Therapeutic and diagnostic applications of monoclonal antibodies often require careful selection of binders that recognize specific epitopes on the target molecule to exert a desired modulation of ...biological function. Here we present a proof-of-concept application for the rational design of an epitope-specific antibody binding with the target protein Keap1, by grafting pre-defined structural interaction patterns from the native binding partner protein, Nrf2, onto geometrically matched positions of a set of antibody scaffolds. The designed antibodies bind to Keap1 and block the Keap1-Nrf2 interaction in an epitope-specific way. One resulting antibody is further optimised to achieve low-nanomolar binding affinity by in silico redesign of the CDRH3 sequences. An X-ray co-crystal structure of one resulting design reveals that the actual binding orientation and interface with Keap1 is very close to the design model, despite an unexpected CDRH3 tilt and V
/V
interface deviation, which indicates that the modelling precision may be improved by taking into account simultaneous CDR loops conformation and V
/V
orientation optimisation upon antibody sequence change. Our study confirms that, given a pre-existing crystal structure of the target protein-protein interaction, hotspots grafting with CDR loop swapping is an attractive route to the rational design of an antibody targeting a pre-selected epitope.
We report phonon densities of states (DOS) of iron measured by nuclear resonant inelastic x-ray scattering to 153 gigapascals and calculated from ab initio theory. Qualitatively, they are in ...agreement, but the theory predicts density at higher energies. From the DOS, we derive elastic and thermodynamic parameters of iron, including shear modulus, compressional and shear velocities, heat capacity, entropy, kinetic energy, zero-point energy, and Debye temperature. In comparison to the compressional and shear velocities from the preliminary reference Earth model (PREM) seismic model, our results suggest that Earth's inner core has a mean atomic number equal to or higher than pure iron, which is consistent with an iron-nickel alloy.
A new disulfide crosslinking method was developed for the preparation of blended hyaluronan (HA)–gelatin hydrogels to form a synthetic, covalently linked mimic of the extracellular matrix (ECM). The ...HA and gelatin were chemically modified using 3,3′-dithiobis(propionic hydrazide) (DTP). After reduction with dithiothreitol (DTT), the thiol derivatives of HA (HA-DTPH) and gelatin (gelatin-DTPH) were obtained and characterized. To minimize interference with biological function, the degree of substitution of HA-DTPH and gelatin-DTPH was kept below 50%. Solutions of HA-DTPH and gelatin-DTPH in varying blends (20%, 40%, 60%, 80% gelatin) were prepared in 1% w/v NaCl and crosslinked by disulfide bond formation in air. Hydrogel films were dried and further crosslinked with dilute hydrogen peroxide. Disulfide crosslinked HA-DTPH, gelatin-DTPH, and blends thereof, were degradable enzymatically by collagenase and by hyaluronidase (HAse). The rapid digestion of the crosslinked 100% gelatin-DTPH film by collagenase was significantly retarded by the presence of 20% or 40% HA-DTPH. Addition of at least 40% w/v gelatin into the 100% HA-DTPH films significantly improved the attachment and spreading of Balb/c 3T3 murine fibroblasts seeded on the surface of the hydrogel. These results demonstrate that disulfide-crosslinked HA–gelatin hydrogels, a new type of covalent synthetic ECM, constitute biocompatible and biodegradable substrata for cell culture in vitro.
Cambrian fossil-Lagerstätten (sites of exceptional fossil preservation), such as those from Chengjiang (Lower Cambrian) and the Burgess Shale (Middle Cambrian), provide our best window into the ...Cambrian 'explosion'. Such faunas are known from about 40 localities, and have yielded a widely disparate series of taxa ranging from ctenophores to agnathan fish. Recent excavations of the Chengjiang fossil-Lagerstätte, known from a series of sites near Kunming in Yunnan, south China, have resulted in the discovery of several new forms. In conjunction with material described earlier, these provide evidence for a new group of metazoans, the vetulicolians. Several features, notably a series of gill slits, suggest that this group can throw light on an early stage of deuterostome diversification.
The microstructure and corrosion behavior of a base metal (BM) and heat-affected zone (HAZ) of Q315NS steel in 0.1 mol L
−1
HCl solution at 60 °C were investigated with a thermal simulation ...technique, surface observation and electrochemical measurements. The transient characteristics, as well as the power spectral density (PSD) and wavelet transform, had been employed to analyze the electrochemical noise (EN) data. It was revealed that the microstructure of BM, the fine grained region (FGHAZ) and the inter critical region (ICHAZ) mainly consisted of ferrite and pearlite, while the coarse grained region (CGHAZ) mainly consisted of granular bainite. The corrosion processes of BM, FGHAZ and ICHAZ were similar and characterized by two stages. The first stage involved the metastable pitting process and the second stage corresponded to the pitting corrosion. On the CGHAZ electrode, metastable pitting occurred in the initial corrosion stage and then the corrosion process turned into uniform corrosion. With increasing corrosion time, the corrosion strength of all electrodes decreased significantly at first and then remained constant. The corrosion rate of CGHAZ was slightly lower than the other electrodes in the second corrosion stage, and there was no obvious difference in corrosion rate among BM, FGHAZ and ICHAZ.
Uniform corrosion occurred on CGHAZ of Q315NS owing to coarse GB while pitting occurs on BM after metastable pitting.
We report on the discovery of three bright, strongly lensed objects behind Abell 1703 and CL0024+16 from a dropout search over 25 arcmin2 of deep NICMOS data, with deep ACS optical coverage. They are ...undetected in the deep ACS images below 8500 A and have clear detections in the J and H bands. Fits to the ACS, NICMOS, and IRAC data yield robust photometric redshifts in the range z ~ 6-7 and largely rule out the possibility that they are low-redshift interlopers. All three objects are extended, and resolved into a pair of bright knots. The bright i-band dropout in Abell 1703 has an H-band AB magnitude of 23.9, which makes it one of the brightest known galaxy candidates at z > 5.5. Our model fits suggest a young, massive galaxy only ~60 million years old with a mass of ~1010 M . The dropout galaxy candidates behind CL0024+16 are separated by 25 (~2 kpc in the source plane), and have H-band AB magnitudes of 25.0 and 25.6. Lensing models of CL0024+16 suggest that the objects have comparable intrinsic magnitudes of AB ~27.3, approximately one magnitude fainter than L* at z ~ 6.5. Their similar redshifts, spectral energy distribution, and luminosities, coupled with their very close proximity on the sky, suggest that they are spatially associated, and plausibly are physically bound. Combining this sample with two previously reported, similarly magnified galaxy candidates at z ~ 6-8, we find that complex systems with dual nuclei may be a common feature of high-redshift galaxies.
The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is ...unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.
Hyaluronan (HA) can be chemically modified to engineer robust materials with pre-selected mechanical properties and resorption rates that can be dictated by the intended clinical use. ...Disulfide-crosslinked HA films were prepared by air oxidation of thiol-modified HA, followed by treatment with 0.3% hydrogen peroxide. The degradation of the disulfide-crosslinked films in vitro was very slow (<10% in 7 days) in buffer alone and shorter (
t
1/2=3–5 days) in the presence of hyaluronidase (HAse). The cytocompatibility of the disulfide-crosslinked HA films was determined using two separate conditions: (i) in vitro culture of mouse fibroblasts in indirect contract with the films, and (ii) in vitro culture of fibroblasts directly on films coated with poly
d-lysine. Excellent cytocompatibility was observed in murine fibroblasts that were cultured in indirect contact with thiolated HA films. Although cells were unable to attach and spread on thiolated HA films, pre-coating the thiolated HA films with poly D-lysine resulted in attachment and spreading equivalent to that observed on polystyrene. Rates of resorption in vivo were obtained by subcutaneous implantation of disulfide-crosslinked HA films into the backs of Wistar rats. Biocompatibility in vivo was determined in both subcutaneous flank and peritoneal cavity implantation of the films in Wistar rats. The disulfide-crosslinked HA films were less than 30% resorbed after 42 days in vivo, and histochemical and cytochemical analysis indicated that the films were well-tolerated with mild inflammatory response at both sites of implantation.
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