Biology subjects

Kong, D.

Publications and source records attributed to Kong, D..

66 records · Page 4Linked to original sources

bioRxiv2019

Mitochondrial Dysfunction Induces Epigenetic Dysregulation by H3K27 Hyperacetylation to Perturb Active Enhancers in Parkinson’s Disease Models

Genetic mutations explain only 10-15% of cases of Parkinsons disease (PD), while an overriding environmental component has been implicated in the etiopathogenesis of PD. But regardless of where the underlying triggers for the onset of familial and sporadic PD fall on the gene-environment axis, mitochondrial dysfunction emerges as a common mediator of dopaminergic neuronal degeneration. Herein, we employ a multidisciplinary approach to convincingly demonstrate that neurotoxicant exposure- and genetic mutation-driven mitochondrial dysfunction share a common mechanism of epigenetic dysregulation. Under both scenarios, lysine 27 acetylation of likely variant H3.2 (H3.2K27ac) increased in dopaminergic neuronal models of PD, thereby opening that region to active enhancer activity via H3K27 hyperacetylation. These vulnerable epigenomic loci represent potential transcription factor motifs for PD pathogenesis. We further confirmed the mitochondrial dysfunction induced H3K27ac during neurodegeneration in ex vivo models of PD. Our results reveal an exciting axis of exposure/mutation-mitochondrial dysfunction-metabolism-H3K27ac-transcriptome for PD pathogenesis. Collectively, the novel mechanistic insights presented here interlinks mitochondrial dysfunction to epigenetic transcriptional regulation in dopaminergic degeneration as well as offer potential new epigenetic intervention strategies for PD.

neuroscience↗

bioRxiv2019

Girdin: an essential component of pre-replicative complex in human cells

A central event in the initiation of DNA replication in eukaryotes is the assembly of pre-replicative complex (pre-RC) on specific chromatin sites known as DNA replication origins. The pre-RC assembly process differs between budding and fission yeasts. In fission yeast, Sap1 directly participates in pre-RC assembly, together with the four initiation factors: ORC, Cdc18/Cdc6, Cdt1, and MCM. In metazoans, the nature of DNA replication origins is not defined and the mechanism of pre-RC assembly remains incompletely known. In this study, Girdin was identified as an essential replication initiation factor in human cells. Similar to the activity of Sap1, human Girdin binds to DNA origins, interacts with ORC, and is required for pre-RC assembly due to its essential role in recruitment of Cdc6 to DNA origins. Thus, DNA origins in human or metazoans are defined as including two elements, one bound by ORC and the other bound by Girdin.

biochemistry↗

bioRxiv2018

Regulation of Cilia Abundance in Multiciliated Cells

Multiciliated cells (MCC) are specialized epithelia that contain hundreds of motile cilia used to propel fluid over the surface of the cell. To template these cilia, each MCC produces hundreds of centrioles by a process termed centriole amplification. Airway progenitor cells initially contain two parental centrioles that nucleate multiple centrioles at once, and structures called deuterosomes that assemble the vast majority of centrioles during amplification. Remarkably, how each cell regulates the precise number of its centrioles and cilia remains unknown. Here, we investigate mechanisms that establish centriole number in MCC using an ex vivo airway culture model. We show that ablation of parental centrioles, via inhibition of Plk4 kinase, does not perturb deuterosome formation and centriole amplification, nor alter the total complement of centrioles per cell. Airway MCC vary in size and surface area, and exhibit a broad range in centriole number. Quantification of centriole abundance in vitro and in vivo identified a direct relationship between cell-surface area and centriole number. By manipulating cell size and shape, we discovered that centriole number scales with increasing surface area. Collectively, our results demonstrate that parental centrioles and Plk4 are dispensable for deuterosome formation, centriole amplification, and establishment of centriole number. Instead, a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells.

cell biology↗

bioRxiv2018

Glycolytic inhibitor 2-deoxyglucose prevents cortical hyperexcitability after traumatic brain injury

Traumatic brain injury (TBI) causes cortical dysfunction and can lead to post-traumatic epilepsy. Multiple studies demonstrate that GABAergic inhibitory network function is compromised following TBI, which may contribute to hyperexcitability and motor, behavioral, and cognitive deficits. Preserving the function of GABAergic interneurons, therefore, is a rational therapeutic strategy to preserve cortical function after TBI and prevent long-term clinical complications. Here, we explored an approach based on the ketogenic diet, a neuroprotective and anticonvulsant dietary therapy which results in reduced glycolysis and increased ketosis. Utilizing a pharmacologic inhibitor of glycolysis (2-deoxyglucose, or 2-DG), we found that acute in vitro glycolytic inhibition decreased the excitability of excitatory neurons, but not inhibitory interneurons, in cortical slices from naive mice. Employing the controlled cortical impact (CCI) model of TBI in mice, we found that in vitro 2-DG treatment rapidly attenuated epileptiform activity seen in acute cortical slices 3-5 weeks after TBI. One week of in vivo 2-DG treatment immediately after TBI prevented the development of epileptiform activity, restored excitatory and inhibitory synaptic activity, and attenuated loss of parvalbumin-positive inhibitory interneurons. In summary, inhibition of glycolysis with 2-DG may have therapeutic potential to restore network function following TBI.\n\nOne Sentence SummaryFollowing traumatic brain injury in mice, in vivo treatment with the glycolytic inhibitor 2-deoxyglucose prevented cortical network pathology including cortical hyperexcitability, changes in synaptic activity, and loss of parvalbumin-expressing GABAergic interneurons.

neuroscience↗

bioRxiv2018

Multi-Center Study of Resectable Lung Lesions by Ultra-Deep Sequencing of Targeted Genes in Plasma Cell-Free DNA to Assess Nodule Malignancy and Detect Lung Cancers

BACKGROUNDEarly detection of lung cancer to allow curative treatment remains challenging. Cell-free circulating tumor DNA (ctDNA) analysis may aid in malignancy assessment and early cancer diagnosis of lung nodules found in screening imagery.\n\nMETHODSThe multi-center clinical study enrolled 192 patients with operable occupying lung diseases. Plasma ctDNA, white blood cell genomic DNA (gDNA) and tumor tissue gDNA of each patient were analyzed by ultra-deep sequencing to an average of 35,000X of the coding regions of 65 lung cancer-related genes.\n\nRESULTSThe cohort consists of a quarter of benign lung diseases and three quarters of cancer patients with all histopathology subtypes. 64% of the cancer patients is at Stage I. Gene mutations detection in tissue gDNA and plasma ctDNA results in a sensitivity of 91% and specificity of 88%. When ctDNA assay was used as the test, the sensitivity was 69% and specificity 96%. As for the lung cancer patients, the assay detected 63%, 83%, 94% and 100%, for Stage I, II, III and IV, respectively. In a linear discriminant analysis, combination of ctDNA, patient age and a panel of serum biomarkers boosted the overall sensitivity to 80% at a specificity of 99%. 29 out of the 65 genes harbored mutations in the lung cancer patients with the largest number found in TP53 (30% plasma and 62% tumor tissue samples) and EGFR (20% and 40%, respectively).\n\nCONCLUSIONPlasma ctDNA was analyzed in lung nodule assessment and early cancer detection while an algorithm combining clinical information enhanced the test performance.

clinical trials↗

bioRxiv2018

Experiments and simulations on short chain fatty acid production in a colonic bacterial community

Understanding how production of specific metabolites by gut microbes is modulated by interactions with surrounding species and by environmental nutrient availability is an important open challenge in microbiome research. As part of this endeavor, this work explores interactions between F. prausnitzii, a major butyrate producer, and B. thetaiotaomicron, an acetate producer, under three different in vitro media conditions in monoculture and coculture. In silico Genome-scale dynamic flux balance analysis (dFBA) models of metabolism in the system using COMETS (Computation of Microbial Ecosystems in Time and Space) are also tested for explanatory, predictive and inferential power. Experimental findings indicate enhancement of butyrate production in coculture relative to F. prausnitzii monoculture but defy a simple model of monotonic increases in butyrate production as a function of acetate availability in the medium. Simulations recapitulate biomass production curves for monocultures and accurately predict the growth curve of coculture total biomass, using parameters learned from monocultures, suggesting that the model captures some aspects of how the two bacteria interact. However, a comparison of data and simulations for environmental acetate and butyrate changes suggest that the organisms adopt one of many possible metabolic strategies equivalent in terms of growth efficiency. Furthermore, the model seems not to capture subsequent shifts in metabolic activities observed experimentally under low-nutrient regimes. Some discrepancies can be explained by the multiplicity of possible fermentative states for F. prausnitzii. In general, these results provide valuable guidelines for design of future experiments aimed at better determining the mechanisms leading to enhanced butyrate in this ecosystem.\n\nImportanceStudies associating butyrate levels with human colonic health have inspired research on therapeutic microbiota consortia that would optimize butyrate production if implanted in the human colon. Faecalibacterium prausnitzii is commonly observed in human fecal samples and produces butyrate as a product of fermentation. Previous studies indicate that Bacteroides thetaiotaomicron, also commonly found in human fecal samples, may enhance butyrate production in F. prausnitzi when the two species are co-localized. This possibility is investigated here under different environmental conditions using experimental methods paired with computer simulations of the whole metabolism of bacterial cells. Initial findings indicate that interactions between these two species result in enhanced butyrate production. However, results also paint a nuanced picture, suggesting the existence of a multiplicity of equivalently efficient metabolic strategies and complex interactions between acetate and butyrate production in these species that appear highly dependent on specific environmental conditions.

systems biology↗

bioRxiv2018

Separation and Loss of Centrioles from Primordidal Germ Cells to Mature Oocytes in the Mouse

Oocytes, including those from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM); concomitantly, a rise in single CETN2 pairs is identified. CETN2 dissolution accelerates following meiosis resumption. Remarkably, a single CETN2 pair is retained in the PCM of most meiotic metaphase-I and -II spindle poles. Partial dissolution of the CETN2 foci occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, much later than previously thought.

developmental biology↗

bioRxiv2018

PLK4 is a microtubule-associated protein that self assembles promoting de novo MTOC formation

Summary statementPLK4 binds to microtubules and self assembles into supramolecular assemblies that recruit tubulin and trigger de novo MTOC formation in Xenopus laevis extracts.\n\nAbstractThe centrosome is an important microtubule-organizing center (MTOCs) in animal cells and it consists of two barrel-shaped structures (centrioles), surrounded by the pericentriolar material (PCM), which nucleates microtubules. PCM components form condensates, supramolecular assemblies that concentrate microtubule nucleators. Centrosomes can form close to an existing structure (canonical duplication) or de novo. How centrosomes form de novo is not known. PLK4 is a master driver of centrosome biogenesis, which is critical to recruit several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where we and others have shown that PLK4 can induce de novo MTOC formation (Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into supramolecular assemblies that recruit /{beta}-tubulin. In Xenopus extracts, PLK4 supramolecular assemblies additionally recruit the PLK4 substrate STIL and the microtubule nucleator, {gamma}-tubulin, and form acentriolar MTOCs de novo. The assembly of these robust microtubule asters is independent of dynein, similarly to centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organizing catalytic scaffold that recruits centriole components, PCM factors and /{beta}-tubulin, leading to MTOC formation.

cell biology↗

bioRxiv2018

Arsenite exposure inhibits histone acetyltransferase p300 for attenuating H3K27ac at enhancers in low-dose exposed mouse embryonic fibroblast cells

Epidemiological investigations have validated the positive relationships between arsenic in drinking water and several cancers, including skin, liver and lung cancers. Besides genotoxicity, arsenic exposure-related pathogenesis of disease is widely considered through epigenetic mechanisms; however, the underlying mechanistic insight remains elusive. Herein we explore the initial epigenetic changes via acute low-dose arsenite exposures of mouse embryonic fibroblast (MEF) cells and Dot1L knockout MEF (Dot1L-/- for abbreviation) cells. Our RNA-seq and Western blot data demonstrated that, in both cell lines, acute low-dose arsenite exposure abolished histone acetyltransferase p300 at the RNA level and subsequent protein level. Consequently, p300-specific main target histone H3K27ac, a marker separating active from poised enhancers, decreased dramatically as validated by both Western blot and ChIP-seq analyses. Concomitantly, H3K4me1 as another well-known marker for enhancers also showed significant decreases, suggesting an underappreciated crosstalk between H3K4me1 and H3K27ac involved in arsenite exposure. Significantly, arsenite exposure-reduced H3K27ac and H3K4me1 inhibit the expression of genes including EP300 itself and Kruppel Like Factor 4(Klf4), a tumor suppressor gene. Collectively, our investigations identify p300 as an internal bridging factor within cells to sense external environmental arsenite exposure to alter chromatin, thereby changing gene transcription for disease pathogenesis.

pharmacology and toxicology↗

bioRxiv2018

PACAP neurons in the ventral premammillary nucleus regulate reproductive function in the female mouse

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuromodulator implicated in anxiety, metabolism and reproductive behavior. PACAP global knockout mice have decreased fertility and PACAP modulates LH release. However, its source and role at the hypothalamic level remain unknown. We demonstrate that PACAP-expressing neurons of the ventral premamillary nucleus of the hypothalamus (PMVPACAP) project to, and make direct contact with, kisspeptin neurons in the arcuate and AVPV/PeN nuclei and a subset of these neurons respond to PACAP exposure. Targeted deletion of PACAP from the PMV through stereotaxic virally mediated cre- injection or genetic cross to LepR-i-cre mice with PACAPfl/fl mice led to delayed puberty onset and impaired reproductive function in female, but not male, mice. We propose a new, sex-specific role for PACAP-expressing neurons in the PMV in the relay of nutritional state information to regulate GnRH release by modulating the activity of kisspeptin neurons, thereby regulating reproduction.

neuroscience↗

bioRxiv2018

Optochemical control of cell contractility at single cell resolution during tissue morphogenesis

AbstractsThe spatial and temporal dynamics of cell contractility plays a key role in tissue morphogenesis, wound healing and cancer invasion. Here we report a simple, single cell resolution, optochemical method to induce reversible minute-scale cell contractions in vivo during morphogenesis. We employed the photolabile Ca2+ chelator o-nitrophenyl EGTA to induce bursts of intracellular free Ca2+ by laser photolysis. Ca2+ bursts appear within seconds and are restricted to individual target cells. Cell contraction reliably followed within a minute, to about half of the cross-sectional area. Increased Ca2+ levels and contraction were reversible and the target cells further participated in tissue morphogenesis. Cell contractions are paralleled with non-muscle myosin-II accumulation in the apico-medial cortex, indicating that Ca2+ bursts trigger non-muscle myosin II activation. Our approach can be easily adapted to many experimental systems and species, as no specific genetic elements are required and a widely used reagent is employed.

developmental biology↗

bioRxiv2017

Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells

Centrioles are composed of long-lived microtubules arranged in nine triplets. In unicellular eukaryotes, loss of the noncanonical tubulins, delta-tubulin and epsilon tubulin, result in loss of the triplet microtubule structure. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Here, we report the first characterization of delta-tubulin and epsilon-tubulin null human cells. Centrioles in cells lacking either delta-tubulin or epsilon-tubulin lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

cell biology↗