Biology subjects

Shi, K.

Publications and source records attributed to Shi, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

m6A methylation dynamically participates in the immune response against Vibrio anguillarum in half-smooth tongue sole (Cynoglossus semilaevis)

N6-methyladenosine (m6A) is the most prevalent RNA modification and a multifaceted regulator capable of affecting various aspects of mRNA metabolism, thereby playing important roles in numerous physiological processes. However, it is still unknown whether, when, and to what extent m6A modulation are implicated in the immune response of an economically important aquaculture fish, half-smooth tongue sole (Cynoglossus semilaevis). Herein, we systematically profiled and characterized the m6A epitranscriptome and transcriptome in C. semilaevis after the infection of Vibrio anguillarum, a serious threat to the aquaculture industry. We demonstrated that m6A could be modulated as early as 4-hour post infection (hpi), and the overall intensity of m6A methylation was enhanced following infection. Both conservative and novel motifs were uncovered from the m6A modification sites. Furthermore, differentially m6A methylated genes (DMGs) and differentially expressed genes (DEGs) were identified, and functional enrichment revealed multiple immune-related pathways, especially the FoxO signaling pathway which showed significance in every comparison. Joint analysis highlighted the remarkedly dynamic role of m6A on gene expression, i.e. early on, m6A mainly prioritized the down-regulation of specific genes, and later, it switched gears to promote expression of another set of genes. Moreover, key immune-related genes, including pdp1, rgs5l, and plk2b, were identified. To our limited knowledge, this is the first study comprehensively characterizing the global m6A atlas in aquaculture fish species. The presented results provide new insights into the dynamics of m6A modifications in the transcriptome of the half-smooth tongue sole following bacterial infection. Further studies are warranted to elucidate the functional significance of these changes and to understand how they affect specific biological processes.

molecular biology↗

bioRxiv2024

Immunomagneto-activated hepatocyte-specific tissue vesicle profiling reveals message diversification from regenerating liver

Background & AimsThe liver possesses a remarkable regenerative capacity involving intricate intercellular communication, the mechanisms of which remain poorly understood. Extracellular vesicles (EVs) emerge as important messengers in diverse pathophysiological conditions. However, tissue-derived, cell-specific functional EV populations in regeneration have not been robustly investigated. MethodsBulk and single-cell RNA sequencing analyses of the regenerating liver after partial hepatectomy (PHx), ultrastructural examinations of in situ liver tissue EVs (LT-EVs), and nanoscale and proteomic profiling of hepatocyte-derived tissue EVs (Hep-EVs) were integrated. Targeted inhibition of Hep-EV release in vivo was performed via AAV-mediated shRNA knockdown of Rab27a, and Hep-EVs were intravenously infused for therapeutically use. Gross, histological, and immunofluorescent examinations of livers with evaluating in vivo and ex vivo hepatocyte proliferation were conducted. ResultsLT-EVs contribute to the liver regenerative process after PHx, and hepatocytes serve as the major origin of tissue EVs in the regenerating liver. Moreover, Hep-EVs play an indispensable role to orchestrate liver regeneration, which is strengthened to release with proliferative messages identified after PHx. Mechanistically, Hep-EVs from the regenerating liver reciprocally promote hepatocyte proliferation, which are hallmarked by and function based on the Cyclin dependent kinase 1 (Cdk1) activity. Importantly, replenishment of Hep-EVs from the regenerating liver holds translational promise and rescues insufficient liver regeneration. ConclusionsOur study establishes a functional and mechanistic framework that release of regenerative Hep-EVs govern rapid hepatocyte proliferation through cell cycle control, shedding light on investigation of physiological and endogenous tissue EV populations in organ regeneration and therapy.

cell biology↗

bioRxiv2024

The Genotype and Phenotypes in Families (GPF) platform manages the large and complex data at SFARI

The exploration of genotypic variants impacting phenotypes is a cornerstone in genetics research. The emergence of vast collections containing deeply genotyped and phenotyped families has made it possible to pursue the search for variants associated with complex diseases. However, managing these large-scale datasets requires specialized computational tools tailored to organize and analyze the extensive data. GPF (Genotypes and Phenotypes in Families) is an open-source platform (https://github.com/iossifovlab/gpf) that manages genotypes and phenotypes derived from collections of families. The GPF interface allows interactive exploration of genetic variants, enrichment analysis for de novo mutations, and phenotype/genotype association tools. In addition, GPF allows researchers to share their data securely with the broader scientific community. GPF is used to disseminate two large-scale family collection datasets (SSC, SPARK) for the study of autism funded by the SFARI foundation. However, GPF is versatile and can manage genotypic data from other small or large family collections. Our GPF-SFARI GPF instance (https://gpf.sfari.org/) provides protected access to comprehensive genotypic and phenotypic data for the SSC and SPARK. In addition, GPF-SFARI provides public access to an extensive collection of de novo mutations identified in individuals with autism and related disorders and to gene-level statistics of the protected datasets characterizing the genes roles in autism. Here, we highlight the primary features of GPF within the context of GPF-SFARI.

