Biology subjects

Nguyen, A.

Publications and source records attributed to Nguyen, A..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

HolistiC: Leveraging Hi-C and Whole Genome Shotgun Sequencing for Double Minute Chromosome Discovery

Double minute chromosomes are acentric extrachromosomal DNA artifacts that are frequently observed in the cells of numerous cancers. They are highly amplified and contain oncogenes and drug resistance genes, making their presence a challenge for effective cancer treatment. Algorithmic discovery of double minutes (DM) can potentially improve bench-derived therapies for cancer treatment. A hindrance to this task is that DMs evolve, yielding circular chromatin that shares segments from progenitor double minutes. This creates double minutes with overlapping amplicon coordinates. Existing DM discovery algorithms use whole genome shotgun sequencing in isolation, which can potentially incorrectly classify DMs that share overlapping coordinates. In this study, we describe an algorithm called " HolistIC" that can predict double minutes in tumor genomes by integrating whole genome shotgun sequencing (WGS) and Hi-C sequencing data. The consolidation of these sources of information resolves ambiguity in double minute amplicon prediction that exists in DM prediction with WGS data used in isolation. We implemented and tested our algorithm on the tandem Hi-C and WGS datasets of three cancer datasets and a simulated dataset. Results on the cancer datasets demonstrated HolistICs ability to predict DMs from Hi-C and WGS data in tandem. The results on the simulated data showed the HolistIC can accurately distinguish double minutes that have overlapping amplicon coordinates, an advance over methods that predict extrachromosomal amplification using WGS data in isolation. AvailabilityOur software is available at http://www.github.com/mhayes20/HolistIC.

bioinformatics↗

bioRxiv2021

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here we use biophysical tools to study N-protein interactions with oligonucleotides of different length, examining the size, composition, secondary structure, and energetics of the resulting states. We observe formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant increase in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within molecular condensates.

biophysics↗

bioRxiv2020

BBrowser: Making single-cell data easily accessible

BioTurings BBrowser is a software solution that helps scientists effectively analyze single-cell omics data. It combines big data with big computation and modern data visualization to create a unique platform where scientists can interact and obtain important biological insights from the massive amounts of single-cell data. BBrowser has three main components: a curated single-cell database, a big-data analytics layer, and a data visualization module. BBrowser is available for download at: https://bioturing.com/bbrowser/download.

bioinformatics↗

bioRxiv2020

Altered functional integration of brain activity is a marker of impaired cognitive performance following sleep deprivation

Sleep deprivation (SD) leads to impairments in cognitive function. Here, we tested the hypothesis that cognitive changes in the sleep-deprived brain can be explained by information processing within and between large-scale cortical networks. We acquired functional magnetic resonance imaging (fMRI) scans of 20 healthy volunteers during attention and executive tasks following a regular night of sleep, a night of sleep deprivation, and a recovery nap containing non-rapid eye movement (NREM) sleep. Overall, sleep deprivation was associated with increased cortex-wide functional integration, driven by a rise of integration within cortical networks. The ratio of within vs between network integration in the cortex increased further in the recovery nap, suggesting that prolonged wakefulness drives the cortex toward a state resembling sleep. This balance of integration and segregation in the sleep-deprived state was tightly associated with deficits in cognitive performance. This was a distinct and better marker of cognitive impairment than conventional indicators of homeostatic sleep pressure, as well as the pronounced thalamo-cortical connectivity changes that occurs towards falling asleep. Importantly, restoration of the balance between segregation and integration of cortical activity was also related to performance recovery after the nap, demonstrating a bi-directional effect. These results demonstrate that intra- and inter-individual differences in cortical network integration and segregation during task performance may play a critical role in vulnerability to cognitive impairment in the sleep deprived state. Significance StatementSleep deprivation has significant negative consequences for cognitive function. Understanding how changes in brain activity underpin changes in cognition is important not only to discover why performance declines following extended periods of wakefulness, but also for answering the fundamental question of why we require regular and recurrent sleep for optimal performance. Finding neural correlates that predict performance following sleep deprivation also has the potential to understand which individuals are particularly vulnerable to sleep deprivation, and what aspects of brain function may protect them from these negative consequences on performance. Finally, understanding how perturbations to regular (well-rested) brain functioning affect cognitive performance, will provide important insight into how underlying principles of information processing in the brain may support cognition generally.

neuroscience↗

bioRxiv2020

Temporal stability of human sperm mosaic mutations results in life-long threat of transmission to offspring

Every newborn harbors scores of new single nucleotide variants (SNVs) that may impact health and disease1-4; the majority of these are contributed by the paternal germ cells5. In some cases, these mutations are identifiable in a subset of the parents cells--a phenomenon called mosaicism, which is capable of producing disease recurrence6-8. Here, we provide a comprehensive analysis of male gonadal mosaic mutations, employing 300x whole genome sequencing (WGS) of blood and sperm in 17 healthy individuals, including assessment across multiple samples and age groups. Approximately 1 in 15 healthy males is predicted to harbor a transmissible, likely pathogenic exonic variant that is mosaic in his sperm. In general, only a third of sperm mosaic mutations were detectable in blood cells, all were remarkably stable over the course of months to years, and 23% were present in 5% or more of sperm cells. There was no evidence of age-dependent clonal expansion or collapse, as seen in hematopoiesis. Thus, despite the observed increase of mutations in offspring of men with advanced paternal age, detectable sperm mosaicism remains stable, represents a life-long transmission risk to offspring, and suggests a testis stem cell niche that prevents widespread clonality.

genomics↗

bioRxiv2020

The Endoplasmic Reticulum Proteostasis Regulator ATF6 is Essential for Human Cone Photoreceptor Development

Dysregulation of the endoplasmic reticulum (ER) Unfolded Protein Response (UPR) is implicated in the pathology of many human diseases associated with ER stress. Inactivating genetic variants in the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital heritable vision loss in patients by an unknown pathomechanism. To investigate this, we generated retinal organoids from patient iPSCs carrying ATF6 disease-causing variants and ATF6 null hESCs generated by CRISPR. Interestingly, we found that cone photoreceptor cells in ATF6 mutant retinal organoids lacked inner and outer segments concomitant with absence of cone phototransduction gene expression; while rod photoreceptors developed normally. Adaptive optics retinal imaging of patients with disease-causing variants in ATF6 also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the phenotypes observed in our retinal organoids. These results reveal that ATF6 is essential for the formation of human cone photoreceptors, and associated absence of cone phototransduction components explains the severe visual impairment in patients with ATF6 -associated retinopathy. Moreover, we show that a selective small molecule ATF6 activator compound restores the transcriptional activity of ATF6 disease-causing variants and stimulates the growth of cone photoreceptors in patient retinal organoids, demonstrating that pharmacologic targeting of ATF6 signaling is a therapeutic strategy that needs to be further explored for blinding retinal diseases.

developmental biology↗

bioRxiv2020

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

Meiosis produces haploid gametes that will give rise to the next diploid generation. Chromosome segregation errors occurring at one or both meiotic divisions result in aneuploidy, which can lead to miscarriages or birth defects in humans. During meiosis I, ring-shaped cohesin complexes play important roles to aid in the proper segregation of homologous chromosomes. While REC8 is a specialized meiosis-specific cohesin that functions to hold sister chromatids together, the role of its vertebrate-specific paralog, RAD21L, is poorly understood. Here we tested if Rad21l1, the zebrafish homolog of human and mouse RAD21L, is required for meiotic chromosome dynamics during oogenesis and spermatogenesis. We found that Rad21l1 is an abundant component of meiotic chromosomes where it localizes to both the chromosome axes and the transverse filament of the synaptonemal complex (SC). Knocking out rad21l1 causes nearly the entire mutant population to develop as fertile males, suggesting the mutation triggers a sex reversal from female to male due to a failure in oocyte production. The rad21l1-/- mutant males display normal fertility at sexual maturity. Sex reversal was partially suppressed in the absence of tp53, suggesting that the rad21l1-/- mutation causes defects leading to a Tp53 dependent response, specifically in females. The rad21l1-/-;tp53-/- double mutant females produced elevated rates of decomposing eggs and deformed offspring compared to tp53-/- controls. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of Spo11 does not suppress the sex reversal phenotype. Overall, our data highlight an exceptional sexually dimorphic phenotype caused by knocking out a meiotic-specific cohesin subunit. We propose that Rad21l1 is required for maintaining the integrity of meiotic chromatin architecture during oogenesis. Author SummaryA prominent symptom of age-linked reproductive decline in women is the increased rate of miscarriage and birth defects due to aneuploidy. Aneuploidy can arise when chromosomes fail to segregate properly during meiosis, the process of creating haploid gametes from a diploid germ cell. Oocyte progression normally arrests prior to anaphase I, after homologous chromosomes have formed crossovers, but before ovulation, which triggers the first round of segregation. This prolonged arrest makes oocytes especially vulnerable to degradation of meiotic chromosome structure and homolog connections over time. Cohesin complexes play a major role in maintaining the meiotic chromosome architecture. Here we assess the role of the vertebrate-specific Rad21l1 cohesin subunit in zebrafish. We find that while males appear mostly unaffected by loss of Rad21l1, oocyte production is massively compromised, leading to sex reversion to males. Sex reversion can be partially prevented in the absence of Tp53, demonstrating that loss or Rad21l1 leads to a Tp53-dependent response in oocytes. Strikingly, double mutant rad21l1 tp53 females produce large numbers of poor quality eggs and malformed offspring. This demonstrates a cohesin-linked vulnerability in female meiosis not present in males and sheds light on a potential mechanism associated with the decline in female reproductive health.

