Multilayered proteomics reveals that JAM-A promotes breast cancer progression via regulation of amino acid transporter LAT1.

Recent studies have shown that transmembrane-type tight junction proteins are upregulated in various cancers compared with their levels in normal tissues and are involved in cancer progression, suggesting that they are potential therapeutic targets. Here, we demonstrated the expression profile and a novel role of junctional adhesion molecule-A (JAM-A) in breast cancer. Immunohistochemistry of surgical specimens showed that JAM-A was highly expressed from carcinoma in situ lesions, as in other adenocarcinomas, with higher expression in invasive carcinomas. High expression of JAM-A contributed to malignant aspects such as lymph node metastasis and lymphatic involvement positivity. In breast cancer cells, JAM-A expression status affects malignant potentials including proliferation and migration. Multilayered proteomics revealed that JAM-A interacts with the amino acid transporter LAT1 in breast cancer cells. JAM-A regulates the expression of LAT1 and interacts with it on the whole cell membrane, leading to enhanced amino acid uptake to promote tumor growth. Double high expression of JAM-A and LAT1 predicts poor prognosis in patients with breast cancer. Of note, an antibody against an extracellular domain of JAM-A suppressed the proliferation of breast cancer cells. Our findings indicate the possibility of JAM-A-targeted therapy ideally combined with LAT1-targeted therapy as a new therapeutic strategy against breast cancer.

Molecular and ultrastructural morphological analyses of highly metamorphosed Aspergillus fumigatus on human formalin-fixed paraffin-embedded tissue.

Invasive fungal infections including invasive pulmonary aspergillosis (IPA) generally have a poor prognosis, because the fungi spread throughout various organs. Therefore, it is important to accurately identify the fungal species for treatment. In this article, we present the results of pathological and molecular morphological analyses that were performed to elucidate the cause of respiratory failure in a patient who died despite suspicion of IPA and treatment with micafungin (MCFG). Pathological analysis revealed the existence of cystic and linear fungi in lung tissue. The fungi were identified as Aspergillus fumigatus (A. fumigatus) by partial sequencing of genomic DNA. Correlative light microscopy and electron microscopy (CLEM) analysis confirmed that fungi observed with light microscopy can also be observed with scanning electron microscopy (SEM) using formalin-fixed paraffin-embedded tissue sections. SEM revealed an atypical ultrastructure of the fungi including inhomogeneous widths, rough surfaces, and numerous cyst-like structures of various sizes. The fungi showed several morphological changes of cultured A. fumigatus treated with MCFG that were previously reported. Our results indicate that integrated analysis of ultrastructural observation by SEM and DNA sequencing may be an effective tool for analyzing fungi that are difficult to identify by conventional pathological analysis.

A novel approach to diagnosing crystal-storing histiocytosis: utility of scanning electron microscopy for formalin-fixed paraffin-embedded tissue specimens.

Crystal-storing histiocytosis (CSH) is a rare disorder that shows infiltration of histiocytes with an aberrant cytoplasmic accumulation of crystalline structures and is often accompanied by lymphoproliferative-plasma cell disorders (LP-PCD) as background diseases. The diagnosis of CSH requires identification of crystalline structures that accumulate in the infiltrating histiocytes, which may be challenging by optical microscopy alone. In this case report, we describe an atypical course of systemic CSH with multifocal fibrosclerosis of an unknown background disease that was diagnosed by ultrastructural observation, including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), in pathological autopsy. In addition, crystalline structures were successfully identified by scanning electron microscopic observations using formalin-fixed and paraffin-embedded (FFPE) tissue from biopsy specimens taken before death. Since CSH was identified by SEM in a tiny biopsy specimen, observation of histiocytic infiltrative lesions by SEM using FFPE tissue may lead to early detection of and initiation of treatment for CSH.

Invasive pulmonary aspergillosis with candidiasis: usefulness of molecular and ultrastructural morphological analysis on FFPE tissue for invasive fungal infections.

Invasive pulmonary aspergillosis (IPA) is one of the most frequent forms of invasive fungal infections (IFI); however, it is often difficult to identify the pathogenic fungal species and to select appropriate treatments for patients with IFI including IPA. Here, we describe the detailed pathophysiology of an autopsy case of severe respiratory failure due to IPA with candidiasis. The patient developed severe respiratory failure after influenza infection and died, and the autopsy revealed a mixed disease of IPA with candidiasis. In this study, in addition to the routine pathological examination, we further examined formalin-fixed paraffin-embedded (FFPE) tissues by scanning electron microscopy (SEM) and partial genomic DNA sequencing. Although optical microscopy alone was insufficient to identify the pathogenic organisms, SEM clearly depicted the characteristic morphology of Aspergillus sp. and Candida sp. as closely overlapping in a nested fashion, providing evidence of mixed infection of both fungal species in a focal site. The technique using FFPE tissue in combination with ultrastructural observation by SEM, elemental analysis by SEM-EDX, and DNA sequencing is promising for analyzing the pathophysiology of IFI.

