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"Waugh, David"
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Mastering primary english
'Mastering Primary English' introduces the primary English curriculum and helps trainees and teachers learn how to plan and teach inspiring lessons that make English learning irresistible.
Book
The Ability to Enhance the Solubility of Its Fusion Partners Is an Intrinsic Property of Maltose-Binding Protein but Their Folding Is Either Spontaneous or Chaperone-Mediated
Escherichia coli maltose binding protein (MBP) is commonly used to promote the solubility of its fusion partners. To investigate the mechanism of solubility enhancement by MBP, we compared the properties of MBP fusion proteins refolded in vitro with those of the corresponding fusion proteins purified under native conditions. We fused five aggregation-prone passenger proteins to 3 different N-terminal tags: His₆-MBP, His₆-GST and His₆. After purifying the 15 fusion proteins under denaturing conditions and refolding them by rapid dilution, we recovered far more of the soluble MBP fusion proteins than their GST- or His-tagged counterparts. Hence, we can reproduce the solubilizing activity of MBP in a simple in vitro system, indicating that no additional factors are required to mediate this effect. We assayed both the soluble fusion proteins and their TEV protease digestion products (i.e., with the N-terminal tag removed) for biological activity. Little or no activity was detected for some fusion proteins whereas others were quite active. When the MBP fusions proteins were purified from E. coli under native conditions they were all substantially active. These results indicate that the ability of MBP to promote the solubility of its fusion partners in vitro sometimes, but not always, results in their proper folding. We show that the folding of some passenger proteins is mediated by endogenous chaperones in vivo. Hence, MBP serves as a passive participant in the folding process; passenger proteins either fold spontaneously or with the assistance of chaperones.
Journal Article
The pattern of brain-size change in the early evolution of cetaceans
Most authors have identified two rapid increases in relative brain size (encephalization quotient, EQ) in cetacean evolution: first at the origin of the modern suborders (odontocetes and mysticetes) around the Eocene-Oligocene transition, and a second at the origin of the delphinoid odontocetes during the middle Miocene. We explore how methods used to estimate brain and body mass alter this perceived timing and rate of cetacean EQ evolution. We provide new data on modern mammals (mysticetes, odontocetes, and terrestrial artiodactyls) and show that brain mass and endocranial volume scale allometrically, and that endocranial volume is not a direct proxy for brain mass. We demonstrate that inconsistencies in the methods used to estimate body size across the Eocene-Oligocene boundary have caused a spurious pattern in earlier relative brain size studies. Instead, we employ a single method, using occipital condyle width as a skeletal proxy for body mass using a new dataset of extant cetaceans, to clarify this pattern. We suggest that cetacean relative brain size is most accurately portrayed using EQs based on the scaling coefficients as observed in the closely related terrestrial artiodactyls. Finally, we include additional data for an Eocene whale, raising the sample size of Eocene archaeocetes to seven. Our analysis of fossil cetacean EQ is different from previous works which had shown that a sudden increase in EQ coincided with the origin of odontocetes at the Eocene-Oligocene boundary. Instead, our data show that brain size increased at the origin of basilosaurids, 5 million years before the Eocene-Oligocene transition, and we do not observe a significant increase in relative brain size at the origin of odontocetes.
Journal Article
Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus
N
-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine
N
-hydroxylase DesB from
Streptomyces sviceus
(
Ss
DesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the
Ss
DesB holoenzyme with FAD and the ternary complex with bound NADP
+
were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment.
Ss
DesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous
N
-hydroxylating FMOs from
Erwinia amylovora
(DfoA),
Pseudomonas aeruginosa
(PvdA), and
Aspergillus fumigatus
(SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with
Ss
DesB.
Ss
DesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent
k
cat
values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s
-1
and 0.24 ± 0.01 s
-1
, respectively. However, in product formation assays used to measure the rate of
N
-hydroxylation, the apparent
k
cat
for NADPH (0.034 ± 0.008 s
-1
) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH K
M
was 33 ± 24 μM. Under saturating FAD and NADPH concentrations, the apparent
k
cat
and K
M
for cadaverine in Csaky assays were 0.048 ± 0.004 s
-1
and 19 ± 9 μM, respectively.
