Biology subjects

Kim, S.

Publications and source records attributed to Kim, S..

At least 289 records · Page 16Linked to original sources

bioRxiv2024

An Innovative Mitochondrial-targeted Gene Therapy for Cancer Treatment

Targeting cancer cell mitochondria holds great therapeutic promise, yet current strategies to specifically and effectively destroy cancer mitochondria in vivo are limited. Here, we introduce mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics gene therapy designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. We synthesize a blue light-gated channelrhodopsin (CoChR) in the IMM and co-express a blue bioluminescence-emitting Nanoluciferase (NLuc) in the cytosol of the same cells. The mLumiOpto genes are selectively delivered to cancer cells in vivo by using adeno-associated virus (AAV) carrying a cancer-specific promoter or cancer-targeted monoclonal antibody-tagged exosome-associated AAV. Induction with NLuc luciferin elicits robust endogenous bioluminescence, which activates mitochondrial CoChR, triggering cancer cell IMM permeability disruption, mitochondrial damage, and subsequent cell death. Importantly, mLumiOpto demonstrates remarkable efficacy in reducing tumor burden and killing tumor cells in glioblastoma or triple-negative breast cancer xenografted mouse models. These findings establish mLumiOpto as a novel and promising therapeutic strategy by targeting cancer cell mitochondria in vivo.

cancer biology↗

bioRxiv2024

Drumming Performance and Underlying Muscle Activities in a Professional Rock Drummer with Lower-Limb Dystonia: A Case Study

Task-specific focal dystonia (TSFD), characterized by the loss of fine motor control and coordination, affects drummers lower-limb movements, yet it remains relatively understudied, leaving a gap in understanding its effects on drumming performance and underlying muscle activities. This study explores lower limb dystonias impact on drumming performance and underlying muscle activity in a professional rock drummer. The drummer executed an eight-beat pattern on a drum kit, emphasizing kicking the bass drum on the initial beat and performing syncopation of the third beat. Dystonia symptoms primarily manifested in the initial beat, with fewer symptoms on syncopation of the third beat. Analysis revealed decreased bass-drum sound peak amplitude during the initial beat and increased (becoming more negative) synchronization error. Electromyographic (EMG) measurements of ten muscles in the affected right lower limb showed significant changes in the Biceps Femoris (BF), Tibialis Anterior (TA), Extensor Digitorum Longus (EDL), and Extensor Digitorum Brevis (EDB) muscles during symptom onset. Notably, we observed 1) earlier overactivation of the TA and EDL muscles during the leg lift-up motion or preparatory phase of pedaling, 2) reduced activation of the EDB muscle, and 3) increased activation of the BF muscle during the final pedaling movement when symptoms occurred. These findings suggest that lower-limb dystonia symptoms are characterized by a reduction in amplitude and an increase in synchronization error, potentially due to premature overactivation of the ankle dorsiflexor muscles.

neuroscience↗

bioRxiv2024

N-glycosylated molecules act as a co-precipitant in RNA purification

A recent ground-breaking study suggested that small RNA from mammalian cells can undergo N-glycan modifications (termed glycoRNA) 1. The discovery relied upon a metabolic glycan labeling strategy in combination with commonly used phase-separation-based RNA isolation. Following the reported procedure, we likewise identified an N-glycosylated species in the RNA fraction. However, our results suggest that the reported RNase sensitivity of the glycosylated species depends on the specific RNA purification method. This suggests the possibility of co-purifying unexpected RNase-insensitive N-glycoconjugates during glycoRNA isolation. The co-existence of two independent, yet highly similar molecular entities, complicates biochemical assays on glycoRNA, and calls for more specific approaches for glycoRNA analysis. To address this, we propose a control experiment that can help distinguish genuine glycoRNA species from co-purified glycoconjugates.

molecular biology↗

bioRxiv2024

Human immunodeficiency virus-1 induces and targets host genomic R-loops for viral genome integration

Although HIV-1 integration sites favor active transcription units in the human genome, high-resolution analysis of individual HIV-1 integration sites has shown that the virus can integrate into a variety of host genomic locations, including non-genic regions. The invisible infection by HIV-1 integrating into non-genic regions, challenging the traditional understanding of HIV-1 integration site selection, is more problematic because they are selected for preservation in the host genome during prolonged antiretroviral therapies. Here, we showed that HIV-1 integrates its viral genome into the vicinity of R-loops, a genomic structure composed of DNA- RNA hybrids. VSV-G-pseudotyped HIV-1 infection initiates the formation of R-loops in both genic and non-genic regions of the host genome and preferentially integrates into R-loop-rich regions. Using a HeLa cell model that can independently control transcriptional activity and R-loop formation, we demonstrated that the exogenous formation of R-loops directs HIV-1 integration-targeting sites. We also found that HIV-1 integrase proteins physically bind to the host genomic R-loops. These findings provide novel insights into the mechanisms underlying retroviral integration and the new strategies for antiretroviral therapy against HIV-1 latent infection.

molecular biology↗

bioRxiv2024

Structure-guided aminoacylation and assembly of chimeric RNAs

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture found in modern tRNA. We demonstrate that the T-loop assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of such chimeric ribozymes would have allowed the greater functionality of amino acids to contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid specific aminoacyl-RNA synthetase enzymes prior to their role in protein synthesis.

biochemistry↗

bioRxiv2024

Monosynaptically-interconnected Network Module (MNM) Approach for High-Resolution Brain Sub-Network Analysis

We introduce the Monosynaptically-interconnected Network Module (MNM) approach, an innovative method designed for efficiently analyzing the anatomical structure and functional dynamics of specific brain network modules in vivo. Utilizing an Intein-mediated split-Cre system combined with bidirectional adeno-associated viruses, this technique precisely targets and manipulates monosynaptically interconnected modular subnetworks in freely moving animals. We demonstrate its utility through anatomical and functional mapping of a specific MNM encompassing the prefrontal cortex (PFC), basolateral amygdala (BLA), and intermediary hub regions. Specifically, the MNM approach with Cre-reporter mice visualizes detailed network architecture and enables the tracing of axonal connections among the nodes in the network. Furthermore, integration of the MNM approach with Cre-dependent Ca2+ indicator and multi-fiber photometry in freely moving mice reveals enhanced correlative network activities in social contexts. This versatile technique offers significant potential for advancing our understanding of network functions that underlie complex behaviors, providing a modular network perspective.

neuroscience↗

bioRxiv2024

A novel truncated CHAP modular endolysin, CHAPSAP26-161, that lyses Staphylococcus aureus, Acinetobacter baumannii, and Clostridioides difficile

BackgroundDevelopment of novel antimicrobial agents is imperative due to the increasing threat of antibiotic-resistant pathogens. This study aimed to validate the enhanced antibacterial activity and in vivo efficacy of a novel truncated endolysin, CHAPSAP26-161, derived from the CHAP domain of LysSAP26, against multidrug-resistant bacteria. MethodsTwo deletion mutants, CHAPSAP26-139 and CHAPSAP26-161, were constructed by deleting the C-terminal portion of LysSAP26. These were cloned and expressed, and their antibacterial activities, together with protein purification efficiency, were evaluated against 12 bacterial species under various environmental conditions. To test the temperature and pH stability of the three recombinant proteins, the antibacterial effects of the proteins at various temperatures (4{degrees}C-60{degrees}C) and pH values (3-10) were measured. Time-kill assay measured the optical density (600 nm) and colony-forming units after incubation for 0, 2, 4, 6, 8, and 24 h. We verified this through in vivo experiments using mouse models to evaluate the therapeutic potential of CHAPSAP26-161 against Acinetobacter baumannii. ResultsCHAPSAP26-161 exhibited higher protein purification efficiency and antibacterial activity than LysSAP26. Moreover, CHAPSAP26-161 showed the highest lytic activity against A. baumannii with a minimal bactericidal concentration (MBC) of 5-10 {micro}g/mL, followed by Staphylococcus aureus with an MBC of 10-25 {micro}g/mL. Interestingly, CHAPSAP26-161 could lyse anaerobic bacteria, such as C. difficile, with an MBC of 25-50 {micro}g/mL. At pH 4-8 and temperatures of 4{degrees}C-45{degrees}C, CHAPSAP26-161 exhibited optimal hydrolase activity. The lytic activity of CHAPSAP26-161 was dependent on divalent metal ions, especially Zn2+, and increased in the presence of ethylenediamine tetraacetic acid. CHAPSAP26-161 demonstrated superior protein purification efficiency and antibacterial activity than LysSAP26. It showed high lytic activity against gram-positive, gram-negative, and anaerobic bacteria, including S. aureus and Clostridioides difficile. Enhanced stability under varied temperatures and pH conditions. In vivo, tests demonstrated promising therapeutic effects of CHAPSAP26-161 in murine systemic A. baumannii infection models. ConclusionsCHAPSAP26-161, a truncated modular endolysin containing only the CHAP domain of LysSAP26, demonstrated higher protein purification efficiency and antibacterial activity than LysSAP26. It also exhibited extended-spectrum antibacterial activity against gram-positive, gram-negative, and anaerobic bacteria, such as S. aureus, A. baumannii, and C. difficile. Its successful in vivo application in murine models highlights its potential as an alternative therapeutic agent in combating antibiotic resistance.

