Advanced biology最新文献

Infections caused by Enterovirus like rhinoviruses, coxsackieviruses, and polioviruses represent a significant public health concern, for which there are no antivirals available yet. The highly conserved viral 3C protease has been the primary target for antiviral development, but competitive inhibitors targeting its active site does not meet expectations in clinical studies. Previously, an unconventional allosteric site is identified on human rhinovirus 14 (HRV14) 3C, representing novel opportunities for pan-enterovirus antivirals development. Here, in silico screening of 143,621 natural products against this allosteric site is performed and 28 candidate molecules are identified, among which dihydromyricetin (DHM) and oridonin-A1 bind to HRV14 3C and allosterically inhibit its protease activity. Moreover, DHM shows minimal cytotoxicity and potent antiviral efficacy against HRV14 infections across different cell models, with selective indexes exceeding 700. Structural analysis and mutagenesis assays further pinpoint key 3C residues essential for DHM binding. Consistent with the high conservation of these residues across Enterovirus genus, DHM broadly binds and efficiently inhibits 3C proteases from not only rhinoviruses, but also coxsackieviruses, enteroviruses and polioviruses. These findings establish DHM as a unique, broad-spectrum allosteric inhibitor of Enterovirus 3C proteases and underscore its potential as a promising candidate for the development of pan-enterovirus antivirals.

Deficiencies in DNA damage repair (DDR), such as poly (ADP-ribose) polymerase (PARP) deficient, cause cancer development by promoting DNA mutations while also exposing the specificity and vulnerability of cancer to afford a treatment option. PARP inhibitor (PARPi) has shown great prospects in the treatment of tumors carrying homologous recombination (HR) deficiencies, such as germline BRCA1/2 mutations. PARPi leads to an increase in the expression of tumor neoantigen, interferon (IFN), and programmed cell death 1/programmed death-ligand 1 (PD-1/PD-L1), which also regulate the tumor microenvironment (TME), promoting a deeper anti-tumor immunotherapy. ICIs targeting PD-1/PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have achieved impressive success in the treatment of malignancies. Considering PARPi do enhance the anti-tumor response of ICIs, the combination of PARPi and ICIs has gradually become an alternative treatment option for individuals not receiving apparent efficacy from ICI monotherapy. In this review, the emphasis will be on the mechanisms and immune responses associated with PARPi, profess the principle, then count the clinical studies of this combination therapy.

Adipose tissue-derived stem cells (ASCs) hold significant potential for treating various clinical conditions. To enhance their regenerative properties, ASCs can be chemically stimulated using various in vitro protocols. However, unsatisfactory results persist, partly due to the relatively costly long-term methods. Furthermore, current culturing techniques often rely on the use of xenogenic fetal bovine serum that can be immunogenic, limiting clinical translations. To facilitate clinical translation of ASCs-derived therapeutics, the effect of different stimulation protocols on human ASCs cultured in a xeno-free medium (PRIME-XV MSC Expansion XSFM) is investigated. The xeno-free medium was supplemented with stimulants (forskolin (FSK), basic fibroblast growth factor, platelet-derived growth factor-AA, neuregulin-1) in combinations or individually. Stimulation for 72 h in FSK alone, or together with the growth factors, enhanced the production of urokinase plasminogen activator (uPA), a serine protease involved in tissue remodeling processes. Conditioned medium derived from stimulated ASCs enhanced in vitro angiogenesis and endothelial cells migration. This study shows that pro-angiogenic responses in human ASCs can be enhanced with a defined short stimulation protocol using a xeno-free medium. The protocol, using readily available manufacturing cell therapy grade molecules, may boost the regenerative properties of ASCs secretome which could enhance their efficacy in clinical treatments.

Rupture of atherosclerotic plaque caps is the cause of many disabling or lethal cardiovascular events, such as stroke and myocardial infarction. Microcalcifications (<50 µm) have been shown, in computational models, to affect the biomechanical stability of the cap. The current study aims to develop a tissue-engineered model of the atherosclerotic fibrous cap with microcalcifications produced by mesenchymal stromal cells (MSCs). Human MSCs are seeded in fibrin gels and cultured for 2 weeks in medium supplemented with TGF-β1 to induce smooth muscle cell differentiation and collagenous matrix formation. Afterward, mineralizing medium stimulates microcalcification formation for an additional 4 weeks. Tissue-engineered structures are imaged after culture with second harmonic generation microscopy with a hydroxyapatite probe, showing collagenous matrix with microcalcifications. Mechanical characterization shows the effect of microcalcifications on global tissue mechanics, as the ultimate stress at rupture of the tissue is significantly lower compared to control tissues. The amount of calcification, determined by histological analysis, is correlated to the decrease in ultimate tensile stress, with a higher amount of microcalcification resulting in weakened mechanical properties. The developed tissue-engineered plaque cap model with biologically formed collagenous matrix and microcalcifications offers valuable insight into the impact of microcalcifications on biomechanical stability.

Bis-(3′-5′)-cyclic diguanylic acid (c-di-GMP), a ubiquitous secondary messenger, affects multiple biological characteristics, including biofilm formation in avian pathogenic Escherichia coli (APEC). C-di-GMP is synthesized by diguanylate cyclase harboring a GGDEF domain and degraded by phosphodiesterase harboring either an EAL or an HD-GYP domain. However, the roles of PdeN, encoding a CSS-EAL domain, are uncharacterized. In this study, it is demonstrated that lacking pdeN significantly promotes biofilm formation and reduces the motility of the clinically isolated APEC O2 serotype strain DE17. In addition, macrocolony morphotypes showed that the ΔpdeN strain exhibits increasing production of curli fibers and cellulose, which is consistent with the results of RNA-seq and qPCR. Further exploration shows that lactose permease LacY and mannose permease subunit ManZ interact with PdeN. Infection experiments show that lacking pdeN significantly reduced the release of LDH in HD-11 cells and adhesion capacity to DF-1 cells. In conclusion, c-di-GMP metabolic gene pdeN involves biofilm formation and pathogenicity of APEC. Besides, it interacts with LacY and ManZ. Those results provide a basis for the prevention and control of APEC from the perspective of biofilm and carbohydrate metabolism.

Chronic kidney disease (CKD) affects over 850 million individuals worldwide, often progressing to stages requiring dialysis or kidney transplants. Central to kidney function is the glomerular filtration barrier (GFB), which selectively filters waste while retaining essential proteins. Traditional models, including animal studies and 2D cell cultures, fail to fully replicate the GFB's complexity, limiting CKD research. Recent developments in microphysiological systems (MPS), particularly microphysiological glomerular filtration barriers (MPGFB), provide more accurate in vitro models for studying kidney diseases and evaluating therapies. MPGFB systems use organ-on-chip technology to integrate podocytes and glomerular endothelial cells within confined microfluidic environments, closely mimicking GFB's dynamic in vivo conditions. This setup enables detailed permeability analysis, aiding in research on disease mechanisms and drug toxicity. Furthermore, using human-induced pluripotent stem cells in MPGFB platforms allows patient-specific studies, enhancing insights into genetic kidney disorders. This review first examines the GFB's structure and function, focusing on its cellular and extracellular matrix components. It then discusses biological and engineering approaches to MPGFB fabrication, covering materials, 3D design, and flow control. The review concludes with MPGFB applications in disease modeling and drug testing, and addresses improvements needed for refining MPGFB as a key tool in kidney disease research and treatment.

Neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by hallmark pathological features such as the accumulation of misfolded proteins and neuroinflammation. Chaperone-mediated autophagy (CMA), a selective lysosomal pathway, facilitates the degradation of proteins containing KFERQ-like motifs via the receptor lysosome-associated membrane protein type 2A (LAMP2A). In the recent review, the pivotal role of CMA in regulating proteostasis and modulating inflammatory responses is highlighted. This commentary explores the multifaceted roles of CMA in neurodegenerative disease progression, emphasizing its involvement in age-related decline, feedback loops between CMA dysregulation and neurodegeneration, and potential as a therapeutic target. Emerging CMA activators and the challenges of modulating CMA for clinical use are also discussed.

