Qualitative Exploration Toward the Development of a Parent-Rated Scale for Insomnia in Children with Autism Spectrum Disorder.

Toward the development of a new parent-rating for insomnia, this multi-site qualitative study explored sleep problems and related impacts in children with autism spectrum disorder (ASD) and their families. To ensure content validity of the measure, we conducted six focus groups with caregivers (N = 25) of 24 children (age 3 to 18 years) with ASD. Based on parent report, all children had a history of mild or greater insomnia. The focus group transcripts were systematically coded to identify major themes. Verbatim comments from caretakers were used to generate 134 candidate items. Further review by the research team and an expert panel followed by individual cognitive interviews with 12 parents reduced the item bank to 40. The thematic analysis of focus group transcripts identified 7 categories: (1) Trouble falling asleep; (2) trouble staying asleep; (3) early morning waking; (4) bedtime routines; (5) parental strategies for bedtime management; (6) impact of sleep problems on the child; and (7) impact of sleep problems on the family. The Flesch Kincaid Grade Level of the 40-item version was 7.2 (seventh grade reading level). Insomnia in children with ASD shares features in common with insomnia in the general pediatric population. However, perhaps owing to autistic features such as insistence on sameness, sensory sensitivities, communication impairments, insomnia in children with ASD appears to have unique behavioral manifestations. Content validity and item clarity of the 40-item bank were supported by expert panel review and cognitive interviews with caregivers of children with ASD.

Feasibility of Actigraphy for Evaluating Sleep and Daytime Physical Activity in Children with Autism Spectrum Disorder.

This research evaluated the feasibility of actigraphy to measure sleep and physical activity in children (ages 2-8 years) with autism spectrum disorder (ASD). We also explored associations between sleep and physical activity. Validated screening measures established eligibility. Questionnaires, diaries, and 5 days and 5 nights of actigraphy monitoring were used to collect data. Of the 32 children enrolled, 27 (84.4%) completed actigraphy monitoring. Based on the median steps per day, children with high physical activity had lower total sleep time and more disruptive behaviors than children with low physical activity. Findings support the feasibility of using actigraphy to measure sleep and physical activity in children with ASD. Larger studies are needed to evaluate interactions of physical activity on sleep in this population.

Drift-kelp suppresses foraging movement of overgrazing sea urchins.

Sea urchins have the capacity to destructively overgraze kelp beds and cause a wholesale shift to an alternative and stable 'urchin barren' reef state. However, destructive grazing appears labile and contingent on behavioural shift. Changes in supply of allochthonous 'drift-kelp' food are hypothesised as a trigger of change in urchin grazing behaviour, yet field tests are lacking. Here we conduct a suite of in situ behavioural surveys and manipulative experiments within kelp beds and on urchin barrens to examine foraging movements and evidence for a behavioural switch to an 'overgrazing mode' by the Australian urchin Heliocidaris erythrogramma (Echinometridae). Tracking urchins using time-lapse photography revealed urchin foraging to conform to a random walk model within kelp beds and on barrens. However, many individuals tended towards local movement within proximal crevices and movement was reduced in kelp beds compared to barrens. Directional movement of urchins toward newly available kelp was experimentally inducible, consistent with locally observed 'mobile-feeding-fronts' that develop at barrens-kelp interfaces. Habitat-specific feeding modes were also evidenced by herbivory assays which revealed urchin grazing rates to be high on both drift-kelp and standing kelp on barren grounds, while drift-kelp but not standing kelp was consumed at high rates within kelp beds. Time-lapse tracking of urchin foraging before/after addition of drift-kelp revealed a reduction in foraging across the reef surface after drift-kelp capture. Collectively, results indicate that the availability of drift-kelp is a pivotal trigger in determining urchin feeding modes which thus mediates the shift between alternative stable states for rocky reef ecosystems.

Reduced resistance to sediment-trapping turfs with decline of native kelp and establishment of an exotic kelp.

