Beta: Bioprinting engineering technology for academia

Higher STEM education is a field of growing potential, but too many middle school and high school students are not testing proficiently in STEM subjects. The BETA team worked to improve biology classroom engagement through the development of technologies for high school biology experiments. The BETA project team expanded functionality of an existing product line to allow for better student and teacher user experience and the execution of more interesting experiments. The BETA project’s first goal was to create a modular incubating Box for the high school classroom. This Box, called the BETA Box was designed with a variety of sensors to allow for custom temperature and lighting environments for each experiment. It was completed with a clear interface to control the settings and an automatic image capture system. The team also conducted a feasibility study on auto calibration and dual-extrusion for SE3D’s existing 3D bioprinter. The findings of this study led to the incorporation of a force sensor for auto calibration and the evidence to support the feasibility of dual extrusion, although further work is needed. These additions to the current SE3D educational product line will increase effectiveness in the classroom and allow the target audience, high school students, to better engage in STEM education activities

The evolution of self-control

This work was supported by the National Evolutionary Synthesis Center (NESCent) through support of a working group led by C.L.N. and B.H. NESCent is supported by the National Science Foundation (NSF) EF-0905606. For training in phylogenetic comparative methods, we thank the AnthroTree Workshop (supported by NSF BCS-0923791). Y.S. thanks the National Natural Science Foundation of China (Project 31170995) and National Basic Research Program (973 Program: 2010CB833904). E.E.B. thanks the Duke Vertical Integration Program and the Duke Undergraduate Research Support Office. J.M.P. was supported by a Newton International Fellowship from the Royal Society and the British Academy. L.R.S. thanks the James S. McDonnell Foundation for Award 220020242. L.J.N.B. and M.L.P. acknowledge the National Institutes of Mental Health (R01-MH096875 and R01-MH089484), a Duke Institute for Brain Sciences Incubator Award (to M.L.P.), and a Duke Center for Interdisciplinary Decision Sciences Fellowship (to L.J.N.B.). E.V. and E.A. thank the Programma Nazionale per la Ricerca–Consiglio Nazionale delle Ricerche (CNR) Aging Program 2012–2014 for financial support, Roma Capitale–Museo Civico di Zoologia and Fondazione Bioparco for hosting the Istituto di Scienze e Tecnologie della Cognizione–CNR Unit of Cognitive Primatology and Primate Centre, and Massimiliano Bianchi and Simone Catarinacci for assistance with capuchin monkeys. K.F. thanks the Japan Society for the Promotion of Science (JSPS) for Grant-in-Aid for Scientific Research 20220004. F. Aureli thanks the Stages in the Evolution and Development of Sign Use project (Contract 012-984 NESTPathfinder) and the Integrating Cooperation Research Across Europe project (Contract 043318), both funded by the European Community’s Sixth Framework Programme (FP6/2002–2006). F. Amici was supported by Humboldt Research Fellowship for Postdoctoral Researchers (Humboldt ID 1138999). L.F.J. and M.M.D. acknowledge NSF Electrical, Communications, and Cyber Systems Grant 1028319 (to L.F.J.) and an NSF Graduate Fellowship (to M.M.D.). C.H. thanks Grant-in-Aid for JSPS Fellows (10J04395). A.T. thanks Research Fellowships of the JSPS for Young Scientists (21264). F.R. and Z.V. acknowledge Austrian Science Fund (FWF) Project P21244-B17, the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP/2007–2013)/ERC Grant Agreement 311870 (to F.R.), Vienna Science and Technology Fund Project CS11-026 (to Z.V.), and many private sponsors, including Royal Canin for financial support and the Game Park Ernstbrunn for hosting the Wolf Science Center. S.M.R. thanks the Natural Sciences and Engineering Research Council (Canada). J.K.Y. thanks the US Department of Agriculture–Wildlife Services–National Wildlife Research Center. J.F.C. thanks the James S. McDonnell Foundation and Alfred P. Sloan Foundation. E.L.M. and B.H. thank the Duke Lemur Center and acknowledge National Institutes of Health Grant 5 R03 HD070649-02 and NSF Grants DGE-1106401, NSF-BCS-27552, and NSF-BCS-25172. This is Publication 1265 of the Duke Lemur Center.Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.PostprintPeer reviewe

Insight into Influenza Manipulation of its Target Cell and the Countermeasures Taken by the Host to Limit Infection

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2012.Influenza A viruses remain a major public health concern, as is evident by the occurrence of annual epidemics and occasional pandemics. Insight into the interactions that occur between influenza and its host will help to uncover targets for novel therapies. One approach is to better understand the cellular signaling events that occur following infection. Our examination of this revealed that mice deficient in the MLK3 kinase disrupted MAPK signaling and resulted in high viral titers present in the lung at late time points following infection. This was a consequence of prolonged survival of infected cells, which in turn were able to produce virus for an extended time. These data implicate MLK3 as a pro-survival regulator during influenza infection. Additionally, it is well established that following infection with influenza a dramatic reduction of cellular protein synthesis ensues, a phenomenon referred to as shutoff. Although evidence in the literature has suggested a role for the viral polymerase complex in this process, how influenza does this remains unknown. Therefore, to investigate the contribution of the polymerase complex to shutoff, we utilized reporter gene assays to evaluate the role of polymerase subunits on host protein expression. In this study, we found that the PA subunit is a critical factor in the initiation of shutoff. Of interest, in the strains that we characterized, PA of avianorigin, such as the pandemic H1N1, were more efficient at inducing shutoff than human-origin PA, such as WSN. This phenotype was confirmed in comparison of protein synthesis following infection with a recombinant WSN virus containing Cal PA and wild-type WSN. Chimeric analysis revealed that the activity of PA responsible for shutoff was localized to the amino terminal domain of this protein, with the lysine at position 134 being critically involved in the phenotype. Although this is the catalytic lysine within the endonuclease active site, endonuclease activity was not required for shutoff. Together, these results implicate PA as a critical determinant in the inhibition of cellular protein synthesis following infection. Overall, these findings uncover novel influenza-host interactions and protein functions that should be considered in the design of future therapeutic agents

Specific residues in the 2009 H1N1 swine-origin influenza matrix protein influence virion morphology and efficiency of viral spread in vitro.

In April 2009, a novel influenza virus emerged as a result of genetic reassortment between two pre-existing swine strains. This highly contagious H1N1 recombinant (pH1N1) contains the same genomic background as North American triple reassortant (TR) viruses except for the NA and M segments which were acquired from the Eurasian swine lineage. Yet, despite their high degree of genetic similarity, we found the morphology of virions produced by the pH1N1 isolate, A/California/04/09 (ACal-04/09), to be predominantly spherical by immunufluorescence and electron microscopy analysis in human lung and swine kidney epithelial cells, whereas TR strains were observed to be mostly filamentous. In addition, nine clinical pH1N1 samples collected from nasal swab specimens showed similar spherical morphology as the ACal-04/09 strain. Sequence analysis between TR and pH1N1 viruses revealed four amino acid differences in the viral matrix protein (M1), a known determinant of influenza morphology, at positions 30, 142, 207, and 209. To test the role of these amino acids in virus morphology, we rescued mutant pH1N1 viruses in which each of the four M1 residues were replaced with the corresponding TR residue. pH1N1 containing substitutions at positions 30, 207 and 209 exhibited a switch to filamentous morphology, indicating a role for these residues in virion morphology. Substitutions at these residues resulted in lower viral titers, reduced growth kinetics, and small plaque phenotypes compared to wild-type, suggesting a correlation between influenza morphology and efficient cell-to-cell spread in vitro. Furthermore, we observed efficient virus-like particle production from cells expressing wild-type pH1N1 M1, but not M1 containing substitutions at positions 30, 207, and 209, or M1 from other strains. These data suggest a direct role for pH1N1 specific M1 residues in the production and release of spherical progeny, which may contribute to the rapid spread of the pandemic virus

Identification of the N-terminal domain of the influenza virus PA responsible for the suppression of host protein synthesis.

Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA's reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix α4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis

TR swine virus but not the pH1N1 strain induces filament formation from infected human lung and swine kidney cells.

<p>Human A549 and swine LLC-PK1 cells infected with the ACal-04/09 or AWisc05 were processed for visualization of viral surface proteins 18 hpi by IF microscopy using anti-H1N1 mouse serum followed by anti-mouse IgG-Texas Red.</p

Electron microscopy analysis of TR or pH1N1-infected A549 cells.

<p>(<b>A</b>) Mock infected A549 cells, and A549 cells infected with AWisc05 or ACal-04/09 were fixed at 18 h and processed for scanning electron microscopy. Bars measure 10 µm (top) and 1 µm (bottom). Arrows point to virions budding from infected cell surfaces. (<b>B</b>) Electron micrograph of negatively stained ACal-04/09 virion released from infected A549 cells.</p

Substitutions at positions 30, 207 and 209 in the ACal-04/09 M1 protein resulted in filament formation in infected cells.

<p>A549 cells were infected with ACal-04/09 viruses containing single or multiple substitutions at positions 30, 142, 207, and 209 in the M1 protein as indicated. Cells were fixed and processed for visualization of viral surface proteins 18 h by IF microscopy as described for Fig. 1.</p

Amino acid differences between classical swine, Eurasian swine and the 2009 pH1N1 M1 proteins.

<p>Number of isolates containing the indicated residues/total number of isolates is indicated.</p

Mutations of ACal-04/09 M1 at residues 30 or 207 and 209 reduced VLP production from transfected cells.

<p>(A) 293T cells were transfected with expression plasmids containing WT or mutant ACal-04/09, or other M1 genes. Purified radiolabeled VLPs in culture supernatants were analyzed by SDS-PAGE and total M1 in cell lysates was determined by Western blot analysis. (B) Band intensities were quantified using BioRad software. VLP production is shown as the amount of M1 released into cell culture supernatant normalized to M1 produced in cell lysates. ACal-04/09 WT was set to 1. Data shown are an average of three individual experiments. Error bars represent standard deviation.</p