Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.     

Overexpression of CD44 in Neural Precursor Cells Improves Trans- Endothelial Migration and Facilitates Their Invasion of Perivascular Tissues In Vivo

Neural precursor (NPC) based therapies are used to restore neurons or oligodendrocytes and/or provide neuroprotection in a large variety of neurological diseases. In multiple sclerosis models, intravenously (i.v) -delivered NPCs reduced clinical signs via immunomodulation. We demonstrated recently that NPCs were able to cross cerebral endothelial cells in vitro and that the multifunctional signalling molecule, CD44 involved in trans-endothelial migration of lymphocytes to sites of inflammation, plays a crucial role in extravasation of syngeneic NPCs. In view of the role of CD44 in NPCs trans-endothelial migration in vitro, we questioned presently the benefit of CD44 overexpression by NPCs in vitro and in vivo, in EAE mice. We show that overexpression of CD44 by NPCs enhanced over 2 folds their trans-endothelial migration in vitro, without impinging on the proliferation or differentiation potential of the transduced cells. Moreover, CD44 overexpression by NPCs improved significantly their elongation, spreading and number of filopodia over the extracellular matrix protein laminin in vitro. We then tested the effect of CD44 overexpression after i.v. delivery in the tail vein of EAE mice. CD44 overexpression was functional in vivo as it accelerated trans-endothelial migration and facilitated invasion of HA expressing perivascular sites. These in vitro and in vivo data suggest that CD44 may be crucial not only for NPC crossing the endothelial layer but also for facilitating invasion of extravascular tissues.     

Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1

Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2?U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2?U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65?°C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60?°C for almost 90?min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.     

Assessment of the elastic properties of amorphous calcium silicates hydrates (I) and (II) structures by molecular dynamics simulation

Based on Hamid model of 11Å tobermorite, amorphous calcium silicates hydrates (or C-S-H) structures (Ca₄Si₆O₁₄(OH)₄?2H₂O as the C-S-H(I) and (CaO)_1.67(SiO₂)(H₂O)_1.75 as the C-S-H(II)) with the Ca/Si ratio of 0.67 and 1.7 are concerned. Then, as the representative ‘globule’ C-S-H, two amorphous C-S-H structures with the size of 5.352 × 4.434 × 4.556 nm³ during the stretch process are simulated at a certain strain rate of 10^?3 ps^?1 by LAMMPS program for molecular dynamics simulation, using ClayFF force field. The tensile stress–strain curves are obtained and analysed. Besides, elastic modulus of the ‘globule’ C-S-H is calculated to assess the elastic modulus of C-S-H phases (the low-density C-S-H – LD C-S-H – and the high-density C-S-H – HD C-S-H), where the porosity is a critical factor for explaining the relationship between ‘globule’ C-S-H at nanoscale and C-S-H phases at microscale. Results show that: (1) The C-S-H(I) structure has transformed from crystalline to amorphous during the annealing process, Young’s moduli in x, y and z directions are almost the same. Besides, the extent of aggregation and aggregation path for water molecules in the structure is different in three directions. (2) Young’s modulus of both amorphous C-S-H(I) and C-S-H(II) structures with a size of about 5 nm under strain rate of 10^?3 ps^?1 at 300 K in three directions is averaged to be equal, of which C-S-H(II) structure is about 60.95 GPa thus can be seen as the elastic modulus of the ‘globule’ C-S-H. (3) Based on the ‘globule’ C-S-H, the LD C-S-H and HD C-S-H can be assessed by using the Self-Consistent Scheme (separately 18.11 and 31.45 GPa) and using the Mori–Tanaka scheme (29.78 and 37.71 GPa), which are close to the nanoindentation experiments by Constantinides et al. (21.7 and 29.4 GPa).     