Understanding the strength and type of interactions among species is vital to anticipate how ecosystems will respond to ongoing anthropogenic stressors. Here, we examine the ecological function of native (Ecklonia radiata) and invasive (Undaria pinnatifida) kelps in resisting shifts to sediment-trapping turf on reefs within the highly urbanized temperate Port Phillip Bay (PPB), Australia. Short-term (30 days) and long-term (232 days) manipulations demonstrated that kelp laminae can clear and maintain the substratum free of turfs, while conversely, removal of kelp leads to a proliferation of turfs. Analyses looking at the relationship between total length of E. radiata and U. pinnatifida and the area cleared of turf algae showed that the clearing effect of E. radiata over a year was greater than that of U. pinnatifida due to the annual die-back of the invasive. A natural experiment (608 days) identified that ongoing sea urchin (Heliocidaris erythrogramma) grazing led to native kelp bed decline, facilitating turf dominance. Even though U. pinnatifida establishes once native beds are disturbed, its ecological function in clearing turf is weaker than E. radiata, given its annual habit. In PPB, turfs represent the more persistent and problematic algal group and are likely changing the structure, function, and energy flows of shallow temperate reefs in this urbanised embayment.

Photon Doppler velocimetry measurements of transverse surface velocities.

The goal of this work was to develop a technique for making transverse surface velocity measures utilizing Photon Doppler Velocimetry (PDV). Such a task is achieved by transmitting light and collecting Doppler-shifted light at an angle relative to the normal axis, where measured velocities are representative of a component of the transverse velocity. Because surface characteristics have an intrinsic effect on light scatter, different surface preparations were explored to direct reflectivity, including diffusion by means of sandpapering, or increasing retroreflectivity by coating with microspheres, milling v-cuts, and electrochemically etching grooves. Testing of these surface preparations was performed using an experiment featuring a 30 mm diameter aluminum disk rotating at 6000 or 6600 RPM. A single PDV collimator was positioned along the rotational axis of the disk at various angles, resolving the apparent transverse velocity. To characterize surface preparations, light return and velocities were recorded as a function of probe angle ranging from 0° to 51° from the surface normal for each preparation. Polished and electrochemically etched surfaces did not provide enough reflected light to resolve a beat frequency; however, sandpapered surfaces, retroreflective microspheres, and milled v-cuts provided adequate reflected light for incidence angles up to 51°. Applications of the surface preparations were then studied in gas gun experiments. Retroreflective microspheres were studied in a planar impact experiment, and milled v-cuts were studied in an oblique impact experiment. A normal and transverse profile of particle velocity was resolved in the oblique impact experiment.

Participation levels in 25 Community-based participatory research projects.

This analysis describes the nature of community participation in National Institutes of Health and Centers for Disease Control and Prevention funded community-based participatory research (CBPR) projects, and explores the scientific and social implications of variation in community participation. We conducted in-depth interviews in 2012 with professional and community researchers from 25 CBPR projects in the Southeast US. Interview topics focused on participants' experiences with the nature and conduct of their CBPR project. Projects were rated on community participation in 13 components of research. Projects varied substantially in community participation. Some projects had community participation in only two to three components; others had participation in every component. Some professional researchers were deliberate in their inclusion of community participation in all aspects of research, others had community participation in some aspects, and others were mainly concerned that community members had the opportunity to participate in the study. Findings suggest a need for a standardized rubric for community-based research that facilitates delineation of approaches and procedures that are effective and efficient. Little actual community participation may also result in negative social impacts for communities.

Using molecular prey detection to quantify rock lobster predation on barrens-forming sea urchins.

We apply qPCR molecular techniques to detect in situ rates of consumption of sea urchins (Centrostephanus rodgersii and Heliocidaris erythrogramma) by rock lobsters (Jasus edwardsii). A non-lethal method was used to source faecal samples from trap-caught lobsters over 2 years within two no-take research reserves. There was high variability in the proportion of lobsters with faeces positive for sea urchin DNA across years and seasons dependent on lobster size. Independent estimates of lobster predation rate on sea urchins (determined from observed declines in urchin abundances in the reserves relative to control sites) suggest that rates of molecular prey detection generally overestimated predation rates. Also, small lobsters known to be incapable of directly predating emergent sea urchins showed relatively high rates of positive tests. These results indicate that some lobsters ingest non-predatory sources of sea urchin DNA, which may include (i) ingestion of C. rodgersii DNA from the benthos (urchin DNA is detectable in sediments and some lobsters yield urchin DNA in faeces when fed urchin faeces or sediment); (ii) scavenging; and/or predation by rock lobsters on small pre-emergent urchins that live cryptically within the reef matrix (although this possibility could not be assessed). While the DNA-based approach and direct monitoring of urchin populations both indicate high predation rates of large lobsters on emergent urchins, the study shows that in some cases absolute predation rates and inferences of predator-prey interactions cannot be reliably estimated from molecular signals obtained from the faeces of benthic predators. At a broad semi-quantitative level, the approach is useful to identify relative magnitudes of predation and temporal and spatial variability in predation.

