Biology subjects

Ross, J.

Publications and source records attributed to Ross, J..

32 records · Page 2Linked to original sources

bioRxiv2021

HOXB13 suppresses de novo lipogenesis through HDAC3-mediated epigenetic reprogramming

HOXB13, a homeodomain transcription factor, critically regulates androgen receptor (AR) function and promotes androgen-dependent prostate cancer (PCa) growth. However, the functions of HOXB13 in an AR-independent context remain elusive. Here we report an essential role of HOXB13 in directly suppressing lipogenic transcriptional programs in both AR-positive and -negative PCa cells. The MEIS domain (aa70-150) of HOXB13 interacts with the histone deacetylase HDAC3, which is disrupted by HOXB13 G84E mutation that has been associated with early-onset PCa. Thus, HOXB13 wildtype (WT), but not G84E mutant, recruits HDAC3 to lipogenic enhancers to catalyze histone de-acetylation and suppress lipogenic programs. HOXB13 knockdown unleashes the expression of key lipogenic regulators such as fatty acid synthase (FASN), requiring HDAC3. Analysis of human tissues revealed that HOXB13 is lost in about 30% of metastatic castration-resistant PCa, at least in part, through DNA hypermethylation. Functionally, loss of HOXB13 leads to massive lipid accumulation in PCa cells, thereby promoting cell motility in vitro and fueling xenograft tumor metastasis in vivo, which is mitigated by pharmaceutical inhibitors of FASN. In summary, our study discovers an essential AR-independent function of HOXB13 in repressing de novo lipogenesis and inhibiting tumor metastasis and defines a subclass of PCa that may benefit from lipogenic pathway inhibitors.

cancer biology↗

bioRxiv2021

Oligomerization of the FliF domains suggests a coordinated assembly of the bacterial flagellum MS ring

The bacterial flagellum is a complex, self-assembling macromolecular machine that powers bacterial motility. It plays diverse roles in bacterial virulence, including aiding in colonization and dissemination during infection. The flagellum consists of a filamentous structure protruding from the cell, and the basal body, a large assembly that spans the cell envelope. The basal body is comprised of over 10 different proteins, forming several concentric ring structures, termed the M- S- L- P- and C-rings, respectively. In particular, the MS rings are formed by a single protein FliF, which consists of two trans-membrane helices anchoring it to the inner membrane and surrounding a large periplasmic domain. Assembly of the MS ring, through oligomerization of FliF, is one of the first steps of basal body assembly. Previous computational analysis had shown that the periplasmic region of FliF consists of three structurally similar domains, termed Ring-Building Motif (RBM)1, RBM2 and RBM3. The structure of the MS-ring has been reported recently, and unexpectedly shown that these three domains adopt different symmetries, with RBM3 having a 34-mer stoichiometry, while RBM2 adopts two distinct positions in the complex, including a 23-mer ring. This observation raises some important question on the assembly of the MS ring, and the formation of this symmetry mis-match within a single protein. In this study, we analyze the oligomerization of the individual RBM domains in isolation, in the Salmonella typhimurium FliF orthologue. We demonstrate that the periplasmic domain of FliF assembles into the MS ring, in the absence of the trans-membrane helices. We also report that the RBM2 and RBM3 domains oligomerize into ring structures, but not RBM1. Intriguingly, we observe that a construct encompassing RBM1 and RBM2 is monomeric, suggesting that RBM1 interacts with RBM2, and inhibits its oligomerization. However, this inhibition is lifted by the addition of RBM3. Collectively, this data suggests a mechanism for the controlled assembly of the MS ring.

biophysics↗

bioRxiv2021

Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment revealed by Cryo-EM and hydrogen/deuterium exchange mass spectrometry

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications. TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible.

