Biochemistry publications

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The 25-Hydroxyvitamin D 24-Hydroxylase
(Academic Press, 1997) Omdahl, John L.; May, B.; School of Molecular and Biomedical Science : Biochemistry
This chapter reviews the role of 25-hydroxyvitamin D 24-hydroxylase in vitamin D metabolism. The 24-hydroxylase enzyme is a widely distributed component of the vitamin D pathway. It is regulated by a spectrum of hormones and functions to synthesise 24-hydroxylated metabolites, with preferential action in promoting bone mineralization and possibly other undisclosed cellular functions that may become evident through the use of gene knockout models. It also plays a central role in directing the metabolic turnover of several 25-hydroxylated vitamin D metabolites.
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A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behaviour
(MARY ANN LIEBERT INC PUBL, 1999) Clarkson, M.; Saint, R.
We have generated Drosophila melanogaster lines carrying a modified genomic fragment which encodes the D. melanogaster variant H2A.F/Z class histone, His2AvD, fused to the green fluorescent protein (GFP) of the jellyfish Aequorea victoria. We show here that the fusion protein consists of functional GFP and functional histone His2AvD. The His2AvD portion of the fusion gene was shown to be functional by rescue of His2AvD mutant lethality. Fluorescence of the fusion protein in vivo was observed in embryonic cleavage stage interphase nuclei and on chromosomes as early as cycle 9, correlating with activation of transcription. Unlike transcription factors, the His2AvDGFP protein remained on transcriptionally inactive chromosomes throughout mitosis. Subsequently, fluorescence was observed in nuclei at all stages of embryonic and larval development and in adult somatic tissues, consistent with the distribution of His2AvD observed by immunohistochemical staining. This functional fusion histone acts as an excellent in vivo marker for chromosomes and chromosome behavior and, given the ability of the fusion gene to prevent null-mutant lethality, without disrupting normal cellular functions. The very high level of conservation of the H2A.F/Z family of variant histones suggests that the equivalent fusion protein construct should function equally well in a wide range of organisms.
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A mouse model of endometriosis that displays vaginal, colon, cutaneous, and bladder sensory comorbidities
(Federation of American Society of Experimental Biology, 2021) Castro, J.; Maddern, J.; Grundy, L.; Manavis, J.; Harrington, A.M.; Schober, G.; Brierley, S.M.
Endometriosis is a painful inflammatory disorder affecting ~10% of women of reproductive age. Although chronic pelvic pain (CPP) remains the main symptom of endometriosis patients, adequate treatments for CPP are lacking. Animal models that recapitulate the features and symptoms experienced by women with endometriosis are essential for investigating the etiology of endometriosis, as well as developing new treatments. In this study, we used an autologous mouse model of endometriosis to examine a combination of disease features and symptoms including: a 10 week time course of endometriotic lesion development; the chronic inflammatory environment and development of neuroangiogenesis within lesions; sensory hypersensitivity and altered pain responses to vaginal, colon, bladder, and skin stimulation in conscious animals; and spontaneous animal behavior. We found significant increases in lesion size from week 6 posttransplant. Lesions displayed endometrial glands, stroma, and underwent neuroangiogenesis. Additionally, peritoneal fluid of mice with endometriosis contained known inflammatory mediators and angiogenic factors. Compared to Sham, mice with endometriosis displayed: enhanced sensitivity to pain evoked by (i) vaginal and (ii) colorectal distension, (iii) altered bladder function and increased sensitivity to cutaneous (iv) thermal and (v) mechanical stimuli. The development of endometriosis had no effect on spontaneous behavior. This study describes a comprehensive characterization of a mouse model of endometriosis, recapitulating the clinical features and symptoms experienced by women with endometriosis. Moreover, it delivers the groundwork to investigate the etiology of endometriosis and provides a platform for the development of therapeutical interventions to manage endometriosis-associated CPP.
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A novel fluorescent probe reveals starvation controls the commitment of amyloid precursor protein to the lysosome
(Elsevier, 2017) Hein, L.; Apaja, P.; Hattersley, K.; Grose, R.; Xie, J.; Proud, C.; Sargeant, T.
Alzheimer's disease is the most important cause of dementia but there is no therapy that has been demonstrated to stop or slow disease progression. Amyloid precursor protein (APP) is the source of amyloid-β (Aβ), which aggregates in Alzheimer's disease to form toxic oligomeric species. The endo-lysosomal system can clear APP and Aβ from the cell if these molecular species are trafficked through to the lysosome. Currently, there are no easy methods available for the analysis of lysosomal APP trafficking. We therefore generated a fusion protein (tandem-fluorescent, or tf-APP) that allows detection of changes in APP trafficking using accessible techniques such as flow cytometry. This permits rapid analysis or screening of genes and compounds that alter APP processing in the cell. Using our novel molecular probe, we determined that starvation induces trafficking of APP and APP-carboxy-terminal fragments (APP-CTFs) to the degradative endo-lysosomal network. In line with this finding, suppression of mTOR signalling using AZD8055 also strongly induced trafficking of APP to the endo-lysosomal system. Remarkably, activation of mTOR signalling via RHEB over-expression inhibited the starvation-induced autophagy but did not affect trafficking of tf-APP. These results show tf-APP can be used to determine how APP is trafficked through the lysosomal system of the cell. This molecular probe is therefore useful for determining the molecular mechanism behind the commitment of APP to the degradative pathway or for screening compounds that can induce this effect. This is important as clearance of APP and APP-CTF provides an important potential therapeutic strategy for Alzheimer's disease.
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A novel method for detecting antigen-specific human regulatory T cells
(Elsevier, 2012) Ebert, L.M.; MacRaild, S.E.; Davis, I.D.; Cebon, J.; Chen, W.
Antigenic epitopes recognized by FoxP3(+) regulatory T cells (Treg) are poorly defined, largely due to a lack of assays for determining Treg specificity. We have developed a novel approach for detecting human Treg specific to peptide antigen, utilizing down-regulation of surface CD3 as a read-out of antigen recognition. Culture conditions and re-stimulation time have been optimized, allowing the detection of even very rare Treg, such as those specific to tumor antigens.
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A novel MOF/graphene oxide composite GrO@MIL-101 with high adsorption capacity for acetone
(Royal Society of Chemistry, 2014) Zhou, X.; Huang, W.; Shi, J.; Zhao, Z.; Xia, Q.; Li, Y.; Wang, H.; Li, Z.
A novel composite material GrO@MIL-101 was synthesized using a solvothermal synthesis method. Then the parent materials (MIL-101 and graphene oxide) and the GrO@MIL-101 were characterized using SEM, TEM, XRD, nitrogen sorption, and Raman. The acetone isotherms on the GrO@MIL-101 and MIL-101 were measured separately. The isosteric heat of adsorption and the desorption activation energies of acetone on the two samples were estimated. The results of characterization confirmed the formation of well-defined GrO@MIL-101 with higher surface area and pore volume compared to the MIL-101, and the crystal size of the MIL-101 in the composite was smaller than that of the parent MIL-101. The acetone isotherms on the GrO@MIL-101 were much higher than those on the MIL-101. The acetone adsorption capacity of the GrO@MIL-101 was up to 20.10 mmol g−1 at 288 K and 161.8 mbar, having an increase of 44.4% in comparison with the MIL-101. The desorption activation energy of acetone on the GrO@MIL-101 was higher than that on the MIL-101, indicating the stronger interaction between acetone molecules and the GrO@MIL-101. Consecutive cycles of acetone adsorption–desorption showed that the desorption efficiency of acetone on the GrO@MIL-101 can reach 91.3%. Acetone adsorption on this composite material was highly reversible.
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A novel series of N-(pyridin-2-yl)-4-(thiazol-5-yl)pyrimidin-2-amines as highly potent CDK4/6 inhibitors
(Future Science, 2017) Tadesse, S.; Zhu, G.; Mekonnen, L.B.; Lenjisa, J.L.; Yu, M.; Brown, M.P.; Wang, S.
Inhibitors of CDK4/6 have emerged as a powerful class of therapeutics for treatment of several malignancies. We herein describe the identification of a new series of molecules that demonstrated excellent selectivity for CDK4/6 over CDKs1, 7 and 9.Medicinal chemistry optimization led to the discovery of 58 and 69 that inhibited CDK4 and CDK4/6, respectively, with high potency and selectivity, and 58 and 69 exhibited potent antiproliferative activities in a panel of human cancer cell lines including leukemia, and cancers of the breast, colon, ovary, pancreas and prostate.Compounds 58 and 69 caused remarkable growth inhibition of melanoma cells, particularly the cells harboring multiple BRAF and NRAS mutations, via a CDK4/6-targeted mechanism of action. [Formula: see text].
Open Access
A novel single-cell based method for breast cancer prognosis
(Public Library of Science, 2020) Li, X.; Liu, L.; Goodall, G.J.; Schreiber, A.; Xu, T.; Li, J.; Le, T.D.; Ioshikhes, I.
Breast cancer prognosis is challenging due to the heterogeneity of the disease. Various computational methods using bulk RNA-seq data have been proposed for breast cancer prognosis. However, these methods suffer from limited performances or ambiguous biological relevance, as a result of the neglect of intra-tumor heterogeneity. Recently, single cell RNA-sequencing (scRNA-seq) has emerged for studying tumor heterogeneity at cellular levels. In this paper, we propose a novel method, scPrognosis, to improve breast cancer prognosis with scRNA-seq data. scPrognosis uses the scRNA-seq data of the biological process Epithelial-to-Mesenchymal Transition (EMT). It firstly infers the EMT pseudotime and a dynamic gene co-expression network, then uses an integrative model to select genes important in EMT based on their expression variation and differentiation in different stages of EMT, and their roles in the dynamic gene co-expression network. To validate and apply the selected signatures to breast cancer prognosis, we use them as the features to build a prediction model with bulk RNA-seq data. The experimental results show that scPrognosis outperforms other benchmark breast cancer prognosis methods that use bulk RNA-seq data. Moreover, the dynamic changes in the expression of the selected signature genes in EMT may provide clues to the link between EMT and clinical outcomes of breast cancer. scPrognosis will also be useful when applied to scRNA-seq datasets of different biological processes other than EMT.
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A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186
(Oxford University Press, 2020) Cutts, E.E.; Egan, J.; Dodd, I.B.; Shearwin, K.E.
The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.
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A structure-function analysis of the left ventricle
(American Physiological Society, 2016) Snelling, E.; Seymour, R.; Green, J.; Meyer, L.; Fuller, A.; Haw, A.; Mitchell, D.; Farrell, A.; Costello, M.; Izwan, A.; Badenhorst, M.; Maloney, S.
Left ventricular external mechanical work rate was calculated from cardiac output and systemic mean arterial blood pressure in resting sheep (Ovis aries; N = 4) and goats (Capra hircus; N = 4) under mild sedation, followed by perfusion-fixation of the left ventricle, and quantification of the cardiac capillary-tissue geometry and cardiomyocyte ultrastructure. The investigation was extended to heavy exercise by increasing cardiac work according to published hemodynamics during sustained treadmill exercise. Left ventricular work rate averaged 0.017 W cm(-3) of tissue at rest, and was estimated to increase to ~0.060 W cm(-3) during heavy exercise. We predicted that oxygen consumption increases from 195 nmol O2 s(-1) cm(-3) at rest, to ~600 nmol O2 s(-1) cm(-3) during heavy exercise, which is within 90% of the demand rate and consistent with work remaining predominantly aerobic. Mitochondria represent 21 - 22% of cardiomyocyte volume and consume oxygen at a rate of 1150 nmol O2 s(-1) cm(-3) of mitochondria at rest, and ~3600 nmol O2 s(-1) cm(-3) during heavy exercise, which is within 80% of maximum in vitro rates and consistent with mitochondria operating near their functional limits. Myofibrils represent 65 - 66% of cardiomyocyte volume, and according to a Laplacian model of the left ventricular chamber, generate peak fiber tensions between ~54 and 62 kPa at rest and during heavy exercise, which is less than maximum tension of isolated cardiac tissue (120 - 140 kPa), and is explained by an apparent reserve capacity for tension development built into the left ventricle.
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A taxonomic tool for identifying needle remains of south-western European Pinus species of the Late Quaternary
(Linnean Society of London, 2014) Alvarez, S.; Juaristi, C.; Paull, R.; Garcia-Amorena, I.
Abstract not available
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A template guided approach to generating cell permeable inhibitors of Staphylococcus aureus biotin protein ligase
(Elsevier, 2018) Paparella, A.; Feng, J.; Blanco-Rodriguez, B.; Feng, Z.; Phetsang, W.; Blaskovich, M.; Cooper, M.; Booker, G.; Polyak, S.; Abell, A.
Abstract not available
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A third fibronectin-type-111 domain in the insulin-family receptors
(ELSEVIER SCI LTD, 1998) Mulhern, T.; Booker, G.; Cosgrove, L.
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A tree-ring based reconstruction of early summer precipitation in southwestern Virginia (1750-1981)
(Inter-Research, 2015) Dawson, A.; Austin, D.; Walker, D.; Appleton, S.; Gillanders, B.; Griffin, S.; Sakata, C.; Trouet, V.