genetics↗

bioRxiv2020

The importance of charge in perturbing the aromatic glue stabilizing the protein-protein interface of homodimeric tRNA-guanine transglycosylase

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each exchange with respect to dimer and thermal stability or enzyme activity applying native mass spectrometry, thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate strong influence of pH on homodimer stability. Obviously, protonation of a histidine within the aromatic cluster promotes the collapse of an essential structural motif within the dimer interface at slightly acidic pH. TOC GraphicFor table of contents use only. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2020

Identification of limb-specific Lmx1b auto-regulatory modules with Nail-Patella Syndrome pathogenicity.

LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly, in mice Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of murine Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. Interestingly, in Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression in the limb and are necessary for Lmx1b-mediated limb dorsalization. Remarkably, we also report on two NPS patient families with normal LMX1B coding sequence, but loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

developmental biology↗

bioRxiv2020

Somatic mosaicism in the mature brain reveals clonal cellular distributions during cortical development

The structure of the human neocortex underlies species-specific features and is a reflection of intricate developmental programs. Here we analyzed neocortical cellular lineages through a comprehensive assessment of brain somatic mosaicism--which acts as a neutral recorder of lineage history. We employed deep whole genome and variant sequencing in a single postmortem neurotypical human brain across 25 anatomic regions and three distinct modalities: bulk geographies, sorted cell types, and single nuclei. We identified 259 mosaic variants, revealing remarkable differences in localization, clonal abundance, cell type specificity, and clade distribution. We identified a set of hierarchical cellular diffusion barriers, whereby the left-right axis separation of the neocortex occurs prior to anterior-posterior and dorsal-ventral axis separation. We also found that stochastic distribution is a driver of clonal dispersion, and that rules regarding cellular lineages and anatomical boundaries are often ignored. Our data provides a comprehensive analysis of brain somatic mosaicism across the human cerebral cortex, deconvolving clonal distributions and migration patterns in the human embryo. One Sentence SummaryComprehensive evaluation of brain somatic mosaicism in the adult human identifies rules governing cellular distribution during embryogenesis.

genomics↗

bioRxiv2020

TLR7 and RIG-I dual-adjuvant loaded nanoparticles drive broadened and synergistic responses in dendritic cells and boost cellular immunity in influenza vaccination

