Nanobodies (Nbs) have been extensively utilized in medical diagnosis and therapies due to their advantages, such as ease of genetic manipulation and efficient soluble expression in prokaryotic systems. However, enhancing their binding affinity to antigens to match or even exceed that of polyclonal antibodies for high-sensitivity bioassays remains a challenge. Therefore, in the present study, a novel strategy to enhance the binding affinity of antibodies to antigens was developed by fusing an anti-C-reactive protein Nb (CRPNb) to the N-terminus of self-assembling peptides such as right-handed coiled coil [RHCC; derived from a right-handed coiled-coil peptide of an archaebacterium (Staphylothermus marinus)], verotoxin (VTB; the B-subunit of Escherichia coli verotoxin) and C4-binding protein (C4bp; derived from human plasma C4-binding protein α-chain). For functional detection, alkaline phosphatase (AP) was further fused to the C-terminus of RHCC, VTB and C4bp, respectively. This approach enabled the formation of CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers. These were then expressed as soluble cytoplasmic proteins in the E. coli strain BL21 (DE3) and purified by imidazole elution. The protein expression was assessed by western blotting. Additionally, the stability of the multimeric constructs was evaluated using a direct ELISA, whilst their sensitivity was assessed using a competitive ELISA. The CRPNb-VTB-AP pentamers and CRPNb-RHCC-AP tetramers demonstrated antigen-specific recognition and enhanced affinity compared with the CRP-AP monomer based on a direct ELISA. Furthermore, they exhibited good thermal stability based on a direct ELISA. At 80˚C, CRPNb-VTB-AP, CRPNb-AP and CRPNb-RHCC-AP retained ~85, 75 and 60% activity, while CRPNb-H only retained ~40%. After incubation at 80˚C for 55 min, the two multimers still maintained ~40% activity. The results suggested them to be suitable for storage and transportation at ambient temperatures. In conclusion, a novel nanobody-fusion protein platform was established in the present study. The fusion proteins functioned as high-affinity binders for target antigens and heat-stable signal tracers for immunoassay detection and quantitative analysis, providing a novel type of thermostable immunoreagent.
The pathogenesis of diabetic kidney disease (DKD) is complex and closely related to ferroptosis and immune dysregulation, but the relevance is unclear. The present study investigates the potential mechanisms of ferroptosis-related genes (FRGs) in DKD and their relationship with the immune-inflammatory response. It searches for new diagnostic biomarkers to help diagnose and treat DKD. Four Gene Expression Omnibus (GEO) datasets, GSE30528, GSE30529 and GSE30122 as the test set, and GSE96804 for validation, were analyzed. FRGs were obtained from GeneCards, and 47 ferroptosis-related differentially expressed genes (FRDEGs) were identified by intersecting with DKD-related differentially expressed genes. Functional enrichment analyses, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Gene Set Enrichment Analysis and Gene Set Variation Analysis, revealed that these FRDEGs are primarily associated with ferroptosis, hypoxia response and immune inflammation. Subsequently, the weighted gene co-expression network analysis (WGCNA) was employed to expand the ferroptosis-related gene network, and intersection of the 47 FRDEGs with key WGCNA module genes yielded 10 key genes. Based on the 10 key genes, the least absolute shrinkage and selection operator and support vector machine algorithms identified three hub genes [chemokine ligand 5 (CCL5), forkhead box C1 (FOXC1) and lactotransferrin (LTF)] for DKD diagnosis. Receiver operating characteristic curves confirmed their diagnostic value, with FOXC1 and LTF validated in the independent dataset. Immune infiltration analysis via CIBERSORT revealed eight immune cell types with significantly different infiltration levels between the DKD and control group in the integrated GEO datasets. Notably, both LTF and CCL5 showed a significant positive correlation with gamma delta T cells (γδT). Quantitative PCR results confirmed differential expression of the three hub genes in the DKD group, with elevated expression observed in DKD mice following intervention with rosiglitazone and hyperoside.
