A Computer-Assisted 3D Model for Analyzing the Aggregation of Tumorigenic Cells Reveals Specialized Behaviors and Unique Cell Types that Facilitate Aggregate Coalescence

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named “facilitators” and “probes.” A third cell type, the “dervish”, is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.     

Evaluating Multi-Level Models to Test Occupancy State Responses of Plethodontid Salamanders

Plethodontid salamanders are diverse and widely distributed taxa and play critical roles in ecosystem processes. Due to salamander use of structurally complex habitats, and because only a portion of a population is available for sampling, evaluation of sampling designs and estimators is critical to provide strong inference about Plethodontid ecology and responses to conservation and management activities. We conducted a simulation study to evaluate the effectiveness of multi-scale and hierarchical single-scale occupancy models in the context of a Before-After Control-Impact (BACI) experimental design with multiple levels of sampling. Also, we fit the hierarchical single-scale model to empirical data collected for Oregon slender and Ensatina salamanders across two years on 66 forest stands in the Cascade Range, Oregon, USA. All models were fit within a Bayesian framework. Estimator precision in both models improved with increasing numbers of primary and secondary sampling units, underscoring the potential gains accrued when adding secondary sampling units. Both models showed evidence of estimator bias at low detection probabilities and low sample sizes; this problem was particularly acute for the multi-scale model. Our results suggested that sufficient sample sizes at both the primary and secondary sampling levels could ameliorate this issue. Empirical data indicated Oregon slender salamander occupancy was associated strongly with the amount of coarse woody debris (posterior mean = 0.74; SD = 0.24); Ensatina occupancy was not associated with amount of coarse woody debris (posterior mean = -0.01; SD = 0.29). Our simulation results indicate that either model is suitable for use in an experimental study of Plethodontid salamanders provided that sample sizes are sufficiently large. However, hierarchical single-scale and multi-scale models describe different processes and estimate different parameters. As a result, we recommend careful consideration of study questions and objectives prior to sampling data and fitting models.     

Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA_257–264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific K^b:OVA_257–264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA_257–264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.     

Participation and Contribution in Crowdsourced Surveys

This paper identifies trends within and relationships between the amount of participation and the quality of contributions in three crowdsourced surveys. Participants were asked to perform a collective problem solving task that lacked any explicit incentive: they were instructed not only to respond to survey questions but also to pose new questions that they thought might-if responded to by others-predict an outcome variable of interest to them. While the three surveys had very different outcome variables, target audiences, methods of advertisement, and lengths of deployment, we found very similar patterns of collective behavior. In particular, we found that: the rate at which participants submitted new survey questions followed a heavy-tailed distribution; the distribution in the types of questions posed was similar; and many users posed non-obvious yet predictive questions. By analyzing responses to questions that contained a built-in range of valid response we found that less than 0.2% of responses lay outside of those ranges, indicating that most participants tend to respond honestly to surveys of this form, even without explicit incentives for honesty. While we did not find a significant relationship between the quantity of participation and the quality of contribution for both response submissions and question submissions, we did find several other more nuanced participant behavior patterns, which did correlate with contribution in one of the three surveys. We conclude that there exists an optimal time for users to pose questions early on in their participation, but only after they have submitted a few responses to other questions. This suggests that future crowdsourced surveys may attract more predictive questions by prompting users to pose new questions at specific times during their participation and limiting question submission at non-optimal times.     

Decoding the Traumatic Memory among Women with PTSD: Implications for Neurocircuitry Models of PTSD and Real-Time fMRI Neurofeedback

Posttraumatic Stress Disorder (PTSD) is characterized by intrusive recall of the traumatic memory. While numerous studies have investigated the neural processing mechanisms engaged during trauma memory recall in PTSD, these analyses have only focused on group-level contrasts that reveal little about the predictive validity of the identified brain regions. By contrast, a multivariate pattern analysis (MVPA) approach towards identifying the neural mechanisms engaged during trauma memory recall would entail testing whether a multivariate set of brain regions is reliably predictive of (i.e., discriminates) whether an individual is engaging in trauma or non-trauma memory recall. Here, we use a MVPA approach to test 1) whether trauma memory vs neutral memory recall can be predicted reliably using a multivariate set of brain regions among women with PTSD related to assaultive violence exposure (N=16), 2) the methodological parameters (e.g., spatial smoothing, number of memory recall repetitions, etc.) that optimize classification accuracy and reproducibility of the feature weight spatial maps, and 3) the correspondence between brain regions that discriminate trauma memory recall and the brain regions predicted by neurocircuitry models of PTSD. Cross-validation classification accuracy was significantly above chance for all methodological permutations tested; mean accuracy across participants was 76% for the methodological parameters selected as optimal for both efficiency and accuracy. Classification accuracy was significantly better for a voxel-wise approach relative to voxels within restricted regions-of-interest (ROIs); classification accuracy did not differ when using PTSD-related ROIs compared to randomly generated ROIs. ROI-based analyses suggested the reliable involvement of the left hippocampus in discriminating memory recall across participants and that the contribution of the left amygdala to the decision function was dependent upon PTSD symptom severity. These results have methodological implications for real-time fMRI neurofeedback of the trauma memory in PTSD and conceptual implications for neurocircuitry models of PTSD that attempt to explain core neural processing mechanisms mediating PTSD.     