Aberrant expression of claudin-6 contributes to malignant potentials and drug resistance of cervical adenocarcinoma.

Recent studies have revealed that aberrant expression of tight junction (TJ) proteins is a hallmark of various solid tumors and it is recognized as a useful therapeutic target. Claudin-6 (CLDN6), a member of the family of TJ transmembrane proteins, is an ideal therapeutic target because it is not expressed in human adult normal tissues. In this study, we found that CLDN6 is highly expressed in uterine cervical adenocarcinoma (ADC) and that high CLDN6 expression was correlated with lymph node metastasis and lymphovascular infiltration and was an independent prognostic factor. Shotgun proteome analysis revealed that cell-cell adhesion-related proteins and drug metabolism-associated proteins (aldo-keto reductase [AKR] family proteins) were significantly increased in CLDN6-overexpressing cells. Furthermore, overexpression of CLDN6 enhanced cell-cell adhesion properties and attenuated sensitivity to anticancer drugs including doxorubicin, daunorubicin, and cisplatin. Taken together, the results indicate that aberrant expression of CLDN6 enhances malignant potentials and drug resistance of cervical ADC, possibly due to increased cell-cell adhesion properties and drug metabolism. Our findings provide an insight into a new therapeutic strategy, a CLDN6-targeting therapy, against cervical ADC.

Disparities in co-payments for influenza vaccine among the elderly, during the COVID-19 pandemic in Japan.

INTRODUCTION: Seasonal influenza vaccination for the elderly is highly recommended during the COVID-19 pandemic. In Japan, the amount of subsidy for influenza differs among municipalities. Thus, we investigated the amount of and variation in subsidy for influenza vaccination for the elderly in 2020. METHODS: This was an ecological study of 1,922 municipalities in Japan. The amount of subsidy for influenza vaccines for the elderly in each municipality was surveyed through websites or via telephone. Geographic and financial data for municipalities and prefectures were obtained from the open data. The amount of co-payment for the influenza vaccine and the geographical and financial status of each municipality were compared, according to the aging rate. Univariate logistic regression analysis was performed to explore factors related to the free influenza vaccine. RESULTS: Municipalities with higher aging rates tended to have higher median co-payments for vaccines in 2020. (0 yen vs 1000 yen, p < 0.001) In addition, they tended to have worse financial conditions and lower per capita incomes. A similar trend was observed in the analysis by prefecture, i.e., a higher influenza mortality rate in prefectures with a higher aging rate. Despite having lower incomes, municipalities and prefectures with higher aging populations had higher mortality rates from influenza and higher co-payments for influenza vaccination. CONCLUSIONS: In Japan, there is a disparity among elderly people; areas with an aging population have higher co-payments for influenza vaccines despite lower incomes, suggesting that the government needs to implement corrective measures to reduce this disparity.

Pathological classification of desmoplastic reaction is prognostic factor in cervical adenocarcinoma.

Desmoplastic reaction (DR) and inflammation are significant pathological manifestations of tumorigenesis in several cancers. However, the correlation between these stromal reactions and cervical adenocarcinoma has been poorly documented. This investigation elucidated whether DR is a prognostic indicator in early cervical adenocarcinoma patients. Fifty-nine patients with early stage cervical adenocarcinoma (stages I/II) were included in the study. DR was divided into three groups, mature, intermediate, and immature, based on the presence of myxoid stroma and hyalinized keloid-like collagen. Inflammatory cell responses were classified as mild, moderate, and severe. Those stromal reactions were separately evaluated in the invasion front stroma and intratumoral stroma. In both the intratumor and invasion front stroma, intermediate/immature DR was correlated with tumor size, T stage, N stage, lymphovascular invasion, and parametrial infiltration (p < 0.001 to p < 0.05). In addition, in the intratumoral stroma, intermediate/immature DR led to short relapse-free survival and overall survival (p < 0.001). In the invasion front stroma, inflammatory cell responses were associated with DR immaturity and FIGO stage (p < 0.01). These results suggest that the classification of DR maturity is a potential prognostic biomarker in early stage cervical adenocarcinoma patients. DR can be evaluated by routine H&E staining without immunohistochemistry, making it convenient and economical in clinical practice.

[AFM1 Content Survey in Dairy Products for Infants].

Infant formula in liquid for childcare can be stored at room temperature for a certain period of time, reducing the burden of childcare and preparing for disasters. Against this background, domestic manufacturing and sales began in March 2019. AFM1 is a metabolite of aflatoxin B1 (AFB1), a carcinogenic mycotoxin, and is contained in the milk of livestock fed a diet contaminated with AFB1. At present, standard values have not been set for infant formula in liquid as well as prepared infant formula in liquid, and infants consume a large amount of dairy products per body weight, so care must be taken in the intake.In this study, we investigated the actual condition of AFM1 content in dairy products with high intake of infants. As a result of the investigation, the AFM1 of the detected dairy products was 0.001 to 0.005 µg/kg, which was extremely small compared to the AFM1 in the dairy products reported so far. Since infant nutrition depends on dairy products, it is undeniable that they may consume more than adults, so continuous research is needed.

Aberrant expression of junctional adhesion molecule-A contributes to the malignancy of cervical adenocarcinoma by interaction with poliovirus receptor/CD155.

Recent studies have shown that aberrant expression of tight junction proteins (TJP) contributes to malignant potential of various cancers. In the present study, we investigated the expression of junctional adhesion molecule-A (JAM-A), one of the transmembrane TJP, in uterine cervical adenocarcinoma and the significance of its expression for malignancy. Immunohistochemistry on human surgical specimens showed that JAM-A was aberrantly expressed in neoplastic regions including adenocarcinoma in situ (AIS). Knockout of JAM-A significantly suppressed cell proliferation and colony-forming and migration abilities. We also showed that an antibody specific to an extracellular region of JAM-A reduced cell proliferation ability and that loss of JAM-A increased drug sensitivity of cervical adenocarcinoma cells. Based on a comprehensive proteome analysis, we found that poliovirus receptor (PVR/CD155) was regulated by JAM-A and formed a physical interaction with JAM-A. In human surgical specimens, PVR/CD155 expression was significantly correlated with some clinicopathological features and prognosis of cervical adenocarcinoma. Interestingly, most of the PVR/CD155-positive cases expressed a high level of JAM-A, and patients with the expression pattern of PVR/CD155 positive/JAM-A high had significantly shorter periods of relapse-free survival (P = .00964) and overall survival (P = .0204) than those for the other patients. Our observations suggest that aberrant expression of JAM-A promotes malignancy of uterine cervical adenocarcinoma by regulation of PVR/CD155, and JAM-A is therefore a potential therapeutic target for this malignancy.

Regulatory roles of claudin-1 in cell adhesion and microvilli formation.

Aberrant expression of tight junction proteins has recently been focused on in the cancer research field. We previously showed that claudin-1 is aberrantly expressed from an early stage of uterine cervical adenocarcinoma and contributes to malignant potentials. To elucidate the molecular mechanisms underlying tumor-promoting roles of claudin-1, we established and analyzed claudin-1 knockout cells. Knockout of claudin-1 suppressed conventional tight junctional functions, barrier and fence functions, and expression of cell adhesion-associated proteins including E-cadherin. Comparative proteome analysis revealed that expression of claudin-1 affected expression of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling. Interactome analysis of the identified proteins revealed that E-cadherin and focal adhesion kinase play central roles in the claudin-1-dependently affected protein network. Moreover, knockout of claudin-1 significantly suppressed microvilli formation and activity of Ezrin/Radixin/Moesin. Taken together, the results indicate that expression of claudin-1 affects not only conventional tight junction function but also expression and activity of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling, to contribute to malignant potentials and microvilli formation in cervical adenocarcinoma cells.

Morphological and biochemical changes of lymphatic vessels in the soleus muscle of mice after hindlimb unloading.

INTRODUCTION/AIMS: Lymphatic vessels are responsible for the removal of metabolic waste from body tissues. They also play a crucial role in skeletal muscle functioning thorough their high-energy metabolism. In this study we investigated whether disuse muscle atrophy induced by hindlimb unloading is associated with an alteration in the number of lymphatic vessels and differential expression of lymphangiogenic factors in the soleus muscle. METHODS: Male C57BL/6 mice were subjected to tail suspension (TS) for 2 or 4 weeks to induce soleus muscle atrophy. After TS, lymphatic and blood capillaries in the soleus muscle were visualized and counted by double staining with LYVE-1 and CD31. The protein and mRNA levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, and vascular endothelial growth factor receptor-3 were measured by Western blotting and real-time reverse transcript polymerase chain reaction, respectively. RESULTS: TS for 2 weeks resulted in a significant decrease in the number of blood capillaries compared with controls. However, there was no significant change in the number of lymphatic capillaries. By contrast, TS for 4 weeks resulted in a significant decrease in the number of lymphatic and blood capillaries. We observed a significant decrease in the mRNA levels of VEGF-C and VEGF-D in mice subjected to TS for 4 weeks. DISCUSSION: The decrease of intramuscular lymphatic vessels may a crucial role in the process of muscle atrophy.

