Biology subjects

Kwon, Y.

Publications and source records attributed to Kwon, Y..

49 records · Page 3Linked to original sources

bioRxiv2021

A new protocol for speed vernalisation of winter cereals

There are many challenges facing the development of high-yielding, nutritious crops for future environments. One limiting factor is generation time, which prolongs research and plant breeding timelines. Recent advances in speed breeding protocols have dramatically reduced generation time for many short-day and long-day species by optimising light and temperature conditions during plant growth. However, winter crops with a vernalisation requirement still require up to 6-10 weeks in low-temperature conditions before transition to reproductive development. Here, we tested a suite of environmental conditions and protocols to investigate if vernalisation can be satisfied more efficiently. We identified a vernalisation method consisting of exposing seeds at the soil surface to an extended photoperiod of 22 h day:2 h night at 10{degrees}C with transfer to speed breeding conditions that dramatically reduces generation time in both winter wheat (Triticum aestivum) and winter barley (Hordeum vulgare). Implementation of this protocol achieved up to five generations per year for winter wheat or barley, instead of the two typically obtained under standard vernalisation and plant growth conditions. The protocol has great potential to enhance training and to accelerate research, pre-breeding, and breeding outcomes focussed on winter crop improvement.

plant biology↗

bioRxiv2021

The obesity-linked human lncRNA AATBC regulates adipocyte plasticity by stimulating mitochondrial dynamics and respiration

Adipocytes are critical regulators of metabolism and energy balance. While white adipocyte dysfunction is a hallmark of obesity-associated disorders, the activation of thermogenic brown and beige adipocytes is linked to improved cardiometabolic health. As adipocytes dynamically adapt to environmental cues by functionally switching between white and thermogenic phenotypes, a molecular understanding of this adipocyte plasticity could help improving energy balance and weight loss. Here, we show that the long non-coding RNA (lncRNA) Apoptosis associated transcript in bladder cancer (AATBC) is a human-specific regulator of adipocyte plasticity. Searching for new human lncRNAs implicated in adipocyte biology we compared transcriptional profiles of human adipose tissues and cultured adipocytes and discovered that AATBC was enriched in thermogenic conditions. Using primary human adipocytes and immortalized human adipocytes we found that gain-of-function of AATBC enhanced the thermogenic phenotype whereas loss-of-function diminished this effect. The AATBC-mediated increase in mitochondrial respiration was linked to a more fragmented mitochondrial network and vice versa. While we found that AATBC is predominantly located in the nucleus, its effect on global transcription was only marginal. As AATBC is specific to humans, we expressed AATBC in adipose tissue of mice to study its systemic impact, which led to lower plasma leptin levels. Interestingly, this association was also present in human subjects, as AATBC in adipose tissue was inversely correlated with plasma leptin levels, body mass index and other measures of metabolic health. In conclusion, AATBC is a novel obesity-linked regulator of adipocyte plasticity and mitochondrial function in humans.

molecular biology↗

bioRxiv2021

RAD51AP1 compensates for the loss of RAD54 in homology-directed DNA repair

Homologous recombination (HR) is a complex DNA damage repair pathway and an attractive target of inhibition in anti-cancer therapy. To help guide the development of efficient HR inhibitors, it is critical to identify compensatory sub-pathways. In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that deletion of both RAD51AP1 and RAD54 synergistically sensitizes human cancer cell lines to treatment with a Poly(adenosine 5 s-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which stalls the progression of DNA replication forks. We infer that HR-directed anti-cancer treatment modalities shall consider this intra-pathway functional overlap, and we hypothesize that in cancerous cells the simultaneous inactivation of both RAD54 and RAD51AP1 will accentuate tumor kill.

