Endogenous pH 6.0 β-Galactosidase Activity Is Linked to Neuronal Differentiation in the Olfactory Epithelium

The histochemical detection of β-galactosidase enzymatic activity at pH 6.0 (β-gal-pH6) is a widely used biomarker of cellular senescence in aging tissues. This histochemical assay also detects the presence of programmed cell senescence during specific time windows in degenerating structures of vertebrate embryos. However, it has recently been shown that this enzymatic activity is also enhanced in subpopulations of differentiating neurons in the developing central nervous system in vertebrates. The present study addressed the histochemical detection of β-gal-pH6 enzymatic activity in the developing postnatal olfactory epithelium in the mouse. This activity was detected in the intermediate layer of the olfactory epithelium. As development progressed, the band of β-gal-pH6 labeling in this layer increased in width. Immunohistochemistry and lectin histochemistry showed the β-gal-pH6 staining to be strongly correlated with the immunolabeling of the olfactory marker protein (OMP) that identifies mature olfactory sensory neurons. The cell somata of a subpopulation of differentiated olfactory neurons that were recognized with the Dolichos biflorus agglutinin (DBA) were always located inside this band of β-gal-pH6 staining. However, the β-gal-pH6 histochemical signal was always absent from the apical region where the cytokeratin-8 positive supporting cells were located. Furthermore, no β-gal-pH6 staining was found in the basal region of the olfactory epithelium where PCNA/pHisH3 immunoreactive proliferating progenitor cells, GAP43 positive immature neurons, and cytokeratin-5 positive horizontal basal cells were located. Therefore, β-gal-pH6 seems to be linked to neuronal differentiation and cannot be regarded as a biomarker of cellular senescence during olfactory epithelium development in mice.

A VIN3-like Protein OsVIL1 Is Involved in Grain Yield and Biomass in Rice

In chromatin remodeling, the post-translational modification of histone proteins is mediated by multimeric protein complexes. VERNALIZATION INSENSITIVE3 (VIN3) forms a complex with Polycomb Repressive Complex 2 (PRC2), which mediates the trimethylation of H3K27 to repress target gene expression. In rice, four genes (OsVIL1-OsVIL4) encoding the VIN3-like proteins are expressed ubiquitously in various tissues. Null mutants of osvil2 display pleiotropic phenotypes such as altered flowering time, floral organ defects, and reduced tiller size. In contrast, osvil1 mutants did not show significant phenotypes except in fertilization compared with the wild type. However, transgenic plants overexpressing OsVIL1 showed phenotypes of increased biomass and grain yield. Cross-sections of the basal region of elongating stems revealed that the increased biomass was mediated by inducing cell proliferation in the meristem. Chromatin immunoprecipitation assay indicated that OsVIL1 repressed expression of cytokinin oxidase/dehydrogenase gene (OsCKX2) by binding to the promoter and genic regions of OsCKX2. We also observed that OsVIL1 modified the levels of H3K27me3 in the OsCKX2 chromatin. Because OsCKX2 encodes an enzyme that degrades active cytokinin, we conclude that OsVIL1 functions in the regulation of endogenous active cytokinin levels, thereby increasing plant height and productivity.

292 Mechanical stress, myocardial deformation abnormalities, and ventricular fibrosis: a fatal cascade in arrhythmic mitral valve prolapse patients

Aims Arrhythmic mitral valve prolapse (MVP) is characterized by left ventricular (LV) fibrosis in the basal inferolateral wall and papillary muscles. We hypothesized that LV fibrosis are driven by excessive mechanical forces acting on myocardial susceptible cells, representing the last step in the MVP-induced myocardial stretch process. We evaluated the LV myocardial deformation, using strain assessed with cardiac magnetic resonance (CMR), in arrhythmic MVP patients with normal LV ejection fraction (LVEF) and absent/trivial mitral regurgitation (MR) and its correlation with the presence of LV fibrosis, detected by late gadolinium enhancement (LGE) in post-contrast CMR images. Methods and results We enrolled consecutive arrhythmic MVP patients with normal LVEF and no/trivial MR. Sixty-nine (39 female; median age: 40 years) patients without MVP, arrhythmias or cardiovascular history served as control group. All patients underwent CMR for identification of LGE and evaluation of LV myocardial deformation. A total of 66 patients were enrolled (47 female; median age: 44 years). In the overall MVP population, LGE was present in 41 patients (62.1%). MVP patients without LGE (25 patients, 37.9%) presented a higher global radial (median: 42.19 vs. 33; P <0.0001) and higher global longitudinal strain (median: −21.61 vs. −18.10; P <0.0001), compared to the control group. A reduction of regional basal posterolateral radial (BPL median: 50.60 vs. 67.30; P = 0.0015) and longitudinal strain (BPL median: −23.50 vs. −26.70; P = 0.0186) were observed in the MVP patients as compared with controls (Figures A–D). Conversely to the basal region, mid anterolateral and posterolateral region presented a higher radial (MAL median: 52.60 vs. 31.10; P < 0.0001 and MPL median: 52.80 vs. 21.50; P < 0.0001) and longitudinal strain (MAL median: −24.80 vs. −18.30; P < 0.0001 and MPL median: −25.30 vs. −14.80; P < 0.0001), when compared to control group. MVP patients with LGE had a lower global radial (median: 36.48 vs. 42.19; P <0.0047), longitudinal (median: −19.18 vs. −21.61; P = 0.0013), and circumferential strain (median: −17.80 vs. −19.28; P = 0.0134) compared with those without fibrosis. According to MVP patients without LGE, the presence of fibrosis is associated with a lower regional radial (BAL median: 64.40 vs. 82.80; P = 0.0481; MAL median: 42.60 vs. 52.60; P = 0.0287) and circumferential strain (BAL median: −21.90 vs. −24.20; P = 0.0174; BPL median: −16.80 vs. −18.80; P = 0.0411; MPL median: −15.50 vs. −19.40; P = 0.0077) in the LV basal-mid lateral walls (Figures E–H). 292 Figures A–D and E-H Conclusions Arrhythmic MVP patients with normal LV systolic function and absent/trivial MR presented an abnormal myocardial deformation pattern. The reduced strain in BPL wall of MVP patients without LGE could be considered as an early marker of MVP-induced myocardial stress, that could promote, time by time, LV fibrosis and arrhythmias in MVP patients.

Oral administration of cystine and theanine attenuates 5-fluorouracil-induced intestinal mucositis and diarrhea by suppressing both glutathione level decrease and ROS production in the small intestine of mucositis mouse model

Background Chemotherapy is frequently used in cancer treatment; however, it may cause adverse events, which must be managed. Reactive oxygen species (ROS) have been reported to be involved in the induction of intestinal mucositis and diarrhea, which are common side effects of treatment with fluoropyrimidine 5-fluorouracil (5-FU). Our previous studies have shown that oral administration of cystine and theanine (CT) increases glutathione (GSH) production in vivo. In the present study, we hypothesized that CT might inhibit oxidative stress, including the overproduction of ROS, and attenuate 5-FU-induced mucositis and diarrhea. Methods We investigated the inhibitory effect of CT administration on mucositis and diarrhea, as well as its mechanism, using a mouse model of 5-FU-induced intestinal mucositis. Results CT administration suppressed 5-FU-induced diarrhea and weight loss in the studied mice. After 5-FU administration, the GSH level and the GSH/GSSG ratio in the small intestine mucosal tissue decreased compared to normal control group; but CT administration improved the GSH/GSSG ratio to normal control levels. 5-FU induced ROS production in the basal region of the crypt of the small intestine mucosal tissue, which was inhibited by CT. CT did not affect the antitumor effect of 5-FU. Conclusions CT administration suppressed intestinal mucositis and diarrhea in a mouse model. This finding might be associated with the antioxidant characteristics of CT, including the improved rate of GSH redox and the reduced rate of ROS production in the small intestine mucosal tissue. CT might be a suitable candidate for the treatment of gastrointestinal mucositis associated with chemotherapy.

The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells†

Here we report on the related TBC/RabGAPs EPI64A and EPI64B and show that they function to organize the apical aspect of epithelial cells. EPI64A binds the scaffolding protein EBP50/NHERF1, which itself binds active ezrin in epithelial cell microvilli. Epithelial cells additionally express EPI64B that also localizes to microvilli. However, EPI64B does not bind EBP50 and both proteins are shown to have a microvillar localization domain that spans the RabGAP domains. CRISPR/Cas9 was used to inactivate expression of each protein individually or both in Jeg-3 and Caco2 cells. In Jeg-3 cells, loss of EPI64B resulted in a reduction of apical microvilli, and a further reduction was seen in the double knockout, mostly likely due to misregulation of Rab8 and Rab35. In addition, apical junctions were partially disrupted in cells lacking EPI64A, and accentuated in the double knock out. In Caco2 loss of EPI64B resulted in wavy junctions, whereas loss of both EPI64A and EPI64B had a severe phenotype often resulting in cells with a stellate apical morphology. In the knockout cells, the basal region of the cell remained unchanged, so EPI64A and EPI64B specifically localize to and regulate the morphology of the apical domain of polarized epithelial cells.

Dynamic Hormone Gradients Regulate Wound-Induced de novo Organ Formation in Tomato Hypocotyl Explants

Plants have a remarkable regenerative capacity, which allows them to survive tissue damage after biotic and abiotic stresses. In this study, we use Solanum lycopersicum ‘Micro-Tom’ explants as a model to investigate wound-induced de novo organ formation, as these explants can regenerate the missing structures without the exogenous application of plant hormones. Here, we performed simultaneous targeted profiling of 22 phytohormone-related metabolites during de novo organ formation and found that endogenous hormone levels dynamically changed after root and shoot excision, according to region-specific patterns. Our results indicate that a defined temporal window of high auxin-to-cytokinin accumulation in the basal region of the explants was required for adventitious root formation and that was dependent on a concerted regulation of polar auxin transport through the hypocotyl, of local induction of auxin biosynthesis, and of local inhibition of auxin degradation. In the apical region, though, a minimum of auxin-to-cytokinin ratio is established shortly after wounding both by decreasing active auxin levels and by draining auxin via its basipetal transport and internalization. Cross-validation with transcriptomic data highlighted the main hormonal gradients involved in wound-induced de novo organ formation in tomato hypocotyl explants.

Daple deficiency causes hearing loss in adult mice by inducing defects in cochlear stereocilia and apical microtubules

The V-shaped arrangement of hair bundles on cochlear hair cells is critical for auditory sensing. However, regulation of hair bundle arrangements has not been fully understood. Recently, defects in hair bundle arrangement were reported in postnatal Dishevelled-associating protein (ccdc88c, alias Daple)-deficient mice. In the present study, we found that adult Daple−/− mice exhibited hearing disturbances over a broad frequency range through auditory brainstem response testing. Consistently, distorted patterns of hair bundles were detected in almost all regions, more typically in the basal region of the cochlear duct. In adult Daple−/− mice, apical microtubules were irregularly aggregated, and the number of microtubules attached to plasma membranes was decreased. Similar phenotypes were manifested upon nocodazole treatment in a wild type cochlea culture without affecting the microtubule structure of the kinocilium. These results indicate critical role of Daple in hair bundle arrangement through the orchestration of apical microtubule distribution, and thereby in hearing, especially at high frequencies.

Abstract 38: Radiation Exposure And Coronary Atherosclerosis: Differential Effect Of The Radiation Site

Background: Thoracic radiation is commonly used in breast cancer, Hodgkin’s lymphoma, head and neck and lung cancer patients. Accelerated coronary artery atherosclerosis is a common complication of thoracic radiation as a result of unintended cardiac radiation. It is unclear however whether specific areas of the heart are more susceptible to the effects of radiation (RT). In this study we hypothesize that accelerated development of lesions post RT is dependent upon differential sensitivity of specific areas of the heart to the effects of RT. Methods: Male Apolipoprotein E knockout mice on a high fat diet received 16Gy cardiac RT targeted to the whole or partial (apical or basal) region of the heart at 9 or 16 weeks of age (n=5 per group). Atherosclerotic lesions in H&E stained slides and inflammatory infiltrates in the hearts by IHC were assessed 8 weeks following RT and compared to control unirradiated mice. Results: Our studies show that: (1) Subendocardial atherosclerotic lesions at the base of heart in mice irradiated at 9 weeks of age after basal irradiation (7.8±2.49) were comparable to whole heart irradiation (12.2±3.29). (2) A greater number of atherosclerotic lesions were present in the basal coronary arteries (29.33±5.48 vs 9±2.70) and basal subendocardial vasculature (6.66±2.07 vs 0.2±0.2) after irradiation of the cardiac base as compared to unirradiated controls in mice irradiated at 16 weeks of age. (3) Apical or whole heart irradiation had no impact on the development of lesions in the basal region of the hearts of older mice. (4) IL-6 was significantly increased in the serum of mice 6 hours post basal cardiac irradiation (105.10±17.56 pg/ml) when compared to unirradiated controls (29.85±11.63 pg/ml), demonstrating an acute inflammatory response. (5) Infiltration of inflammatory cells (CD45 and CD3) and enhanced expression of endothelial adhesion molecules (CD31) were differentially and locally regulated based upon the site of irradiation. Conclusion: Our results indicate that the base of the heart is more prone to development of RT induced atherosclerotic lesions likely due to acute and delayed inflammatory responses. Avoiding this area from direct radiation exposure may improve the quality of life for cancer patients receiving thoracic RT.

INTERMITTENT TINNITUS IN ADULTS: AN INSIGHT INTO COVERT HEARING LOSS

Objectives: Association of high frequency hearing loss/minor damage in peripheral auditory system in continuous chronic tinnitus with normal PTA is well established.The purpose of the study was to verify whether this finding is true for intermittent unilateral or bilateral tinnitus patients with normal PTA using EHF audiometry and conventional DPOAEs. Materials and method:This study was conducted on 45 normal hearing adults between the age ranges of 18-30 years. Among them 30 adults comprised of study group with intermittent tinnitus which varies in laterality.Tinnitus evaluation was done on these population followed by THI administration. DPOAE and EHF audiometry was completed on all subjects after conventional hearing assessment program. Result and Discussion:Kruskal Wallis H test & Wilcoxon signed rank test was used to compare OAE amplitude & EHF thresholds.Spearman's correlation was used to evaluate the correlation between DPOAE amplitude with EHF threshold. Reduced hearing sensitivity in the extended high frequency region may be early predictor of outer hair cell dysfunction in the most basal area.Findings of this study suggest that intermittent tinnitus may also lead to subtle lesion at the basal region of cochlea which would result in a significant hearing loss with continuous tinnitus in future. Conclusion:Intermittent tinnitus may increase the fragility of peripheral auditory system which may lead to permanent lesions and would be evident as elevated thresholds in conventional PTA.

Comparison of myocardial T1 mapping during breath-holding and free-breathing

Background: T1 mapping is a recently developed imaging analysis method that allows quantitative assessment of myocardial T1 values obtained using MRI. In children, MRI is performed under free-breathing. Thus, it is important to know the changes in T1 values between free-breathing and breath-holding. This study aimed to compare the myocardial T1 mapping during breath-holding and free-breathing. Methods: Thirteen patients and eight healthy volunteers underwent cardiac MRI, and T1 values obtained during breath-holding and free-breathing were examined and compared. Statistical differences were determined using the paired t-test. Results: The mean T1 values during breath-holding were 1211.1 ± 39.0 ms, 1209.7 ± 37.4 ms, and 1228.9 ± 52.5 ms in the basal, mid, and apical regions, respectively, while the mean T1 values during free-breathing were 1165.1 ± 69.0 ms, 1103.7 ± 55.8 ms, and 1112.0 ± 81.5 ms in the basal, mid, and apical regions, respectively. The T1 values were lower during free-breathing than during breath-holding in almost all segments (basal: p = 0.008, mid: p < 0.001, apical: p < 0.001). The mean T1 values in each cross section were 3.1, 7.8, and 7.7% lower during free-breathing than during breath-holding in the basal, mid, and apical regions, respectively. Conclusions: We found that myocardial T1 values during free-breathing were about 3–8% lower in all cross sections than those during breath-holding. In free-breathing, it may be difficult to assess myocardial T1 values, except in the basal region, because of underestimation; thus, the findings should be interpreted with caution, especially in children.