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Control of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis. We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry) ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs. The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis. • PP1 inhibitors show angiogenic effects in zebrafish embryos and human ECs. • MLCK inhibitors show anti- angiogenic effects in zebrafish embryos and ECs. • PP1 and MLCK inhibitors affect EC elongation, but not cell viability or migration. • Elongated-shaped ECs show co-localization of pMLC2 and F-actin. • Knockdown of the MYL9 gene encoding MLC2 inhibits EC migration and morphological activity and suppresses angiogenesis. [ABSTRACT FROM AUTHOR]

Eosinophilic granulomatosis with polyangiitis (EGPA) is systemic vasculitis caused by eosinophilia affecting small to medium-sized blood vessels, which damages the organs. Antineutrophil cytoplasmic antibody-associated vasculitis EGPA treatment guidelines added anti-interleukin-5 antibody mepolizumab to the standard treatment protocol for active-non-severe EGPA based on the MIRRA study. Nevertheless, the role of mepolizumab in treating patients with active severe EGPA has not been established. We treated a patient with EGPA complicated with small intestine perforation using steroid pulse intravenous, high-dose glucocorticoids, intravenous high-dose immunoglobulin therapy, and mepolizumab without immunosuppression agents; the patient went into remission, suggesting that mepolizumab is an effective therapeutic agent that could lead to remission in severe EGPA. [ABSTRACT FROM AUTHOR]

Summary: Osteocytes act as mechanosensors to control local bone volume. However, their roles in the homeostasis of remote organs are largely unknown. We show that ablation of osteocytes in mice (osteocyte-less [OL] mice) leads to severe lymphopenia, due to lack of lymphoid-supporting stroma in both the bone marrow and thymus, and complete loss of white adipose tissues. These effects were reversed when osteocytes were replenished within the bone. In contrast, neither in vivo supply of T cell progenitors and humoral factors via shared circulation with a normal parabiotic partner nor ablation of specific hypothalamic nuclei rescued thymic atrophy and fat loss in OL mice. Furthermore, ablation of the hypothalamus in OL mice led to hepatic steatosis, which was rescued by parabiosis with normal mice. Our results define a role for osteocytes as critical regulators of lymphopoiesis and fat metabolism and suggest that bone acts as a central regulator of multiple organs. [Copyright &y& Elsevier]

Abstract: CXCL12 (stromal cell-derived factor-1, SDF-1), produced by stromal and endothelial cells including cells of the bone marrow, binds to its receptor CXCR4 and this axis regulates hematopoietic cell trafficking. Recently, osteoclast precursor cells were found to express CXCR4 and a potential role for the CXCL12–CXCR4 axis during osteoclast precursor cell recruitment/retention and development was proposed as a regulator of bone resorption. We examined the role of canonical Wnt signaling in regulating the expression of CXCL12 in bone marrow stromal cells. In mouse stromal ST2 cells, CXCL12 mRNA was expressed, while its expression was reduced in Wnt3a over-expressing ST2 (Wnt3a-ST2) cells or by treatment with lithium chloride (LiCl). Wnt3a decreased CXCL12 levels in culture supernatants from mouse bone marrow stromal cells. The culture supernatant from Wnt3a-ST2 cells also reduced migratory activity of bone marrow-derived cells in a Transwell migration assay. Silencing of glycogen synthase kinase-3β decreased CXCL12 expression, suggesting that the canonical Wnt signaling pathway regulates CXCL12 expression. In a transfection assay, LiCl down-regulated the activity of a reporter gene, a 1.8kb fragment of the 5′-flanking region of the CXCL12 gene. These results show that canonical Wnt signaling regulates CXCL12 gene expression at the transcriptional level, and this is the first study linking chemokine expression to canonical Wnt signaling. [Copyright &y& Elsevier]

Wilson disease is an autosomal recessive disorder with copper metabolism. In Japan, the standard treatment is the administration of copper chelating agents, such as D-penicillamine and trientine. In this study, the authors used zinc acetate to treat Japanese patients with Wilson disease and investigated its efficacy. The 37 patients that comprise this study were found to have Wilson disease using clinical and biochemical tests and were administrated zinc acetate for 48 weeks. The authors followed the clinical symptoms and laboratory findings of the patients by assessing their complete blood counts, biochemical findings, as well as the results of urinalysis and special laboratory tests for copper and zinc metabolism. We also examined side effects of the treatment. Zinc acetate did not aggravate the hepatic or neurological symptoms of any of the patients. Blood biochemical analysis also did not reveal elevation of alanine aminotransferase, aspartate aminotransferase, and γ-glutamyltranspeptidase levels. Zinc treatment did not aggravate the patients’ clinical signs and/or laboratory findings. However, it did improve some clinical symptoms of the Wilson disease patients. Although this agent had some side effects, none of them were severe. The authors measured spot urinary copper excretion, which gave an indication of the efficacy of treatment and of the sufficient dosage of zinc. We recommend maintaining a spot urinary copper excretion less than 0.075-μg/mg creatinine. The authors conclude that zinc acetate is an effective and safe treatment for Japanese patients with Wilson disease. [ABSTRACT FROM AUTHOR]