microbiology↗

bioRxiv2024

Modulation of stress granule dynamics by phosphorylation and ubiquitination in plants

The Arabidopsis tandem CCCH zinc finger 1 (TZF1) is an RNA-binding protein that plays a crucial role in plant growth and stress response. TZF1 can localize to ribonucleoprotein (RNP) granules in response to various abiotic stresses. However, very little is known about the composition, function, and assembly mechanism of plant RNP granules. In this report, we show that TZF1 contains two intrinsically disordered regions (IDRs) necessary for its localization to stress granules (SGs), a subclass of RNP granules. TZF1 recruits mitogen-activated protein kinase (MAPK) signaling components and an E3 ubiquitin ligase KEEP-ON-GOING (KEG) to SGs. TZF1 is phosphorylated by MPKs and ubiquitinated by KEG. The phosphorylation sites of TZF1 were mapped by mass spectrometry. Mutant studies revealed that phosphorylation and ubiquitination of specific residues played differential roles in enhancing or reducing TZF1 SG assembly and protein-protein interaction with mitogen-activated kinase kinase 5 (MKK5) in SGs. TZF1 is extremely unstable, and its accumulation can be enhanced by proteosome inhibitor MG132. We showed that TZF1 was ubiquitinated in vivo and in vitro by KEG and TZF1 accumulated at a much lower level in gain-of-function mutant keg-4, compared to the WT. Ubiquitination appeared to play a positive role in TZF1 SG assembly, because either single or higher order mutations caused reduced number of SGs per cell, while enhanced the coalescence of small SGs into a large nucleus-like SG encompassing the nucleus. Together, our results demonstrate that the assembly of TZF1 SGs is distinctively regulated by ubiquitination and phosphorylation.

cell biology↗

bioRxiv2024

Leaf abaxial and adaxial surfaces differentially affect plant-fungal pathogen interactions

Eudicot plant species have bifacial leaves with each surface varying in a diversity of components, resulting in potentially different microhabitats for pathogens. We tested how Botrytis cinerea, a necrotroph fungal pathogen, interacts with the two different leaf surfaces across 16 crop species and 20 Arabidopsis genotypes. This showed that the abaxial surface is generally more susceptible to the pathogen than the adaxial surface. In Arabidopsis, the differential lesion area between leaf surfaces was associated to jasmonic acid (JA) and salicylic acid (SA) signaling and differential induction of defense chemistry. When infecting the adaxial surface, leaves mounted stronger defenses by producing more glucosinolates and camalexin defense compounds, partially explaining the differential susceptibility across surfaces. Testing a collection of 96 B. cinerea strains showed genetic heterogeneity of growth patterns, with a few strains preferring the adaxial surface while most are more virulent on the abaxial surface. Overall, we show that leaf-Botrytis interactions are complex with host-specific, surface-specific, and strain-specific behaviors. Within Arabidopsis, this mechanistically links to potential variation in JA/SA signaling across the two surfaces.

plant biology↗

bioRxiv2024

Discovery of Highly Active Kynureninases for Cancer Immunotherapy through Protein Language Model

Overcoming the immunosuppressive tumor microenvironment is a promising strategy in anticancer therapy. L-kynurenine, a strong immunosuppressive metabolite can be degraded through kynureninases. Through homology searches and protein language models, we identified and then experimentally determined the efficacy of four top-ranked kynureninases. The catalytically most active one nearly doubles turnover number over the prior best, reducing tumor weight by 3.42 times in mouse model comparisons, and thus, presenting substantial therapeutic potential.

cancer biology↗

bioRxiv2024

miRNA family miR-29 inhibits PINK1-PRKN dependent mitophagy via ATG9A

Loss-of-function mutations in the genes encoding PINK1 and PRKN result in early-onset Parkinson disease (EOPD). Together the encoded enzymes direct a neuroprotective pathway that ensures the elimination of damaged mitochondria via autophagy. We performed a genome-wide high content imaging miRNA screen for inhibitors of the PINK1-PRKN pathway and identified all three members of the miRNA family 29 (miR-29). Using RNAseq we identified target genes and found that siRNA against ATG9A phenocopied the effects of miR-29 and inhibited the initiation of PINK1-PRKN mitophagy. Furthermore, we discovered two rare, potentially deleterious, missense variants (p.R631W and p.S828L) in our EOPD cohort and tested them experimentally in cells. While expression of wild-type ATG9A was able to rescue the effects of miR-29a, the EOPD-associated variants behaved like loss-of-function mutations. Together, our study validates miR-29 and its target gene ATG9A as novel regulators of mitophagy initiation. It further serves as proof-of-concept of finding novel, potentially disease-causing EOPD-linked variants specifically in mitophagy regulating genes. The nomination of genetic variants and biological pathways is important for the stratification and treatment of patients that suffer from devastating diseases, such as EOPD.

neuroscience↗

bioRxiv2024

Rapid and label-free exosome analysis by surface-enhanced Raman spectroscopy using silver nanoparticle substrate based on selective laser ablation and melting of silver nanowire

Early diagnostics of breast cancer is crucial to reduce the risk of cancer metastasis and late relapse. Exosome, which contains distinct information of its origin, can be the target object as a liquid biopsy. However, its low sensitivity and inadequate diagnostic tools interfere with the point-of-care testing (POCT) of the exosome. Recently, Surface-enhanced Raman spectroscopy (SERS), which enables the detection of Raman scattering, has been proved as a promising tool for exosome detection but the fabrication process of SERS probe or substrate is still inefficient and far from large-scale production. This study proposes rapid and label-free detection of breast cancer-derived exosomes by statistical analysis of Raman spectra using silver nanoparticle SERS substrate fabricated by selective laser ablation and melting. Employing silver nanowire and optimizing pulse repetition rate power enable rapid and energy-efficient fabrication of SERS substrate. The functionality including sensitivity, reproducibility, stability, and renewability was evaluated using rhodamine 6G as a probe molecule. Then, the feasibility of POCT was examined by the statistical analysis of Raman spectra of exosomes from malignant breast cancer cells and non-tumorigenic breast epithelial cells. The presented framework is anticipated to be utilized in other biomedical applications, facilitating cost-effective and large-scale production performance.

bioengineering↗

bioRxiv2024

Thermal Tolerance Plasticity and Dynamics of Thermal Tolerance in Eublepharis macularius: Implications for Future Climate-Driven Heat Stress

The intensity and duration of heat waves, as well as average global temperatures, are expected to increase due to climate change. Heat waves can cause physiological stress and reduce fitness in animals. Species can reduce overheating risk through phenotypic plasticity, which allows them to raise their thermal tolerance limits over time. This mechanism could be important for ectotherms whose body temperatures are directly influenced by available environmental temperatures. Geckos are a large, diverse group of ectotherms that vary in their thermal habitats and times of daily activity, which could affect how they physiologically adjust to heat waves. Data on thermal physiology are scarce for reptiles, with only one study in geckos. Understanding thermal tolerance and plasticity, and their relationship, is essential for understanding how some species are able to adjust or adapt to changing temperatures. In this study, we estimated thermal tolerance and plasticity, and their interaction, in the crepuscular gecko, Eublepharis macularius, a species that is emerging as a model for reptile biology. After estimating basal thermal tolerance for 28 geckos, thermal tolerance was measured for each individual a second time at several timepoints (3, 6, or 24 h) to determine thermal tolerance plasticity. We found that thermal tolerance plasticity (1) does not depend on the basal thermal tolerance of the organism, (2) was highest after 6 hours from initial heat shock, and (3) was negatively influenced by individual body mass. Our findings contribute to the increasing body of work focused on understanding the influence of biological and environmental factors on thermal tolerance plasticity in organisms and provide phenotypic data to further investigate the molecular basis of thermal tolerance plasticity in organisms.

zoology↗

bioRxiv2024

Inositol polyphosphate multikinase regulates Th1 and Th17 cell differentiation by controlling Akt-mTOR signaling

Activated proinflammatory T helper (Th) cells, such as Th1 and Th17 cells, mediate immune responses against intra- and extra-cellular pathogens as well as cause the development of various autoimmune diseases. Inositol polyphosphate multikinase (IPMK) is a key enzyme essential for inositol phosphate and phosphoinositide metabolism, which is known to control major biological events such as growth; however, its role in the function of Th cells remains unclear. Here we show that the expression of IPMK is highly induced in distinct Th1 and Th17 subsets. Further, while conditional deletion of IPMK in CD4+ T cells is dispensable for Th2-dependent immune responses, both Th1- and Th17-mediated immune responses are markedly diminished when this enzyme is absent resulting in reduced resistance to Leishmania major infection and attenuation of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. In addition, IPMK-deficient naive CD4+ T cells display aberrant T cell activation and impaired differentiation into Th17 cells, which is associated with reduced activation of Akt, mechanistic target of rapamycin (mTOR), and STAT3. Mechanistically, IPMK as a phosphatidylinositol 3-kinase (PI3-kinase) controls the production of phosphatidylinositol (3,4,5)-trisphosphate, thereby promoting T cell activation, differentiation, and effector functions. Our findings suggest that IPMK acts as a critical regulator of Th1 and Th17 differentiation, highlighting the physiological importance of IPMK in Th1- and Th17-mediated immune homeostasis.

immunology↗

bioRxiv2024

Detailed mechanisms for unintended large DNA deletions with CRISPR, base editors, and prime editors

CRISPR-Cas9 nucleases are versatile tools for genetic engineering cells and function by producing targeted double-strand breaks (DSBs) in the DNA sequence. However, the unintended production of large deletions (>100 bp) represents a challenge to the effective application of this genome-editing system. We optimized a long-range amplicon sequencing system and developed a k-mer sequence-alignment algorithm to simultaneously detect small DNA alteration events and large DNA deletions. With this workflow, we determined that CRISPR-Cas9 induced large deletions at varying frequencies in cancer cell lines, stem cells, and primary T cells. With CRISPR interference screening, we determined that end resection and the subsequent TMEJ [DNA polymerase theta-mediated end joining] repair process produce most large deletions. Furthermore, base editors and prime editors also generated large deletions despite employing mutated Cas9 "nickases" that produce single-strand breaks. Our findings reveal an important limitation of current genome-editing tools and identify strategies for mitigating unwanted large deletion events.

genetics↗

bioRxiv2024

Hidden route of protein damage through confined oxygen gas

Oxidative modifications can severely impair protein structure, fold, and function, closely linked to human aging and diseases. Conventional oxidation pathways typically involve the free diffusion of reactive oxygen species (ROS), followed by chemical attacks on the protein surface. Here, we report a hidden route of protein oxidative damage, which we refer to as O2-confinement oxidation pathway. This pathway starts with the initial trapping of dissolved molecular oxygen (O2) within protein cavity spaces, followed by interaction with photosensitizing tryptophan residues. The trapped O2 is then converted to singlet oxygen (1O2), a powerful ROS, through spin-flip electron transfer mechanism under blue light. The generated 1O2 within the protein ultimately attacks the protein core residues through constrained diffusion, accelerating the oxidative damage. This alternative photooxidation pathway through the initial O2 trapping would bypass the antioxidant defense systems which target freely-diffusing ROS, constituting an additional layer of protein oxidative damage in cells and tissues.

biochemistry↗

bioRxiv2023

Spatial dissection of cellular heterogeneity and functional crosstalk in the tumor microenvironments of gastric cancers

Spatial transcriptomics offers unprecedented challenges in characterizing tumor ecosystems, encompassing tumor cells and their surrounding tumor microenvironments (TME) along with cellular interactions therein. Here, we conducted Visium-based spatial transcriptomics on nine primary gastric cancers (GC) to translate the spatial organization of GC ecosystems into the functional landscape of cellular crosstalk across malignant, stromal and immune cells. We identified three distinct GC subtypes of 3 immunogenic, 3 epithelial and 3 stromal GCs with elevated infiltration of immune, malignant cells and fibroblasts reflecting the heterogeneity of the cell types and their interactions in GC TME. GC spatial architecture was further delineated into 6 regional compartments with varying degree of TME infiltration, among which the fibroblast-enriched TME was associated with the upregulation of epithelial-to-mesenchymal transformation (EMT) and immune response in malignant and immune cells, respectively. Cell type-specific transcriptional dynamics representing cellular crosstalk between TME cells were further identified, e.g., the infiltration of malignant and endothelial cells promote the cellular proliferations of TME cells whereas the fibroblasts and immune cells are associated with pro-cancer and anti-cancer immunity, respectively. Ligand-receptor analysis further revealed that CCL2-expressing fibroblasts promote the pro-cancer signaling of immune cells including JAK-STAT3 signaling and inflammatory response in tumor-infiltration macrophages. The CCL2+fibroblasts and macrophages with activated JAK-STAT3 signals are co-localized in spatial GC architecture and their co-abundance was associated with unfavorable clinical outcomes. Taken together, GC spatial transcriptomes can be regionally and functionally delineated into functional cellular crosstalk involving multiple cell types with potential clinical relevance, particularly the interaction between CCL2+ fibroblasts and JAK-STAT3+macrophages contributing to cancer-favoring immune contexture in GC TME.

cancer biology↗

bioRxiv2023

Neutrophil extracellular traps formation is associated with postoperative complications in neonates and infants undergoing congenital cardiac surgery

Pediatric patients with congenital heart diseases (CHD) often undergo surgical repair on cardiopulmonary bypass (CPB). Despite a significant medical and surgical improvement, the mortality of neonates and infants remains high. Damage-associated molecular patterns (DAMPs) are endogenous molecules released from injured/damaged tissues as danger signals. We examined 101 pediatric patients who underwent congenital cardiac surgery on CPB. The mortality rate was 4.0%, and the complication rate was 31.6%. We found that neonates/infants experienced multiple complications most, consistent with the previous knowledge. Neonates and infants in the complication group had received more transfusion intraoperatively than the non-complication arm with lower maximum amplitude (MA) on rewarming CPB thromboelastography (TEG). Despite TEG profiles were comparable at ICU admission between the two groups, the complication arm had higher postoperative chest tube output, requiring more blood transfusion. The complication group showed greater neutrophil extracellular traps (NETs) formation at the end of CPB and postoperatively. Plasma histones and high mobility group box 1 (HMGB1) levels were significantly higher in the complication arm. Both induced NETs in vitro and in vivo. As histones and HMGB1 target Toll-like receptor (TLR)2 and TLR4, their mRNA expression in neutrophils was upregulated in the complication arm. Taken together, NETs play a major role in postoperative complication in pediatric cardiac surgery and would be considered a target for intervention. Key pointsO_LINeonates and infants showed highest postoperative complications with more upregulation of inflammatory transcriptomes of neutrophils. C_LIO_LINeonates and infants with organ dysfunction had more NETs formation with higher plasma histones and HMGB1 levels. C_LI

immunology↗