Generation of human antibody fragments against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>using a phage display chain shuffling approach

Background  

Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials.     

Spatial range of autocrine signaling: modeling and computational analysis

Autocrine loops formed by growth factors and their receptors have been identified in a large number of developmental, physiological, and pathological contexts. In general, the spatially distributed and recursive nature of autocrine signaling systems makes their experimental analysis, and often even their detection, very difficult. Here, we combine Brownian motion theory, Monte Carlo simulations, and reaction-diffusion models to analyze the spatial operation of autocrine loops. Within this modeling framework, the ability of autocrine cells to recapture the endogenous ligand and the distances traveled by autocrine ligands are explicitly related to ligand diffusion coefficients, density of surface receptors, ligand secretion rate, and rate constants of ligand binding and endocytic internalization. Applying our models to study autocrine loops in the epidermal growth factor receptor system, we find that autocrine loops can be highly localized--even at the level of a single cell. We demonstrate how the variations in molecular and cellular parameters may "tune" the spatial range of autocrine signals over several orders of magnitude: from microns to millimeters. We argue that this versatile regulation of the spatial range of autocrine signaling enables autocrine cells to perceive a broad spectrum of environmental information.     

Metformin attenuates post-epidural fibrosis by inhibiting the TGF-β1/Smad3 and HMGB1/TLR4 signaling pathways

Excessive post-epidural fibrosis is a common cause of recurrent back pain after spinal surgery. Though various treatment methods have been conducted, the safe and effective drug for alleviating post-epidural fibrosis remains largely unknown. Metformin, a medicine used in the treatment of type 2 diabetes, has been noted to relieve fibrosis in various organs. In the present study, we aimed to explore the roles and mechanisms of metformin in scar formation in a mouse model of laminectomy. Post-epidural fibrosis developed in a mouse model of laminectomy by spinous process and the T12-L2 vertebral plate with a rongeur. With the administration of metformin, post-epidural fibrosis was reduced, accompanied with decreased collagen and fibronectin in the scar tissues. Mechanistically, metformin decreased fibronectin and collagen deposition in fibroblast cells, and this effect was dependent on the HMGB1/TLR4 and TGF-β1/Smad3 signalling pathways. In addition, metformin influenced the metabolomics of the fibroblast cells. Taken together, our study suggests that metformin may be a potential option to mitigate epidural fibrosis after laminectomy.     

Gα<Subscript>16</Subscript> interacts with tetratricopeptide repeat 1 (TPR1) through its β3 region to activate Ras independently of phospholipase Cβ signaling

Background  

G protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα₁₆ as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1) binds to the same region as its canonical effector, phospholipase Cβ (PLCβ).     

Lactoferricin mediates anti‐inflammatory and anti‐catabolic effects via inhibition of IL‐1 and LPS activity in the intervertebral disc

The catabolic cytokine interleukin‐1 (IL‐1) and endotoxin lipopolysaccharide (LPS) are well‐known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL‐1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti‐catabolic and anti‐inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL‐1 and LPS‐mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL‐1 and LPS‐mediated proteoglycan (PG) depletion, matrix‐degrading enzyme production, and enzyme activity in long‐term (alginate beads) and short‐term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL‐1 and LPS‐mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage‐degrading enzymes, including MMP‐1, MMP‐3, MMP‐13, ADAMTS‐4, and ADAMTS‐5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor‐induced stimulation of oxidative and inflammatory factors such as iNOS, IL‐6, and toll‐like receptor‐2 (TLR‐2) and TLR‐4. Finally, the ability of LfcinB to antagonize IL‐1 and LPS‐mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. J. Cell. Physiol. 228: 1884–1896, 2013. © 2013 Wiley Periodicals, Inc.     

Induction of immunostimulatory cytokine genes expression in human PBMCs by a novel semiquinone glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1

