Biology subjects

Russell, A.

Publications and source records attributed to Russell, A..

32 records · Page 2Linked to original sources

bioRxiv2021

A novel tubulin binding molecule drives differentiation of acute myeloid leukaemia cells

Acute Myeloid Leukaemia (AML) continues to have a poor prognosis, especially in the elderly. One reason for this is that many treatment regimens are not well tolerated by elderly patients. Much current focus is on the development of therapies that can target specific vulnerabilities of AML while having fewer toxic side effects. However, despite much recent progress in developing better drugs, many patients with AML still die within a year of diagnosis, partly due to the fact that it is difficult to identify therapeutic targets that are effective across multiple AML subtypes. One common factor across AML subtypes is the presence of a block in differentiation. Thus screening for compounds that can overcome this block in genetically diverse AML models should allow for the identification of agents that are not dependent on a specific mutation for their efficacy. Here, we used a phenotypic screen to identify novel compounds that stimulate differentiation in several AML cell lines. Lead compounds were shown to decrease tumour burden and to increase survival in vivo. Using multiple complementary target deconvolution approaches, these compounds were revealed to be anti-mitotic tubulin disruptors that cause differentiation by inducing a G2-M mitotic arrest. Together, these results reveal a novel function for tubulin disruptors in causing differentiation of AML cells.

cancer biology↗

bioRxiv2021

Transmission of vibrations in buzz-pollinated plant species with disparate floral morphologies

In buzz-pollinated plants, bees apply vibrations produced by their thoracic muscles to the flower, causing pollen release from anthers, often through small apical pores. During floral buzzing, bees grasp one or more anthers with their mandibles, and vibrations are transmitted to the focal anther(s), adjacent anthers, and the whole flower. Because pollen release depends on the vibrations experienced by the anther, the transmission of vibrations through flowers with different morphologies may determine patterns of release, affecting both bee foraging and plant fitness. Anther morphology and intra-floral arrangement varies widely among buzz-pollinated plants. Here, we compare the transmission of vibrations among focal and non-focal anthers in four species with contrasting anther morphologies: Cyclamen persicum (Primulaceae), Exacum affine (Gentianaceae), Solanum dulcamara and S. houstonii (Solanaceae). We used a mechanical transducer to apply bee-like artificial vibrations to focal anthers, and simultaneously measured the vibration frequency and displacement amplitude at the tips of focal and non-focal anthers using high-speed video analysis (6,000 frames per second). In flowers in which anthers are tightly held together (C. persicum and S. dulcamara), vibrations in focal and non-focal anthers are indistinguishable in both frequency and displacement amplitude. In contrast, flowers with loosely arranged anthers (E. affine) including those in which stamens are morphologically differentiated within the same flower (heterantherous S. houstonii), show the same frequency but higher displacement amplitude in non-focal anthers compared to focal anthers. Our results suggest that stamen arrangement affects vibration transmission with potential consequences for pollen release and bee behaviour.

biophysics↗

bioRxiv2020

A phenotypic screen identifies a compound series that induces differentiation of acute myeloid leukemia cells in vitro and shows anti-tumour effects in vivo

Induction of differentiation is a promising therapeutic strategy against acute myeloid leukemia. However, current differentiation therapies are effective only to specific patient populations. To identify novel differentiation agents with wider efficacy, we developed a phenotypic high-throughput screen with a range of genetically diverse cell lines. From the resulting hits, one chemical scaffold was optimised in terms of activity and physicochemical properties to yield OXS007417, which was also able to decrease tumour volume in a murine in vivo xenograft model.

cancer biology↗

bioRxiv2020

Cdc42 reactivation at growth sites is regulated by cell-cycle-dependent removal of its GAP Rga4 in fission yeast

In fission yeast, polarized cell growth stops during division and resumes after cell separation. We uncoupled these sequential events by delaying cytokinesis with a temporary Latrunculin A treatment. Mitotic cells recovering from treatment initiate end growth without cell separation, displaying a polar elongation sans separation (PrESS) phenotype. PrESS cell ends reactivate Cdc42, a major regulator of polarized growth, before cell separation, but at a fixed time after anaphase B. A candidate screen implicates Rga4, a negative regulator of Cdc42, in this process. We show that Rga4 appears punctate at the cell sides during G2, but is diffuse during mitosis, extending to the ends. While the Morphogenesis Orb6 (MOR) pathway is known to promote cell separation and growth by activating protein synthesis, we find that for polarized growth, removal of Rga4 from the ends is also necessary. Therefore, we propose that growth resumes after division once the MOR pathway is activated and the ends lose Rga4 in a cell-cycle-dependent manner.

cell biology↗

bioRxiv2020

Loss-of-function of Fbxo10, encoding a post-translational regulator of BCL2 in lymphomas, has no discernible effect on BCL2 or B lymphocyte accumulation in mice

