by presenting a highly sensitive and selective surface-enhanced Raman scattering (SERS) nanosensor specifically designed for quantifying •OH within water microdroplets. Utilizing a phthalhydrazide (Phth) probe, the SERS technique enables rapid, interference-free detection of •OH at nanomolar concentrations. It achieves a linear detection range from 2 nM to 2 μM and a limit of detection as low as 0.34 nM. Importantly, the SERS sensor demonstrates robustness and accuracy within water microdroplets, paving the way for comprehensive mechanistic studies of H2O2 generation in the atmosphere. This innovative approach not only offers a powerful tool for environmental research but also holds potential for advancing our understanding of atmospheric H2O2 formation and its impact on air quality and pollutant degradation.

BACKGROUND: RNA sequencing (RNA-seq) is widely used for gene expression profiling and quantification. Quantitative RNA sequencing usually requires cell counting and spike-in, which is not always applicable to many samples. Here, we present a novel quantitative RNA sequencing method independent of spike-ins or cell counting, named siqRNA-seq, which can be used to quantitatively profile gene expression by utilizing gDNA as an internal control. Single-stranded library preparation used in siqRNA-seq profiles gDNA and cDNA with equal efficiency.
RESULTS: To quantify mRNA expression levels, siqRNA-seq constructs libraries for total nucleic acid to establish a model for expression quantification. Compared to Relative Quantification RNA-seq, siqRNA-seq is technically reliable and reproducible for expression profiling but also can sequence reads from gDNA which can be used as an internal reference for accurate expression quantification. Applying siqRNA-seq to investigate the effects of actinomycin D on gene expression in HEK293T cells, we show the advantages of siqRNA-seq in accurately identifying differentially expressed genes between samples with distinct global mRNA levels. Furthermore, we analyzed factors influencing the downward trend of gene expression regulated by ActD using siqRNA-seq and found that mRNA with m6A modification exhibited a faster decay rate compared to mRNA without m6A modification. Additionally, applying this technique to the quantitative analysis of seven tumor cell lines revealed a high degree of diversity in total mRNA expression among tumor cell lines.
CONCLUSIONS: Collectively, siqRNA-seq is a spike-in independent quantitative RNA sequencing method, which creatively uses gDNA as an internal reference to absolutely quantify gene expression. We consider that siqRNA-seq provides a convenient and versatile method to quantitatively profile the mRNA landscape in various samples.

Variations in herb dosage due to species adulteration and dosing inaccuracies can substantially affect clinical safety and efficacy. Accurate species quantification remains challenging, as current methods often yield inconsistent results. This study introduces a novel pyrosequencing-based technique, termed herb molecular quantification (Herb-Q), designed to precisely quantify herbal products. We evaluated its effectiveness using Pinellia ternata and five of its adulterants. Initially, we assessed commonly used DNA barcodes with sequences from a public database, identifying two candidate regions, Maturase K (matK) and internal transcribed spacer 2 (ITS2), for screening specific single nucleotide polymorphism (SNP) loci, allowing for species-specific identification. These loci were validated by amplifying and sequencing genomic material from collected samples. Our validation studies showed that Herb-Q demonstrated excellent linearity, accuracy, repeatability, and detection limits. We established quantitative standard curves with high R2 values (> 0.99) to enable precise species quantification, which were combined with external standards to provide clear and accurate visual quantification results. The average bias in quantifying the tuber of P. ternata was 2.38%, confirming that Herb-Q can accurately identify and quantify herbal product constituents. Moreover, the entire quantification process took less than 4 h. This study presents a novel, rapid method for accurately quantifying species in herbal products and advances the application of DNA barcoding from species identification to quantitative detection.

In recent years, serum hepatitis B virus (HBV) RNA has been identified as a promising noninvasive surrogate biomarker of intrahepatic covalently closed circular DNA (cccDNA), detection of which requires an invasive liver biopsy in patients with chronic HBV infection. It is impractical to detect intrahepatic cccDNA as a routine diagnosis for chronic hepatitis B (CHB) patients in clinical management. Here, we describe a detailed protocol for serum HBV RNA quantification, which can reflect the activity of intrahepatic cccDNA. The procedure includes three major steps: (1) Simultaneous isolation of HBV DNA and RNA from patients\' serum, (2) DNase I digestion for removing HBV DNA contamination, and (3) HBV RNA quantification by one-step reverse transcription qPCR.

The diterpene ent-copalol is an important precursor to the synthesis of andrographolide and is found only in green chiretta (Andrographis paniculata). De novo biosynthesis of ent-copalol has not been reported, because the catalytic activity of ent-copalyl diphosphate synthase (CPS) is very low in microorganisms. In order to achieve the biosynthesis of ent-copalol, Saccharomyces cerevisiae was selected as the chassis strain, because its endogenous mevalonate pathway and dephosphorylases could provide natural promotion for the synthesis of ent-copalol. The strain capable of synthesizing diterpene geranylgeranyl pyrophosphate was constructed by strengthening the mevalonate pathway genes and weakening the competing pathway. Five full-length ApCPSs were screened by transcriptome sequencing of A. paniculata and ApCPS2 had the best activity and produced ent-CPP exclusively. The peak area of ent-copalol was increased after the ApCPS2 saturation mutation and its configuration was determined by NMR and ESI-MS detection. By appropriately optimizing acetyl-CoA supply and fusion-expressing key enzymes, 35.6 mg/L ent-copalol was generated. In this study, de novo biosynthesis and identification of ent-copalol were achieved and the highest titer ever reported. It provides a platform strain for the further pathway analysis of andrographolide and derivatives and provides a reference for the synthesis of other pharmaceutical intermediates.

BACKGROUND: Allii Macrostemonis Bulbus is also named Xiebai in China. It is an edible vegetable, and also a famous herb for treating coronary heart disease. Allium chinense G. Don (ACGD) and Allium macrostemon Bunge (AMB) are it botanical sources. The aim of this study was to explore the cardioprotective effects, and decipher the visual spatial distribution and absolute content of primary metabolites derived from these two herbs.
METHODS: H9c2 cells were used to perform the hypoxia-reoxygenation (H/R)-induced myocardial injury model. Their protective effects were evaluated by apoptosis levels. Furthermore, matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry imaging approach (MALDI-TOF MSI) was carried out to present the spatial location of primary metabolites including fatty acids, amino acids, carotenoids, and vitamins in these two Allium herbs. Multiple analytical methods were applied to perform quantitative analysis of these primary metabolites in AMB and ACGD bulbs by liquid chromatography tandem mass spectrometry (LC-MS).
RESULTS: First, AMB and ACGD extracts both could increase the cell viability in H9c2 cells, and attenuate H/R-induced injury. They markedly decreased apoptosis, accompanied by activating the BCL-2/BAX pathway. Further, MALDI-TOF MSI-based relative quantification results showed several amino acids, fatty acids, carotenoids, and vitamins were largely rich in the tunics and outside scales of fresh bulbs, while some primary metabolites were abundant in their developing flower buds. Absolute quantification results displayed total contents of amino acids in ACGD bulbs were higher than those in AMB, while total contents of fatty acids and vitamins provides opposite trends in these two Allium herbs. The total contents of carotenoids and trace elements showed no significant differences between AMB and ACGD samples.
CONCLUSIONS: This study would be helpful to understand the myocardial injury protection effects of these two Allium herbs, and the spatial accumulation and quantitative content levels of their main nutrients.

UNASSIGNED: The study aims to develop a finite element model of the pelvic floor and thighs of elderly men to quantitatively assess the impact of different pelvic floor muscle trainings and the urinary and defecation control ability.
UNASSIGNED: A finite element model of the pelvic floor and thighs of elderly men was constructed based on MRI and CT. Material properties of pelvic floor tissues were assigned through literature review, and the relative changes in waistline, retrovesical angle (RVA) and anorectad angulation (ARA) to quantitatively verify the effectiveness of the model. By changing the material properties of muscles, the study analyzed the muscle strengthening or impairment effects of the five types of rehabilitation training for four types of urination and defecation dysfunction. The changes in four outcome indicators, including the retrovesical angle, anorectad angulation, stress, and strain, were compared.
UNASSIGNED: This study indicates that ARA and RVA approached their normal ranges as material properties changed, indicating an enhancement in the urinary and defecation control ability, particularly through targeted exercises for the levator ani muscle, external anal sphincter, and pelvic floor muscles. This study also emphasizes the effectiveness of personalized rehabilitation programs including biofeedback, exercise training, electrical stimulation, magnetic stimulation, and vibration training and advocates for providing optimized rehabilitation training methods for elderly patients.
UNASSIGNED: Based on the results of computational biomechanics, this study provides foundational scientific insights and practical recommendations for rehabilitation training of the elderly\'s urinary and defecation control ability, thereby improving their quality of life. In addition, this study also provides new perspectives and potential applications of finite element analysis in elderly men, particularly in evaluating and designing targeted rehabilitation training.

Microplastics (MPs), especially polystyrene microplastics (PS-MPs), have emerged a new worldwide pollutant, prompting significant public concern regarding their detection in environmental media. Analysis of PS-MPs in soil remains as a challenging task for analysts due to the highly intricate matrices. This work presents a practical approach for detecting PS-MPs in soil, which involves dilute HCl-assisted extraction and gel permeation chromatography- ultraviolet detection (GPC-UV) analysis. The presence of MPs in soil was confirmed through the use of a scanning electron microscope in conjunction with energy dispersive spectroscopy investigation. PS-MPs was isolated from soil, by agitating it with a diluted HCl solution, filtering the resulting liquid, and dissolving the residue on the filter with THF. The extractant was subsequently determined by GPC-UV. The introduction of a small amount of HCl into the extraction system was found to greatly expedite the settling of soil in water and enhance the efficacy of extracting PS-MPs in about 30 min. The linear range of PS-MPs was from 1.0 to 100 μg/mL with R2 > 0.999. Good reproducibility was obtained with the intra-day relative standard deviation (RSD, n = 3) of 1.36 % and the inter-day RSD (n = 3) of 4.78 %. The concentration of PS-MPs in soil samples were N.D. - 2.33 μg/g, and the good recoveries were 76.7-100.3 %. The corresponding AFGEEprer score was calculated to be 0.59, indicating the concept of green analytical chemistry for the pretreatment method. These results indicated that this method has a powerful potential for the accurate and rapid determination of PS-MPs in soil.

BACKGROUND: To investigate the accuracy of three model formulae for ultrasound quantification of pleural effusion (PE) volume in patients in supine position.
METHODS: A prospective study including 100 patients with thoracentesis and drainage of PE was conducted. Three model formulae (single section model, two section model and multi-section model) were used to calculate the PE volume. The correlation and consistency analyses between calculated volumes derived from three models and actual PE volume were performed.
RESULTS: PE volumes calculated by three models all showed significant linear correlations with actual PE volume in supine position (all p < 0.001). The reliability of multi-section model in predicting PE volume was significantly higher than that of single section model and slightly higher than that of two section model. When compared with actual drainage volume, the intra-class correlation coefficients (ICCs) of single section model, two section model and multi-section model were 0.72, 0.97 and 0.99, respectively. Significant consistency between calculated PE volumes by using two section model and multi-section model existed for full PE volume range (ICC 0.98).
CONCLUSIONS: Based on the convenience and accuracy of ultrasound quantification of PE volume, two section model is recommended for pleural effusion assessment in routine clinic, though different model formulae can be selected according to clinical needs.