Human stool sample collection is a non-invasive method of assessing the gut microbiome. Rapid changes in microbial composition upon defecation limit the use of fresh stool for microbial community analysis. This investigation tests the effects of stool sample storage and shipment conditions on bacterial DNA quality and microbial community structure to establish and validate a methodology for stool collection in human population-based studies. The goal is to establish a protocol that minimizes bias due to shipping conditions. Fresh stool samples were collected from three participants, homogenized, and aliquoted with or without a preservative (RNALater) within 48 hours of collection. To mimic sample transport conditions, we incubated samples from three individuals under the following conditions: -80°C without RNALater, 37°C, 22°C, 4°C, -80°C -5°C, or frozen samples packed in ice left at 22°C for 48 hours. Samples were then stored at -80°C for 48 hours after which fecal DNA was extracted. We used 16S rRNA gene sequencing of the V1-V3 region (Illumina Mi-seq) to measure the community composition. Multivariate analysis was used to test the effect of sample storage and shipping regime on the microbiome. On average, the phylum level distribution was 76% Firmicutes, 20% Bacteroidetes, 3% Actinobacteria, and <1% Lentisphaerae, Tenericutes, and Actinobacteria. A perMANOVA (F=2.25, p< 0.1) showed that there was no statistically significant difference in the phyla due to temperature and handling. Regardless of treatment, samples from each individual clustered together suggesting that between individual variation exceeds any differences due to shipping regimen. Our current protocol for stool collection and preservation shows no significant loss of bacterial DNA quality, nor does it significantly affect microbial community representation and is adequate for investigations on microbiome composition, structure and activity in human studies.

Nanoparticle delivery to specific locations within the brain requires the ability to control transport properties of the nanoparticle platform. Neurological injury or disease is not generally limited to one location or one cell type. A therapeutic or diagnostic platform must have the ability to move from the point of access in the brain to diffuse disease-related cells. Nanoparticles with no electrostatic, hydrophobic, or hydrogen bonding interactions within the brain microenvironment are classified as brain-penetrating. These brain-penetrating nanoparticles (BPN) can determine the steric limitations of nanoparticle transport within the brain microenvironment, and can characterize microrheological properties of the brain extracellular space. Therefore, adequate characterization of nanoparticle physicochemical properties in physiologically relevant solutions is critical to understanding nanoparticle behavior within the brain microenvironment. Here, we developed a methodology to assay nanoparticle size, surface charge, and polydispersity in the presence of cerebrospinal fluid (CSF), the primary medium that nanoparticles are exposed to in the brain. Typically nanoparticles are characterized in low concentration salt solutions (i.e 10mM NaCl) or in serum, which does not contain any unbound divalent ions, unlike CSF. We demonstrated that the hydrodynamic diameter measured in CSF is 10-20 nm larger than that measured in NaCl or serum, and that surface charge is more negative in CSF. In addition, we saw a decrease in nanoparticle stability in CSF compared to NaCl or serum. This methodology for characterization of nanoparticles in CSF, with comparison to literature standards, will be useful in determining critical nanoparticle physicochemical properties for brain penetration in the presence of brain microenvironment mediums like CSF.

Developmental phenotypic plasticity can be a vital means to buffer the sudden and persistent effects of anthropogenic climate change, through facilitation of species persistence and possible facilitation of species range shifts. Integrating our understanding of plastic physiological growth mechanisms with functional traits may prove useful for interpretation of the ecological impacts of climate change. We investigated the influence of water-stress and light limitation on leaf mass per area (LMA), leaf anatomy, and leaf gas exchange of juvenile Populus tremuloides trees. Leaves that developed while water-stressed had significantly elevated LMA, due to increased leaf density from significantly higher spongy mesophyll surface area per leaf area (A_smes/A) and reduced stomatal conductance (g_s) but only marginally reduced photosynthesis (Pmax). In shade, leaves had significantly reduced LMA, due to reduced thickness, density, and combined A_mes/A, but maintained the same A_smes/A, and reduced palisade mesophyll surface area per leaf area (A_pmes/A), which was coupled with a reduction of g_s and P_max. Furthermore, water-stressed leaves had elevated intrinsic water use efficiency (WUE_i: P_max/g_s) while the shaded leaves, despite reduced g_s, maintained similar WUE_i. Our results suggest that, under water-stress and full sunlight, greater A_smes/A conferred maintenance of photosynthetic rate possibly through reduction of mesophyll resistance (r_m), at the cost of reducing expansive leaf area (and increasing density) as leaves were more dense but were 20 % the area of control leaves. In shade, suppressing A_pmes/A while maintaining A_smes/A may be a plastic strategy to increase laminar light capture, as leaves had reduced density and thickness but were still 50 % the area of control leaves . Together, in P. tremuloides saplings, these stresses induced developmental plasticity in LMA with differing plastic underlying anatomy and function; we discuss future implications specifically in the context of developmental plasticity, growth trade-offs, and the ecological impacts of climate change.

