The glycocalyx is a carbohydrate-rich layer formed by glycoproteins, glycolipids, proteoglycans, and glycosaminoglycans at the outer cell surface. In cancer, this layer is not a passive coating. Its thickness, composition, charge, and nanoscale organization are altered by oncogenic signaling, dysregulated glycosyltransferases, protease-mediated shedding, and reciprocal interactions with stromal and immune cells. These changes affect how tumor cells sense force, bind the extracellular matrix, cluster receptors, evade immune surveillance, survive in circulation, and arrest at distant vascular beds. Studies in the past decade have linked a bulky glycocalyx to integrin activation and growth factor signaling, identified hyaluronan-CD44 and selectin-dependent steps in metastatic dissemination, and placed the sialoglycan-Siglec axis among the immune suppressive circuits operating in the tumor microenvironment. Meanwhile, pathology, imaging, glycoproteomics, and liquid-biopsy studies have started to define glycocalyx-derived biomarkers with diagnostic and prognostic value, especially in hepatocellular carcinoma, pancreatic cancer, breast cancer, and hematologic malignancies. This review summarizes the structure of the cancer glycocalyx, the molecular programs that remodel it, and the consequences of these changes for tumor progression. It also discusses analytical approaches, biomarker development, and therapeutic strategies aimed at mucins, proteoglycans, glycosyltransferases, hyaluronan signaling, heparanase, and tumor-associated sialoglycans. A central theme is that the glycocalyx should be viewed as an integrated biophysical and signaling interface, and its clinical value will depend on spatially resolved, context-specific analysis rather than single-marker measurements alone.

62 Background: The cell-surface glycocalyx (GCX) is a carbohydrate-rich layer covering epithelial cells and plays an important role in tumor–microenvironment interactions and immune modulation. In breast cancer, GCX remodeling has been implicated in tumor heterogeneity and disease progression. However, integrated analyses linking GCX-associated morphological features with underlying molecular expression programs at the single-cell level across breast cancer subtypes remain limited. We have previously investigated GCX alterations in breast cancer using lectin staining and scanning electron microscopy (SEM). Methods: Frozen sections of resected breast cancer tissues and adjacent normal mammary epithelium were analyzed using multiple fluorescently labeled lectins to profile cell-surface glycan patterns. GCX ultrastructure was further evaluated by SEM using lanthanum nitrate staining. To elucidate the molecular basis underlying GCX phenotypes, publicly available breast cancer single-cell RNA sequencing (scRNA-seq) datasets were analyzed. Tumor epithelial cell populations were extracted, and expression programs of GCX-associated molecules, including glycosylation-related enzymes, mucins, and proteoglycans, were evaluated in a subtype-specific manner. Transcriptomic findings were integrated with lectin-based glycan patterns observed in frozen tissue sections. Results: Lectin staining demonstrated altered cell-surface glycan distribution and marked intratumoral heterogeneity in breast cancer tissues compared with normal mammary epithelium. Reduced binding of O-glycan–associated lectins was observed in tumor regions, indicating GCX remodeling, with patterns differing among breast cancer subtypes. SEM analysis revealed thinning, discontinuity, and surface irregularities of the GCX in tumor areas, consistent with lectin-based observations. Analysis of public scRNA-seq data revealed subtype-associated differences in the expression of GCX-related genes within tumor epithelial cell populations, including genes involved in glycosylation, mucin production, and proteoglycan biology. These transcript-level differences paralleled the subtype-dependent glycan distributions identified by lectin staining. Conclusions: By integrating lectin-based GCX profiling and SEM with public single-cell transcriptomic analysis, we mapped cell-surface glycocalyx phenotypes to corresponding molecular expression programs in breast cancer. GCX remodeling identified by lectin staining was associated with subtype-specific transcriptional programs at the single-cell level. This integrative approach provides a framework for understanding GCX-related tumor heterogeneity and may facilitate future studies on subtype-specific tumor biology and tumor–immune interactions in breast cancer.

