Advanced biology最新文献

Accurate detection of heparin-induced thrombocytopenia (HIT) antibodies is crucial for diagnosing and managing thrombotic events. Conventional immunoassays, however, often lack specificity and require confirmatory testing with fresh human platelets. To address this limitation, we optimized our previously developed cell-based enzyme-linked immunosorbent assay (ELISA) for improved HIT detection under various experimental conditions. Platelet factor 4 was immobilized on breast cancer cells (MDA-MB-231) to capture monoclonal HIT-like (KKO) and non-HIT (RTO) antibodies, which served as models to evaluate assay performance under different pH levels, ionic strengths (NaCl), and fixation methods (ethanol, paraformaldehyde, glutaraldehyde). To identify the most suitable substrate, additional cancer cell lines (HCT-116, MCF-7, HepG2) were tested under live and fixed conditions, with selected conditions validated using human HIT sera. Optimal detection tested with monoclonal antibodies was achieved using 50 mM NaCl and 4% paraformaldehyde fixation. Notably, live MDA-MB-231 and HCT-116 cells demonstrated superior sensitivity and specificity compared to fixed cells. Furthermore, these cell lines enable the efficient detection of HIT antibodies using flow cytometry, a robust and platelet-free diagnostic method. Our findings establish live MDA-MB-231 and HCT-116 cells as highly promising platforms for clinical applications in HIT antibody detection.

The S-phase kinase-associated protein 1 (Skp1)-Cullin-F-box protein E3 ligase adaptor F-box-only protein 31 (FBXO31) regulates genomic stability and cell signaling in normal, genotoxic, and tumor cells by recognizing and ubiquitinating multiple downstream substrates. The stability and role of FBXO31 may be regulated by specific residual modification. In this study, five FBXO31 phosphorylation sites are identified in HEK293T cells using biochemical and biological techniques. Liquid chromatography–tandem mass spectrometry identifies phosphorylated residues, including threonine-28 and -37 and serine-33, -400, and -523. The PyMOL crystal structure reveals the location of these residues on FBXO31 and assesses whether they interact with the reported kinases. Western blotting and fluorescence-activated cell sorting demonstrate that the phosphorylation of Thr-37 and Ser-523 contributes to FBXO31 protein stabilization, which is further confirmed by cycloheximide experiments. The regulatory roles of Thr-37 and Ser-523 in FBXO31 stability are associated with variations in phosphorylation levels and degradation pathways. These results demonstrate that phosphorylation regulates FBXO31 turnover, and phosphorylation at Thr-37 or Ser-523 may help identify upstream kinases and enhance the understanding of the physiological role of FBXO31.

Mesenchymal stem cells (MSCs) migrate to injured tissues, aiding tissue repair, remodeling, and wound healing. As tumors are often considered to have traits of “injured tissues,” MSCs are recruited to tumor microenvironments where they can have pro- and antitumorigenic influences. This study assesses whether human mesenchymal stem cells (hMSCs) of shared ancestry exhibit similar tumorigenic properties. Bone marrow-derived (hBM-MSCs) and umbilical cord-derived (hUC-MSCs) MSCs embedded in collagen are cultured in conditioned media from lung adenocarcinoma (A549) cells to mimic the extracellular matrix and soluble cues of the cancer microenvironment. Cell viability, proliferation, and immunofluorescence analyses evaluate MSC behavior under these conditions. Further, A549 cells are exposed to conditioned media from cancer-stimulated MSCs to simulate indirect co-culture, and their response is assessed through viability, immunofluorescence, and flow cytometric analysis. Results show increased viability and proliferation of hBM-MSCs, morphological changes, and elevated alpha-smooth muscle actin expression, suggesting a transition toward cancer-associated fibroblasts. In contrast, hUC-MSCs display reduced viability and no morphological alterations. Conditioned media from cancer-exposed hUC-MSCs induce apoptosis in A549 cells, whereas hBM-MSCs support A549 growth. These findings demonstrate that, despite their common origin, hUC-MSCs and hBM-MSCs exhibit opposing responses to tumor cues and influence lung cancer cell behavior differently.

Label-free optical stimulation of brain cells with infrared (IR) light provides a powerful tool for spatially targeted neuromodulation. However, lingering questions about the off-target effects of IR stimulation on non-neuronal cells remain sparsely explored. It is shown that rat astroglial cultures are independently sensitive to single pulses of infrared light, evoking calcium signaling and osmoregulatory phenomena in vitro. Recent studies highlight that astrocytes respond differently to electromagnetic and laser stimulation, recruiting different pathways. The impact of three different IR stimulation time courses on astrocyte calcium and water transport dynamics is explored with widefield fluorescence microscopy and pharmacology to fill this gap. Results show that different stimulation methods can evoke astrocyte calcium responses, resulting from distinct biomolecular signaling processes. Notably, swelling and shrinkage are also differently evoked by short-term and long-term stimulation pulses. It is shown that specific IR stimulation can drive selective water and calcium dynamics in astrocytes. The work uniquely reports label-free optical modulation techniques to drive astroglial homeostatic machinery, a crucial process in healthy brain function that lacks tools for spatially precise modulation. More broadly, the results demonstrate the need to consider off-target effects with neuromodulation strategies and how to use such effects to study brain physiology.

Spheroid Cell Aggregation

In article number 2500092, Kenjiro Takemura and co-workers demonstrate that ultrasound-based, enzyme-free cell detachment accelerates spheroid formation by preserving surface proteins, reducing variability, and enhancing cell aggregation. Comparable engraftment and improved spatial organization in co-cultured spheroids were observed. These findings suggest ultrasound detachment as a robust alternative for efficient, high-quality spheroid production in tissue engineering and related biomedical applications.