Ss
DesB also
N
-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that
Ss
DesB has wider substrate scope compared to other well-studied ornithine and lysine
N
-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other
N
-substituted products.
Journal Article
Making the most of affinity tags
Proteins do not naturally lend themselves to high-throughput analysis because of their diverse physiochemical properties. Consequently, affinity tags have become indispensable tools for structural and functional proteomics initiatives. Although originally developed to facilitate the detection and purification of recombinant proteins, in recent years it has become clear that affinity tags can have a positive impact on the yield, solubility and even the folding of their fusion partners. However, no single affinity tag is optimal with respect to all of these parameters; each has its strengths and weaknesses. Therefore, combinatorial tagging might be the only way to harness the full potential of affinity tags in a high-throughput setting.
Journal Article
Validation of Next Generation Sequencing Technologies in Comparison to Current Diagnostic Gold Standards for BRAF, EGFR and KRAS Mutational Analysis
Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis. Thirteen clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analysed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analysed using closed platform-specific proprietary bioinformatics software as well as open third party applications. Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology before consideration for diagnostic use.
Journal Article
Critical Thinking Skills for your Education Degree
Critical Thinking Skills for your Education Degree provides you with a sound knowledge and understanding of:
the nature of critical thinking, and its relevance and importance in HE
how to adopt a critical approach to all aspects of your studies within education
the importance of active, critical reading, and how it allows you an efficient, principled, effective assessment of the literature in your field
the need to adopt a critical approach to writing, characterised by analytical and evaluative use of sources and the development of your own 'voice'
If you are embarking on a university education or teaching degree, the books in this series will help you acquire and develop the knowledge, skills and strategies you need to achieve your goals. They provide support in all areas important for university study, including institutional and disciplinary policy and practice, self-management, and research and communication. Tasks and activities are designed to foster aspects of learning which are valued in higher education, including learner autonomy and critical thinking, and to guide you towards reflective practice in your study and work life.
eBook
Communication skills for your education degree
Communication Skills for your Education Degree will help you to:
improve your oral and written communication skills in a range of academic and educational settings
improve your public speaking, including academic presentations
improve your practical writing and speaking skills
If you are embarking on a university education or teaching degree, the books in this series will help you acquire and develop the knowledge, skills and strategies you need to achieve your goals. Tasks and activities are designed to foster aspects of learning which are valued in higher education, including learner autonomy and critical thinking, and to guide you towards reflective practice in your study and work life.
eBook
A really big fossil whale
A newly discovered fossil of an extinct whale from Peru indicates that the animal’s skeleton was unexpectedly enormous. This finding challenges our understanding of body-size evolution.
Fossil evidence challenges our understanding of body-size evolution.
Journal Article
Validation of Growth Layer Group (GLG) depositional rate using daily incremental growth lines in the dentin of beluga (Delphinapterus leucas (Pallas, 1776)) teeth
Counts of Growth Layer Groups (GLGs) in the dentin of marine mammal teeth are widely used as indicators of age. In most marine mammals, observations document that GLGs are deposited yearly, but in beluga whales, some studies have supported the view that two GLGs are deposited each year. Our understanding of beluga life-history differs substantially depending on assumptions regarding the timing of GLG deposition; therefore, resolving this issue has important considerations for population assessments. In this study, we used incremental lines that represent daily pulses of dentin mineralization to test the hypothesis that GLGs in beluga dentin are deposited on a yearly basis. Our estimate of the number of daily growth lines within one GLG is remarkably close to 365 days within error, supporting the hypothesis that GLGs are deposited annually in beluga. We show that measurement of daily growth increments can be used to validate the time represented by GLGs in beluga. Furthermore, we believe this methodology may have broader applications to age estimation in other taxa.
Journal Article