Contributors: Buha, Aleksandra, Jugdaohsingh, Ravin, Matovic, Vesna, Bulat, Zorica, Antonijevic, Biljana, Kerns, Jemma G, Goodship, Allen, Hart, Alister, Powell, Jonathan
... Exposure to cadmium (Cd) is recognised as one of the risk factors for osteoporosis, although critical exposure levels and exact mechanisms are still unknown. Here, we first confirmed that in male Wistar rats challenged orally with 7 different levels of Cd (0.3-10 mg/kg b.w.), over 28 days, there was a direct dose relationship to bone Cd concentration. Moreover, bone mineral content was significantly diminished by ~ 15% (p <0.0001) plateauing already at the lowest exposure level. For the other essential bone elements zinc (Zn) loss was most marked. Having established the sensitive metrics (measures of Cd exposure), we then applied them to 20 randomly selected human femoral head bone samples from 16 independent subjects. Bone Cd concentration was inversely proportional to trabecular bone mineral density and mineral (calcium) content and Zn content of bone, but not the donor’s age. Our findings, through direct bone analyses, support the emerging epidemiological view that bone health, adjudged by mineral density, is extremely sensitive to even background levels of environmental Cd. Importantly, however, our data also suggest that Cd may play an even greater role in compromised bone health than prior indirect estimates of exposure could reveal. Environmental Cd may be a substantially determining factor in osteoporosis and large cohort studies with direct bone analyses are now merited.
Evaluation of Associations between Genetically Predicted Circulating Protein Biomarkers and Breast Cancer Risk
Contributors: Easton, Douglas, Bolla, Manjeet, Wang, qin
... A small number of circulating proteins have been reported to be associated with breast cancer risk, with inconsistent results. Herein, we attempted to identify novel protein biomarkers for breast cancer via the integration of genomics and proteomics data. In the Breast Cancer Association Consortium (BCAC), with 122,977 cases and 105,974 controls of European descendants, we evaluated the associations of the genetically predicted concentrations of >1,400 circulating proteins with breast cancer risk. We used data from a large-scale protein quantitative trait loci (pQTL) analysis as our study instrument. Summary statistics for these pQTL variants related to breast cancer risk were obtained from the BCAC and used to estimate odds ratios (OR) for each protein using the inverse-variance weighted method. We identified 56 proteins significantly associated with breast cancer risk by instrumental analysis (false discovery rate < 0.05). Of these, the concentrations of 32 were influenced by variants close to a breast cancer susceptibility locus (ABO, 9q34.2). Many of these proteins, such as insulin receptor, insulin-like growth factor receptor 1 and other membrane receptors (OR: 0.82 to 1.18, P values: 6.96×10-4 to 3.28×10-8), are linked to insulin resistance and estrogen receptor signaling pathways. Proteins identified at other loci include those involved in biological processes such as alcohol and lipid metabolism, proteolysis, apoptosis, immune regulation, and cell motility and proliferation. Consistent associations were observed for 22 proteins in the UK Biobank data (P < 0.05). The study identifies potential novel biomarkers for breast cancer, but further investigation is needed to replicate our findings.
Structure of KAP1 tripartite motif identifies molecular interfaces required for retroelement silencing
Contributors: Modis, Yorgo, Stoll, Guido, Oda, Shun, Chong, Zheng, Yu, Minmin, McLaughlin, Stephen H
... Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site, in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.
Contributors: King, Martin S, Kunji, Edmund
... Saccharomyces cerevisiae is one of the most popular expression systems for eukaryotic membrane proteins. Here, we describe protocols for the expression and purification of mitochondrial membrane proteins developed in our laboratory during the last 15 years. To optimize their expression in a functional form, different promoter systems, as well as codon-optimization and complementation strategies were established. Purification approaches were developed that remove the membrane protein from the affinity column by specific proteolytic cleavage rather than by elution. This strategy has several important advantages, most notably improving the purity of the sample, as contaminants stay bound to the column, thus eliminating the need for a secondary purification step, such as size exclusion chromatography. This strategy also avoids dilution of the sample, which would occur as a consequence of elution, precluding the need for concentration steps, and thus preventing detergent concentration.