Regulation of the anti-apoptotic BCL2 protein determines cell survival and is frequently abnormal in B cell lymphomas. An evolutionarily conserved post-translational mechanism for over-expression of BCL2 in human B cell lymphomas and the BCL2 paralogue CED-9 in Caenorhabditis elegans results from loss-of-function mutations in human FBXO10 and its C.elegans paralogue DRE-1, a BCL2/CED-9-binding subunit of the SKP-CULLIN-FBOX (SCF) ubiquitin ligase. Here, we tested the role of FBXO10 in BCL2 regulation by producing mice with two different CRISPR/Cas9-engineered Fbxo10 mutations: an Asp54Lys (E54K) missense mutation in the FBOX domain and a Cys55SerfsTer55 frameshift (fs) truncating mutation. Mice homozygous for either mutant allele were born at the expected Mendelian frequency and appeared normal in body weight and appearance as adults. Spleen B cells from homozygous mutant mice did not have increased BCL2 protein, nor were the numbers of mature B cells or germinal centre B cells increased as would be expected if BCL2 was increased. Other lymphocyte subsets that are also regulated by BCL2 levels also displayed no difference in frequency in homozygous Fbxo10 mutant mice. These results support one of two conclusions: either FBXO10 does not regulate BCL2 in mice, or it does so redundantly with other ubiquitin ligase complexes. Possible candidates for the latter include FBXO11 or ARTS-XIAP. The difference between the role of FBXO10 in regulating BCL2 protein levels in C. elegans and in human DLBCL, relative to single-gene deficient mouse leukocytes, should be further investigated.

immunology↗

bioRxiv2020

Evolutionary transition to XY sex chromosomes associated with Y-linked duplication of a male hormone gene in a terrestrial isopod

Sex chromosomes are highly variable in some taxonomic groups, but the evolutionary mechanisms underlying this diversity are not well understood. In terrestrial isopod crustaceans, evolutionary turnovers in sex chromosomes are frequent, possibly caused by Wolbachia, a vertically-transmitted endosymbiont causing male-to-female sex reversal. Here, we use surgical manipulations and genetic crosses, plus genome sequencing, to examine sex chromosomes in the terrestrial isopod Trachelipus rathkei. Although an earlier cytogenetics study suggested a ZZ/ZW sex chromosome system in this species, we surprisingly find multiple lines of evidence that in our study population, sex is determined by an XX/XY system. Consistent with a recent evolutionary origin for this XX/XY system, the putative male-specific region of the genome is small. The genome shows evidence of Y-linked duplications of the gene encoding the androgenic gland hormone, a major component of male sexual differentiation in isopods. Our analyses also uncover sequences horizontally acquired from past Wolbachia infections, consistent with the hypothesis that Wolbachia may have interfered with the evolution of sex determination in T. rathkei. Overall, these results provide evidence for the co-occurrence of multiple sex chromosome systems within T. rathkei, further highlighting the relevance of terrestrial isopods as models for the study of sex chromosome evolution.

evolutionary biology↗

bioRxiv2020

Pex11 β knockdown decreases peroxisome abundance and reverses the inhibitory effect of palmitate on pancreatic beta-cell function.

AimsReactive oxygen species generated by the peroxisomes and mitochondria contribute to lipotoxicity in pancreatic beta-cells. Through targeted Pex11{beta} knockdown and peroxisome depletion, our aim was to investigate the specific contribution of peroxisomes to palmitate mediated pancreatic beta-cell dysfunction. MethodsMIN6 cells were transfected with probes targeted against Pex11{beta}, a regulator of peroxisome abundance, or with scrambled control probes. Peroxisome abundance was measured by PMP-70 protein expression. 48hrs post transfection, cells were incubated with or without 250M palmitate for a further 48hrs before measurement of reactive oxygen species, mitochondrial respiratory function, and glucose stimulated insulin secretion. ResultsPex11{beta} knockdown decreased target gene expression by more than 80% compared with the scrambled control (P<0.001), leading to decreased PMP-70 expression (p<0.01). Pex11{beta} knockdown decreased palmitate mediated generation of reactive oxygen species (P<0.001), but with no effect on mitochondrial respiratory function. At 25mM glucose, palmitate treatment decreased insulin secretion in the control cells (2.54{+/-}0.25 vs 7.07{+/-}0.83 [mean{+/-}SEM] ng/hr/g protein; P<0.001), with a similar pattern in the Pex11{beta} knockdown cells. However, in the presence of palmitate, insulin secretion was significantly higher in the Pex11{beta} knockdown versus control cells (4.04{+/-}0.46 vs 2.54{+/-}0.25 ng/hr/g protein; p<0.05). ConclusionPex11{beta} knockdown decreased peroxisome abundance, decreased palmitate mediated ROS generation, and reversed the inhibitory effect of palmitate on insulin secretion. These findings highlight a specific and independent role for peroxisomes in pancreatic beta-cell lipotoxicity.

cell biology↗

bioRxiv2019

The use of CRISPR for variant specificity in the genetic diagnosis of primary immunodeficiency disease (PID)

The functional validation of genetic variants of uncertain significance (VUS) found in PID patients by next-generation sequencing has traditionally been carried out in model systems that are susceptible to artefact. We use CRISPR correction of primary human T lymphocytes to demonstrate that a specific variant in an IL-6R deficient patient is causative for their condition. This methodology can be adapted and used for variant assessment of the heterogeneous genetic defects that affect T lymphocytes in PID.

immunology↗

bioRxiv2019

GenEditID: an open-access platform for the high-throughput identification of CRISPR edited cell clones

CRISPR-Cas9-based gene editing is a powerful tool to reveal genotype-phenotype relationships, but identifying cell clones carrying desired edits remains challenging. To address this issue we developed GenEditID, a flexible, open-access platform for sample tracking, analysis and integration of multiplexed deep sequencing and proteomic data, and intuitive plate-based data visualisation to facilitate gene edited clone identification. To demonstrate the scalability and sensitivity of this method, we identified KO clones in parallel from multiplexed targeting experiments, and optimised conditions for single base editing using homology directed repair. GenEditID enables non-specialist groups to expand their gene targeting efforts, facilitating the study of genetically complex human disease.

genomics↗

bioRxiv2019

A rapid and automated sample-to-result Candida auris real-time PCR assay for high throughput testing of surveillance samples with BD MAX™ open system

The multidrug-resistant yeast pathogen Candida auris continues to cause outbreaks and clusters of clinical cases worldwide. Previously, we developed a real-time PCR assay for the detection of C. auris from surveillance samples (Leach et al. JCM. 2018: 56, e01223-17). The assay played a crucial role in the ongoing investigations of C. auris outbreak in New York City. To ease the implementation of the assay in other laboratories, we developed an automated sample-to-result real-time C. auris PCR assay using BD MAX open system. We optimized sample extraction at three different temperatures and four incubation periods. Sensitivity was determined using eight pools of patient samples, and specificity was calculated using four clades of C. auris, and closely and distantly related yeasts. Three independent extractions and testing of two patient sample pools in the quadruplicate yielded assay precision. BD MAX optimum assay conditions were: DNA extraction at 75{degrees}C for 20 min, and the use of PerfeCTa Multiplex qPCR ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR reaction. We detected all four clades of C. auris without cross-reactivity to other yeasts. Of the 110 patient surveillance samples tested, 50 were positive for C. auris using the BD MAX System with 96% clinical sensitivity and 94% accuracy compared to the manual assay. BD MAX assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-hour workday.

microbiology↗

bioRxiv2019

Phospho-tuning immunity through Denisovan, modern human, and mouse TNFAIP3 gene variants

Resisting or tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here, genomic analyses of anatomically modern humans, extinct Denisovan hominins, and mice revealed a series of missense variants in the immune response inhibitor A20 (encoded by TNFAIP3), substituting non-catalytic residues of the ubiquitin protease domain to diminish I{kappa}B-dependent phosphorylation and activation of A20. Two A20 variants with partial phosphorylation deficits appeared beneficial: one originating in Denisovans and introgressed in modern humans throughout Oceania, and another in a mouse strain resistant to Coxsackievirus. By contrast, a variant with 95% loss of phosphorylation caused spontaneous inflammatory disease in humans and mice. Analysis of the partial phosphorylation variant in mice revealed diminished tolerance of bacterial lipopolysaccharide or to poxvirus inoculation as trade-offs for enhanced immunity.\n\nOne Sentence SummaryModern and ancient variants reveal a genetically tunable element for balancing immunity and microbial tolerance.

immunology↗

bioRxiv2019

The Malaria Cell Atlas: a comprehensive reference of single parasite transcriptomes across the complete Plasmodium life cycle

Malaria parasites adopt a remarkable variety of morphological life stages as they transition through multiple mammalian host and mosquito vector environments. Here we profile the single-cell transcriptomes of thousands of individual parasites, deriving the first high-resolution transcriptional atlas of the entire Plasmodium berghei life cycle. We then use our atlas to precisely define developmental stages of single cells from three different human malaria parasite species, including parasites isolated directly from infected individuals. The Malaria Cell Atlas provides both a comprehensive view of gene usage in a complex eukaryotic parasite and an open access reference data set for the study of malaria parasites.

genomics↗

bioRxiv2018

Poor coherence in older people’s speech is explained by impaired semantic and executive processes

The ability to speak coherently is essential for effective communication but declines with age: older people more frequently produce tangential, off-topic speech. The cognitive factors underpinning this decline are poorly understood. We predicted that maintaining coherence relies on effective regulation of activated semantic knowledge about the world, and particularly on the selection of currently relevant semantic representations to drive speech production. To test this, we collected 840 speech samples along with measures of executive and semantic ability from 60 young and older adults, using a novel computational method to quantify coherence. Semantic selection ability predicted coherence, as did level of semantic knowledge and a measure of domain-general executive ability. These factors fully accounted for the age-related coherence deficit. Our results indicate that maintaining coherence in speech becomes more challenging as people age because they accumulate more knowledge but are less able to effectively regulate how it is activated and used.

neuroscience↗

bioRxiv2017

CRISPRi is not strand-specific and redefines the transcriptional landscape

CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, binding of sgRNA/dCas9 creates an environment that is permissive for transcription initiation and termination, thus generating novel sense and antisense transcripts. At HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9 binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.

genetics↗