Osteoporosis is characterized by reduced bone strength and an increased risk of fracture. Low-magnitude mechanical stress (LMMS) has been investigated as a non-invasive stimulus capable of modulating mechanotransduction in bone cells. This review synthesizes evidence concerning integrin-cytoskeletal signaling, linker of nucleoskeleton and cytoskeleton-mediated nuclear force transmission, connexin 43-mediated intercellular communication and the downstream regulation of bone marrow mesenchymal stem cells, osteoblasts and osteoclasts. Preclinical studies indicate that LMMS can activate the Wnt/β-catenin, MAPK/ERK, PI3K/AKT and receptor activator of nuclear factor-κB ligand/osteoprotegerin pathways in a context-dependent manner. However, clinical responses to whole-body vibration remain heterogeneous, vary according to the applied dose and skeletal site, and have not yet demonstrated fracture-prevention efficacy. Pulsed electromagnetic fields and therapeutic exercise are therefore discussed as related biophysical or mixed-magnitude interventions rather than as forms of LMMS itself. The present review highlights the gap between simplified cellular models and the three-dimensional bone microenvironment and proposes priorities for reporting standards and trial design to facilitate clinical translation.
The present study describes the clinical, histopathological and immunohistochemical features of a case of neuroendocrine carcinoma (NEC) incidentally discovered in hemorrhoidal tissue of a 46-year-old female patient, and also reviewes the relevant literature. The patient was admitted because a colonoscopy revealed hemorrhoids, accompanied by diarrhea. Following surgical resection, pathological examination revealed that the majority of the specimen was hemorrhoidal tissue, with intravascular tumor thrombus and a focus of poorly differentiated NEC (maximum diameter of ~0.5 cm) in the mucosa and submucosa of a single tissue. Furthermore, immunohistochemical results showed a 95% Ki-67 proliferation index and positivity for pan-cytokeratin, chromogranin A and synaptophysin. However, special stains such as Alcian blue and periodic acid-Schiff were negative. Subsequently, the patient received chemotherapy. The 6-month postoperative follow-up computed tomography revealed suspicious metastatic lesions in the perirectal fat space. Subsequent pathological examination of percutaneous needle biopsy specimens confirmed metastatic poorly differentiated NEC. Following confirmation of the diagnosis, the patient underwent radical resection for anal canal carcinoma. The patient reported multiple metastases and signs of systemic failure at 23 months of follow-up. Based on this case and limited literature reports, poorly differentiated NEC of the anal canal incidentally detected in hemorrhoidal tissue-particularly those accompanied by intravascular tumor thrombus and a high Ki-67 index-may indicate high aggressiveness. Long-term and close postoperative monitoring is recommended. Owing to the rarity of such cases, their universal invasive characteristics and the optimal treatment regimen still require validation with more clinical data. The findings of this study can provide a reference for clinical diagnosis and management.
The present study developed a long-term asymmetric force (AF)-induced degenerative lumbar scoliosis (DLS) model using bipedal upright rats and aimed to elucidate the role of AF in DLS pathogenesis. Sprague-Dawley rats underwent humeral head resection and tail amputation, with elevated food placement to encourage bipedal standing and feeding. The present training protocol continued for 16 weeks to establish the bipedal rat model. Subsequently, an AF-induced DLS rat model was established by implanting a nickel-titanium spring with anchor screws for 24, 36 and 48 weeks, respectively. X-ray imaging, micro-computed tomography, transmission electron microscopy, hematoxylin and eosin staining and safranin-O-fast green staining were performed. AF was associated with structural lumbar scoliosis in bipedal rats. Prolonged AF exposure reduced bone volume fraction, trabecular number, trabecular thickness, bone mineral density and tissue mineral density in the L3 lower vertebral endplate, ultimately leading to bone loss, microarchitectural deterioration, diminished mechanical strength and DLS development, although structure model index and trabecular separation remained unaffected. Prolonged asymmetric stress also caused structural damage to the medullary nerve fibers, degeneration and necrosis of intervertebral disc cartilage endplate cells, disorder of annulus fibrosus and reduction of nucleus pulposus cells. Additionally, long-term AF stimulation may have induced compensatory chondrocyte proliferation, contributing to early pathological repair processes, but appeared to reach a saturation point with extended exposure. The present study therefore successfully established a novel AF-induced DLS aging bipedal rat model, providing a basis for exploring therapeutic strategies targeting mechanical stress intervention in DLS.