The effect of disease on the urinary purine metabolite concentrations in dogs.

This prospective study was designed to determine the urinary concentrations of purine metabolites in healthy and diseased dogs. The goals were to test the hypothesis that urine concentrations of terminal purine metabolites will identify dogs with diseases that disturb purine degradation. Five hundred and sixty-three client-owned dogs admitted sequentially to the veterinary medical centre were included. Dogs were divided into groups on the basis of their disease. Urine concentrations of xanthine, uric acid, allantoin and creatinine were measured by high-pressure liquid chromatography. Xanthine and uric acid to creatinine ratios were significantly increased in dogs with chronic kidney disease (p = 0.01). The uric acid to creatinine ratio was significantly increased in dogs with cancer compared with clinically healthy dogs (p = 0.04), and significantly increased in dogs receiving chemotherapy for their disease (p < 0.01). Compared to clinically healthy dogs, xanthine and uric acid to creatinine ratios were significantly increased in dogs with hyperadrenocorticism (p < 0.01, and 0.04, respectively). Therefore, the results of this study found that the urinary concentrations of purine metabolites in dogs are significantly impacted by systemic disease. Cancer, chronic kidney disease, and hyperadrenocorticism are associated with altered concentrations of urinary purine metabolites in dogs.

Evaluation of reproductive and economic outcomes of dairy heifers inseminated at induced estrus or at fixed time after a 5-day or 7-day progesterone insert-based ovulation synchronization protocol.

The objectives of the current experiment were to evaluate the reproductive performance and economic outcome of 3 synchronization strategies for first artificial insemination (AI) of dairy heifers. Holstein heifers from 2 herds (site A, California, n=415; site B, Idaho, n=425) were assigned to 1 of 3 treatments. Heifers assigned to the AI on estrus (AIE) treatment received an injection of 25mg of PGF(2α) at enrollment (d 0) and every 11 d thereafter until AI occurred. Heifers assigned to the CIDR5 treatment received a controlled internal drug release insert (CIDR) containing 1.38 g of progesterone, which was removed 5 d later concomitantly with an injection of 25mg of PGF(2α), and received fixed-time AI (TAI) concomitantly with an injection of 100 µg of GnRH 53 to 60 h later. Heifers assigned to the CIDR7 treatment received a CIDR insert, which was removed 7 d later concomitantly with an injection of 25mg of PGF(2α), and received TAI concomitantly with an injection of 100 µg of GnRH 53 to 60 h later. Heifers were observed for estrus and inseminated up to 98 and 73 d after enrollment in sites A and B, respectively. Thereafter, heifers were moved to pens with bulls and considered failure to conceive to AI if still not pregnant at the end of the observation period. Economic outcomes were based on cost of synchronization protocol (CIDR treatment=$11, PGF(2α) or GnRH treatments=$2.5/treatment, estrous detection=$0.80/heifer per day), rearing cost ($2.75/heifer per day), and economic loss if a heifer did not conceive to first AI ($150). Input cost of the reproductive programs=synchronization protocol cost + semen cost + rearing cost + replacement cost. Pregnancy per AI (P/AI) 38 ± 3 d after first AI was greatest for AIE heifers (61.1%) followed by CIDR5 (44.8%) and CIDR7 (35.7%) heifers. Furthermore, P/AI 73 ± 7 d after first AI was greatest for AIE (58.8%) and tended to be greater for CIDR5 (42%) than for CIDR7 (34.1%) heifers. The percentage of heifers that had spontaneous luteolysis from CIDR insertion to CIDR removal was reduced for CIDR5 compared with CIDR7 (13.8 vs 31.8%). Pregnancy rate was greatest for AIE heifers but did not differ between CIDR5 [adjusted hazard ratio (95% confidence interval)=0.75 (0.63, 0.90)] and CIDR7 [adjusted hazard ratio (95% confidence interval)=0.65 (0.54, 0.77)] heifers. Consequently, rearing cost and input cost of AIE heifers ($67.1 ± 4.4 and -$107.1 ± 7.0, respectively) were reduced compared with CIDR5 ($86.9 ± 5.1 and -$143.4 ± 8.1, respectively) and CIDR7 ($98.3 ± 5.1 and -$156.5 ± 8.2, respectively) heifers, but no differences were observed between CIDR5 and CIDR7 heifers.

