Time-lapse Video Recording Research Articles

Evaluating the toxic mechanism of 1,2-diacetylbenzene in neural cells/tissues: The favorable impact of silibinin

1,2-diacetylbenzene (1,2-DAB) is a neurotoxic component of aromatic solvents commonly used in industrial applications that induces neuropathological changes in animals. This study unraveled the toxic impact of 1,2-DAB in nerve tissues, explant cultures, and neuron-glial cultures, and explored whether herbal products can mitigate its toxicity. The effects of DAB on axonal transport were studied in retinal explant cultures grown in a micro-patterned dish. The mitochondrial movement in the axons was captured using time-lapse video recordings. The results showed that 1,2-DAB, but not 1,3-DAB inhibited axonal outgrowth and mitochondrial movement in a dose-dependent manner. The toxicity of 1,2-DAB was further studied in spinal cord tissues and cultures. 1,2-DAB selectively induced modifications of microtubules and neurofilaments in spinal cord tissues. 1,2-DAB also potently induced cell damage in both neuronal and glial cultures. Further, 1,2-DAB-induced cellular ATP depletion precedes cell damage in glial cells. Interestingly, treatment with the herbal products silibinin or silymarin effectively mitigated 1,2-DAB-induced toxicity in spinal cord tissues and neuronal/glial cultures. Collectively, the molecular toxicity of 1,2-DAB in neural tissues involves protein modification, ATP depletion, and axonal transport defects, leading to cell death. Silibinin and silymarin show promising neuroprotective effects against 2-DAB-induced toxicity.

A novel method for quantifying the attractiveness of lobster trap baits

We developed a new method for quantifying the attractiveness of bait in traps used for crustacean fisheries by using accelerometers in bait containers to assess the amount of time animals spend interacting with the baits. We validated our approach in the laboratory by using time-lapse video recordings to verify that the accelerometry data accurately reflected when, and how much, American lobsters (Homarus americanus) interacted with bait. We then conducted a field experiment comparing traps baited with traditional herring, crushed green crabs (Carcinus maenas), or an inert control. We found that during 48-h trap sets, the number of minutes in which the accelerometers recorded movements of the bait bags was correlated with lobster catch. Although the traps baited with herring did not catch significantly more lobsters than traps baited with crushed green crabs, the accelerometer data revealed differences in bait attractiveness over time. Lobsters interacted less with the green crab bait on the second day of the set, while no such decline was observed with the herring bait. This technique is inexpensive, easy to use, and the data obtained can be more rapidly analyzed than the video data traditionally used for these types of studies, making it well suited for future work on bait choice and attractiveness.

RS6077 induces mitotic arrest and selectively activates cell death in human cancer cell lines and in a lymphoma tumor in vivo

We synthesized a new inhibitor of tubulin polymerization, the pyrrole (1-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrrol-3-yl)(3,4,5-trimethoxy-phenyl)methanone 6 (RS6077). Compound 6 inhibited the growth of multiple cancer cell lines, with IC50 values in the nM range, without affecting the growth of non-transformed cells. The novel agent arrested cells in the G2/M phase of the cell cycle in both transformed and non-transformed cell lines, but single cell analysis by time-lapse video recording revealed a remarkable selectivity in cell death induction by compound 6: in RPE-1 non-transformed cells mitotic arrest induced was not necessarily followed by cell death; in contrast, in HeLa transformed and in lymphoid-derived transformed AHH1 cell lines, cell death was effectively induced during mitotic arrest in cells that fail to complete mitosis. Importantly, the agent also inhibited the growth of the lymphoma TMD8 xenograft model. Together these findings suggest that derivative 6 has a selective efficacy in transformed vs non-transformed cells and indicate that the same compound has potential as novel therapeutic agent to treat lymphomas. Compound 6 showed good metabolic stability upon incubation with human liver microsomes.

Transitioning a Research Protocol for Videosomnography to Assess Sleep and Nighttime Caregiving Activities in School-Aged Children With Developmental Disabilities During the COVID-19 Pandemic

The COVID-19 pandemic significantly affected children with developmental disabilities (DDs)' sleep. Videosomnography is a noninvasive, portable time-lapse video recording system to objectively obtain a child's sleep-wake behaviors and parents' caregiving activities in a natural environment. From September 2020 to February 2021, we conducted a feasibility study using actigraphy (in mothers) and videosomnography in children with DDs for seven consecutive nights to assess sleep and nighttime caregiving activities. Because of the pandemic, we developed and implemented alternative data collection strategies, such as delivering a "study package" with easy-to-follow written instructions and emailed video-recorded instructions on recording a child's sleep. We aimed to enroll 10 mothers and 10 school-aged children with DDs and achieved this goal. Nine out of 10 mothers completed video recordings of their child's sleep, with only 10% missing data for videosomnography. This paper shared adaptations to our videosomnography protocol and lessons learned.

Improving natural ventilation in renovated free-stall barns for dairy cows: Optimized building solutions by using a validated computational fluid dynamics model

Natural ventilation is the most used system to create suitable conditions, removing gases, introducing oxygen in livestock buildings. Its efficiency depends on several factors and above all on the number, the dimensions and the position of wall openings and internal layout of livestock buildings. The aim of this research was to develop optimized layout solutions for improving natural ventilation effectiveness in free-stall barns for dairy cows by using a CFD approach. A validated computational fluid dynamics (CFD) model was applied in a case study which is highly representative of building interventions for renovating the layout of free-stall barns for dairy cows located in an area of the Mediterranean basin. Firstly, dairy cow behaviour was analysed by visual examination of time-lapse video-recordings. Then, simulations were carried out by using the validated CFD model and changing the position of internal and external building elements (i.e., internal office and external buildings for milking) in order to find the best condition for the thermal comfort of the animals. The results showed that the best conditions were recorded for a new configuration of the building in terms of air velocity distribution within the resting area, the service alley and the feeding alley for dairy cows, and in the pens for calves. In this new layout, the office areas and the north-west wall openings were located by mirroring them along the transversal axis of the barn. Therefore, the CFD approach proposed in this study could be used during the design phase, as a decision support system aimed at improving the natural ventilation within the barn.

Long-term study of behaviors of two cohabiting sea urchin species, Mesocentrotus nudus and Strongylocentrotus intermedius, under conditions of high food quantity and predation risk in situ.

BackgroundIn the predator–sea urchin–macrophyte trophic cascade, the ecological effect of sea urchins as grazers depends both on their density and the changes in foraging activity, which are influenced by various disturbing factors. However, the complete duration of the alarm reactions of echinoids has not been studied until now. Here, we tested a hypothesis that two cohabiting sea urchins, Mesocentrotus nudus and Strongylocentrotus intermedius, which differ morphologically, might display different behavioral responses to high hydrodynamic activity and predation.MethodsWe used continuous time-lapse video recording to clarify behavioral patterns of M. nudus and S. intermedius in presence of a large quantity of food (the kelp Saccharina japonica) but under different weather conditions and different types of predation threat: (1) calm weather conditions, (2) stormy weather conditions, (3) predation risk associated with the presence of several sea star species and (4) predation risk associated with an alarm stimulus (crushed conspecifics or heterospecifics). Three separate video recording experiments (134 days in total) were conducted under field conditions. Video recording analysis was performed to determine the number of specimens of each sea urchin species in the cameras’ field of view, size of sea urchins’ groups, movement patterns and the duration of the alarm responses of both sea urchin species.ResultsWe showed that in the presence of kelp, M. nudus and S. intermedius exhibited both similar and different behavioral responses to hydrodynamics and predation threat. Under calm weather, movement patterns of both echinoids were similar but M. nudus exhibited the higher locomotion speed and distance traveled. Furthermore, S. intermedius but not M. nudus tended to group near the food substrate. The stormy weather caused a sharp decrease in movement activity followed by escape response in both echinoids. Six starfish species failed to predate on healthy sea urchins of either species and only a few attacks on ailing S. intermedius specimens were successful. The alarm response of S. intermedius lasted approximately 90 h and 20 h for starfish attacks on ailing conspecifics and for simulated attacks (crushed conspecifics or heterospecifics), respectively and involved several phases: (1) flight response, (2) grouping close to the food, (3) leaving the food and (4) return to the food. Phase three was the more pronounced in a case of starfish attack. M. nudus only responded to crushed conspecifics and exhibited no grouping behavior but displayed fast escape (during 4 h) and prolonged (up to 19 days) avoidance of the food source. This outcome is the longest alarm response reported for sea urchins.DiscussionThe most interesting finding is that two cohabiting sea urchin species, M. nudus and S. intermedius, display different alarm responses to predation threat. Both alarm responses are interpreted as defensive adaptations against visual predators.

