Biology subjects

Alexander, D.

Publications and source records attributed to Alexander, D..

6 recordsLinked to original sources

bioRxiv2022

Mutation Rate Evolution Drives Immune Escape In Mismatch Repair-Deficient Cancer

Mutation rate optimisation drives evolution and immune evasion of bacteria and lentiviral strains, including HIV. Whether evolving cancer lineages similarly adapt mutation rates to increase tumour cell fitness is unknown. Here, by mapping the clonal topography of mismatch repair-deficient (MMRd) colorectal cancer, we show that genomic MMRd mutability co-evolves with neoantigen selection to drive intratumour diversification and immune escape. Mechanistically, we find that microsatellite instability modulates subclonal DNA repair by toggling two hypermutable mononucleotide homopolymer runs in the mismatch repair genes MSH6 and MSH3 (C8 and A8, respectively) through stochastic frameshift switching. Spontaneous mutation and reversion at these evolvability switches modulates subclonal mutation rate, mutation bias, and clonal HLA diversity during MMRd cancer evolution. Combined experimental and simulation studies demonstrate that subclonal immune selection favours incremental MMR mutations. MMRd cancers thus fuel intratumour heterogeneity by adapting subclonal mutation rate and mutation bias to immune selection, revealing a conserved co-evolutionary arms race between neoantigen selection and adaptive genomic mutability. Our work reveals layers of mutational complexity and microsatellite biology in MMRd cancer evolution previously hidden in bulk analyses.

cancer biology↗

bioRxiv2020

Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

Across eukaryotes, checkpoints maintain the order of cell cycle events in the face of DNA damage or incomplete replication. Although a wide array of DNA lesions activates the checkpoint kinases, whether and how this response differs in different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in the budding yeast Saccharomyces cerevisiae is caused by Rad53 kinase-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M phase, preventing inappropriate gene amplification events. In addition we show that inhibition of Sld3 and Dbf4 after DNA damage in G1 phase prevents premature replication initiation at all origins at the G1/S transition. This study redefines the scope and specificity of the S-phase checkpoint with implications for understanding the roles of this checkpoint in the majority of cancers that lack proper cell cycle controls.

cell biology↗

bioRxiv2019

Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time

The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate development in Aspergillus flavus, and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time. Two field isolates of A. flavus, AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15mM H2O2, and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points. The moderate aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H2O2-stressed A. flavus and drought-stressed hosts suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering.\n\nAuthor SummaryAspergillus flavus is a fungal pathogen of several important crops including maize and peanut. This pathogen produces carcinogenic mycotoxins known as aflatoxins during infection of plant materials, and is particularly severe under drought stress conditions. This results in significant losses in crop value and poses a threat to food safety and security globally. To combat this, understanding how this fungus responds to environmental stresses related to drought can allow us to identify novel methods of mitigating aflatoxin contamination. Here, we analyzed the accumulation of a broad series of metabolites over time in two isolates of A. flavus with differing stress tolerance and aflatoxin production capabilities in response to drought-related oxidative stress. We identified several metabolites and mechanisms in A. flavus which allow it to cope with environmental oxidative stress and may influence aflatoxin production and fungal growth. These may serve as potential targets for selection in breeding programs for the development of new cultivars, or for alteration using genetic engineering approaches to mitigate excessive aflatoxin contamination under drought stress.

microbiology↗

bioRxiv2018

Abnormal microstructural development of the cerebral cortex in neonates with congenital heart disease is associated with impaired cerebral oxygen delivery

BackgroundAbnormal macrostructural development of the cerebral cortex has been associated with hypoxia in infants with congenital heart disease (CHD). Animal studies have suggested that hypoxia results in cortical dysmaturation at the cellular level. New magnetic resonance imaging (MRI) techniques offer the potential to investigate the relationship between cerebral oxygen delivery and microstructural development of the cortex in newborn infants with CHD.\n\nMethodsWe measured macrostructural and microstructural properties of the cortex in 48 newborn infants with complex CHD and 48 age-matched healthy controls. Cortical volume and gyrification index were calculated from high resolution structural MRI. Neurite density and orientation dispersion indices were modelled using high angular resolution diffusion MRI. Cerebral oxygen delivery was estimated in infants with CHD using phase contrast MRI and pre-ductal pulse oximetry. We used tract-based spatial statistics to examine voxel-wise group differences in cortical microstructure.\n\nResultsMicrostructural development of the cortex was abnormal in 48 infants with CHD, with regions of increased fractional anisotropy (FA) and reduced orientation dispersion index (ODI) compared to 48 healthy controls, correcting for gestational age at birth and scan (FWE-corrected for multiple comparisons at P<0.05). Regions of reduced cortical ODI in infants with CHD were related to impaired cerebral oxygen delivery (R2=0.637, n=39). Cortical ODI was associated with gyrification index (R2=0.589, P<0.0001, n=48).\n\nConclusionsThis study suggests that the primary component of cerebral cortex dysmaturation in CHD is impaired dendritic arborisation, which may underlie abnormal macrostructural findings reported in this population. The degree of impairment was related to cerebral oxygen delivery, supporting the hypothesis that maternal oxygen therapy may be beneficial in this population.

neuroscience↗

bioRxiv2018

Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine for production

The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10-100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of low expression, high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. We also show that the efficiency of this process can be greatly improved with robotic selection. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years.

bioengineering↗

bioRxiv2017

Image Processing and Quality Control for the first 10,000 Brain Imaging Datasets from UK Biobank

UK Biobank is a large-scale prospective epidemiological study with all data accessible to researchers worldwide. It is currently in the process of bringing back 100,000 of the original participants for brain, heart and body MRI, carotid ultrasound and low-dose bone/fat x-ray. The brain imaging component covers 6 modalities (T1, T2 FLAIR, susceptibility weighted MRI, Resting fMRI, Task fMRI and Diffusion MRI). Raw and processed data from the first 10,000 imaged subjects has recently been released for general research access. To help convert this data into useful summary information we have developed an automated processing and QC (Quality Control) pipeline that is available for use by other researchers. In this paper we describe the pipeline in detail, following a brief overview of UK Biobank brain imaging and the acquisition protocol. We also describe several quantitative investigations carried out as part of the development of both the imaging protocol and the processing pipeline.

neuroscience↗