Biology subjects

Koh, J.

Publications and source records attributed to Koh, J..

28 records · Page 2Linked to original sources

bioRxiv2021

Marked differences in local bone remodeling in response to different marrow stimulation techniques in a large animal

Marrow stimulation, including subchondral drilling and microfracture, is the most commonly performed cartilage repair strategy, whereby the subchondral bone plate is perforated to release marrow-derived cells into a cartilage defect to initiate repair. Novel scaffolds and therapeutics are being designed to enhance and extend the positive short-term outcomes of this marrow stimulation. However, the translation of these newer treatments is hindered by bony abnormalities, including bone resorption, intralesional osteophytes, and bone cysts, that can arise after marrow stimulation. In this study, three different marrow stimulation approaches -- microfracture, subchondral drilling, and needle-puncture - were evaluated in a translationally relevant large animal model, the Yucatan minipig. The objective of this study was to determine which method of marrow access (malleted awl, drilled Kirschner wire, or spring-loaded needle) best preserved the underlying subchondral bone. Fluorochrome labels were injected at the time of surgery and 2 weeks post-surgery to capture bone remodeling over the first 4 weeks. Comprehensive outcome measures included cartilage indentation testing, histological grading, microcomputed tomography, and fluorochrome imaging. Our findings indicated that needle-puncture devices best preserved the underlying subchondral bone relative to other marrow access approaches. This may relate to the degree of bony compaction occurring with marrow access, as the Kirschner wire approach, which consolidated bone most, induced the most significant bone damage with marrow stimulation. This study provides basic science evidence in support of updated marrow stimulation techniques for preclinical and clinical practice.

bioengineering↗

bioRxiv2021

Identification of the Molecular Targets of Disulfide Bond Disrupting Agents

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

pharmacology and toxicology↗

bioRxiv2020

Early growth response 2 (EGR2) is a novel regulator of the senescence program

Senescence, a state of stable growth arrest, plays an important role in ageing and age-related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programs, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up-regulated during senescence and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to the ARF promoter. Loss of EGR2 downregulates p16 levels and increases the pool of p16- p21- reversed cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a regulator of the p16/pRB and direct transcriptional activator of the ARF/p53/p21 pathways in senescence and a novel marker of senescence.

cell biology↗

bioRxiv2020

Fungi.guru: comparative genomic and transcriptomic database for the Fungi kingdom

The fungi kingdom is composed of eukaryotic heterotrophs, which are responsible for balancing the ecosystem and play a major role as decomposers. They also produce a vast diversity of secondary metabolites, which have antibiotic or pharmacological properties. However, our lack of knowledge of gene function in fungi precludes us from tailoring them to our needs and tapping into their metabolic diversity. To remedy this, we gathered genomic and gene expression data of 19 most widely-researched fungi to build a database, fungi.guru, which contains tools for cross-species identification of conserved pathways, functional gene modules, and gene families. We exemplify how our database can elucidate the molecular function, biological process and cellular component of genes involved in various biological processes, by identifying a secondary metabolite pathway producing gliotoxin in Aspergillus fumigatus, the catabolic pathway of cellulose in Coprinopsis cinerea and the conserved DNA replication pathway in Fusarium graminearum and Pyricularia oryzae. The database is available at www.fungi.guru.

bioinformatics↗

bioRxiv2020

Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing.

Biomaterial scaffolds represent a promising approach for material-based tissue regeneration. We previously developed microporous annealed particle (MAP) hydrogels - a flowable, microparticle-based hydrogel in which neighboring hydrogel particles are linked in situ to form a porous scaffold that accelerates wound healing. To promote more extensive tissue ingrowth before scaffold degradation, we aimed to slow scaffold degradation by switching the chirality of the crosslinking peptides from L-peptides to D-peptides. Unexpectedly, despite showing the predicted slower enzymatic degradation in vitro, D-peptide crosslinked MAP hydrogel (D-MAP) hastened material degradation in vivo and imparted significant tissue regeneration to healed cutaneous wounds, including increased tensile strength and hair neogenesis. By themselves, D-chiral peptides were poor activators of macrophage innate immune signaling in vivo, but MAP particles elicit IL-33 type 2 myeloid cell recruitment which is amplified in vivo in the presence of D-peptides. Remarkably, D-MAP elicited significant antigen-specific immunity against the D-chiral peptides, and an intact adaptive immune system was required for the hydrogel-induced skin regeneration. These findings demonstrate that the generation of an adaptive immune response from a biomaterial is sufficient to induce cutaneous regenerative healing despite faster scaffold degradation.