Short communication: characterization of early postpartum estrous behavior and ovulation in lactating dairy cows using radiotelemetry.

The objective of this study was to describe early postpartum estrous behavior and ovulation in lactating dairy cows using radiotelemetry. Cows (n=50) were continuously monitored for behavioral estrus with a radiotelemetric system, HeatWatch II (CowChips LLC, Manalapan, NJ), from d 14 to approximately d 49 postpartum. Blood collection for analysis of progesterone and ovarian ultrasonography were performed once weekly starting on d 14. First ovulation was associated with behavioral estrus in 5 cows and occurred at 28.2±10.8 d (mean±SD; range 17 to 40 d). The average duration of estrus was 6.0±4.9 h (range 3 to 12.2 h), and the mean number of standing events was 18.4±8.9 (range 4 to 26). Based on progesterone concentrations of ≥1 ng/mL, estimated first postpartum ovulation occurred at 25.1±10.4 d (range 10 to 49 d) for 38 animals without evidence of behavioral estrus. The interval to estimated first ovulation without behavioral estrus was not different from the interval to first ovulation associated with behavioral estrus. Level of milk production and body condition score loss did not affect the interval to estimated first ovulation without estrus or first ovulation associated with estrus. Six animals did not show evidence of ovulation based on progesterone concentration, whereas 1 cow showed evidence of estrous behavior on the day before removal from the study. The majority of first postpartum ovulations (38/43; 88.4%) were not associated with behavioral estrus.

Marine reserves reduce risk of climate-driven phase shift by reinstating size- and habitat-specific trophic interactions.

Spatial closures in the marine environment are widely accepted as effective conservation and fisheries management tools. Given increasing human-derived stressors acting on marine ecosystems, the need for such effective action is urgently clear. Here we explore mechanisms underlying the utility of marine reserves to reinstate trophic dynamics and to increase resilience of kelp beds against climate-driven phase shift to sea urchin barrens on the rapidly warming Tasmanian east coast. Tethering and tagging experiments were used to examine size- and shelter-specific survival of the range-extending sea urchin Centrostephanus rodgersii (Diadematidae) translocated to reefs inside and outside no-take Tasmanian marine reserves. Results show that survival rates of C. rodgersii exposed on flat reef substratum by tethering were approximately seven times (small urchins 10.1 times; large urchins 6.1 times) lower on protected reef within marine reserve boundaries (high abundance of large predatory-capable lobsters) compared to fished reef (large predatory lobsters absent). When able to seek crevice shelter, tag-resighting models estimated that mortality rates of C. rodgersii were lower overall but remained 3.3 times (small urchins 2.1 times; large urchins 6.4 times) higher in the presence of large lobsters inside marine reserves, with higher survival of small urchins owing to greater access to crevices relative to large urchins. Indeed, shelter was 6.3 times and 3.1 times more important to survival of small and large urchins, respectively, on reserved relative to fished reef. Experimental results corroborate with surveys throughout the range extension region, showing greater occurrence of overgrazing on high-relief rocky habitats where shelter for C. rodgersii is readily available. This shows that ecosystem impacts mediated by range extension of such habitat-modifying organisms will be heterogeneous in space, and that marine systems with a more natural complement of large and thus functional predators, as achievable within no-take reserves, will minimize local risk of phase shifts by reinstating size and habitat-specific predator-prey dynamics eroded by fishing. Importantly, our findings also highlight the crucial need to account for the influence of size dynamics and habitat complexity on rates of key predator-prey interactions when managing expectations of ecosystem-level responses within marine reserve boundaries.

Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): standardised reporting for model reproducibility, interoperability, and data sharing.

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.

Microfluidic laminate-based phantom for diffusion tensor-magnetic resonance imaging (DT-MRI).

This paper reports fabrication of a magnetic resonance imaging (MRI) phantom created by stacking of multiple thin polydimethylsiloxane (PDMS) layers. PDMS is spin coated on SU-8 molds to obtain the desired layer thickness and imprints of the microchannel patterns that define the phantom geometry. This paper also identifies the unique challenges related to the fabrication and assembly of multiple thin layers and reports for the first time assembly of a large number of thin laminates of this nature. Use of photolithography techniques allows us to create a wide range of phantom geometries. The target dimensions of the phantoms reported here are (i) a stack of 30 thin PDMS layers of 10 µm thickness (ii) curved 5 µm × 5 µm microchannels with 8.7 µm spacing, and (iii) straight 5 µm × 5 µm microchannels with 3.6 µm spacing. SEM scans of the assembled phantoms show open microchannels and a monolithic cross-section with no visible interface between PDMS layers. Based on the results of diffusion tensor magnetic resonance imaging (DT-MRI) scan, the anisotropic diffusion of water molecules due to the physical restriction of the microchannels was detected, which means that the phantom can be used to calibrate and optimize MRI instrumentation.

