A genetically encoded reporter of synaptic activity in vivo

To image synaptic activity within neural circuits, we tethered the genetically encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detected the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: it identified the locations of synapses and had a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicated that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrated that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum and graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses

Experimental Demonstration of Five-photon Entanglement and Open-destination Teleportation

Universal quantum error-correction requires the ability of manipulating entanglement of five or more particles. Although entanglement of three or four particles has been experimentally demonstrated and used to obtain the extreme contradiction between quantum mechanics and local realism, the realization of five-particle entanglement remains an experimental challenge. Meanwhile, a crucial experimental challenge in multi-party quantum communication and computation is the so-called open-destination teleportation. During open-destination teleportation, an unknown quantum state of a single particle is first teleported onto a N-particle coherent superposition to perform distributed quantum information processing. At a later stage this teleported state can be readout at any of the N particles for further applications by performing a projection measurement on the remaining N-1 particles. Here, we report a proof-of-principle demonstration of five-photon entanglement and open-destination teleportation. In the experiment, we use two entangled photon pairs to generate a four-photon entangled state, which is then combined with a single photon state to achieve the experimental goals. The methods developed in our experiment would have various applications e.g. in quantum secret sharing and measurement-based quantum computation.Comment: 19 pages, 4 figures, submitted for publication on 15 October, 200

Tripartite interactions between two phase qubits and a resonant cavity

The creation and manipulation of multipartite entangled states is important for advancements in quantum computation and communication, and for testing our fundamental understanding of quantum mechanics and precision measurements. Multipartite entanglement has been achieved by use of various forms of quantum bits (qubits), such as trapped ions, photons, and atoms passing through microwave cavities. Quantum systems based on superconducting circuits have been used to control pair-wise interactions of qubits, either directly, through a quantum bus, or via controllable coupling. Here, we describe the first demonstration of coherent interactions of three directly coupled superconducting quantum systems, two phase qubits and a resonant cavity. We introduce a simple Bloch-sphere-like representation to help one visualize the unitary evolution of this tripartite system as it shares a single microwave photon. With careful control and timing of the initial conditions, this leads to a protocol for creating a rich variety of entangled states. Experimentally, we provide evidence for the deterministic evolution from a simple product state, through a tripartite W-state, into a bipartite Bell-state. These experiments are another step towards deterministically generating multipartite entanglement in superconducting systems with more than two qubits

Disposition of Federally Owned Surpluses

PDZ domains are scaffolding modules in protein-protein interactions that mediate numerous physiological functions by interacting canonically with the C-terminus or non-canonically with an internal motif of protein ligands. A conserved carboxylate-binding site in the PDZ domain facilitates binding via backbone hydrogen bonds; however, little is known about the role of these hydrogen bonds due to experimental challenges with backbone mutations. Here we address this interaction by generating semisynthetic PDZ domains containing backbone amide-to-ester mutations and evaluating the importance of individual hydrogen bonds for ligand binding. We observe substantial and differential effects upon amide-to-ester mutation in PDZ2 of postsynaptic density protein 95 and other PDZ domains, suggesting that hydrogen bonding at the carboxylate-binding site contributes to both affinity and selectivity. In particular, the hydrogen-bonding pattern is surprisingly different between the non-canonical and canonical interaction. Our data provide a detailed understanding of the role of hydrogen bonds in protein-protein interactions

Adhesion of <i>Pseudomonas fluorescens</i> biofilms to glass, stainless steel and cellulose

Objectives: The adhesion of colloidal probes of stainless steel, glass and cellulose to Pseudomonas fluorescens biofilms was examined using atomic force microscopy (AFM) to allow comparisons between surfaces to which biofilms might adhere. Results: Biofilm was grown on a stainless steel substrate and covered most of the surface after 96 h. AFM approach and retraction curves were obtained when the biofilm was immersed in a tryptone/soy medium. On approach, all the colloidal probes experienced a long non-contact phase more than 100 nm in length, possibly due to the steric repulsion by extracellular polymers from the biofilm and hydrophobic effects. Retraction data showed that the adhesion varied from position to position on the biofilm. The mean value of adhesion of glass to the biofilm (48 ± 7 nN) was the greatest, followed by stainless steel (30 ± 7 nN) and cellulose (7.8 ± 0.4 nN). Conclusion: The method allows understanding of adhesion between the three materials and biofilm, and development of a better strategy to remove the biofilm from these surfaces relevant to different industrial applications

The instantaneous helical axis of the subtalar and talocrural joints: a non-invasive in vivo dynamic study

<p>Abstract</p> <p>Background</p> <p>An understanding of rear-foot (talocrural and subtalar joints) kinematics is critical for diagnosing foot pathologies, designing total ankle implants, treating rear-foot injuries and quantifying gait abnormalities. The majority of kinematic data available have been acquired through static cadaver work or passive <it>in vivo </it>studies. The applicability of these data to dynamic <it>in vivo </it>situations remains unknown. Thus, the purpose of this study was to fully quantify subtalar, talocrural and calcaneal-tibial <it>in vivo </it>kinematics in terms of the instantaneous helical axis (IHA) in twenty-five healthy ankles during a volitional activity that simulated single-leg toe-raises with partial-weight support, requiring active muscle control.</p> <p>Methods</p> <p>Subjects were each placed supine in a 1.5 T MRI and asked to repeat this simulated toe-raise while a full sagittal-cine-phase contrast (dynamic) MRI dataset was acquired. From the cine-phase contrast velocity a full kinematic description for each joint was derived.</p> <p>Results</p> <p>Nearly all motion quantified at the calcaneal-tibial joint was attributable to the talocrural joint. The subtalar IHA orientation and position were highly variable; whereas, the talocrural IHA orientation and position were extremely consistent.</p> <p>Conclusion</p> <p>The talocrural was well described by the IHA and could be modeled as a fixed-hinge joint, whereas the subtalar could not be.</p

A comparison of cryopreservation methods: Slow-cooling vs. rapid-cooling based on cell viability, oxidative stress, apoptosis, and CD34+ enumeration of human umbilical cord blood mononucleated cells

<p>Abstract</p> <p>Background</p> <p>The finding of human umbilical cord blood as one of the most likely sources of hematopoietic stem cells offers a less invasive alternative for the need of hematopoietic stem cell transplantation. Due to the once-in-a-life time chance of collecting it, an optimum cryopreservation method that can preserve the life and function of the cells contained is critically needed.</p> <p>Methods</p> <p>Until now, slow-cooling has been the routine method of cryopreservation; however, rapid-cooling offers a simple, efficient, and harmless method for preserving the life and function of the desired cells. Therefore, this study was conducted to compare the effectiveness of slow- and rapid-cooling to preserve umbilical cord blood of mononucleated cells suspected of containing hematopoietic stem cells. The parameters used in this study were differences in cell viability, malondialdehyde content, and apoptosis level. The identification of hematopoietic stem cells themselves was carried out by enumerating CD34⁺in a flow cytometer.</p> <p>Results</p> <p>Our results showed that mononucleated cell viability after rapid-cooling (91.9%) was significantly higher than that after slow-cooling (75.5%), with a <it>p </it>value = 0.003. Interestingly, the malondialdehyde level in the mononucleated cell population after rapid-cooling (56.45 μM) was also significantly higher than that after slow-cooling (33.25 μM), with a <it>p </it>value < 0.001. The apoptosis level in rapid-cooling population (5.18%) was not significantly different from that of the mononucleated cell population that underwent slow-cooling (3.81%), with a <it>p </it>value = 0.138. However, CD34⁺enumeration was much higher in the population that underwent slow-cooling (23.32 cell/μl) than in the one that underwent rapid-cooling (2.47 cell/μl), with a <it>p </it>value = 0.001.</p> <p>Conclusions</p> <p>Rapid-cooling is a potential cryopreservation method to be used to preserve the umbilical cord blood of mononucleated cells, although further optimization of the number of CD34⁺cells after rapid-cooling is critically needed.</p

CAR-T cell. the long and winding road to solid tumors

Adoptive cell therapy of solid tumors with reprogrammed T cells can be considered the "next generation" of cancer hallmarks. CAR-T cells fail to be as effective as in liquid tumors for the inability to reach and survive in the microenvironment surrounding the neoplastic foci. The intricate net of cross-interactions occurring between tumor components, stromal and immune cells leads to an ineffective anergic status favoring the evasion from the host's defenses. Our goal is hereby to trace the road imposed by solid tumors to CAR-T cells, highlighting pitfalls and strategies to be developed and refined to possibly overcome these hurdles

Application of PCR amplicon sequencing using a single primer pair in PCR amplification to assess variations in Helicobacter pylori CagA EPIYA tyrosine phosphorylation motifs

<p>Abstract</p> <p>Background</p> <p>The presence of various EPIYA tyrosine phosphorylation motifs in the CagA protein of <it>Helicobacter pylori </it>has been suggested to contribute to pathogenesis in adults. In this study, a unique PCR assay and sequencing strategy was developed to establish the number and variation of <it>cagA </it>EPIYA motifs.</p> <p>Findings</p> <p>MDA-DNA derived from gastric biopsy specimens from eleven subjects with gastritis was used with M13- and T7-sequence-tagged primers for amplification of the <it>cagA </it>EPIYA motif region. Automated capillary electrophoresis using a high resolution kit and amplicon sequencing confirmed variations in the <it>cagA </it>EPIYA motif region. In nine cases, sequencing revealed the presence of AB, ABC, or ABCC (Western type) <it>cagA </it>EPIYA motif, respectively. In two cases, double <it>cagA </it>EPIYA motifs were detected (ABC/ABCC or ABC/AB), indicating the presence of two <it>H. pylori </it>strains in the same biopsy.</p> <p>Conclusion</p> <p>Automated capillary electrophoresis and Amplicon sequencing using a single, M13- and T7-sequence-tagged primer pair in PCR amplification enabled a rapid molecular typing of <it>cagA </it>EPIYA motifs. Moreover, the techniques described allowed for a rapid detection of mixed <it>H. pylori </it>strains present in the same biopsy specimen.</p