A 67-year-old man with stage IV B lung adenocarcinoma was treated with pembrolizumab. The patient was admitted to the hospital because of influenza on the day of the second cycle of pembrolizumab treatment. He was diagnosed with pneumonia and was treated with antiviral drugs and steroids. However, the patient eventually died. In this case, treatment with immune checkpoint inhibitors might have affected the immune response caused by influenza virus infection, that might have caused lung injury, which is an immune-related adverse event (irAE). Hence, it is important that, caution should be taken to prevent transmission of viral infection, and Therefore, it is important to prevent viral infections, but caution should also be paid to the possibility that infections may cause irAEs in patients with lung cancer. [ABSTRACT FROM AUTHOR]

A 69-year-old woman visited our outpatient clinic due to abdominal pain and anorexia. She received initial diagnostic workup, and was admitted to the hospital as an inpatient because of ileus. CT showed tumor with a diameter of 3cm in upper left abdomen, liver metastasis, peritoneal fluid localized in the pelvic cavity, and peritoneal node. Upper and lower gastrointestinal endoscopy showed no abnormalities, and percutaneous liver biopsy confirmed adenocarcinoma. PET showed a focal abnormal uptake in proximal jejunum, but no abnormalities in pancreas and biliary tract. We diagnosed the patient with small bowel cancer. It was considered unresectable, and FOLFOX chemotherapy was initiated. However, during FOLFOX chemotherapy after 1st course, CT showed that exacerbation of primary lesion and liver mass, and significant increase in ascites. She subsequently died of pneumonia day 32 after the start of chemotherapy. Autopsy findings revealed primary small bowel adenocarcinoma with proximal jejunum. Histological response was Grade1a. The incidence of small bowel adenocarcinoma (SBA) is very low, accounting for 0.1%-0.3% of all gastrointestinal malignant neoplasms. Many cases have been reported to be within 50 cm of Treitz ligament or Bauhin valve. For diagnosis of SBA, some reports suggest small bowel series, gastrointestinal endoscope, CT, and PET are useful. However, in many advanced cases it is difficult to apply curative therapy at the time of discovery. There is no standard treatment especially for unresectable small bowel cancer, and the prognosis is poor. In this case SBA was confirmed by metastatic lesion biopsy and exclusion diagnosis. Because the disease was already advanced at the time of diagnosis and furthermore time was necessary for diagnostic workup, it caused deterioration of the general condition. As a result, adequate chemotherapy could not be performed. There is a need for further case studies to establish diagnosis and treatment of SBA. [ABSTRACT FROM AUTHOR]

Distinct oxygenases and their oxylipin products have been shown to participate in thermogenesis by mediating physiological adaptations required to sustain body temperature. Since the role of the lipoxygenase (LOX) family in cold adaptation remains elusive, we aimed to investigate whether, and how, LOX activity is required for cold adaptation and to identify LOX-derived lipid mediators that could serve as putative cold mimetics with therapeutic potential to combat diabetes. By utilizing mass-spectrometry-based lipidomics in mice and humans, we demonstrated that cold and β3-adrenergic stimulation could promote the biosynthesis and release of 12-LOX metabolites from brown adipose tissue (BAT). Moreover, 12-LOX ablation in mouse brown adipocytes impaired glucose uptake and metabolism, resulting in blunted adaptation to the cold in vivo. The cold-induced 12-LOX product 12-HEPE was found to be a batokine that improves glucose metabolism by promoting glucose uptake into adipocytes and skeletal muscle through activation of an insulin-like intracellular signaling pathway. • Cold exposure increases 12-LOX activity in brown adipose tissue • 12-LOX activity in BAT contributes to cold adaptation • 12-HEPE is a cold-induced and BAT-secreted oxylipin • 12-HEPE promotes glucose uptake in vivo and in vitro Leiria et al. uncover a cold-induced response involving the activation of the enzyme 12-lipoxygenase in brown adipose tissue, which subsequently produces an omega-3 oxylipin 12-HEPE to promote glucose uptake into tissue via an insulin-like intracellular signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: On Earth, it is common to employ laboratory animals such as the nematode Caenorhabditis elegans to help understand human health concerns. Similar studies in Earth orbit should help understand and address the concerns associated with spaceflight. The “International Caenorhabditis elegans Experiment FIRST” (ICE FIRST), was carried out onboard the Dutch Taxiflight in April of 2004 by an international collaboration of laboratories in France, Canada, Japan and the United States. With the exception of a slight movement defect upon return to Earth, the result of altered muscle development, no significant abnormalities were detected in spaceflown C. elegans. Work from Japan revealed apoptosis proceeds normally and work from Canada revealed no significant increase in the rate of mutation. These results suggest that C. elegans can be used to study non-lethal responses to spaceflight and can possibly be developed as a biological sensor. To further our understanding of C. elegans response to spaceflight, we examined the gene transcription response to the 10 days in space using a near full genome microarray analysis. The transcriptional response is consistent with the observed normal developmental timing, apoptosis, DNA repair, and altered muscle development. The genes identified as altered in response to spaceflight are enriched for genes known to be regulated, in C. elegans, in response to altered environmental conditions (Insulin and TGF-β regulated). These results demonstrate C. elegans can be used to study the effects of altered gravity and suggest that C. elegans responds to spaceflight by altering the expression of at least some of the same metabolic genes that are altered in response to differing terrestrial environments. [Copyright &y& Elsevier]