Mutualistic Coupling Between Vocabulary and Reasoning in Young Children: A Replication and Extension of the Study by Kievit et al. (2017).
Contributors: Kievit, Rogier, Hofman, Abe D, Nation, Kate
... Recent work suggests that the positive manifold of individual differences may arise, or be amplified, by a mechanism called mutualism. Kievit et al. (2017) showed that a latent change score implementation of the mutualism model outperformed alternative models, demonstrating positive reciprocal interactions between vocabulary and reasoning during development. Here, we replicated these findings in a cohort of children ( N = 227, 6-8 years old) and expanded the findings in three directions. First, a third wave of data was included, and the findings were robust to alternative model specifications. Second, a simulation demonstrated that data sets of similar magnitude and distributional properties could have, in principle, favored alternative models with close to 100% power. Third, we found support for the hypothesis that mutualistic-coupling effects are stronger and self-feedback parameters weaker in younger children. Together, these findings replicated the work of Kievit et al. (2017) and further support the hypothesis that mutualism supports cognitive development.
Data of 'Terahertz Spectroscopy: An investigation of the Structural Dynamics of Freeze-Dried Poly Lactic-co-glycolic Acid Microspheres'
Contributors: Shmool, T, Hooper, Philippa, Kaminski, Gabriele, van der Walle, Christopher, Zeitler, Jochen
... All data provided for the manuscript, this includes terahertz time-domain spectra over a range of temperature, mid-infrared FTIR spectra, DSC data and SEM images of microspheres used for drug delivery of protein drugs based on poly lactic-co-glycolic acid (PLGA) formulations.
Transcriptomic profiling reveals three molecular phenotypes of adenocarcinoma at the gastroesophageal junction.
Contributors: Bornschein, Jan, Wernisch, Lorenz, Secrier, Maria, Miremadi, Ahmad, Perner, Juliane, MacRae, Shona, O'Donovan, Maria, Newton, Richard, Menon, Suraj, Bower, Lawrence
... Cancers occurring at the gastroesophageal junction (GEJ) are classified as predominantly esophageal or gastric, which is often difficult to decipher. We hypothesized that the transcriptomic profile might reveal molecular subgroups which could help to define the tumor origin and behavior beyond anatomical location. The gene expression profiles of 107 treatment-naïve, intestinal type, gastroesophageal adenocarcinomas were assessed by the Illumina-HTv4.0 beadchip. Differential gene expression (limma), unsupervised subgroup assignment (mclust) and pathway analysis (gage) were undertaken in R statistical computing and results were related to demographic and clinical parameters. Unsupervised assignment of the gene expression profiles revealed three distinct molecular subgroups, which were not associated with anatomical location, tumor stage or grade (p > 0.05). Group 1 was enriched for pathways involved in cell turnover, Group 2 was enriched for metabolic processes and Group 3 for immune-response pathways. Patients in group 1 showed the worst overall survival (p = 0.019). Key genes for the three subtypes were confirmed by immunohistochemistry. The newly defined intrinsic subtypes were analyzed in four independent datasets of gastric and esophageal adenocarcinomas with transcriptomic data available (RNAseq data: OCCAMS cohort, n = 158; gene expression arrays: Belfast, n = 63; Singapore, n = 191; Asian Cancer Research Group, n = 300). The subgroups were represented in the independent cohorts and pooled analysis confirmed the prognostic effect of the new subtypes. In conclusion, adenocarcinomas at the GEJ comprise three distinct molecular phenotypes which do not reflect anatomical location but rather inform our understanding of the key pathways expressed.
The quantification of pregnancy-induced bone mineral mobilisation in the maternal appendicular skeleton with novel peripheral quantitative computed tomography (pQCT) techniques.
