Iron deposition in gastric black spots: Clinicopathological insights and NanoSuit-correlative light and electron microscopy analysis.

Objectives: Black spots (BSs) are lentiginous findings observed in the gastric body and fundus during upper gastrointestinal endoscopy and are predominantly seen in patients undergoing Helicobacter pylori eradication treatment. However, the detailed patient background and exact composition are poorly understood. This study aims to clarify the clinicopathological features of BSs, examine patient demographics, and use the NanoSuit-correlative light and electron microscopy (CLEM) method combined with scanning electron microscopy-energy dispersive X-ray spectroscopy for elemental analysis. Methods: Patients who underwent upper gastrointestinal endoscopy between 2017 and 2022 were included. Data on age, medications, blood tests, and H. pylori infection status were retrospectively gathered from medical records. Univariate analysis was conducted to examine BS presence, with results then used in a multivariate model to identify associated risk factors. Additionally, pathological specimens from patients with BSs were analyzed for elemental composition using the NanoSuit-CLEM method combined with scanning electronmicroscopy-energy dispersive X-ray spectroscopy. Results: An analysis of 6778 cases identified risk factors for BSs, including older age and using proton pump inhibitors, statins, corticosteroids, and antithrombotic drugs. Endoscopically, BSs correlated with higher gastric atrophy and lower active H. pylori infection. Iron deposition at BS sites was specifically identified using NanoSuit-CLEM. Conclusions: BSs on gastrointestinal endoscopy may indicate an absence of active H. pylori inflammation. The discovery of iron deposition within BSs using the NanoSuit-CLEM method has offered new insights into the possible causative factors and advances our understanding of the etiology of BSs, bringing us closer to unraveling the underlying mechanisms of their formation.

Effect of solvent chain length on the colloidal behavior of alkylamine-protected silver nanoparticles in alkanes: Insights from solvent relaxation NMR.

HYPOTHESIS: The dispersion of apolar-ligand-protected nanoparticles (NPs) in alkanes is a complex process diverging from the "like dissolves like" principle, making its prediction beyond the capability of the Hansen solubility parameter (HSP) sphere method. This necessitates experimental investigation at the molecular level to understand dispersion behavior, particularly the role of solvent-ligand interactions. EXPERIMENTS: Solvent relaxation NMR was applied for the first time to investigate solvent-ligand interactions in the dispersion/agglomeration of hexadecylamine-protected silver nanoparticles (C16-Ag NPs) in alkanes. The dispersibilities in different alkanes were determined from the localized surface plasmon resonance (LSPR) and compared with those predicted from the HSPs. FINDINGS: The colloidal behavior of C16-Ag NPs in alkanes was notably affected by the length of the solvent chain. LSPR analysis demonstrated that while C16-Ag NPs remained dispersed in pentane, hexane, and octane, they exhibited agglomeration in decane, dodecane, and tetradecane, contradicting the HSP theory predictions. Solvent relaxation NMR revealed that this unexpected agglomeration stems from the strong bonding of longer-chain solvents to surface C16 ligands, leading to significant interaction. In contrast, shorter-chain solvents exhibited weaker bonding, promoting better dispersion. These findings emphasize the importance of solvent choice in NP applications and offer valuable insights into ligand-shell dynamics, furthering the development of NP technologies.

A Case of Brunner's Gland Hyperplasia Accompanied by an Increase in Endocrine Cells and Endocrine Cell Micronests.

Endocrine cell micronests (ECMs) are aggregates of endocrine cells known as enterochromaffin-like cells, typically measuring approximately 50 µm and usually observed in the mucosal layer of atrophic gastric fundic glands associated with hypergastrinemia. Although there are numerous reports on gastric ECMs, reports on duodenal ECMs are exceedingly rare. We report a rare case of Brunner's gland hyperplasia with increased endocrine cells and ECMs. An approximately 40 mm polyp was found in the duodenal bulb of a 57-year-old Japanese male patient during an upper gastrointestinal endoscopy, and a polypectomy was performed. Microscopic examination revealed hyperplasia of Brunner's glands in the duodenal polyp. Compared to normal Brunner's glands, hyperplastic Brunner's glands exhibited more endocrine cells. Additionally, many ECMs were observed in the fibromuscular connective tissue, comprising smooth muscle cells and myofibroblasts, adjacent to the hyperplastic Brunner's glands. The patient presented with hypergastrinemia (2,500 pg/mL; normal range: 30-140 pg/mL), and the ECMs were considered related to this condition. This case represents the first instance of a benign duodenal lesion with an increase in endocrine cells and the presence of ECMs.

Cell-free DNA analysis for recurrent respiratory papillomatosis: A case report.

A 35-year-old male presented with recurrent respiratory papillomatosis. Human papillomavirus type 11 was detected from all sites of tumor tissue DNA by PCR. The pre-surgery cell-free DNA (cfDNA) viral load (3.33 × 103 copies/ng DNA) fell below the post-surgical detection limits on achieving remission, suggesting cfDNA's potential as a biomarker.

Enhancing cervical cancer cytology screening via artificial intelligence innovation.

A double-check process helps prevent errors and ensures quality control. However, it may lead to decreased personal accountability, reduced effort, and declining quality checks. Introducing an artificial intelligence (AI)-based system in such scenarios could effectively address the risk of oversights. This study introduces an innovative AI-integrated workflow for cervical cytology screening that substantially improves efficiency and reduces the burden on cytologists. The AI model prioritizes cases for review based on anomaly scores and streamlines the first screening process to approximately 10 s per case. The model enhances the identification of high-risk cases via detailed microscopic observation, high anomaly scores cases, and a targeted review of low-score cases. The workflow highlights its capability for rapid, accurate, and less labor-intensive evaluations, demonstrating the potential to transform cervical cancer screening. This study highlights the importance of AI in modern medical diagnostics, particularly in areas with a high demand for accuracy and efficiency.

Effect of Gold Nanoparticle Size on Regulated Catalytic Activity of Temperature-Responsive Polymer-Gold Nanoparticle Hybrid Microgels.

Gold nanoparticles (AuNPs) possess attractive electronic, optical, and catalytic properties, enabling many potential applications. Poly(N-isopropyl acrylamide) (PNIPAAm) is a temperature-responsive polymer that changes its hydrophilicity upon a slight temperature change, and combining PNIPAAm with AuNPs allows us to modulate the properties of AuNPs by temperature. In a previous study, we proposed a simpler method for designing PNIPAAm-AuNP hybrid microgels, which used an AuNP monomer with polymerizable groups. The size of AuNPs is the most important factor influencing their catalytic performance, and numerous studies have emphasized the importance of controlling the size of AuNPs by adjusting their stabilizer concentration. This paper focuses on the effect of AuNP size on the catalytic activity of PNIPAAm-AuNP hybrid microgels prepared via the copolymerization of N-isopropyl acrylamide and AuNP monomers with different AuNP sizes. To quantitatively evaluate the catalytic activity of the hybrid microgels, we monitored the reduction of 4-nitrophenol to 4-aminophenol using the hybrid microgels with various AuNP sizes. While the hybrid microgels with an AuNP size of 13.0 nm exhibited the highest reaction rate and the apparent reaction rate constant (kapp) of 24.2 × 10-3 s-1, those of 35.9 nm exhibited a small kapp of 1.3 × 10-3 s-1. Thus, the catalytic activity of the PNIPAAm-AuNP hybrid microgel was strongly influenced by the AuNP size. The hybrid microgels with various AuNP sizes enabled the reversibly temperature-responsive on-off regulation of the reduction reaction.

Development of an Undifferentiated Pleomorphic Sarcoma After Aortic Aneurysm Graft Replacement: A Case Report and Literature Review.