Mass Timber Productivity- the Significance of Reduction in Non-Value Add Activities during On-Site Installation Sequence.

The recent uptake of Mass Timber (M.T.), a prefabricated timber panelised form of construction, provides a potential sustainable resource to facilitate improved productivity outcomes to the construction industry. However, in Australia as well as U.S. and Canada, M.T. is in its infancy and there is a lack of empirical information available to industry. This consequently has resulted in reluctance by contractors and professionals to uptake this new innovative system. The aim of this paper is to undertake a comprehensive review of the on-site productivity outcomes. A quantitative Case Study approach was implemented by way of time-lapse digital video recording of three M.T. multi-storey buildings located in NSW, Australia. Crane cycles of the installation of the M.T. prefabricated panels were found to be the most representative and repeatable process and as a consequence were used to measure the M.T. productivity. Discussion is provided on potential areas of process and consequential productivity improvement. The M.T. crane cycles productivity at an Activity Level compared to M.T. productivity at Project Level revealed large differential between the two levels. The quantum of Non-Value Add activities was found to be a significant factor in the overall Project Level productivity outcome. This review paper undertakes a review of the outcomes of the case studies on the M.T. installation on three multi-storey buildings, the factors found that affected the resultant on-site construction productivity and its resultant beneficial implications to the construction industry.

Does polycystic ovary syndrome affect morphokinetics or abnormalities in early embryonic development?

ObjectivesThis study aimed to investigate whether oocyte retrieval from PCOS patients affects the morphokinetics or the incidence of abnormalities in early embryonic development, using time-lapse imaging analysis.MethodsThis was a prospective study in total of 39 PCOS patients and 67 women with normal ovarian function, underwent a GnRH antagonist protocol of controlled ovarian stimulation and fertilization by ICSI. 402 zygotes from the PCOS group and 449 zygotes from the control group were observed by time-lapse monitoring for 48 h following sperm injection.ResultsPatients with PCOS showed a significantly higher number of retrieved oocytes, and a significantly higher number of metaphase II oocytes per cycle than that of the non-PCOS group (18.85 ± 9.41 vs. 11.48 ± 5.51 and 14.97 ± 7.43 vs. 9.51 ± 4.7, respectively). However, oocyte maturation rate and morphologically assessed embryo quality did not differ between two groups. After 48 h of the embryo culture using time-lapse video recording, most of the embryos in both groups had reached the 4-cell stage (353/449 vs. 314/402 embryos). There were no significant differences between the control and PCOS group regarding the time-points of the successive events in early embryonic development from the appearance of 2 polar bodies to the 6-cell stage. However, the percentage of t2 stages which fell in “optimal range” (>24 h and ≤28 h) was significantly lower in the PCOS group than in the control group (37.8% vs. 48.1%, P = 0.004). The proportion of embryos manifesting abnormal fertility, multinucleation, direct cleavage and reverse cleavage were not significantly different between the two groups.ConclusionOur data indicated that PCOS does not affect the morphokinetics or incidence of abnormalities in early embryonic development.

Breeding of carnations (Dianthus caryophyllus L.) for long vase life.

Carnation (Dianthus caryophyllus L.) is one of the main floricultural crops in Japan and worldwide. The vase life of cut ornamental flowers, including carnations, is important in determining their quality and consumers’ preference. To improve the vase life of carnation flowers, my group started a breeding research program in 1992 using conventional cross-breeding techniques. We repeatedly crossed and selected promising offspring with long vase life for seven generations, from 1992 to 2008. In 2005, we developed two cultivars, ‘Miracle Rouge’ and ‘Miracle Symphony’, with genetically determined long vase lives of 17.7 to 20.7 days (3.2 to 3.6 times that of ‘White Sim’) under standard conditions (23°C, 70% RH, 12-h photoperiod). Line 532-6 showed an ultra-long vase life averaging 27.8 to 32.7 days (4.6 to 5.4 times that of ‘White Sim’). We evaluated changes in ethylene sensitivity with flower senescence simply and accurately using a time-lapse video recorder. In 2010, we selected line 806-46b with both ultra-long vase life (27.1 days, 4.4 times that of ‘White Sim’) and ethylene resistance. Analyses using six cultivars and 123 selected lines from the 1st to the 7th generations revealed that the long vase life was strongly associated with a decrease in ethylene production.