Highlights • ASEM of primary culture neurons and their networks in aqueous liquid. • Imaging of fine structures, 50 nm buttons and spines, of synapses in solution. • Line of mitochondria imaged on neurite backbone including microtubule. • ASEM visualized fatty liver tissues of ob/ob type 2 diabetes model mouse. • Osmium tetroxide staining revealed lipid droplets pressing the liver sinusoids. Abstract Neural networking, including axon targeting and synapse formation, is the basis of various brain functions, including memory and learning. Diabetes-mellitus affects peripheral nerves and is known to cause fatty liver disease. Electron microscopy (EM) provides the resolution required to observe changes in fine subcellular structures caused by such physiological and pathological processes, but samples are observed in vacuum. Environmental capsule EM can directly observe cells in a more natural aqueous environment, but the size-limited capsules restrict cell culturability. The recently developed atmospheric scanning electron microscope (ASEM) has an open, 35 mm sample dish, allowing the culture of primary cells, including neurons, on the electron-transparent film window fabricated in its base. The system's inverted scanning electron microscope observes aldehyde-fixed cells or tissues from below through the silicon nitride film; the optical microscope located above allows direct correlation of fluorescence labeling. To observe fixed biological samples, damage due to low dose electron radiation is minimized in three ways. First, knock on damage that pushes out atoms is decreased by the low accelerating voltage of 10–30 kV. Second, increased radical generation due to the decreased acceleration voltage is countered by the addition of a radical scavenger, glucose or ascorbic acid, to the sample solution. Third, the large volume (max. 2 ml) of aqueous buffer surrounding the sample has a high specific heat capacity, minimizing the temperature increase caused by irradiation. Using ASEM, we have developed protocols for heavy metal staining in solution to selectively visualize intracellular structures. Various EM staining methods served as a starting point. Uranyl acetate preferably stains proteins and nucleic acid, and prior tannic acid treatment enhances membranes. Osmium tetroxide is suggested to enhance lipids, especially oil droplets. Imaging primary-culture neurons stained with platinum blue or uranyl acetate revealed growth cones, synapses, and 50–500 nm spines, together with neurite backbones and their associated structures. Correlative microscopy with immuno-fluorescence labeling suggested that these were mainly microtubule associated objects; some showed signs of a fission process and were, thus, possibly mitochondria. Liver tissue excised from the ob/ob type 2 diabetes model mouse, was stained with osmium tetroxide and observed using ASEM. Swollen bright balls occupied a large area of the cytoplasm and could be distinguished from vacuoles, suggesting that they are oil droplets. In some of the images, oil-like droplets were pressing surrounding structures, including sinusoids, significant for blood circulation in the liver. Based on these studies, ASEM combined with metal staining methods promises to allow the study of various mesoscopic-scale phenomena of cells and tissues immersed in natural aqueous environment in the near future. The quick nature of ASEM could facilitate not only the precise imaging for neuroscience but also the diagnosis of fatty liver disease and related diseases. [ABSTRACT FROM AUTHOR]