Cessation of electrically-induced muscle contraction activates autophagy in cultured myotubes.

Exercise is known to improve skeletal muscle function. The mechanism involves muscle contraction-induced activation of the mTOR pathway, which plays a central role in protein synthesis. However, mTOR activation blocks autophagy, a recycling mechanism with a critical role in cellular maintenance/homeostasis. These two responses to muscle contraction look contradictory to the functional improvement of exercise. Herein, we investigate these paradoxical muscle responses in a series of active-inactive phases in a cultured myotube model receiving electrical stimulation to induce intermittent muscle contraction. Our model shows that (1) contractile activity induces mTOR activation and muscle hypertrophy but blocks autophagy, resulting in the accumulation of damaged proteins, while (2) cessation of muscle contraction rapidly activates autophagy, removing damaged protein, yet a prolonged inactive state results in muscle atrophy. Our findings provide new insights into muscle biology and suggest that not only muscle contraction, but also the subsequent cessation of contraction plays a substantial role for the improvement of skeletal muscle function.

Leucine supplementation after mechanical stimulation activates protein synthesis via L-type amino acid transporter 1 in vitro.

Branched-chain amino acid supplements consumed following exercise are widely used to increase muscle mass. Although both exercise (ie, mechanical stimulation) and branched-chain amino acid leucine supplementation have been reported to stimulate muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) signaling pathway independently, the mechanisms underlying their synergistic effects are largely unknown. Utilizing cultured differentiated C2C12 myotubes, we established a combination treatment model in which the cells were subjected to cyclic uniaxial mechanical stretching (4 h, 15%, 1 Hz) followed by stimulation with 2 mM leucine for 45 min. Phosphorylation of p70 S6 kinase (p70S6K), an mTOR-regulated marker of protein translation initiation, was significantly increased following mechanical stretching alone but returned to the baseline after 4 h. Leucine supplementation further increased p70S6K phosphorylation, with a greater increase observed in the stretched cells than in the non-stretched cells. Notably, the expression of L-type amino acid transporter 1 (LAT1), a stimulator of the mTOR pathway, was also increased by mechanical stretching, and siRNA-mediated knockdown partially attenuated leucine-induced p70S6K phosphorylation. These results suggest that mechanical stretching promotes LAT1 expression and, consequently, amino acid uptake, leading to enhanced leucine-induced activation of protein synthesis. LAT1 has been demonstrated to be a point of crosstalk between exercise- and nutrition-induced skeletal muscle growth.

Attenuation of Pulse Pressure Amplification in Patients with Complete Transposition of the Great Arteries After an Arterial Switch Operation in Children.

Although the arterial switch operation has become the standard procedure for infants with complete transposition of the great arteries, possible late adverse events after surgery have not been fully elucidated. One such problem may be the postoperative function of the aorta that is radically manipulated. The current study enrolled 12 patients aged 4-9 years who had undergone an arterial switch operation. The ascending and descending aortic pressure waveforms were recorded by a catheter-mounted pressure sensor. The pressure values were compared with those of 28 age-matched controls. The mean patient age was 6.5 ± 1.0 years, and the mean age at the time of surgery was 15.2 ± 8.7 days. The pulse pressure in the ascending aorta was greater in the patients than in the controls (37.7 ± 5.7 vs. 33.5 ± 5.3 mmHg, p = 0.042), while no difference was observed at the descending aorta between the two groups (39.5 ± 5.1 vs. 37.4 ± 5.4 mmHg, respectively, p = 0.27). The pulse pressure amplification, defined as the pulse pressure in the descending aorta minus that in the ascending aorta, was significantly lower in patients who had undergone the arterial switch operation than in control patients (1.8 ± 1.6 vs. 4.0 ± 2.3 mmHg, p = 0.0052). The augmented pulse pressure in the ascending aorta and attenuated pulse pressure amplification observed in children treated with arterial switch surgery for complete transposition of the great arteries may implicate the procedure as a cause of future cardiovascular disease.

Training at non-damaging intensities facilitates recovery from muscle atrophy.

INTRODUCTION: Resistance training promotes recovery from muscle atrophy, but optimum training programs have not been established. We aimed to determine the optimum training intensity for muscle atrophy. METHODS: Mice recovering from atrophied muscles after 2 weeks of tail suspension underwent repeated isometric training with varying joint torques 50 times per day. RESULTS: Muscle recovery assessed by maximal isometric contraction and myofiber cross-sectional areas (CSAs) were facilitated at 40% and 60% maximum contraction strength (MC), but at not at 10% and 90% MC. At 60% and 90% MC, damaged and contained smaller diameter fibers were observed. Activation of myogenic satellite cells and a marked increase in myonuclei were observed at 40%, 60%, and 90% MC. CONCLUSIONS: The increases in myofiber CSAs were likely caused by increased myonuclei formed through fusion of resistance-induced myofibers with myogenic satellite cells. These data indicate that resistance training without muscle damage facilitates efficient recovery from atrophy. Muscle Nerve 55: 243-253, 2017.

Neuroanatomic pathways associated with monoaminergic dysregulation after stroke.

OBJECTIVE: We examined the complex relationship between lesion location, symptoms of depression (affective and apathetic), and monoamine dysfunction after stroke. METHODS: Magnetic resonance imaging was performed on 48 post-stroke patients that had been assessed for affective and apathetic symptoms using the Hospital Anxiety and Depression Scale and the Apathy Scale, respectively. Noradrenalin (NA), dopamine (DA), their metabolites, and a metabolite of serotonin (5-HT) were measured using 24-h urine samples, and 5-HT and 3-methoxy-4-hydroxyphenylglycol were measured using blood samples. We developed a statistical parametric map that displayed the associations between lesion location and both positive and negative alterations of monoamines and their metabolites. RESULTS: Multivariate analysis indicated that basal ganglia lesions and 5-HT showed relationships with affective symptoms, whereas homovanillic acid was related to apathetic symptoms. Univariate analysis showed no such relationships. However, decreases in NA and DA and increases in NA and DA turnover were related to lesions in the brainstem, whereas increases in NA and DA as well as decreases in NA and DA turnover were related to cortical and/or striatum lesions. 5-HT turnover data showed a pattern opposite to that seen for NA and DA turnover. CONCLUSIONS: Monoaminergic neuronal pathways are controlled by both receptor-mediated feedback mechanisms and turnover; thus, depletion of monoamines is not the only cause of depression and apathy. Moreover, the monoamine neuronal network might be divided into two branches, catecholamine (NA and DA) and 5-HT, both of which are anatomically and functionally interconnected and could respectively influence apathetic and affective symptoms of depression.

Effects of popping on nutrient contents of amaranth seed.

Amaranth seeds can be popped by heating. The traditional method of popping in a skillet is simple, but it is difficult to control the heating time and temperature. To overcome these disadvantages, we developed a fluidized bed continuous processing system based on hot air heating for producing popped amaranth seeds in bulk. Using this system, we evaluated the effects of heat treatment at 260 °C for 15 s on the contents of B-group vitamins and essential and trace elements in amaranth seeds. The results showed that the treatment did not affect the content of B-group vitamins, and the recovery for essential and trace elements was 97-196%. This popping system is useful for processing amaranth seeds in terms of the product quality and nutrition.

[Selective coil embolization of a distal anterior choroidal artery aneurysm associated with moyamoya disease: a case report].

Intracranial hemorrhage in patients with moyamoya disease is often caused by rupture of the associated aneurysms. Of these aneurysms, distal anterior choroidal artery (AChoA) aneurysms are rare. In patients with moyamoya disease, the AChoA constitutes collateral vessels and the aneurysm requires careful treatment strategy. However, reported cases of distal AChoA aneurysms include various procedures including conservative therapy, direct surgery, and endovascular therapy. Herein, we report a case of coil embolization of a distal AChoA aneurysm associated with moyamoya disease and discuss the treatment strategy. A 39-year-old female presented with severe headache and subsequent deep coma. Computed tomography (CT) revealed thick intraventricular hemorrhage, and three-dimensional CT angiography revealed a right distal AChoA aneurysm. Bilateral ventricular drainage was performed and subsequent ventriculoperitoneal (VP)shunt was performed. The persisting distal AChoA aneurysm was coil embolized without any complication. Rebleeding did not occur during the 1-year follow-up period. Endovascular treatment is effective for distal AChoA aneurysms associated with moyamoya disease to preserve collateral circulation.