Contributors: Ó Breasail, Mícheál
... The quantification of pregnancy-induced bone mineral mobilisation in the maternal appendicular skeleton with novel peripheral quantitative computed tomography (pQCT) techniques. Adaptations in maternal calcium (Ca) economy occur during pregnancy to meet fetal demand and by birth a typical new-born contains 25-30g Ca. At the individual-level if Ca is mobilised it is important to identify where this occurs to identify any potential impact on fracture risk. At present, pregnancy is not considered a risk factor for osteoporosis but data are few. Trabecular bone is more metabolically active than cortical bone in part due to its structure, orientation and much greater surface area. During lactation, studies have consistently reported significant transient bone mineral mobilisation from trabecular-rich axial sites such as the hip and spine, while newer peripheral quantitative computed tomography (pQCT) techniques have found trabecular microarchitectural change in the appendicular skeleton also. My primary objective was to explore whether a similar pregnancy-induced bone mineral mobilisation could be observed in two distinct cohorts of pregnant women using novel pQCT techniques from mid- to late-pregnancy. These techniques provide non-invasive measurements of bone density, mass, geometry, distribution and strength at the radius and tibia. This thesis aims to determine: whether pregnancy-induced changes occur in a) the trabecular compartment, b) the cortical compartment, c) any potential predictors of a) and b); d) to explore the correlation and agreement of pQCT techniques in-vivo. Single-slice and high-resolution (HR) pQCT data were used to characterise maternal appendicular skeletal change during pregnancy in two studies in contrasting populations: 1) a resource poor subsistence farming Sub-Saharan African community with an habitually low Ca intake and high parity; 2) an affluent Cambridge based population, where Ca intake is higher but parity much lower. In The Gambia, existing pQCT data obtained at 14 and 30 weeks of pregnancy from women (n=811), aged 18-45 and accustomed to low habitual Ca intake, were analysed. In Cambridge, UK, I designed a study where pregnant women (n=53) and non-pregnant non-lactating (NPNL, n=37) controls aged 30-45 years were scanned with pQCT and HRpQCT at 14 and 36 weeks. These data were modelled to explore the maternal response to pregnancy in these distinct populations. Contrary to my primary hypothesis there was no evidence of trabecular bone mobilisation in Gambian women, however in contrast in the UK study a -0.5 SD decrease in trabecular vBMD was observed. In The Gambia total vBMD did not change at the radius or tibia, while in Cambridge both techniques detected decreases in total vBMD at the distal tibia. Changes in cortical bone were documented during pregnancy in both populations. In Gambian women small but significant increases in cortical vBMD and BMC were observed at the radius and tibia. In Cambridge at the tibia HRpQCT showed a decrease in cortical vBMD and cortical thickness with an increase in cortical porosity. There were fewer pregnancy-induced changes at the radius but at the cortical- rich diaphysis cortical thickness decreased and endosteal circumference increased. No consistent predictors of these changes were found in either population. These data from two contrasting populations show a “one size fits all” approach cannot be applied to the maternal skeletal response to pregnancy. In Gambian women with a habitually low Ca intake we have observed the conservation of bone mineral in the appendicular skeleton into the third trimester. In contrast in the Cambridge women evidence of trabecular and cortical mobilisation was observed at the distal tibia. At the radius changes were confined to the cortical-rich proximal radius and supported endosteal resorption during pregnancy. These data suggest that the maternal skeleton is a significant source of fetal Ca in a population accustomed to higher dietary Ca intakes. Further work is needed to determine what this mobilisation means at the individual level and to determine if the mobilised bone mineral is restored postpartum or whether there are lasting consequences for the woman’s bone health.
Contributors: Dickinson, Nicholas Alexander
... Active-source marine seismic reflection records have been used to construct acoustic images of oceanic thermohaline structure. In the water column, acoustic boundaries are produced by variations in temperature and, to a lesser extent, salinity. Acoustic waves generated by an array of airguns suspended behind a vessel are reflected from these boundaries and recorded by long cables of hydrophones which are towed beneath the sea surface. Careful signal processing of these records yields acoustic images of thermohaline structure with both horizontal and vertical resolutions of $O(10)$~m. In this dissertation, a three-dimensional seismic survey from the Faroe-Shetland Channel and a two-dimensional seismic survey from the Gulf of Mexico are presented and analysed. In the Faroe-Shetland Channel, 54 seismic transects were acquired over 25 days in July-August 1997. These transects were recorded sequentially, with adjacent transects separated by $\sim 0.3$~km and $\sim 1$~day. The survey thus provides time-lapse sampling of local thermohaline structure within an area of $\sim 150$~km$^2$. Processed acoustic images are dominated by a prominent band of bright reflections at depth. Calibration using nearby hydrographic measurements shows that this band corresponds to a pycnocline which separates warm near-surface waters from cold deep waters. The depth of the pycnocline varies by $\sim 350$~m over $\sim 10$~days. Acoustic reflections within the pycnocline are tilted by up to 1\textdegree \ with respect to the horizontal, suggesting currents in geostrophic balance. Temporal variations in pycnocline depth and in reflection tilt are consistent with northeastward advection of a mesoscale anticyclonic vortex. Comparison with contemporaneous satellite measurements of sea-surface temperature and sea-surface height suggests that this vortex is caused by meandering of the Continental Slope Current. These results have significant implications for our understanding of how mesoscale activity in the Faroe-Shetland Channel affects flux of heat and salt to the Nordic Seas. At finer scales, the seismic images show clearly defined internal waves. The geometry of these waves is compared with theoretical descriptions of linear and nonlinear waves in a two-layer fluid. The form of the internal wave field and of localised turbulence is investigated by spectrally analysing the vertical displacements of automatically tracked reflections. Internal wave spectra differ markedly from the open-ocean Garrett-Munk model spectrum. Diapycnal diffusivities are estimated from clear turbulent spectral subranges. Temporal variations in the form of spectra are related to changes in shear and stratification associated with passage of the mesoscale vortex. In the Gulf of Mexico, a single seismic image shows clear reflections in the uppermost $\sim 1000$~m adjacent to and overlying the continental slope. Spectral analysis of these reflections shows that the local internal wave field is accurately described by the Garrett-Munk spectrum. Diapycnal diffusivities are estimated using a semi-empirical parametrisation of internal wave energy. Comparison with diffusivities estimated from nearby hydrographic records and with previous measurements indicates that this method is robust. The analyses presented in this dissertation suggest that automated signal processing and interpretation of existing seismic reflection records has the potential to strengthen our understanding of the dynamical links between oceanic mesoscale activity, internal waves and turbulence.
Contributors: Rowitch, David, Schirmer, Lucas, Velmeshev, Dmitry, Holmqvist, Staffan, Kaufmann, Max, Sebastian, Werneburg, Jung, Diane, Vistnes, Stephanie, Stockley, John, Young, Adam
... Multiple sclerosis (MS) is a neuroinflammatory disease with a relapsing-remitting disease course at early stages, distinct lesion characteristics in cortical gray versus subcortical white matter, and neurodegeneration at chronic stages. We assessed multilineage cell expression changes using single-nucleus RNA sequencing (snRNA-seq) and validated results using multiplex in situ hybridization in MS lesions. We found selective vulnerability and loss of excitatory CUX2-expressing projection neurons in upper cortical layers underlying meningeal inflammation; such MS neuron populations showed upregulation of stress pathway genes and long non-coding RNAs. Signatures of stressed oligodendrocytes, reactive astrocytes and activated phagocytosing cells mapped most strongly to the rim of MS plaques. Interestingly, snRNA-seq identified phagocytosing microglia and/or macrophages by their ingestion and perinuclear import of myelin transcripts, confirmed by functional mouse and human culture assays. Our findings indicate lineage- and region-specific transcriptomic changes associated with selective cortical neuron damage and glial activation contributing to MS lesion progression.