In the present study, a tandem-repeat type galectin was characterized from an embryonic cell line (Bge) and circulating hemocytes of the snail Biomphalaria glabrata, intermediate host of the human ...blood fluke Schistosoma mansoni. The predicted B. glabrata galectin (BgGal) protein of 32 kDa possessed 2 carbohydrate recognition domains, each displaying 6 of 8 conserved amino acids involved in galactoside-binding activity. A recombinant BgGal (rBgGal) demonstrated hemagglutinating activity against rabbit erythrocytes, which was specifically inhibited by galactose-containing sugars (lacNAc/lac>galNAc/gal). Although native galectin was immunolocalized in the cytoplasm of Bge cells and the plasma membrane of a subset of snail hemocytes (60%), it was not detected in cell-free plasma by Western blot analysis. The findings that rBgGal selectively recognizes the schistosome-related sugar, lacNAc, and strongly binds to hemocytes and the tegument of S. mansoni sporocysts in a sugar-inhibitable fashion suggest that hemocyte-bound galectin may be serving as a pattern recognition receptor for this, or other pathogens possessing appropriate sugar ligands. Based on molecular and functional features, BgGal represents an authentic galectin, the first to be fully characterized in the medically-important molluscan Class Gastropoda.
Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed ...VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.
A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four ...human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified U-15N-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified U-15N-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.
Functional magnetic resonance imaging (fMRI) techniques involve studying the brain activity of an experimental subject in response to a mental stimulus, such as a picture or a video shown to the ...subject. The design problem in fMRI studies is to come up with the best sequence of stimuli to be shown to subjects which enables the precise estimation of the brain activity. In previous analytical studies concerning fMRI designs, it has been assumed that the errors are independent over time. The current state-of-the-art method to find optimal designs for the situations when the errors are correlated is to use the genetic algorithm. In this paper, we analytically obtain the optimal designs in a subclass for the situations when the errors over time are assumed to have an auto-regressive (AR) structure. Since such optimal designs might not exist, in practice, we advocate the use of what we call g-lag orthogonal designs. We show that these g-lag orthogonal designs perform reasonably well under a wide range of conditions under the models with correlated errors.
•(k-1)-lag orthogonal designs for event-related fMRI studies with one stimulus type, which are universally optimal for estimation when errors are uncorrelated, can be efficient when errors are correlated.•Under an AR(1) error structure, A-optimal (k-1)-lag orthogonal designs are identified.•Under an AR(1) error structure, k-lag orthogonal designs are highly efficient for most values of the AR(1) parameter.•Under an AR(2) error structure, (k+1)-lag orthogonal designs are highly efficient for many values of the AR(2) parameters.•Efficient designs for larger design sequences can be constructed from those for smaller design sequences.
The LOFAR Two-metre Sky Survey Shimwell, T. W.; Hardcastle, M. J.; Tasse, C. ...
Astronomy and astrophysics (Berlin),
03/2022, Letnik:
659
Journal Article
Recenzirano
Odprti dostop
In this data release from the ongoing LOw-Frequency ARray (LOFAR) Two-metre Sky Survey we present 120–168 MHz images covering 27% of the northern sky. Our coverage is split into two regions centred ...at approximately 12h45m +44°30′ and 1h00m +28°00′ and spanning 4178 and 1457 square degrees respectively. The images were derived from 3451 h (7.6 PB) of LOFAR High Band Antenna data which were corrected for the direction-independent instrumental properties as well as direction-dependent ionospheric distortions during extensive, but fully automated, data processing. A catalogue of 4 396 228 radio sources is derived from our total intensity (Stokes
I
) maps, where the majority of these have never been detected at radio wavelengths before. At 6″ resolution, our full bandwidth Stokes
I
continuum maps with a central frequency of 144 MHz have: a median rms sensitivity of 83 μJy beam
−1
; a flux density scale accuracy of approximately 10%; an astrometric accuracy of 0.2″; and we estimate the point-source completeness to be 90% at a peak brightness of 0.8 mJy beam
−1
. By creating three 16 MHz bandwidth images across the band we are able to measure the in-band spectral index of many sources, albeit with an error on the derived spectral index of > ± 0.2 which is a consequence of our flux-density scale accuracy and small fractional bandwidth. Our circular polarisation (Stokes
V
) 20″ resolution 120–168 MHz continuum images have a median rms sensitivity of 95 μJy beam
−1
, and we estimate a Stokes
I
to Stokes
V
leakage of 0.056%. Our linear polarisation (Stokes
Q
and Stokes
U
) image cubes consist of 480 × 97.6 kHz wide planes and have a median rms sensitivity per plane of 10.8 mJy beam
−1
at 4′ and 2.2 mJy beam
−1
at 20″; we estimate the Stokes
I
to Stokes
Q
/
U
leakage to be approximately 0.2%. Here we characterise and publicly release our Stokes
I
,
Q
,
U
and
V
images in addition to the calibrated
uv
-data to facilitate the thorough scientific exploitation of this unique dataset.
The interaction between IgM and C1q represents the first step of the classical pathway of the complement system in higher vertebrates. To identify the significance of particular IgM/C1q interactions, ...recombinant IgMs were used in both hexameric and pentameric configurations and with two different specificities, along with C1q derived from human serum (sC1q) and two recombinant single-chain variants of the trimeric globular region of C1q. Interaction and complement activation assays were performed using the ELISA format, and bio-layer interferometry measurements to study kinetic behavior. The differences between hexameric and pentameric IgM conformations were only slightly visible in the interaction assay, but significant in the complement activation assay. Hexameric IgM requires a lower concentration of sC1q to activate the complement compared to pentameric IgM, leading to an increased release of C4 compared to pentameric IgM. The recombinant C1q mimetics competed with sC1q in interaction assays and were able to inhibit complement activation. The bio-layer interferometry measurements revealed K
values in the nanomolar range for the IgM/C1q interaction, while the C1q mimetics exhibited rapid on and off binding rates with the IgMs. Our results make C1q mimetics valuable tools for developing recombinant C1q, specifically its variants, for further scientific studies and clinical applications.
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