An algorithm-based technique for counting mitochondria in cells using immunohistochemical staining of formalin-fixed and paraffin-embedded sections.

PURPOSE: Visualizing mitochondria in cancer cells from human pathological specimens may improve our understanding of cancer biology. However, using immunohistochemistry to evaluate mitochondria remains difficult because almost all cells contain mitochondria and the number of mitochondria per cell may have important effects on mitochondrial function. Herein, we established an objective system (Mito-score) for evaluating mitochondria using machine-based processing of hue, saturation, and value color spaces. METHODS: The Mito-score was defined as the number of COX4 (mitochondrial inner membrane) immunohistochemistry-positive pixels divided by the number of nuclei per cell. The system was validated using four lung cancer cell lines, normal tissues, and lung cancer tissues (199 cases). RESULTS: The Mito-score correlated with MitoTracker, a fluorescent dye used to selectively label and visualize mitochondria within cells under a microscope (R2 = 0.68) and with the number of mitochondria counted using electron microscopy (R2 = 0.79). Histologically, the Mito-score of small cell carcinoma (57.25) was significantly lower than that of adenocarcinoma (147.5, p < 0.0001), squamous cell carcinoma (120.6, p = 0.0004), and large cell neuroendocrine carcinoma (111.8, p = 0.002). CONCLUSION: The Mito-score method enables the analysis of the mitochondrial status of human formalin-fixed paraffin-embedded specimens and may provide insights into the metabolic status of cancer.

Integrative Systematics and Biogeography of the Hydrozoans (Leptothecata: Eirenidae) Eirene menoni Kramp, 1953 and Eirene lacteoides Kubota and Horita, 1992 from Japan and China with Comments on Pacific Ocean Distributions.

The hydrozoan family Eirenidae is known scientifically for its morphological plasticity and challenges in species identification. We used an integrative taxonomic approach based on morphological, molecular and life history evidence to systematically assess field-collected medusae of Eirene menoni Kramp 1953 and captive raised polyps of both E. menoni and E. lacteoides Kubota and Horita 1992. Following morphological review, we updated the genus description to include the presence of rudimentary bulbs (warts) on the ring canal in at least eight of the 24 valid Eirene species. We propose the potential for the mature E. menoni hydrotheca to develop into a gonotheca. However, this proposal will require additional study for verification. We provide validated distribution records from the Indo-Pacific Ocean for E. menoni,and updated collection records for E. lacteoides from the Yellow and East China Seas, and public aquaria-cultured specimens from Japan and Hawaii, using cytochrome c oxidase I (COI) sequences that we generated and compared with those from GenBank. The COI gene reliably separated four species, each forming a monophyletic clade with strong bootstrap support and low mean intraspecific molecular divergences (≤ 1%) within clades. However, some of the deeper nodes of the tree remained poorly resolved, and our analysis failed to demonstrate monophyly among eirenid genera Eirene and Tima. Our integrative taxonomic approach is essential in confirming species identity within the family Eirenidae and genus Eirene,and we have also identified a likely range expansion of E. lacteoides to Hawaii.

CDC7 inhibition induces replication stress-mediated aneuploid cells with an inflammatory phenotype sensitizing tumors to immune checkpoint blockade.

Serine/threonine kinase, cell division cycle 7 (CDC7) is critical for initiating DNA replication. TAK-931 is a specific CDC7 inhibitor, which is a next-generation replication stress (RS) inducer. This study preclinically investigates TAK-931 antitumor efficacy and immunity regulation. TAK-931 induce RS, generating senescence-like aneuploid cells, which highly expressed inflammatory cytokines and chemokines (senescence-associated secretory phenotype, SASP). In vivo multilayer-omics analyses in gene expression panel, immune panel, immunohistochemistry, RNA sequencing, and single-cell RNA sequencing reveal that the RS-mediated aneuploid cells generated by TAK-931 intensively activate inflammatory-related and senescence-associated pathways, resulting in accumulation of tumor-infiltrating immune cells and potent antitumor immunity and efficacy. Finally, the combination of TAK-931 and immune checkpoint inhibitors profoundly enhance antiproliferative activities. These findings suggest that TAK-931 has therapeutic antitumor properties and improved clinical benefits in combination with conventional immunotherapy.

Analysis of micrognathia with obstructive sleep apnea syndrome improved by a combination of Le Fort I with horseshoe osteotomies, mandibular distraction osteogenesis, and genioplasty.

The relationship between microgenia and obstructive sleep apnea syndrome is well known. 27-year-old woman. She underwent a combination of Le Fort I with horseshoe osteotomies and mandibular distraction osteogenesis and genioplasty. She was satisfied with the aesthetics of her face, with an AHI of 7.8/h.

Bone marrow-derived vasculogenesis leads to scarless regeneration in deep wounds with periosteal defects.

Deep skin wounds with periosteal defects, frequently caused by traffic accidents or radical dissection, are refractory. Transplant surgery is frequently performed, but patients are subjected to stress for long operation periods, the sacrifice of donor regions, or several complications, such as flap necrosis or intractable ulcers. Even if the defects are covered, a scar composed of fibrous tissue remains in the body, which can cause itching, dysesthesia, or repeated ulcers because of the lack of distribution of peripheral nerves or hair follicles. Thus, treatments with the aim of regenerating lost tissue for deep wounds with periosteal defects are needed. Here, we show that the use of gelatin sponges (GS), which have been used as haemostatic materials in clinical practice, allowed the regeneration of heterogeneous tissues, including periosteum, skin, and skin appendages, when used as scaffolds in deep wounds with periosteal defects in rats. Bone marrow transplantation in rats revealed the mechanism by which the microenvironment provided by GS enabled bone marrow-derived cells (BMDCs) to form a vascular niche, followed by regeneration of the periosteum, skin, or skin appendages such as hair follicles by local cells. Our findings demonstrated that vascular niche formation provided by BMDCs is crucial for heterogeneous tissue regeneration.

Use of bronchoalveolar lavage in diagnosing angioimmunoblastic T-cell lymphoma: A case report.

Angioimmunoblastic T-cell lymphoma (AITL) is a type of peripheral T-cell tumour that belongs to the group of non-Hodgkin's lymphomas. Pulmonary lesions can be found in 7%-10% of AITL cases. Imaging findings of the lungs varied; however, immunoblastic infiltration in the lungs is rare. Our patient was a 73-year-old man who received repeated chemotherapy for AITL. Fourth-line therapy using romidepsin controlled the illness, but the patient was hospitalized for dyspnoea and an infiltrative shadow. We performed bronchoalveolar lavage (BAL), and the culture was positive for Haemophilus influenzae. The patient was initially discharged with antibiotic therapy, but hospitalized again. Antibiotics were ineffective and the patient required mechanical ventilation. BAL was performed again, after which fluid cytology revealed immunoblast-like atypical cells. Therefore, the patient was diagnosed with pulmonary infiltration due to AITL. Steroid therapy proved ineffective, and the patient died. BAL was used to effectively diagnose pulmonary AITL infiltration.

Comparison of the wound-healing efficacy of gelatin sponge dressings and that of artificial dermis using atelocollagen in a rat cranial periosteal defect model.

In oral surgery, tissue loss may occur in some cases, resulting in bone exposure and subsequent wound infection and possible scar formation during secondary healing. In this study, Terudermis® Artificial Dermis (AD-T), a dermal defect graft made from processed bovine dermis collagen and gelatin sponge (GS) were used as dressings on 100-mm2 wounds with exposed bone on the heads of rats. For the control group, the wound was left exposed. The wound-healing efficacy of the treatment was compared macroscopically and histologically among the three groups at 1, 2, and 4 weeks after surgery. Complete wound healing was achieved faster in the AD-T group than in the GS group, and osteoblasts appeared on the bone surface, indicating accelerated bone remodeling. Furthermore, in the AD-T group, there was an increased production of newly formed blood vessels, fibroblasts and osteoblasts positive for anti-cortactin antibodies, which are believed to contribute to wound healing. Our findings suggest that AD-T is better than GS as a wound dressing material.

First-line osimertinib in elderly patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer: a retrospective multicenter study (HOT2002).

Osimertinib is a standard of care therapy for previously untreated epidermal growth factor receptor mutation-positive non-small cell lung cancer. However, limited data exist regarding the efficacy and safety of osimertinib as a first-line therapy for elderly patients aged 75 years or older. To assess the potential clinical benefits of osimertinib in this population, this retrospective multi-institutional observational study included 132 patients with non-small cell lung cancer (age ≥ 75 years), who received osimertinib as first-line treatment. The proportion of patients with 1-year progression-free survival was 65.8% (95% confidence interval 57.1-73.5). The median progression-free survival was 19.4 (95% confidence interval 15.9-23.9) months. The median overall survival was not reached (95% confidence interval 24.6-not reached). The frequency of pneumonitis was 17.4%, with a grade 3 or higher rate of 9.1%. More than two-thirds of treatment discontinuations due to pneumonitis occurred within 3 months of starting osimertinib, and the prognosis of patients with pneumonitis was unsatisfactory. Osimertinib is one of the effective first-line therapeutic options for patients aged 75 years or older; however, special caution should be exercised due to the potential development of pneumonitis.