Monitoring associations between clade-level variation, overall community structure and ecosystem function in enhanced biological phosphorus removal (EBPR) systems using terminal-restriction fragment length polymorphism (T-RFLP).

The role of Candidatus "Accumulibacter phosphatis" (Accumulibacter) in enhanced biological phosphorus removal (EBPR) is well established but the relevance of different Accumulibacter clades to the performance of EBPR systems is unknown. We developed a terminal-restriction fragment length polymorphism (T-RFLP) technique to monitor changes in the relative abundance of key members of the bacterial community, including Accumulibacter clades, in four replicate mini-sequencing batch reactors (mSBRs) operated for EBPR over a 35-day period. The ability of the T-RFLP technique to detect trends was confirmed using fluorescence in situ hybridisation (FISH). EBPR performance varied between reactors and over time; by day 35, performance was maintained in mSBR2 whilst it had deteriorated in mSBR1. However, reproducible trends in structure-function relationships were detected in the mSBRs. EBPR performance was strongly associated with the relative abundance of total Accumulibacter. A shift in the ratio of the dominant Accumulibacter clades was also detected, with Type IA associated with good EBPR performance and Type IIC associated with poor EBPR performance. Changes in ecosystem function of the mSBRs in the early stages of the experiment were more closely associated with changes in the abundance of (unknown) members of the flanking community than of either Accumulibacter or Candidatus "Competibacter phosphatis". This study therefore reveals a hitherto unrecorded and complex relationship between Accumulibacter clades, the flanking community and ecosystem function of laboratory-scale EBPR systems.

Overfishing reduces resilience of kelp beds to climate-driven catastrophic phase shift.

A key consideration in assessing impacts of climate change is the possibility of synergistic effects with other human-induced stressors. In the ocean realm, climate change and overfishing pose two of the greatest challenges to the structure and functioning of marine ecosystems. In eastern Tasmania, temperate coastal waters are warming at approximately four times the global ocean warming average, representing the fastest rate of warming in the Southern Hemisphere. This has driven range extension of the ecologically important long-spined sea urchin (Centrostephanus rodgersii), which has now commenced catastrophic overgrazing of productive Tasmanian kelp beds leading to loss of biodiversity and important rocky reef ecosystem services. Coincident with the overgrazing is heavy fishing of reef-based predators including the spiny lobster Jasus edwardsii. By conducting experiments inside and outside Marine Protected Areas we show that fishing, by removing large predatory lobsters, has reduced the resilience of kelp beds against the climate-driven threat of the sea urchin and thus increased risk of catastrophic shift to widespread sea urchin barrens. This shows that interactions between multiple human-induced stressors can exacerbate nonlinear responses of ecosystems to climate change and limit the adaptive capacity of these systems. Management actions focused on reducing the risk of catastrophic phase shift in ecosystems are particularly urgent in the face of ongoing warming and unprecedented levels of predator removal from the world's oceans.

Subject-specific, multiscale simulation of electrophysiology: a software pipeline for image-based models and application examples.

Many simulation studies in biomedicine are based on a similar sequence of processing steps, starting from images and running through geometric model generation, assignment of tissue properties, numerical simulation and visualization of the results--a process known as image-based geometric modelling and simulation. We present an overview of software systems for implementing such a sequence both within highly integrated problem-solving environments and in the form of loosely integrated pipelines. Loose integration in this case indicates that individual programs function largely independently but communicate through files of a common format and support simple scripting, so as to automate multiple executions wherever possible. We then describe three specific applications of such pipelines to translational biomedical research in electrophysiology.

A molecular approach to identify prey of the southern rock lobster.

We demonstrate the use of molecular techniques to detect specific prey consumed by the southern rock lobster (Jasus edwardsii). A quick and non-lethal method was used to collect rock lobster faecal material and a molecular protocol was employed to isolate prey DNA from faecal samples. The isolated DNA was amplified using the polymerase chain reaction (PCR) with PCR primers designed to target specific prey items. Feeding experiments determined that DNA from black-lipped abalone (Haliotis rubra) and sea urchins (Centrostephanus rodgersii and Heliocidaris erythrogramma) can be detected in rock lobster faecal samples within seven hours and remains present for up to 60 h after ingestion.

A randomised controlled trial of the reciprocating syringe in arthrocentesis.

OBJECTIVE: To evaluate the outcomes of arthrocentesis with the new highly controllable, one handed reciprocating procedure syringe compared with a conventional syringe. METHODS: 100 arthrocentesis procedures were randomised between the reciprocating syringe and the conventional syringe. Outcome measures included patient pain, procedure duration, operator satisfaction, synovial fluid volume, cell counts, and complications. RESULTS: 50 arthrocentesis procedures with the conventional syringe resulted in a mean (SD) procedure time of 3.39 (1.88) minutes, a mean VAPS (patient pain) score of 5.35 (3.15), and a mean VASS (operator satisfaction) score of 4.88 (1.92); 30 of the 50 subjects experienced moderate to severe pain (VAPS score 5 or greater) during arthrocentesis. In contrast, the reciprocating syringe resulted in a reduced procedure time of 1.94 (1.14) minutes (p<0.001), a reduced VAPS (patient pain) score of 2.54 (1.60) (p<0.001), and an increased VASS (operator satisfaction) score of 8.91 (0.79) (p<0.001). Only five of the 50 of subjects experienced moderate to severe pain with the reciprocating syringe. Synovial cell counts were similar between the two syringes (p>0.05), but there was a trend toward greater volume (greater synovial fluid yield) and fewer red blood cells with the reciprocating syringe. CONCLUSIONS: Arthrocentesis with a conventional syringe results in moderate to severe pain in 60% of subjects. The reciprocating syringe prevents significant pain, reduces procedure time, and improves physician performance of arthrocentesis. The reciprocating syringe is superior to the conventional syringe in arthrocentesis.

Caspase-9 regulation: an update.

The complex sequence of events leading to apoptotic cell death is governed by an elaborate regulatory scheme involving the actions of both initiator and executioner proteases. Among the most intensively studied of the initiator caspases is caspase-9, an essential throughput element in the so-called intrinsic or mitochondrially gated pathway of apoptosis. Previous reviews have described the proteolytic processing and activation of this protease in much detail; here we provide an update on caspase-9 regulation. A comprehensive description of the intra- and intermolecular events involved in modulating protein expression and activity are presented. Particular emphasis is placed on the role alternative splicing plays in the expression of functionally divergent protein isoforms, as well as, the participation of specific post-translational events in regulating caspase-9 activity. Such discrete modulation in reported activity characterizes, not only the pivotal role of this protease in the final commitment process itself, but also emphasizes the more general interplay that exists between mutually opposing cytotoxic and cytoprotective influences in maintaining cellular homeostasis.

Requirement for SAPK-JNK signaling in the induction of apoptosis by ribosomal stress in REH lymphoid leukemia cells.

The present studies examined performance of SAPK cascades and apoptotic commitment following ribosomal trauma in REH lymphoid leukemia cells. Ribostatic insults included disruption of ribosomal activity by mechanistically dissimilar agents such as blasticidin-S (BCS) (which binds 28S-rRNA to block peptidyl bond formation), kasugamycin (KSM) (which binds 18S-rRNA to prevent translational initiation), and cycloheximide (CHX) (which blocks A-site to P-site translocation of peptidyl-tRNA). Exposure of REH cells to BCS elicited DNA degradation and apoptotic cytolysis. BCS stimulated JNK1/JNK2 and p38, and their shared targets c-Jun and ATF2. Inhibition of JNK1/JNK2 (but not of p38) antagonized blasticidin-induced apoptosis, whereas targeting alternative ribosomal sites with KSM or CHX limited translation, but failed to activate the SAPK cascade or initiate apoptosis. Our findings indicate that interference with 28S-rRNA by BCS initiates apoptosis in REH cells through recruitment of SAPK-JNK signaling. Disparities between the lethal actions of BCS, KSM, and CHX appear to reflect established differences in the subribosomal targets of these agents. We propose that the SAPK cascade comprises an essential mechanism for the transduction of specific lethal stress signals emanating from active ribosomes, and that interference with the 28S-rRNA, rather than the peptidyl transfer center of the large subunit, is critical to apoptotic commitment.