Ferroptosis, an iron-dependent form of regulated cell death driven by excessive lipid peroxidation, has been increasingly implicated in the development and progression of liver cancer. Despite sanguinarine chloride (SC) exhibiting antitumor activity in a number of cancer models, whether it modulates ferroptosis in liver cancer remains unclear. The present study systematically investigated the ferroptosis-inducing effects of SC and the underlying molecular mechanisms in liver cancer. Molecular docking and molecular dynamics simulations suggested that SC interacts with glutathione peroxidase 4 (GPX4) with favorable affinity and forms a stable complex. Functional assays showed that SC significantly inhibited proliferation and reduced the viability of HepG2 and Huh7 cells. Mechanistically, SC markedly decreased GPX4 protein expression, leading to the accumulation of lipid reactive oxygen species and malondialdehyde, together with characteristic mitochondrial changes associated with ferroptosis, including increased membrane density and loss of cristae. SC-induced cell death was partially rescued by the ferroptosis inhibitors ferrostatin-1 and deferoxamine, further supporting the involvement of ferroptosis. Consistently, in a xenograft model, SC suppressed GPX4 expression, promoted ferroptosis and significantly inhibited tumor growth. Collectively, these findings indicate that SC exerts antitumor effects in liver cancer, at least in part, by inducing ferroptosis through GPX4 downregulation, supporting further investigation of SC as a potential therapeutic candidate.
Chlamydia psittaci pneumonia (CPP) is a zoonotic disease with non-specific clinical and radiographic features, complicating timely diagnosis. Although not common, pleural effusion may indicate more severe disease. Conventional diagnostic methods lack the sensitivity and speed. The present report highlights the utility of multiplex PCR-based targeted next-generation sequencing (mp-tNGS) for rapid etiological diagnosis of CPP, particularly in patients with pleural effusion. A 52-year-old woman and a 64-year-old woman both presented with high fever, cough and systemic symptoms. Inflammatory markers (hs-CRP, IL-6 and ESR) were found to be markedly elevated with normal or mildly elevated white blood cell counts. A chest CT exhibited unilateral consolidations with small ipsilateral pleural effusions. Conventional microbiological tests (stains, cultures and serology) were negative. Bronchoalveolar lavage fluid mp-tNGS rapidly identified Chlamydia psittaci (142 and 251 reads per 100,000 sequencing reads, respectively), with a mean turnaround time of 11.35 h (from laboratory receipt to result). Epidemiological history revealed exposure to pigeons and a bird market. Doxycycline therapy led to rapid clinical improvement, fever resolution and notable radiographic clearance. Therefore, pleural effusion can be a feature of CPP and may indicate more severe disease. mp-tNGS proved rapid and decisive when conventional methods failed, enabling timely targeted therapy. A high index of suspicion combined with mp-tNGS is thus key in accurately diagnosing and improving prognosis.
Perianal fistulas (PAF) represent a complex anorectal condition characterized by marked therapeutic and surveillance challenges. Due to its high propensity for recurrence, sustained long-term follow-up is as important for patient management as the initial intervention. The present analysis included 245 patients who underwent surgical treatment for PAF. Patient data were examined from the early postoperative period onward. Follow-up data at 1, 6 and 24 months were obtained by reviewing operating room, inpatient ward and outpatient clinic records. Patients lost to follow up, whether due to non-attendance at scheduled outpatient visits or inability to be contacted, were not excluded from the analysis; rather, this subgroup was categorized separately to assess the characteristics and potential impact of the missing data. Results indicated that MRIs demonstrated a high diagnostic success rate for PAF (90.95%). By contrast, retrospective clinical examination proved insufficient for accurately identifying the internal fistula opening. In long-term assessment, both fistulectomy and seton placement yielded comparable success rates. A progressive increase in missing data was observed throughout the follow-up period. Overall, MRI remains an established diagnostic modality for PAF. Long-term therapeutic outcomes are not determined solely by surgical technique, but rather by a complex interplay of factors. These include gaps in key anatomical data, socioeconomic barriers contributing to patient attrition, cultural perspectives on healthcare and the growing influence of social media in shaping patient expectations and health-associated anxieties. All these elements markedly influence the clinical pathway and ultimate success of PAF management.
The present study reports, to the best of our knowledge, the first case of neonatal bloodstream infection caused by Malassezia furfur in mainland China identified using metagenomic next-generation sequencing (mNGS). Traditional microbiological methods have failed to identify causative organisms, highlighting the diagnostic limitations of neonatal sepsis with atypical presentations. Using this case as a clinical entry point, a systematic review was conducted to consolidate the epidemiological, clinical and prognostic characteristics of previously reported neonatal Malassezia infections and critically evaluate current diagnostic challenges. This case suggests the need to expand the pathogen spectrum of neonatal sepsis, particularly among extremely preterm and extremely low-birth-weight infants. Furthermore, it demonstrates the transformative potential of mNGS as an adjunctive diagnostic modality capable of identifying rare, fastidious organisms that evade conventional detection. Integrating mNGS into routine clinical workflows may not only facilitate early and precise pathogen identification but also redefine clinical decision-making paradigms in neonatal infectious disease management.
