Biological Procedures Online最新文献

Archived clinical formalin-fixed paraffin-embedded tissue (FFPE) is valuable for the study of tumor epigenetics. Although protocol of chromatin immunoprecipitation coupled with next generation sequencing (NGS) (ChIP-seq) using FFPE samples has been established, removal of interference signals from non-target cell components in the samples is still needed. In this study, the protocol of ChIP-seq with purified cells from FFPE lymphoid tissue of nodal T follicular helper cell lymphoma, angioimmunoblastic type (nTFHL-AI) after fluorescence-activated cell sorting (FACS) was established and optimized. Essential steps included single cell preparation, heat treatment enhancing antigen retrieval and labeling, cell sorting, chromatin shearing, ChIP and NGS. Through assistance of FACS, we successfully isolated tumor cells from FFPE lymph node samples of nTFHL-AI and profiled super-enhancers (SEs) mapping by enrichment of H3K27ac signals. The data indicated that the SEs mapping of the sorted cells was different from that of the entire unsorted tissue sample. The H3K27ac signals with cell lineage specificity from background cell components were successfully removed, and the remaining SEs mapping was more similar to T follicular helper cell in an unsupervised clustering analysis, rather than the primary tissue. In addition, we also evaluated the protocol using cultured pure cell lines, and the results indicated that the sequencing data obtained through this protocol had high fidelity and reproducibility. These results show that ChIP-seq for H3K27ac profiling and SEs mapping assisted by FACS with pathological FFPE tissue is available for research of histone modification. Precise epigenetic characteristics of the tumor cell can be described with this protocol.

Strychni Semen, characterized by its bitter taste and warm properties, has been confirmed to possess anti-tumor properties. However, the molecular mechanism of Strychni Semen in treating non-small cell lung cancer (NSCLC) needs further study. This study aimed to explore the molecular mechanism of Strychni Semen in treating NSCLC based on network pharmacology and molecular docking. The active components and targets of Strychni Semen were retrieved from the TCMSP, supplemented by the HERB database and the related literature. NSCLC-related targets were retrieved from the GeneCards, OMIM and DisGenet databases. The intersection targets of Strychni Semen in treating NSCLC were obtained via an online platform. The Protein-Protein Interaction (PPI) network was subsequently constructed to deeply analyse the interrelationship of the intersection targets via the String database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were carried out via the Metascape database. The interactive networks between Strychni Semen and NSCLC were constructed via Cytoscape 3.9.1. Molecular docking detected interactions between the key components and the core targets. The core targets were validated via GEO datasets. 21 active components and 67 targets were identified, with 47 associated with NSCLC. The key active components were Stigmasterol, IcarideA, 2-Hydroxymethylanthraquinone, (+)-catechin, (2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one, (S)-Stylopine, Brucine and Isobrucine. The core targets were PTGS2, NR3C1, ESR1, CASP3 and PRKACA. Molecular docking revealed that these compounds undergo strong binding affinity with the core genes. GEO database indicated that PTGS2 was the most promising core target. In addition, Strychni Semen's effects on NSCLC involved mainly the Calcium pathway, the Estrogen pathway, and the cGMP-PKG and cAMP pathways. This study visually demonstrated the mechanism of the therapeutic effect of Strychni Semen in NSCLC through multiple components, targets and pathways which provides a basis for clinical treatment and further experimental research.

Chang-Wei-Qing (CWQ) is a widely recognized Traditional Chinese Medicine (TCM) formulation composed of Astragalus, Codonopsis, Atractylodes, Poria, Coix seed, Akebia trifoliata Koidz, Sargentodoxa cuneata, and Vitis quinquangularis Rehd. This formulation has garnered significant interest for its positive effects in mitigating colorectal cancer, and when combined with PD-1, it affects some gut microbiota associated with tumor infiltrating lymphocytes cells. However, the biological rationale underlying the suppression of colitis-associated colorectal cancer (CAC) in AOM/DSS-treated mice by CWQ combined with PD-1 inhibitor remains to be explored. Our aim is to explore the chemopreventive effect of CWQ combined with PD-1 inhibitor on CAC, with a focus on modulating the gut microbiota. A mouse model of CAC was established using azoxymethane (AOM) and dextran sulfate sodium (DSS) treatment. Pathological evaluation of tissue samples included immunohistochemistry and hematoxylin and eosin staining. Intestinal barrier function was assessed by transmission electron microscopy. Fecal microbiota and metabolites were analyzed through 16 S rRNA gene sequencing and liquid chromatography-mass spectrometry, respectively. Mice treated with antibiotics served as models for fecal microbiota transplantation. CWQ combined with PD-1 inhibitor suppressed CAC in AOM/DSS-treated mice. This combined therapy effectively alleviated gut dysbiosis in the CAC model by increasing microbial diversity, enriching probiotic populations such as Limosilactobacillus and Bifidobacterium, and reducing pathogenic bacteria like Desulfovibrio. Additionally, CWQ combined with PD-1 inhibitor downregulated metabolites associated with the NF-kappa B signaling pathway. The combined treatment also significantly improved intestinal barrier function in CAC mice. Transmission electron microscopy of the CWQ combined with PD-1 inhibitor group showed enhanced cellular integrity, a relatively normal mitochondrial structure with intact membranes, and a more abundant, unexpanded endoplasmic reticulum, underscoring the protective effects of this combination on intestinal barrier integrity. Transcriptomic analysis further demonstrated that the combined therapy upregulated genes involved in tight and adherens junctions, while downregulating genes linked to innate immune responses. CWQ combined with PD-1 inhibitor can ameliorate dysbiosis in the AOM/DSS mouse model, with the metabolites of the gut microbiome potentially possessing anti-inflammatory activity. Moreover, CWQ combined with PD-1 inhibitor improves intestinal barrier function, thereby effectively inhibiting the occurrence and development of CAC.