biochemistry↗

bioRxiv2020

Mean-field models for EEG/MEG: from oscillations to waves

Neural mass models have been actively used since the 1970s to model the coarse-grained activity of large populations of neurons. They have proven especially fruitful for understanding brain rhythms. However, although motivated by neurobiological considerations they are phenomeno-logical in nature, and cannot hope to recreate some of the rich repertoire of responses seen in real neuronal tissue. Here we consider a simple spiking neuron network model that has recently been shown to admit to an exact mean-field description for both synaptic and gap-junction interactions. The mean-field model takes a similar form to a standard neural mass model, with an additional dynamical equation to describe the evolution of population synchrony. As well as reviewing the origins of this next generation mass model we discuss its extension to describe an idealised spatially extended planar cortex. To emphasise the usefulness of this model for EEG/MEG modelling we show how it can be used to uncover the role of local gap-junction coupling in shaping large scale synaptic waves.

neuroscience↗

bioRxiv2020

Sublethal concentrations of clothianidin affect honey bee colony behavior and interact with landscapes to affect colony growth

Honey bee colonies were exposed to sublethal concentrations (5 and 20 ppb) of clothianidin in sugar syrup, while control colonies were fed syrup with no pesticide. In addition to standard colony assessments of adult bees and brood, hive weight and internal temperature were monitored on a continuous basis at all sites. Experiments were conducted twice in Arizona, in successive years at the same site, and once in Mississippi, to examine the concomitant effects of weather and landscape. Adult bee masses at the Arizona site were significantly affected by clothianidin concentration. Newly-emerged bee dry weights, measured only at the Arizona site, were significantly lower for colonies fed 5 ppb clothianidin compared to the other groups. CO2 concentration, also only measured at the Arizona site, was higher in colonies fed 20 ppb clothianidin. Neither daily hive weight change nor colony thermoregulation were affected by clothianidin exposure. The Mississippi site had higher rainfall, more diverse land use, and a different temperature regime, and bee colonies there did not show any effects of clothianidin. These results suggest that bee colonies in more stressful environments, such as the Sonoran desert of southern Arizona, are affected more by clothianidin exposure than colonies at sites with higher rainfall and more forage. Clothianidin was also found to be, like imidacloprid, highly stable in honey in the hive environment at least over several months. These results also showed that CO2 concentration within the hive is potentially valuable in measuring the effects of stressors on bee health.

ecology↗

bioRxiv2019

Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins

Encapsulated ferritins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question, due to differences in organization of the ferroxidase catalytic site and secondary metal binding sites vicinal to this. We have previously identified a metal binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this proposed iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site.

biochemistry↗

bioRxiv2019

Novel engraftment and T cell differentiation of human hematopoietic cells in Art-/- IL2RG-/ SCID pigs

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (Artemis)-/- genetic background. We confirmed Art-/- IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we and successfully performed a bone marrow transplant on one Art-/- IL2RG-/Y male SCID pig with a bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal Art-/- IL2RG-/Y SCID pigs. At birth, human CD45+ CD3{varepsilon}+ cells were detected in peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.\n\nOne sentence summaryWe have generated a T- B- NK- SCID pig model through site directed mutagenesis of IL2RG in a naturally occurring Artemis null background and show successful engraftment of human T and B cells in blood and lymphoid organs after in utero injection of human hematopoietic stem cells.

immunology↗

bioRxiv2019

The ebb and flow of heteroplasmy during intra-species hybridization in Caenorhabditis briggsae

