A simple and sensitive LC-MS/MS method for the determination of loganic acid in rat plasma and its pharmacokinetic analysis.

Loganic acid is an iridoid compound extracted from Gentianaceae plant Gentiana macrophylla Pall. It can effectively inhibit inflammation and tumor migration and has antioxidant activity. In this paper, we establish a simple, fast, sensitive and validated LC-MS method with the purpose of quantification of loganic acid in rat plasma with gliclazide as an internal standard (IS). Methanol was used to precipitate the protein in the plasma sample, and a C18 column (2.1 × 50 mm, 1.7 µm) was used for the separation of the target compound. Meanwhile, 0.1% formic acid water-methanol was employed as the mobile phase. Multiple reaction monitoring detection mode was adopted in detection with m/z 375.1 > 213.2 for loganic acid and m/z 322.1 > 169.9 for the IS, respectively, in negative ion scan mode. The linear range of calibration curve was 5.77-11,540.00 ng/ml, and the lower limit of detedtion was 2.89 ng/ml. The inter-day and intra-day precision and accuracy were <15% for lower limit of quantitation, low, middle and high quality control samples. This method was successfully used for the pharmacokinetic study of loganic acid in rat plasma at a dose range of 50-150 mg/kg for oral administration and 2 mg/kg for intravenous administration. The pharmacokinetic results showed that the oral bioavailability of loganic acid was low (2.71-5.58%).

The application of THz-TDS in the characterization of Bayan Obo magnetite ore composition.

The application of terahertz time-domain spectroscopy (THz-TDS) in the quantitative analysis of major minerals in Bayan Obo magnetite ore was explored. The positive correlation between the optical parameters of the original ore and its iron content is confirmed. The detections of three main iron containing minerals, including magnetite, pyrite, and hematite, were simulated using corresponding reagents. The random forest algorithm is used for quantitative analysis, and FeS2 is detected with precision of R2 = 0.7686 and MAE = 0.6307% in ternary mixtures. The experimental results demonstrate that THz-TDS can distinguish specific iron containing minerals and reveal the potential application value of this testing method in exploration and mineral processing fields.

Crystallographic phase identifier of a convolutional self-attention neural network (CPICANN) on powder diffraction patterns.

Spectroscopic data, particularly diffraction data, are essential for materials characterization due to their comprehensive crystallographic information. The current crystallographic phase identification, however, is very time consuming. To address this challenge, we have developed a real-time crystallographic phase identifier based on a convolutional self-attention neural network (CPICANN). Trained on 692 190 simulated powder X-ray diffraction (XRD) patterns from 23 073 distinct inorganic crystallographic information files, CPICANN demonstrates superior phase-identification power. Single-phase identification on simulated XRD patterns yields 98.5 and 87.5% accuracies with and without elemental information, respectively, outperforming JADE software (68.2 and 38.7%, respectively). Bi-phase identification on simulated XRD patterns achieves 84.2 and 51.5% accuracies, respectively. In experimental settings, CPICANN achieves an 80% identification accuracy, surpassing JADE software (61%). Integration of CPICANN into XRD refinement software will significantly advance the cutting-edge technology in XRD materials characterization.

The therapeutic effect of sufficient oxygen-rich PRP injection in facial rejuvenation by multiple objective evaluations in 26 cases.

Background: Ozone can enhance the expression of some growth factors (GFs) in platelet rich plasma (PRP), recent study showed oxygen-rich PRP (ozonized PRP) have better therapeutic effects on bone and joint diseases. PRP injection has been widely used in the treatment of facial rejuvenation, but the efficacy of sufficient oxygen-rich PRP in facial rejuvenation has not been studied. Objective: Firstly, we examined whether ozone treatment can increase the concentration of GFs of PRP in vitro. And then a variety of subjective and objective detection methods were used to evaluate the effect of sufficient(10-12 mL each time for the injection of face and neck) oxygen-rich (ozonized PRP) PRP injection in facial rejuvenation by follow-up for 6 months. At last, we investigated the satisfaction, side effects and pain score of the treatment through a questionnaire survey. Methods: The concentration of main GFs in PRP treated with different dose of ozone in vitro was measured by ELISA. Clinical picture, the collagen thickness of dermis by reflectance confocal microscope(RCM), skin conditions (including spots, ultraviolet (UV) spots, brown spots, red area, pores, wrinkles, texture and porphyrin) by VISIA were collected before treatment and each month follow-up visit after treatment until 6-month follow-up period was finished. Patients' satisfaction, side effects and pain score were collected at the end of follow-up period. Results: PRP treated by high-dose ozone (57 µg/mL, ozone/PRP volume ratio:1/1) in vitro showed a significant increase in endothelial growth factor (EGF) and transforming growth factor-ß (TGF-ß) compared to baseline(P < 0.05). Collagen thickness of forehead, cheek and neck improved significantly compare to the baseline until to the 6 months after treatment. Spots, UV spots, brown spots, red area and texture improved significantly compare to the baseline(P < 0.05). All of participants reported improvement and have a median pain score of 4.19. No serious adverse events were observed. Conclusions: Ozone treatment can increase the concentration of GFs such as EGF and TGF-ß in PRP in vitro. Sufficient oxygen-rich PRP injection may be an effective and promising method to treat facial rejuvenation.

0.01% Atropine Eye Drops in Children With Myopia and Intermittent Exotropia: The AMIXT Randomized Clinical Trial.

Importance: Exotropia and myopia are commonly coexistent. However, evidence is limited regarding atropine interventions for myopia control in children with myopia and intermittent exotropia (IXT). Objective: To evaluate the efficacy and safety of 0.01% atropine eye drops on myopia progression, exotropia conditions, and binocular vision in individuals with myopia and IXT. Design, Setting, and Participants: This placebo-controlled, double-masked, randomized clinical trial was conducted from December 2020 to September 2023. Children aged 6 to 12 years with basic-type IXT and myopia of -0.50 to -6.00 diopters (D) after cycloplegic refraction in both eyes were enrolled. Intervention: Participants were randomly assigned in a 2:1 ratio to 0.01% atropine or placebo eye drops administered in both eyes once at night for 12 months. Main Outcomes and Measures: The primary outcome was change in cycloplegic spherical equivalent from baseline at 1 year. Secondary outcomes included change in axial length (AL), accommodative amplitude (AA), exotropia conditions, and binocular vision at 1 year. Results: Among 323 screened participants, 300 children (mean [SD] age, 9.1 [1.6] years; 152 male [50.7%]) were included in this study. A total of 200 children (66.7%) were in the atropine group, and 100 (33.3%) were in the placebo group. At 1 year, the 0.01% atropine group had slower spherical equivalent progression (-0.51 D vs -0.75 D; difference = 0.24 D; 95% CI, 0.11-0.37 D; P < .001) and AL elongation (0.31 mm vs 0.42 mm; difference = -0.11 mm; 95% CI, -0.17 to -0.06 mm; P < .001) than the placebo group. The mean AA change was -3.06 D vs 0.12 D (difference = -3.18 D; 95% CI, -3.92 to -2.44 D; P < .001) in the atropine and placebo groups, respectively. The 0.01% atropine group had a decrease in near magnitude of exodeviation whereas the placebo group had an increase (-1.25 prism diopters [PD] vs 0.74 PD; difference = -1.99 PD; 95% CI, -3.79 to -0.19 PD; P = .03). In the atropine vs placebo group, respectively, the incidence of study drug-related photophobia was 6.0% (12 of 200 participants) vs 8.0% (8 of 100 participants; difference = -2.0%; 95% CI, -9.4% to 3.7%; P = .51) and for blurred near vision was 6.0% (12 of 200 participants) vs 7.0% (7 of 100 participants) (difference = -1.0%; 95% CI, -8.2% to 4.5%; P = .74). Conclusions and Relevance: The findings of this randomized clinical trial support use of 0.01% atropine eye drops, although compromising AA to some extent, for slowing myopia progression without interfering with exotropia conditions or binocular vision in children with myopia and IXT. Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR2000039827.

Traditional Chinese Medicine JianPiHuaTan formula improving quality of life and survival in patients with colorectal cancer through RAS/RAF downstream signaling pathways.