Successful rechallenge with cetuximab after progression with nivolumab for recurrent cervical lymph node metastasis from carcinoma of the tongue.

We can infer that the immunostimulatory effect of nivolumab and reactivation of cetuximab enhance the antitumor effect of the therapy.

Maxillofacial Injuries in Cyclists: A Biomechanical Approach for the Analysis of Mechanisms of Mandible Fractures.

PURPOSE: The investigators characterized the occurrence of maxillofacial injuries in cyclists and biomechanically analyzed the mechanisms of mandible fractures. METHODS: We retrospectively analyzed injury data and performed biomechanical analyses with finite element models. Hospital records from 2011 through 2019 were reviewed to identify patients who had sustained oral and maxillofacial injuries while riding a bicycle. Patients with maxillofacial fractures were compared to those without. Logistic regression analysis was performed to identify which variables were independently associated with the occurrence of maxillofacial fractures. To reconstruct the injury scenario (one in which a person falls from a bicycle and contacts the road surface with their face), computer simulations using The Total Human Model for Safety model were performed. RESULTS: The hospital records of 94 patients (62 men, 32 women; 26.1 ± 17.3 years of age) who sustained oral and maxillofacial injuries while riding a bicycle were reviewed. Twenty patients (21.3%) sustained maxillofacial fractures; mandible fractures were most common (16 patients). Patients with maxillofacial fractures were significantly older and had higher severity injuries; however, logistic regression analysis showed that only age was an independent predictor of the occurrence of maxillofacial fracture (odds ratio, 1.03; P = .025). In simulations, higher von Mises stresses were found in the mandible when the cyclist fell with the neck extended and the body horizontal, and consequently, the center of mandibular body strikes the road surface. Contact forces were approximately 8 kN. High tensile stresses occurred laterally and high compressive stresses occurred medially in the mandibular ramus, which indicated that the mandibular ramus deformed in the transverse plane. CONCLUSION: Biomechanical analyses show that mandible fractures can occur when a cyclist falls from a bicycle and their lower face strikes the road's surface.

Similarities and differences in metabolites of tongue cancer cells among two- and three-dimensional cultures and xenografts.

Metabolic programming of cancer cells is an essential step in transformation and tumor growth. We established two-dimensional (2D) monolayer and three-dimensional (3D) cultures, the latter called a "tissueoid cell culture system", using four types of tongue cancer cell lines. We also undertook a comprehensive metabolome analysis of three groups that included xenografts created by transplanting the cell lines into nude mice. In addition, we undertook a functional analysis of the mitochondria, which plays a key role in cancer metabolism. Principal component analysis revealed the plots of the four cell lines to be much narrower in 2D culture than in 3D culture and xenograft groups. Moreover, compared to xenografts, the 2D culture had significantly lower levels of most metabolites. These results suggest that the unique characteristics of each cell disappeared in 2D culture, and a type of metabolism unique to monolayer culture took over. Conversely, ATP production, biomass synthesis, and maintenance of redox balance were shown in 3D culture using sufficient nutrients, which closely resembled the metabolic activity in the xenografts. However, there were several differences between the metabolic activity in the 3D culture and xenografts. In vivo, the cancer tissue had blood flow with stromal cells present around the cancer cells. In the xenografts, we detected metabolized and degraded products in the liver and other organs of the host mice. Furthermore, the 3D system did not show impairment of mitochondrial function in the cancer cells, suggesting that cancer cells produce energy simultaneously through mitochondria, as well as aerobic glycolysis.

Sensitive Method for the Identification of Potential Sensitizing Impurities in Reaction Mixtures by Fluorescent Nitrobenzoxadiazole-Labeled Glutathione.