Mitochondria are typically maternally inherited. In many species, this transmission pattern is produced by sperm-borne mitochondria being eliminated either from sperm before fertilization or from the embryo after fertilization. In the nematode Caenorhabditis briggsae, repeatedly backcrossing hybrids to genetically diverse males can elicit paternal mitochondrial transmission. Studies of other taxa also suggest that hybridization increases paternal mitochondrial transmission. Thus, hybrid genotypes might disrupt the systems that normally prevent paternal mitochondrial transmission. Given the reliance of a number of genetic analyses on the assumption of purely maternal mitochondrial inheritance, it would be broadly valuable to learn more about the processes embryos employ to prevent sperm-borne mitochondria from persisting in offspring, as well as the circumstances under which paternal transmission might be expected to occur. To quantify the tempo of paternal mitochondrial transmission in hybrids, we assessed the presence of paternal mitotypes in replicate lines at three timepoints spanning fifteen generations. All lines exhibited paternal mitochondrial transmission. However, this heteroplasmy always then resolved to homoplasmy for the maternal mitotype. Additionally, one nuclear locus exhibited allele transmission ratio distortion that might reflect mito-nuclear co-evolution. This study frames the genetic architecture of a hybrid genetic incompatibility that leads to paternal mitochondrial transmission and to a reduction in hybrid fitness.

evolutionary biology↗

bioRxiv2019

Optimised prophylactic vaccination in metapopulations

A highly effective method for controlling the spread of an infectious disease is vaccination. However, there are many situations where vaccines are in limited supply. The ability to determine, under this constraint, a vaccination strategy which minimises the number of people that become infected over the course of a potential epidemic is essential. Two questions naturally arise: when is it best to allocate vaccines, and to whom should they be allocated? We address these questions in the context of metapopulation models of disease spread. We discover that it is optimal to distribute all vaccines prophylactically, rather than withholding until infection is introduced. For small metapopulations, we provide a method for determining the optimal allocation. As the optimal strategy becomes computationally intensive to obtain when the population size increases, we detail an approximation method to determine an approximately optimal vaccination scheme. Through comparisons with other strategies in the literature, we find that our approximate strategy is superior.

epidemiology↗

bioRxiv2018

Conservation of the structural and functional architecture of encapsulated ferritins in bacteria and archaea

Iron is an essential element for many biological processes; however, due to its high reactivity iron can also be very toxic, producing reactive oxygen species through Fenton chemistry. Ferritins protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing the resulting iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. We recently characterised the Rhodospirillum rubrum EncFtn, showing that although enzymatically active, due to its open structure it requires the association with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether the structure and catalytic activity is conserved across the family. Here we structurally characterise two EncFtn members from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the same distinct annular decamer topology observed in R. rubrum EncFtn, with the ferroxidase centre (FOC) formed between one of the dimer interfaces. Solution and native mass spectrometr analyses show that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The catalytic role of the EncFtn proteins was confirmed by biochemical assays, and we show that Zn(II) ions inhibit the ferroxidase activity of the EncFtn proteins to varying degrees. Our results represent a further step in the characterisation of the recently discovered EncFtn ferritin-like sub-family, indicating a common structural organisation and catalytic activity, despite diverse host environments.

biochemistry↗

bioRxiv2018

IDH1R132H acts as a tumor suppressor in glioma via epigenetic upregulation of the DNA damage response

One sentence summaryMutant IDH1 acts as a tumor suppressor when co-expressed together with TP53 and ATRX inactivating mutations in glioma, inducing genomic stability, DNA repair and resistance to genotoxic therapies.\n\nAbstractGlioma patients whose tumors carry a mutation in the Isocitrate Dehydrogenase 1 (IDH1R132H) gene are younger at the time of diagnosis and survive longer. The molecular glioma subtype which we modelled, harbors IDH1R132H, tumor protein 53 (TP53) and alpha-thalassemia/mental retardation syndrome X-linked (ATRX) loss. The impact of IDH1R132H on genomic stability, DNA damage response (DDR) and DNA repair in this molecular glioma subtype is unknown. We discovered that IDH1R132H expression in the genetic context of ATRX and TP53 inactivation: (i) increases median survival (MS), (ii) enhances DDR activity via epigenetic upregulation of Ataxia-telangiectasia mutated (ATM) signaling, and (iii) elicits tumor radioresistance. Pharmacological inhibition of ATM or checkpoint kinase 1 and 2 (CHK1/2), two essential kinases in the DDR pathways, restored tumors radiosensitivity. Translation of these findings for mlDH1 glioma patients could significantly improve the therapeutic efficacy of radiotherapy, and thus have a major impact on patient survival.

