Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM

Over the last three decades, Cryo-TEM has developed into a powerful technique for high-resolution imaging of biological macromolecules in their native vitrified state. However, the method for vitrifying specimens onto EM grids is essentially unchanged - application of ～3 μL sample to a grid, followed by blotting and rapid plunge freezing into liquid ethane. Several trials are often required to obtain suitable thin (few hundred nanometers or less) vitrified layers amenable for cryo-TEM imaging, which results in waste of precious sample and resources. While commercially available instruments provide some level of automation to control the vitrification process in an effort to increase quality and reproducibility, obtaining satisfactory vitrified specimens remains a bottleneck in the Cryo-TEM pipeline. We describe here a completely novel method for EM specimen preparation based on small volume (picoliter to nanoliter) dispensing using inkjet technology. A first prototype system (Spotiton v0.5) demonstrates feasibility of this new approach for specimen vitrification. A piezo-electric inkjet dispenser is integrated with optical real-time cameras (100 Hz frame rate) to analyze picoliter to nanoliter droplet profiles in-flight and spreading dynamics on the grid, and thus provides a method to optimize timing of the process. Using TEM imaging and biochemical assays we demonstrate that the piezo-electric inkjet mechanism does not disrupt the structural or functional integrity of macromolecules. These preliminary studies provide insight into the factors and components that will need further development to enable a robust and repeatable technique for specimen vitrification using this novel approach.     

Consultation for Teachers and Children's Language and Literacy Development during Pre-Kindergarten

MyTeachingPartner (MTP) is a teacher professional development program designed to improve the quality of teacher-child interactions in pre-kindergarten classrooms and children's language and literacy development. The program includes language/literacy activities and two Web-based resources—video exemplars of effective interactions and individualized consultation—designed to support teachers' high quality implementation of these activities. This study examined the impacts of the MTP Web-based resources on the language and literacy development of 1,165 children during pre-kindergarten. Children whose teachers were randomly assigned to receive access to both the video exemplars and participated in consultation (MTP Consultancy n = 65) made greater gains in receptive language skills during pre-kindergarten compared to children whose teachers were randomly assigned to receive access to the video exemplars only (MTP Video Library n = 69). Further, among MTP Consultancy teachers, more hours of participating in the consultation process was positively associated with children's receptive language development, and more hours implementing the language/literacy activities was positively associated with children's language and literacy development. Implications for improving children's school readiness and promoting teachers' participation in professional development programs are discussed.     

Investigation of Genetic Structure between Deep and Shallow Populations of the Southern Rock Lobster,Jasus edwardsii in Tasmania,Australia

The southern rock lobster, Jasus edwardsii, shows clear phenotypic differences between shallow water (red coloured) and deeper water (pale coloured) individuals. Translocations of individuals from deeper water to shallower waters are currently being trialled as a management strategy to facilitate a phenotypic change from lower value pale colouration, common in deeper waters, to the higher value red colouration found in shallow waters. Although panmixia across the J. edwardsii range has been long assumed, it is critical to assess the genetic variability of the species to ensure that the level of population connectivity is appropriately understood and translocations do not have unintended consequences. Eight microsatellite loci were used to investigate genetic differentiation between six sites (three shallow, three deep) across southern Tasmania, Australia, and one from New Zealand. Based on analyses the assumption of panmixia was rejected, revealing small levels of genetic differentiation across southern Tasmania, significant levels of differentiation between Tasmania and New Zealand, and high levels of asymmetric gene flow in an easterly direction from Tasmania into New Zealand. These results suggest that translocation among Tasmanian populations are not likely to be problematic, however, a re-consideration of panmictic stock structure for this species is necessary.     

Modeling Long-Term Host Cell-Giardia lamblia Interactions in an In Vitro Co-Culture System

Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions.     

Rapid Accumulation of Endogenous Tau Oligomers in a Rat Model of Traumatic Brain Injury: POSSIBLE LINK BETWEEN TRAUMATIC BRAIN INJURY AND SPORADIC TAUOPATHIES*

Traumatic brain injury (TBI) is a serious problem that affects millions of people in the United States alone. Multiple concussions or even a single moderate to severe TBI can also predispose individuals to develop a pathologically distinct form of tauopathy-related dementia at an early age. No effective treatments are currently available for TBI or TBI-related dementia; moreover, only recently has insight been gained regarding the mechanisms behind their connection. Here, we used antibodies to detect oligomeric and phosphorylated Tau proteins in a non-transgenic rodent model of parasagittal fluid percussion injury. Oligomeric and phosphorylated Tau proteins were detected 4 and 24 h and 2 weeks post-TBI in injured, but not sham control rats. These findings suggest that diagnostic tools and therapeutics that target only toxic forms of Tau may provide earlier detection and safe, more effective treatments for tauopathies associated with repetitive neurotrauma.