In a closed-canopy forest, stand dynamics play an important role in shaping the forest, and it has been hypothesized that dense forests are not sufficiently limited by climate to warrant climate reconstruction. We collected Quercus prinus tree-ring data from a dense forest in the Appalachians, and after removal of stand dynamics and age trends we found strong influence of early summer precipitation on annual tree growth. We used the new Q. prinus chronology in a nested principal component analysis (PCA) of southeastern US Q. prinus chronologies and further strengthened the early summer precipitation signal in the tree-growth proxy, with favorable assessment of reconstruction skill. Our reconstruction was modeled using Bayesian regression, which allowed uncertainty to be quantified. The May–June precipitation reconstruction covered the period 1750-1981 and extended the instrumental record by 150 yr. It showed key drought years identified by other regional reconstructions, as well as an 11 yr quasi-periodicity that may be related to solar variability. This reconstruction has established a baseline precipitation record that can be used to measure changes brought about by global climate change.
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ABC50 mutants modify translation start codon selection
(Portland Press, 2015) Stewart, J.; Cowan, J.; Perry, L.; Coldwell, M.; Proud, C.
ATP-binding cassette 50 (ABC50; also known as ABCF1) binds to eukaryotic initiation factor 2 (eIF2) and is required for efficient translation initiation. An essential step of this process is accurate recognition and selection of the initiation codon. It is widely accepted that the presence and movement of eIF1, eIF1A and eIF5 are key factors in modulating the stringency of start-site selection, which normally requires an AUG codon in an appropriate sequence context. In the present study, we show that expression of ABC50 mutants, which cannot hydrolyse ATP, decreases general translation and relaxes the discrimination against the use of non-AUG codons at translation start sites. These mutants do not appear to alter the association of key initiation factors to 40S subunits. The stringency of start-site selection can be restored through overexpression of eIF1, consistent with the role of that factor in enhancing stringency. The present study indicates that interfering with the function of ABC50 influences the accuracy of initiation codon selection.
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Abnormal concentrations of Cu-Co in Haumaniastrum katangense, Haumaniastrum robertii and Aeolanthus biformifolius: contamination or hyperaccumulation?
(Royal Society of Chemistry, 2019) van der Ent, A.; Malaisse, F.; Erskine, P.D.; Mesjasz-Przybyłowicz, J.; Przybyłowicz, W.J.; Barnabas, A.D.; Sośnicka, M.; Harris, H.H.
The Central African Copperbelt of the DR Congo and Zambia hosts more than 30 known Cu-Co hyperaccumulator plant species. These plants can accumulate extraordinarily high concentrations of Cu and Co in their living tissues without showing any signs of toxicity. Haumaniastrum robertii is the most extreme Co hyperaccumulator (able to accumulate up to 1 wt% Co), whereas Aeolanthus biformifolius is the most extreme Cu hyperaccumulator (with up to 1 wt% Cu). The phenomenon of Cu-Co hyperaccumulator plants was studied intensively in the 1970s through to the 1990s, but doubts arose regarding earlier observations due to surficial contamination of plant material with mineral particles. This study set out to determine whether such extraneous contamination could be observed on herbarium specimens of Haumaniastrum robertii and Aeolanthus biformifolius using scanning electron microscopy with energy-dispersive spectroscopy (SEM-EDS). Further, synchrotron X-ray absorption spectroscopy (XAS) was used to identify the chemical forms of Cu and Co in newly collected Haumaniastrum katangense plant material from the DR Congo. The results show that surficial contamination is not the cause for abnormal Cu-Co concentrations in the plant material, but rather that Cu-Co enrichment is endogenous. The chemical form of Cu and Co (complexation with carboxylic acids) provides additional evidence that genuine hyperaccumulation, and not soil mineral contamination, is responsible for extreme tissue concentrations of Cu and Co in Haumaniastrum katangense.
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Accelerated loss of hypoxia response in zebrafish with familial Alzheimer's disease-like mutation of Presenilin 1
(Oxford University Press (OUP), 2020) Newman, M.; Moussavi Nik, S.H.; Sutherland, G.T.; Hin, N.; Kim, W.S.; Halliday, G.M.; Jayadev, S.; Smith, C.; Laird, A.; Lucas, C.; Kittipassorn, T.; Peet, D.J.; Lardelli, M.
