Biology subjects

Shin, D.

Publications and source records attributed to Shin, D..

46 records · Page 3Linked to original sources

bioRxiv2020

Collective nuclear behavior shapes bilateral nuclear symmetry for subsequent left-right asymmetric morphogenesis in Drosophila

Proper organ development often requires nuclei to move to a specific position within the cell. To determine how nuclear positioning affects left-right (LR) development in the Drosophila anterior midgut (AMG), we developed a surface-modeling method to measure and describe nuclear behavior at stages 13-14, captured in three-dimensional time-lapse movies. We describe the distinctive positioning and a novel collective nuclear behavior by which nuclei align LR-symmetrically along the anterior-posterior axis in the visceral muscles that overlie the midgut and are responsible for this organs LR-asymmetric development. Wnt4 signaling is crucial for the collective behavior and proper positioning of the nuclei, as are myosin II and LINC complex, without which the nuclei failed to align LR-symmetrically. The LR-symmetric positioning of the nuclei is important for the subsequent LR-asymmetric development of the AMG. We propose that the bilaterally symmetrical positioning of these nuclei may be mechanically coupled with subsequent LR-asymmetric morphogenesis.

cell biology↗

bioRxiv2020

Combination of kdr mutation and detoxification gene expression associated with high tolerance for permethrin in a resistant Aedes aegypti population

Aedes aegypti, as one of the vectors transmitting several arboviruses, is a main target in mosquito control programs. Permethrin remains the major adulticide used to control these mosquitoes. The increasing percentage of permethrin resistant Aedes aegypti has become an important issue around the world. Knockdown resistance (kdr) is one of the major mechanisms related to permethrin resistance. On the other hand, detoxification genes including cytochrome P450 monooxygenases (P450) and glutathione S-transferases (GSTs) are also suggested as permethrin resistance apparatus. Here we selected a permethrin resistant (p-s) Aedes aegypti population from Florida and compared its mortality after exposure, median lethal dose (LD50), adult survivorship and larval development to several field populations. We used allele-specific PCR genotyping of the S989P, V1016I and F1534C sites in the sodium channel gene and gene expression analysis of several p450 and GSTs genes before and after permethrin exposure to determine their involvement in permethrin sensitivity between Ae. aegypti populations. Results indicated the p-s population had the highest resistance to permethrin based on LD50 and the mortality test. The larval development time did not significantly differ between the populations, however the p-s adults survived longer than the other populations. In the genotype study, p-s population had mostly homozygous mutations in all three mutant sites of the sodium channel gene. Detoxification gene expression studies showed that two p450 genes, AAEL009124 (CYP6N12) and, AAEL009127 (CYP6M11), were upregulated and, accession # AAEL006802, AAEL014891 (CYP6P12) and AAEL014619 (CYP9J22) were downregulated after 120 minutes of permethrin exposure in the p-s population. These results suggest that in highly permethrin resistant Aedes aegypti populations both kdr mutations and xenobiotic metabolism might be involved. Involvement of multiple mechanisms to achieve resistance to permethrin supports the need for implementing comprehensive mosquito control measures, such as an integrated pest management strategy, so that selection pressure for resistance is decreased without compromising control efforts while new methodologies are being developed. Author summaryPyrethroids have been applied as a major type of insecticide targeted at Aedes aegypti, a key vector in the transmission of several flaviviruses. Resistance to pyrethroids has emerged and has become a worldwide threat to mosquito control. Pyrethroid resistance usually occurs with knockdown resistance (kdr) where the voltage gated sodium channel is mutated. We selected a permethrin resistant (p-s) Aedes aegypti population from Florida and, along with two other field populations, examined three mutation sites, S989P, V1016I and F1534C. The data showed the p-s population had the most homozygous mutations which correlated to the permethrin resistance level. Besides kdr, detoxification genes also have been identified to have pyrethroid metabolizing abilities. We found two cytochrome P450 monooxygenases genes, CYP6N12 and CYP6M11, were overexpressed in the p-s population after permethrin exposure, suggesting a role in resistance to permethrin. Together, our results provide information about potential mechanisms used in major vectors with high insecticide resistance.

molecular biology↗

bioRxiv2020

Sensitivity of radio-photoluminescence glass dosimeters to accumulated doses

BackgroundThis study investigated the effect of accumulated doses on radio-photoluminescence glass dosimeters (RPLGDs) from measurements involving mega-voltage photons. MethodsForty-five commercially available RPLGDs were irradiated to estimate their dose responses. Photon beams of 6, 10, and 15 MV were irradiated onto the RPLGDs inside a phantom, which were divided into five groups with different doses and energies. Groups 1 and 2 were irradiated at 1, 5, 10, 50, and 100 Gy in a sequential manner; Group 3 was irradiated 10 times with a dose of 10 Gy; and Groups 4 and 5 followed the same method as that of Group 3, but with doses of 50 Gy and 100 Gy, respectively. ResultsFor the annealed Group 1, RPLGD exhibited a linearity response with variance within 5%. For the non-annealed Group 2, readings demonstrated hyperlinearity at 6 MV and 10 MV, and linearity at 15 MV. Following the 100 Gy irradiation, the readings for Group 2 were 118.7 {+/-} 1.9%, 112.2 {+/-} 2.7%, and 101.5 {+/-} 2.3% at 6, 10, and 15 MV, respectively. For Groups 3, 4, and 5, the responsiveness of the RPLGDs gradually decreased as the number of repeated irradiations increased. The percentage readings for the 10th beam irradiation with respect to the readings for the primary beam irradiation were 84.6 {+/-} 1.9%, 87.5 {+/-} 2.4%, and 93.0 {+/-} 3.0% at 6 MV, 10 MV, and 15 MV, respectively. ConclusionsThe non-annealed RPLGD response to dose was hyperlinear for the 6 MV and 10 MV photon beams but not for the 15 MV photon beam. Additionally, the annealed RPLGD exhibited a fading phenomenon when the measurement was repeated several times and demonstrated a relatively large fading effect at low energies than at high energies.

cancer biology↗

bioRxiv2020

Simeprevir suppresses SARS-CoV-2 replication and synergizes with remdesivir

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients. One Sentence SummaryDiscovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.

microbiology↗

bioRxiv2020

Novel class of OTU deubiquitinases regulate substrate ubiquitinationupon Legionella infection

Legionella pneumophila is a gram-negative pathogenic bacterium that causes Legionaries disease. The Legionella genome codes more than 300 effector proteins able to modulate host-pathogen interactions during infection. Among them are also enzymes altering the host-ubiquitination system including bacterial ligases and deubiquitinases. In this study, based on homology-detection screening on 305 Legionella effector proteins, we identified two Legionella OTU-like deubiquitinases (LOT; LotB (Lpg1621/Ceg23) and LotC (Lpg2529), LotA (Lpg2248/Lem21) is already known). A crystal structure of LotC catalytic core (LotC14-310) was determined at 2.4 [A] and compared with other OTU deubiquitinases, including LotB. Unlike the classical OTU-family, the structures of Legionella OTU-family (LotB and LotC) shows an extended helical lobe between the Cys-loop and the variable loop, which define a novel class of OTU-deubiquitinase. Despite structural differences in their helical lobes, both LotB and LotC interact with ubiquitin. LotB has an additional ubiquitin binding site (S1) enabling specific cleavage of Lys63-linked poly-ubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of catalytically inactive LotB and LotC identified different categories of host-substrates for these two related DUBs. Together, our results provide new structural insights of bacterial OTU deubiquitinases and indicate distinct roles of bacterial deubiquitinases in host-pathogen interactions.

molecular biology↗

bioRxiv2020

Human microglia upregulate cytokine signatures and accelerate maturation of neural networks

Microglia are the resident macrophages of the brain that emerge in early development and play vital role disease states, as well as in normal development. Many fundamental questions about microglia diversity and function during human brain development remain unanswered, as we currently lack cellular-resolution datasets focusing on microglia in developing primary tissue, or experimental strategies for interrogating their function. Here, we report an integrative analysis of microglia throughout human brain development, which reveals molecular signatures of stepwise maturation, as well as human-specific cytokine-associated subtype that emerges around the onset of neurogenesis. To demonstrate the utility of this atlas, we have compared microglia across several culture models, including cultured primary microglia, pluripotent stem cell-derived microglia. We identify gene expression signatures differentially recruited and attenuated across experimental models, which will accelerate functional characterization of microglia across perturbations, species, and disease conditions. Finally, we identify a role for human microglia in development of synchronized network activity using a xenotransplantation model of human microglia into cerebral organoids.

developmental biology↗

bioRxiv2020

Brainstem development requires galactosylceramidase and is critical for the pathogenesis of Krabbe Disease

Krabbe disease (KD) is caused by a deficiency of galactosylceramidase (GALC), which induces demyelination and neurodegeneration due to accumulation of cytotoxic psychosine. Hematopoietic stem cell transplantation (HSCT) improves clinical outcomes in KD patients only if delivered pre-symptomatically. We hypothesized that the restricted temporal efficacy of HSCT reflects a requirement for GALC in early brain development. Using a novel Galc floxed allele, we induced ubiquitous GALC ablation (Galc-iKO) at various postnatal timepoints and identified a critical period of vulnerability to GALC ablation between P4-6. Early Galc-iKO induction caused a worse KD phenotype, higher psychosine levels, and a significantly shorter life-span. Intriguingly, GALC expression peaks during this critical developmental period. Further analysis revealed a novel cell autonomous role for GALC in the development and maturation of immature T-box-brain-1 positive brainstem neurons. These data identify a perinatal developmental period, in which neuronal GALC expression influences brainstem development that is critical for KD pathogenesis.

neuroscience↗

bioRxiv2018

Multiplexed single-cell RNA-seq via transient barcoding for drug screening

To simultaneously analyze multiple samples of various conditions with scRNA-seq, we developed a universal sample barcoding method through transient transfection of SBOs. A 48-plex drug treatment experiment of pooled samples analyzed by a single run of Drop-Seq revealed a unique transcriptome response for each drug and target-specific gene expression signatures at the single-cell level. Our cost-effective method is widely applicable for single-cell profiling of multiple experimental conditions.

genomics↗

bioRxiv2018

Differential Profiles of Soluble and Cellular Toll-like Receptors (TLRs) 2 and 4 in Chronic Periodontitis.

Chronic periodontitis is a common inflammatory disease initiated by a complex microbial biofilm and mediated by the host response causing destruction of the supporting tissues of the teeth. Host recognition of pathogens is mediated by toll-like receptors (TLRs) that bind conserved molecular patterns shared by groups of microorganisms. The oral epithelial cells respond to most periodontopathic bacteria via TLR-2 and TLR-4. Many studies have previously reported the presence of elevated numbers of viable exfoliated epithelial cells (SEC) in the saliva of patients with chronic periodontitis. In addition to the membrane-associated receptors, soluble forms of TLR-2 (sTLR-2) and TLR-4 (sTLR-4) have been identified and are thought to play a regulatory role by binding microbial ligands. sTLR-2 has been shown to arise from ectodomain shedding of the extracellular domain of the membrane receptor and sTLR-4 is thought to be an alternate spliced form. The objective of this study was to investigate the potential value of salivary sTLR-2/4 and the paired epithelial cell-associated TLR-2/4 mRNA as diagnostic markers for chronic periodontitis. Unstimulated whole saliva was collected after obtaining informed consent from 40 individuals in either periodontitis or gingivitis cohorts. The levels of sTLR-2/4 were measured by enzyme-linked immunosorbent assay (ELISA). SEC TLR-2/4 transcripts were quantitated by real time polymerase chain reaction. While levels of sTLR-2 exhibited an inverse correlation, sTLR-4 positively correlated with clinical parameters in the gingivitis cohort. Interestingly, both correlations were lost in the periodontitis cohort indicating a dysregulated host response. On the other hand, while sTLR-2 and the paired SEC associated TLR-2 mRNA exhibited a direct correlation (r2=0.62), that of sTLR4 and SEC TLR-4 mRNA exhibited an inverse correlation (r2=0.53) in the periodontitis cohort. Collectively, assessments of salivary sTLR2 and sTLR4 together with the respective transcripts in SECs could provide clinically relevant markers of disease progression from gingivitis to periodontitis.

immunology↗

bioRxiv2017

Dissecting the Causal Mechanism of X-Linked Dystonia-Parkinsonism by Integrating Genome and Transcriptome Assembly

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease endemic to the Philippines. We integrated genome and transcriptome assembly with induced pluripotent stem cell-based modeling to identify the XDP causal locus and potential pathogenic mechanism. Genome sequencing identified novel variation that was shared by all probands and three recombination events that narrowed the causal locus to a genomic segment including TAF1. Transcriptome assembly in neural derivative cells discovered novel TAF1 transcripts, including a truncated transcript exclusively observed in probands that involved aberrant splicing and intron retention (IR) associated with a SINE-VNTR-Alu (SVA)-type retrotransposon insertion. This IR correlated with decreased expression of the predominant TAF1 transcript and altered expression of neurodevelopmental genes; both the IR and aberrant TAF1 expression patterns were rescued by CRISPR/Cas9 excision of the SVA. These data suggest a unique genomic cause of XDP and may provide a roadmap for integrative genomic studies in other unsolved Mendelian disorders.\n\nHighlights O_LIGenome assembly narrows the XDP causal locus to a segment including TAF1\nC_LIO_LIXDP-specific SVA insertion induces intron retention and down-regulation of TAF1\nC_LIO_LICRISPR/Cas9 excision of SVA rescues aberrant splicing and cTAF1 expression in XDP\nC_LIO_LIGene networks perturbed in proband cells associate to synapse and neurodevelopment\nC_LI

genomics↗