Reassembly of adult human testicular cells: can testis cord-like structures be created in vitro?

Can enzymatically dispersed testicular cells from adult men reassemble into seminiferous cord-like structures in vitro? Adult human testicular somatic cells reassembled into testicular cord-like structures via dynamic interactions of Sertoli and peritubular cells. In vitro approaches using dispersed single cell suspensions of human testes to generate seminiferous tubule structures and to initiate their functionality have as yet shown only limited success. Testes from 15 adult gender dysphoria patients (mean ± standard deviation age 35 ± 9.3 years) showing spermatogonial arrest became available for this study after sex-reassignment surgery. In vitro primary testicular somatic cell cultures were generated to explore the self-organizing ability of testicular somatic cells to form testis cords over a 2-week period. Morphological phenotype, protein marker expression and temporal dynamics of cell reassembly were analyzed. Cell suspensions obtained by two-step enzymatic digestion were plated onto glass coverslips in 24-well plates. To obtain adherent somatic cells, the supernatant was discarded on Day 2. The culture of the attached cell population was continued. Reassembly into cord-like structures was analyzed daily by microscopic observations. Endpoints were qualitative changes in morphology. Cell types were characterized by phase-contrast microscopy and immunohistochemistry. Dynamics of cord formation were recorded by time-lapse microscopy. Primary adult human testicular cells underwent sequential morphological changes including compaction and reaggregation resulting in round or elongated cord-like structures. Time-lapse video recordings within the first 4 days of culture revealed highly dynamic processes of migration and coalescence of reaggregated cells. The cellular movements were mediated by peritubular cells. Immunohistochemical analysis showed that both SRY-related high mobility box 9-positive Sertoli and α-smooth muscle actin-positive peritubular myoid cells interacted and contributed to cord-like structure formation. Not applicable. Owing to scarcity of normal human testicular tissue, testes from gender dysphoria patients were used in the study. The regressed status might influence the experimental responses of primary cells. We observed basic morphological features resembling in vivo testicular cords, however, the proof of functionality (e.g. support of germ cells) will need further studies. The proposed in vitro culture system may open opportunities for examination of testicular cell interactions during testicular tubulogenesis. Further refinement of our approach may enable initiation of ex vivo spermatogenesis. The work was supported by EU-FP7-PEOPLE-2013-ITN 603568: 'Growsperm'. No conflict of interests is declared.

Biocompatible silica-gelatin hybrid aerogels covalently labeled with fluorescein

Silica-gelatin hybrid aerogels, introduced recently as drug delivery devices, were investigated for biocompatibility with SCC tumor cells by a time-lapse video-microscopy technique. The particles proved to be not only non-toxic in nature, but the cells migrated towards them. Fluorescently labeled hybrid aerogels were synthesized by covalently binding fluorescein isothiocyanate either to the amino groups of gelatin molecules, or to the amine-functionalized silica backbone prior to sol-gel polycondensation and co-gelation steps. Scanning electron microscopy and fluorescence spectroscopy characterization of pristine and labeled aerogels showed identical structures and spectra, while fluorescence lifetimes were 10% shorter than that of the free fluorescein, due to covalent attachment. Migration plots based on time-lapse video recordings showed that the cells exhibited a directive migration nearby the aerogel particles. No signs of apoptosis or necrosis were observed at any phases of the cell cycle in the presence of the silica-gelatin aerogel particles, indicating that the hybrid aerogels are biocompatible materials, and may be used as drug delivery matrices in living organisms.

Elevated Na+/H+ exchanger-1 expression enhances the metastatic collective migration of head and neck squamous cell carcinoma cells

Cancer cells can migrate as collectives during invasion and/or metastasis; however, the precise molecular mechanisms of this form of migration are less clear compared with single cell migration following epithelial-mesenchymal transition. Elevated Na+/H+ exchanger1 (NHE1) expression has been suggested to have malignant roles in a number of cancer cell lines and in vivo tumor models. Furthermore, a metastatic human head and neck squamous cell carcinoma (HNSCC) cell line (SASL1m) that was isolated based on its increased metastatic potential also exhibited higher NHE1 expression than its parental line SAS. Time-lapse video recordings indicated that both cell lines migrate as collectives, although with different features, e.g., SASL1m was much more active and changed the direction of migration more frequently than SAS cells, whereas locomotive activities were comparable. SASL1m cells also exhibited higher invasive activity than SAS in Matrigel invasion assays. shRNA-mediated NHE1 knockdown in SASL1m led to reduced locomotive and invasive activities, suggesting a critical role for NHE1 in the collective migration of SASL1m cells. SASL1m cells also exhibited a higher metastatic rate than SAS cells in a mouse lymph node metastasis model, while NHE1 knockdown suppressed in vivo SASL1m metastasis. Finally, elevated NHE1 expression was observed in human HNSCC tissue, and Cariporide, a specific NHE1 inhibitor, reduced the invasive activity of SASL1m cells, implying NHE1 could be a target for anti-invasion/metastasis therapy.

Behavior of Mouse Spermatozoa in the Female Reproductive Tract from Soon after Mating to the Beginning of Fertilization.

Using transgenic mice with spermatozoa expressing enhanced green fluorescent protein in their acrosome and red fluorescent protein in their midpiece mitochondria, we followed the behavior of spermatozoa within the female genital tract after natural mating. When examined 15 min after coitus, many spermatozoa were around the opening of the uterotubal junction. Spermatozoa that entered the uterotubal junction were seemingly not moving, yet they steadily migrated toward the isthmus at a speed only time-lapse video recording could demonstrate. Many spermatozoa reaching the lower isthmus were motile. The site where spermatozoa attached and detached from the isthmus epithelium shifted from the lower to the upper segment of the isthmus with time. Virtually all the live spermatozoa within the lower isthmus were acrosome intact, whereas many of the actively motile spermatozoa in the upper isthmus were acrosome reacted. As far as we could observe, all the spermatozoa we found within the lumen of the ampulla and the cumulus oophorus were acrosome reacted. Even though we saw only a very few spermatozoa within the ampulla during fertilization, all were associated with, or were already within, oocytes, indicating that mouse fertilization in vivo is extremely efficient.

Chloroplast and nuclear photorelocation movements.

Chloroplasts move toward weak light to increase photosynthetic efficiency, and migrate away from strong light to protect chloroplasts from photodamage and eventual cell death. These chloroplast behaviors were first observed more than 100 years ago, but the underlying mechanism has only recently been identified. Ideal plant materials, such as fern gametophytes for photobiological and cell biological approaches, and Arabidopsis thaliana for genetic analyses, have been used along with sophisticated methods, such as partial cell irradiation and time-lapse video recording under infrared light to study chloroplast movement. These studies have revealed precise chloroplast behavior, and identified photoreceptors, other relevant protein components, and novel actin filament structures required for chloroplast movement. In this review, our findings regarding chloroplast and nuclear movements are described.

Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality

Noninvasive markers of embryo quality are being sought to improve IVF success. The present study aimed to discover possible associations between embryo division kinetics in the cleavage stage, the subsequent ability of human embryos to reach the blastocyst stage and the resulting blastocyst morphology. A retrospective cohort study analysed 834 embryos from 165 oocyte donation couples using a time-lapse monitoring system that allowed the recording of the exact timings for key events related to embryo development. Timing parameters were categorized into four quartiles. The probability of an embryo developing to a blastocyst was linked to a strict chronology of development. To further evaluate the relationships between these morphokinetic parameters and subsequent blastocyst formation, the ensuing blastocyst morphology was compared with a viability score based on a hierarchical classification of the cleavage-stage morphokinetic parameters. It is concluded that the kinetics of early embryo development and the potential for human embryos to develop to the blastocyst stage on day 5 are closely related and that time-lapse-based evaluation of the exact timing of early events in embryo development is a promising tool for the prediction of blastocyst formation and quality according to the proposed model.Assisted reproduction treatment has helped several thousand infertile couples to start a family. Even though treatment is presently very successful, there is still scope for improvement in the process of selecting the embryos for transfer that have the highest implantation potential. An improved selection procedure may be able to increase the pregnancy rates, or alternatively, to reduce the number of embryos transferred while maintaining high pregnancy rates.We report the results after using an incubator with time-lapse video recording and image analysis to determine the timing of key events during embryo development. The events were assessed as potential markers of embryo morphology and development. One of the major factors that has already helped to increase success rates is extended embryo culture and blastocyst transfer on day 5 of development, because at this stage the embryo has stronger developmental competence. Also embryo/uterine synchrony is better achieved by a blastocyst-stage than a cleavage-stage embryo. However, extended culture in vitro is time consuming and extended exposure to artificial culture conditions in vitro may stress the embryo and could have negative side effects. For that reason, we sought evidence in the data obtained from the time-lapse monitoring system for a link between timing of early embryo cell divisions and the probability of subsequent development of embryos into blastocysts. The purpose of this was to avoid extended culture if blastocyst development could instead be reliably predicted from time-lapse images of the early cleavage states.

Hearing Preservation and Nanotechnology-based Targeted Drug Delivery Future in Cochlear Implantation?

Cochlear Implantation (CI) remains as one of the greatest medical achievements in modern medicine. New and innovative strategies continue to be developed to optimize and improve the functional results of CI surgery. Preservation of residual hearing through a-traumatic surgical techniques and electrode arrays may alter indications. Conditions with profound SNHL with preserved low tone hearing may have several causes and pathology may vary accordingly. In patients with progressive adult-onset SNHL neurons may be conserved even after long duration of deafness. IHCs and OHCs, supporting cells, ganglion cells and dendrites may be preserved in the apical region while in the lower turn despite atrophic organ of Corti and loss of lamina fibers ganglion cells can be present even after 28 years duration of deafness. These spiral ganglion cells may be excellent targets for electric stimulation using EAS technique that combines electric and acoustic stimulation in the same ear and utilizes both low frequency acoustic hearing and electric stimulation of preserved neurons. At the moment we are trying to elucidate the mechanism responsible for this preservation in humans and to use this knowledge for future therapy. Nano-technology may offer new possibilities for focused release of drugs and possibly genes to the inner ear. European project“NanoEar”is a concerted action to develop 3rd generation of nanoparticles (NP) for treatment of inner ear deafness. One goal is to target drugs and genes to specific inner ear cells through so-called multifunctional NP which are degradable, non-toxic, traceable and can be released in a controlled and biocompatible way. The small size of the NP may give new properties for technical advancement but risks must also be thoroughly evaluated. Uppsala is a Swedish partner to evaluate NP uptake in vitro in both human and animal spiral ganglion neurons. In my presentation I will show the system of culture spiral ganglion cells and demonstrate their locomotive behaviour and NP intracellular uptake using time lapse video recording and combined immunofluorescence and confocal microscopy.

P102 CLEVER-1 mediates the transmigration of B cells across human hepatic sinusoidal endothelium

IntroductionLymphocytes are recruited via the unique hepatic sinusoidal channels during chronic inflammatory liver diseases. This low shear vascular bed is lined by hepatic sinusoidal endothelium (HSEC) which lacks certain conventional...

Identification and evolutionary implications of neurotransmitter–ciliary interactions underlying the behavioral response to hypoxia inLymnaea stagnalisembryos