Background: Shoulder pain and disability from rotator cuff tears remain challenging clinical problem despite advancements in surgical techniques and materials. To advance our understanding of injury progression and develop effective therapeutics using tissue engineering and regenerative medicine approaches, it is crucial to develop and utilize animal models that closely resemble the anatomy and display the pathophysiology of the human rotator cuff. Among various animal models, the rabbit shoulder defect model is particularly favored due to its similarity to human rotator cuff pathology. However, a standardized protocol for creating a massive rotator cuff defect in the rabbits is not well defined. Therefore, the objective of our study was to establish a robust and reproducible model of a rotator cuff defect to evaluate the regenerative efficacy of scaffolds.

Results: In our study, we successfully developed a rabbit model with a massive supraspinatus tendon defect that closely resembles the common rotator cuff injuries observed in humans. This defect involved a complete transection of the tendon, spanning 10 mm in length and encompassing its full thickness and width. To ensure stable scaffolding, we employed an innovative bridging suture technique that utilized a modified Mason-Allen suture as a structural support. Moreover, to assess the therapeutic effectiveness of the model, we utilized different scaffolds, including a bovine tendon extracellular matrix (ECM) scaffold and a commercial acellular dermal matrix (ADM) scaffold. Throughout the observation period, no scaffold damage was observed. Notably, comprehensive histological analysis demonstrated that the regenerative tissue in the tendon ECM scaffold group exhibited an organized and aligned fiber structure, indicating tendon-like tissue regeneration while the tissue in the ADM group showed comparatively less organization.

Conclusions: This study presents a comprehensive description of the implemented procedures for the development of a highly reproducible animal model that induces massive segmental defects in rotator cuff tendons. This protocol can be universally implemented with alternative scaffolds to investigate extensive tendon defects and evaluate the efficacy of regenerative treatments. The application of our animal model offers a standardized and reproducible platform, enabling researchers to systematically evaluate, compare, and optimize scaffold designs. This approach holds significant importance in advancing the development of tissue engineering strategies for effectively repairing extensive tendon defects.

Background: Astrocytes have recently gained attention as key players in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease. Numerous differentiation protocols have been developed to study human astrocytes in vitro. However, the properties of the resulting glia are inconsistent, making it difficult to select an appropriate method for a given research question. Therefore, we compared three approaches for the generation of iPSC-derived astrocytes. We performed a detailed analysis using a widely used long serum-free (LSFP) and short serum-free (SSFP) protocol, as well as a TUSP protocol using serum for a limited time of differentiation.

Results: We used RNA sequencing and immunochemistry to characterize the cultures. Astrocytes generated by the LSFP and SSFP methods differed significantly in their characteristics from those generated by the TUSP method using serum. The TUSP astrocytes had a less neuronal pattern, showed a higher degree of extracellular matrix formation, and were more mature. The short-term presence of FBS in the medium facilitated the induction of astroglia characteristics but did not result in reactive astrocytes. Data from cell-type deconvolution analysis applied to bulk transcriptomes from the cultures assessed their similarity to primary and fetal human astrocytes.

Conclusions: Overall, our analyses highlight the need to consider the advantages and disadvantages of a given differentiation protocol for solving specific research tasks or drug discovery studies with iPSC-derived astrocytes.

Background: To investigate the survival outcome of "radical" GreenLight photoselective vaporization of bladder tumor (RPVBT) in conjunction with postoperative chemotherapy for patients with single, < 3 cm in diameter, T2 stage muscle invasive bladder cancer (MIBC).

Methods: Thirty-eight patients with single, < 3 cm, T2 stage bladder cancer were treated with RPVBT combined with chemotherapy and were included in the RPVBT group. To compare the differences in survival outcome, 80 patients with Ta/T1 bladder cancer and 30 patients with T2 bladder cancer were included as controls. The 80 patients with Ta/T1 bladder cancer underwent GreenLight photoselective vaporization of bladder tumors(PVBT), while 30 patients with T2 bladder cancer underwent radical cystectomy (RC) combined with pelvic lymph node dissection (PLND). Tumor recurrence and death were recorded, and recurrence-free survival (RFS) and overall survival (OS) curves were plotted to compare the survival difference between the RPVBT and control groups.

Results: No significant differences were observed in comorbidities or living habits between the RPVBT and control groups. Blood loss [RPVBT: 20 (IQR10, 20) vs. RC: 100 (IQR90, 150) mL] and postoperative hospital stay [RPVBT: 5.5 (IQR5, 6), vs. RC: 10 (IQR8, 12) days] in the RPVBT group were significantly lower than that in the RC group. Urinary tract infection [RPVBT: 6 (15.8%) vs. PVBT: 14 (17.5%)] and bladder irritation sign [RPVBT: 11 (28.9%) vs. PVBT: 23 (28.8%) ] were the most common short-term complications in the RPVBT group, with no statistical difference between the RPVBT and PVBT group. The median follow-up time for survival endpoints was 22 (16, 27) months for the included patients after surgery. The outcomes of tumor recurrence at 12, 24, and 36 months were 2 (5.3%), 3 (7.9%), and 5 (13.2%) patients in the RPVBT groups, 13 (16.3%) and 3 (10%) patients experienced recurrence in the PVBT and RC groups at 36 months. No significant differences were noted among the three groups (P = 0.778). Additionally, Kaplan-Meier survival analysis revealed no statistically significant differences in RFS (P = 0.791) and OS (P = 0.689) among the three groups.

Conclusions: Our findings indicate that RPVBT combined with chemotherapy is a simple and feasible treatment option with fewer complications and satisfactory survival outcomes in patients with single, < 3 cm, T2 stage bladder cancer.