Objective: JianPiHuaTan Formula (JPHTF), a traditional Chinese medicine (TCM), has been utilized as an adjunctive therapy for colorectal cancer (CRC). The study aims to evaluate the potential clinical benefits of JPHTF and its effectiveness in inhibiting tumor growth. Methods: 300 stage II/III CRC patients and 412 advanced CRC patients were enrolled to verify the clinical value of JPHTF in CRC treatment. Furthermore, CRC patient-derived xenograft (PDX) mice were utilized to investigate the regulatory mechanisms of JPHTF. Results: JPHTF significantly improved abdominal distension, shortness of breath, drowsiness, loss of appetite, sleep, and tiredness in stage II/III CRC patients, thereby improving their quality of life. Simultaneously, JPHTF served as a supportive therapy in extending the overall survival (OS) of stage IV CRC patients with RAS/RAF mutations undergoing chemotherapy. Additionally, JPHTF effectively impeded tumor progression in CRC PDX models with RAS mutation, accompanied by a reduction in tumor cell content in the JPHTF group. Transcriptomic analysis revealed the involvement of the Hippo and Hedgehog signaling pathways in JPHTF-mediated CRC inhibition. Furthermore, mice in the JPHTF group exhibited increased immune cell infiltration. Conclusion: These findings suggested that JPHTF may inhibits tumor growth in CRC with RAS mutation by modulating RAS/RAF downstream signaling pathways, specifically the Hippo and Hedgehog signaling, leading to increased immune cell infiltration.

Establishment of a transgene-free iPS cell line (SDCHi007-A) from a young patient bearing a ATP1A2 mutation and suffering from Epilepsy.

Epilepsy is a chronic neurological disease. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient diagnosed as epilepsy caused by ATP1A2 gene mutation. Induced pluripotent stem cells (iPSCs) were developed using non-integrating episomal vectors containing OCT4, SOX2, KLF4, BCL-XL and C-MYC. The established iPSC line (SDCHi007-A) displayed pluripotent cell morphology, high expression levels of pluripotency markers, differentiation potential in vitro, normal karyotype, and remaining the original ATP1A2 gene mutation.

Sea urchin inspired ultrafast response low humidity sensor based on ionic liquid modified UiO-66 with advanced applications.

Numerous applications require low humidity sensors that not only sensitive but also stable, small hysteresis, high resolution and fast response. However, most reported low humidity sensors cannot possess these properties at the same time. In this work, inspired by sea urchin, we developed an ionic liquid (IL) modified metal organic framework (UiO-66) based low humidity sensor. Owing to the synergistic effect of the hydrophilicity and ionic conductivity of IL and the steric hindrance effects of UiO-66, the optimized low humidity sensor simultaneously exhibits high response (47.5), small hysteresis (0.3 % RH), ultrafast response speed (0.2 s), high resolution (1 % RH), and excellent long-term stability (>120 days). In particular, the sensor has been proved to have potential applications in visual humidity detection and water source location. This work provides a preliminary design principle that will contribute to the preparation of high-performance low humidity sensing materials.

Biodegradable Poly (L-Lactic acid) Fibrous Membrane with Ribbon-Structured Fibers and Ultrafine Nanofibers Enhances Air Filtration Performance.

Here, the poly (l-lactic acid) (PLLA) membrane with multi-structured networks (MSN) is successfully prepared by electrospinning technology for the first time. It is composed of micron-sized ribbon-structured fibers and ultrafine nanofibers with a diameter of tens of nanometers, and they are connected to form the new network structure. Thanks to the special fiber morphology and structure, the interception and electrostatic adsorption ability for against atmospheric particulate matter (PM) are significantly enhanced, and the resistance to airflow is reduced due to the "slip effect" caused by ultrafine nanofibers. The PLLA MSN membrane shows excellent filtration performance with ultra-high filtration efficiency (>99.9% for PM2.5 and >99.5% for PM0.3) and ultra-low pressure drop (≈20 Pa). It has demonstrated filtration performance that even exceeds current non-biodegradable polymer materials, laying the foundation for future applications of biodegradable PLLA in the field of air filtration. In addition, this new structure also provides a new idea for optimizing the performance of other polymer materials.

Overview and Evolution of Insect Fibroin Heavy Chain (FibH).

The FibH gene, crucial for silk spinning in insects, encodes a protein that significantly influences silk fiber mechanics. Due to its large size and repetitive sequences, limited known sequences of insect FibH impede comprehensive understanding. Here, we analyzed 114 complete FibH gene sequences from Lepidoptera (71 moths, 24 butterflies) and 13 Trichoptera, revealing single-copy FibH in most species, with 2-3 copies in Hesperinae and Heteropterinae (subfamily of skippers). All FibH genes are structured with two exons and one intron (39-45 bp), with the second exon being notably longer. Moths exhibit higher GC content in FibH compared to butterflies and Trichoptera. The FibH composition varies among species, with moths and butterflies favoring Ala, Gly, Ser, Pro, Gln, and Asn, while Trichoptera FibH is enriched in Gly, Ser, and Arg, and has less Ala. Unique to Trichoptera FibH are Tyr, Val, Arg, and Trp, whereas Lepidoptera FibH is marked by polyAla (polyalanine), polySer (polyserine), and the hexapeptide GAGSGA. A phylogenetic analysis suggests that Lepidoptera FibH evolved from Trichoptera, with skipper FibH evolving from Papilionoidea. This study substantially expands the FibH repertoire, providing a foundation for the development of artificial silk.

In Situ Growth of COF/PVA-Carrageenan Hydrogel Using the Impregnation Method for the Purpose of Highly Sensitive Ammonia Detection.

Flexible ammonia (NH3) gas sensors have gained increasing attention for their potential in medical diagnostics and health monitoring, as they serve as a biomarker for kidney disease. Utilizing the pre-designable and porous properties of covalent organic frameworks (COFs) is an innovative way to address the demand for high-performance NH3 sensing. However, COF particles frequently encounter aggregation, low conductivity, and mechanical rigidity, reducing the effectiveness of portable NH3 detection. To overcome these challenges, we propose a practical approach using polyvinyl alcohol-carrageenan (κPVA) as a template for in the situ growth of two-dimensional COF film and particles to produce a flexible hydrogel gas sensor (COF/κPVA). The synergistic effect of COF and κPVA enhances the gas sensing, water retention, and mechanical properties. The COF/κPVA hydrogel shows a 54.4% response to 1 ppm NH3 with a root mean square error of less than 5% and full recovery compared to the low response and no recovery of bare κPVA. Owing to the dual effects of the COF film and the particles anchoring the water molecules, the COF/κPVA hydrogel remained stable after 70 h in atmospheric conditions, in contrast, the bare κPVA hydrogel was completely dehydrated. Our work might pave the way for highly sensitive hydrogel gas sensors, which have intriguing applications in flexible electronic devices for gas sensing.

Single-cell EpiChem jointly measures drug-chromatin binding and multimodal epigenome.

Studies of molecular and cellular functions of small-molecule inhibitors in cancer treatment, eliciting effects by targeting genome and epigenome associated proteins, requires measurement of drug-target engagement in single-cell resolution. Here we present EpiChem for in situ single-cell joint mapping of small molecules and multimodal epigenomic landscape. We demonstrate single-cell co-assays of three small molecules together with histone modifications, chromatin accessibility or target proteins in human colorectal cancer (CRC) organoids. Integrated multimodal analysis reveals diverse drug interactions in the context of chromatin states within heterogeneous CRC organoids. We further reveal drug genomic binding dynamics and adaptive epigenome across cell types after small-molecule drug treatment in CRC organoids. This method provides a unique tool to exploit the mechanisms of cell type-specific drug actions.

Recent Advances in Whiskers: Properties and Clinical Applications in Dentistry.

Whiskers are nanoscale, high-strength fibrous crystals with a wide range of potential applications in dentistry owing to their unique mechanical, thermal, electrical, and biological properties. They possess high strength, a high modulus of elasticity and good biocompatibility. Hence, adding these crystals to dental composites as reinforcement can considerably improve the mechanical properties and durability of restorations. Additionally, whiskers are involved in inducing the value-added differentiation of osteoblasts, odontogenic osteocytes, and pulp stem cells, and promoting the regeneration of alveolar bone, periodontal tissue, and pulp tissue. They can also enhance the mucosal barrier function, inhibit the proliferation of tumor cells, control inflammation, and aid in cancer prevention. This review comprehensively summarizes the classification, properties, growth mechanisms and preparation methods of whiskers and focuses on their application in dentistry. Due to their unique physicochemical properties, excellent biological properties, and nanoscale characteristics, whiskers show great potential for application in bone, periodontal, and pulp tissue regeneration. Additionally, they can be used to prevent and treat oral cancer and improve medical devices, thus making them a promising new material in dentistry.