bioinformatics↗

bioRxiv2024

Increased regional P2X7R expression detected by GSK1482160 PET in a tauopathy mouse model

Neuroinflammation plays an important role in Alzheimers disease and primary tauopathies. The aim of the current study was to map [18F]GSK1482160 for imaging of purinergic P2X7R in Alzheimers disease and primary tauopathy mouse models. MicroPET was performed using [18F]GSK1482160 in widely used mouse models of Alzheimers disease (APP/PS1, 5xFAD and 3xTg), 4-repeat tauopathy (rTg4510) mice and age-matched wild-type mice. Increased uptake of [18F]GSK1482160 was observed in the cortex and basal forebrain of 7-month-old rTg4510 mice compared to age-matched wild-type mice and compared to 3-month-old rTg4510 mice. Nonparametric Spearmans rank analysis revealed a positive correlation between tau [18F]APN-1607 uptake and [18F]GSK1482160 in the hippocampus of rTg4510 mice. No significant differences in the uptake of [18F]GSK1482160 were observed between wild-type mice and APP/PS1 mice (5, 10 months), 5xFAD mice (3, 7 months) or 3xTg mice (10 months). Immunofluorescence staining further indicated the distribution of P2X7Rs in the brains of 7-month-old rTg4510 mice with accumulation of tau inclusion compared to wild-type mice. These findings provide in vivo imaging evidence for increased P2X7R in the brains of tauopathy model mice.

neuroscience↗

bioRxiv2023

Prothoracicotropic hormone controls female sexual receptivity through the function of ecdysone in pC1 neurons of Drosophila

Female sexual receptivity is essential for reproduction of a species. Neuropeptides play the main role in regulating female receptivity. However, whether neuropeptides regulate female sexual receptivity during the neurodevelopment is unknown. Here we found the peptide hormone prothoracicotropic hormone (PTTH), which belongs to the insect PG axis, negatively regulated virgin female receptivity through ecdysone during neurodevelopment in Drosophila melanogaster. We identified PTTH neurons as doublesex-positive neurons, they regulated virgin female receptivity before the metamorphosis during the 3rd-instar larval stage. PTTH deletion resulted in the increased EcR-A expression in the whole newly formed prepupae. Furthermore, the ecdysone receptor EcR-A in pC1 neurons positively regulated virgin female receptivity during metamorphosis. The decreased EcR-A in pC1 neurons induced abnormal morphological development of pC1 neurons without changing neural activity. Among all subtypes of pC1 neurons, the function of EcR-A in pC1b neurons was necessary for virgin female copulation rate. These suggested that the changes of synaptic connections between pC1b and other neurons decreased female copulation rate. Moreover, female receptivity significantly decreased when the expression of PTTH receptor Torso was reduced in pC1 neurons. This suggested that PTTH not only regulates female receptivity through ecdysone but also through affecting female receptivity associated neurons directly. The PG axis has similar functional strategy as the HPG axis in mammals to trigger the juvenile-adult transition. Our work suggests a general mechanism underlying which the neurodevelopment during maturation regulates female sexual receptivity.

neuroscience↗

bioRxiv2023

Reduced SV2A and GABAA receptor levels in the brains of type 2 diabetic rats revealed by flumazenil PET

PurposeType 2 diabetes mellitus (T2DM) is associated with a greater risk of Alzheimers disease. Synaptic impairment and protein aggregates have been reported in the brains of T2DM models. Here, we assessed whether neurodegenerative changes in synaptic vesicle 2A (SV2A), {gamma};-aminobutyric acid type A (GABAA) receptor, amyloid-{beta}, tau and receptor for advanced glycosylation end product (RAGE) can be detected in vivo in T2DM rats. MethodsPositron emission tomography (PET) using [18F]SDM-8 (SV2A), [18F]flumazenil (GABAA receptor), [18F]florbetapir (amyloid-{beta}), [18F]PM-PBB3 (tau), and [18F]FPS-ZM1 (RAGE) was carried out in 12-month-old diabetic Zucker diabetic fatty (ZDF) and Sprague{square}Dawley (SD) rats. Proteomic profiling and pathway analysis of the hippocampus of ZDF and SD rats were performed. ResultsReduced cortical [18F]SDM-8 and cortical and hippocampal [18F]flumazenil uptake were observed in 12-month-old ZDF rats compared to SD rats. [18F]florbetapir and [18F]PM-PBB3 uptake were comparable in the brains of 12-month-old ZDF rats and SD rats. ConclusionThe findings provide in vivo evidence for regional reductions in SV2A and GABAA receptor levels in the brains of aged T2DM ZDF rats.