Tendon injuries are frequently occurring disorders; it is clinically important to enhance tendon regeneration and prevent functional impairment post-injury. While tendon injuries in children heal quickly with minimal scarring, those in adults heal slowly and are accompanied by fibrotic scarring. Therefore, investigating the healing mechanisms after tendon injury, and identifying the factors that regulate the inherent regenerative capacity of tendons are promising approaches to promoting tendon regeneration. Here, we identify that the PI3K-Akt signalling pathway is preferentially upregulated in injured neonatal murine Achilles tendons. Inhibition of PI3K-Akt signalling in a neonatal murine Achilles tendon rupture model decreases cell proliferation and migration in both Scx-lineage intrinsic tenocytes and Tppp3-lineage extrinsic paratenon sheath cells. Moreover, the inhibition of PI3K-Akt signalling decreases stemness and promotes mature tenogenic differentiation in both Scx- and Tppp3-lineage cells. Collectively, these results suggest that PI3K-Akt signalling plays a pivotal role in neonatal tendon regeneration.

Secretory carcinomas (SCs) of the salivary gland have recently been recognized as low-grade, malignant tumors. Before this designation, most SCs were diagnosed as variants of acinic cell carcinomas (AciCCs). SCs harbor the t(12;15)(p13;q25) translocation that generates an oncogenic fusion gene, ETS variant transcription factor 6/Neurotrophic tyrosine receptor kinase(ETV6::NTRK3). However, detecting fusion genes in a clinical setting is time-consuming and costly. In this study, we examined 31 cases previously diagnosed as AciCC and SC using pathological analyses with detection of fusion genes using ETV6 break-apart fluorescence in-situ hybridization and reverse transcription-polymerase chain reaction. After re-analysis, we found that these 31 cases actually comprised 21 SCs and 10 AciCCs. We examined the diagnostic utility of immunohistochemistry by comparing results with the fusion gene, Pan-Trk, which despite having recently been reported as effective for diagnosis of SC, was not universally accurate. However, combining mammaglobin and S-100 could be particularly useful in diagnosing SC. This practical method will contribute to accurate diagnosis of SCs, while saving time in daily clinical practice.

OBJECTIVES:The receptor tyrosine kinase Kit is expressed in cells derived from the trunk neural crest (NC), such as melanocytes; however, its role in cranial NC cell development is not fully understood. METHODS:We investigated the effects of the heterozygous loss of Kit in NC cells during embryonic development by mating Kit2lox/+ mice with Wnt1-Cre mice to produce Wnt1-Cre; Kit2lox/+ embryos. In addition, Wnt1-Cre mice were mated with Rosa26R-yellow fluorescent protein (YFP) mice to visualize the tissue regions expressing Cre recombinase. Histological studies of the craniofacial regions of these mice were performed using samples from embryonic day (E) 12.5 and postnatal day (P) 1. Cellular apoptosis and proliferation were both analyzed through the immunostaining of tissue sections collected on E13.5 and E14.5 using anti-cleaved caspase 3 (CC3) to detect apoptosis and anti-Ki67 to detect proliferation. Cells from YFP-positive tissue regions of the facial areas of Wnt1-Cre; Kit+/+; Rosa26R-YFP embryos and Wnt1-Cre; Kit2lox/+; Rosa26R-YFP embryos collected on E12.5 and E15.5 were cultured and evaluated for cell proliferation. RESULTS:Compared with control littermates, Wnt1-Cre; Kit2lox/+ embryos exhibited midline cleft lip and bifid nose deformities. Substantial early (P1) postnatal lethality was observed in Wnt1-Cre; Kit2lox/+ mice, with none surviving to 3 weeks of age. YFP-positive cells from the maxillary regions of Wnt1-Cre; Kit2lox/+; Rosa26R-YFP embryos exhibited defective cell growth and self-renewal in vitro. CONCLUSION:Conditional heterozygous loss of Kit in Wnt1-Cre; Kit2lox/+ embryos is associated with craniofacial dysplasia and exhibit defective NC development in vitro and in vivo.

Immunohistochemistry (IHC) is the basis of histological or pathological analysis and is widely used to enable the detection and characterization of proteins in various organ tissues, including brain tissues. IHC is commonly performed on formalin-fixed paraffin-embedded (FFPE) tissues because of their easy storage and versatility. IHC is a key method for providing more accurate analysis of localization and function of neurons, neuroendocrine cells, and neural stem cells in the brain and other nervous systems. The related cells such as glial cells and neurovascular units have also been analyzed by IHC. Visualization of antibody-antigen interactions can be performed primarily in one of the following ways: chromogenically stained IHC and fluorescently stained IHC. In chromogenically stained IHC, an antibody is chemically conjugated to an enzyme, such as peroxidase, that can be reacted with a suitable substrate to give a colored product. In fluorescently stained IHC, the antibodies are finally tagged with fluorescent chemicals such as fluorescein isothiocyanate (FITC) or rhodamine. Here, we describe the standard methods of IHC applied to brain slice sections. Furthermore, an automated immunostainer is presented as another option for standardized immunohistochemistry.

Introduction:This study aimed to investigate the clinical and radiological outcome of "indirect decompression" using lateral-posterior combined surgery for osteoporotic vertebral fracture (OVF) with neurological symptoms. Methods:A total of 17 patients who underwent lateral and posterior combined indirect decompressive spinal reconstruction (LP-IDR) for single-level OVF with neurological symptoms were included in this study. The neurological symptoms (sensory disturbance and muscle weakness) and imaging findings (local angle and height of the fracture segment and bone fragment occupancy in the spinal canal) were investigated preoperatively, postoperatively, and at the 1-year follow-up. Results:Muscle weakness was observed preoperatively in ten patients. Nine patients had complete recovery of muscle weakness (p<0.001), whereas one had residual muscle weakness at the 1-year follow-up. The presence of sensory disturbance was observed in 16 patients preoperatively, and it was significantly reduced to 8 patients at the 1-year follow-up (p=0.003). The bony fragment occupancy rate in the spinal canal was decreased from 44.0% to 40.2% postoperatively (p=0.04) and to 33.1% at 1 year (p=0.002). The local angle was corrected from 8.3° to -2.6° postoperatively (p=0.003) and to 1.2° at 1 year. The local height was corrected from 26.7 to 32.0 mm postoperatively (p<0.001) and to 29.8 mm at 1 year. Conclusions:LP-IDR for OVF with neurological symptoms provided sufficient neurological improvement with expansion of the spinal canal over time.

Spinopelvic sagittal alignment is crucial for assessing balance and determining treatment efficacy in patients with adult spinal deformity (ASD). Only a limited number of reports have addressed spinopelvic parameters and lumbosacral transitional vertebrae (LSTV). Our primary objective was to study spinopelvic sagittal parameter changes in patients with LSTV. A secondary objective was to investigate clinical symptoms and quality of life (QOL) in patients with LSTV. In this study, we investigated 371 participants who had undergone medical check-ups for the spine. LSTV was evaluated using Castellvi’s classification, and patients were divided into LSTV+ (type II-IV, L5 vertebra articulated or fused with the sacrum) and LSTV- groups. After propensity score matching for demographic data, we analyzed spinopelvic parameters, sacroiliac joint degeneration, clinical symptoms, and QOL for these two participant groups. Oswestry Disability Index (ODI) scores and EQ-5D (EuroQol 5 dimensions) indices were compared between the two groups. Forty-four patients each were analyzed in the LSTV + and LSTV- groups. The LSTV + group had significantly greater pelvic incidence (52.1 ± 11.2 vs. 47.8 ± 10.0 degrees, P = 0.031) and shorter pelvic thickness (10.2 ± 0.9 vs. 10.7 ± 0.8 cm, P = 0.018) compared to the LSTV- group. The “Sitting” domain of ODI (1.1 ± 0.9 vs. 0.6 ± 0.7, P = 0.011) and “Pain/Discomfort” domain of EQ-5D (2.0 ± 0.8 vs. 1.6 ± 0.7, P = 0.005) were larger in the LSTV + group. There was a robust association between LSTV and pelvic sagittal parameters. Clinical symptoms also differed between the two groups in some domains. Surgeons should be aware of the relationship between LSTV assessment, radiographic parameters and clinical symptoms. 3.

Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including congenital myasthenic syndromes (CMS). Germline mutations in CHRNE encoding the acetylcholine receptor (AChR) ε subunit are the most common cause of CMS. An active form of vitamin D, calcitriol, binds to vitamin D receptor (VDR) and regulates gene expressions. We found that calcitriol enhanced MuSK phosphorylation, AChR clustering, and myotube twitching in co-cultured C2C12 myotubes and NSC34 motor neurons. RNA-seq analysis of co-cultured cells showed that calcitriol increased the expressions of Rspo2, Rapsn, and Dusp6. ChIP-seq of VDR revealed that VDR binds to a region approximately 15 ​kbp upstream to Rspo2. Biallelic deletion of the VDR-binding site of Rspo2 by CRISPR/Cas9 in C2C12 myoblasts/myotubes nullified the calcitriol-mediated induction of Rspo2 expression and MuSK phosphorylation. We generated Chrne knockout (Chrne KO) mouse by CRISPR/Cas9. Intraperitoneal administration of calcitriol markedly increased the number of AChR clusters, as well as the area, the intensity, and the number of synaptophysin-positive synaptic vesicles, in Chrne KO mice. In addition, calcitriol ameliorated motor deficits and prolonged survival of Chrne KO mice. In the skeletal muscle, calcitriol increased the gene expressions of Rspo2, Rapsn, and Dusp6. We propose that calcitriol is a potential therapeutic agent for CMS and other diseases with defective neuromuscular signal transmission.

Purpose This study aimed to compare the radiological tumor (T)-category using multiparametric MRI with the pathological T category in patients with oral tongue squamous cell carcinoma (OTSCC) and to examine which is a better predictor of prognosis.Methods This retrospective study included 110 consecutive patients with surgically resected primary OTSCC who underwent preoperative contrast-enhanced MRI. T categories determined by maximum diameter and depth of invasion were retrospectively assessed based on the pathological specimen and multiparametric MRI. The MRI assessment included the axial and coronal T1-weighted image (T1WI), axial T2-weighted image (T2WI), coronal fat-suppressed T2WI, and axial and coronal fat-suppressed contrast-enhanced T1WI (CET1WI). Axial and coronal CET1WI measurements were divided into two groups: measurements excluding peritumoral enhancement (MEP) and measurements including peritumoral enhancement. The prognostic values for recurrence and disease-specific survival after radiological and pathological T categorization of cases into T1/T2 and T3/T4 groups were compared.Results The T category of MEP on coronal CET1WI was the most relevant prognostic factor for recurrence [hazard ratio (HR) = 3.30, p = 0.001] and the HR was higher than the HR for pathological assessment (HR = 2.26, p = 0.026). The T category determined by MEP on coronal CET1WI was also the most relevant prognostic factor for disease-specific survival (HR = 3.12, p = 0.03), and the HR was higher than the HR for pathological assessment (HR = 2.02, p = 0.20).Conclusion The T category determined by MEP on the coronal CET1WI was the best prognostic factor among all radiological and pathological T category measurements.

Transgenic mice, including those created using Bacterial Artificial Chromosomes (BACs), are artificial manipulations that have become critical tools for studying gene function. While conventional transgenic techniques face challenges in achieving precise expression of foreign genes in specific cells and tissues, BAC transgenic mice offer a solution by incorporating large DNA segments that can include entire expression units with tissue-specific enhancers. This review provides a thorough examination of BAC transgenic mouse technology, encompassing both traditional and humanized models. We explore the benefits and drawbacks of BAC transgenesis compared to other techniques such as knock-in and CRISPR/Cas9 technologies. The review emphasizes the applications of BAC transgenic mice in various disciplines, including neuroscience, immunology, drug metabolism, and disease modeling. Additionally, we address crucial aspects of generating and analyzing BAC transgenic mice, such as position effects, copy number variations, and strategies to mitigate these challenges. Despite certain limitations, humanized BAC transgenic mice have proven to be invaluable tools for studying the pathogenesis of human diseases, drug development, and understanding intricate gene regulatory mechanisms. This review discusses current topics on BAC transgenic mice and their evolving significance in biomedical research.

In vivo dynamic nuclear polarization-MRI (DNP-MRI, also called OMRI, PEDRI) using carbamoyl-PROXYL(CmP) as a redox sensitive DNP probe enables the accurate monitoring of the tissue redox status. We found that the redox status decreases 1 day after radiation treatment, and the decay of redox status occurs before any micro- or macroscopic changes in tumor morphology and pyruvate metabolism based on the Warburg effect. This decay of redox status can also be associated with the decreased production of intratumor reducing redox molecules such as GSH and AsA.

BACKGROUND:The glycocalyx (GCX) is a glycan structure on the vascular endothelium and cancer cells. It is crucial for blood flow regulation, tumor invasion, and cancer drug resistance. Understanding the role of GCX in human tumors could help develop new cancer biomarkers and therapies. AIM:This study aimed to demonstrate microstructural changes in human primary and metastatic liver tumors (henceforth termed liver tumors) by visualizing GCX using surgical specimens and comparing formalin-fixed paraffin-embedded sections (FFPEs) with frozen sections. The results of lectin staining were also compared between frozen and FFPE specimens to determine which was more useful for accurately assessing GCX structure and composition. METHODS:Liver tumors and normal tissue samples from three patients were collected and processed into FFPEs and frozen sections, respectively. Lanthanum nitrate staining and scanning electron microscopy (SEM) were used to assess the GCX structures. Twenty lectins were analyzed for their glycan components in the samples. RESULTS:SEM revealed significant differences in GCX morphology among the cancer specimens. Frozen sections provided a more accurate GCX evaluation than FFPEs, showing distinct glycan compositions in hepatocellular carcinoma, colorectal carcinoma liver metastases, and melanoma liver metastases. Hepatocellular carcinoma samples exhibited a loss of N-acetylgalactosamine-related lectins. CONCLUSION:The results revealed that liver tumors have distinct and bulky GCX compared to normal liver tissue, while frozen sections are more reliable for GCX evaluation. These findings highlight glycan alterations in liver tumors and contribute to the development of new cancer therapies targeting GCX on tumor cell surfaces.

Introduction: After posterior lumbar interbody fusion (PLIF), trabecular bone remodeling (TBR) occurs in the vertebral body. This study aimed to investigate whether imaging findings obtained with PLIF are applicable to lateral lumbar interbody fusion (LLIF). Methods: A total of 53 cases who underwent one- or two-level LLIF with polyether ether ketone cage and posterior spinal fixation/fusion (PSF) were retrospectively included in this study. TBR, vertebral endplate cyst (VEC), facet union, and pseudarthrosis were investigated on computed tomography (CT) images at 3 months, 1 year, and 2 years postoperatively. Of the 53 patients, 36 (68%) who underwent CT examination at approximately 5 years postoperatively were subanalyzed. Results: TBR was commonly observed anterior to the cage on CT sagittal images. The TBR-positive rate was 21%, 67%, and 73% at 3 months, 1 year, and 2 years postoperatively, respectively. The 3-month TBR-positive segments showed significantly less VEC (0% vs. 29%, P=0.029) at 1 year postoperatively. The 1-year TBR-positive segments showed a significantly higher facet union rate (83% vs. 57%, P=0.019) and less pseudoarthrosis (0% vs. 13%, P=0.041) at 2 years postoperatively. At 5 years postoperatively, 50% of the 2-year TBR-positive segments turned negative with solid intervertebral bony fusion. Conclusions: TBR-positive segments had significantly lower future VEC positivity, higher future facet union rates, and lower future pseudarthrosis rates. In LLIF-PSF, TBR suggests the establishment of intervertebral stability and allows consideration of intervertebral biomechanics.

Scanning electron microscopy (SEM) is used to observe the surface structure of an object by irradiating an electron beam onto the sample and detecting the reflected and emitted electrons. Because of its large depth of focus, SEM can provide the three-dimensional structure of small surfaces that cannot be observed using an optical microscope. Furthermore, the cross-sectional structure of the tissue can be observed by freeze-cracking. Observing the ultrastructure of organisms that contain large amounts of water in their bodies while maintaining high resolution is challenging; however, this has recently become possible. Here, we explain the fixation and freeze-cracking method for mouse brain samples.

The mesothelium is a non-adhesive protective surface that lines the serosal cavities and organs within the body. The glycocalyx is a complex structure that coats the outer layer of the mesothelium. However, due to the limitations of conventional fixation techniques, studies on glycans are limited. In this study, lectin staining of frozen tissues was performed to investigate the diversity of glycans in the glycocalyx of mesothelial cells in mice. Datura stramonium lectin (DSL), which recognizes lactosamine and binds to Galectin-3 and -1, was broadly bound to the mesothelial cells of the visceral and parietal peritoneum but not to the pancreas, liver, intestine, or heart. Furthermore, human mesothelial cells in the omentum and parietal peritoneum were positive for DSL. Erythrina cristagalli lectin binding was specific to mesothelial cells in the parietal peritoneum, that is, the pleura, diaphragm, and peritoneum. Intriguingly, surface sialylation, the key element in reducing peritoneal dissemination and implantation, and promoting ascites formation by ovarian carcinoma cells, was much higher in the parietal peritoneum than in the omentum. These findings revealed slight differences in the glycans of mesothelial cells of different organs, which may be related to clinical diseases. These results also suggest that there may be differences in the functions of parietal and visceral mesothelial cells.

Owing to its mitogenic and angiogenic characteristics, the use of basic fibroblast growth factor (bFGF) to promote wound healing has been investigated. However, its clinical efficacy has fallen short of expectations due to its instability. Heparin has been reported to stabilize bFGF. Therefore, we hypothesized that the combination of these agents would more effectively promote wound healing than bFGF alone; a single-center, two-arm parallel, single-blind, and a prospective randomized controlled pilot study was therefore performed involving 12 patients who underwent split-thickness skin graft harvesting. To ensure a feasible clinical treatment model, commercially available agents were used. The patients were randomly assigned to either the control group treated with bFGF (n = 6) or the intervention group treated with bFGF and heparin (n = 6) in a 1:1 ratio. The wound area and the wound area variation was assessed each week postoperatively, as was the number of days required for epithelialization. As a supplementary analysis, the least-squares means were calculated using a linear mixed-effects model. The results of this study indicate that the combination of bFGF and heparin may more effectively promote wound healing than bFGF alone, consistent with our hypothesis. A multicenter trial based on these data is ongoing.

Oxaliplatin, a platinum-based anticancer drug, is associated with peripheral neuropathy (oxaliplatin-induced peripheral neuropathy, OIPN), which can lead to worsening of quality of life and treatment interruption. The endothelial glycocalyx, a fragile carbohydrate-rich layer covering the luminal surface of endothelial cells, acts as an endothelial gatekeeper and has been suggested to protect nerves, astrocytes, and other cells from toxins and substances released from the capillary vessels. Mechanisms underlying OIPN and the role of the glycocalyx remain unclear. This study aimed to define changes in the three-dimensional ultrastructure of capillary endothelial glycocalyx near nerve fibers in the hind paws of mice with OIPN. The mouse model of OPIN revealed disruption of the endothelial glycocalyx in the peripheral nerve compartment, accompanied by vascular permeability, edema, and damage to the peripheral nerves. To investigate the potential treatment interventions, nafamostat mesilate, a glycocalyx protective agent was used in tumor-bearing male mice. Nafamostat mesilate suppressed mechanical allodynia associated with neuropathy. It also prevented intra-epidermal nerve fiber loss and improved vascular permeability in the peripheral paws. The disruption of endothelial glycocalyx in the capillaries that lie within peripheral nerve bundles is a novel finding in OPIN. Furthermore, these findings point toward the potential of a new treatment strategy targeting endothelial glycocalyx to prevent vascular injury as an effective treatment of neuropathy as well as of many other diseases. PERSPECTIVE: OIPN damages the endothelial glycocalyx in the peripheral capillaries, increasing vascular permeability. In order to prevent OIPN, this work offers a novel therapy approach that targets endothelial glycocalyx.