Suction feeding is a valuable process to study since most aquatic vertebrates, which make up nearly half of all vertebrates, eat this way. Having a better understanding of this may give important insight into the evolution of vertebrates as a whole. During suction feeding the cranium elevates, causing bending in the vertebral column, resulting in a rotation of the cranium and body plane about an axis of rotation located on the vertebral column. We hypothesized that the vertebral column behaves like a flexible rod and accordingly asked if axial rotation is located more posteriorly with larger degrees of cranial elevation. Using video reconstruction of moving morphology and joint coordinate systems, which use coordinates to measure the 3D motions of bones in a anatomicaly meaningful coordinate space, we compared movement of the cranium in relation to the body in striped surfperch, largemouth bass, and Pacific staghorn sculpin. We found no significant correlation between degree of cranial elevation and location of axial rotation. However, the degree of cranial elevation and location of axial rotation varied among these species in a consistent way. These species’ unique ways of performing these movements could be related to their differences in morphologies.

This study investigates the effects of anthropogenic pollution and mitigation efforts in Elliot Bay, Puget Sound, by analyzing the relationships between pollutants and benthic foraminiferal assemblages from 1998, 2007 and 2013. Foraminifera are single-celled microorganisms, which are typically encased within secreted shells (tests) comprised of either calcium carbonate (calcareous) or cemented sediment grains (agglutinated). Foraminifera are commonly found in marine environments and are a valuable tool for monitoring environmental conditions of the past and present. Elliot Bay has an extensive history of pollution, beginning with the European settlers establishing lumber mills in the 1850’s, and continues to have many industrial and recreational sources of pollution today. For this study, a total of 45 samples collected by the Washington State Department of Ecology in 1998, 2007 and 2013 were analyzed to identify any changes or trends in foraminiferal assemblages. From these samples, 21 species were recovered and identified, most of which are stress-tolerant species found in estuaries around the world. Some species are more tolerant of pollution and stress, and so have been more informative of the environmental conditions in Elliott Bay. For example, the agglutinate species Eggerella advena, found abundantly in some 2013 samples, is an opportunistic colonizer of industrial and sewage outfalls. In addition, the calcareous species Buliminella elegantissima and Cribroelphidium excavatum, also widely present in the bay, are tolerant of low oxygen environments. Correlations of species density and diversity with heavy metals such as copper, zinc, lead, arsenic, cadmium, and mercury result in a better understanding of the impacts of pollution and mitigation in Elliott Bay.

 There have been major advancements in the use of Virtual Reality (VR) technologies in military, medical, and educational applications. However, it is not well understood how representations of spatial memory are encoded by the bi-modal, visual and audio sensory stimulation inherent to VR interactions. Previous studies have identified the hippocampus and entorhinal cortex as structures involved in the formation of spatial memory. Because Alzheimer’s pathology affects these medial temporal structures and is characterized by deficits in memory, spatiotemporal reasoning, and route finding, better understanding of spatial representations in medial temporal areas would help create VR with potentially therapeutic effects in patients with memory deficits. Using VR, non-human primates will utilize a joystick to move a first-person avatar and perform a foraging task wherein they must collect virtual objects to obtain food rewards. We hypothesize that monkeys will use a demonstrable strategy to optimize behavior in our VR task and propose experiments and computational methods to identify unique strategies wherein memory and route planning are being used. Preliminary results indicate that the three monkeys fully trained on this task utilize unique strategies to obtain food rewards. Furthermore, the evolution of individual strategies over the training period suggests that memory and other factors may play a role in route planning. Corresponding and ongoing research is aimed at identifying neural representations of complex spaces and encoded memory and planning elicited by our task.

 For a nanoparticle to efficiently penetrate within the brain microenvironment, it must avoid any steric limitation it would experience as it moves between cells, vessels, and extracellular matrix (ECM) components. The particle must avoid any adhesive interactions, via hydrogen bonding, hydrophobic interactions, or electrostatic interactions between the particle surface and a cell membrane, protein, or ECM component. Steric limitations are influenced by the brain’s volume fraction and the tortuosity. To maximize diffusion within the brain, it is important to have a bio-inert particle that will avoid adhesive interactions to accurately assess the steric limitations that occur due to changes in volume fraction and tortuosity. However, nanoparticles in the brain microenvironment are exposed to a 300 mM ion concentration in cerebral spinal fluid (CSF), which contains multiple divalent cations. Using dynamic light scattering, we sought to characterize nanoparticle size and surface charge in the presence of CSF as a function of particle size, surface chemistry, and exposure time. We demonstrate that the presence of these divalent cations can disrupt the electrostatic double layers that provide nanoparticle stability and prevent nanoparticle aggregation in the brain. Our results demonstrate that within one hour after exposure to physiological CSF, nanoparticles that are electrostatically stabilized begin to aggregate, increasing in hydrodynamic size and polydispersity. Nanoparticles that are sterically stabilized (i.e with polyethylene glycol) aggregated in the presence of CSF, but on a much slower time scale. This aggregation behavior may be one reason why nanoparticles that are otherwise stable tend to aggregate, and therefore do not penetrate, once in the brain microenvironment. Characterizing nanoparticle behavior in the brain can have significant implications for nanoparticle design for drug delivery to brain diseases.

