Fast Benchtop Fabrication of Laminar Flow Chambers for Advanced Microscopy Techniques

Background: Fluid handling technology is acquiring an ever more prominent place in laboratory science whether it is in simple buffer exchange systems, perfusion chambers, or advanced microfluidic devices. Many of these applications remain the providence of laboratories at large institutions with a great deal of expertise and specialized equipment. Even with the expansion of these techniques, limitations remain that frequently prevent the coupling of controlled fluid flow with other technologies, such as coupling microfluidics and high-resolution position and force measurements by optical trapping microscopy. Method: Here we present a method for fabrication of multiple-input laminar flow devices that are optically clear [glass] on each face, chemically inert, reusable, inexpensive, and can be fabricated on the benchtop in approximately one hour. Further these devices are designed to allow flow regulation by a simple gravity method thus requiring no specialized equipment to drive flow. Here we use these devices to perform total internal reflection fluorescence microscopy measurements as well as position sensitive optical trapping experiments. Significance: Flow chamber technology needs to be more accessible to the general scientific community. The method presented here is versatile and robust. These devices use standard slides and coverslips making them compatible with nearly all types and models of light microscopes. These devices meet the needs of groups doing advanced optical trapping experiments, but could also be adapted by nearly any lab that has a function for solution flow coupled with microscopy

Dynamics of Dynamics within a Single Data Acquisition Session: Variation in Neocortical Alpha Oscillations in Human MEG

Background Behavioral paradigms applied during human recordings in electro- and magneto- encephalography (EEG and MEG) typically require 1–2 hours of data collection. Over this time scale, the natural fluctuations in brain state or rapid learning effects could impact measured signals, but are seldom analyzed. Methods and Findings We investigated within-session dynamics of neocortical alpha (7–14 Hz) rhythms and their allocation with cued-attention using MEG recorded from primary somatosensory neocortex (SI) in humans. We found that there were significant and systematic changes across a single ~1 hour recording session in several dimensions, including increased alpha power, increased differentiation in attention-induced alpha allocation, increased distinction in immediate time-locked post-cue evoked responses in SI to different visual cues, and enhanced power in the immediate cue-locked alpha band frequency response. Further, comparison of two commonly used baseline methods showed that conclusions on the evolution of alpha dynamics across a session were dependent on the normalization method used. Conclusions These findings are important not only as they relate to studies of oscillations in SI, they also provide a robust example of the type of dynamic changes in brain measures within a single session that are overlooked in most human brain imaging/recording studies.National Institutes of Health (U.S.) (P41RR14075)National Institutes of Health (U.S.) (K25MH072941)National Institutes of Health (U.S.) (K01AT003459)National Institutes of Health (U.S.) (1RO1-NS045130-01)National Institutes of Health (U.S.) (T32GM007484)National Science Foundation (U.S.) (0316933)Osher Lifelong Learning Institute

Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen

This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O(2)) level of 3% and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O(2), where they acquire a neuroepithelial identity over two weeks. This timescale is comparable to that at 20% O(2), but survival is enhanced. Sequential application of retinoic acid (RA) and purmorphamine (PM), from day 14 to 28, directs differentiation towards spinal motor neurons. Alternatively, addition of FGF-8 and PM generates midbrain dopaminergic neurons. OLIG2 induction in motor neuron precursors is 2-fold greater than at 20% O(2), whereas EN1 is 5-fold enhanced. 3% NPCs can be differentiated into all three neural lineages, and such cultures can be maintained long-term in the absence of neurotrophins. The ability to generate defined cell types at 3% O(2) should represent a significant advance for in vitro disease modelling and potentially cell-based therapies

Treatment results for hypopharyngeal cancer by different treatment strategies and its secondary primary- an experience in Taiwan

<p>Abstract</p> <p>Purpose</p> <p>The aim of this study was to evaluate treatment results in our hypopharyngeal cancer patients.</p> <p>Patients and Methods</p> <p>A total of three hundred and ninety five hypopharyngeal cancer patients received radical treatment at our hospital; 96% were male. The majority were habitual smokers (88%), alcohol drinkers (73%) and/or betel quid chewers (51%). All patients received a CT scan or MRI for tumor staging before treatment. The stage distribution was stage I: 2 (0.5%); stage II: 22 (5.6%); stage III: 57 (14.4%) and stage IV: 314 (79.5%). Radical surgery was used first in 81 patients (20.5%), and the remaining patients (79.5%) received organ preservation-intended treatment (OPIT). In the OPIT group, 46 patients received radiotherapy alone, 156 patients received chemotherapy followed by radiotherapy (CT/RT) and 112 patients received concomitant chemo-radiotherapy (CCRT).</p> <p>Results</p> <p>The five-year overall survival rates for stages I/II, III and IV were 49.5%, 47.4% and 18.6%, respectively. There was no significant difference in overall and disease-specific survival rates between patients who received radical surgery first and those who received OPIT. In the OPIT group, CCRT tended to preserve the larynx better (p = 0.088), with three-year larynx preservation rates of 44.8% for CCRT and 27.2% for CT/RT. Thirty-seven patients developed a second malignancy, with an annual incidence of 4.6%.</p> <p>Conclusions</p> <p>There was no survival difference between OPIT and radical surgery in hypopharyngeal cancer patients at our hospital. CCRT may offer better laryngeal preservation than RT alone or CT/RT. However, prospective studies are still needed to confirm this finding. Additionally, second primary cancers are another important issue for hypopharyngeal cancer management.</p

De novo domestication of wild tomato using genome editing

Breeding of crops over millennia for yield and productivity1 has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost2. We devised a CRISPR–Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. We report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium. Engineered S. pimpinellifolium morphology was altered, together with the size, number and nutritional value of the fruits. Compared with the wild parent, our engineered lines have a threefold increase in fruit size and a tenfold increase in fruit number. Notably, fruit lycopene accumulation is improved by 500% compared with the widely cultivated S. lycopersicum. Our results pave the way for molecular breeding programs to exploit the genetic diversity present in wild plants

Ensemble Modeling of the Likely Public Health Impact of a Pre-Erythrocytic Malaria Vaccine

Using an ensemble modeling approach, Thomas Smith and colleagues find that targeted mass vaccination with a pre-erythrocytic malaria vaccine RTS,S in low-transmission settings might have better health effects than vaccination through national EPI programs

Enhancing volunteer engagement to achieve desirable outcomes: what can non-profit employers do?

Abstract Engagement is a positive psychological state that is linked with a range of beneficial individual and organizational outcomes. However, the factors associated with volunteer engagement have rarely been examined. Data from 1064 volunteers of a wildlife charity in the United Kingdom revealed that both task- and emotion-oriented organizational support were positively related to volunteer engagement, and volunteer engagement was positively related to volunteer happiness and perceived social worth and negatively related to intent to leave the voluntary organization. Consistent with theory, engagement acted as a mediator between these factors. The implications for future research and the relevance of the findings for voluntary organizations are discussed

Neural Stem/Progenitor Cells from the Adult Human Spinal Cord Are Multipotent and Self-Renewing and Differentiate after Transplantation

Neural stem/progenitor cell (NSPC) transplantation is a promising therapy for spinal cord injury (SCI). However, little is known about NSPC from the adult human spinal cord as a donor source. We demonstrate for the first time that multipotent and self-renewing NSPC can be cultured, passaged and transplanted from the adult human spinal cord of organ transplant donors. Adult human spinal cord NSPC require an adherent substrate for selection and expansion in EGF (epidermal growth factor) and FGF2 (fibroblast growth factor) enriched medium. NSPC as an adherent monolayer can be passaged for at least 9 months and form neurospheres when plated in suspension culture. In EGF/FGF2 culture, NSPC proliferate and primarily express nestin and Sox2, and low levels of markers for differentiating cells. Leukemia inhibitory factor (LIF) promotes NSPC proliferation and significantly enhances GFAP expression in hypoxia. In differentiating conditions in the presence of serum, these NSPC show multipotentiality, expressing markers of neurons, astrocytes, and oligodendrocytes. Dibutyryl cyclic AMP (dbcAMP) significantly enhances neuronal differentiation. We transplanted the multipotent NSPC into SCI rats and show that the xenografts survive, are post-mitotic, and retain the capacity to differentiate into neurons and glia

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages