25 research outputs found
Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration
Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase α
In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo(−)) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment
The Murchison Widefield Array
It is shown that the excellent Murchison Radio-astronomy Observatory site
allows the Murchison Widefield Array to employ a simple RFI blanking scheme and
still calibrate visibilities and form images in the FM radio band. The
techniques described are running autonomously in our calibration and imaging
software, which is currently being used to process an FM-band survey of the
entire southern sky.Comment: Accepted for publication in Proceedings of Science [PoS(RFI2010)016].
6 pages and 3 figures. Presented at RFI2010, the Third Workshop on RFI
Mitigation in Radio Astronomy, 29-31 March 2010, Groningen, The Netherland
The Murchison Widefield Array: Design Overview
The Murchison Widefield Array (MWA) is a dipole-based aperture array
synthesis telescope designed to operate in the 80-300 MHz frequency range. It
is capable of a wide range of science investigations, but is initially focused
on three key science projects. These are detection and characterization of
3-dimensional brightness temperature fluctuations in the 21cm line of neutral
hydrogen during the Epoch of Reionization (EoR) at redshifts from 6 to 10,
solar imaging and remote sensing of the inner heliosphere via propagation
effects on signals from distant background sources,and high-sensitivity
exploration of the variable radio sky. The array design features 8192
dual-polarization broad-band active dipoles, arranged into 512 tiles comprising
16 dipoles each. The tiles are quasi-randomly distributed over an aperture
1.5km in diameter, with a small number of outliers extending to 3km. All
tile-tile baselines are correlated in custom FPGA-based hardware, yielding a
Nyquist-sampled instantaneous monochromatic uv coverage and unprecedented point
spread function (PSF) quality. The correlated data are calibrated in real time
using novel position-dependent self-calibration algorithms. The array is
located in the Murchison region of outback Western Australia. This region is
characterized by extremely low population density and a superbly radio-quiet
environment,allowing full exploitation of the instrumental capabilities.Comment: 9 pages, 5 figures, 1 table. Accepted for publication in Proceedings
of the IEE
Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host
Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm (“codon harmonization”) for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the “recoded” genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli
The giant lobes of Centaurus A observed at 118MHz with the Murchison Widefield Array
We present new wide-field observations of Centaurus A (Cen A) and the surrounding region at 118MHz with the Murchison Widefield Array (MWA) 32-tile prototype, with which we investigate the spectral-index distribution of Cen A’s giant radio lobes. We compare our images to 1.4 GHz maps of Cen A and compute spectral indices using temperature–temperature plots and spectral tomography. We find that the morphologies at 118MHz and 1.4 GHz match very closely apart from an extra peak in the southern lobe at 118 MHz, which provides tentative evidence for the existence of a southern counterpart to the northern middle lobe of Cen A. Our spatially averaged spectral indices for both the northern and southern lobes are consistent with previous analyses, however we find significant spatial variation of the spectra across the extent of each lobe. Both the spectral-index distribution and the morphology at low radio frequencies support a scenario of multiple outbursts of activity from the central engine. Our results are consistent with inverse-Compton modelling of radio and gamma-ray data that support a value for the lobe age of between 10 and 80 Myr
First Spectroscopic Imaging Observations of the Sun at Low Radio Frequencies with the Murchison Widefield Array Prototype
We present the first spectroscopic images of solar radio transients from the prototype for the Murchison Widefield Array, observed on 2010 March 27. Our observations span the instantaneous frequency band 170.9– 201.6 MHz. Though our observing period is characterized as a period of “low” to “medium” activity, one broadband emission feature and numerous short-lived, narrowband, non-thermal emission features are evident. Our data represent a significant advance in low radio frequency solar imaging, enabling us to follow the spatial, spectral, and temporal evolution of events simultaneously and in unprecedented detail. The rich variety of features seen here reaffirms the coronal diagnostic capability of low radio frequency emission and provides an early glimpse of the nature of radio observations that will become available as the next generation of low-frequency radio interferometers come online over the next few years
Mammalian microRNA: an important modulator of host-pathogen interactions in human viral infections
MicroRNAs (miRNAs), which are small non-coding RNAs expressed by almost all metazoans, have key roles in the regulation of cell differentiation, organism development and gene expression. Thousands of miRNAs regulating approximately 60æ% of the total human genome have been identified. They regulate genetic expression either by direct cleavage or by translational repression of the target mRNAs recognized through partial complementary base pairing. The active and functional unit of miRNA is its complex with Argonaute proteins known as the microRNA-induced silencing complex (miRISC). De-regulated miRNA expression in the human cell may contribute to a diverse group of disorders including cancer, cardiovascular dysfunctions, liver damage, immunological dysfunction, metabolic syndromes and pathogenic infections. Current day studies have revealed that miRNAs are indeed a pivotal component of host-pathogen interactions and host immune responses toward microorganisms. miRNA is emerging as a tool for genetic study, therapeutic development and diagnosis for human pathogenic infections caused by viruses, bacteria, parasites and fungi. Many pathogens can exploit the host miRNA system for their own benefit such as surviving inside the host cell, replication, pathogenesis and bypassing some host immune barriers, while some express pathogen-encoded miRNA inside the host contributing to their replication, survival and/or latency. In this review, we discuss the role and significance of miRNA in relation to some pathogenic viruses
Profiling Early Lung Immune Responses in the Mouse Model of Tuberculosis
Tuberculosis (TB) is caused by the intracellular bacteria Mycobacterium tuberculosis, and kills more than 1.5 million people every year worldwide. Immunity to TB is associated with the accumulation of IFNγ-producing T helper cell type 1 (Th1) in the lungs, activation of M.tuberculosis-infected macrophages and control of bacterial growth. However, very little is known regarding the early immune responses that mediate accumulation of activated Th1 cells in the M.tuberculosis-infected lungs. To define the induction of early immune mediators in the M.tuberculosis-infected lung, we performed mRNA profiling studies and characterized immune cells in M.tuberculosis-infected lungs at early stages of infection in the mouse model. Our data show that induction of mRNAs involved in the recognition of pathogens, expression of inflammatory cytokines, activation of APCs and generation of Th1 responses occurs between day 15 and day 21 post infection. The induction of these mRNAs coincides with cellular accumulation of Th1 cells and activation of myeloid cells in M.tuberculosis-infected lungs. Strikingly, we show the induction of mRNAs associated with Gr1+ cells, namely neutrophils and inflammatory monocytes, takes place on day 12 and coincides with cellular accumulation of Gr1+ cells in M.tuberculosis-infected lungs. Interestingly, in vivo depletion of Gr1+ neutrophils between days 10–15 results in decreased accumulation of Th1 cells on day 21 in M.tuberculosis-infected lungs without impacting overall protective outcomes. These data suggest that the recruitment of Gr1+ neutrophils is an early event that leads to production of chemokines that regulate the accumulation of Th1 cells in the M.tuberculosis-infected lungs
First spectroscopic imaging observations of the sun at low radio frequencies with the Murchison Widefield Array Prototype
We present the first spectroscopic images of solar radio transients from the prototype for the Murchison Widefield Array, observed on 2010 March 27. Our observations span the instantaneous frequency band 170.9- 201.6 MHz. Though our observing period is characterized as a period of "low" to "medium" activity, one broadband emission feature and numerous short-lived, narrowband, non-thermal emission features are evident. Our data represent a significant advance in low radio frequency solar imaging, enabling us to follow the spatial, spectral, and temporal evolution of events simultaneously and in unprecedented detail. The rich variety of features seen here reaffirms the coronal diagnostic capability of low radio frequency emission and provides an early glimpse of the nature of radio observations that will become available as the next generation of low-frequency radio interferometers come online over the next few years