cell biology↗

bioRxiv2021

Genomic consequences of domestication of the Siamese fighting fish

Siamese fighting fish, commonly known as betta, are among the worlds most popular and morphologically diverse pet fish, but the genetic processes leading to their domestication and phenotypic diversification are largely unknown. We assembled de novo the genome of a wild Betta splendens and whole-genome sequenced multiple individuals across five species within the B. splendens species complex, including wild populations and domesticated ornamental betta. Given our estimate of the mutation rate from pedigrees, our analyses suggest that betta were domesticated at least 1,000 years ago, centuries earlier than previously thought. Ornamental betta individuals have variable contributions from other Betta species and have also introgressed into wild populations of those species. We identify dmrt1 as the main sex determination gene in ornamental betta but not in wild B. splendens, and find evidence for recent directional selection at the X-allele of the locus. Furthermore, we find genes with signatures of recent, strong selection that have large effects on color in specific parts of the body, or the shape of individual fins, and are almost all unlinked. Our results demonstrate how simple genetic architectures paired with anatomical modularity can lead to vast phenotypic diversity generated during animal domestication, and set the stage for using betta as a modern system for evolutionary genetics. One-Sentence SummaryGenomic analyses reveal betta fish were domesticated more than 1,000 years ago and the genes that changed in the process.

evolutionary biology↗

bioRxiv2021

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 {micro}M). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a "BL2 groove" that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

microbiology↗

bioRxiv2020

Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination

Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55- Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re- assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA-bound states, the rate of which is dynamically controlled though the opposing actions of Rad55-Rad57 and Srs2.

biophysics↗

bioRxiv2020

Exosomes Facilitate Transmission of SARS-CoV-2 Genome into Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

ABSTRACTThe novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into a worldwide pandemic. Early data suggest that the prevalence and severity of COVID-19 appear to be higher among patients with underlying cardiovascular risk factors. Despite the expression of angiotensin-converting enzyme 2 (ACE2), a functional receptor for SARS-CoV-2 infection, in cardiomyocytes, there has been no conclusive evidence of direct viral infection although the presence of inflammation and viral genome within the hearts of COVID-19 patients have been reported. Here we transduced A549 lung epithelial cells with lentivirus overexpressing selected genes of the SARS-CoV-2. We then isolated extracellular vesicles (EVs) from the supernatant of A549 cells and detected the presence of viral RNA within the purified EVs. Importantly, we observed that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were able to actively uptake these EVs and viral genes were subsequently detected in the cardiomyocytes. Accordingly, uptake of EVs containing viral genes led to an upregulation of inflammation-related genes in hiPSC-CMs. Thus, our findings indicate that SARS-CoV-2 RNA-containing EVs represent an indirect route of viral RNA entry into cardiomyocytes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

Structural Basis of CD4 Downregulation by HIV-1 Nef

The HIV-1 protein Nef suppresses multiple immune surveillance mechanisms to promote viral pathogenesis1. Individuals infected with HIV-1 encoding defective nef genes do not develop AIDS for decades2,3. A key target of Nef is the cellular receptor CD4. Although essential for viral entry into host cells, CD4 is problematic for the virus later in its replication cycle: CD4 disrupts processing of the viral glycoprotein, Env, inhibiting infectivity4; it interferes with the release of new virions5,6; and it causes vulnerability to superinfection, causing premature cell death and limiting viral productivity7. Furthermore, binding of CD4 to Env exposes otherwise-concealed Env epitopes, rendering infected cells more susceptible to antibody-dependent cellular cytotoxicity and virus particles more susceptible to neutralizing antibodies8-10. HIV-1 has evolved strategies to mitigate these problems. Newly synthesized CD4 is targeted in the endoplasmic reticulum by the viral Vpu protein for proteasomal degradation11. Surface-expressed CD4, in contrast, is targeted by Nef for endocytosis and lysosomal degradation12-15. Nefs effect on CD4 involves hijacking of clathrin adaptor complex 2 (AP2)-dependent endocytosis16,17. Although how Nef associates with a part of the tetrameric AP2 is understood18, a complete understanding of the interaction, especially how CD4 is sequestered by Nef into a complex with AP2, has remained elusive. Here, we present a high-resolution crystal structure that describes the underlying mechanism. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. Strikingly, a pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nefs activities and sensitize the virus to immune clearance

biochemistry↗

bioRxiv2020

PARP1 and MGMT interaction-based sensitivity to DNA damage in Ewing sarcoma

The Ewing family of sarcomas comprises the fourth most common highly aggressive bone tumor. Four of five Ewing sarcoma chemotherapeutics induce DNA damage, as does radiation therapy. At relapse, two additional DNA-damaging agents are routinely used to re-induce remission, indicating that Ewing sarcoma is intrinsically sensitive to DNA damage. However, current treatment regimens are relatively ineffective, specifically for relapsed or metastatic disease. Several preclinical studies, including our study in the Pediatric Preclinical Testing Program (PPTP), provide evidence for the synthetic lethal combination of PARP1 inhibitor talazoparib with a DNA-methylating agent, temozolomide, for Ewing sarcoma. Nevertheless, in both preclinical studies and clinical trials, doses of temozolomide were significantly reduced because of toxicity of the drug combination. Temozolomide-induced DNA lesions are repaired via poly(ADP) ribose polymerase I (PARP1)-dependent base excision repair and by O6-methylguanine-DNA methyltransferase (MGMT) in a single-step adduct removal. Here, we provide evidence that the two DNA repair pathways act in an epistatic manner in lesion removal. Further, we demonstrate that PARP1 and MGMT physically interact, and that this association is stimulated upon DNA damage. Protein co-immunoprecipitation and microscale thermophoresis analyses revealed that PARP1/MGMT complex formation is DNA and PARylation-independent. Collectively, our results show that: 1) DNA damage response pathways mediated by PARP1 and MGMT work epistatically to eliminate temozolomide-induced DNA adducts; 2) PARP1 and MGMT physically interact; and 3) PARP1/MGMT interaction is increased in response to DNA damage. We discuss how our findings may affect therapeutic advancement for Ewing sarcoma and potentially other cancer types.

biochemistry↗

bioRxiv2018

Clathrin-coated Pits are Shredded Organelles Presorting Receptors in the Plasma Membrane

A myriad of receptor types crowds the plasma membrane of cells. Here, we revealed that clathrin-coated pits (CCPs) presort receptors to modulate receptor activation on the plasma membrane. We visualized individual molecules of receptors, including epidermal growth factor receptor (EGFR), ErbB2, transferrin receptor (TfR), and beta2-adrenergic receptors ({beta}2-AR), inside a single CCP in a living cell using single-molecule diffusivity-based colocalization analysis. The spatially distinct subsets of CCPs selectively allocated for these receptors were observed in a resting state. The EGFR pre-allocated CCP subset was partially shared with that for ErbB2, whereas the EGFR and TfR pre-allocated CCP subsets were mutually exclusive. PICALM was necessary for the pre-allocation of CCPs for EGFR. Furthermore, EGFR dimerization was markedly elevated inside the pre-allocated CCP subset after dynamin recruitment. The pre-sorting function of CCPs provides an efficient mechanism to control competition and cooperation between receptors on the crowded plasma membrane.\n\nOne Sentence SummaryClathrin-coated pits are small-sized organelles pre-sorting receptor kinds to modulate receptor activation on the crowded plasma membrane.

cell biology↗

bioRxiv2018

PTK2 regulates the UPS impairment via p62 phosphorylation in TDP-43 proteinopathy

TDP-43 proteinopathy is a common feature in a variety of neurodegenerative disorders including Amyotrophic lateral sclerosis (ALS) cases, Frontotemporal lobar degeneration (FTLD), and Alzheimers disease. However, the molecular mechanisms underlying TDP-43-induced neurotoxicity are largely unknown. In this study, we demonstrated that TDP-43 proteinopathy induces impairment in ubiquitin-proteasome system (UPS) evidenced by an accumulation of ubiquitinated proteins and reduction of proteasome activity in neuronal cells. Through kinase inhibitor screening, we identified PTK2 as a suppressor of neurotoxicity induced by UPS impairment. Importantly, PTK2 inhibition significantly reduces ubiquitin aggregates and attenuated TDP-43-induced cytotoxicity in Drosophila model of TDP-43 proteinopathy. We further identified that phosphorylation of p62 at serine 403 (p-p62S403), a key component in the autophagic degradation of poly-ubiquitinated proteins, is increased upon TDP-43 overexpression and dependent on activation of PTK2 in neuronal cells. Moreover, expressing a non-phosphorylated form of p62 (p62S403A) significantly represses accumulation of polyubiquitinated proteins and neurotoxicity induced by TDP-43 overexpression in neuronal cells. In addition, inhibition of TBK1, a kinase which phosphorylates S403 of p62, ameliorates neurotoxicity upon UPS impairment in neuronal cells. Taken together, our data suggest that activation of PTK2-TBK1-p62 axis plays a critical role in the pathogenesis of TDP-43 by regulating neurotoxicity induced by UPS impairment. Therefore, targeting PTK2-TBK1-p62 axis may represent a novel therapeutic intervention for neurodegenerative diseases with TDP-43 proteinopathy.

neuroscience↗

bioRxiv2018

Loop-mediated isothermal amplification assay for Enterococcus sp., E. coli and S. aureus in chicken

Bacterial chondronecrosis with osteomyelitis (BCO) is a major cause of lameness in broiler chicken, and results in serious economic losses worldwide. Although the pathogenesis mechanism leading to lameness is not entirely understood, some strains of Enterococcus sp., avian pathogenic Escherichia coli, or Staphylococcus aureus have been long recognized as important causative pathogens. To prevent the progression of Enterococcus sp., avian pathogenic E. coli, or S. aureus infections, we developed rapid, sensitive, and convenient diagnostic assays using loop-mediated isothermal amplification (LAMP). Entero-Common-LAMP assays were developed for a simultaneous detection of eight Enterococcus species. To target specific microorganisms, seven Entero-Specific-LAMP assays for E. faecalis, E. faecium, E. hirae, E. gallinarum, E. avium, E. durans and E. cecorum, and E. coli-LAMP and S. aureus-LAMP assays, were developed. Considering the prevalence and economic impact of Enterococcus sp., E. coli, and S. aureus, the developed ten different LAMP assays have a considerable potential as routine diagnostic methods in the field or in resource-limited environments.

microbiology↗

bioRxiv2018

Tasman-PCR: A genetic diagnostic assay for Tasmanian devil facial tumour diseases

Tasmanian devils have spawned two transmissible cancer clones, known as devil facial tumour 1 (DFT1) and devil facial tumour 2 (DFT2). DFT1 and DFT2 are transmitted between animals by the transfer of allogeneic contagious cancer cells by biting, and both cause facial tumours. DFT1 and DFT2 tumours are grossly indistinguishable, but can be differentiated using histopathology, cytogenetics or genotyping of polymorphic markers. However, standard diagnostic methods require specialist skills and equipment and entail long processing times. Here, we describe Tasman-PCR: a simple PCR-based diagnostic assay that distinguishes DFT1 and DFT2 by amplification of DNA spanning tumour-specific interchromosomal translocations. We demonstrate the high sensitivity and specificity of this assay by testing DNA from 557 tumours and 818 normal devils. A temporal-spatial screen confirmed the reported geographic ranges of DFT1 and DFT2 and did not provide evidence of additional DFT clones. DFT2 affects disproportionately more males than females, and devils can be co-infected with DFT1 and DFT2. Overall, we present a PCR-based assay that delivers rapid, accurate and high-throughput diagnosis of DFT1 and DFT2. This tool provides an additional resource for devil disease management and may assist with ongoing conservation efforts.

genetics↗