The period of time before, during, and after birth (the perinatal period) is associated with the greatest risk of permanent brain injury, due in large part to perinatal asphyxia, where newborn infants suffer lack of oxygen and blood flow to the brain. Unfortunately, perinatal asphyxia, which can cause a hypoxic-ischemic insult (HI), is difficult to prevent or predict, and rates of perinatal asphyxia have remained around 2-4 per 1,000 live births, despite excellent obstetric care. Currently, therapeutic hypothermia (TH) is the only treatment that has been shown to provide robust improvement in newborns after moderate or severe perinatal asphyxia. Unfortunately, TH is not universally neuroprotective, and more effective therapeutic methods are necessary to reduce mortality and lead to symptom free survival of newborns with brain injury. In this study, we develop a curcumin-loaded poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticle for treatment of newborn HI. Currently, we are synthesizing the nanoparticles and doing characterization tests to determine if certain parameters are within the desired ranges. After this procedure, we will continue on and measure the drug loading and release of the nanoparticle. The results of this study are important because this nanoparticle has the ability to achieve high loading, sustained release of curcumin, and rapid diffusion within the brain microenvironment. Curcumin has recently been shown to prevent reduction of omega 3 fatty acids, which are critical to brain function and recovery after injury, particularly in the newborn brain after an HI event. Enhanced delivery of curcumin to the brain via a nanoparticle platform can have significant implications for treatment of HI in the newborn brain.

Nationally, twenty-two percent of discharged heart failure patients are readmitted within 30 days. Readmission places the patient at increased risk for complications and has financial repercussions for the hospital due to Medicare penalties under the Affordable Care Act. Previous studies indicate a multi-level approach to the discharge process—including discharge protocols, patient education, and supported transition to home or care facility—reduces readmission rates. Teach-Back is one approach to patient education that includes an assessment of patient learning about self-care, allowing them to demonstrate their knowledge and understanding to their nurse or other healthcare professional. The purpose of this study was to understand the nurses’ perceptions of the benefits and barriers to using Teach-Back during discharge. A Quality Improvement survey was distributed to approximately 1100 nurses at University of Washington Medical Center with a 28% response rate. Nurses indicated that primary barriers to using Teach-Back were increased time requirements (58%), burden of documentation requirements (52%) and lack of knowledge on how to perform Teach-Back (36%). Preliminary content analysis of two open ended questions related to Teach-Back integration and implementation identified four themes: efficiency, support, checklists and training. We interpreted these results to suggest that increasing demands on nurses’ time requires efficiencies in other areas to rebalance the workload. Recommendations include a more efficient system of prompts and documentation of Teach-Back. In-service training should be carried out first on units with the lowest rates of Teach-Back. Additionally, we recommend streamlining discharge documentation in the electronic medical records with the aim of time reduction by way of fewer screens and fewer clicks. One limitation of this study is non-respondent bias; since we do not know why 72% of nurses did not respond, we cannot rule out the possibility that the shortage may have been due to lack of confidence with Teach-Back.

The cultivation of tomatoes is currently a 58 billion dollar industry worldwide. Over the past few years, tomato plants have been subjected to more severe and abrupt climatic fluctuations than ever before. In-depth understanding of the tomato leaf anatomy and physiology can have significant consequences in agriculture. Fluxes (fluid flow rate) in leaf water and sugar-conducting tissues, xylem and phloem, are challenging to visualize in real time. It is important to attempt this measurement using intact leaves as excision interrupts control of normal flow paths. The possibility that vein density and flux influence photosynthetic rate is being investigated here.  Optical Coherence Tomography (OCT) is a non-invasive imaging modality that performs high-resolution cross-sectional imaging, allowing to visualize the structure and motion in biological samples in 3D.  In this study, we have used a state-of-the-art OCT system with 7 μm resolution, operating at 1300 nm wavelength, to investigate leaf structure, venation and vein flow in live tomato leaves for the first time. The low intensity imaging beam (3 mW) is in the near IR spectral region and imaging is performed at high speed (4 ms/frame) hence, does not interfere with the leaf’s natural physiological processes. We have used Rutgers’ tomatoes for this study. To improve the image contrast in the leaf veins, we are investigating using dye and lipid particles to be taken up by the leaves