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.     

Physiological determinants of the candidate physical ability test in firefighters

The purpose of this study was to examine the relative importance of physiological characteristics during firefighting performance, as assessed by the Candidate Physical Ability Test (CPAT). Subjects included career and volunteer firefighters aged 18-39 (N = 33). Upper- and lower-body strength, muscle endurance, lower body muscle power, body composition analysis, aerobic capacity, anaerobic fitness, and the heart rate (HR) and blood pressure response to stair climbing were assessed to determine the physiological characteristics of the subjects. To quantify firefighting performance, the CPAT was administered by members of the fire service. Absolute and relative mean power during the Wingate anaerobic cycling test (WAnT), relative peak power during the WAnT, and absolute maximal oxygen uptake (VO2max) were significantly higher in those who passed the CPAT (N = 18), compared to those who failed (N = 15; p < 0.01). Mean power during the WAnT, fatigue index during WAnT, absolute VO2max, upper body strength, grip strength, and the HR response to stair climbing were significantly related to CPAT performance time (p < 0.01). Absolute VO2max and anaerobic fatigue resistance during WAnT best predicted CPAT performance (Adj. R2 = 0.817; p < 0.001). Performance on the ceiling breach and pull was the only CPAT task that was not significantly related to the physiological characteristics assessed. Measures of anaerobic and cardiovascular fitness best predict overall CPAT performance, and individual task performance. Remedial programs aimed at improving firefighting performance should target anaerobic and aerobic fitness qualities.     

The effect of helmets on the risk of head and neck injuries among skiers and snowboarders: a meta-analysis

Background

The prevention of head injuries in alpine activities has focused on helmets. However, no systematic review has examined the effect of helmets on head and neck injuries among skiers and snowboarders.

Methods

We searched electronic databases, conference proceedings and reference lists using a combination of the key words “head injury or head trauma,” “helmet” and “skiing or snowboarding.” We included studies that used a control group; compared skiers or snowboarders with and without helmets; and measured at least one objectively quantified outcome (e.g., head injury, and neck or cervical injury).

Results

We included 10 case–control, 1 case–control/case-crossover and 1 cohort study in our analysis. The pooled odds ratio (OR) indicated that skiers and snowboarders with a helmet were significantly less likely than those without a helmet to have a head injury (OR 0.65, 95% confidence interval [CI] 0.55–0.79). The result was similar for studies that used controls without an injury (OR 0.61, 95% CI 0.36–0.92), those that used controls with an injury other than a head or neck injury (OR 0.63, 95% CI 0.52–0.80) and studies that included children under the age of 13 years (OR 0.41, 95% CI 0.27–0.59). Helmets were not associated with an increased risk of neck injury (OR 0.89, 95% CI 0.72–1.09).

Interpretation

Our findings show that helmets reduce the risk of head injury among skiers and snowboarders with no evidence of an increased risk of neck injury.Skiing and snowboarding are popular winter activities.¹ Estimates from numerous countries indicate that head injuries account for 9% to 19%, and neck injuries for 1% to 4%, of all injuries reported by ski patrols and emergency departments.^2–11 Rates of head and neck injuries have been reported between 0.09 and 0.46 per 1000 outings.¹² Head and neck injuries are disproportionately represented in cases of severe trauma, and traumatic brain injury is the leading cause of death and serious injury among skiers and snow-boarders.¹³ As far back as 1983, Oh and Schmid recommended mandatory helmet use for children while skiing.¹⁴Many studies of the relation between helmet use and head injuries among skiers and snowboarders have found a protective effect.^15–24 It has been suggested that the use of helmets may increase the risk of neck injury in a crash or fall.²⁵ This may be more evident among children because they have a greater head:body ratio than adults, and the additional size and weight of the helmet may increase the risk of neck injury in an otherwise routine fall.²⁶ We conducted a systematic review of the effect of helmets on head and neck injuries among skiers and snowboarders.