Although the existing flu vaccines elicit strong antigen-specific antibody responses, they fail to provide effective, long term protection - partly due to the absence of robust cellular memory immunity. We hypothesized that co-administration of combination adjuvants, mirroring the flu-virus related innate signaling pathways, could elicit strong cellular immunity. Here, we show that the small molecule adjuvant R848 and the RNA adjuvant PUUC, targeting endosomal TLR7s and cytoplasmic RLRs respectively, when delivered together in polymer nanoparticles (NP), elicits a broadened immune responses in mouse bone marrow-derived dendritic cells (mBMDCs) and a synergistic response in both mouse and human plasmacytoid dendritic cells (pDCs). In mBMDCs, NP-R848-PUUC induced both NF-{kappa}B and interferon signaling. Interferon responses to co-delivered R848 and PUUC were additive in human peripheral blood mononuclear cells (PBMCs) and synergistic in human FLT3-differentiated mBMDCs and CAL-1 pDCs. Vaccination with NPs loaded with H1N1 Flu antigen, R848, and PUUC increased percentage of CD8+ T-cells in the lungs, percentage of antigen-specific CD4+T-cells in the spleen, and enhanced overall cytokine-secreting T cell percentages upon antigen restimulation. Also in the spleen, T lymphopenia, especially after in vitro restimulation, was observed. Our results demonstrate that simultaneous engagement of TLR7 and RIG-I pathways using particulate carriers is a potential approach to improve cellular immunity in flu vaccination. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/207423v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1599111org.highwire.dtl.DTLVardef@1431395org.highwire.dtl.DTLVardef@baa40dorg.highwire.dtl.DTLVardef@16877cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Microchip based microrheology via Acoustic Force Spectroscopy shows that endothelial cell mechanics follows a fractional viscoelastic model

Active microrheology is one of the main methods to determine the mechanical properties of cells and tissue, and the modelling of the viscoelastic properties of cells and tissue is under heavy debate with many competing approaches. Most experimental methods of active microrheology such as optical tweezers or atomic force microscopy based approaches rely on single cell measurements, and thus suffer from a low throughput. Here, we present a novel method for cell based microrheology using acoustic forces which allows multiplexed measurements of several cells in parallel. Acoustic Force Spectroscopy (AFS) is used to generate multi-oscillatory forces in the range of pN-nN on particles attached to primary human umbilical vein endothelial cells (HUVEC) cultivated inside a microfluidic chip. While the AFS was introduced as a single-molecule technique to measure mechanochemical properties of biomolecules, we exploit the AFS to measure the dynamic viscoelastic properties of cells exposed to different conditions, such as flow shear stresses or drug injections. By controlling the force and measuring the position of the particle, the complex shear modulus G*(ω) can be measured continuously over several hours. The resulting power-law shear moduli are consistent with fractional viscoelastic models. In our experiments we confirm a decrease in shear modulus after perturbing the actin cytoskeleton via cytochalasin B. This effect was reversible after washing out the drug. Although these measurements are possible, we provide critical information regarding the AFS as a measurement tool showing its capabilities and limitations. A key result is that for performing viscoelastic measurements with the AFS, a thorough calibration and careful data analysis is crucial, for which we provide protocols and guidelines.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.View Full Text

developmental biology↗

bioRxiv2020

Dopamine neurons promote arousal and wakefulness via Dop1R receptor in the Drosophila mushroom body

Neural circuits involved in regulation of sleep play a critical role in sleep-wake transition and ability of an organism to engage in other behaviors critical for survival. The fruit fly, Drosophila melanogaster is a powerful system for the study of sleep and circuit mechanisms underlying sleep and co-regulation of sleep with other behaviors. In Drosophila, two neuropils in the central brain, mushroom body (MB) and central complex (CX) have been shown to influence sleep homeostasis and receive neuromodulator input critical to sleep-wake switch. Dopamine neurons (DANs) are the primary neuromodulator inputs to the MB but the mechanisms by which they regulate sleep- and wake-promoting neurons within MB are unknown. Here we investigate the role of subsets of DANs that signal wakefulness and project to wake-promoting compartments of the MB. We find that inhibition of specific subsets of PAM and PPL1 DANs projecting to the MB increase sleep in the presence of strong wake-inducing stimuli that reduces GABA transmission, although activity of these neurons is not directly modulated by GABA signaling. Of these subsets we find that DANs innervating the {gamma}5 and {beta}2 MB compartments require both DopR1 and DopR2 receptors located in downstream Kenyon cells and mushroom body output neurons (MBONs). Further, we report that unlike the activity of wake-promoting MBONs and KCs, whose activity is modulated by sleep-need and PAM-DAN activity is independent of sleep-need. We have characterized a dopamine mediated sleep-circuit providing an inroad into understanding how common circuits within MB regulate sleep, wakefulness and behavioral arousal.

neuroscience↗

bioRxiv2020

Role of c-Met/β1 integrin complex in the metastatic cascade

Metastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. While individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a structural complex between receptor tyrosine kinase c-Met and {beta}1 integrin in metastases. Using novel cell culture and in vivo assays, we found that c-Met/{beta}1 complex induction promotes breast cancer intravasation and adhesion to the vessel wall, but does not increase extravasation. These effects may be driven by the ability of the c-Met/{beta}1 complex to increase mesenchymal and stem cell characteristics. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/{beta}1 complex induction. A {beta}1 integrin point mutation that prevented binding to c-Met reduced intravasation. OS2966, a therapeutic antibody disrupting c-Met/{beta}1 binding, decreased invasion and mesenchymal gene expression and morphology of breast cancer cells. Bone-seeking breast cancer cells exhibited higher c-Met/{beta}1 complex levels than parental controls and preferentially adhere to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/{beta}1 levels than brain metastases. Thus, the c-Met/{beta}1 complex drives breast cancer cell intravasation and preferential affinity for bone tissue-specific matrix. Pharmacological targeting of the complex may prevent metastases, particularly osseous metastases.

cancer biology↗

bioRxiv2020

MK2-deficient mice are bradycardic and display delayed hypertrophic remodelling in response to a chronic increase in afterload

MAP kinase-activated protein kinase-2 (MK2) is protein serine/threonine kinase activated by p38/{beta}. Herein we examined the cardiac phenotype of pan MK2-null (MK2-/-) mice. Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P = 0.580). At 12-weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, ageing was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3 were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+-sensitivity to mitochondrial permeability transition pore (mPTP) opening. Finally, the pressure overload-induced increase in heart weight/tibia length and decrease in systolic function were attenuated in MK2-/- mice two weeks, but not eight weeks, after constriction of the transverse aorta. Collectively, these results implicate MK2 in (i) autonomic regulation of heart rate, (ii) cardiac mitochondrial function, and (iii) the early stages of myocardial remodeling in response to chronic pressure overload. Key points summaryThe cardiac characterization of pan MK2-null mice showed: O_LIaltered autonomic regulation of heart rate C_LIO_LIincreased expression of key metabolic genes C_LIO_LIdecreased Ca2+-sensitivity for MPTP opening C_LIO_LIdelayed hypertrophic remodeling in response to increased afterload C_LI

physiology↗

bioRxiv2019

Multiplexed direct detection of barcoded protein reporters on a nanopore array

Genetically encoded reporter proteins are a cornerstone of molecular biology. While they are widely used to measure many biological activities, the current number of uniquely addressable reporters that can be used together for one-pot multiplexed tracking is small due to overlapping detection channels such as fluorescence. To address this, we built an expanded library of orthogonally-barcoded Nanopore-addressable protein Tags Engineered as Reporters (NanoporeTERs), which can be read and demuxed by nanopore sensors at the single-molecule level. By adapting a commercially available nanopore sensor array platform typically used for real-time DNA and RNA sequencing (Oxford Nanopore Technologies MinION), we show direct detection of NanoporeTER expression levels from unprocessed bacterial culture with no specialized sample preparation. These results lay the foundations for a new class of reporter proteins to enable multiplexed, real-time tracking of gene expression with nascent nanopore sensor technology.

synthetic biology↗

bioRxiv2019

PCK1 and DHODH drive colorectal cancer liver metastatic colonization and hypoxic pyrimidine nucleotide anabolic biosynthesis

Colorectal cancer (CRC) is a major cause of human death. Mortality is primarily due to metastatic organ colonization, with liver being the primary organ affected. We modeled metastatic CRC (mCRC) liver colonization using patient-derived primary and metastatic tumor xenografts (PDX). Such PDX modeling predicted patient survival outcomes. In vivo selection of multiple PDXs for enhanced metastatic capacity upregulated the gluconeogenic enzyme PCK1, which enhanced metastatic hypoxic survival by driving anabolic pyrimidine nucleotide biosynthesis. Consistently, highly metastatic tumors upregulated multiple pyrimidine biosynthesis intermediary metabolites. Therapeutic inhibition of the pyrimidine biosynthetic enzyme DHODH with oral leflunomide substantially impaired CRC liver metastatic colonization and hypoxic survival. Our findings provide a potential mechanistic basis for the epidemiologic association of anti-gluconeogenic drugs with improved CRC metastasis outcomes, reveal the exploitation of a gluconeogenesis enzyme for pyrimidine biosynthesis during hypoxia, and implicate DHODH and PCK1 as metabolic therapeutic targets in colorectal cancer metastasis.

cancer biology↗