Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.

Intermittent electrical stimuli for guidance of human mesenchymal stem cell lineage commitment towards neural-like cells on electroconductive substrates

In the context of the role of multiple physical factors in dictating stem cell fate, the present paper demonstrates the effectiveness of the intermittently delivered external electric field stimulation towards switching the stem cell fate to specific lineage, when cultured in the absence of biochemical growth factors. In particular, our findings present the ability of human mesenchymal stem cells (hMSCs) to respond to the electric stimuli by adopting extended neural-like morphology on conducting polymeric substrates. Polyaniline (PANI) is selected as the model system to demonstrate this effect, as the electrical conductivity of the polymeric substrates can be systematically tailored over a broad range (10(-9) to 10 S/cm) from highly insulating to conducting by doping with varying concentrations (10(-5) to 1 M) of HCl. On the basis of the culture protocol involving the systematic delivery of intermittent electric field (dc) stimulation, the parametric window of substrate conductivity and electric field strength was established to promote significant morphological extensions, with minimal cellular damage. A time dependent morphological change in hMSCs with significant filopodial elongation was observed after 7 days of electrically stimulated culture. Concomitant with morphological changes, a commensurate increase in the expression of neural lineage commitment markers such as nestin and PI tubulin was recorded from hMSCs grown on highly conducting substrates, as revealed from the mRNA expression analysis using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) as well as by immune-fluorescence imaging. Therefore, the present work establishes the key role of intermittent and systematic delivery of electric stimuli as guidance cues in promoting neural-like differentiation of hMSCs, when grown on electroconductive substrates. (C) 2014 Elsevier Ltd. All rights reserved.

Turbulence and cooling in galaxy cluster cores

We study the interplay between turbulent heating, mixing, and radiative cooling in an idealized model of cool cluster cores. Active galactic nuclei (AGN) jets are expected to drive turbulence and heat cluster cores. Cooling of the intracluster medium (ICM) and stirring by AGN jets are tightly coupled in a feedback loop. We impose the feedback loop by balancing radiative cooling with turbulent heating. In addition to heating the plasma, turbulence also mixes it, suppressing the formation of cold gas at small scales. In this regard, the effect of turbulence is analogous to thermal conduction. For uniform plasma in thermal balance (turbulent heating balancing radiative cooling), cold gas condenses only if the cooling time is shorter than the mixing time. This condition requires the turbulent kinetic energy to be a parts per thousand(3) the plasma internal energy; such high velocities in cool cores are ruled out by observations. The results with realistic magnetic fields and thermal conduction are qualitatively similar to the hydrodynamic simulations. Simulations where the runaway cooling of the cool core is prevented due to mixing with the hot ICM show cold gas even with subsonic turbulence, consistent with observations. Thus, turbulent mixing is the likely mechanism via which AGN jets heat cluster cores. The thermal instability growth rates observed in simulations with turbulence are consistent with the local thermal instability interpretation of cold gas in cluster cores.

Subexponential Algorithm for d-Cluster Edge Deletion: Exception or Rule?

The correlation clustering problem is a fundamental problem in both theory and practice, and it involves identifying clusters of objects in a data set based on their similarity. A traditional modeling of this question as a graph theoretic problem involves associating vertices with data points and indicating similarity by adjacency. Clusters then correspond to cliques in the graph. The resulting optimization problem, Cluster Editing (and several variants) are very well-studied algorithmically. In many situations, however, translating clusters to cliques can be somewhat restrictive. A more flexible notion would be that of a structure where the vertices are mutually ``not too far apart'', without necessarily being adjacent. One such generalization is realized by structures called s-clubs, which are graphs of diameter at most s. In this work, we study the question of finding a set of at most k edges whose removal leaves us with a graph whose components are s-clubs. Recently, it has been shown that unless Exponential Time Hypothesis fail (ETH) fails Cluster Editing (whose components are 1-clubs) does not admit sub-exponential time algorithm STACS, 2013]. That is, there is no algorithm solving the problem in time 2 degrees((k))n(O(1)). However, surprisingly they show that when the number of cliques in the output graph is restricted to d, then the problem can be solved in time O(2(O(root dk)) + m + n). We show that this sub-exponential time algorithm for the fixed number of cliques is rather an exception than a rule. Our first result shows that assuming the ETH, there is no algorithm solving the s-Club Cluster Edge Deletion problem in time 2 degrees((k))n(O(1)). We show, further, that even the problem of deleting edges to obtain a graph with d s-clubs cannot be solved in time 2 degrees((k))n(O)(1) for any fixed s, d >= 2. This is a radical contrast from the situation established for cliques, where sub-exponential algorithms are known.

Large carbon cluster thin film gauges for measuring aerodynamic heat transfer rates in hypersonic shock tunnels

Different types of Large Carbon Cluster (LCC) layers are synthesized by a single-step pyrolysis technique at various ratios of precursor mixture. The aim is to develop a fast responsive and stable thermal gauge based on a LCC layer which has relatively good electrical conduction in order to use it in the hypersonic flow field. The thermoelectric property of the LCC layer has been studied. It is found that these carbon clusters are sensitive to temperature changes. Therefore suitable thermal gauges were developed for blunt cone bodies and were tested in hypersonic shock tunnels at a flow Mach number of 6.8 to measure aerodynamic heating. The LCC layer of this thermal gauge encounters high shear forces and a hostile environment for test duration in the range of a millisecond. The results are favorable to use large carbon clusters as a better sensor than a conventional platinum thin film gauge in view of fast responsiveness and stability.

DEEP WASHINGTON PHOTOMETRY OF INCONSPICUOUS STAR CLUSTER CANDIDATES IN THE LARGE MAGELLANIC CLOUD

We present deep Washington photometry of 45 poorly populated star cluster candidates in the Large Magellanic Cloud (LMC). We have performed a systematic study to estimate the parameters of the cluster candidates by matching theoretical isochrones to the cleaned and dereddened cluster color-magnitude diagrams. We were able to estimate the basic parameters for 33 clusters, out of which 23 are identified as single clusters and 10 are found to be members of double clusters. The other 12 cluster candidates have been classified as possible clusters/asterisms. About 50% of the true clusters are in the 100-300 Myr age range, whereas some are older or younger. We have discussed the distribution of age, location, and reddening with respect to field, as well as the size of true clusters. The sizes and masses of the studied sample are found to be similar to that of open clusters in the Milky Way. Our study adds to the lower end of cluster mass distribution in the LMC, suggesting that the LMC, apart from hosting rich clusters, also has formed small, less massive open clusters in the 100-300 Myr age range.

Chemical Functionalization of Graphene To Augment Stem Cell Osteogenesis and Inhibit Biofilm Formation on Polymer Composites for Orthopedic Applications

Toward designing the next generation of resorbable biomaterials for orthopedic applications, we studied poly(epsilon-caprolactone) (PCL) composites containing graphene. The role, if any, of the functionalization of graphene on mechanical properties, stem cell response, and biofilm formation was systematically evaluated. PCL composites of graphene oxide (GO), reduced GO (RGO), and amine-functionalized GO (AGO) were prepared at different filler contents (1%, 3%, and 5%). Although the addition of the nanoparticles to PCL markedly increased the storage modulus, this increase was largest for GO followed by AGO and RGO. In vitro cell studies revealed that the AGO and GO particles significantly increased human mesenchymal stem cell proliferation. AGO was most effective in augmenting stem cell osteogenesis leading to mineralization. Bacterial studies revealed that interaction with functionalized GO induced bacterial cell death because of membrane damage, which was further accentuated by amine groups in AGO. As a result, AGO composites were best at inhibiting biofilm formation. The synergistic effect of oxygen containing functional groups and amine groups on AGO imparts the optimal combination of improved modulus, favorable stem cell response, and biofilm inhibition in AGO-reinforced composites desired for orthopedic applications. This work elucidates the importance of chemical functionalization of graphene in polymer composites for biomedical applications.

Magnetic field assisted stem cell differentiation - role of substrate magnetization in osteogenesis

Among the multiple modulatory physical cues explored to regulate cellular processes, the potential of magneto-responsive substrates in magnetic field stimulated stem cell differentiation is still unperceived. In this regard, the present work demonstrates how an external magnetic field can be applied to direct stem cell differentiation towards osteogenic commitment. A new culture methodology involving periodic delivery of 100 mT static magnetic field (SMF) in combination with HA-Fe3O4 magnetic substrates possessing a varying degree of substrate magnetization was designed for the study. The results demonstrate that an appropriate combination of weakly ferromagnetic substrates and SMF exposure enhanced cell viability, DNA synthesis and caused an early switchover to osteogenic lineage as supported by Runx2 immunocytochemistry and ALP expression. However, the mRNA expression profile of early osteogenic markers (Runx2, ALP, Col IA) was comparable despite varying substrate magnetic properties (diamagnetic to ferromagnetic). On the contrary, a remarkable upregulation of late bone development markers (OCN and OPN) was explicitly detected on weak and strongly ferromagnetic substrates. Furthermore, SMF induced matrix mineralization with elevated calcium deposition on similar substrates, even in the absence of osteogenic supplements. More specifically, the role of SMF in increasing intracellular calcium levels and in inducing cell cycle arrest at G0/G1 phase was elucidated as the major molecular event triggering osteogenic differentiation. Taken together, the above results demonstrate the competence of magnetic stimuli in combination with magneto-responsive biomaterials as a potential strategy for stem cell based bone tissue engineering.

A novel Monoclonal Antibody against Notch1 Targets Leukemia-associated Mutant Notch1 and Depletes Therapy Resistant Cancer Stem Cells in Solid Tumors

Higher Notch signaling is known to be associated with hematological and solid cancers. We developed a potential immunotherapeutic monoclonal antibody (MAb) specific for the Negative Regulatory Region of Notch1 (NRR). The MAb604.107 exhibited higher affinity for the ``Gain-offunction'' mutants of Notch1 NRR associated with T Acute lymphoblastic Leukemia (T-ALL). Modeling of the mutant NRR with 12 amino-acid insertion demonstrated ``opening'' resulting in exposure of the S2-cleavage site leading to activated Notch1 signaling. The MAb, at low concentrations (1-2 mu g/ml), inhibited elevated ligand-independent Notch1 signaling of NRR mutants, augmented effect of Thapsigargin, an inhibitor of mutant Notch1, but had no effect on the wild-type Notch1. The antibody decreased proliferation of the primary T-ALL cells and depleted leukemia initiating CD34/CD44 high population. At relatively high concentrations, (10-20 mu g/ml), the MAb affected Notch1 signaling in the breast and colon cancer cell lines. The Notch-high cells sorted from solid-tumor cell lines exhibited characteristics of cancer stem cells, which were inhibited by the MAb. The antibody also increased the sensitivity to Doxorubucinirubicin. Further, the MAb impeded the growth of xenografts from breast and colon cancer cells potentiated regression of the tumors along with Doxorubucin. Thus, this antibody is potential immunotherapeutic tool for different cancers.

Hemagglutinin sequence conservation guided stem immunogen design from influenza A H3 subtype

Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential ``universal'' vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, ``isoleucine-zipper'', or ``foldon''. These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.

COOL CORE CYCLES: COLD GAS AND AGN JET FEEDBACK IN CLUSTER CORES

Using high-resolution 3D and 2D (axisymmetric) hydrodynamic simulations in spherical geometry, we study the evolution of cool cluster cores heated by feedback-driven bipolar active galactic nuclei (AGNs) jets. Condensation of cold gas, and the consequent enhanced accretion, is required for AGN feedback to balance radiative cooling with reasonable efficiencies, and to match the observed cool core properties. A feedback efficiency (mechanical luminosity approximate to epsilon(M) over dot(acc)c(2); where (M) over dot(acc). is the mass accretion rate at 1 kpc) as small as 6 x 10(-5) is sufficient to reduce the cooling/accretion rate by similar to 10 compared to a pure cooling flow in clusters (with M-200 less than or similar to 7 x 10(14) M-circle dot). This value is much smaller compared to the ones considered earlier, and is consistent with the jet efficiency and the fact that only a small fraction of gas at 1 kpc is accreted onto the supermassive black hole (SMBH). The feedback efficiency in earlier works was so high that the cluster core reached equilibrium in a hot state without much precipitation, unlike what is observed in cool-core clusters. We find hysteresis cycles in all our simulations with cold mode feedback: condensation of cold gas when the ratio of the cooling-time to the free-fall time (t(cool)/t(ff)) is less than or similar to 10 leads to a sudden enhancement in the accretion rate; a large accretion rate causes strong jets and overheating of the hot intracluster medium such that t(cool)/t(ff) > 10; further condensation of cold gas is suppressed and the accretion rate falls, leading to slow cooling of the core and condensation of cold gas, restarting the cycle. Therefore, there is a spread in core properties, such as the jet power, accretion rate, for the same value of core entropy t(cool)/t(ff). A smaller number of cycles is observed for higher efficiencies and for lower mass halos because the core is overheated to a longer cooling time. The 3D simulations show the formation of a few-kpc scale, rotationally supported, massive (similar to 10(11) M-circle dot) cold gas torus. Since the torus gas is not accreted onto the SMBH, it is largely decoupled from the feedback cycle. The radially dominant cold gas (T < 5 x 10(4) K; vertical bar v(r)vertical bar >vertical bar v(phi vertical bar)) consists of fast cold gas uplifted by AGN jets and freely infalling cold gas condensing out of the core. The radially dominant cold gas extends out to 25 kpc for the fiducial run (halo mass 7 x 10(14) M-circle dot and feedback efficiency 6 x 10(-5)), with the average mass inflow rate dominating the outflow rate by a factor of approximate to 2. We compare our simulation results with recent observations.

Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-alpha and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-alpha treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs - LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival. (C) 2015 The Authors. Published by Elsevier B.V.