Anticrossing Behavior of Surface Plasmon Polariton Dispersions in Metal-Insulator-Metal Structures

The anticrossing behavior of dispersion curves of the surface plasmon polaritons supported by metal-insulator-metal structures are studied experimentally and theoretically. Samples consisting of a poly(methyl methacrylate) layer sandwiched by Ag films are prepared and their angle- and wavelength-scan attenuated total reflection spectra are measured. From an analysis of the angle-scan spectrum, the coupled-mode nature of the surface plasmon polariton modes is suggested. The dispersion relations obtained from the wavelength-scan spectra exhibit clearly the anticrossing behavior that arises from the coupling of the modes. The experimental dispersion relations are in good agreement with theoretical ones.     

Exercise training decreases plasma leptin levels and the expression of hepatic leptin receptor-a, -b, and, -e in rats

In addition to acting in the central nervous system, leptin also acts on peripheral tissues such as liver to provide a protection against lipid accretion. Previous evidence from human and animal model indicates that exercise training reduces circulating leptin levels beyond the changes in adiposity levels. Because liver is one of the main peripheral organs for leptin action, this present study was designed to determine whether leptin receptors expression in liver is changed by exercise training. Female rats trained (TR) or kept sedentary (Sed) for 8 weeks were submitted either to a standard (SD) diet for 8 weeks or for 6 weeks followed by 2 weeks of high-fat (HF) or high-carbohydrate (HC) feeding. Food intake, adiposity levels, circulating plasma leptin and insulin concentrations along with the hepatic expression of leptin receptors (ObR-a, -b, and -e) and peroxisome proliferator-activated receptor α (PPARα) and peroxisome proliferator-activated receptor-gamma co-activator-1α (PGC-1α), were measured in all the animals. Intra-abdominal fat depots were increased under the HF but not under the HC diet. As expected, exercise training decreases intra-abdominal adiposity in animals fed with the SD and the HF diet, and to a lesser extent in HC-fed rats. Plasma leptin levels either expressed in absolute values or in values relative to adiposity levels were significantly (P < 0.05) increased with the HF diet and significantly decreased in TR animals, independently of the diet. Moreover, a significant (P < 0.01) reduction in hepatic gene expression of ObR-a, -b and -e was found in TR animals in all the three diet conditions. PPARα and PGC-1α mRNAs were also decreased (P < 0.05) in TR animals in two out of three diet conditions. The present findings indicate that exercise training-induced decrease in plasma leptin levels is accompanied by a reduction in gene expression of three different isoforms of leptin receptors in liver.     

Silencing NaTPI Expression Increases Nectar Germin,Nectarins, and Hydrogen Peroxide Levels and Inhibits Nectar Removal from Plants in Nature

