Biology subjects

Walker, C.

Publications and source records attributed to Walker, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Ventromedial Frontoinsular Connectivity is Associated with Long-term Smoking Behavior Change in Aging

A central question in the field of cognitive aging and behavioral neuroscience is what enables some individuals to successfully change their behavior more than others? Smoking is a significant risk factor for cognitive decline, particularly in vulnerable populations, including those who are at an elevated risk for Alzheimers disease (AD). Developing effective smoking reduction strategies is therefore a public health priority. The goal of the current study is to better understand the brain mechanisms underlying long-term smoking behavior change in cognitively normal, but at-risk, older adults. Neuroimaging and human lesional studies have implicated the insula and its functional network in subjective interoceptive awareness of cigarette craving and smoking-cue reactivity. We sought to characterize the extent to which anterior insular resting-state functional connectivity MRI predicted long-term smoking reduction (mean: 2.7 years, range 8 months - 4 years) using a seed-to-voxel approach. Twenty-three (18 women; 26% APOE4 carriers; 61.5 years, SD = 3.7) cognitively unimpaired older individuals who smoked cigarettes at their baseline visit and have a first-degree family history of AD (at least one parent or multiple siblings affected) were included from a prospective longitudinal cohort, PREVENT-AD (Pre-symptomatic Evaluation of Experimental or Novel Treatments for Alzheimer Disease) in the current study. We found that reduced long-term smoking behavior was associated with diminished functional connectivity between bilateral anterior insula and ventromedial prefrontal cortex (vmPFC). In a second pre-registered replication study within a larger, independent sample of one hundred and eighteen cognitively normal older adults who smoked cigarettes at baseline from the UK Biobank (73 women; 27.9 % APOE4 carriers; 60.3 years, SD = 2.7), we found that baseline diminished resting-state functional connectivity between anterior insula and vmPFC predicted long-term smoking reduction (mean 5.2 years; ranging from 3 years to 7 years). To our knowledge, this is the largest study to examine the neural substrates of long-term smoking cessation in human aging. Our results suggest that frontoinsular circuits may be a therapeutic target for smoking reduction and disease prevention in older adults at risk for AD.

neuroscience↗

bioRxiv2023

The αC-β4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA). Not only this long-range synergistic action is involved in substrate recognition and fidelity, but it is also likely to regulate PKA association with regulatory subunits and other binding partners. To date, a complete understanding of the molecular determinants for this intramolecular mechanism is still lacking. Here, we integrated NMR-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of the catalytic subunit of protein kinase A (PKA-C). We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the C helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the C-{beta}4 loop. To experimentally validate the second excited state, we mutated F100 into alanine (F100A) and used NMR spectroscopy to characterize the structural response of the kinase to ATP and substrate binding. While the catalytic efficiency of PKA-CF100A with a canonical peptide substrate remains unaltered, this mutation rearranges the C-{beta}4 loop conformation, interrupting the structural coupling of the two lobes and abolishing the allosteric binding cooperativity of the enzyme. The highly conserved C-{beta}4 loop emerges as a pivotal element able to control the synergistic binding between nucleotide and substrate. These results may explain how mutations or insertions near or within this motif affect the function and drug sensitivity in other homologous kinases.

biophysics↗

bioRxiv2023

A Molecular Basis of Human Brain Connectivity

Neuroimaging is commonly used to infer human brain connectivity, but those measurements are far-removed from the molecular underpinnings at synapses. To uncover the molecular basis of human brain connectivity, we analyzed a unique cohort of 98 individuals who provided neuroimaging and genetic data contemporaneous with dendritic spine morphometric, proteomic, and gene expression data from the superior frontal and inferior temporal gyri. Through cellular contextualization of the molecular data with dendritic spine morphology, we identified hundreds of proteins related to synapses, energy metabolism, and RNA processing that explain between-individual differences in functional connectivity and structural covariation. By integrating data at the genetic, molecular, subcellular, and tissue levels, we bridged the divergent fields of molecular biology and neuroimaging to identify a molecular basis of brain connectivity. One-Sentence SummaryDendritic spine morphometry and synaptic proteins unite the divergent fields of molecular biology and neuroimaging.

neuroscience↗

bioRxiv2023

A DRG genetic toolkit reveals molecular, morphological, and functional diversity of somatosensory neuron subtypes

Mechanical and thermal stimuli acting on the skin are detected by morphologically and physiologically distinct sensory neurons of the dorsal root ganglia (DRG). Achieving a holistic view of how this diverse neuronal population relays sensory information from the skin to the central nervous system (CNS) has been challenging with existing tools. Here, we used transcriptomic datasets of the mouse DRG to guide development and curation of a genetic toolkit to interrogate transcriptionally defined DRG neuron subtypes. Morphological analysis revealed unique cutaneous axon arborization areas and branching patterns of each subtype. Physiological analysis showed that subtypes exhibit distinct thresholds and ranges of responses to mechanical and/or thermal stimuli. The somatosensory neuron toolbox thus enables comprehensive phenotyping of most principal sensory neuron subtypes. Moreover, our findings support a population coding scheme in which the activation thresholds of morphologically and physiologically distinct cutaneous DRG neuron subtypes tile multiple dimensions of stimulus space.

neuroscience↗

bioRxiv2023

Proteomes Reveal Metabolic Capabilities of Yarrowia lipolytica for Biological Upcycling of Polyethylene into High-Value Chemicals

Polyolefins derived from plastic wastes are recalcitrant for biological upcycling. However, chemical depolymerization of polyolefins can generate depolymerized plastic (DP) oil comprising of a complex mixture of saturated, unsaturated, even and odd hydrocarbons suitable for biological conversion. While DP oil contains a rich carbon and energy source, it is inhibitory to cells. Understanding and harnessing robust metabolic capabilities of microorganisms to upcycle the hydrocarbons in DP oil, both naturally and unnaturally occurring, into high-value chemicals are limited. Here, we discovered that an oleaginous yeast Yarrowia lipolytica undergoing short-term adaptation to DP oil robustly utilized a wide range of hydrocarbons for cell growth and production of citric acid and neutral lipids. When growing on hydrocarbons, Y. lipolytica partitioned into planktonic and oil-bound cells with each exhibiting distinct proteomes and amino acid distributions invested into establishing these proteomes. Significant proteome reallocation towards energy and lipid metabolism, belonging to two of the 23 KOG (Eukaryotic Orthologous Groups) classes C and I, enabled robust growth of Y. lipolytica on hydrocarbons, with n-hexadecane as the preferential substrate. This investment was even higher for growth on DP oil where both the KOG classes C and I were the top two, and many associated proteins and pathways were expressed and upregulated including the hydrocarbon degradation pathway, Krebs cycle, glyoxylate shunt and, unexpectedly, propionate metabolism. However, a reduction in proteome allocation for protein biosynthesis, at the expense of the observed increase towards energy and lipid metabolisms, might have caused the inhibitory effect of DP oil on cell growth. MPORTANCESustainable processes for biological upcycling plastic wastes in a circular bioeconomy are needed to promote decarbonization and reduce environmental pollution due to increased plastic consumption, incineration, and landfill storage. Strain characterization and proteomic analysis revealed the robust metabolic capabilities of Y. lipolytica to upcycle polyethylene into high-value chemicals. Significant proteome reallocation towards energy and lipid metabolisms was required for robust growth on hydrocarbons with n-hexadecane as the preferential substrate. However, an apparent over-investment in these same categories to utilize complex DP oil came at the expense of protein biosynthesis, limiting cell growth. Taken together, this study elucidates how Y. lipolytica activates its metabolism to utilize DP oil and establishes Y. lipolytica as a promising host for the upcycling of plastic wastes.

systems biology↗

bioRxiv2023

ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation by forming large transmembrane pores upon cleavage by inflammatory caspases. Here we report the surprising finding that GSDMD cleavage is not sufficient for its pore formation. Instead, GSDMD is lipidated by S-palmitoylation at Cys191 upon inflammasome activation, and only palmitoylated GSDMD N-terminal domain (GSDMD-NT) is capable of membrane translocation and pore formation, suggesting that palmitoylation licenses GSDMD activation. Treatment by the palmitoylation inhibitor 2-bromopalmitate and alanine mutation of Cys191 abrogate GSDMD membrane localization, cytokine secretion, and cell death, without affecting GSDMD cleavage. Because palmitoylation is formed by a reversible thioester bond sensitive to free thiols, we tested if GSDMD palmitoylation is regulated by cellular redox state. Lipopolysaccharide (LPS) mildly and LPS plus the NLRP3 inflammasome activator nigericin markedly elevate reactive oxygen species (ROS) and GSDMD palmitoylation, suggesting that these two processes are coupled. Manipulation of cellular ROS by its activators and quenchers augment and abolish, respectively, GSDMD palmitoylation, GSDMD pore formation and cell death. We discover that zDHHC5 and zDHHC9 are the major palmitoyl transferases that mediate GSDMD palmitoylation, and when cleaved, recombinant and partly palmitoylated GSDMD is 10-fold more active in pore formation than bacterially expressed, unpalmitoylated GSDMD, evidenced by liposome leakage assay. Finally, other GSDM family members are also palmitoylated, suggesting that ROS stress and palmitoylation may be a general switch for the activation of this pore-forming family. One-Sentence SummaryGSDMD palmitoylation is induced by ROS and required for pore formation

immunology↗

bioRxiv2022

Chronic inflammation suppresses del(5q)-like MDS HSCs via p53

Inflammation is associated with the pathogenesis of Myelodysplastic syndromes (MDS). Emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPCs) exhibit an altered response to systemic low-grade inflammation, which contributes to their competitive advantage over wild-type HSPCs and ensuing hematopoietic defects. Deletion of the long arm of chromosome 5 (del(5q)) is the most common chromosomal abnormality in patients with MDS. Although this subtype of MDS contains several haploinsufficient genes that directly impact innate immune signaling, the effects of an inflammatory milieu on del(5q) MDS HSPCs remains poorly defined. Utilizing a model of del(5q)-like MDS, wherein two 5q genes, miR-146a and TIFAB, are deleted, we found that chronic low-grade inflammation impaired the function of del(5q)-like MDS HSPCs and contributed to a more severe disease. The del(5q)-like MDS HSPCs exposed to chronic inflammation became less quiescent, but without changes in cell viability. In response to inflammation, mouse and human del(5q) MDS HSPCs activated a partial p53 response. The impaired function and reduced cellular quiescence of del(5q) MDS HSPCs exposed to inflammation could be restored by deletion of p53. Since TP53 mutations are highly enriched in del(5q) AML patients following an initial MDS diagnosis, increased p53 activation in del(5q) MDS HSPCs due to inflammation may create a selective pressure for genetic inactivation of p53. These findings uncover the contribution of systemic inflammation on dyshematopoiesis in del(5q) MDS and provide a potential explanation for acquired p53 mutations in myeloid malignancies with del(5q).

cancer biology↗

bioRxiv2022

Gene Co-expression Connectivity Predicts Gene Targets Underlying High Ionic Liquid Tolerance in Yarrowia lipolytica

Microbial tolerance to organic solvents such as ionic liquids (ILs) is a robust phenotype beneficial for novel biotransformation. While most microbes become inhibited in 1%-5% (v/v) IL (e.g., 1-ethyl-3-methylimidazolium acetate), we engineered a robust Yarrowia lipolytica (YlCW001) that tolerates a record high of 18% (v/v) IL via adaptive laboratory evolution. Yet, genotypes conferring high IL tolerance in YlCW001 remain to be discovered. Using dynamic RNA-Seq data, we shed light on the underlying cellular processes that Y. lipolytica responds to IL. We introduced Gene Co-expression Connectivity (GeCCo) to discover genotypes conferring desirable phenotypes that might not be found by the conventional differential expression (DE) approaches. GeCCo selects genes based on their number of co-expressed genes in a sub-network of upregulated genes by the target phenotype. We experimentally validated GeCCo by reversely engineering high IL-tolerant phenotype in wildtype Y. lipolytica. We found that gene targets selected by both DE and GeCCo exhibited the best statistical chance at increasing IL tolerance when individually overexpressed. Remarkably, the best combination of dual-overexpressed genes were genes selected by GeCCo alone. This non-intuitive combination of genes, BRN1 and OYE2, are involved in guiding/regulating mitotic cell division, chromatin segregation/condensation, microtubule and cytoskeletal organization, and Golgi vesicle transport.

systems biology↗

bioRxiv2022

Per-Pathogen Virulence of HIV-1 subtypes A, C and D

HIV-1 subtypes differ, among other things, in their clinical manifestations and the speed in which they spread. In particular, the frequency of subtype C is increasing relative to subtype A and D. We aim to investigate whether HIV-1 subtype A, C and D differ in their per-pathogen virulence and to what extend this can explain the difference in spread between these subtypes. We use data from the Hormonal Contraception and HIV-1 Genital Shedding and Disease Progression among Women with Primary HIV Infection (GS) Study. For each study participant, we determine the set-point viral load value, CD4+ T cell level after primary infection and CD4+ T cell decline. Based on both the CD4+ T cell count after primary infection and CD4+ T cell decline, we estimate the time until AIDS for each individual. We then obtain our newly introduced measure of virulence as the inverse of the estimated time until AIDS. This new measure of virulence has an improved correlation with the set-point viral load compared to the decline of CD4+ T cells alone. After fitting a model to the measured virulence and set-point viral load values, we tested if this relation varies per subtype. We found that subtype C has a significantly higher per-pathogen virulence than subtype A. Based on an evolutionary model, we then hypothesize that differences in the primary length of infection period cause the observed variation in the speed of spread of the subtypes. Author summaryHIV-1 subtype C is currently spreading relatively fast in various parts of the world. Data from a study that followed many women infected with different HIV-1 subtypes (A, C and D) before they started treatment shows that neither their viral load nor the disease duration are increased for subtype C compared to subtype A and D. Thus, it seems that subtype C does not have a transmission advantage, neither per contact nor due to longer infection, making the observed relative rise in subtype C a puzzle. We used the same data to test if subtype C has optimized its potential to spread by decreasing the disease duration per unit of viral load (per-pathogen virulence) compared to subtype A and D. However, we find that subtype C has a significantly higher per-pathogen virulence than subtype A and D. This result makes the rise of subtype C even more counter-intuitive. In a last step, we develop an evolutionary model, in which we synthesize all our empirical results. With this model we can show that the most likely explanation for the global spread of subtype C is a difference in the duration of primary infection between the subtypes.

evolutionary biology↗

bioRxiv2021

Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveal Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation

Yarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504, which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing non-preferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica. To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, {beta}-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation and degradation in biomass hydrolysates. IMPORTANCEYarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates. Our investigation into the genetic diversity of undomesticated Y. lipolytica strains, coupled with detailed strain characterization and proteomic analysis, revealed metabolic processes and regulatory elements conferring desirable phenotypes for growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates by these natural variants. This study provides a better understanding of the robust metabolism of Y. lipolytica and suggests potential metabolic engineering strategies to enhance its performance.

systems biology↗

bioRxiv2021

Autism spectrum disorder: understanding the impact of SNPs on biological pathways in the fetal and adult cortex.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by significant and complex genetic etiology. GWAS studies have identified genetic variants associated with ASD, but the functional impacts of these variants remain unknown. Here, we integrated four distinct levels of biological information (GWAS, eQTL, spatial genome organization and protein-protein interactions) to identify potential regulatory impacts of ASD-associated SNPs (p < 5x10-8) on biological pathways within fetal and adult cortical tissues. We found 80 and 58 SNPs that mark regulatory regions (i.e. expression quantitative trait loci or eQTLs) in the fetal and adult cortex, respectively. These eQTLs were also linked to other psychiatric disorders (e.g. schizophrenia, ADHD, bipolar disorder). Functional annotation of ASD-associated eQTLs revealed that they are involved in diverse regulatory processes. In particular, we found significant enrichment of eQTLs within regions repressed by Polycomb proteins in the fetal cortex compared to the adult cortex. Furthermore, we constructed fetal and adult cortex-specific protein-protein interaction networks and identified that ASD-associated regulatory SNPs impact on immune pathways, fatty acid metabolism, ribosome biogenesis, aminoacyl-tRNA biosynthesis and spliceosome in the fetal cortex. By contrast in the adult cortex, they largely affect immune pathways. Overall, our findings highlight potential regulatory mechanisms and pathways important for the etiology of ASD in early brain development and adulthood. This approach, in combination with clinical studies on ASD, will contribute to individualized mechanistic understanding of ASD development.

genomics↗

bioRxiv2020

Evaluation of a polymeric composite bone filler scaffold for local antibiotic delivery to prevent Staphylococcus aureus infection in a contaminated bone defect

We previously reported the development of an osteogenic bone filler scaffold consisting of degradable polyurethane (dPU), nano-sized hydroxyapatite (nHA), and decellularized bovine bone particles (DBP). In this report we describe the results of studies aimed at evaluating the use of this scaffold as a means of local antibiotic delivery for the prevention of infection in a segmental bone defect contaminated with Staphylococcus aureus. We evaluated two different scaffold formulations that contained the same components in the same ratios but differed from each other with respect to overall porosity and therefore surface area. Studies done with vancomycin, daptomycin, and gentamicin confirmed that antibiotic uptake was concentration dependent and that increased porosity was correlated with increased uptake and prolonged release of all three antibiotics. Vancomycin could be passively loaded into either scaffold formulation in an amount sufficient to prevent infection, as evidenced by the complete eradication of viable bacteria from the surgical site of most animals in a rabbit model of a contaminated mid-radial segmental bone defect. Even in those few cases in which complete eradication was not achieved, the number of viable bacteria present in the bone was significantly reduced comparison to untreated controls. There was also no radiographic evidence of osteomyelitis in any rabbit treated with vancomycin-loaded scaffold. Microcomputed tomography (CT) of bone defects up to 84 days of exposure to scaffolds with and without vancomycin also demonstrated that the addition of vancomycin even in the highest concentration did not significantly diminish the osteogenic properties of either scaffold formulation. Together, these results demonstrate the potential utility of our bone regeneration scaffold for local antibiotic delivery.

microbiology↗

bioRxiv2020

Molecular determinants for α-tubulin methylation by SETD2

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. A recently identified tubulin modification, methylation, occurs on mitotic spindle microtubules during cell division, and is enzymatically added to tubulin by the histone methyltransferase SETD2. We used a truncated version of human SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2 Rpb1 interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 has a higher activity towards tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrate that methylation is restricted to lysine 40 (K40) of -tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain, R1625C, bound to tubulin but could not methylate it whereas a mutation in the SRI domain, R2510H, caused loss of both tubulin binding and methylation. We thus further probed a role for the SRI domain in substrate binding and found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus RNA Polymerase II substrates, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Lastly, we found that substrate recognition also requires the negatively-charged C-terminal tail of -tubulin. Together, this work provides a framework for understanding how SETD2 serves as a dual methyltransferase for histone and tubulin methylation.

biochemistry↗

bioRxiv2020

Persistent broken chromosome inheritance drives genome instability

Persistent DNA damage arising from unrepaired broken chromosomes or telomere loss can promote DNA damage checkpoint adaptation, and cell cycle progression, thereby increasing cell survival but also genome instability. However, the nature and extent of such instability is unclear. We show, using Schizosaccharomyces pombe, that inherited broken chromosomes, arising from failed homologous recombination repair, are subject to cycles of segregation, DNA replication and extensive end-processing, termed here SERPent cycles, by daughter cells, over multiple generations. Following Chk1 loss these post-adaptive cycles continue until extensive processing through inverted repeats promotes annealing, fold-back inversion and a spectrum of chromosomal rearrangements, typically isochromosomes, or chromosome loss, in the resultant population. These findings explain how persistent DNA damage drives widespread genome instability, with implications for punctuated evolution, genetic disease and tumorigenesis. One Sentence SummaryReplication and processing of inherited broken chromosomes drives chromosomal instability.

molecular biology↗

bioRxiv2020

A phylodynamic workflow to rapidly gain insights into the dispersal history and dynamics of SARS-CoV-2 lineages

Since the start of the COVID-19 pandemic, an unprecedented number of genomic sequences of the causative virus (SARS-CoV-2) have been generated and shared with the scientific community. The unparalleled volume of available genetic data presents a unique opportunity to gain real-time insights into the virus transmission during the pandemic, but also a daunting computational hurdle if analysed with gold-standard phylogeographic approaches. We here describe and apply an analytical pipeline that is a compromise between fast and rigorous analytical steps. As a proof of concept, we focus on the Belgium epidemic, with one of the highest spatial density of available SARS-CoV-2 genomes. At the global scale, our analyses confirm the importance of external introduction events in establishing multiple transmission chains in the country. At the country scale, our spatially-explicit phylogeographic analyses highlight that the national lockdown had a relatively low impact on both the lineage dispersal velocity and the long-distance dispersal events within Belgium. Our pipeline has the potential to be quickly applied to other countries or regions, with key benefits in complementing epidemiological analyses in assessing the impact of intervention measures or their progressive easement.

