Biology subjects

Jing, Y.

Publications and source records attributed to Jing, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Effects of Electric Field Direction on TMS-based Motor Cortex Mapping

BackgroundTranscranial magnetic stimulation (TMS) modulates brain activity by inducing electric fields (E-fields) that can elicit action potentials in cortical neurons. Neuronal responses to TMS depend not only on the magnitude of the induced E-field but also on various physiological factors. In this study, we incorporated a novel average response model that efficiently estimates the firing threshold of neurons based on their orientation relative to the applied E-field, thereby advancing TMS mapping for motor function. MethodsWe conducted a regression-based TMS mapping experiment with fourteen subjects to localize cortical origins of motor evoked potential (MEP) on the first dorsal interosseous (FDI) muscle. Firing thresholds were estimated for excitatory neurons in cortical layers 2/3 and 5 via an average response model. Regression was performed between MEPs and three E-field quantities: the magnitude (magnitude model), the normal component (cosine model), and the effective E-field, which scales the E-field magnitude based on the firing thresholds specific to the neuronal orientation (neuron model). To validate, we applied TMS to ten subjects with optimized coil placements based on these three models to determine which model could yield the highest MEPs. ResultsThe magnitude and neuron models performed similarly, while the cosine model showed lower explained variance in regression results, required more TMS trials for stable mapping, and yielded the lowest MEP in the validation. ConclusionThis study is the first to advance TMS modeling by incorporating neuron-specific factors at the individual level. Results show that on the motor cortex, the magnitude model is-as expected-a good approximation of cortical TMS effects as it shows similar results as the neuron model. In contrast, the classic cosine model exhibited lower performance and required more TMS trials for stable results, and is not recommended for future studies.

neuroscience↗

bioRxiv2024

Efficient Genome Editing with Chimeric Oligonucleotide-Directed Editing

Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report new nCas9-DNA polymerase fusion proteins to create chimeric oligonucleotide-directed editing (CODE) systems for search-and-replace genome editing. Through successive rounds of engineering, we developed CODEMax and CODEMax(exo+) editors that achieve efficient genome modifications in human cells with low unintended edits. CODEMax and CODEMax(exo+) contain an engineered Bst DNA polymerase derivative known for its robust strand displacement ability. Additionally, CODEMax(exo+) features a 5 to 3 exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.

bioengineering↗

bioRxiv2024

Time Resolved Inspection of Ionizable-Lipid Facilitated Lipid Nanoparticle Disintegration and Cargo Release at an Endosomal Membrane Mimic

This study investigates pH-triggered fusion dynamics of lipid nanoparticles (LNPs) with an endosomal membrane mimic, addressing mechanistic aspects of a crucial yet elusive process for effective mRNA delivery. Utilizing time-resolved total internal reflection fluorescence (TIRF) imaging, we observed a delayed onset of LNP fusion upon pH drop, lasting seconds to minutes depending on pH and LNP size. Once initiated, LNP fusion and cargo escape occurred rapidly within tens to hundreds of milliseconds. While LNP disintegration is observed to lead to release of a significant portion of mRNA into the acidic environment, some mRNA molecules remained mobile on the endosomal membrane mimic due to deprotonation-resistant complex salt formation. Comparison of the fusion efficiency of two LNP formulations correlated with protein translation in human primary cell transfection data, emphasizing the importance of biophysical investigations in understanding ionizable-lipid-containing LNP-assisted mRNA delivery mechanisms and providing insights for optimizing mRNA-LNP design for enhanced endosomal escape.

biophysics↗

bioRxiv2024

LSD1 inhibition suppresses ASCL1 and de-represses YAP1 to drive potent activity against neuroendocrine prostate cancer

Progression to lethal metastatic castration-resistant prostate cancer (mCRPC) is driven in part by epigenetic modulators such as LSD1 (KDM1A), a lysine-specific demethylase. Yet, mCRPC is increasingly recognized as a highly heterogeneous disease whose classification into subtypes is defined by the extent of androgen receptor (AR) and/or neuroendocrine (NE) characteristics. Meanwhile, the role of LSD1 in driving the different subtypes of mCRPC has remained unclear. Here, we assess the necessity of LSD1 in driving progression of mCRPC subtypes including AR+/NE- (ARPC), AR-/NE+ (NEPC), AR+/NE+ (amphicrine; AMPC), and AR-/NE- (double-negative; DNPC) through the use of LSD1 inhibitors in clinical development. LSD1 inhibition (LSD1i) was observed to be highly effective in restricting growth of NEPC, and efficacy was associated with TP53 loss-of-function. Mice bearing NEPC patient-derived xenografts treated with the LSD1 inhibitors, bomedemstat (MK-3543) or iadademstat (ORY-1001), exhibited suppression of the NE transcriptional profile, including ASCL1. LSD1i also induced expression and activity of YAP1, a non-NE transcription factor canonically silenced in NEPC (YAPOFF cancer), thereby switching NEPC from a YAPOFF to a YAPON cancer class. Therapeutically-induced YAPON NEPC tumors exhibited cell cycle arrest and repression of proliferative transcriptional programs. Importantly, the LSD1i-mediated YAPON state induced sensitivity to an inhibitor of YAP/TEAD function, IAG933, which extended antitumor efficacy against NEPC. Altogether, these findings indicate that patients diagnosed with NEPC may obtain greater relative benefit from LSD1-targeted therapies compared to those with other mCRPC subtypes and that dual inhibition of LSD1 and YAP/TEAD function demonstrates a promising treatment strategy potentially extending to other YAPOFF cancers. SignificanceAcross prostate cancer subtypes, NEPC is exceptionally responsive to LSD1 inhibition and this response is enhanced in combination with a YAP/TEAD disruptor which may improve patient selection and outcomes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=171 HEIGHT=200 SRC="FIGDIR/small/576106v2_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@77332eorg.highwire.dtl.DTLVardef@1c127d0org.highwire.dtl.DTLVardef@1cec77org.highwire.dtl.DTLVardef@e87ed7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Epithelial N-methyl-D-aspartate (NMDA) receptors mediate renal vasodilation by affecting kidney autoregulation.

BackgroundN-methyl-D-aspartate receptor (NMDAR) are amino acid receptors that are well studied in brain physiology; however, their role in kidney is poorly understood. Nonetheless, NMDAR inhibitors can increase serum K+ and reduce GFR, which suggests they have an important physiological role in the kidney. We hypothesized that NMDARs in the distal nephron induce afferent-arteriole vasodilation through the vasodilator mechanism connecting-tubule-glomerular feedback (CNTGF) that involves ENaC activation. Methods and resultsUsing a tubule-specific transcriptome database combined with molecular biology and microscopy techniques, we showed kidney expression of NMDAR subunits along the nephron and specifically in ENaC-positive cells. This receptor is expressed in both male and female mice, with higher abundance in females (p=0.02). Microperfusing NMDAR agonists into the connecting tubule induced afferent-arteriole vasodilation (EC50 10.7 vs. 24.5 mM; p<0.001) that was blunted or eliminated with the use of NMDAR blocker MK-801 or with the ENaC inhibitor Benzamil, indicating a dependence on CNTGF of the NMDAR-induced vasodilation. In vivo, we confirmed this CNTGF-associated vasodilation using kidney micropuncture (Stop-flow pressure 37.9{+/-}2.6 vs. 28.6{+/-}1.9 mmHg, NMDAR agonist vs vehicle; p<0.01). We explored NMDAR and ENaC channel interaction by using mpkCCD cells and split-open connecting tubules. We observed increased amiloride-sensitive current following NMDAR activation that was prevented by MK-801 (1.14 vs. 0.4 Amp; p=0.03). In split-open tubules, NMDAR activation increased ENaC activity (Npo Vehicle vs. NMDA; p=0.04). ConclusionNMDARs are expressed along the nephron, including ENaC-positive cells, with higher expression in females. Epithelial NMDAR mediates renal vasodilation through the connecting-tubule-glomerular feedback, by increasing ENaC activity.

physiology↗

bioRxiv2023

Integrated genomics provides insights for the evolution of the polyphosphate accumulation trait of Ca. Accumulibacter

Candidatus Accumulibacter plays a major role in enhanced biological phosphorus removal (EBPR), but the key genomic elements in metagenome assembled genomes enabling their phosphorus cycling ability remain unclear. Pangenome analyses were performed to systematically compare the genomic makeup of Ca. Accumulibacter and non-Ca. Accumulibacter members within the Rhodocyclaceae family. Metatranscriptomic analyses of an enrichment culture of Ca. Accumulibacter clade IIC strain SCUT-2 were performed to investigate gene transcription characteristics in a typical anaerobic-aerobic cycle. Two hundred ninety-eight core genes were shown to be obtained by Ca. Accumulibacter at their least common ancestor. One hundred twenty-four of them were acquired via horizontal gene transfer (HGT) based on best-match analysis against the NCBI database. Fourty-four laterally derived genes were actively transcribed in a typical EBPR cycle, including the polyphosphate kinase 2 (PPK2) gene. Genes in the phosphate regulon (Pho) were poorly transcribed. Via a systematical analysis of the occurrences of these genes in closely related Dechloromonas-polyphosphate accumulating organisms (PAOs) and Propionivibrio-non-PAOs, a Pho dysregulation hypothesis is proposed to explain the mechanism of EBPR. It states that the PhoU acquired by HGT fails in regulating the high-affinity phosphate transport (Pst) system. To avoid phosphate poisoning, the laterally acquired PPK2 is employed to condense excess phosphate into polyphosphate. Alternatively, genes encoding PhoU and PPK2 are obtained from different donor bacteria, leading to unmatched phosphate concentration thresholds for their activation/inactivation. PPK2 tends to reduce the intracellular phosphate to concentration levels perceived by PhoU as low-phosphate states. PhoU is not activated to turn off the Pst system, resulting in continuous phosphate uptake. In conclusion, based on integrated genomic analyses, the HGT of phoU and ppk2 and the resultant Pho dysregulation may have triggered the development and evolution of the P cycling trait in Ca. Accumulibacter.