neuroscience↗

bioRxiv2023

Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase

Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

bioengineering↗

bioRxiv2023

Relationship between reactive astrocytes, by SMBT-1 imaging, with amyloid-beta, tau, glucose metabolism, and microgliosis in mouse models of Alzheimer's disease

PurposeReactive astrocytes play an important role in the development of Alzheimers disease (AD). Here, we aim to investigate the temporospatial relationship between reactive astrocytes, tau and amyloid-{beta}, glucose metabolism, and microgliosis by using multitracer imaging in AD transgenic mouse models. MethodsPositron emission tomography (PET) imaging with [18F]SMBT-1 (monoamine oxidase-B), [18F]florbetapir (A{beta}), [18F]PM-PBB3 (tau), [18F]FDG, and [18F]DPA-714 (translocator protein) was carried out in 5- and 10-month-old APP/PS1, 11-month-old 3xTg mice, and aged-matched wild-type mice. The brain regional referenced standard uptake value (SUVR) was computed with the cerebellum as the reference region. Immunofluorescence staining was performed in mouse brain tissue slices. Results[18F]SMBT-1 and [18F]florbetapir SUVRs were higher in the cortex and hippocampus of 10-month-old APP/PS1 mice than in 5-month-old APP/PS1 mice and wild-type mice. Reduced [18F]FDG SUVR was observed in the thalamus and midbrain of 5-month-old APP/PS1 mice compared to wild-type mice. No significant difference in brain regional [18F]DPA-714 SUVR was observed in 5- and 10-month-old APP/PS1 mice compared to wild- type mice. No significant difference in the SUVRs of any tracers was observed in 11-month-old 3xTg mice compared to age-matched wild-type mice. A positive correlation between the SUVRs of [18F]SMBT-1 and [18F]DPA-714 in the cortex was observed. Immunostaining validated the distribution of MAO-B and TSPO, amyloid and tau inclusions in brain tissue from 10-month-old APP/PS1 mice and limited changes in 11-month- old 3xTg mice. ConclusionThe findings provide in vivo evidence for reactive astrocytes along with amyloid plaque and tau deposition preceding microgliosis in animal models of AD pathologies.

neuroscience↗

bioRxiv2023

Population coding of time-varying sounds in the non-lemniscal Inferior Colliculus

The inferior colliculus (IC) of the midbrain is important for complex sound processing, such as discriminating conspecific vocalizations and human speech. The ICs non-lemniscal, dorsal "shell" region is likely important for this process, as neurons in these layers project to higher-order thalamic nuclei that subsequently funnel acoustic signals to the amygdala and non-primary auditory cortices; forebrain circuits important for vocalization coding in a variety of mammals, including humans. However, the extent to which shell IC neurons transmit acoustic features necessary to discern vocalizations is less clear, owing to the technical difficulty of recording from neurons in the ICs superficial layers via traditional approaches. Here we use 2-photon Ca2+ imaging in mice of either sex to test how shell IC neuron populations encode the rate and depth of amplitude modulation, important sound cues for speech perception. Most shell IC neurons were broadly tuned, with a low neurometric discrimination of amplitude modulation rate; only a subset were highly selective to specific modulation rates. Nevertheless, neural network classifier trained on fluorescence data from shell IC neuron populations accurately classified amplitude modulation rate, and decoding accuracy was only marginally reduced when highly tuned neurons were omitted from training data. Rather, classifier accuracy increased monotonically with the modulation depth of the training data, such that classifiers trained on full-depth modulated sounds had median decoding errors of [~]0.2 octaves. Thus, shell IC neurons may transmit time-varying signals via a population code, with perhaps limited reliance on the discriminative capacity of any individual neuron. Significance StatementThe ICs shell layers originate a "non-lemniscal" pathway whose first- and second-order targets are thought important for perceiving conspecific vocalizations and human speech. However, prior studies suggest that individual shell IC neurons are broadly tuned and have high response thresholds, implying a limited reliability of efferent signals. Here we use Ca2+ imaging to test how shell IC neurons encode amplitude modulation, a key sound cue for speech perception and stream segregation. We show that the rate and depth of amplitude modulation is accurately represented in the ensemble activity of shell IC neuron populations. Thus, downstream targets can read out a sounds temporal envelope from a distributed rate code transmitted by populations of broadly tuned neurons.