Sarcopenia is a specific disease that commonly occurs in the elderly and patients with consumptive diseases. Sarcopenia can cause a severe decline in strength and movement, and even lead to prolonged bed rest. Nutritional support is an emerging approach to mitigate sarcopenia and is currently the gold standard for clinical management of sarcopenia. However, complex nutritional composition may affect the effect of treatment. Herein, the pathogenesis of sarcopenia is first introduced. Then, the diagnostic methods of sarcopenia are described in detail, including grip strength, chair stand test, dual-energy X-ray Absorptiometry, bioelectrical impedance analysis, gait speed, balance tests, and biomarkers. Then, the latest progress of the nutritional support (including protein quantity and quality, bioactive peptides, amino acids and their metabolites, antioxidant/ anti-inflammatory nutrients, vitamins and minerals, microbiome-targeted interventions, metabolite-derived molecules, and Chinese herbal medicine) on the mitigation of sarcopenia is elaborated. This review may contribute to the understanding of the mechanism and importance of diagnosis and nutritional support in alleviating sarcopenia, so as to promote the development of sarcopenia treatment.

Type I interferons （IFN-I） are key proteins in antiviral response and immunomodulation. Negative regulators avoid abnormal activation of the interferon pathway or overactivation of interferon-activating proteins through multiple mechanisms. Loss-of-function mutations in negative regulator genes lead to the development of a variety of type I interferonopathy. It is of great significance in theory and clinic to discover new negative regulators and study their functions, but there is no effective screening system at present. Here, a screening system is established through a resistance reporter, which is designed to be expressed only in cells with interferon pathway activation. In conjunction with CRISPR knockout library, cells are screened for interferon pathway activation due to knockout of negatively regulators, which led to the identification of negatively regulator candidate genes such as PCGF3/5, UCK2, and ITPKA. The expression products of these genes functioned by targeting MAVS and promoted EMCV (encephalomyocarditis virus) infection by inhibiting interferon pathway activation. This study deepens the understanding of the regulatory network of the interferon pathway and provides a new theoretical basis for the study of the pathogenesis of autoimmune diseases.

The invasive pest Halyomorpha halys (Hemiptera: Pentatomidae) poses a significant threat to agriculture and requires control methods beyond chemical pesticides. This study investigates RNA interference (RNAi) as a targeted gene silencing approach to manage H. halys populations. However, RNAi efficacy varies between insect orders, including hemipterans, due to factors such as the rapid degradation of double-stranded RNA (dsRNA) by a DNA/RNA non-specific nuclease (HhNSE) present in the saliva of H. halys. Notably, this study proves that double-stranded DNA (dsDNA) can stabilise dsRNA in saliva, probably by competitively inhibiting HhNSE, which is highly expressed in salivary glands. In vivo tests targeting the clathrin heavy chain gene (HhCHC) demonstrate that a mixture of dsRNA-CHC and dsDNA result in enhanced gene silencing when fed to H. halys, compared to dsRNA alone. While dsRNA-CHC injection causes almost complete mortality, the dsDNA formulation do not significantly increase mortality when fed together with dsRNA-CHC. These findings highlight the need to further investigate factors beyond nucleases such as dsRNA uptake and release mechanisms in the insect gut. Nevertheless, this study provides promising insights for improving RNAi delivery in H. halys, and perhaps other pests with such nucleases, in support of sustainable pest management solutions.

Oesophageal squamous cell carcinoma (ESCC) is the most prevalent type of oesophageal cancer. It is an aggressive malignancy with a poor prognosis, and recent studies have revealed the critical role of the microbiota in its occurrence and development. In this review, the current understanding of the impact of microbiota is summarized on the tumour immune microenvironment (TIME) in ESCC, focusing on intratumoural microbes and the oral-gut microbiota axis as potential therapeutic targets. The mechanisms are discussed by which specific bacterial species, such as Fusobacterium nucleatum (F. nucleatum), Porphyromonas gingivalis (P. gingivalis), Streptococcus spp., and Lactobacillus spp., influence immune responses and contribute to the progression of ESCC. Additionally, the potential of the microbiota is highlighted as a biomarker for early detection, prognosis, and prediction of treatment responses, and explore emerging strategies in microbiota-based immunotherapy that exploit the tumour-targeting properties of bacteria to improve cancer treatment outcomes. Despite these promising developments, the complex interactions between the microbiota and the immune system remain unclarified, and translating research findings into clinical practice is a significant challenge. In this review, the current advancements and emphasise the need for further investigation is summarized into the mechanisms of microbiota-mediated immunotherapy, while outlining future directions for developing personalised treatments for ESCC

Each year, ≈1.6 million women are diagnosed with breast cancer worldwide. Of these cases, 0.5 million result in death, with over 90% of these deaths resulting from metastasis. Since it is one of the top 5 cancers with the highest mortality rates, the development of breast cancer models that are physiologically relevant to the human body is severely needed. This includes models of the breast tissue microenvironment, the microenvironment of metastatic sites (e.g., brain, lungs, bone, liver), and models specifically mimicking each individual step of the cancer metastatic cascade. This review focuses on models of the primary tumor environment for studying cell invasion and spread within the mammary tissue, prior to metastatic outgrowth. Using a combination of methods such as tumor spheroids, 3D printed biomaterials, and microfluidic designs, a variety of 3D in vitro modeling methods exist to recapitulate specific aspects of the tumor microenvironment and understand sources of tumor heterogeneity. An effective model can be specified for each patient, eliminating the need for human trials. Subsequently, as the mechanisms involved in breast cancer metastasis are studied utilizing more effective in vitro models, targeted therapeutics can be discovered, thus advancing clinical treatment strategies.