Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant

Abstract We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.http://deepblue.lib.umich.edu/bitstream/2027.42/109454/1/13075_2000_Article_409.pd

Evaluating the Bioactivity of a Novel Broad-Spectrum Antimicrobial Peptide Brevinin-1GHa from the Frog Skin Secretion of Hylarana guentheri and Its Analogues

Many antimicrobial peptides (AMPs) have been identified from the skin secretion of the frog Hylarana guentheri (H.guentheri), including Temporin, Brevinin-1, and Brevinin-2. In this study, an antimicrobial peptide named Brevinin-1GHa was identified for the first time by using ‘shotgun’ cloning. The primary structure was also confirmed through mass spectral analysis of the skin secretion purified by reversed-phase high-performance liquid chromatography (RP-HPLC). There was a Rana-box (CKISKKC) in the C-terminal of Brevinin-1GHa, which formed an intra-disulfide bridge. To detect the significance of Rana-box and reduce the hemolytic activity, we chemically synthesized Brevinin-1GHb (without Rana-box) and Brevinin-1GHc (Rana-box in central position). Brevinin-1GHa exhibited a strong and broad-spectrum antimicrobial activity against seven microorganisms, while Brevinin-1GHb only inhibited the growth of Staphylococcus aureus (S. aureus), which indicates Rana-box was necessary for the antimicrobial activity of Brevinin-1GHa. The results of Brevinin-1GHc suggested transferring Rana-box to the central position could reduce the hemolytic activity, but the antimicrobial activity also declined. Additionally, Brevinin-1GHa demonstrated the capability of permeating cell membrane and eliminating biofilm of S. aureus, Escherichia coli (E. coli), and Candida albicans (C. albicans). The discovery of this research may provide some novel insights into natural antimicrobial drug desig

Enzymatic Degradation of Alginate and in Vitro Immunological Activity of Its Degraded Products

In this study, changes in the molecular mass of alginate were investigated during its enzymatic degradation and the processing parameters for the enzymatic preparation of alginate oligosaccharides were explored. Furthermore, the in vitro immunological activity of degraded products with different molecular mass was evaluated. The results showed that the molecular mass of alginate decreased significantly after degradation by alginate lyase, and three degradation products with different molecular mass were obtained through gradient ethanol fractionation. Their weight-average molecular masses were 13.4, 5.73 and 3.85 kDa, respectively. Using single factor experiments, the optimal processing parameters were determined as pH 7.0, alginate lyase dosage 15 U/g substrate, and hydrolysis time 24 h, giving a yield of 28.05%. All alginate and its degraded products had immunoenhancing activity in mouse macrophages, and among them, the effect of the product with a weight-average molecular mass of 5.73 kDa was most pronounced and more pronounced than that of alginate oligosaccharides. By adding TAK-242, a blocker of macrophage Toll-like receptor 4 (TLR4), it was verified that the degraded products of alginate regulated macrophage immune activity by inducing the secretion of TLR4 and consequently causing cascade reactions to increase the secretion of NO, TNF-α and IL-6. These results can provide a theoretical basis for the high-value utilization of alginate

Aurora A–Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform–selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A–selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition–associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A–selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent

Placement By Marriage

As the field programmable gate array (FPGA) industry grows device capacity with Moore's law and expands its market to high performance computing, scalability of its key CAD algorithms emerges as a new priority to deliver a user experience competitive to parallel processors. Among the many walls to overcome, placement stands out due to its critical impact on both frontend synthesis and backend routing. To construct a scalable placement flow, we present three innovations in detailed placement: a legalizer that works well under low whitespace; a wirelength optimizer based on bipartite matching; and a cache-aware annealer. When applied to the hundred-thousand cell IBM benchmark suite, our detailed placer can achieve 27% better wirelength and 8X faster runtime against FastDP, the fastest academic detailed placer reported, and our full placement flow can achieve 101X faster runtime, with 5% wirelength overhead, against VPR, the de facto standard in FPGA placements.MAS

The Defensive Role of Endogenous H2S in Brassica rapa against Mercury-Selenium Combined Stress

Plants are always exposed to the environment, polluted by multiple trace elements. Hydrogen sulfide (H2S), an endogenous gaseous transmitter in plant cells, can help plant combat single elements with excess concentration. Until now, little has been known about the regulatory role of H2S in response to combined stress of multiple elements. Here we found that combined exposure of mercury (Hg) and selenium (Se) triggered endogenous H2S signal in the roots of Brasscia rapa. However, neither Hg nor Se alone worked on it. In roots upon Hg + Se exposure, the defensive role of endogenous H2S was associated to the decrease in reactive oxygen species (ROS) level, followed by alleviating cell death and recovering root growth. Such findings extend our knowledge of plant H2S in response to multiple stress conditions

Contrasting photosynthesis, photoinhibition and oxidative damage in honeysuckle (Lonicera japonica Thunb.) under iso-osmotic salt and drought stresses

Honeysuckle (Lonicera japonica Thunb.) is a traditional Chinese medicinal crop and belongs to the glycophyte with certain salt tolerance. This study aimed to deeply dissect its salt adaptability by contrasting photosynthesis, photoinhibition and oxidative damage under moderate and severe iso-osmotic salt (150 and 300 mM NaCl) and drought (19.3 % and 28 % PEG-6000) stresses with hydroponic protocol. Photosynthesis was more susceptible to drought stress than iso-osmotic salt stress in honeysuckle according to drought-induced greater decrease in photosynthetic rate. In contrast to salt-induced mild PSII and PSI photoinhibition, severe photosystem II (PSII) and photosystem I (PSI) photoinhibition arose upon iso-osmotic drought stress, indicated by greater decreased the maximal photochemical efficiency of PSII and PSI and remarkable loss of their reaction center proteins. However, PSII and PSI interaction hardly contributed to salt stability of photosynthetic apparatus because of salt-induced finite restriction on electron flow from PSII to PSI. Consistent with photosystems photoinhibition, leaf lipid peroxidation, H2O2 production and electrolyte leakage were elevated much greater by drought stress than iso-osmotic salt stress, confirming drought-induced severe oxidative stress in honeysuckle. Furthermore, the principal components analysis comprehensively showed higher salt adaptability in honeysuckle due to larger cluster separation upon drought stress than iso-osmotic salt stress. As an apparent reason, honeysuckle could prevent drought-induced tremendous leaf water loss upon iso-osmotic salt stress, and had a capacity to dispose accumulated Na+. Therefore, honeysuckle resembles halophytes in this respect and seems appropriate for planting in coastal saline land