This study aimed to identify trends in frequency, serotype, and antimicrobial susceptibility of Streptococcus pneumoniae and Haemophilus influenzae isolated from middle ear fluid specimens of children aged≤15 years (mean, 2 years), before and after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) and the H. influenzae type b vaccine, at a pediatric facility in Japan. Sixty-six S. pneumoniae and 88 H. influenzae strains were isolated from 820 middle ear fluid samples. Serotyping and antimicrobial susceptibility testing were performed. The study time-frame was divided into period 1 (2007–2010) and period 2 (2011–2014), according to the availability of vaccine public funding. The S. pneumoniae detection rate decreased from 9.6% in period 1–6.1% in period 2 (p = 0.042). PCV7 serotypes decreased from 56.8% to 9.1% (p = 0.0002). No significant change was observed for the 13-valent pneumococcal conjugate vaccine (PCV13) serotypes: 72.7% in period 1 and 59.1% in period 2. Penicillin-resistant strains (penicillin G-MIC ≥2 μg/mL) decreased from 25% to 4.5% (p = 0.038). Detection rates for H. influenzae did not change significantly: 10.3% in period 1 and 11.3% in period 2. Serotypes were mostly non-typeable: 97.9% in period 1 and 90.2% in period 2, and only one serotype b strain was isolated in each period. The frequency of ampicillin-resistant strains (MIC ≥4 μg/mL) did not change. These results show a preventative effect of PCV7 on otitis media due to S. pneumoniae . PCV7 was replaced with PCV13 in 2013 in Japan; therefore, a further decrease in pneumococcal otitis media is anticipated in the future. [ABSTRACT FROM AUTHOR]

Background: The Great East Japan Earthquake and subsequent tsunami devastated the northeastern part of Japan. Low back pain is thought to increase after a natural disaster and is related to various factors. The aim of this study was to examine the influencing factors of "Living environment" and "Subjective economic hardship" on new-onset of low back pain in the chronic phase for the survivors of the earthquake evaluated by a self-report questionnaire.Methods: A panel study was conducted with the Great East Japan Earthquake survivors at 2 and 3 years after the disaster. New-onset of low back pain was defined as low back pain absent at the 1st period (2 years after the earthquake) and present at the 2nd period (3 years after the earthquake). Living environment was divided into 4 categories (1. Living in the same house as before the earthquake, 2. Living in a prefabricated house, 3. Living in a new house, 4. Others: Living in an apartment, house of relatives or acquaintance). Subjective economic hardship was obtained using the following self-report question: "How do you feel about the current economic situation of your household?" The response alternatives were "Normal", "A little bit hard", "Hard", and "Very hard". A univariate and multivariate logistic regression models were used.Results: 1357 survivors consented to join this study. There was no significant association between new-onset of low back pain and living environment. There was significant association between new-onset of low back pain and "A little hard" (OR = 1.6, 95% CI = 1.07-2.40), "Hard" (OR = 2.2, 95% CI = 1.56-3.74), and "Very hard" (OR = 3.19, 95% CI = 1.84-5.53) in subjective economic hardship.Conclusions: Subjective economic hardship was significantly associated with new-onset of low back pain in the chronic phase for survivors of the Great East Japan Earthquake. [ABSTRACT FROM AUTHOR]

Background: The Great East Japan Earthquake and devastating Tsunami caused irreparable damage on the northeastern coast of Japan. This study aimed to examine the influencing factors of "Living environment" and "Working status" on low back pain for the survivors of the earthquake evaluated by a self-report questionnaire.Methods: Between 2011 and 2013, survivors replied to the self-report questionnaire, and 986 people consented to join this study. The living environment was divided into 3 categories (1. Living in the same house as before the earthquake, 2. Living in a safe shelter or temporary small house, 3. Living in a house of relatives or apartment house) and working status was divided into 5 categories (1. Unemployed before the earthquake, 2. Unemployed after the earthquake, 3. Decrease in income, 4. Different occupation after the earthquake, 5. The same occupation as before the earthquake). Age, gender, living areas, past history of arthritis, arthropathy, osteoporosis, sleep disturbance, psychological distress, and economic status were considered as confounding factors. Generalized estimating regression models with logit link function were used because outcome variables are repeatedly measured and binomial. We evaluated the correlation between the presence/severity of low back pain over time and housing status/working status at 1 year after the earthquake.Results: There were no significant differences between age, gender, living areas, working status, or living environment before or after the earthquake. There was no significant difference in the risk of having low back pain in living environment or gender. There was significant difference in the risk of having low back pain in those with "Decrease in income" (OR = 1.93, 95% CI = 1.23-3.03) and "The same occupation as before the earthquake" (OR = 1.67, 95% CI = 1.1-2.52).Conclusions: Though living environment has little effect, "Decrease in income" and "The same occupation as before the earthquake" have strong influences on low back pain. [ABSTRACT FROM AUTHOR]

Abstract: Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. [Copyright &y& Elsevier]