evolutionary biology↗

bioRxiv2019

Transgene-mediated skeletal phenotypic variation in zebrafish

When considering relationships between genotype and phenotype we frequently ignore the fact that the genome of a typical animal, notably including that of a fish and a human, harbors a huge amount of foreign DNA. Some of it, including the DNA of \"autonomous\" transposable elements, can spontaneously mobilize to occupy new chromosomal sites and take on new functions, presenting a challenge to the host organism and also possibly introducing new fuel for evolutionary change. Transposable elements are useful for introducing transgenes, integrating them into host genomes with high efficiency. Transgenesis has become very widespread in biological research, and in our society at large. This year the governments of both Canada and the United States have approved the first use of genetically engineered animals in food production, Atlantic salmon, Salmo salar. With the recent advent of amazing gene-editing technology, there is no doubt that the transgene industry will grow explosively in the coming years. The biology of transgenes needs to be included in our understanding of the genome. It is in this spirit that we have investigated an unexpected and novel phenotypic effect of the chromosomally integrated transgene fli1a-F-hsp70l:Gal4VP16. We examine larval fras1 mutant zebrafish (Danio rerio). Gal4VP16 is a potent transcriptional activator, and already well known for toxicity and mediating unusual transcriptional effects. In the presence of the transgene, phenotypes in the neural crest-derived craniofacial skeleton, notably fusions and shape changes associated with loss of function fras1 mutations, are made more severe, as we quantify by scoring phenotypic penetrance, the fraction of mutants expressing the trait. A very interesting feature is that the enhancements are highly specific for fras1 mutant phenotypes - occurring in the apparent absence of more wide-spread changes. Except for the features due to the fras1 mutation, the transgene-bearing larvae appear generally healthy and to be developing normally. The transgene behaves as a genetic partial dominant: A single copy is sufficient for the enhancements, yet, for some traits, two copies may exert a stronger effect. We made new strains bearing independent insertions of the fli1a-F-hsp70l:Gal4VP16 transgene in new locations in the genome, and observed increased severities of the same phenotypes as observed for the original insertion. This finding suggests that sequences within the transgene, e.g. Gal4VP16, are responsible for the enhancements, rather than effect on neighboring host sequences (such as an insertional mutation). The specificity, and biological action underlying the traits, are subjects of considerable interest for further investigation, as we discuss. Our findings show that work with transgenes needs to be undertaken with caution and attention to detail.

developmental biology↗

bioRxiv2019

Understanding and eliminating the detrimental effect of endogenous thiamine auxotrophy on metabolism of the oleaginous yeast Yarrowia lipolytica

Thiamine is an essential vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism for all living cells. While most plants, fungi and bacteria can synthesize thiamine de novo, the oleaginous yeast, Yarrowia lipolytica, cannot. In this study, we used proteomics together with physiological characterization to understand key metabolic processes influenced and regulated by thiamine availability and identified the genetic basis of thiamine auxotrophy in Y. lipolytica. Specifically, we found thiamine depletion results in decreased protein abundance of the lipid biosynthesis pathways and energy metabolism (i.e., ATP synthase), attributing to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing gene scTHI13, encoding the 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase for the de novo thiamine synthesis in Y. lipolytica, and discovered an exceptional promoter, P3, that exhibits strong activation or tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene, we engineered the first thiamine-prototrophic Y. lipolytica reported to date. By comparing this engineered strain to the wildtype, we unveiled the tight relationship linking thiamine availability to lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica.\n\nIMPORTANCEThiamine plays a crucial role as an essential cofactor for enzymes in carbon and energy metabolism of all living cells. Thiamine deficiency has detrimental consequences on cellular health. Yarrowia lipolytica, a non-conventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph, whose effects on cellular metabolism are not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host to study thiamine metabolism, especially as mammalian cells are also thiamine-auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamentals of thiamine deficiency on cellular metabolism of Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica. Furthermore, discovery of thiamine-regulated elements enables development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.

microbiology↗

bioRxiv2019

The 14q32.31 DLK1-DIO3 MIR300 tumor suppressor promotes leukemogenesis by inducing cancer stem cell quiescence and inhibiting NK cell anti-cancer immunity.

Drug-resistance of tumor-initiating cells, impaired NK cell immune-response, PP2A loss-of-function and aberrant miRNA expression are cancer features resulting from microenvironmental- and tumor-specific signals. Here we report that genomic-imprinted MIR300 is a cell context-independent dual function tumor suppressor which is upregulated in quiescent leukemic stem (LSC) and NK cells by microenvironmental signals to induce quiescence and impair immune-response, respectively, but inhibited in CML and AML proliferating blasts to prevent PP2A-induced apoptosis. MIR300 anti-proliferative and PP2A-activating functions are differentially activated through dose-dependent CCND2/CDK6 and SET inhibition, respectively. LSCs escape PP2A-mediated apoptosis through TUG1 lncRNA that uncouples and limits MIR300 functions to cytostasis by regulating unbound-MIR300 levels. Halting MIR300 homeostasis restores NK cell activity and suppresses leukemic but not normal hematopoiesis by eradicating nearly all LSCs. Thus, MIR300 tumor suppressor activity is essential and therapeutically important for LSC-driven leukemias.

cancer biology↗