The role of mTORC1 in mesenchymal stem cell fate determination, osteoblast differentiation and skeletal development

As we age, skeletal integrity becomes compromised due to a decrease in bone mineral density. Bone formation is mediated by osteoblast (OB) cells which originate from mesenchymal stem cells (MSC). MSCs, a rare stem cell population within the bone marrow, possess self-renewal and multi-lineage differentiation potential. In aging or diseased states, such as osteoporosis, there is a reduction in osteoblastic differentiation potential of MSCs in favour of differentiation toward fat storing adipocytes (AdC). Several signalling pathways have been shown to regulate osteogenic commitment of MSCs as well as the subsequent processes of osteoblast differentiation and skeletal formation. The mammalian target of rapamycin complex 1 (mTORC1) has been implicated as a master regulator of cell metabolism that integrates signals which control MSC commitment and OB function. However, the direct role of this complex in these functions remains to be determined. To definitively address the role of mTORC1 in MSC fate determination, OB differentiation and bone accrual, this project utilised the Cre-loxP system of targeted transgenesis, which enabled the tissue-specific and temporal inactivation of Rptor, the gene encoding for the mTORC1 complex-specific protein raptor. In this study Rptor-deficient MSCs cultured under osteogenic and adipogenicinductive conditions displayed a reduced capacity to form lipid-laden AdCs and an increased capacity to form a mineralised matrix. Consistent with the increased osteogenic differentiation, deletion of Rptor in MSC resulted in an up-regulation in the expression of the osteogenic growth factor BMP2, a known inducer of RUNX2 expression, OB maturation and mineral formation. To examine the role of raptor in skeletal development, Rptor was deleted in preosteoblastic cells marked by their expression of the osterix (Osx) gene, by crossing Rptorfl/fl mice with Osx-cre transgenic mice. Deletion of Rptor lead to a reduction in limb length at birth and post-natally and was associated with smaller epiphyseal growth plates. Deletion of Rptor caused a marked reduction in pre- and post-natal bone acquisition in both intramembranous and endochondral ossification leading to skeletal fragility. The decrease in bone acquisition was not due to a reduction in OB numbers but a reduction in OB function. In vitro, primary OBs from knockout animals failed to respond to extracellular factors that promote bone formation including insulin and BMP2 and assessment of bone development markers in Rptor knockout OBs revealed a transcriptional profile consistent with an immature OB phenotype suggesting that OB differentiation was hindered early in osteogenic development. These findings demonstrate that mTORC1 plays an important role in skeletal development by controlling OB differentiation and hence function. Taken together, these studies show that mTORC1 plays an important role in MSC fate determination and bone accrual. Notably, deletion of raptor in pre-osteoblasts blocked osteoblast differentiation causing defective intramembranous and endochondral ossification resulting in a low bone mass phenotype and skeletal fragility.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201

Income Elasticities of Food Demand in Africa: A Meta-Analysis

In order to combat malnutrition, economists and policymakers need to understand how food demand will change, as the continent further develops. Especially, a better understanding of, first, the factors underlying the relation between income and food demand, and, second, how this relation is changing according to the income level and/or characteristics of the country under study, may help improve the design and implementation of nutrition policies. There are a number of studies that have estimated the relation between income growth and food demand in Africa, but the resulting estimates are highly heterogeneous. This report provides a systematic review of the existing literature on income elasticities of food demand in Africa. Using a meta-analysis approach, this report identifies the factors determining the relation between food demand and income. Further research could usefully explore in greater detail some of the patterns identified and, in doing so, contribute to the design of policies aimed at addressing malnutrition.JRC.J.4-Agriculture and Life Sciences in the Econom

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO

Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory

The Auger Engineering Radio Array (AERA) is part of the Pierre Auger Observatory and is used to detect the radio emission of cosmic-ray air showers. These observations are compared to the data of the surface detector stations of the Observatory, which provide well-calibrated information on the cosmic-ray energies and arrival directions. The response of the radio stations in the 30 to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of the incoming electric field. For the latter, the energy deposit per area is determined from the radio pulses at each observer position and is interpolated using a two-dimensional function that takes into account signal asymmetries due to interference between the geomagnetic and charge-excess emission components. The spatial integral over the signal distribution gives a direct measurement of the energy transferred from the primary cosmic ray into radio emission in the AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air shower arriving perpendicularly to the geomagnetic field. This radiation energy -- corrected for geometrical effects -- is used as a cosmic-ray energy estimator. Performing an absolute energy calibration against the surface-detector information, we observe that this radio-energy estimator scales quadratically with the cosmic-ray energy as expected for coherent emission. We find an energy resolution of the radio reconstruction of 22% for the data set and 17% for a high-quality subset containing only events with at least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO

Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy

We measure the energy emitted by extensive air showers in the form of radio emission in the frequency range from 30 to 80 MHz. Exploiting the accurate energy scale of the Pierre Auger Observatory, we obtain a radiation energy of 15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV arriving perpendicularly to a geomagnetic field of 0.24 G, scaling quadratically with the cosmic-ray energy. A comparison with predictions from state-of-the-art first-principle calculations shows agreement with our measurement. The radiation energy provides direct access to the calorimetric energy in the electromagnetic cascade of extensive air showers. Comparison with our result thus allows the direct calibration of any cosmic-ray radio detector against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI. Supplemental material in the ancillary file

Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. Video Abstract [Figure presented] Development of the bioinformatics tool LOHHLA allows precise measurement of allele-specific HLA copy number, improves the accuracy in neoantigen prediction, and uncovers insights into how immune escape contributes to tumor evolution in non-small-cell lung cancer

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies