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Recent clinical studies have suggested that inhalation of incense smoke (IS) may result in impaired lung function and asthma. However, there is little experimental evidence to link IS with airway hyperresponsiveness (AHR) and bronchial epithelial barrier function. Using mouse and cell culture models, we evaluated the effects of IS exposure on AHR, expression of multiple epithelial tight junction (TJ)- and adherens junction-associated mRNAs and proteins in the lungs, and the barrier function of bronchial epithelial cells assessed by transepithelial electronic resistance (TEER). Exposure of BALB/c mice to IS increased AHR and inflammatory macrophage recruitment to BALF; reduced claudin-1, -2, -3, -7, -10b, -12, -15, and -18, occludin, zonula occludens-1 [ZO-1], and E-cadherin mRNA expression; and caused discontinuity of claudin-2 and ZO-1 protein immunostaining in lung tissue. IS extract dose-dependently decreased TEER and increased reactive oxygen species production in bronchial epithelial cell cultures. Treatment with N-acetyl-l-cysteine, but not glucocorticosteroids or long-acting β2-agonists, prevented the detrimental effects of IS. IS exposure can be problematic for respiratory health, as evidenced by AHR, increased recruitment of inflammatory macrophages and disruption of TJ proteins in the lung, and damage to epithelial barrier function. However, antioxidants may be useful for the treatment of IS-induced airway dysfunction. [ABSTRACT FROM AUTHOR]

Highlights: [•] We found that Gap43 and Ntn1 were up-regulated by IGF-1 in the process of hair cell protection. [•] The expression of Gap43 and Ntn1 was dependent on both the PI3K/Akt and MEK/ERK pathways. [•] The up-regulation of Gap43 and Ntn1 transcripts were transient. [Copyright &y& Elsevier]

As an initial step for using technology derived from induced pluripotent stem cells (iPSCs) in the field of inner ear therapeutics, we examined the potential of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc, which are employed in the generation of iPSCs, for dedifferentiating cochlear epithelial cells. Otospheres, which are sphere-forming cells derived from dissociated cochlear epithelial cells of neonatal mice, were used as a cell source. The four transcription factors were introduced into otospheres using retroviral vectors. Virally transduced otospheres formed embryonic stem cell-like colonies that expressed markers for pluripotent stem cells and were capable of differentiating into the three germ layers in vivo and in vitro. These findings illustrate that viral transduction of four transcription factors can lead to reprogramming of cochlear epithelial cells, which may contribute to future studies of dedifferentiation of cochlear epithelial cells in tissue and identification of key molecules for otic induction. [ABSTRACT FROM AUTHOR]

Abstract: Sensorineural hearing loss, which is mainly caused by cochlear hair cell damage, is an intractable disease, as cochlear hair cells and supporting cells are unable to proliferate in postnatal mammals. As a novel and potent treatment for sensorineural hearing loss, we have studied IGF-1 and found that it protects cochlear hair cells from the damage caused by noise and ischemic trauma. Through a clinical trial, we have also confirmed that IGF-1 is an effective treatment for idiopathic sudden sensorineural hearing loss. In the current study, we attempted to identify the downstream pathways of the IGF-1 signal and the mechanisms by which IGF-1 protects the neonatal mouse cochlear hair cells that have been damaged by neomycin. IGF-1 activated both the PI3K/Akt and MEK/ERK pathways to maintain the hair cell numbers in the injured cochlea. The PI3K/Akt pathway specifically protected the cochlear inner hair cells through the inhibition of apoptosis. In contrast, the MEK/ERK pathway induced the cell cycle promotion of Hensen's and Claudius' cells, the supporting cells that are located lateral to the outer hair cells of the cochlea. This cell cycle promotion of the supporting cells resulted in the maintenance of the outer hair cell numbers. These results indicate that IGF-1 is a growth factor that efficiently regulates different mechanisms through different downstream cascades, thereby protecting cochlear hair cells. [Copyright &y& Elsevier]

Abstract: Septins are a family of GTP binding proteins that are well conserved in eukaryotic species except plants. Septins contribute to the lateral compartmentalization of membranes, cortical rigidity, and the regulation of membrane trafficking by associating with membrane lipids, actin, and microtubules. The organ of Corti in the cochlea has pivotal roles in auditory perception and includes two kinds of highly polarized cells, hair and supporting cells, both of which are rich in actin and microtubules. To identify the roles of septins in the cochlea, we analyzed the localization of three septin proteins, septin 4 (SEPT4), septin 5 (SEPT5), and septin 7 (SEPT7) that are abundantly expressed in brain tissues, and also examined auditory functions of Sept4 and Sept5 null mice. SEPT4, SEPT5, and SEPT7 were expressed in inner and outer pillar cells and Deiters'' cells but the distribution patterns of each protein in Deiters'' cells were different. SEPT4 and SEPT7 were expressed in the phalangeal process where SEPT5 was not detected. In addition to these cells SEPT5 and SEPT7 were co-localized with presynaptic vesicles of efferent nerve terminals. Only SEPT7 was expressed in the cochlea at embryonic stages. Although expression patterns of septin proteins suggested their important roles in the function of the cochlea, both Sept4 and Sept5 null mice had similar auditory functions to their wild type littermates. Immunohistochemical analysis of Sept4 null mice showed that compensatory expression of SEPT5 in the phalangeal process of Deiters'' cells may have caused functional compensation of hearing ability in Sept4 null mice. [Copyright &y& Elsevier]

Abstract: This study explored the physiological roles of PGE-type receptor 4 (EP4) in auditory function. EP4-deficient mice exhibited slight hearing loss and a reduction of distortion-product otoacoustic emissions (DPOAEs) with loss of outer hair cells (OHCs) in cochleae. After exposure to intense noise, these mice showed significantly larger threshold shifts of auditory brain–stem responses (ABRs) and greater reductions of DPOAEs than wild-type mice. A significant increase of OHC loss was confirmed morphologically in the cochleae of EP4-deficient mice. Pharmacological inhibition of EP4 had a similar effect to genetic deletion, causing loss of both hearing and OHCs in C57BL/6 mice, indicating a critical role for EP4 signaling in the maintenance of auditory function. Pharmacological activation of EP4 significantly protected OHCs against noise trauma, and attenuated noise-induced hearing loss in C57BL/6 mice. These findings suggest that EP4 signaling is necessary for the maintenance of cochlear physiological function and for cochlear protection against noise-induced damage, in particular OHCs. EP4 might therefore be an effective target for cochlear disease therapeutics. [Copyright &y& Elsevier]

Abstract: Aims: Overproduction of nitric oxide (NO) from inducible NO synthase (iNOS) plays many pivotal roles in various inflammatory diseases. In this study, we examined the effects of 6 flavonoids on lipopolysaccharide (LPS)-induced NO generation in macrophage (Mφ), and addressed molecular mechanisms of cardamonin. Main methods: Suppressive effects on NO generation in vitro were assayed in LPS-stimulated macrophages (Mφ). In vivo anti-inflammatory activity was evaluated with LPS-challenged ICR mice. Mechanistic analyses were done by ELISA, Western blot, RT-PCR, etc. Key findings: Cardamonin, a chalcone, exhibited pronounced suppressive activity, while pinocembrin, a counterpart flavanone, was much less active. Administration of cardamonin (0.02–2mg/kg body weight) significantly decreased NOx concentrations in the sera from LPS-challenged ICR mice. This efficacy was superior to that of curcumin, a well-known anti-inflammatory agent present in turmeric. Cardamonin down-regulated iNOS mRNA expression and suppressed activation of STAT-1, but not nuclear factor kappaB, both of which are transcription factors for the iNOS gene in peritoneal Mφ of ICR mice. Interferon (IFN)-γ was identified as the major cytokine which mediates LPS-induced STAT-1 activation and resultant iNOS expression. Intriguingly, cardamonin suppressed the activation of not only STAT-1 but also STATs-2, 3 and 4. The involvement of the JAK/STATs pathway in NO generation was suggested because its specific inhibitor, AG490, decreased NO generation. Significance: Cardamonin was identified to be a unique phytochemical that targets the production of IFN- and thereby suppresses the STAT pathway for mitigating inflammation. [Copyright &y& Elsevier]

Imaging with fluorescent proteins has been revolutionary and has led to the new field of in vivo cell biology. Many new applications of this technology have been developed. Green fluorescent protein (GFP)-labeled or red fluorescent protein (RFP)-labeled HT-1080 human fibrosarcoma cells were used to determine clonality of metastasis by imaging of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. Dual-color fluorescent cancer cells expressing GFP in the nucleus and RFP in the cytoplasm were engineered. The dual-color cancer cells enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo, including mitosis and apoptosis. The nuclear and cytoplasmic behavior of dual-color cancer cells in real time in blood vessels was observed as they trafficked by various means or extravasated in an abdominal skin flap. Dual-color cancer cells were also visualized trafficking through lymphatic vessels where they were imaged via a skin flap. Seeding and arresting of single dual-color cancer cells in the lung, accumulation of cancer-cell emboli, cancer-cell viability, and metastatic colony formation were imaged in real time in an open-chest nude mouse model using assisted ventilation. Novel treatment was evaluated in these imageable models. UVC irradiation killed approximately 70% of the dual-color cancer cells in a nude mouse model. An RFP-expressing glioma was transplanted to the spinal cord of transgenic nude mice expressing nestin-driven green fluorescent protein (ND-GFP). In ND-GFP mice, GFP is expressed in nascent blood vessels and neural stem cells. ND-GFP cells staining positively for neuronal class III-β-tubulin or CD31 surrounded the tumor, suggesting that the tumor stimulated both neurogenesis and angiogenesis. The tumor caused paralysis and also metastasized to the brain. The Salmonella typhimurium A1-R tumor-targeting bacterial strain was administered in the orthotopic spinal cord glioma model. The treated animals had a significant increase in survival and decrease in paralysis. S. typhimurium A1-R was effective against primary bone tumor and lung metastasis expressing RFP in a nude mouse model. S. typhimurium A1-R was effective against both axillary lymph and popliteal lymph node metastases of human dual-color pancreatic cancer and fibrosarcoma cells, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. Imaging with fluorescent proteins will reveal mechanisms of cancer progression and provide visual targets for novel therapeutics. [ABSTRACT FROM AUTHOR]

[Display omitted] • Microglia and behavior were studied using an early postnatal asthma model. • Long- but not short-term asthma induced more dystrophic, inflammatory microglia. • Long-term asthma induced excitatory synapse surplus compared with short-term asthma. • Long-term asthma induced autism-like behavior compared with short-term asthma. • Long-term asthma increased corticoids but reduced corticoid receptors in microglia. Microglia play key roles in synaptic pruning, which primarily occurs from the postnatal period to adolescence. Synaptic pruning is essential for normal brain development and its impairment is implicated in neuropsychiatric developmental diseases such as autism spectrum disorders (ASD). Recent epidemiological surveys reported a strong link between ASD and atopic/allergic diseases. However, few studies have experimentally investigated the relationship between allergy and ASD-like manifestations, particularly in the early postnatal period, when allergic disorders occur frequently. Therefore, we aimed to characterize how allergic inflammation in the early postnatal period influences microglia and behavior using mouse models of short- and long-term airway allergy. Male mice were immunized by an intraperitoneal injection of aluminum hydroxide and ovalbumin (OVA) or phosphate-buffered saline (control) on postnatal days (P) 3, 7, and 11, followed by intranasal challenge with OVA or phosphate-buffered saline solution twice a week until P30 or P70. In the hippocampus, Iba-1-positive areas, the size of Iba-1-positive microglial cell bodies, and the ramification index of microglia by Sholl analysis were significantly smaller in the OVA group than in the control group on P30 and P70, although Iba-1-positive microglia numbers did not differ significantly between the two groups. In Iba-1-positive cells, postsynaptic density protein 95 (PSD95)-occupied areas and CD68-occupied areas were significantly decreased on P30 and P70, respectively, in the OVA group compared with the control group. Immunoblotting using hippocampal tissues demonstrated that amounts of PSD95, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor 2, and N-methyl-D-aspartate (NMDA) receptor 2B were significantly increased in the OVA group compared with the control group on P70, and a similar increasing trend for PSD95 was observed on P30. Neurogenesis was not significantly different between the two groups on P30 or P70 by doublecortin immunohistochemistry. The social preference index was significantly lower in the three chamber test and the number of buried marbles was significantly higher in the OVA group than in the control group on P70 but not on P30, whereas locomotion and anxiety were not different between the two groups. Compared with the control group, serum basal corticosterone levels were significantly elevated and hippocampal glucocorticoid receptor (GR) amounts and nuclear GR translocation in microglia, but not in neurons or astrocytes, were significantly decreased in the OVA group on P70 but not on P30. Gene set enrichment analysis of isolated microglia revealed that genes related to immune responses including Toll-like receptor signaling and chemokine signaling pathways, senescence, and glucocorticoid signaling were significantly upregulated in the OVA group compared with the control group on P30 and P70. These findings suggest that early postnatal allergic airway inflammation induces dystrophic microglia that exhibit defective synaptic pruning upon short- and long-term allergen exposure. Furthermore, long-term allergen exposure induced excitatory postsynaptic surplus and ASD-like behavior. Hypothalamo-pituitary-adrenal axis activation and the compensatory downregulation of microglial GR during long-term allergic airway inflammation may also facilitate these changes. [ABSTRACT FROM AUTHOR]

Objective: Rapid epithelialization is crucial to maintain tracheal patency and prevent potential graft failure in tracheal reconstruction after tracheal resection for cancer with tracheal infiltration or tracheal stenosis. Insulin-like growth factor 1 is a liver-secreted endocrine molecule that controls cell proliferation, differentiation, and apoptosis and has been reported to promote epithelialization in several organs. Here, we utilized mouse tracheal organ cultures to examine the effect of insulin-like growth factor 1 on tracheal epithelialization.Methods: The trachea was resected from thirteen-week-old female ICR mice, and cut into small plate-shaped tracheal sections. First, the expression of insulin-like growth factor 1 receptor was assessed by immunohistochemistry. Secondly, the tracheal sections were cultured for seven days in the culture medium, and the morphological change during the seven-day culture was assessed by immunohistochemistry, hematoxylin and eosin staining, and scanning electron microscopy. Moreover, the tracheal sections were cultured for 48 h with different concentration of insulin-like growth factor 1 (0, 0.1, 1 and 10 µg/mL) in the culture medium, and the extension length of the tracheal epithelium during culture was measured in order to assess the effect of topical IGF1 on tracheal epithelialization.Results: Immunohistochemistry showed that insulin-like growth factor 1 receptor was expressed in tracheal epithelium. Immunohistochemistry, hematoxylin and eosin staining, and scanning electron microscopy showed that the tracheal organ cultures were stable for at least seven days without apparent morphological damage. The effect of insulin-like growth factor 1 on tracheal epithelialization was examined in plate-shaped tracheal sections cultured in medium supplemented with or without insulin-like growth factor 1 for 48 h. We also found that the epithelial edge of plate-shaped tracheal sections extended further along the surface of the tracheal section in culture medium containing insulin-like growth factor 1 compared with that in culture medium without insulin-like growth factor 1.Conclusion: The current study using an in vitro mouse tracheal organ culture model demonstrated that topical insulin-like growth factor 1 treatment promoted the extension of tracheal epithelium, suggesting the potential utility of insulin-like growth factor 1 in aiding rapid tracheal epithelialization in patients requiring tracheal reconstruction using tissue-engineered tracheas. [ABSTRACT FROM AUTHOR]

Abstract: 3-Hydroxybutyric acid (3HB) is a ketone body and acts as an indicator of energy balance and a central regulator of energy homeostasis. We report the application of a sensitive fluorometric assay for the quantitative determination of 3HB. The assay is based on the oxidation of 3HB by 3HB dehydrogenase and on the diaphorase–resazurin amplifying system. This simple assay enables the measurement of changes in 3HB levels in the blood of normal mice by very small volume sample collection. Therefore, this assay will be useful for in vivo studies of small animals. [Copyright &y& Elsevier]

We have previously established a patient-derived orthotopic xenograft (PDOX) model of undifferentiated spindle cell sarcoma (USCS). Recombinant methioninase (rMETase) has previously demonstrated efficacy in PDOX mouse models of human cancers. In the present study, we determined if rMETase in combination with doxorubicin (DOX) can overcome first-line DOX resistance in a PDOX models of USCS. The USCS PDOX mouse models were randomized into the following groups when tumor volume reached 100 mm3: G1, control without treatment; G2, doxorubicin (DOX) (3 mg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks); G3, rMETase (100 units/mouse, i.p., daily, for 2 weeks); G4, DOX (3 mg/kg, i.p., weekly, for 2 weeks) combined with rMETase (100 units/mouse, i.p., daily, for 2 weeks). Tumor size and body weight were measured twice a week. On day 14 after initiation, the USCS PDOX tumor sizes were (G1): 360 ± 85 mm3; DOX (G2): 355 ± 111 mm3, p = .927; rMETase (G3): 182 ± 57 mm3, p = .0003; DOX + rMETase (G4): 134 ± 29 mm3, p = .00001. These results indicate that rMETase can overcome USCS resistance to DOX, which is first line therapy for this disease. The body weight of treated mice was not significantly different in any group. The present results demonstrate the power of the PDOX model to identify effective therapy for recalcitrant cancer and the potential of rMETase to overcome DOX resistance. [ABSTRACT FROM AUTHOR]

Undifferentiated soft tissue sarcoma (USTS) is a recalcitrant and heterogeneous subgroup of soft tissue sarcoma with high risk of metastasis and recurrence. Due to heterogeneity of USTS, there is no reliably effective first-line therapy. We have generated tumor-targeting Salmonella typhimurium A1-R ( S. typhimurium A1-R), which previously showed strong efficacy on single patient-derived orthotopic xenograft (PDOX) models of Ewing's sarcoma and follicular dendritic cell sarcoma. In the present study, tumor resected from 4 patients with a biopsy-proven USTS (2 undifferentiated pleomorphic sarcoma [UPS], 1 undifferentiated sarcoma not otherwise specified [NOS] and 1 undifferentiated spindle cell sarcoma [USS]) were grown orthotopically in the biceps femoris muscle of mice to establish PDOX models. One USS model and one UPS model were doxorubicin (DOX) resistant. One UPS and the NOS model were partially sensitive to DOX. DOX is first-line therapy for these diseases. S. typhimurium A1-R arrested tumor growth all 4 models. In addition to arresting tumor growth in each case, S. typhimurium A1-R was significantly more efficacious than DOX in each case, thereby surpassing first-line therapy. These results suggest that S. typhimurium A1-R can be a general therapeutic for USTS and possibly sarcoma in general. [ABSTRACT FROM AUTHOR]

Transcription factors are believed to play key roles in determining cell fate in inner ear development. Olig genes, which are basic helix–loop–helix transcription factors, have been reported to play important roles in the development of the central nervous system. However, members of this family have not previously been implicated in inner ear development, despite the similarity between otocyst and neural tube development. Olig1 begins to be expressed at the ventral domain of the otocyst at embryonic day (E) 9.5, and Olig1 expression in the epithelium of the developing inner ear persists to E15.5. Olig2 expression is localized to the cochleovestibular ganglia from E12.5 through E14.5. Olig3 has a diffuse expression pattern in the developing inner ear from E12.5 through the postnatal stage. Furthermore, at early stages of inner ear development, the Olig1 expression domain overlaps a region that is positive for Sox2 and Jagged1. This observation indicates that Olig1 may play an important role in the specification of the prosensory domain in the developing inner ear. As Olig genes are expressed in the mouse developing inner ear in a temporospatially distinct fashion, they may play substantial roles in the regulation of mammalian inner ear development. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the absorption and metabolism of 4-hydroxyderricin and xanthoangelol, we established an analytical method based on liquid chromatography-tandem mass spectrometry and measured these compounds in the plasma, urine, feces, liver, kidney, spleen, muscle and white adipose tissues of mice orally administered with Ashitaba extract (50–500mg/kg body weight). 4-Hydroxyderricin and xanthoangelol were quickly absorbed into the plasma, with time-to-maximum plasma concentrations of 2 and 0.5h for 4-hydroxyderricin and xanthoangelol, respectively. Although these compounds have similar structures, the total plasma concentration of 4-hydroxyderricin and its metabolites was approximately 4-fold greater than that of xanthoangelol and its metabolites at 24h. 4-Hydroxyderricin and xanthoangelol were mostly excreted in their aglycone forms and related metabolites (glucuronate and/or sulfate forms) in urine between 2 and 4h after oral administration of Ashitaba extract. On the other hand, these compounds were only excreted in their aglycone forms in feces. When tissue distribution of 4-hydroxyderricin and xanthoangelol was estimated 2h after administration of Ashitaba extract, both compounds were detected in all of the tissues assessed, mainly in their aglycone forms, except in the mesenteric adipose tissue. These results suggest that 4-hydroxyderricin and xanthoangelol are rapidly absorbed and distributed to various tissues. [Copyright &y& Elsevier]

Abstract: Neurogenesis persists in restricted regions of the adult vertebrate brain. However, the molecular mechanisms supporting adult neurogenesis are not fully understood. Here we demonstrated that C cell-specific deletion of RBP-J in the adult subventricular zones (SVZs) caused reduction in numbers of mature granule cells in the olfactory bulbs (OBs) with concomitant increase in Olig2 + oligodendroglial progenitors, although generation of immature neurons was enhanced in the SVZs. Adenovirus-mediated Cre introduction to the SVZs of RBP-J-floxed mice indicated that Olig2 + cells in the OBs can be generated from RBP-J-deficient SVZs, although no oligodendroglial cells in the OBs are derived from the normal SVZs. This preferential differentiation to oligodendroglial progenitor cells and reduction in differentiation of mature neurons were also confirmed by in vitro culture of RBP-J-deficient SVZ-derived neural progenitor cells, in addition to defects in the maintenance of adult neural stem cell population. The defects in maturation of RBP-J-deficient neurons could be partly rescued by knockdown of Olig2 in vivo. Our findings suggest that RBP-J might regulate neuronal maturation at least in part through transcriptional repression of Olig2. [Copyright &y& Elsevier]