Ageing is the major risk factor for Alzheimer's disease (ad), a condition involving brain hypoxia. The majority of early onset familial ad (EOfAD) cases involve dominant mutations in the gene PSEN1. PSEN1 null mutations do not cause EOfAD. We exploited putative hypomorphic and EOfAD-like mutations in the zebrafish psen1 gene to explore the effects of age and genotype on brain responses to acute hypoxia. Both mutations accelerate age-dependent changes in hypoxia-sensitive gene expression supporting that ageing is necessary, but insufficient, for ad occurrence. Curiously, the responses to acute hypoxia become inverted in extremely aged fish. This is associated with an apparent inability to upregulate glycolysis. Wild type PSEN1 allele expression is reduced in post-mortem brains of human EOfAD mutation carriers (and extremely aged fish), possibly contributing to EOfad pathogenesis. We also observed that age-dependent loss of HIF1 stabilisation under hypoxia is a phenomenon conserved across vertebrate classes.
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Accelerated sonochemical synthesis of calcium deficient hydroxyapatite nanoparticles: structural and morphological evolution
(American Scientific Publishers, 2014) Varadarajan, N.; Balu, R.; Sampath Kumar, T.; Rana, D.; Ramalingam, M.; Ramalingam, M.; Ramalingam, M.
Calcium deficient hydroxyapatite (CDHA) nanoparticles with a Ca/P ratio of 1.6 were synthesized by accelerated sonochemical process. The synthesis was carried out using calcium nitrate and diammonium hydrogen phosphate in an ultrasonic bath operated at a fixed frequency of 135 kHz and 250 Watts power. The effect of ultrasonic radiation as a function of time over the formation and structure of nanoparticles were investigated using X-ray diffraction, spectroscopy and microscopy methods. The synthesized nanocrystals showed X-ray powder diffraction pattern corresponding to that of hydroxyapatite stoichiometry with CDHA characteristics. HPO2– 4 Fourier transform infrared vibration band observed at 875 cm–1. Transmission electron microscopic analysis confirmed the nanocrystalline nature and growth of acicular, rod and needle-like CDHA nanocrystals morphology with increasing irradiation time.
Open Access
Adaptive traits to improve durum wheat yield in drought and crown rot environments
(MDPI, 2020) Alahmad, S.; Kang, Y.; Dinglasan, E.; Mazzucotelli, E.; Voss-Fels, K.P.; Able, J.A.; Christopher, J.; Bassi, F.M.; Hickey, L.T.
Durum wheat (Triticum turgidum L. ssp. durum) production can experience significant yield losses due to crown rot (CR) disease. Losses are usually exacerbated when disease infection coincides with terminal drought. Durum wheat is very susceptible to CR, and resistant germplasm is not currently available in elite breeding pools. We hypothesize that deploying physiological traits for drought adaptation, such as optimal root system architecture to reduce water stress, might minimize losses due to CR infection. This study evaluated a subset of lines from a nested association mapping population for stay-green traits, CR incidence and yield in field experiments as well as root traits under controlled conditions. Weekly measurements of normalized difference vegetative index (NDVI) in the field were used to model canopy senescence and to determine stay-green traits for each genotype. Genome-wide association studies using DArTseq molecular markers identified quantitative trait loci (QTLs) on chromosome 6B (qCR-6B) associated with CR tolerance and stay-green. We explored the value of qCR-6B and a major QTL for root angle QTL qSRA-6A using yield datasets from six rainfed environments, including two environments with high CR disease pressure. In the absence of CR, the favorable allele for qSRA-6A provided an average yield advantage of 0.57 t·ha−1, whereas in the presence of CR, the combination of favorable alleles for both qSRA-6A and qCR-6B resulted in a yield advantage of 0.90 t·ha−1. Results of this study highlight the value of combining above- and belowground physiological traits to enhance yield potential. We anticipate that these insights will assist breeders to design improved durum varieties that mitigate production losses due to water deficit and CR.
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Adaptors as the regulators of HECT ubiquitin ligases
(Springer, 2021) Shah, S.S.; Kumar, S.
The HECT (homologous to E6AP C-terminus) ubiquitin ligases (E3s) are a small family of highly conserved enzymes involved in diverse cellular functions and pathological conditions. Characterised by a C-terminal HECT domain that accepts ubiquitin from E2 ubiquitin conjugating enzymes, these E3s regulate key signalling pathways. The activity and functional regulation of HECT E3s are controlled by several factors including post-translational modifications, inter- and intramolecular interactions and binding of co-activators and adaptor proteins. In this review, we focus on the regulation of HECT E3s by accessory proteins or adaptors and discuss various ways by which adaptors mediate their regulatory roles to affect physiological outcomes. We discuss common features that are conserved from yeast to mammals, regardless of the type of E3s as well as shed light on recent discoveries explaining some existing enigmas in the field.

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