Causes of industrial protein a column degradation, explored using Raman spectroscopy.

Monoclonal antibodies (mAbs) are used extensively as biotherapeutics for chronic and acute conditions. Production of mAbs is lengthy and expensive, with protein A affinity capture the most costly step, due both to the nature of the resin and its marked reduction in binding capacity with repeated use. Our previous studies using in situ ATR-FTIR spectroscopy indicated that loss in protein A binding capacity is not the result of leaching or degradation of protein A ligand, suggesting fouling is the principal cause. Here we explore binding behavior and resin capacity loss using Raman spectroscopy. Our data reveal a distinct Raman spectral fingerprint for mAb bound to the protein A ligand of MabSelect SuRe. The results show that the drop in static binding capacity (SBC) previously observed for used protein A resin is discernible by Raman spectroscopy in combination with partial least-squares regression. The SBC is lowest (35.76 mg mL-1) for used inlet resin compared to used outlet (40.17 mg mL-1) and unused resin samples (70.35 mg mL-1). Depth profiling by Raman spectroscopy indicates that at below saturating concentrations (∼18 mg mL-1), binding of mAb is not homogeneous through used resin beads with protein binding preferentially to the outer regions of the bead, in contrast to fully homogeneous distribution through unused control MabSelect SuRe resin beads. Analysis of the Raman spectra indicates that one foulant is irreversibly bound mAb. The presence of irreversibly bound mAb and host cell proteins was confirmed by mass spectrometric analysis of used resin beads

Efficient assembly of very short oligonucleotides using T4 DNA Ligase

<p>Abstract</p> <p>Background</p> <p>In principle, a pre-constructed library of all possible short oligonucleotides could be used to construct many distinct gene sequences. In order to assess the feasibility of such an approach, we characterized T4 DNA Ligase activity on short oligonucleotide substrates and defined conditions suitable for assembly of a plurality of oligonucleotides.</p> <p>Findings</p> <p>Ligation by T4 DNA Ligase was found to be dependent on the formation of a double stranded DNA duplex of at least five base pairs surrounding the site of ligation. However, ligations could be performed effectively with overhangs smaller than five base pairs and oligonucleotides as small as octamers, in the presence of a second, complementary oligonucleotide. We demonstrate the feasibility of simultaneous oligonucleotide phosphorylation and ligation and, as a proof of principle for DNA synthesis through the assembly of short oligonucleotides, we performed a hierarchical ligation procedure whereby octamers were combined to construct a target 128-bp segment of the beta-actin gene.</p> <p>Conclusions</p> <p>Oligonucleotides as short as 8 nucleotides can be efficiently assembled using T4 DNA Ligase. Thus, the construction of synthetic genes, without the need for custom oligonucleotide synthesis, appears feasible.</p

B Cell: T Cell Interactions Occur within Hepatic Granulomas during Experimental Visceral Leishmaniasis

Hepatic resistance to Leishmania donovani infection in mice is associated with the development of granulomas, in which a variety of lymphoid and non-lymphoid populations accumulate. Although previous studies have identified B cells in hepatic granulomas and functional studies in B cell-deficient mice have suggested a role for B cells in the control of experimental visceral leishmaniasis, little is known about the behaviour of B cells in the granuloma microenvironment. Here, we first compared the hepatic B cell population in infected mice, where ≈60% of B cells are located within granulomas, with that of naïve mice. In infected mice, there was a small increase in mIgMlomIgD+ mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production. Using 2-photon microscopy to quantify the entire intra-granuloma B cell population, in conjunction with the adoptive transfer of polyclonal and HEL-specific BCR-transgenic B cells isolated from L. donovani-infected mice, we demonstrated that B cells accumulate in granulomas over time in an antigen-independent manner. Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment. These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site

Minimum joint space width and tibial cartilage morphology in the knees of healthy individuals: A cross-sectional study

<p>Abstract</p> <p>Background</p> <p>The clinical use of minimum joint space width (mJSW) and cartilage volume and thickness has been limited to the longitudinal measurement of disease progression (i.e. change over time) rather than the diagnosis of OA in which values are compared to a standard. This is primarily due to lack of establishment of normative values of joint space width and cartilage morphometry as has been done with bone density values in diagnosing osteoporosis. Thus, the purpose of this pilot study is to estimate reference values of medial joint space width and cartilage morphometry in healthy individuals of all ages using standard radiography and peripheral magnetic resonance imaging.</p> <p>Design</p> <p>For this cross-sectional study, healthy volunteers underwent a fixed-flexion knee X-ray and a peripheral MR (pMR) scan of the same knee using a 1T machine (ONI OrthOne™, Wilmington, MA). Radiographs were digitized and analyzed for medial mJSW using an automated algorithm. Only knees scoring ≤1 on the Kellgren-Lawrence scale (no radiographic evidence of knee OA) were included in the analyses. All 3D SPGRE fat-sat sagittal pMR scans were analyzed for medial tibial cartilage morphometry using a proprietary software program (Chondrometrics GmbH).</p> <p>Results</p> <p>Of 119 healthy participants, 73 were female and 47 were male; mean (SD) age 38.2 (13.2) years, mean BMI 25.0 (4.4) kg/m². Minimum JSW values were calculated for each sex and decade of life. Analyses revealed mJSW did not significantly decrease with increasing decade (p > 0.05) in either sex. Females had a mean (SD) medial mJSW of 4.8 (0.7) mm compared to males with corresponding larger value of 5.7 (0.8) mm. Cartilage morphometry results showed similar trends with mean (SD) tibial cartilage volume and thickness in females of 1.50 (0.19) μL/mm²and 1.45 (0.19) mm, respectively, and 1.77 (0.24) μL/mm²and 1.71 (0.24) mm, respectively, in males.</p> <p>Conclusion</p> <p>These data suggest that medial mJSW values do not decrease with aging in healthy individuals but remain fairly constant throughout the lifespan with "healthy" values of 4.8 mm for females and 5.7 mm for males. Similar trends were seen for cartilage morphology. Results suggest there may be no need to differentiate a t-score and a z-score in OA diagnosis because cartilage thickness and JSW remain constant throughout life in the absence of OA.</p

Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells

Citation: Hand, E. S., Haller, S. L., Peng, C., Rothenburg, S., & Hersperger, A. R. (2015). Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells. Plos One, 10(3), 15. doi:10.1371/journal.pone.0119189As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV

Subliminal Semantic Priming in Speech

Numerous studies have reported subliminal repetition and semantic priming in the visual modality. We transferred this paradigm to the auditory modality. Prime awareness was manipulated by a reduction of sound intensity level. Uncategorized prime words (according to a post-test) were followed by semantically related, unrelated, or repeated target words (presented without intensity reduction) and participants performed a lexical decision task (LDT). Participants with slower reaction times in the LDT showed semantic priming (faster reaction times for semantically related compared to unrelated targets) and negative repetition priming (slower reaction times for repeated compared to semantically related targets). This is the first report of semantic priming in the auditory modality without conscious categorization of the prime

Quantifying garnet-melt trace element partitioning using lattice-strain theory: New crystal-chemical and thermodynamic constraints

Many geochemical models of major igneous differentiation events on the Earth, the Moon, and Mars invoke the presence of garnet or its high-pressure majoritic equivalent as a residual phase, based on its ability to fractionate critical trace element pairs (Lu/Hf, U/Th, heavy REE/light REE). As a result, quantitative descriptions of mid-ocean ridge and hot spot magmatism, and lunar, martian, and terrestrial magma oceans require knowledge of garnet-melt partition coefficients over a wide range of conditions. In this contribution, we present new crystal-chemical and thermodynamic constraints on the partitioning of rare earth elements (REE), Y and Sc between garnet and anhydrous silicate melt as a function of pressure (P), temperature (T), and composition (X). Our approach is based on the interpretation of experimentally determined values of partition coefficients D using lattice-strain theory. In this and a companion paper (Draper and van Westrenen this issue) we derive new predictive equations for the ideal ionic radius of the dodecahedral garnet X-site,

Corticosterone Alters AMPAR Mobility and Facilitates Bidirectional Synaptic Plasticity

Background: The stress hormone corticosterone has the ability both to enhance and suppress synaptic plasticity and learning and memory processes. However, until today there is very little known about the molecular mechanism that underlies the bidirectional effects of stress and corticosteroid hormones on synaptic efficacy and learning and memory processes. In this study we investigate the relationship between corticosterone and AMPA receptors which play a critical role in activity-dependent plasticity and hippocampal-dependent learning. Methodology/Principal Findings: Using immunocytochemistry and live cell imaging techniques we show that corticosterone selectively increases surface expression of the AMPAR subunit GluR2 in primary hippocampal cultures via a glucocorticoid receptor and protein synthesis dependent mechanism. In agreement, we report that corticosterone also dramatically increases the fraction of surface expressed GluR2 that undergo lateral diffusion. Furthermore, our data indicate that corticosterone facilitates NMDAR-invoked endocytosis of both synaptic and extra-synaptic GluR2 under conditions that weaken synaptic transmission. Conclusion/Significance: Our results reveal that corticosterone increases mobile GluR2 containing AMPARs. The enhanced lateral diffusion properties can both facilitate the recruitment of AMPARs but under appropriate conditions facilitate the loss of synaptic AMPARs (LTD). These actions may underlie both the facilitating and suppressive effects of corticosteroid hormones on synaptic plasticity and learning and memory and suggest that these hormones accentuate synaptic efficacy

Subliminal Semantic Priming in Speech

Numerous studies have reported subliminal repetition and semantic priming in the visual modality. We transferred this paradigm to the auditory modality. Prime awareness was manipulated by a reduction of sound intensity level. Uncategorized prime words (according to a post-test) were followed by semantically related, unrelated, or repeated target words (presented without intensity reduction) and participants performed a lexical decision task (LDT). Participants with slower reaction times in the LDT showed semantic priming (faster reaction times for semantically related compared to unrelated targets) and negative repetition priming (slower reaction times for repeated compared to semantically related targets). This is the first report of semantic priming in the auditory modality without conscious categorization of the prime

Search for pair-produced long-lived neutral particles decaying to jets in the ATLAS hadronic calorimeter in ppcollisions at √s=8TeV

The ATLAS detector at the Large Hadron Collider at CERN is used to search for the decay of a scalar boson to a pair of long-lived particles, neutral under the Standard Model gauge group, in 20.3fb−1of data collected in proton–proton collisions at √s=8TeV. This search is sensitive to long-lived particles that decay to Standard Model particles producing jets at the outer edge of the ATLAS electromagnetic calorimeter or inside the hadronic calorimeter. No significant excess of events is observed. Limits are reported on the product of the scalar boson production cross section times branching ratio into long-lived neutral particles as a function of the proper lifetime of the particles. Limits are reported for boson masses from 100 GeVto 900 GeV, and a long-lived neutral particle mass from 10 GeVto 150 GeV