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellant, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H₂O₂) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H₂O₂ compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H₂O₂ and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H₂O₂ levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H₂O₂ to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.Floral nectar is an innovative feature of plants that is thought to have evolved as a reward for pollen-transporting floral visitors. Sugars (e.g. Glc, Fru, and Suc), amino acids, and lipids (Baker and Baker, 1982, 1986) provide nutritional rewards that are essential for many pollinators. But nectar is also known to contain other compounds, such as volatile organic compounds (VOCs), alkaloids, phenolics, and nonprotein amino acids (Baker, 1977, 1978; Raguso, 2004; Kessler and Baldwin, 2007), which do not increase the nutritional value of nectar. Nectar is also exploited as a food source by nectar robbers and nectar-infesting microorganisms, which do not provide mutualistic services to the plant and are known to directly reduce a plant''s fitness either by competing with pollinators or by infesting reproductive organs (Traveset et al., 1998; Irwin and Brody, 1999; Maloof and Inouye, 2000; Farkas et al., 2007). Therefore, flowers must solve the dilemma of repelling nectar thieves or florivores that provide no pollination services while simultaneously attracting fitness-enhancing pollinators.Most of the defensive compounds in nectar have been reported to act selectively (i.e. only on antagonists). For example, the floral nectar of Catalpa speciosa contains iridoid glycosides that fend off nectar robbers but not the plant''s specific pollinators (Stephenson, 1981). Similarly, the presence of phenols in the floral nectar of Aloe vryheidensis lowers its palatability to generalist floral visitors like sunbirds or honey bees while not affecting the attractiveness of the nectar to a specialist bird, the dark-capped bulbul (Johnson et al., 2006). In its native habitat, Nicotiana attenuata (Solanaceae) maximizes its maternal and paternal reproductive success while repelling herbivores, florivores, and nectar robbers by producing a sophisticated blend of both repellants (nicotine) and attractants (benzylacetone) in its nectar and floral head space (Kessler et al., 2008) as well as by changing its floral phenology in response to herbivore attack, so as to switch from the use of night-active hawkmoth pollinators, which oviposit herbivores on the plants they pollinate, to day-active hummingbird pollinators, which do not (Kessler et al., 2010). While this sophisticated use of chemical attractants and repellants is likely a common solution to the dilemma, very little is known about the function of most secondary metabolites found in nectar (Thornburg, 2007).Similar chemically mediated strategies are used to solve a similar problem when plants use a combination of direct and indirect defenses to protect their leaves from herbivore attack (Halitschke et al., 2008). In N. attenuata, attack by the specialist herbivore Manduca sexta elicits a remarkable array of direct and indirect defenses, most of which are elicited by the jasmonate signaling pathway in response to herbivore-specific elicitors (Baldwin, 2001; Kessler and Baldwin, 2002; Wu and Baldwin, 2009). These herbivory-elicited responses include the accumulation of toxins and digestibility reducers, which function as direct defenses, as well as the release of a complicated blend of VOCs (Gaquerel et al., 2009), which repel further oviposition by M. sexta moths and attract predacious bugs that feed on M. sexta eggs or larvae, thereby functioning as an indirect defense (Kessler and Baldwin, 2001).Once herbivores start feeding on N. attenuata leaves, they are frequently repelled by a suite of locally and systemically elicited direct defenses (Steppuhn et al., 2008). Trypsin protease inhibitors (TPIs) are an effective component of this inducible defensive system that reduces the performance of folivores by targeting their main proteolytic digestive enzymes and is strongly induced by herbivore attack (van Dam et al., 2000; Glawe et al., 2003; Zavala et al., 2004b, 2008; Horn et al., 2005). However, in N. attenuata, the biosynthesis of TPIs incurs substantial fitness costs (Zavala et al., 2004a); silencing the TPI gene in N. attenuata abolishes the plant''s capacity to produce TPIs and allows it to grow faster, flower earlier, and produce more seed capsules compared with TPI-producing genotypes (Zavala et al., 2004a). Similarly, restoring TPI production by transforming an ecotype of N. attenuata naturally deficient in TPI production (Wu et al., 2007) reduces lifetime seed production (Zavala et al., 2004a). TPIs are not only restricted to leaves but accumulates in reproductive organs, where they may protect these fitness-valuable tissues against attack from florivores and microbes. Atkinson et al. (1993) and Johnson et al. (2007) elegantly demonstrated that TPIs dramatically accumulate in Nicotiana alata stigmas to become the most abundant protein in these tissues. PIs have been reported to accumulate in Solanum americanum seeds, where they were shown to play an important role in seed development (Suk-Fong et al., 2006). These studies highlight that while it is clear that TPIs occur at high levels in reproductive organs, their role in floral function has not been thoroughly explored.As part of a research program to study the defensive functions of nicotine and TPIs against folivores, we planted N. attenuata plants that had been transformed with RNA interference constructs to silence their nicotine (ir-pmt), TPI (ir-pi), or both (ir-pmt/pi) in the plant''s native habitats in Utah during four field seasons. Serendipitously, we noticed that the amount of nectar removed by the native community of floral visitors from ir-pmt/pi plants did not differ from that removed from wild-type plants, although we had recently discovered that silencing nicotine alone (ir-pmt) consistently increased nectar removal (Kessler and Baldwin, 2007). These observations suggested that silencing TPIs alone might impede nectar removal by the native community of floral visitors. During two field seasons (2007 and 2009), we compared the amount of nectar removed from wild-type plants and from TPI-silenced plants (ir-pi) and found that, indeed, less nectar was consistently removed from ir-pi plants. To understand these observations, we compared the floral chemistry of wild-type and ir-pi plants, including floral volatiles, nectar sugar, nicotine, and proteomes. We found that silencing TPIs increased the accumulation of nectar proteins, especially the nectar germin-like proteins (GLPs) and nectarins, which are known to participate in the nectar redox cycle and generate hydrogen peroxide (H₂O₂; Carter and Thornburg, 2000). Consistent with data on nectar proteins, we also found significantly higher levels of H₂O₂ in the nectar of ir-pi plants compared with those of wild-type plants. To test whether the differences in the accumulation of H₂O₂ in the nectar of ir-pi and wild-type plants could explain the nectar removal observations in the field, we experimentally increased H₂O₂ in the nectar of wild-type plants to the levels found in ir-pi nectar using a mixture of Glc oxidase (GOX) and Glc and compared nectar removal by the native community of floral visitors.     

