Biological Procedures Online最新文献

Background: Membrane-bound vesicles called apoptotic bodies (ApoBDs) formed during apoptosis are being recognized as non-invasive biomarkers for neurological diseases like dementia and ischemic stroke. Centrifugation-based isolation of these vesicles from blood was suggested in a recent paper by Serrano-Heras et al. (2020), which asserted their use for clinical diagnosis. This initial study provides a foundational methodology for ApoBD diagnostics. Albeit, centrifugation parameters employed present technical challenges that require optimization to ensure the purity and reproducibility of ApoBD preparations for clinical utility.

Results: Due to the use of improper centrifugation speeds (160×g and 700×g) not consistent with accepted norms for the separation of platelets (ideally 2,000-5,000×g), running the risk of being seriously contaminated by platelets and platelet-derived EVs (P-EVs), which make up 70-90% of EVs in circulation. Such contamination can render functional studies problematic, cloud proteomic and size-distribution analysis, and lower the specificity of ApoBDs enumeration on the basis of Annexin V/PI labeling. Even more extreme centrifugation has little chance to eliminate platelets completely, according to comparative literature data. Increased centrifugal pressure, density gradient separation, size-exclusion chromatography (SEC), and confirmation with platelet-specific markers are among some procedural changes recommended to improve sample purity in attempts to solve such problems.

Conclusions: Even as the Serrano-Heras et al. study pinpoints the potential of ApoBDs as biomarkers, constraints to the procedure under consideration pose the threat of bias in future studies. Proper isolation of ApoBDs requires multimodal confirmation, optimized platelet removal protocols and clinically feasible methods like SEC are essential to enhance ApoBDs isolation for neurological disease diagnostics. Rectification of such issues enhance significantly the practicality of ApoBDs in clinical practice for the detection of neurological illnesses.

Background: Western blotting is a fundamental technique for protein detection and quantification, with critical dependence on antibody for its sensitivity and specificity. Antibody solutions are frequently reused to conserve the limited stock and compensate for their high cost. However, storing antibody solutions is often challenged by protein aggregation and loss of immunoreactivity, especially when skim milk is added. Recent advancements in Western blot include microfluidic techniques which minimize antibody consumption. To facilitate these techniques, a storage strategy is needed for diluted antibody solutions to properly preserve their immunoreactivity.

Results: We found that Tris-buffered saline with 0.1% Tween 20 (TBST), an ordinary washing buffer for Western blot, provides protection to diluted antibodies for extended period of storage at 4 °C. We discovered that TBST-diluted commercial antibodies retained their immunoreactivity for 281 days at ambient temperature and 1,690 days at 4 °C. In addition, we demonstrated that Tween 20 plays a critical role in stabilizing the diluted antibody.

Conclusions: We propose that TBST is an effective and practical buffer to preserve diluted antibodies, which can be utilized as an integral part of strategies to reduce antibody consumption in laboratories.

Background: Advances in nanomedicine have spurred interest in antibody-conjugated carbon nanotubes for targeted cancer therapy. CD133, a marker enriched in chemoresistant cancer stem cells, presents a strategic target for precision drug delivery. This study explores functionalized single-walled carbon nanotubes (SWCNTs) as dual-action platforms for enhanced doxorubicin (DXR) delivery and CD133-specific targeting.

Methods: Two nano formulations-SWCNT-Ab/DXR (antibody-functionalized) and SWCNT-PEG-Ab/DXR (pegylated antibody-functionalized)-were engineered to encapsulate DXR. Release profiles, cytotoxicity, and apoptosis were assessed in CD133⁺ HT-29 colorectal cancer cells and CD133^- CHO control cells. Computational modeling included DFT-D structural optimization, Monte Carlo adsorption simulations for DXR binding analysis, and molecular docking to evaluate carrier-receptor interactions.

Results: PEGylation markedly enhanced colloidal stability and drug-loading capacity, with SWCNT-PEG-Ab/DXR achieving 92% DXR encapsulation vs. 78% for non-PEGylated counterparts. In vitro, the PEGylated system showed amplified cytotoxicity (IC50: 2.1 µM vs. 3.8 µM for SWCNT-Ab/DXR) and 1.7-fold higher apoptosis induction in HT-29 cells. Computational data aligned with experimental findings: DXR adsorption energy was strongest on PEG-NH₂-modified SWCNTs (- 32.6 kcal/mol) versus carboxylated variants (- 24.8 kcal/mol), confirming PEG's role in stabilizing drug-carrier interactions.

Conclusions: By integrating PEG-mediated stealth properties, charge-modified surfaces, and antibody targeting, SWCNT-PEG-Ab/DXR emerges as a multifunctional nanoplatform with enhanced tumor selectivity and therapeutic payload delivery. This dual experimental-computational approach underscores the potential of rationally engineered nanotubes to overcome limitations in conventional chemotherapy.

Non-small cell lung cancer (NSCLC) frequently develops acquired resistance to immune checkpoint inhibitors (ICIs), necessitating innovative strategies to remodel the immunosuppressive tumor microenvironment (TME). This study engineered an inhalable pH-responsive nano-liposome co-delivering Astragaloside IV (AS-IV) and Polyphyllin VII (Pol VII) (AS-IV/Pol VII-Lipo) to overcome anti-PD-1 resistance via spatiotemporal-controlled dual-drug delivery. AS-IV/Pol VII-Lipo (1:1 mass ratio) exhibited optimal physicochemical properties: high drug loading and pH-triggered release. Nebulized inhalation achieved 3.4-fold higher lung accumulation than oral administration. Suppressed orthotopic LLC-Luc tumor growth by 54% and reduced exhausted CD8⁺ T cells while increasing cytotoxic CD8⁺Granzyme B⁺ T cells. Combination therapy further inhibited tumor metastasis and elevated survival. Transcriptomics (RNA-seq) identified suppression of IL-2/STAT5/BLIMP1 pathway and T-cell exhaustion genes. AS-IV/Pol VII-Lipo reprograms the immunosuppressive TME through three synergistic mechanisms: (1) enhanced lung-targeted drug delivery via inhalation; (2) reversal T-cell exhaustion through IL-2/STAT5/BLIMP1 pathway inhibition; (3) synergizing with αPD-1 therapy to overcome ICI resistance. This inhalable nanoplatform presents a promising clinical strategy for NSCLC patients with acquired immunotherapyresistance.

Introduction: Magnesium homeostasis is critical for cellular growth and metabolism, yet its pan-cancer implications remain poorly characterized. This study aims to comprehensively analyze magnesium homeostasis across 33 cancer types, exploring its role in tumorigenesis, immune regulation, and therapeutic potential. Key magnesium homeostasis-related genes (e.g., ANK3, CNNM2) were significantly downregulated in most tumors, correlating with improved prognosis. Magnesium homeostasis scores (MHS) were reduced in cancers and linked to lower tumor mutational burden (TMB), microsatellite instability (MSI), immune dysfunction, and checkpoint gene expression. Single-cell sequencing revealed elevated MHS in CD8 + T cells, suggesting immune modulation roles.

Conclusion: These findings highlight magnesium homeostasis as a regulator of tumor progression and immunity, with MHS serving as a prognostic biomarker. Targeting magnesium pathways may offer novel therapeutic strategies, warranting further clinical validation to advance personalized cancer therapies.

Introduction: Gastric cancer (GC) remains a formidable global health issue with limited therapeutic options. ShenXia KuanZhong Decoction (SXKZD), a classical traditional Chinese medicine (TCM) formula, is used to manage GC; however, its anti-tumor mechanisms remain poorly understood.

Material and methods: The anti-GC effects of SXKZD were investigated in a GC model using dose-weighted network pharmacology, molecular docking, molecular dynamics (MD) simulations, and pharmacokinetic profiling. Its impacts on tumor metabolism, immunity, and gut microbiota were assessed. A gut microbiota-substrate-metabolite (GM-S-M) network was constructed, and key targets and pathways were analyzed using computational and experimental methods.

Results: SXKZD treatment significantly alleviated tumor progression in GC models. Network analysis revealed upregulated TNF and IL6 expression in GC, which SXKZD reduced, alongside enrichment in IL-17 and TNF signaling pathways. Molecular docking and MD simulations confirmed stable binding of Ginsenoside Rh2 and 3-Indolepropionic acid to TNF, with binding energies of -147.63 kJ/mol and - 98.63 kJ/mol, respectively. Pharmacokinetic profiling showed 3-Indolepropionic acid's high bioavailability, while GM-S-M analysis identified key microbial taxa (e.g., Lactobacillus plantarum, Akkermansia muciniphila) modulated by SXKZD, enhancing anti-tumor immunity and metabolism.To further confirm these computational predictions, in vitro CCK-8 assays revealed that GRh2 and IPA inhibited AGS cell growth in a concentration-dependent manner, with IC₅₀ values of 68.74 ± 1.27 µg/mL and 780.60 ± 24.40 µg/mL at 24 hours, respectively. Western blot analysis demonstrated that GRh2 more effectively suppressed TNFα expression, whereas CETSA showed that IPA provided superior thermal stabilization of TNFα.

Conclusions: SXKZD mitigates GC by modulating gut microbiota and inhibiting TNF signaling, offering a mechanistic basis for its therapeutic potential in GC management.

Background: Understanding the role of causal genes of prostate cancer (PrCa) can reveal key biological pathways and identify potential targets for treatment.

Methods: We investigated associations between genetically predicted gene expression levels and PrCa risk using cis-eQTL summary-based Mendelian randomization (SMR) and colocalization analysis. Findings were replicated using two independent PrCa GWAS. We then intersected the identified genes with differentially expressed genes (DEGs) identified from TCGA-PRAD dataset to obtain key genes. Furthermore, enrichment, protein-molecule network, immune infiltration, and epigenetic analyses were conducted to explore their biological pathways. Lastly, phenome-wide association study (PheWAS), drug prediction, and molecular docking simulation analysis were utilized to identify potential drugs.

Results: We identified 15 genes in blood whose expression levels are putatively associated with PrCa, validated in at least one replication GWAS dataset. Using open-access mRNA-sequencing data, we found that ZNF217 and BNIP2 were key genes potentially important in PrCa pathogenesis. Single-cell RNA-sequencing analysis revealed that BNIP2 was predominantly expressed in a subset of endothelial cells, whereas ZNF217 was mainly enriched in epithelial cells. Downstream analysis revealed their involvement in epigenetic modulation-related pathways, while upstream analysis showed that upregulation of ZNF217 notably correlated with increased CpG methylation. Molecular docking simulation suggested doxorubicin, alsterpaullone, and camptothecin as potential drugs targeting these key genes.

Conclusions: These findings provide robust leads for understanding pathogenic mechanisms and developing therapeutic interventions for PrCa.

Background: The mouse fetal intrauterine wound healing model is crucial and commonly used for investigating mechanisms and evaluating potential therapies for scarless skin regeneration compared to fibrotic healing. However, traditional intrauterine surgery remains technically challenging and understudied, which is associated with high maternal mortality and pregnancy loss, prompting us to refine the surgical protocol. Here, we report how the choice of surgical and mating procedure impact outcomes obtained.

Methods: Pregnant mice underwent fetal surgery at embryonic days 15.5, 16.5 (E15.5, E16.5, scarless) and 18.5 (e18.5, fibrotic). Two surgical protocols were used: traditional method involved purse-string sutures, microsurgical scissors, amniotic fluid supplementation, and suture closure (Traditional); and our modified method omitting purse-string sutures, replacing scissors with needle puncture for uterine and fetal incisions, eliminating amniotic fluid supplementation, and employing skin staples for abdominal closure (Modified).

Results: The modified protocol significantly increased the likelihood of successful pregnancy, reduced operative time, decreased abortion rates, and enabled earlier modeling compared to the traditional method. At 48 h, 7 days, and 9 days post-surgery, E15.5 wounds healed scarlessly, displaying regenerated hair follicles and organized collagen. Conversely, E18.5 wounds formed typical fibrotic scars, characterized by dense, disorganized collagen without hair follicles.

Conclusion: The optimized surgical protocol presented here provides a simplified, reliable fetal mouse model with improved pregnancy success, reduced fetal loss, earlier implementation, and consistent phenotypic outcomes. This refined model enhances experimental efficiency, reproducibility, and animal welfare, having a major impact on mechanistic studies and therapeutic exploration for scarless skin regeneration.