Allergic contact dermatitis is a critical issue in the development of new chemicals. Minor impurities with strong skin-sensitizing properties can be generated as byproducts. However, it is very difficult to identify these skin sensitizers in product mixtures. In this study, fluorescent nitrobenzoxadiazole-labeled glutathione (NBD-GSH) was synthesized to identify small amounts of skin sensitizers in reaction mixtures. Twelve known skin sensitizers and three nonsensitizers were reacted with NBD-GSH. Adducts formed only with the skin sensitizers, which allowed for their detection by a fluorescence detector. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that NBD-GSH reacted with the skin sensitizers via its thiol and amino groups. An adduct of NBD-GSH with the strong skin sensitizer 1-chloro-2,4-dinitrobenzene was detected with a limit of detection of 6 × 10-8 mol/L by high-performance liquid chromatography with fluorescence detection. When a reaction mixture from primary alcohol oxidation was incubated with NBD-GSH, a NBD-GSH adduct formed with skin-sensitizing aldehyde impurities and could be specifically detected by LC-MS with fluorescence detection. This method will be useful for detection and identification of small amounts of skin sensitizers in raw materials, intermediates, reaction mixtures, and end products in the chemical industry.

Pulmonary capillary hemangiomatosis-predominant vasculopathy in a patient with rheumatoid arthritis-associated interstitial lung disease: An autopsy report.

Pulmonary capillary hemangiomatosis (PCH) is a rare cause of pulmonary hypertension (PH) associated with poor prognosis. Clinically, it is characterized by severe hypoxemia, centrilobular ground-glass opacities on computed tomography, and pulmonary congestion triggered by pulmonary vasodilating therapy. In some cases, PCH has been reported to develop with other disorders including connective tissue disease; however, to date, no reports have described PCH in a patient with rheumatoid arthritis. We report a case of a 59-year-old male PCH patient with rheumatoid arthritis and associated pulmonary fibrosis. He was initially diagnosed with severe group 3 PH and received sildenafil, which generated a favorable hemodynamic response. However, 5 years later, his pulmonary hemodynamics deteriorated, and he died at the age of 67. An autopsy was performed, and thickening of alveolar septa and capillary proliferation, pathological features of PCH, were extensively observed in both lungs. We discuss when PCH developed, how sildenafil improved his hemodynamics, and how PCH could be clinically detected by noninvasive evaluations.

Application of "Tissueoid Cell Culture System" Using a Silicate Fiber Scaffold for Cancer Research.

BACKGROUND: We developed a 3-dimensional (3D) culture system using a high-purity silica fiber scaffold of unwoven sheets called CellbedTM. METHODS: We used adherent colon and esophagogastric junction adenocarcinoma cells, tongue squamous cell carcinoma (SqCC) cells, and nonadherent gastric cancer cells. These cells were subjected to staining with various substances and observed by electron microscopy. To evaluate the effects of extracellular matrix in carcinoma tissues, SqCC cells were cultured in Cellbed coated with collagens I, III, and IV. RESULTS: Especially well-differentiated carcinoma cells cultured in this 3D system showed their own unique characteristics: luminal formation in adenocarcinoma cells and cell stratification and keratinization in SqCC cells. Scanning electron microscopy revealed the proliferation of cancer cells with cytoplasm entwined in Cellbed. Intercellular desmosomes in squamous epithelia were detected by transmission electron microscopy of vertical cross sections. SqCC cells cultured in Cellbed coated with collagen IV showed enhanced invasive and proliferative abilities. CONCLUSION: Because the morphology of cancer cells cultured in this 3D culture system is similar to that in living organisms, we called the system a "tissueoid cell culture system." Coating with collagen IV enables the modification of cell-matrix interactions as well as recapitulation of the in vivo microenvironment.

Expressions of ezrin, ERK, STAT3, and AKT in tongue cancer and association with tumor characteristics and patient survival.

BACKGROUND: Ezrin, ERK, STAT3, and AKT are proteins that are overexpressed in various types of cancer, although their expressions in tongue cancer has received less focus. This study aimed to address associations between the expression levels of these proteins and with characteristics of the tumor and patient survival. METHODS: We performed immunohistochemical staining of ezrin, ERK, STAT3, and AKT in tumors from patients with tongue carcinoma in situ (CIS, n = 17) and tongue squamous cell carcinoma (SCC, n = 46). Statistical differences between the SCC versus the CIS cohorts were estimated by calculations of bivariate odds ratios of low versus high expression of the proteins. Fisher's exact tests were used to appraise interassociations between the proteins, as well as expression levels versus patient and tumor characteristics. Survival based on Kaplan-Meier statistics in combination log-rank tests were used to address potential effects of the patient and tumor characteristics versus 5-year survival rate. RESULTS: The relative high: low expression of all four proteins in the two cohorts differed, and particularly ERK was markedly overexpressed in the SCC versus the CIS cohort (odds ratio = 45.3, p < .01). The relative high: low expression each protein versus patient and tumor characteristics; showed associations between AKT expression and T stage (p = .002) plus node metastases (p = .12), and between ERK expression and drinking (p = .01) and smoking history (p = .01). There was no significant difference observed between ERK and the three other molecules, nor any significant difference between the degree of expression of each protein and the 5-year disease-specific survival rate. CONCLUSION: Ezrin, ERK, STAT3, and AKT appear to be involved in the progress from carcinoma in situ in the tongue into squamous cell carcinoma. ERK in particular is overexpressed, suggesting that ERK may be a novel therapeutic target for preventing tongue cancer.

ERK phosphorylation functions in invadopodia formation in tongue cancer cells in a novel silicate fibre-based 3D cell culture system.

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase (ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet CellbedTM with a structure resembling the loose connective tissue morphology in a novel 3D culture system. We confirmed that the 3D system using CellbedTM accurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3D-cultured tongue cancer cell lines than in 2D cultures. Typically, under conventional 2D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells. However, in the 3D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3D culture systems using Cellbed™ are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Comparison of the wound healing efficacy of polyglycolic acid sheets with fibrin glue and gelatin sponge dressings in a rat cranial periosteal defect model.

Oral surgical procedures occasionally require removal of the periosteum due to lesions, and these raw bone surfaces are prone not only to infection but also to scar formation during secondary healing. The objective of this study was to identify successful methods for reconstruction using periosteal defect dressings. We created 1-cm2 defects in the skin and cranial periosteum of 10-week-old male Wistar rats under isoflurane anesthesia. The animals were assigned to three defect treatment groups: (1) polyglycolic acid sheets with fibrin glue dressing (PGA-FG), (2) Spongel® gelatin sponge dressing (GS), and (3) open wound (control). Postoperative wound healing was histologically evaluated at 2, 4, and 6 weeks. The moist conditions maintained by the GS and PGA-FG treatments protected the bone surface from the destructive effects of drying and infection. Complete wound healing was observed in the GS group but not for all animals in the PGA-FG and control groups. Histologically, osteoblast proliferation on bone surfaces and complete epithelialization with adnexa were observed in the GS group at 6 weeks after surgery. In contrast, PGA sheets that had not been absorbed inhibited osteoblast proliferation and delayed wound healing in the PGA-FG group. Wound surface dressings maintain a moist environment that promotes wound healing, but PGA materials may not be suitable for cases involving exposed periosteum or bone surfaces due to the observed scar formation and foreign-body reaction.

Continuous taurocholic acid exposure promotes esophageal squamous cell carcinoma progression due to reduced cell loss resulting from enhanced vascular development.

BACKGROUND: Refluxogenic effects of smoking and alcohol abuse may be related to the risk of esophageal squamous cell carcinoma (ESCC). The present study attempts to clarify the effects of continuous taurocholic acid (TCA) exposure, which is neither mutagenic nor genotoxic, on ESCC progression. METHODS: A squamous carcinoma cell line (ESCC-DR) was established from a tumor induced in a rat model of gastroduodenal reflux. ESCC-DR cells were incubated with 2 mM TCA for ≥2 months. The effects of continuous TCA exposure were evaluated in vitro on cell morphology, growth, and invasion and in vivo on xenograft tumor growth in nude mice. Moreover, the mean level of secreted transforming growth factor (TGF)-ß1 and vascular endothelial growth factor (VEGF) proteins in cell culture supernatants and mRNA synthesis of TGF-ß1 and VEGF-A of ESCC cells were measured. The angiogenic potential was further examined by a migration assay using human umbilical vein endothelial cells (HUVECs). RESULTS: Continuous TCA exposure induced marked formation of filopodia in vitro. Expression levels of angiogenic factors were significantly higher in the cells treated with TCA than in control cells. Tumor xenografts derived from cells pre-exposed to TCA were larger and more vascularized than those derived from control cells. In addition, TCA exposure increased HUVEC migration. CONCLUSION: Continuous TCA exposure enhanced ESCC progression due to reduced cell loss in vivo. Cell loss was inhibited by TCA-induced vascular endothelial cell migration, which was mediated by TGF-ß1 and VEGF-A released from ESCC cells.