Aortic sarcomas are extremely rare. Sarcomas associated with aortic graft replacement are even rarer; only 17 cases have been examined through immunohistochemical staining to date, most of which were either angiosarcomas or intimal sarcomas. Here, we report the case of an 88-year-old man with an undifferentiated pleomorphic sarcoma (UPS) that developed after aortic graft replacement and was diagnosed through postmortem autopsy. To the best of our knowledge, this is the first case of graft-associated sarcoma diagnosed as an undifferentiated pleomorphic type following detailed immunohistochemical staining with sufficient antibodies and fluorescencein situ hybridization (FISH).

Goblet Cell Adenocarcinoma in the Stomach: A Case Report.

Goblet cell adenocarcinoma (GCA) is known as an amphicrine tumor often seen in the appendix. Here, we report a rare case of GCA in the stomach. An 80-year-old man underwent gastroscopy due to epigastric pain and was diagnosed with gastric cancer. He received total gastrectomy and histology showed a mixture of a moderately-differentiated tubular adenocarcinoma, a mucinous adenocarcinoma, and a tumor composed of goblet-like mucinous cells with neuroendocrine differentiation. The tumor volume ratio was about 4:1:5, respectively, and a final diagnosis of GCA was made. The metastasis of the regional lymph node was occupied by only the component of goblet-like cells. GCA should be recognized as a rare histologic subtype of gastric cancer.

Human induced pluripotent stem cells are resistant to human cytomegalovirus infection primarily at the attachment level due to the reduced expression of cell-surface heparan sulfate.

Cytomegalovirus (CMV), a type of herpes virus, is the predominant cause of congenital anomalies due to intrauterine infections in humans. Adverse outcomes related to intrauterine infections with human cytomegalovirus (HCMV) vary widely, depending on factors such as fetal infection timing, infection route, and viral virulence. The precise mechanism underlying HCMV susceptibility remains unclear. In this study, we compared the susceptibility of neonatal human dermal fibroblast cells (NHDFCs) and human induced pluripotent stem cells (hiPSCs) derived from NHDFCs, which are genetically identical to HCMV, using immunostaining, microarray, in situ hybridization, quantitative PCR, and scanning electron microscopy. These cells were previously used to compare CMV susceptibility, but the underlying mechanisms were not fully elucidated. HCMV susceptibility of hiPSCs was significantly lower in the earliest phase. No shared gene ontologies were observed immediately post-infection between the two cell types using microarray analysis. Early-stage expression of HCMV antigens and the HCMV genome was minimal in immunostaining and in in situ hybridization in hiPSCs. This strongly suggests that HCMV does not readily bind to hiPSC surfaces. Scanning electron microscopy performed using the NanoSuit method confirmed the scarcity of HCMV particles on hiPSC surfaces. The zeta potential and charge mapping of the charged surface in NHDFCs and hiPSCs exhibited minimal differences when assessed using zeta potential analyzer and scanning ion conductance microscopy; however, the expression of heparan sulfate (HS) was significantly lower in hiPSCs compared with that in NHDFCs. Thus, HS expression could be a primary determinant of HCMV resistance in hiPSCs at the attachment level. IMPORTANCE: Numerous factors such as attachment, virus particle entry, transcription, and virus particle egress can affect viral susceptibility. Since 1984, pluripotent cells are known to be CMV resistant; however, the exact mechanism underlying this resistance remains elusive. Some researchers suggest inhibition in the initial phase of HCMV binding, while others have suggested the possibility of a sufficient amount of HCMV entering the cells to establish latency. This study demonstrates that HCMV particles rarely attach to the surfaces of hiPSCs. This is not due to limitations in the electrostatic interactions between the surface of hiPSCs and HCMV particles, but due to HS expression. Therefore, HS expression should be recognized as a key factor in determining the susceptibility of HCMV in congenital infection in vitro and in vivo. In the future, drugs targeting HS may become crucial for the treatment of congenital CMV infections. Thus, further research in this area is warranted.

Activated platinum in gallium-based room-temperature liquid metals for enhanced reduction reactions.

Room-temperature gallium-based liquid metals (LMs) have recently attracted significant attention worldwide for application in catalysis because of their unique combination of fluidic and catalytic properties. Platinum loading in LMs is expected to enhance the catalytic performance of various reaction systems. However, Pt-loaded methods for Ga-based LMs have not yet been sufficiently developed to improve the catalytic performance and Pt utilization efficiency. In this study, a novel method for the fabrication of Pt-incorporated LMs using Pt sputter deposition (Pt(dep)-LMs) was developed. The Pt(dep)-LMs contained well-dispersed Pt flakes with diameters of 0.89 ± 0.6 µm. The catalytic activity of the Pt(dep)-LM with a Pt loading of ∼0.7 wt% was investigated using model reactions such as methylene blue (MB) reduction and hydrogen production in an acidic aqueous solution. The Pt(dep)-LMs showed a higher MB reduction rate (three times) and hydrogen production (three times) than the LM loaded with conventional Pt black (∼0.7 wt%). In contrast to the Pt(dep)-LMs, solid-based Ga with a Pt loading of ∼0.7 wt% did not catalyze the reactions. These results demonstrate that Pt activation occurred in the Pt(dep)-LMs fabricated by Pt sputtering, and that the fluidic properties of the LMs enhanced the catalytic reduction reactions. Thus, these findings highlight the superior performance of the Pt deposition method and the advantages of using Pt-LM-based catalysts.

A Review on Gold Nanoclusters for Cancer Phototherapy.

Cancer phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has been extensively studied in recent years because of its noninvasive properties, high efficiency, improved selectivity, and reduced side effects. Gold nanoclusters (AuNCs) have the advantages of high biocompatibility, high biosafety, excellent photoresponse, and high tumor penetration ability. This review analyzes the use of AuNCs in tumor phototherapy in recent years from three aspects, namely, AuNCs in PDT, AuNCs in PTT, and AuNCs in combination therapy, and presents the high potential of AuNCs in cancer phototherapy. This review aims to provide readers with the unique advantages, diversified application approaches, and bright application prospects of AuNCs in phototherapy and to provide insights into strategies for applying AuNCs to tumor phototherapy.

An autopsy case of disseminated carcinomatosis of the bone marrow from esophageal adenocarcinoma.

Key Clinical Message: Disseminated carcinomatosis of the bone marrow is rare. We present such a case, which is useful for raising awareness about the importance of early diagnosis and treatment of carcinomas complicated by disseminated carcinomatosis of the bone marrow. Abstract: This is the first autopsy report of disseminated carcinomatosis of the bone marrow (DCBM) in esophageal adenocarcinoma. Advanced poorly differentiated adenocarcinoma with signet ring cell carcinoma arising in Barrett's esophagus caused disseminated intravascular coagulation (DIC) with extensive bone marrow metastasis, resulting in death from cerebral hemorrhage. Although DCBM due to malignancy is rare with poor prognosis, it should be considered in malignancies associated with DIC, and prompt initiation of chemotherapy is the only way to improve the patient's prognosis.

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

Dry liquid metals stabilized by silica particles: Synthesis and application in photothermoelectric power generation.

HYPOTHESIS: Gallium-based room-temperature liquid metals (LMs) have unique physicochemical properties; however, their high surface tension, low flowability, and high corrosiveness to other materials limit their advanced processing (including precise shaping) and application. Consequently, LM-rich free-flowing powders, named "dry LMs" that offer the inherent advantages of dry powders, should play a critical role in expanding the application scope of LMs. EXPERIMENTS: A general method of preparing silica-nanoparticle-stabilized LMs in the form of LM-rich powders (>95 wt% LM) is developed. FINDINGS: Dry LMs can be simply prepared by mixing LMs with silica nanoparticles in a planetary centrifugal mixer in the absence of solvents. As a sustainable dry-process route alternative to wet-process routes, this ecofriendly and simple method of dry LM fabrication has several advantages, e.g., high throughput, scalability, and low toxicity owing to the lack of organic dispersion agents and milling media. Moreover, the unique photothermal properties of dry LMs are used for photothermal electric power generation. Thus, dry LMs not only pave the way for the use of LMs in powder form but also provide a new opportunity for expanding their application scope in energy conversion systems.

Accurate deep learning model using semi-supervised learning and Noisy Student for cervical cancer screening in low magnification images.

Deep learning technology has been used in the medical field to produce devices for clinical practice. Deep learning methods in cytology offer the potential to enhance cancer screening while also providing quantitative, objective, and highly reproducible testing. However, constructing high-accuracy deep learning models necessitates a significant amount of manually labeled data, which takes time. To address this issue, we used the Noisy Student Training technique to create a binary classification deep learning model for cervical cytology screening, which reduces the quantity of labeled data necessary. We used 140 whole-slide images from liquid-based cytology specimens, 50 of which were low-grade squamous intraepithelial lesions, 50 were high-grade squamous intraepithelial lesions, and 40 were negative samples. We extracted 56,996 images from the slides and then used them to train and test the model. We trained the EfficientNet using 2,600 manually labeled images to generate additional pseudo labels for the unlabeled data and then self-trained it within a student-teacher framework. Based on the presence or absence of abnormal cells, the created model was used to classify the images as normal or abnormal. The Grad-CAM approach was used to visualize the image components that contributed to the classification. The model achieved an area under the curve of 0.908, accuracy of 0.873, and F1-score of 0.833 with our test data. We also explored the optimal confidence threshold score and optimal augmentation approaches for low-magnification images. Our model efficiently classified normal and abnormal images at low magnification with high reliability, making it a promising screening tool for cervical cytology.

Microwave-Assisted Incorporation of AgNP into Chitosan-Alginate Hydrogels for Antimicrobial Applications.

Herein, improving the antibacterial activity of a hydrogel system of sodium alginate (SA) and basic chitosan (CS) using sodium hydrogen carbonate by adding AgNPs was investigated. SA-coated AgNPs produced by ascorbic acid or microwave heating were evaluated for their antimicrobial activity. Unlike ascorbic acid, the microwave-assisted method produced uniform and stable SA-AgNPs with an optimal reaction time of 8 min. Transmission electron microscopy (TEM) confirmed the formation of SA-AgNPs with an average particle size of 9 ± 2 nm. Moreover, UV-vis spectroscopy confirmed the optimal conditions for SA-AgNP synthesis (0.5% SA, 50 mM AgNO3, and pH 9 at 80 °C). Fourier transform infrared (FTIR) spectroscopy confirmed that the -COO- group of SA electrostatically interacted with either the Ag+ or -NH3+ of CS. Adding glucono-δ-lactone (GDL) to the mixture of SA-AgNPs/CS resulted in a low pH (below the pKa of CS). An SA-AgNPs/CS gel was formed successfully and retained its shape. This hydrogel exhibited 25 ± 2 mm and 21 ± 1 mm inhibition zones against E. coli and B. subtilis and showed low cytotoxicity. Additionally, the SA-AgNP/CS gel showed higher mechanical strength than SA/CS gels, possibly due to the higher crosslink density. In this work, a novel antibacterial hydrogel system was synthesized via 8 min of microwave heating.

Phosphatidylcholine in bile-derived small extracellular vesicles as a novel biomarker of cholangiocarcinoma.

BACKGROUND: Owing to the lack of definite diagnostic modalities, it is challenging to distinguish malignant cases of cholangiocarcinoma (CCA), which often causes biliary tract obstruction, from benign ones. Here, we investigated a novel lipid biomarker of CCA in bile-derived small extracellular vesicles (sEVs) and developed a simple detection method for clinical application. METHODS: Bile samples from seven patients with malignant diseases (hilar CCA = 4, distal CCA = 3) and eight patients with benign diseases (gallstones = 6, primary sclerosing cholangitis = 1, autoimmune pancreatitis = 1) were collected through a nasal biliary drainage tube. sEVs were isolated via serial ultracentrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting (with CD9, CD63, CD81, and TSG101). Comprehensive lipidomic analysis was performed using liquid chromatography-tandem mass spectrometry. Using a measurement kit, we further confirmed whether lipid concentrations could be used as a potential CCA marker. RESULTS: Lipidomic analysis of bile sEVs in the two groups identified 209 significantly increased lipid species in the malignant group. When focusing on lipid class, phosphatidylcholine (PC) level was 4.98-fold higher in the malignant group than in the benign group (P = 0.037). The receiver operating characteristic (ROC) curve showed a sensitivity of 71.4%, a specificity of 100%, and an area under the curve (AUC) of 0.857 (95% confidence interval [CI]:0.643-1.000). Using a PC assay kit, the ROC curve showed a cutoff value of 16.1 µg/mL, a sensitivity of 71.4%, a specificity of 100%, and an AUC of 0.839 (95% CI: 0.620-1.000). CONCLUSION: PC level in sEVs from human bile is a potential diagnostic marker for CCA and can be assessed by a commercially available assay kit.

Primary Cilia Are Frequently Present in Small Cell Lung Carcinomas but Not in Non-Small Cell Lung Carcinomas or Lung Carcinoids.

Most human malignant neoplasms show loss of primary cilia (PC). However, PC are known to be retained and involved in tumorigenesis in some types of neoplasms. The PC status in lung carcinomas remains largely uninvestigated. In this study, we comprehensively assessed the PC status in lung carcinomas. A total of 492 lung carcinomas, consisting of adenocarcinomas (ACs) (n = 319), squamous cell carcinomas (SCCs) (n = 152), and small cell lung carcinomas (SCLCs) (n = 21), were examined by immunohistochemical analysis using an antibody against ARL13B, a marker of PC. The PC-positive rate was markedly higher in SCLCs (81.0%) than in ACs (1.6%) and SCCs (7.9%). We subsequently performed analyses to characterize the PC-positive lung carcinomas further. PC-positive lung carcinomas were more numerous and had longer PC than normal cells. The presence of PC in these cells was not associated with the phase of the cell cycle. We also found that the PC were retained even in metastases from PC-positive lung carcinomas. Furthermore, the hedgehog signaling pathway was activated in PC-positive lung carcinomas. Because ARL13B immunohistochemistry of lung carcinoids (n = 10) also showed a statistically significantly lower rate (10.0%) of PC positivity than SCLCs, we searched for a gene(s) that might be upregulated in PC-positive SCLCs compared with lung carcinoids, but not in PC-negative carcinomas. This search, and further cell culture experiments, identified HYLS1 as a gene possessing the ability to regulate ciliogenesis in PC-positive lung carcinomas. In conclusion, our findings indicate that PC are frequently present in SCLCs but not in non-SCLCs (ACs and SCCs) or lung carcinoids, and their PC exhibit various specific pathobiological characteristics. This suggests an important link between lung carcinogenesis and PC.

Association between human papillomavirus particle production and the severity of recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) has a wide range of severity. We investigate the relationship between human papillomavirus (HPV) particle production and severity of RRP. From September 2005 to June 2021, 68 RRP samples (from 29 patients) were included. HPV type was determined. HPV viral load, physical status, and demographic and clinical characteristics were assessed. Immunohistochemistry (IHC) was performed for p16, Ki-67, L1, and E4. We used NanoSuit-CLEM (correlative light and electron microscopy) and transmission electron microscopy (TEM) to examine the samples. The total number of surgeries in HPV-positive and HPV-negative cases were 3.78 (n = 55/68, range: 1-16) and 1.30 (n = 13/68, range: 1-3), respectively (p = 0.02). IHC showed that L1 and E4 were correlated and expressed on the tumour surface. NanoSuit-CLEM and TEM revealed HPV particles in L1-positive nuclei. L1 IHC-positive cases had a shorter surgical interval (p < 0.01) and more frequent surgeries (p = 0.04). P16 IHC, viral load, and physical status were not associated with disease severity. This study visualised HPV particle production in RRP for the first time. Persistent HPV particle infection was associated with severity. We suggest L1 IHC for evaluating RRP severity in addition to the Derkay score.

Identifying Active Progeny Virus Particles in Formalin-Fixed, Paraffin-Embedded Sections Using Correlative Light and Scanning Electron Microscopy.

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3'-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3'-diaminobenzidine-stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3'-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases.