Maturation of autophagosomes and endosomes: A key role for Rab7

AbstractMacroautophagy is an important route in cellular maintenance, in the breakdown and reuse of intracellular materials. It is closely related to endocytosis, the means by which the cell can absorb extracellular material, as both macroautophagy and endocytosis have converging steps and common participating molecules. The point where autophagosomes and endosomes fuse with lysosomes to permit for the final degradation of their contents is important. One of the most substantial molecules in the maturation of autophagosomes/endosomes is Rab7, a member of small GTPases. Rab7 designates the maturation of endosomes and also autophagosomes, directing the trafficking of cargos along microtubules, and finally, participating in the fusion step with lysosomes. Rab7 is an effective multifunctional regulator of autophagy and endocytosis. Since many aggregation-based diseases, e.g. age-related macular degeneration of the eye (AMD) and Alzheimer's disease are due of malfunctioning in the autophagic process, the management of Rab7 activity might hold potential as a therapeutic target against these diseases

Unusually Strong Temperature Dependence of P2X3 Receptor Traffic to the Plasma Membrane

ATP-gated P2X3 receptors are expressed by nociceptive neurons and participate in transduction of pain. Responsiveness of P2X3 receptors is strongly reduced at low temperatures, suggesting a role for these receptors in analgesic effects of cooling. Since sustained responsiveness depends on receptor trafficking to the plasma membrane, we employed total internal reflection fluorescence (TIRF) microscopy to highlight perimembrane pool of DsRed-tagged P2X3 receptors and studied the effects of temperature on perimembrane turnover of P2X3-DsRed. Patch-clamp recordings confirmed membrane expression of functional, rapidly desensitizing P2X3-DsRed receptors. By combining TIRF microscopy with the technique of fluorescence recovery after photobleaching (FRAP), we measured the rate of perimembrane turnover of P2X3-DsRed receptors expressed in hippocampal neurons. At room temperature, the P2X3-DsRed perimembrane turnover as measured by TIRF–FRAP had a time constant of ∼2 min. At 29°C, receptor turnover was strongly accelerated (0.6 min), yielding an extremely high temperature dependence coefficient Q10 ∼4.5. In comparison, AMPA receptor turnover measured with TIRF–FRAP was only moderately sensitive to temperature (Q10 ∼1.5). The traffic inhibitor Brefeldin A selectively decelerated P2X3-DsRed receptor turnover at 29°C, but had no effect at 21°C (Q10 ∼1.0). This indicates that receptor traffic to plasma membrane is the key temperature-sensitive component of P2X3 turnover. The selective inhibitor of the RhoA kinase Y27632 significantly decreased the temperature dependence of P2X3-DsRed receptor turnover (Q10 ∼2.0). In summary, the RhoA kinase-dependent membrane trafficking of P2X3 receptors to plasma membrane has an exceptionally high sensitivity to temperature. These findings suggest an important role of P2X3 receptor turnover in hypothermia-associated analgesia

Attenuated Semliki Forest virus for cancer treatment in dogs : safety assessment in two laboratory Beagles

Background: Dogs suffer from spontaneous tumors which may be amenable to therapies developed for human cancer patients, and dogs may serve as large-animal cancer models. A non-pathogenic Semliki Forest virus vector VA7-EGFP previously showed promise in targeting human tumor xenografts in mice, but the oncolytic capacity of the virus in canine cancer cells and the safety of the virus in higher mammals such as dogs, are not known. We therefore assessed the oncolytic potency of VA7-EGFP against canine cancer cells by infectivity and viability assays in two dog solid tumor cell lines. Furthermore we performed a 3-week safety study in two adult Beagles which received a single intravenous injection of similar to 2 x 10(5) plaque forming units of parental A7(74) strain. Results: VA7-EGFP was able to replicate in and kill both canine cancer cell lines tested. No adverse events were observed in either of the two virus-injected adult Beagles and no infective virus could be recovered from any of the biological samples collected over the course of the study. Neutralizing antibodies to Semliki Forest virus became detectable in the dogs at 5 days post infection and remained elevated until study termination. Conclusions: Based on these results, testing of the oncolytic potential of attenuated Semliki Forest virus in canine cancer patients appears feasible.Peer reviewe

Rapid synthesis and enhancement in down conversion emission properties of BaAl2O4:Eu2+,RE3+ (RE3+=Y, Pr) nanophosphors

[EN] BaAl2O4:Eu2+,RE3+ (RE3+=Y, Pr) down conversion nanophosphors were prepared at 600 °C by a rapid gel combustion technique in presence of air using boron as flux and urea as a fuel. A comparative study of the prepared materials was carried out with and without the addition of boric acid. The boric acid was playing the important role of flux and reducer simultaneously. The peaks available in the XPS spectra of BaAl2O4:Eu2+ at 1126.5 and 1154.8 eV was ascribed to Eu2+(3d5/2) and Eu2+(3d3/2) respectively which confirmed the presence of Eu2+ ion in the prepared lattice. Morphology of phosphors was characterized by tunneling electron microscopy. XRD patterns revealed a dominant phase characteristics of hexagonal BaAl2O4 compound and the presence of dopants having unrecognizable effects on basic crystal structure of BaAl2O4. The addition of boric acid showed a remarkable change in luminescence properties and crystal size of nanophosphors. The emission spectra of phosphors had a broad band with maximum at 490–495 nm due to electron transition from 4f65d1 → 4f7 of Eu2+ ion. The codoping of the rare earth (RE3+=Y, Pr) ions help in the enhancement of their luminescent properties. The prepared phosphors had brilliant optoelectronic properties that can be properly used for solid state display device applications.The authors gratefully recognize the financial support from the University Grant Commission (UGC), New Delhi [MRP-40-73/2011(SR)] and the European Commission through Nano CIS project (FP7-PEOPLE-2010-IRSES ref. 269279).Singh, D.; Tanwar, V.; Simantilke, AP.; Marí, B.; Kadyan, PS.; Singh, I. (2016). Rapid synthesis and enhancement in down conversion emission properties of BaAl2O4:Eu2+,RE3+ (RE3+=Y, Pr) nanophosphors. Journal of Materials Science: Materials in Electronics. 27(3):2260-2266. https://doi.org/10.1007/s10854-015-4020-1S22602266273J.S. Kim, P.E. Jeon, J.C. Choi, H.L. Park, S.I. Mho, G.C. Kim, Appl Phys Lett 84, 2931 (2004)D. Jia, D.N. Hunter, J Appl Phys 100, 1131251 (2006)H. Aizawa, T. Katsumata, J. Takahashi, K. Matsunaga, S. Komuro, T. Morikawa, E. Toba, Rev Sci Instrum 74, 1344 (2003)C.N. Xu, X.G. Zheng, M. Akiyama, K. Nonaka, T. Watanabe, Appl Phys Lett 76, 179 (2000)C. Feldmann, T. Justel, C.R. Ronda, P.J. Schmidt, Adv Funct Mater 13, 511 (2004)P.J. Saines, M.M. Elcombe, B.J. Kennedy, J Solid State Chem 179, 613 (2006)R. Sakai, T. Katsumata, S. Komuro, T. Morikawa, J Lumin 85, 149 (1999)T. Aitasalo, P. Deren, J Solid State Chem 171, 114 (2003)S. Nakamura, T. Mukai, M. Senoh, J Appl Phys 76, 8189 (1994)S.H.M. Poort, G. Blasse, J Lumin 72, 247 (1997)P. Mingying, H. Guangyan, J Lumin 127, 735 (2007)X. Linjiu, H. Mingrui, T. Yanwen, C. Yongjie, K. Tomoaki, Z. Liqing, W. Ning, Jap J Applied Physics 46, 5871 (2007)T. Aitasalo, J. Hölsä, H. Jungner, M. Lastusaari, J. Niittykoski, J Phys Chem B 110, 4589 (2006)R. Stefani, L.C.V. Rodrigues, C.A.A. Carvalho, M.C.F.C. Felinto, H.F. Brito, M. Lastusaari, J. Hölsä, Opt Mater 31, 1815 (2009)M. Peng, G. Hong, J Lumin 127, 735 (2007)V. Singh, V. Natarajan, J.J. Zhu, Opt Mater 29, 1447 (2007)X.Y. Chen, C. Ma, X.X. Li, C.W. Shi, X.L. Li, D.R. Lu, J Phys Chem C 113, 2685 (2009)A.J. Zarur, J.Y. Ying, Nature 403, 65 (2000)J. Chen, F. Gu, C. Li, Cry Growth Des 8, 3175 (2008)J. Zhang, M. Yang, H. Jin, X. Wang, X. Zhao, X. Liu, L. Peng, Mater Res Bull 47, 247 (2012)P. Maślankiewicz, J. Szade, A. Winiarski, Ph Daniel, Cryst Res Technol 40, 410 (2005)Y.J. Chen, G.M. Qiu, Y.B. Sun et al., J Rare Earths 20, 50 (2002)F.C. Palilla, A.K. Levine, M.R. Tomkus, J Electrochem Soc 115, 642 (1968)J. Niittykoski, T. Aitasalo, J. Holsa, H. Jungner, M. Lastusaari, M. Parkkinen, M. Tukia, J Alloys Compd 374, 108 (2004)A. Nag, T.R.N. Kutty, J Alloys Compd 354, 221 (2003)D. Haranath, P. Sharma, H. Chander, J Phys D Appl Phys 38, 371 (2005

Real-Time Dynamics of Ca2+, Caspase-3/7, and Morphological Changes in Retinal Ganglion Cell Apoptosis under Elevated Pressure

Quantitative information on the dynamics of multiple molecular processes in individual live cells under controlled stress is central to the understanding of the cell behavior of interest and the establishment of reliable models. Here, the dynamics of the apoptosis regulator intracellular Ca2+, apoptosis effector caspase-3/7, and morphological changes, as well as temporal correlation between them at the single cell level, are examined in retinal gangling cell line (differentiated RGC-5 cells) undergoing apoptosis at elevated hydrostatic pressure using a custom-designed imaging platform that allows long-term real-time simultaneous imaging of morphological and molecular-level physiological changes in large numbers of live cells (beyond the field-of-view of typical microscopy) under controlled hydrostatic pressure. This examination revealed intracellular Ca2+ elevation with transient single or multiple peaks of less than 0.5 hour duration appearing at the early stages (typically less than 5 hours after the onset of 100 mmHg pressure) followed by gradual caspase-3/7 activation at late stages (typically later than 5 hours). The data reveal a strong temporal correlation between the Ca2+ peak occurrence and morphological changes of neurite retraction and cell body shrinkage. This suggests that Ca2+ elevation, through its impact on ion channel activity and water efflux, is likely responsible for the onset of apoptotic morphological changes. Moreover, the data show a significant cell-to-cell variation in the onset of caspase-3/7 activation, an inevitable consequence of the stochastic nature of the underlying biochemical reactions not captured by conventional assays based on population-averaged cellular responses. This real-time imaging study provides, for the first time, statistically significant data on simultaneous multiple molecular level changes to enable refinements and testing of models of the dynamics of mitochondria-mediated apoptosis. Further, the platform developed and the approach has direct significance to the study of a variety of signaling pathway phenomena

Views of 9th class students in Vähäkyrö on vocational education

Työn tavoitteena oli selvittää yhteishaussa 2006 hakeneiden vähäkyröläisten oppilaiden mielikuvaa ammatillisesta koulutuksesta sekä opiskelupaikan valintaan vaikuttavia tekijöitä. Lisäksi hankkeessa selvitettiin kuinka nuoret olivat saaneet tietoa ammatilliseta koulutuksesta sekä heidän tietojaan ammatillisten alojen työllisyystilanteesta. Tutkimusmenetelmänä käytettiin kyselyä, jonka avulla selvitettiin opiskelijoiden näkemyksiä. Työ osoitti, että nuoret pitävät ammatillista kolutusta nopeana tienä ammattiin ja ammatillista koulutusta käytännönläheisenä. Työn tulosten perusteella voidaan päätellä, että ammatilliseen koulutukseen hakeneille työllistyminen oli vaikuttavin seikka opiskelupaikkaa valitessa. Lukioon hakeneet pitivät jatkoopintomahdollisuutta tärkeimpänä perusteena valinnalleen. Tämän aineiston mukaan ammatillisesta koulutuksesta nuoret olivat saaneet tietoa hyvin tai melko hyvin. Oppilaanohjaajan osuus tiedon jakajana oli suurin. Ammatilliseen koulutukseen hakeneista sekä tytöistä että pojista suurin osa tiesi ennalta hakemallaan alalla vallitsevan työtilanteen ja piti työllisyystilannetta melko hyvänä.The object of the work was to study views of students in Vähäkyrö on vocational education and factors affecting choice of study place. In addition to that, it was studied how young people gather information about vocational education and their knowledge about the employment rate in vocational jobs. The research method was a survey, with which students` views were studied. The work showed that young people view vocational education as a quick way to a profession, and that vocational education is seen as very practical. The results of the stud y imply that employment situation is the strongest factor affecting choice of study place for young people who applied to vocational education. The ones who applied to upper secondary school considered possibility for further studies as the most important factor in their choice. According to this study material, young people had received information about vocational education either well or fairly well. The study advisor was the biggest single source of this information. Of those having applied to vocational education, the majority of both girls and boys knew beforehand about the employment situation in the field and considered the employment situation rather good

The initiation knot is a signaling center required for molar tooth development

Signaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determines tooth patterning. Preceding the primary EK, transient epithelial thickening appears, the significance of which remains debated. Using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK), that is required for the progression of tooth development. IK cell dynamics demonstrate the hallmarks of a signaling center: cell cycle exit, condensation and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of cells with high Wnt activity to Shh-expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers.Peer reviewe