Krabbe disease (KD) is a lysosomal storage disorder characterized by severe neurodegeneration and demyelination. It is caused by mutations in the galactosylceramidase (GALC) gene, leading to the accumulation of psychosine, a neurotoxic metabolite. This study presents an optimized workflow for the production and characterization of recombinant murine GALC (rm-GALC) from HEK293T cells, aiming to improve the feasibility of enzyme replacement therapy (ERT) for KD. An affinity chromatography protocol is refined to purify His-tagged rm-GALC, followed by buffer exchange and concentration steps to produce a stable and active enzyme suitable for subsequent in vitro applications. The purified rm-GALC is characterized for enzymatic activity, purity, and stability using SDS-PAGE, immunoblotting, and dynamic light scattering (DLS). In vitro assays reveal dose-dependent enzymatic activity recovery in KD primary cells upon rm-GALC administration, with no adverse effects on cell viability up to the physiological GALC dose. Additionally, GALC treatment at the physiological dose restored autophagic function in KD cells, as shown by LC3 and p62 marker analyses, confirming its compatibility with lysosomal-autophagic pathways. Conversely, supra-physiological GALC administration resulted in decreased viability and autophagy impairment. Finally, the feasibility of loading GALC into a polymeric nanovector based on stabilized reverse micelles is investigated. These findings highlight the critical importance of precise GALC dose regulation in developing a safe and effective enzyme replacement therapy (ERT) strategy for Krabbe disease (KD), further supporting the potential of a nanovector-mediated ERT approach.

This study evaluated the therapeutic effects of fecal microbiota transplantation (FMT) on lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) in rats. The study focused on the balance of T-helper 17 (Th17) and regulatory T (Treg) cells, as well as the modulation of the JAK/STAT pathway. This study established a rat ARDS model using intranasal LPS instillation, administering interventions such as FMT, Treg cell depletion, and JAK inhibitors. Assessments included histopathological examination of lung and intestinal tissues, flow cytometry for Th17 and Treg cell proportions, qPCR and Western blot for gene and protein expression, ELISA for inflammatory cytokines, and correlation analysis using Spearman's method for cytokine-immune cell interactions. Results indicated that FMT and JAK inhibitors significantly reduce lung damage induced by LPS, reduced alveolar destruction and inflammation, restored Th17/Treg balance, and inhibited JAK/STAT pathway activity. Notably, FMT decreased pro-inflammatory cytokines (IL-2, IL-6, IL-8, IL-17A, IL-23, TGF-β1) and increased anti-inflammatory cytokines (IL-10, IL-35) in serum. Spearman correlation analysis indicated that FMT restored immune balance by modulating the interactions between cytokines and immune cells. In conclusion, FMT effectively alleviates lung and intestinal injury in LPS-induced ARDS rat models by modulating Th17/Treg balance and inhibiting JAK/STAT pathway activity, demonstrating promising therapeutic potential for ARDS treatment.

The receptor for advanced glycation end products (RAGE) is a multifunctional cell surface receptor implicated in aging and the progression of chronic diseases, including cancer and Alzheimer's disease. Its interaction with advanced glycation end products (AGEs) promotes cellular stress and inflammation, underscoring the diagnostic and therapeutic relevance of targeting RAGE. In this study, we explored the potential of nanobodiessingle-domain antibodies known for high specificity, strong affinity, and deep tissue penetrationas molecular tools for RAGE-targeted applications. Using a phage display library, a panel of RAGE-specific nanobodies were isolated and characterized. Binding activity and affinity were evaluated through enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assays. Among them, nanobody NbF8 demonstrated the highest affinity and specificity toward RAGE. In vitro, NbF8 selectively bound RAGE-expressing cells, while in vivo imaging in renal carcinoma and Alzheimer's disease mouse models confirmed its targeted accumulation in RAGE-overexpressing tumors and brain tissues. These findings highlight NbF8 as a promising molecular imaging agent for RAGE-associated diseases. This study supports the potential of RAGE-targeting nanobodies in both diagnostic imaging and therapeutic development, offering a novel approach for precision medicine in conditions driven by RAGE signaling.