Leptin regulates MMP-2, TIMP-1 and collagen synthesis via p38 MAPK in HL-1 murine cardiomyocytes

A clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells. Gelatin zymographic analysis of MMP activity in conditioned media showed that leptin enhanced MMP-2 activity in a dose- and time-dependent manner. Leptin is known to stimulate phosphorylation of p38 MAPK in cardiac cells and utilization of the p38 MAPK inhibitor, SB203580, demonstrated that this kinase also plays a role in regulating several extracellular matrix components, such that inhibition of p38 MAPK signaling prevented the leptin-induced increase in MMP-2 activation. We also observed that leptin enhanced collagen synthesis determined by both proline incorporation and picrosirius red staining of conditioned media. Pro-collagen type-I and pro-collagen type-III expression, measured by real-time PCR and Western blotting were also increased by leptin, effects which were again attenuated by SB203580. In summary, these results demonstrate the potential for leptin to play a role in mediating myocardial ECM remodeling and that the p38 MAPK pathway plays an important role in mediating these effects.     

A simplified 2-D electrophoresis protocol with the aid of an organic disulfide

2-DE is still a relatively cumbersome and labor intensive method. Given the successful cysteinyl protection concept with hydroxyethyl disulfide (specific oxidation) during the first dimension separation, the possibility for a simplified equilibration procedure was investigated. This was achieved by maintaining the S-mercaptoethanol modified cysteinyls throughout the 2-D workflow including second dimension separation, spot handling, protein digestion, and protein identification. The traditional equilibration protocol encompassing thiol reduction and alkylation was compared with a one-step protocol employing continuous exposure to hydroxyethyl disulfide. Both equilibration protocols gave equally well-resolved spot maps with analytical protein loads regardless of IPG strip pH range. Using preparative protein loads, narrow range IPG strips gave comparable results for the two protocols while preparative load on wide range IPG strips was the only condition where classical reduction/alkylation outperformed hydroxyethyl disulfide equilibration. Moreover, with analytical protein loads, the hydroxyethyl disulfide equilibration time could be significantly reduced without apparent loss of spot map quality or quantitative protein transfer from the first- to the second dimension gel. MALDI-TOF mass spectrometric protein identification was successfully performed with either iodoacetamide or hydroxyethyl disulfide as the cysteine modifier, yielding comparable identification results with high confidence in protein assignment, sequence coverage, and detection of cysteine-containing peptides. The results provide a novel and simplified protocol for 2-DE where the concept of hydroxyethyl disulfide as the cysteinyl protecting agent is extended to cover the entire 2-D work flow.