Biology subjects

Kim, H.

Publications and source records attributed to Kim, H..

At least 487 records · Page 27Linked to original sources

bioRxiv2020

Multiomics Characterization of Potential Therapeutic Vulnerabilities in Low-grade Serous Ovarian Carcinoma

BackgroundLow-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates. As such, there is a pressing need to develop more effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use a multiomics approach to interrogate a collection of LGSOC patient-derived cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. MethodsFourteen LGSOC cell lines were interrogated using whole exome sequencing, RNA sequencing, and mass spectrometry-based proteomics. Somatic mutation, copy-number aberrations, gene and protein expression were analyzed and integrated using different computational approaches. LGSOC cell line data was compared to publicly available LGSOC tumor data (AACR GENIE cohort), and also used for predictive biomarker identification of MEK inhibitor (MEKi) efficacy. Protein interaction databases were evaluated to identify novel therapeutic targets. ResultsKRAS mutations were exclusively found in MEKi-sensitive and NRAS mutations mostly in MEKi-resistant cell lines. Analysis of COSMIC mutational signatures revealed distinct patterns of nucleotide substitution mutations in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes (chromosome 9p21) were much more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. For in-vitro MEKi efficacy prediction, proteomic data provided better discrimination than gene expression data. Condensin, MCM, and RFC protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. ConclusionsOur LGSOC cell lines are representative models of the most common molecular aberrations found in LGSOC tumors. This study highlights the importance of using proteomic data in multiomics assessment of drug prediction and identification of potential therapeutic targets. CDKN2A/B and MTAP deficiency offer an opportunity to find synthetically lethal candidates for novel treatments. Multiomics approaches are crucial to improving our understanding of the molecular aberrations in LGSOC, establishing effective drug prediction programs and identifying novel therapeutic targets in LGSOC.

cancer biology↗

bioRxiv2020

Convergence of distinct subpopulations of mechanosensory neurons onto a neural circuit that elicits grooming

Diverse subpopulations of mechanosensory neurons detect different mechanical forces and influence behavior. How these subpopulations connect with central circuits to influence behavior remains an important area of study. We previously discovered a neural circuit that elicits grooming of the Drosophila melanogaster antennae that is activated by an antennal mechanosensory chordotonal organ, the Johnstons organ (JO) (Hampel et al., 2015). Here, we describe anatomically and physiologically distinct JO mechanosensory neuron subpopulations and define how they interface with the circuit that elicits antennal grooming. We show that the subpopulations project to distinct zones in the brain and differ in their responses to mechanical stimulation of the antennae. Each subpopulation elicits grooming through direct synaptic connections with a single interneuron in the circuit, the dendrites of which span the different mechanosensory afferent projection zones. Thus, distinct JO subpopulations converge onto the same neural circuit to elicit a common behavioral response.

neuroscience↗

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

XMAP215 and γ-tubulin additively promote microtubule nucleation in purified solutions

Microtubule nucleation is spatiotemporally regulated in cells by several molecules, including the template {gamma}-tubulin and the polymerase XMAP215. Recently, XMAP215 and the {gamma}-tubulin ring complex were reported to function synergistically, and this synergy was hypothesized to be due to direct binding between XMAP215 and {gamma}-tubulin. Here, we address this hypothesis by 1) probing domain requirements for XMAP215 to promote microtubule nucleation and 2) testing whether XMAP215 functions synergistically with {gamma}-tubulin in the absence of the other ring complex proteins. We confirm that {gamma}-tubulin and XMAP215 are classically defined nucleators that reduce the nucleation lag seen in bulk tubulin assembly. Then, using deletion constructs, we show that XMAP215s ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of TOG domains. Finally, we show that XMAP215 and {gamma}-tubulin promote {beta}-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct, and the synergy reported between XMAP215 and the {gamma}-tubulin ring complex is not due to {gamma}-tubulin alone.

biochemistry↗

bioRxiv2020

SDE2 Integrates into the TIMELESS-TIPIN Complex to Protect Stalled Replication Forks

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances TIM stability and its localization to replication forks, thereby aiding the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

cell biology↗

bioRxiv2020

Mitochondrial fission is a critical modulator of mutant APP-induced neural toxicity

Alterations in mitochondrial fission may contribute to the pathophysiology of several neurodegenerative diseases, including Alzheimers disease (AD). However, we understand very little about the normal functions of fission, or how fission disruption may interact with AD-associated proteins to modulate pathogenesis. Here we show that loss of the central mitochondrial fission protein dynamin-related 1 (Drp1) in CA1 and other forebrain neurons markedly worsens the learning and memory of mice expressing mutant human amyloid-precursor protein (hAPP) in neurons. In cultured neurons, Drp1KO and hAPP converge to produce mitochondrial Ca2+ (mitoCa2+) overload, despite decreasing mitochondria-associated ER membranes (MAMs) and cytosolic Ca2+. This mitoCa2+ overload occurs independently of ATP levels. These findings reveal a potential mechanism by which mitochondrial fission protects against hAPP-driven pathology.

neuroscience↗

bioRxiv2020

Irisin directly stimulates osteoclastogenesis and bone resorption in vitro and in vivo

The myokine irisin facilitates muscle-bone crosstalk and skeletal remodeling in part by its action on osteoblasts and osteocytes. In the current study we investigated whether irisin also directly regulates osteoclasts. In vitro, irisin (2-10 ng/mL) increased osteoclast differentiation in C57BL/6J bone marrow progenitors; this increase was blocked by a neutralizing antibody to integrin V{beta}5. Irisin also increased resorption on several substrates in situ. RNAseq revealed differential gene expression induced by irisin including upregulation of markers for osteoclast differentiation and resorption, as well as osteoblast-stimulating clastokines. In vivo, forced expression of the irisin precursor Fndc5 in murine muscle resulted in low bone mass and increased number of osteoclasts. Taken together, our work demonstrates that irisin acts directly on cultured osteoclast progenitors to increase differentiation and promote bone resorption. These actions support the tenet that irisin not only stimulates bone remodeling but may also be an important counter-regulatory hormone during exercise.

cell biology↗

bioRxiv2020

The DMCdrive: practical 3D-printable micro-drive system for reliable chronic multi-tetrode recording and optogenetic application in freely behaving rodents

Electrophysiological recording and optogenetic control of neuronal activity in behaving animals have been integral to the elucidation of how neurons and circuits modulate network activity in the encoding and causation of behavior. However, most current electrophysiological methods require substantial economical investments and prior expertise. Further, the inclusion of optogenetics with electrophysiological recordings in freely moving animals remains a general challenge. Expansion of the technological repertoire across laboratories, research institutes, and countries, demands open access to high-quality devices that can be built with little or no prior expertise from easily accessible parts of low cost. We here present a very affordable, truly easy-to-assemble micro-drive for electrophysiology in combination with optogenetics in freely moving mice and rats. The DMCdrive is particularly suited for reliable long-term recordings of neurons and network activities, and simplify optical tagging and manipulation of neurons in the recorded brain region. The highly functional and practical drive design has been optimized for accurate tetrode movement in brain tissue, and remarkably reduced build time. We provide a complete overview of the drive design, its assembly and use, and proof-of-principle demonstration of long-term recordings paired with cell-type-specific optogenetic manipulations in the prefrontal cortex (PFC) of freely moving transgenic mice and rats.

neuroscience↗

bioRxiv2020

DNA micro-disk for the management of DNA-based data storage with index and write-once-read-many (WORM) memory features

DNA-based data storage has attracted attention because of its higher physical density of the data and longer retention time than those of conventional digital data storage1-7. However, previous DNA-based data storage lacked index features and the data quality of storage after a single access is not preserved, obstructing its industrial use. Here, we propose DNA micro-disks, quick response (QR)-coded micro-sized disks that harbour data-encoded DNA molecules for the efficient management of DNA-based data storage. We demonstrate the two major features that previous DNA-based data storage studies could not achieve. One feature is accessing data items efficiently by indexing the data-encoded DNA library. Another is achieving write-once-read-many (WORM) memory through the immobilization of DNA molecules on the disk and their enrichment through in situ DNA production. Through these features, the reliability of DNA-based data storage was increased by allowing multiple accession of data-encoded DNA without data loss.

bioengineering↗

bioRxiv2020

Tuning Hsp104 specificity to selectively detoxify alpha-synuclein

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of -synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity, and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress -synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, -synuclein-specific Hsp104 variants emerge that mitigate -synuclein toxicity via distinct ATPase-dependent mechanisms, involving -synuclein disaggregation or detoxification of -synuclein conformers without disaggregation. Importantly, both types of -synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinsons disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.

bioengineering↗

bioRxiv2020

Global trends in biodiversity and ecosystem services from 1900 to 2050

Despite the scientific consensus on the extinction crisis and its anthropogenic origin, the quantification of historical trends and of future scenarios of biodiversity and ecosystem services has been limited, due to the lack of inter-model comparisons and harmonized scenarios. Here, we present a multi-model analysis to assess the impacts of land-use and climate change from 1900 to 2050. During the 20th century provisioning services increased, but biodiversity and regulating services decreased. Similar trade-offs are projected for the coming decades, but they may be attenuated in a sustainability scenario. Future biodiversity loss from land-use change is projected to keep up with historical rates or reduce slightly, whereas losses due to climate change are projected to increase greatly. Renewed efforts are needed by governments to meet the 2050 vision of the Convention on Biological Diversity. One Sentence SummaryDevelopment pathways exist that allow for a reduction of the rates of biodiversity loss from land-use change and improvement in regulating services but climate change poses an increasing challenge.

ecology↗

bioRxiv2020

MicroRNA arm switching regulated by uridylation

Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may alter depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find mir-324 as one of the strongly regulated miRNAs by arm switching, and identify terminal uridylyl transferases TUT4 and TUT7 as the key regulators. Uridylation of pre-mir-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus, from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated while the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.

molecular biology↗

bioRxiv2020

Flexible linear mixed models for structured multiple traits

Many genetic studies collect structured multivariate traits containing rich information across traits. We present a flexible multivariate linear mixed model for quantitative trait loci mapping (FlxQTL) for multiple correlated traits that adjusts for genetic relatedness and that models information on multiple environments or multiple timepoints using trait covariates. FlxQTL handles genetic mapping of multivariate traits faster with greater flexibility compared to previous implementations.

genetics↗

bioRxiv2020

Therapeutic genetic variation revealed in diverse Hsp104 homologs

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. We have engineered potentiated Hsp104 variants to antagonize proteotoxic misfolding linked to human neurodegenerative diseases. However, these Hsp104 variants can exhibit off-target toxicity, which may limit their therapeutic utility. Hsp104 is conserved among all nonmetazoan eukaryotes, which raises the possibility that natural variants might exist with enhanced, selective activity against neurodegenerative disease substrates. To assess this possibility, we screened a cross-kingdom collection of Hsp104 homologs in several yeast proteotoxicity models. We uncovered therapeutic genetic variation among several Hsp104 homologs that specifically antagonize TDP-43 or -synuclein condensate formation and toxicity in yeast, human cells, and C. elegans. Surprisingly, this variation manifested as increased passive chaperone activity, distinct from disaggregase activity, which neutralizes proteotoxicity of specific substrates. Thus, by exploring natural tuning of passive chaperone activity we elucidated enhanced, substrate-specific agents to counter proteotoxicity underlying neurodegenerative disease.

biochemistry↗

bioRxiv2020

A dual-mechanism antibiotic targets Gram-negative bacteria and avoids drug resistance

The rise of antibiotic resistance and declining discovery of new antibiotics have created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. In an animal host model, SCH-79797 reduces pathogenesis of Acinetobacter baumannii, a drug-resistant Gram-negative pathogen. To characterize the MoA of SCH-79797 we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline shows that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments with other antifolates and membrane disruptors in killing MRSA persisters. Thus, SCH-79797 represents a promising lead antibiotic and suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to target challenging bacterial pathogens.

microbiology↗

bioRxiv2020

MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.

cancer biology↗

bioRxiv2020

Dose-dependent phorbol 12-myristate-13-acetate-1 mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP-1 cells

Macrophages are sentinels of the innate immune system, and the human monocytic cell line THP-1 is one of the widely used in vitro models to study immune responses. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the widely used and accepted method. However, the concentrations and duration of PMA treatment vary widely in the published literature and their effect on protein expression is not fully deciphered. In this study, we employed a dimethyl labeling-based quantitative proteomics approach to determine the changes in the protein repertoire of macrophage-like cells differentiated from THP-1 monocytes by three commonly used PMA-based differentiation protocols. Our analysis shows that variations in PMA concentration and duration of rest post-stimulation result in downstream differences in the protein expression and cellular processes. We demonstrate that these differences result in altered gene expression of cytokines upon stimulation with various TLR agonists. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics with one of the most common in vitro models for macrophages, which in turn has a profound impact on the immune responses being studied.

immunology↗

bioRxiv2020

New functional identity of the essential inner membrane protein YejM: the cardiolipin translocator is also a metalloenzyme

Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability by translocating and increasing the cardiolipin lipid concentration. How the inner membrane protein YejM with its periplasmic domain acts as a cardiolipin translocator remains unknown. Despite overwhelming structural similarity of the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its cardiolipin translocation properties. Understanding the molecular mechanism by which YejM is involved in OM remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

biophysics↗