Distributions, interactions, and dynamics of prokaryotes and phages in a hybrid biological wastewater treatment system.

BACKGROUND: Understanding the interactions and dynamics of microbiotas within biological wastewater treatment systems is essential for ensuring their stability and long-term sustainability. In this study, we developed a systematic framework employing multi-omics and Hi-C sequencing to extensively investigate prokaryotic and phage communities within a hybrid biofilm and activated sludge system. RESULTS: We uncovered distinct distribution patterns, metabolic capabilities, and activities of functional prokaryotes through the analysis of 454 reconstructed prokaryotic genomes. Additionally, we reconstructed a phage catalog comprising 18,645 viral operational taxonomic units (vOTUs) with high length and contiguity using hybrid assembly, and a distinct distribution of phages was depicted between activated sludge (AS) and biofilm. Importantly, 1340 host-phage pairs were established using Hi-C and conventional in silico methods, unveiling the host-determined phage prevalence. The majority of predicted hosts were found to be involved in various crucial metabolic processes, highlighting the potential vital roles of phages in influencing substance metabolism within this system. Moreover, auxiliary metabolic genes (AMGs) related to various categories (e.g., carbohydrate degradation, sulfur metabolism, transporter) were predicted. Subsequent activity analysis emphasized their potential ability to mediate host metabolism during infection. We also profiled the temporal dynamics of phages and their associated hosts using 13-month time-series metagenomic data, further demonstrating their tight interactions. Notably, we observed lineage-specific infection patterns, such as potentially host abundance- or phage/host ratio-driven phage population changes. CONCLUSIONS: The insights gained from this research contribute to the growing body of knowledge surrounding interactions and dynamics of host-phage and pave the way for further exploration and potential applications in the field of microbial ecology. Video Abstract.

Diagnostic performance of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in bronchoalveolar lavage fluid for pulmonary tuberculosis in HIV-infected patients.

Introduction: Pulmonary tuberculosis (PTB) remains a significant health concern, particularly in individuals infected with human immunodeficiency virus (HIV) who are more susceptible to developing active TB disease. Early and accurate diagnosis of TB is crucial for effective treatment and prevention of transmission. This study aims to evaluate the potential of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of bronchoalveolar lavage fluid (BALF) for diagnosis of suspected PTB in HIV-infected patients. Methods: This retrospective study recruited 60 HIV-infected patients with suspected PTB presenting with respiratory symptoms and abnormal chest radiographs between January 2022 and June 2023. BALF samples were collected and subjected to analysis using MALDI-TOF MS, GeneXpert, acid-fast bacilli (AFB) smear and culture. And their diagnostic performance was compared. Results: The sensitivity of MALDI⁃TOFMS for diagnosing PTB was 83.3 %, which was better than that of smear 11.9 %, culture 40.5 % or Xpert38.1 % (all p < 0.01). The area under the curve (AUC) value of MALDI⁃TOFMS was 0.889, which was better than that of smear 0.532, culture 0.675 or Xpert 0.690 (all p < 0.01). The katG315 and rpoB-RRDR 511 mutations were detected by the MALDI⁃TOFMS in two patients. Conclusion: Nucleotide MALDI-TOFMS has a good clinical performance for rapid diagnosis of PTB from BALF samples in HIV infected patients, and detects mutations of TB simultaneously.

Development and validation of a clinical predictive model for high-volume lymph node metastasis of papillary thyroid carcinoma.