neuroscience↗

bioRxiv2023

Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo

BackgroundAbnormal alpha-synuclein and iron accumulation in the brain play an important role in Parkinsons disease (PD). Herein, we aim at visualizing alpha-synuclein inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. MethodsFluorescently labelled pyrimidoindole-derivative THK-565 was characterized by using recombinant fibrils and brains from 10-11 months old M83 mice, which subsequently underwent in vivo concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging. The in vivo results were verified against structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 Tesla and scanning transmission X-ray microscopy (STXM) of perfused brains. Brain slice immunofluorescence and Prussian blue staining were further performed to validate the detection of alpha-synuclein inclusions and iron deposition in the brain, respectively. ResultsTHK-565 showed increased fluorescence upon binding to recombinant alpha-synuclein fibrils and alpha-synuclein inclusions in post-mortem brain slices from patients with Parkinsons disease and M83 mice. i.v. administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 minutes post-injection by wide-field fluorescence compared to non-transgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. ConclusionWe demonstrated in vivo mapping of alpha-synuclein by means of non-invasive epifluorescence and vMSOT imaging assisted with a targeted THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.

neuroscience↗

bioRxiv2023

Understanding ATP binding to DosS catalytic domain with a short ATP-lid

DosS is a heme-sensor histidine kinase that responds to redox-active stimuli in mycobacterial environments by triggering dormancy transformation. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases suggests that it possesses a rather short ATP-lid. This feature has been thought to inhibit DosS kinase activity by blocking ATP binding in the absence of interdomain interactions with the dimerization and histidine phospho-transfer (DHp) domain of full-length DosS. Here, we use a combination of computational modeling, structural biology, and biophysical studies to re-examine ATP-binding modalities in DosSs CA domain. We show that the closed lid conformation observed in protein crystal structures of DosS CA is caused by the presence of a zinc cation in the ATP binding pocket that coordinates with a glutamate residue on the ATP-lid. Furthermore, circular dichroism (CD) studies and comparisons of DosS CA crystal structure with its AlphaFold model and homologous DesK reveal that a key N-box alpha-helix turn of the ATP pocket manifests as a random coil in the zinc-coordinated protein crystal structure. We note that this closed lid conformation and the random-coil transformation of an N-box alpha-helix turn are artifacts arising from the millimolar zinc concentration used in DosS CA crystallization conditions. In contrast, in the absence of zinc, we find that the short ATP-lid of DosS CA has significant conformational flexibility and can bind ATP (Kd = 53 {+/-} 13 M). We conclude that DosS CA is almost always bound to ATP under physiological conditions (1-5 mM ATP, sub-nanomolar free zinc) in the bacterial environment. Our findings elucidate the conformational adaptability of the short ATP-lid, its relevance to ATP binding in DosS CA and provide insights that extends to 2988 homologous bacterial proteins containing such ATP-lids.

biochemistry↗

bioRxiv2023

Selective S1PR1 activation improves brain infarction, neurological outcome, and cerebrovascular endothelial health following experimental ischemic injury

Sphingosine-1-phosphate receptor 1 (S1PR1) is highly expressed in endothelial cells and receptor activation plays an important role in mediating endothelial function and health, thus showing promise as a pharmacologic target for acute ischemic stroke (AIS) treatment. Here, we examined the effect of a selective S1PR1 ligand, RP101075, on infarct volume and neurological outcome in adult male mice subjected to transient middle cerebral artery occlusion (tMCAO). Concomitantly, we examined S1PR1 expression profile in the ischemic mouse brain, as well as S1PR1 expression and impact of receptor activation on human brain microvascular endothelial cell (HBMEC) proliferation and survival following hypoxia plus glucose deprivation (HGD). We observed that RP101075 administration at onset of reperfusion reduced infarct volume and lessened neurological deficits in tMCAO mice and these responses were S1PR1 dependent. Additionally, we observed that tMCAO increased brain S1PR1 protein levels and flow cytometry revealed increases in S1PR1 levels are greatest in brain endothelial cells compared to other brain cell types (astrocyte, neuron, microglia). In cultured HBMECs, RP101075 increased cell proliferation and ozanimod, parent compound of RP1010175, increased live cell count during HGD; this response was S1PR1 dependent. In conclusion, S1PR1 activation improves neuroprotection/outcome post-stroke and preserves brain endothelial cell survival following ischemia-like injury.