cancer biology↗

bioRxiv2018

Complete Disruption of Autism-Susceptibility Genes by Gene-Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons

Autism Spectrum Disorder is phenotypically and genetically heterogeneous, but genomic analyses have identified candidate susceptibility genes. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of 10 ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NEUROG2)-directed differentiation of iPSCs allowed production of cortical excitatory neurons, and mutant proteins were not detectable. RNAseq revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory post-synaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2- and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.

neuroscience↗

bioRxiv2018

Autometa: Automated extraction of microbial genomes from individual shotgun metagenomes

MotivationShotgun metagenomics is a powerful, high-resolution technique enabling the study of microbial communities in situ. However, species-level resolution is only achieved after a process of \"binning\" where contigs predicted to originate from the same genome are clustered. Such culture-independent sequencing frequently unearths novel microbes, and so various methods have been devised for reference-free binning. Existing methods, however, suffer from: (1) reliance on human pattern recognition, which is inherently unscalable; (2) requirement for multiple co-assembled metagenomes, which degrades assembly quality due to strain variance; and (3) assumption of prior host genome removal not feasible for non-model hosts. We therefore devised a fully-automated pipeline, termed \"Autometa,\" to address these issues. Results: Autometa implements a method for taxonomic partitioning of contigs based on predicted protein homology, and this was shown to vastly improve binning in host-associated and complex metagenomes. Autometas method of automated clustering, based on Barnes-Hut Stochastic Neighbor Embedding (BH-tSNE) and DBSCAN, was shown to be highly scalable, outperforming other binning pipelines in complex simulated datasets.\n\nAvailability and implementationAutometa is freely available at https://bitbucket.org/jasonckwan/autometa and as a docker image at https://hub.docker.com/r/jasonkwan/autometa under the GNU Affero General Public License 3 (AGPL 3).\n\nContactjason.kwan@wisc.edu\n\nSupplementary informationSupplementary data are available attached to this article at https://biorxiv.org

bioinformatics↗

bioRxiv2017

Sex Chromosome Dosage Effects On Gene Expression In Humans

A fundamental question in the biology of sex-differences has eluded direct study in humans: how does sex chromosome dosage (SCD) shape genome function? To address this, we developed a systematic map of SCD effects on gene function by analyzing genome-wide expression data in humans with diverse sex chromosome aneuploidies (XO, XXX, XXY, XYY, XXYY). For sex chromosomes, we demonstrate a pattern of obligate dosage sensitivity amongst evolutionarily preserved X-Y homologs, and update prevailing theoretical models for SCD compensation by detecting X-linked genes whose expression increases with decreasing X- and/or Y-chromosome dosage. We further show that SCD-sensitive sex chromosome genes regulate specific co-expression networks of SCD-sensitive autosomal genes with critical cellular functions and a demonstrable potential to mediate previously documented SCD effects on disease. Our findings detail wide-ranging effects of SCD on genome function with implications for human phenotypic variation.\n\nSIGNIFICANCE STATEMENTSex chromosome dosage (SCD) effects on human gene expression are central to the biology of sex differences and sex chromosome aneuploidy syndromes, but challenging to study given the co-segregation of SCD and gonadal status. We address this obstacle by systematically modelling SCD effects on genome wide expression data from a large and rare cohort of individuals with diverse SCDs (XO, XX, XXX, XXXX, XY, XXY, XYY, XXYY, XXXXY). Our findings update current models of sex chromosome biology by (i) pinpointing a core set of X- and Y-linked genes with \"obligate\" SCD sensitivity, (ii) discovering several non-canonical modes of X-chromosome dosage compensation, and (iii) dissecting complex regulatory effects of X-chromosome dosage on large autosomal gene networks with key roles in cellular functioning.

genomics↗