Biology subjects

Park, D.

Publications and source records attributed to Park, D..

89 records · Page 5Linked to original sources

bioRxiv2020

Absence of Sac2/INPP5F enhances the phenotype of a Parkinson's disease mutation of synaptojanin 1

Many genes whose mutations cause, or increase the risk of, Parkinsons disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol phosphatase domain of synaptojanin 1 (SJ1/PARK20), a phosphoinositide phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain containing protein, was identified as a PD risk locus by GWAS. Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurological defects. We report a "synthetic" effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 weeks, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1RQKI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1RQKI neurons was more severe in double mutant. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.

neuroscience↗

bioRxiv2019

A FIJI Macro for quantifying pattern in extracellular matrix

Diverse extracellular matrix patterns are observed in both normal and pathological tissue. However, most current tools for quantitative analysis focus on a single aspect of matrix patterning. Thus, an automated pipeline that simultaneously quantifies a broad range of metrics and enables a comprehensive description of varied matrix patterns is needed. To this end we have developed an ImageJ plugin called TWOMBLI, which stands for The Workflow Of Matrix BioLogy Informatics. TWOMBLI is designed to be quick, versatile and easy-to-use particularly for non-computational scientists. TWOMBLI can be downloaded from https://github.com/wershofe/TWOMBLI together with detailed documentation. Here we present an overview of the pipeline together with examples from a wide range of contexts where matrix patterns are generated.

cancer biology↗

bioRxiv2019

Development of group-specific nosZ quantification method targeting active nitrous oxide reducing population in complex environmental samples

Substantial N2O emission results from activated sludge nitrogen removal processes. The importance of N2O-reducers possessing NosZ-type N2O reductases have been recognized as the only N2O sink in situ key to determination of the net N2O emissions; however, reliable quantification methods for nosZ genes and transcripts have yet to be developed. Here, nosZ genes and transcripts in activated sludge tank microbiomes were analyzed with the group-specific qPCR assays designed de novo combining culture-based and computational approach. A sewage sample was enriched in a batch reactor fed continuous stream of N2 containing 20-10,000 ppmv N2O, where 14 genera of potential N2O-reducers were identified. All available amino acid sequences of NosZ affiliated to these taxa were grouped into five subgroups (two clade I and three clade II groups), and primer/probe sets exclusively and comprehensively targeting the subgroups were designed and validated with in silico PCR. Four distinct activated sludge samples from three different wastewater treatment plants in Korea were analyzed with the qPCR assays and the results were validated by comparison with the shotgun metagenome analysis results. With the validated qPCR assays, the nosZ genes and transcripts of six additional activated sludge samples were analyzed and the results of the analyses clearly indicated the dominance of two clade II nosZ subgroups (Flavobacterium-like and Dechloromonas-like) among both nosZ gene and transcript pools.

microbiology↗

bioRxiv2019

IQSEC3 Mediates Npas4-dependent GABAergic Synaptic Transmission and Depressive-like Behavior

Organization of mammalian inhibitory synapses is thought to be crucial for normal brain functions, but the underlying molecular mechanisms have been still incompletely understood. IQSEC3 (IQ motif and Sec7 domain 3) is a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF-GEF) that directly interacts with gephyrin. Here, we show that GABAergic synapse-specific transcription factor, Npas4 (neuronal PAS domain protein 4) directly binds to the promoter of Iqsec3 and regulates its transcription. Strikingly, an enriched environment (EE) induced Npas4 upregulation and concurrently increased IQSEC3 protein levels specifically in mouse CA1 stratum oriens layer somatostatin (SST)-expressing GABAergic interneurons, which are compromised in Npas4-knockout (KO) mice. Moreover, expression of wild-type (WT) IQSEC3, but not a dominant-negative (DN) ARF-GEF-inactive mutant, rescued the decreased GABAergic synaptic transmission in Npas4-deficient SST interneurons. Concurrently, expression of IQSEC3 WT normalized the altered GABAergic synaptic transmission in dendrites, but not soma, of Npas4-deficient CA1 pyramidal neurons. Furthermore, expression of IQSEC3 WT, but not IQSEC3 DN, in SST-expressing interneurons in CA1 SST Npas4-KO mice rescued the altered anxiety-like behavior. Collectively, our results suggest that IQSEC3 is a key GABAergic synapse component that is directed by Npas4 activity- and ARF activity-dependent gene programs in SST-expressing interneurons to orchestrate the functional excitation-to-inhibition balance.

neuroscience↗

bioRxiv2019

Cohesin promotes interactions across domain boundaries to regulate gene expression

The mammalian genome can be segmented into thousands of topologically associated domains (TADs) based on chromosome conformation capture studies, such as Hi-C. TADs have been proposed to act as insulated neighborhoods, spatially sequestering and insulating the enclosed genes and regulatory elements through chromatin looping and self-association. Recent results indicate that inter-TAD interactions can also occur, suggesting boundaries may be semi-permissible. However, the nature, extent, and function, if any, of these inter-TAD interactions remains unclear. Here, we combine super-and high-resolution microscopy with Oligopaint technology to precisely quantify the interaction frequency within and between neighboring domains in human cells. We find that intermingling across domain boundaries is a widespread feature of the human genome, with varying levels of interactions across different loci that correlate with their differing boundary strengths by Hi-C. Moreover, we find that cohesin depletion, which is known to abolish TADs at the population-average level, does not induce ectopic interactions but instead reduces both intra- and inter-domain interactions to a similar extent. Reduced chromatin intermixing due to cohesin loss affects domain incorporation and transcriptional bursting frequencies of genes close to architectural boundaries, potentially explaining the gene expression changes observed in the cohesinopathy Cornelia de Lange syndrome. Together, our results provide a mechanistic explanation for stochastic domain intermingling, arguing that cohesin partially bypasses boundaries to promote alternating incorporation of boundary-proximal genes into neighboring regulatory domains.

cell biology↗

bioRxiv2019

Population imaging of neural activity in awake behaving mice in multiple brain regions

A longstanding goal in neuroscience has been to image membrane voltage, with high temporal precision and sensitivity, in awake behaving mammals. Here, we report a genetically encoded voltage indicator, SomArchon, which exhibits millisecond response times and compatibility with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, and number of neurons observable, by manyfold over previous reagents. SomArchon only requires conventional one-photon microscopy to achieve these high performance characteristics. These improvements enable population analysis of neural activity, both at the subthreshold and spiking levels, in multiple brain regions - cortex, hippocampus, and striatum - of awake behaving mice. Using SomArchon, we detect both positive and negative responses of striatal neurons during movement, highlighting the power of voltage imaging to reveal bidirectional modulation. We also examine how the intracellular subthreshold theta oscillations of hippocampal neurons govern spike output, finding that nearby cells can exhibit highly correlated subthreshold activities, even as they generate highly divergent spiking patterns.

neuroscience↗

bioRxiv2019

The Effects of Mutations are Modified by Genetic Background in Mice

BackgroundThe majority of quantitative genetic models used to map complex traits assume that alleles have similar effects across all individuals. Significant evidence suggests, however, that epistatic interactions modulate the impact of many alleles. Nevertheless, identifying epistatic interactions remains computationally and statistically challenging. In this work, we address some of these challenges by developing a statistical test for polygenic epistasis that determines whether the effect of an allele is altered by the global genetic ancestry proportion from distinct progenitors. ResultsWe applied our method to data from mice and yeast. For the mice, we observed 49 significant genotype-by-ancestry interaction associations across 14 phenotypes as well as over 1,400 Bonferroni-corrected genotype-by-ancestry interaction associations for mouse gene expression data. For the yeast, we observed 92 significant genotype-by-ancestry interactions across 38 phenotypes. Given this evidence of epistasis, we test for and observe evidence of rapid selection pressure on ancestry specific polymorphisms within one of the cohorts, consistent with epistatic selection. ConclusionsUnlike our prior work in human populations, we observe widespread evidence of ancestry-modified SNP effects, perhaps reflecting the greater divergence present in crosses using mice and yeast. Author SummaryMany statistical tests which link genetic markers in the genome to differences in traits rely on the assumption that the same polymorphism will have identical effects in different individuals. However, there is substantial evidence indicating that this is not the case. Epistasis is the phenomenon in which multiple polymorphisms interact with one another to amplify or negate each others effects on a trait. We hypothesized that individual SNP effects could be changed in a polygenic manner, such that the proportion of as genetic ancestry, rather than specific markers, might be used to capture epistatic interactions. Motivated by this possibility, we develop a new statistical test that allowed us to examine the genome to identify polymorphisms which have different effects depending on the ancestral makeup of each individual. We use our test in two different populations of inbred mice and a yeast panel and demonstrate that these sorts of variable effect polymorphisms exist in 14 different physical traits in mice and 38 phenotypes in yeast as well as in murine gene expression. We use the term "polygenic epistasis" to distinguish these interactions from the more conventional two- or multi-locus interactions.

genomics↗

bioRxiv2019

JuLI: accurate detection of DNA fusions in clinical sequencing for precision oncology

Accurate detection of genomic fusions by high-throughput sequencing in clinical samples with inadequate tumor purity and formalin-fixed paraffin embedded (FFPE) tissue is an essential task in precise oncology. We developed the fusion detection algorithm Junction Location Identifier (JuLI) for optimization of high-depth clinical sequencing. We implemented novel filtering steps to minimize false positives and a joint calling function to increase sensitivity in clinical setting. We comprehensively validated the algorithm using high-depth sequencing data from cancer cell lines and clinical samples and whole genome sequencing data from NA12878. We showed that JuLI outperformed state-of-the-art fusion callers in cases with high-depth clinical sequencing and rescued a driver fusion from false negative in plasma cell-free DNA. JuLI is freely available via GitHub (https://github.com/sgilab/JuLI).

bioinformatics↗

bioRxiv2019

Efficient estimation and applications of cross-validated genetic predictions

Large-scale cohorts with combined genetic and phenotypic data, coupled with methodological advances, have produced increasingly accurate genetic predictors of complex human phenotypes called polygenic risk scores (PRS). In addition to the potential translational impacts of identifying at-risk individuals, PRS are being utilized for a growing list of scientific applications including causal inference, identifying pleiotropy and genetic correlation, and powerful gene-based and mixed model association tests. Existing PRS approaches rely on external large-scale genetic cohorts that have also measured the phenotype of interest. They further require matching on ancestry and genotyping platform or imputation quality. In this work we present a novel reference-free method to produce PRS that does not rely on an external cohort. We show that naive implementations of reference-free PRS either result in substantial over-fitting or prohibitive increases in computational time. We show that our algorithm avoids both of these issues, and can produce informative in-sample PRS over any existing cohort without over-fitting. We then demonstrate several novel applications of reference-free PRS including detection of pleiotropy across 246 metabolic traits and efficient mixed-model association testing.

genetics↗

bioRxiv2018

Genetic predisposition to hypouricemia on whole-exome sequencing analysis and its utilities in primary screening purposes

Differentiating between inherited renal hypouricemia and transient hypouricemia is challenging. Here, we aimed to describe the genetic predisposition of hypouricemia patients using whole-exome sequencing (WES) and assess the feasibility for genetic diagnosis in primary screening. WES was performed for the discovery of diagnostic markers in discovery cohorts (N=31). Two known genetic markers SLC22A12 c.774G>A (p.Trp258*) and SLC22A12 c.269G>A (p.Arg90His) were identified, We genotyped for the 2 SLC22A12 SNPs among screened 50 hypouricemia subjects for the replication cohorts; 47 carried known SLC22A12 markers; three unexplained hypouricemic cases were analyzed by using WES. We used 46 healthy internal controls for the variant discovery. Four novel variants of SLC22A12, c.408C>A (p.Asn136Lys), c.674C>A (p.Thr225Lys), c.851G>A (p.Arg284Gln), and c.1285G>A (p.Glu429Lys), and one novel variant of SLC2A9, c. 376A>G (p.Met155Val), were identified. After filtering out known genes (SLC22A12 and SLC2A9), the p.Arg78His variant in ASB12 was overlapped in two unexplained conditions. This is the first attempt to investigate the effectiveness of integrating exome sequencing and genotype into the clinical care for hypouricemia and determine the value of genetic diagnostic screening for hypouricemia in the clinical setting. Screening of just two SNPs (p.Trp258* and p.Arg90His) identified 87.7% (71/81) of patients with hypouricemia. Early identification and intervention of hypouricemia is feasible using genetic screening to prevent acute kidney injury, especially for soldiers and athletics.

genetics↗

bioRxiv2018

Evolutionary exploitation of PD-L1 expression in hormone receptor positive breast cancer

Based on clinical data from hormone positive breast cancer patients, we determined that there is a potential tradeoff between reducing tumor burden and altering metastatic potential when administering combination therapy of aromatase inhibitors and immune checkpoint inhibitors. While hormone-deprivation therapies serve to reduce tumor size in the neoadjuvant setting pre-surgery, they may induce tumors to change expression patterns towards a metastatic phenotype. We used mathematical modeling to explore how the timing of the therapies affects tumor burden and metastatic potential with an eye toward developing a dynamic prognostic score and reducing both tumor size and risk of metastasis.

cancer biology↗

bioRxiv2018

Visualization of the type III secretion mediated Salmonella-host cell interface using cryo-electron tomography

Many important gram-negative bacterial pathogens use highly sophisticated type III secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and host cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella-host cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the host cell membrane, and the establishment of an intimate association between the bacteria and the target cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.

microbiology↗

bioRxiv2018

Climate Change Influences the Japanese Cedar (Cryptomeria japonica) Pollen Count and Sensitization Rate in South Korea

BackgroundJapanese cedar pollen (JCP) is the major outdoor allergen for spring pollinosis and seasonal allergic rhinitis (SAR) caused by JCP is the most common disease in Jeju Island, South Korea and in Japan. Prior to our research, JCP counts were strongly temperature dependent and were significantly associated with the JCP sensitization rate and JC pollinosis. This event may still be ongoing due to the effects of global climate change, such as increasing temperature.\n\nMethods and FindingFor these reasons, we are studying the correlation among increasing temperatures, the JCP counts in the atmosphere and the JCP sensitization rate.\n\nConclusionsIn this study, our data show that increasing temperatures in January and April might lead to earlier and longer JCP seasons and that earlier and longer JCP seasons lead to an increase in the JCP sensitization rate, which influences the prevalence of JC pollinosis.

epidemiology↗

bioRxiv2017

Comparative Analysis of Gene Expression Identifies Distinct Molecular Signatures of Bone Marrow- and Periosteal-Skeletal Stem/Progenitor Cells

Periosteum and bone marrow (BM) both contain skeletal stem/progenitor cells (SSCs) that participate in fracture repair. However, the functional difference and selective regulatory mechanisms of SSCs in different location are unknown due to the lack of specific markers. Here, we report a comprehensive gene expression analysis of bone marrow SSCs (BM-SSCs), periosteal SSCs (P-SSCs), and more differentiated osteoprogenitors by using reporter mice expressing Interferon-inducible Mx1 and NestinGFP, previously known SSC markers. We first defined that the BM-SSCs can be enriched by the combination of Mx1 and NestinGFP expression, while endogenous P-SSCs can be isolated by positive selection of Mx1, CD105 and CD140a (known SSC markers) combined with the negative selection of CD45, CD31, and osteocalcinGFP (a mature osteolineage marker). Comparative gene experession analysis with FACS-sorted BM-SSCs, P-SSCs, Osterix+ (OSX) preosteoblasts, CD51+ stroma cells and CD45+ hematopoietic cells as controls revealed that BM-SSCs and P-SSCs have high similarity with few potential differences without statistical significance. We also found that CD51+ cells are highly heterogeneous and little overlap with SSCs. This was further supported by the microarray cluster analysis, and the two populations clustered together. However, when comparing SSC population to controls, we found several genes that were uniquely upregulated in endogenous SSCs. Amongst these genes, we found KDR (aka Flk1 or VEGFR2) to be most interesting and discovered that it is highly and selectively expressed in P-SSCs. This finding suggests that endogenous P-SSCs are functionally very similar to BM-SSCs with undetectable significant differences in gene expression but there are distinct molecular signatures in P-SSCs, which can be useful to specify P-SSC subset in vivo.

cell biology↗

bioRxiv2017

Beyond Autoantibodies: Biological Roles Of Human Autoreactive B Cells In Rheumatoid Arthritis Revealed By Whole Transcriptome Profiling

Although the contribution of B-cell derived autoreactive antibodies to rheumatoid arthritis (RA) has been studied extensively, the autoantibody-independent roles of B cells in the progression of the disease is not well-defined. Here we present the first comprehensive transcriptome profile of human autoreactive B cells in an autoimmune disease by performing RNA-sequencing of citrulline-specific B cells from RA patients. In order to facilitate a comprehensive understanding of the profile of these citrulline-specific (RA-CCPPOS) B cells, we performed comparative analyses to both citrulline-negative (RA-CCPNEG) B cells from the same donors, and identified 431 differentially expressed genes (DEGs); and hemagglutinin-specific (HA) B cells from healthy individuals and identified 1658 DEGs. Three-way comparisons of these B cell populations demonstrated that RA-CCPPOS B cells, in comparison to the RA-CCPNEG B cells, demonstrate a potential role in protein citrullination and inflammation; RA-CCPPOS B cells in comparison to HA-specific B cells demonstrate RA-specific signatures like the expression of pro-inflammatory cytokines, chemokines, costimulatory molecules and B-cell activation cascades; and all B cells from RA patients demonstrated a significant impact of the multitude of TNF signaling pathways. Furthermore, transcription factor profiling suggested that cyclic AMP (cAMP) related pathways and downstream signaling molecules are selectively enriched in RA-CCPPOS cells in comparison to the other two B cell subsets. We advanced the understanding of the citrulline reactive B cells in RA pathophysiology by documenting and validating two novel observations in independent cohorts of patients: (1) the expression of IL15R is restricted to citrulline-specific cells within RA patients and the concentration of soluble IL15R is elevated in the sera of RA patients, (2) B cells from RA patients are capable of producing epidermal growth factor ligand, amphiregulin (AREG) which in turn has a direct impact on the mechanistic effectors of RA, osteoclasts and fibroblastlike synoviocytes (FLS). Overall, our comprehensive dataset identifies several existing FDA-approved drugs that can potentially be repurposed for RA and can serve as a foundation for studying the multi-faceted roles of B cells in other autoimmune diseases.

immunology↗

bioRxiv2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 {degrees}C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

bioengineering↗

bioRxiv2017

Multiplexed confocal and super-resolution fluorescence imaging of cytoskeletal and neuronal synapse proteins

Neuronal synapses contain dozens of protein species whose expression levels and localizations are key determinants of synaptic transmission and plasticity. The spectral properties of fluorophores used in conventional microscopy limit the number of measured proteins to four species within a given sample. The ability to perform high-throughput confocal or super-resolution imaging of many proteins simultaneously without limitation in target number imposed by this spectral limit would enable large-scale characterization of synaptic protein networks in situ. Here, we introduce PRISM: Probe-based Imaging for Sequential Multiplexing, a method that sequentially utilizes either high affinity Locked Nucleic Acid (LNA) or low affinity DNA probes to enable diffraction-limited confocal and PAINT-based super-resolution imaging. High-affinity LNA probes offer high-throughput, confocal-based imaging compared with PAINT, which uses low affinity probes to realize localization-based super-resolution imaging. Simultaneous immunostaining of all targets is performed prior to imaging, followed by sequential LNA/DNA probe exchange that requires only minutes under mild wash conditions. We apply PRISM to quantify the co-expression levels and nanometer-scale organization of one dozen cytoskeletal and synaptic proteins within individual neuronal synapses. Our approach is scalable to dozens of target proteins and is compatible with high-content screening platforms commonly used to interrogate phenotypic changes associated with genetic and drug perturbations in a variety of cell types.

bioengineering↗