In the present study, a semiquinone glucoside derivative (SQGD) isolated from a radioresistant bacterium Bacillus sp. INM-1 was evaluated for its immunostimulatory activities. Human peripheral blood mononuclear cells (PBMCs) were stimulated by different doses (30–90 μg/ml) of SQGD for different time (3–12 h) intervals at 37 °C, and IL-12p40, IL-23p19, IL-10, RelA and c-Jun gene expression analysis was carried out by qRT-PCR method. SQGD dose dependent cytokines protein expression kinetic analysis was carried out using western blotting. As the results of SQGD (30 μg/ml) stimulation for 3 h at 37 °C, significant induction in IL-12p40, IL-23p19 and RelA gene expression was observed in PBMCs compared to unstimulated control cells. However, no such induction in IL-10 and c-Jun gene expression was observed. Time dependent protein expression study indicated significant increase in IL-12p40, IL-12p35, IL-23p19 and RelA protein expression at 3–6 h, which was found decrease at 12 h upon SQGD treatment. In contrast, IL-10 protein expression was found to enhance significantly at 12 h after SQGD treatment to the PBMCs. SQGD dose dependent study showed approximately similar level of induction in IL-12p40, IL-12p35, IL-23p19 and RelA proteins expression at all tested concentration (30–90 μg/ml) compared to control. However, no significant change in the IL-10 and c-Jun protein expression was observed at any SQGD concentration. SQGD treatment (0.25 mg/kg b wt.) was also found to enhance anti-keyhole Limpet Hemocynin (KLH) IgM antibodies significantly in the mice immunized by KLH.Thus, SQGD fraction stimulates cellular immunity by inducing immunostimulatory cytokines and humoral immunity by enhancing IgM antibodies and could be a promising immunostimulant. Further studies related to molecular mechanisms offering immunostimulation is underway, will certainly helpful to unravel its mode of action in the biological system.     

Dopamine and cAMP‐regulated phosphoprotein 32kDa (DARPP‐32), protein phosphatase‐1 and cyclin‐dependent kinase 5 expression in ovarian cancer

Dopamine and cyclic‐AMP activated phosphoprotein Mr32kDa (DARPP‐32) is a central signalling protein in neurotransmission. Following DARPP‐32 phosphorylation by protein kinase A (PKA), DARPP‐32 becomes a potent protein phosphatase 1 (PP1) inhibitor. DARPP‐32 can itself inhibit PKA following DARPP‐32 phosphorylation by cyclin‐dependent kinase 5 (Cdk5). Increasing evidence indicates a role for DARPP‐32 and its associated signalling pathways in cancer; however, its role in ovarian cancer remains unclear. Using immunohistochemistry, expression of DARPP‐32, PP1 and Cdk5 was determined in a large cohort of primary tumours from ovarian cancer patients (n = 428, 445 and 434 respectively) to evaluate associations between clinical outcome and clinicopathological criteria. Low cytoplasmic and nuclear DARPP‐32 expression was associated with shorter patient overall survival and progression‐free survival (P = .001, .001, .004 and .037 respectively). Low nuclear and cytoplasmic DARPP‐32 expression remained significantly associated with overall survival in multivariate Cox regression (P = .045, hazard ratio (HR) = 0.734, 95% confidence interval (CI) = 0.542‐0.993 and P = .001, HR = 0.494, 95% CI = 0.325‐0.749, respectively). High cytoplasmic and nuclear PP1 expression was associated with shorter patient overall survival and high cytoplasmic PP1 expression with shorter progression‐free survival (P = .005, .033, and .037, respectively). High Cdk5 expression was associated with shorter progression‐free survival (P = .006). These data suggest a role for DARPP‐32 and associated signalling kinases as prognostic markers with clinical utility in ovarian cancer.     

Crop rotations differ in soil carbon stabilization efficiency,but the response to quality of structural plant inputs is ambiguous

Plant and Soil - There is a trend of increasing woody biomass in tropical savannas. Here we ask what effect this increase may have on soil carbon pools and fluxes. Using a field experiment we...     

Plant-made vaccines in support of the Millennium Development Goals

Vaccines are one of the most successful public health achievements of the last century. Systematic immunisation programs have reduced the burden of infectious diseases on a global scale. However, there are limitations to the current technology, which often requires costly infrastructure and long lead times for production. Furthermore, the requirement to keep vaccines within the cold-chain throughout manufacture, transport and storage is often impractical and prohibitively expensive in developing countries—the very regions where vaccines are most needed. In contrast, plant-made vaccines (PMVs) can be produced at a lower cost using basic greenhouse agricultural methods, and do not need to be kept within such narrow temperature ranges. This increases the feasibility of developing countries producing vaccines locally at a small-scale to target the specific needs of the region. Additionally, the ability of plant-production technologies to rapidly produce large quantities of strain-specific vaccine demonstrates their potential use in combating pandemics. PMVs are a proven technology that has the potential to play an important role in increasing global health, both in the context of the 2015 Millennium Development Goals and beyond.