Acceleration of embryonic rotation is a common response to hypoxia among pond snails. It was first characterized in Helisoma trivolvis embryos, which have a pair of sensorimotor neurons that detect hypoxia and release serotonin onto postsynaptic ciliary cells. The objective of the present study was to determine how the hypoxia response is mediated in Lymnaea stagnalis, which differ from H. trivolvis by having both serotonergic and dopaminergic neurons, and morphologically distinct ciliated structures at comparative stages of embryonic development. Time-lapse video recordings of the rotational behavior in L. stagnalis revealed similar rotational features to those previously observed in H. trivolvis, including rotational surges and rotational responses to hypoxia. Serotonin and dopamine increased the rate of rotation with similar potency. In contrast, serotonin was more potent than dopamine in stimulating the ciliary beat frequency of isolated pedal cilia. Isolated apical plate cilia displayed an irregular pattern of ciliary beating that precluded the measurement of ciliary beat frequency. A qualitative assessment of ciliary beating revealed that both serotonin and dopamine were able to stimulate apical plate cilia. The ciliary responses to dopamine were reversible in both pedal and apical plate cilia, whereas the responses to serotonin were only reversible at concentrations below 100 μmol l(-1). Mianserin, a serotonin receptor antagonist, and SKF83566, a dopamine receptor antagonist, effectively blocked the rotational responses to serotonin and dopamine, respectively. The rotational response to hypoxia was only partially blocked by mianserin, but was fully blocked by SKF83566. These data suggest that, despite the ability of serotonin to stimulate ciliary beating in L. stagnalis embryos, the rotational response to hypoxia is primarily mediated by the transient apical catecholaminergic neurons that innervate the ciliated apical plate.

Immobilized Pool of NCAM180 in the Postsynaptic Membrane Is Homeostatically Replenished by the Flux of NCAM180 from Extrasynaptic Regions

Homeostatic mechanisms maintaining high levels of adhesion molecules in synapses over prolonged periods of time remain incompletely understood. We used fluorescence recovery after photobleaching experiments to analyze the steady state turnover of the immobile pool of green fluorescent protein-labeled NCAM180, the largest postsynaptically accumulating isoform of the neural cell adhesion molecule (NCAM). We show that there is a continuous flux of NCAM180 to the postsynaptic membrane from nonsynaptic regions of dendrites by diffusion. In the postsynaptic membrane, the newly delivered NCAM180 slowly intermixes with the immobilized pool of NCAM180. Preferential immobilization and accumulation of NCAM180 in the postsynaptic membrane is reduced after disruption of the association of NCAM180 with the spectrin cytoskeleton and in the absence of the homophilic interactions of NCAM180 in synapses. Our observations indicate that the homophilic interactions and binding to the cytoskeleton promote immobilization of NCAM180 and its accumulation in the postsynaptic membrane. Flux of NCAM180 from extrasynaptic regions and its slow intermixture with the immobile pool of NCAM180 in the postsynaptic membrane may be important for the continuous homeostatic replenishment of NCAM180 protein at synaptic contacts without compromising the long term synaptic contact stability.

Effect of doublecortin on self-renewal and differentiation in brain tumor stem cells.

Analysis of microarray probe data from glioma patient samples, in conjunction with patient Kaplan-Meier survival plots, indicates that expression of a glioma suppressor gene doublecortin (DCX) favors glioma patient survival. From neurosphere formation in culture, time-lapse microscopic video recording, and tumor xenograft, we show that DCX synthesis significantly reduces self-renewal of brain tumor stem cells (BTSC) in human primary glioma (YU-PG, HF66) cells from surgically removed human glioma specimens and U87 cells in vitro and in vivo. Time-lapse microscopic video recording revealed that double transfection of YU-PG, HF66, and U87 cells with DCX and neurabin II caused incomplete cell cycle with failure of cytokinesis, that is, endomitosis by dividing into three daughter cells from one mother BTSC. Activation of c-jun NH2-terminal kinase 1 (JNK1) after simvastatin (10 nM) treatment of DCX(+) neurabin II(+) BTSC from YU-PG, HF66, and U87 cells induced terminal differentiation into neuron-like cells. dUTP nick end labeling data indicated that JNK1 activation also induced apoptosis only in double transfected BTSC with DCX and neurabin II, but not in single transfected BTSC from YU-PG, HF66, and U87 cells. Western blot analysis showed that procaspase-3 was induced after DCX transfection and activated after simvastatin treatment in YU-PG, HF66, and U87 BTSC. Sequential immunoprecipitation and Western blot data revealed that DCX synthesis blocked protein phosphatase-1 (PP1)/caspase-3 protein-protein interaction and increased PP1-DCX interaction. These data show that DCX synthesis induces apoptosis in BTSC through a novel JNK1/neurabin II/DCX/PP1/caspase-3 pathway.