bioengineering↗

bioRxiv2020

Prediction of novel components of bacterial ribosomes by crowd-sourced analysis of RNA sequencing data

Bacterial resistance to antibiotics is a growing problem that is projected to cause more deaths than cancer in 2050. Consequently, novel antibiotics are urgently needed. Since more than half of the available antibiotics target the bacterial ribosomes, proteins that are involved in protein synthesis are thus prime targets for the development of novel antibiotics. However, experimental identification of these potential antibiotic target proteins can be labor-intensive and challenging, as these proteins are likely to be poorly characterized and specific to few bacteria. In order to identify these novel proteins, we established a Large-Scale Transcriptomic Analysis Pipeline in Crowd (LSTrAP-Crowd), where 285 individuals processed 26 terabytes of RNA-sequencing data of the 17 most notorious bacterial pathogens. In total, the crowd processed 26,269 RNA-seq experiments and used the data to construct gene co-expression networks, which were used to identify more than a hundred uncharacterized genes that were transcriptionally associated with protein synthesis. We provide the identity of these genes together with the processed gene expression data. The data can be used to identify other vulnerabilities or bacteria, while our approach demonstrates how the processing of gene expression data can be easily crowdsourced.

bioinformatics↗

bioRxiv2019

Postnatal expansion, maturation and functionality of MR1 T cells in humans

MR1-restricted T (MR1T) cells are defined by their recognition of metabolite antigens presented by the monomorphic MHC class 1-related molecule, MR1, the most highly conserved MHC class I related molecule in mammalian species. Mucosal-associated invariant T (MAIT) cells are the predominant subset of MR1T cells expressing an invariant TCR -chain, TRAV1-2. These cells comprise a T cell subset that recognizes and mediates host immune responses to a broad array of microbial pathogens, including Mycobacterium tuberculosis. Here, we sought to characterize development of circulating human MR1T cells as defined by MR1-5-OP-RU tetramer labelling and of the TRAV1-2+ MAIT cells defined by expression of TRAV1-2 and high expression of CD26 and CD161 (TRAV1-2+CD161++CD26++ cells). We analysed postnatal expansion, maturation and functionality of peripheral blood MR1-5-OP-RU tetramer+ MR1T cells in cohorts from three different geographic settings with different tuberculosis (TB) vaccination practices, levels of exposure to and infection with M. tuberculosis. Early after birth, frequencies of MR1-5-OP-RU tetramer+ MR1T cells increased rapidly by several fold. This coincided with the transition from a predominantly CD4+ and TRAV1-2- population in neonates, to a predominantly TRAV1-2+CD161++CD26++ CD8+ population. We also observed that tetramer+ MR1T cells that expressed TNF upon mycobacterial stimulation were very low in neonates, but increased [~]10-fold in the first year of life. These functional MR1T cells in all age groups were MR1-5-OP-RU tetramer+TRAV1-2+ and highly expressed CD161 and CD26, markers that appeared to signal phenotypic and functional maturation of this cell subset. This age-associated maturation was also marked by the loss of naive T cell markers on tetramer+ TRAV1-2+ MR1T cells more rapidly than tetramer+TRAV1-2- MR1T cells and non-MR1T cells. These data suggest that neonates have infrequent populations of MR1T cells with diverse phenotypic attributes; and that exposure to the environment rapidly and preferentially expands the MR1-5-OP-RU tetramer+TRAV1-2+ population of MR1T cells, which becomes the predominant population of functional MR1T cells early during childhood.

immunology↗

bioRxiv2018

Ribosomal stress-induced senescence as a novel pro-senescence strategy for p16 positive basal-like breast cancer

Re-engaging the senescent programme represents an attractive yet underexplored strategy for cancer therapy, particularly for those tumour subtypes where targeted agents are limited or unavailable. Here, we identify a specific subset of ribosomal proteins as novel pro-senescence therapeutic targets for a highly aggressive subtype of breast cancer, p16 positive basal-like breast cancer. Mechanistically, ribosomal stress-induced senescence generates a stable cell cycle arrest, is dependent on endogenous p16 triggering a resensitisation to the p16/RB tumour suppressor axis, followed by establishment of a senescence-associated secretory phenotype, and is independent of DNA damage. Conversely, ribosomal protein knockdown in a p16 negative breast cancer model results in caspase-mediated apoptosis. Importantly, individual ribosomal protein loss is well tolerated by a panel of normal human cells. We demonstrate a reciprocal feedback loop between loss of RPS3A and RPS7 at both the transcriptional and post-transcriptional level during ribosomal stress-induced senescence. Further, our ribosomal hits are co-ordinately dysregulated in breast cancer, with elevated expression associated with a poor prognosis. Clinical relevance is demonstrated in tissue microarrays, and a RPS3AHIGHRPS7HIGH signature is associated with an earlier disease onset and synergises with p16 to further worsen patient outcome. We conclude that dysregulation of ribosomal proteins constitutes a cancer cell-specific mechanism of senescence evasion and that engaging ribosomal stress-induced senescence may be relevant for future pro-senescence therapies.

cancer biology↗

bioRxiv2017

Dynamic expression of tRNA-derived small RNAs define cellular states

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Here we report the identification of a set of tsRNAs upregulated in differentiating mouse embryonic stem cells (mESCs). Mechanistic analyses revealed primary functions of tsRNAs in regulating polysome assembly and translation. Notably, interactome studies with differentially-enriched tsRNAs revealed a switch in associations with effector RNPs and target mRNAs in different cell-states. We also demonstrate that a specific pool of tsRNAs can interact with Igf2bp1, an RNA-binding protein, to influence the expression of the pluripotency-promoting factor-c-Myc, thereby providing evidence for tsRNAs in modulating stem cell-states in mESCs. Finally, tsRNA expression analyses in distinct, heterologous cell and tissue models of stem/transformed versus differentiated/normal states reveal that tsRNA-mediated regulation of protein translation may represent a global biological phenomenon associated with cell-state transitions.\n\nOne Sentence SummaryIdentification and functional characterization of tRNA-derived small RNAs (tsRNAs) in cell state switches.

molecular biology↗

bioRxiv2017

Wakefulness state modulates conscious access: Suppression of auditory detection in the transition to sleep

Mapping the reports of awareness and its neural underpinnings is instrumental to understand the limits of human perception. The capacity to become aware of objects in the world may be studied by suppressing faint target stimuli with strong masking stimuli, or - alternatively - by manipulating the level of wakefulness from full alertness to mild drowsiness. By combining these two approaches, we studied how perceptual awareness is modulated by decreasing wakefulness. We found dynamic changes in behavioural and neural signatures of conscious access in humans between awake and drowsy states. Behaviourally, we show a decrease in the steepness of the psychophysical function for conscious access in drowsy trials. Neural mapping showed delayed processing of target-mask interaction as the consciousness transition progressed, suggesting that the brain resolution of conscious access shifts from early sensory/perceptual to decision-making stages of processing. Once the goal to report the awareness of a target is set, the system behaviourally adapts to rapid changes in wakefulness, revealing the flexibility of the neural signatures of conscious access, and its suppression, to maintain performance. Significance statementMaintaining full alertness for long periods of time in attentionally demanding situations is challenging and may lead to a decrease in performance. We show the effect of wakefulness fluctuations on behaviour and brain dynamics that humans use to maintain performance. We reveal the neural strategies we have to cope with drowsiness by shifting the weights to more flexible brain processes and relaxing the precision of the decisions we take.

neuroscience↗