neuroscience↗

bioRxiv2023

Efficient characterization of multiple binding sites of small molecule imaging ligands on amyloid-beta, 4-repeat/full-length tau and alpha-synuclein

AimThere is an unmet need for compounds that detect alpha-synuclein (Syn) and 4-repeat tau, which are critical in many neurodegenerative diseases for diagnostic and therapeutic purposes. Here, we aim to develop an efficient surface plasmon resonance (SPR)-based method to facilitate the characterization of small molecule ligands/compounds to these fibrils. MethodsSPR measurements were conducted to characterize the binding properties of fluorescent ligands/compounds towards recombinant A{beta}42, K18 4-repeat/full-length tau and Syn fibrils. In silico modelling was performed to examine the binding pockets of ligands on Syn fibrils. Immunofluorescence staining with fluorescence ligands and specific antibodies on postmortem brain tissue slices from patients with Parkinsons disease and disease mouse models was performed. ResultsWe optimized the protocol for immobilizing A{beta}42, K18 tau, full-length tau and Syn fibrils in a controlled aggregation state on SPR sensor chips. The results from the analysis of binding kinetics suggested the presence of at least two binding sites for all fibrils, including luminescent conjugated oligothiophenes (HS-169, HS-84, h-FTAA and q-FTAA), pyridine derivative PBB5, nonfluorescent methylene blue and lansoprazole. In silico modelling studies for Syn (6H6B) showed four binding sites with preference to S4. Immunofluorescence staining validated the detection of pS129-positive Syn in brain tissue from Parkinsons disease patients, Syn PFF-injected mice, 6E10-positive A{beta} in arcA{beta} mice, and AT-8/AT-100-positive in tau pR5 tau mice, respectively. ConclusionsSPR measurements of ligands and small molecules binding to A{beta}42, 4R and full-length tau and Syn fibrils suggest the existence of multiple binding sites. This approach may provide efficient characterization of compound binding properties towards these fibrils important in neurodegenerative diseases.

pharmacology and toxicology↗

bioRxiv2023

Molecular determinants for Rous sarcoma virus intasome assemblies

Integration of retroviral DNA into the host genome involves formation of integrase (IN)-DNA complexes termed intasomes. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate. The STC structure had an overall resolution of 3.36 [A] and 3 [A] in the conserved intasome core (CIC) region. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the CIC, similar to previously shown with the RSV octameric cleaved synaptic complex (CSC) intasome produced with IN and viral DNA only. An extensive analysis of higher-resolution STC structure helped in identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246 and S124 in CSC and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.

biochemistry↗

bioRxiv2023

Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein

Double homeobox (DUX) genes are unique to eutherian mammals and normally expressed transiently during zygotic genome activation. The canonical member, DUX4, is involved in facioscapulohumeral muscular dystrophy (FSHD) and cancer, when misexpressed in other contexts. We evaluate the 3 human DUX genes and the ancestral single homeobox gene sDUX from the non-eutherian mammal, platypus, and find that DUX4 activities are not shared with DUXA or DUXB, which lack transcriptional activation potential, but surprisingly are shared with platypus sDUX. In human myoblasts, platypus sDUX drives cytotoxicity, inhibits myogenesis, and induces DUX4 target genes, particularly those associated with zygotic genome activation (ZGA), by binding DNA as a homodimer in a way that overlaps the DUX4 homeodomain crystal structure. DUXA lacks transcriptional activity but has DNA-binding and chromatin accessibility overlap with DUX4 and sDUX, including on ZGA genes and LTR elements, and can actually be converted into a DUX4-like cytotoxic factor by fusion to a synthetic transactivation domain. DUXA competition antagonizes the activity of DUX4 on its target genes, including in FSHD patient cells. Since DUXA is an early DUX4 target gene, this activity potentiates feedback inhibition, constraining the window of DUX4 activity. The DUX gene family therefore comprises cross-regulating members of opposing function, with implications for their roles in ZGA, FSHD, and cancer. HIGHLIGHTSO_LIPlatypus sDUX is toxic and inhibits myogenic differentiation. C_LIO_LIDUXA targets overlap substantially with those of DUX4. C_LIO_LIDUXA fused to a synthetic transactivation domain acquires DUX4-like toxicity. C_LIO_LIDUXA behaves as a competitive inhibitor of DUX4. C_LI

molecular biology↗

bioRxiv2022

Isolation and characterization of a novel phage of Vibrio parahaemolyticus with biocontrol potential

Vibrio parahaemolyticus is a major foodborne pathogen that contaminates aquatic products and causes great economic losses to aquaculture. Because of the emergence of multidrug-resistant V. parahaemolyticus strains, bacteriophages are considered promising agents for their biocontrol as an alternative or supplement to antibiotics. Here, a lytic vibriophage, vB_VpaM_R16F (R16F), was isolated from sewage from a seafood market by infecting V. parahaemolyticus 1.1997T. R16F was found to infect V. parahaemolyticus, but not nine other Vibrio spp. The phage belongs to the myovirus morphotype and lysed host cells with a short latent period (<10 min) and a small burst size (13 plaque-forming units). R16F has a linear double-stranded DNA with genome size 139,011 bp and a G+C content of 35.21%. Phylogenetic and intergenomic nucleotide sequence similarity analysis revealed that R16F is distinct from currently known vibriophages and belongs to a novel genus. Several genes (e.g., encoding ultraviolet damage endonuclease and endolysin) that may enhance environmental competitiveness were found in the genome of R16F, while no antibiotic resistance- or virulence factor-related gene was detected. In consideration of its biological and genetic properties, R16F is suggested to be a candidate biocontrol agent for use against V. parahaemolyticus.

microbiology↗

bioRxiv2022

P bodies coat germ granules to promote transgenerational gene silencing in C. elegans

The formation of biomolecular condensates has emerged as a critical mechanism for compartmentation in living cells. Despite interactions between distinct condensates having been reported, the biological relevance of these interactions remains elusive. In germ cells, small RNA silencing factors are enriched in germ granule condensates, where distinct factors are organized into sub-compartments with specific functions linked to genome surveillance or transgenerational gene silencing. Here we showed that perinuclear germ granules are coated by P body condensates, which are known for housing translationally-inactive mRNAs and mRNA degradation factors. Disruption of P body factors, including CGH-1/DDX6 and CAR-1/LSM14, lead to dispersal of small RNA factors from perinuclear germ granules and disorganization of sub-compartments within germ granules. We further found that CAR-1 promotes the interaction between CGH-1 and germ granule factors, and these interactions are critical for the ability of CGH-1 to promote piRNA-mediated gene silencing. Importantly, we observed that cgh-1 mutants are competent in triggering gene silencing but exhibit defects in maintaining gene silencing in subsequent generations. Small RNA sequencing further showed that cgh-1 mutants exhibit defects in amplifying secondary small RNAs, known carriers of gene silencing memories. Together, our results uncover the function of P body factors in small RNA-mediated transgenerational gene silencing and highlight how the formation and function of one condensate can be regulated by an adjacent, interacting condensate in cells.

cell biology↗

bioRxiv2022

Targeting N-Myc in Neuroblastoma with Selective Aurora Kinase A Degraders

Summary ParagraphMYCN amplification is the most frequent genetic driver in high-risk neuroblastoma (NB) and strongly associated with poor prognosis.1,2 The N-Myc transcription factor, which is encoded by MYCN, is a mechanistically validated, yet challenging target for NB therapy development.3,4 In normal neuronal progenitors, N-Myc undergoes rapid degradation, while in MYCN-amplified NB cells, Aurora kinase A (Aurora-A) binds to and stabilizes N-Myc, resulting in elevated protein levels.5,6 Allosteric Aurora-A inhibitors that displace N-Myc from binding can promote N-Myc degradation, but with limited efficacy.7-10 Here, we report a chemical approach to decrease N-Myc levels through the targeted protein degradation of Aurora-A. A first-in-class Aurora-A/N-Myc degrader, HLB-0532259 (compound 4), was developed from a novel Aurora-A-binding ligand that engages the Aurora-A/N-Myc complex. HLB-0532259 promotes the degradation of both Aurora-A and N-Myc with nanomolar potency and excellent selectivity and surpasses the cellular efficacy of established allosteric Aurora-A inhibitors. HLB-0532259 exhibits favorable pharmacokinetics properties and elicits tumor reduction in murine xenograft NB models. More broadly, this study delineates a novel strategy for targeting "undruggable" proteins that are reliant on accessory proteins for cellular stabilization.

cancer biology↗