The central lymph node metastasis (CLNM) status in the cervical region serves as a pivotal determinant for the extent of surgical intervention and prognosis in papillary thyroid carcinoma (PTC). This paper seeks to devise and validate a predictive model based on clinical parameters for the early anticipation of high-volume CLNM (hv-CLNM, > 5 nodes) in high-risk patients. A retrospective analysis of the pathological and clinical data of patients with PTC who underwent surgical treatment at Medical Centers A and B was conducted. The data from Center A was randomly divided into training and validation sets in an 8:2 ratio, with those from Center B serving as the test set. Multifactor logistic regression was harnessed in the training set to select variables and construct a predictive model. The generalization ability of the model was assessed in the validation and test sets. The model was evaluated through the receiver operating characteristic area under the curve (AUC) to predict the efficiency of hv-CLNM. The goodness of fit of the model was examined via the Brier verification technique. The incidence of hv-CLNM in 5897 PTC patients attained 4.8%. The occurrence rates in males and females were 9.4% (128/1365) and 3.4% (156/4532), respectively. Multifactor logistic regression unraveled male gender (OR = 2.17, p < .001), multifocality (OR = 4.06, p < .001), and lesion size (OR = 1.08 per increase of 1 mm, p < .001) as risk factors, while age emerged as a protective factor (OR = 0.95 per an increase of 1 year, p < .001). The model constructed with four predictive variables within the training set exhibited an AUC of 0.847 ([95%CI] 0.815-0.878). In the validation and test sets, the AUCs were 0.831 (0.783-0.879) and 0.845 (0.789-0.901), respectively, with Brier scores of 0.037, 0.041, and 0.056. Subgroup analysis unveiled AUCs for the prediction model in PTC lesion size groups (≤ 10 mm and > 10 mm) as 0.803 (0.757-0.85) and 0.747 (0.709-0.785), age groups (≤ 31 years and > 31 years) as 0.778 (0.720-0.881) and 0.837 (0.806-0.867), multifocal and solitary cases as 0.803 (0.767-0.838) and 0.809 (0.769-0.849), and Hashimoto's thyroiditis (HT) and non-HT cases as 0.845 (0.793-0.897) and 0.845 (0.819-0.871). Male gender, multifocality, and larger lesion size are risk factors for hv-CLNM in PTC patients, whereas age serves as a protective factor. The clinical predictive model developed in this research facilitates the early identification of high-risk patients for hv-CLNM, thereby assisting physicians in more efficacious risk stratification management for PTC patients.

Metatranscriptomics-guided genome-scale metabolic reconstruction reveals the carbon flux and trophic interaction in methanogenic communities.

BACKGROUND: Despite rapid advances in genomic-resolved metagenomics and remarkable explosion of metagenome-assembled genomes (MAGs), the function of uncultivated anaerobic lineages and their interactions in carbon mineralization remain largely uncertain, which has profound implications in biotechnology and biogeochemistry. RESULTS: In this study, we combined long-read sequencing and metatranscriptomics-guided metabolic reconstruction to provide a genome-wide perspective of carbon mineralization flow from polymers to methane in an anaerobic bioreactor. Our results showed that incorporating long reads resulted in a substantial improvement in the quality of metagenomic assemblies, enabling the effective recovery of 132 high-quality genomes meeting stringent criteria of minimum information about a metagenome-assembled genome (MIMAG). In addition, hybrid assembly obtained 51% more prokaryotic genes in comparison to the short-read-only assembly. Metatranscriptomics-guided metabolic reconstruction unveiled the remarkable metabolic flexibility of several novel Bacteroidales-affiliated bacteria and populations from Mesotoga sp. in scavenging amino acids and sugars. In addition to recovering two circular genomes of previously known but fragmented syntrophic bacteria, two newly identified bacteria within Syntrophales were found to be highly engaged in fatty acid oxidation through syntrophic relationships with dominant methanogens Methanoregulaceae bin.74 and Methanothrix sp. bin.206. The activity of bin.206 preferring acetate as substrate exceeded that of bin.74 with increasing loading, reinforcing the substrate determinantal role. CONCLUSION: Overall, our study uncovered some key active anaerobic lineages and their metabolic functions in this complex anaerobic ecosystem, offering a framework for understanding carbon transformations in anaerobic digestion. These findings advance the understanding of metabolic activities and trophic interactions between anaerobic guilds, providing foundational insights into carbon flux within both engineered and natural ecosystems. Video Abstract.

A rapid quantitative UPLC-MS/MS method for analysis of key regulatory oxysterols in biological samples for liver cancer.

An UPLC-APCI-MS/MS method was developed for the simultaneous determination of cholesterol, 7-dehydrocholesterol (7DHC) and eight oxysterols including 27-hydroxycholesterol (27OHC), 7α-hydroxycholesterol (7αOHC), 7ß-hydroxycholesterol (7ßOHC), 24S-hydroxycholesterol (24SOHC), 25-hydroxycholesterol (25OHC), 7α,24S-dihydroxycholesterol (7α,24SdiOHC), 7α,25-dihydroxycholesterol (7α,25diOHC), and 7α,27-dihydroxycholesterol (7α,27diOHC). It has been used for quantitative analysis of cholesterol, 7DHC and eight oxysterols in hepatocellular carcinoma (HCC) cells, plasma and tumor tissue samples. And the above compounds were extracted from the biological matrix (plasma and tissue) using liquid-liquid extraction with hexane/isopropanol after saponification to cleave the steroids from their esterified forms without further derivatization. Then cholesterol, 7DHC and oxysterols were separated on a reversed phase column (Agilent Zorbax Eclipse plus, C18) within 8 min using a gradient elution with 0.1 % formic acid in H2O and methanol and detected by an APCI triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) of the cholesterol, 7DHC and oxysterols ranged from 3.9 ng/mL to 31.25 ng/mL, and the recoveries ranged from 83.0 % to 113.9 %. Cholesterol, 7DHC and several oxysterols including 27OHC, 7αOHC and 7ßOHC were successfully quantified in HCC cells, plasma, tissues and urine of HCC mice. Results showed that 27OHC was at high levels in three kind of HCC cells and tumor tissues as well as plasma samples from both HepG2 and Huh7 bearing mice model，and the high levels of 27OHC in tumors were associated with HCC development. Moreover, the levels of cholesterol in HCC cells and tumor issues varied in different HCC cells and mice model. Oxysterols profiling in biological samples might provide complementary information in cancer diagnosis.

SARS-CoV-2-specific T-cell responses are induced in people living with human immunodeficiency virus after booster vaccination.

BACKGROUND: T-cell-mediated immunity is crucial for the effective clearance of viral infection, but the T-cell-mediated immune responses that are induced by booster doses of inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in people living with human immunodeficiency virus (PLWH) remain unclear. METHODS: Forty-five PLWH who had received antiretroviral therapy (ART) for more than two years and 29 healthy controls (HCs) at Beijing Youan Hospital were enrolled to assess the dynamic changes in T-cell responses between the day before the third vaccine dose (week 0) and 4 or 12 weeks (week 4 or week 12) after receiving the third dose of inactivated SARS-CoV-2 vaccine. Flow cytometry, enzyme-linked immunospot (ELISpot), and multiplex cytokines profiling were used to assess T-cell responses at the three timepoints in this study. RESULTS: The results of the ELISpot and activation-induced marker (AIM) assays showed that SARS-CoV-2-specific T-cell responses were increased in both PLWH and HCs after the third dose of the inactivated SARS-CoV-2 vaccine, and a similar magnitude of immune response was induced against the Omicron (B.1.1.529) variant compared to the wild-type strain. In detail, spike-specific T-cell responses (measured by the ELISpot assay for interferon γ [IFN-γ] release) in both PLWH and HCs significantly increased in week 4, and the spike-specific T-cell responses in HCs were significantly stronger than those in PLWH 4 weeks after the third vaccination. In the AIM assay, spike-specific CD4+ T-cell responses peaked in both PLWH and HCs in week 12. Additionally, significantly higher spike-specific CD8+ T-cell responses were induced in PLWH than in HCs in week 12. In PLWH, the release of the cytokines interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-α), and IL-22 by peripheral blood mononuclear cells (PBMCs) that were stimulated with spike peptides increased in week 12. In addition, the levels of IL-4 and IL-5 were higher in PLWH than in HCs in week 12. Interestingly, the magnitude of SARS-CoV-2-specific T-cell responses in PLWH was negatively associated with the extent of CD8+ T-cell activation and exhaustion. In addition, positive correlations were observed between the magnitude of spike-specific T-cell responses (determined by measuring IFN-γ release by ELISpot) and the amounts of IL-4, IL-5, IL-2 and IL-17F. CONCLUSIONS: Our findings suggested that SARS-CoV-2-specific T-cell responses could be enhanced by the booster dose of inactivated COVID-19 vaccines and further illustrate the importance of additional vaccination for PLWH.