bioinformatics↗

bioRxiv2023

Mapping the Genetic Interaction Network of PARP inhibitor Response

Genetic interactions have long informed our understanding of the coordinated proteins and pathways that respond to DNA damage in mammalian cells, but systematic interrogation of the genetic network underlying that system has yet to be achieved. Towards this goal, we measured 147,153 pairwise interactions among genes implicated in PARP inhibitor (PARPi) response. Evaluating genetic interactions at this scale, with and without exposure to PARPi, revealed hierarchical organization of the pathways and complexes that maintain genome stability during normal growth and defined changes that occur upon accumulation of DNA lesions due to cytotoxic doses of PARPi. We uncovered unexpected relationships among DNA repair genes, including context-specific buffering interactions between the minimally characterized AUNIP and BRCA1-A complex genes. Our work thus establishes a foundation for mapping differential genetic interactions in mammalian cells and provides a comprehensive resource for future studies of DNA repair and PARP inhibitors.

genomics↗

bioRxiv2023

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

biochemistry↗

bioRxiv2023

Functional divergences of natural variations of TaNAM-A1 controlling leaf senescence initiation during wheat grain filling

Leaf senescence is an essential physiological process related to grain yield potential and nutritional quality. Green leaf duration (GLD) after anthesis directly reflects the leaf senescence process and exhibits large genotypic differences in common wheat; however, the underlying gene regulatory mechanism is still lacking up to now. Here, we report TaNAM-A1 as the causal gene of major loci qGLD-6A for GLD during grain filling by map-based cloning. The role of TaNAM-A1 in regulating leaf senescence, spike length, and grain size was proved by transgenic assay and TILLING mutants analyses. Furthermore, the functional divergences among TaNAM-A1 three haplotypes were systematically evaluated. Wheat varieties with TaNAM-A1d (containing two mutations in CDS of TaNAM-A1) had longer GLD and advantages in yield-related traits than those with the wild type TaNAM-A1a. All three haplotypes were functional in transactivating the expression of genes involved in macromolecular degradation and mineral nutrient remobilization, with TaNAM-A1a the strongest activity and TaNAM-A1d the weakest. TaNAM-A1 modulates the expression of TaNAC016-3A and TaNAC-S-7A to trigger senescence initiation. TaNAC016-3A enhances TaNAM-A1 transcriptional activation ability by protein-protein interaction. Our study provides new insights into fine-tuning the leaf functional period and grain yield formation for wheat breeding under different geographical climatic conditions.

plant biology↗

bioRxiv2023

Triptolide ameliorates renal tubulointerstitial fibrosis through EZH2

Renal fibrosis is the final pathological pathway of various kidney disease in progression to the end stage of renal failure. Recent studies showed that the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is an important epigenetic regulator of renal fibrosis by promoting epithelial-mesenchymal transition (EMT) and activation of TGF-{beta}/Smad3 signaling pathway in fibrotic kidneys. Triptolide is component extracted from radix tripterygium wilfordii, which provides renal benefits to patients with renal diseases in China. Recently, triptolide was identified as an inhibitor of EZH2. Thus, we hypothesized that triptolide inhibits renal fibrosis through EZH2. In this study, we found that triptolide reduced the deposition of extracellular matrix in the kidney of unilateral ureteral obstruction (UUO) mice. The anti-fibrotic effect of triptolide was further confirmed in TGF-{beta} stimulated HK2 cells, a human renal epithelial cell line. Moreover, treatment of triptolide blocked the up-regulation of EZH2 in UUO kidneys and reduced the expression of EZH2 in TGF-{beta} stimulated HK2 cells. Down-regulation of EZH2 by triptolide was correlated with reduced expression of EMT markers and phosphorylation of Smad3 in UUO kidneys and TGF-{beta} stimulated HK2 cells. Finally, we showed that inhibition of EZH2 by 3-DZNep attenuated the inhibitory effect of triptolide on the expression of extracellular matrix protein, EMT markers and activation of Smad3 in TGF-{beta} stimulated HK2 cells. In conclusion, triptolide inhibits renal tubulointerstitial fibrosis through EZH2 in obstructive kidneys.

molecular biology↗

bioRxiv2023

Increased TCR signaling in regulatory T cells is disengaged from TCR affinity

Foxp3+ regulatory T cells (Tregs) are capable suppressors of aberrant self-reactivity. However, TCR affinity and specificities that support Treg function, and how these compare to autoimmune T cells remain unresolved. In this study, we used antigen agnostic and epitope-focused analyses to compare TCR repertoires of regulatory and effector T cells that spontaneously infiltrate pancreatic islets of non-obese diabetic mice. We show that effector and regulatory T cell-derived TCRs possess similar wide-ranging reactivity for self-antigen. Treg-derived TCRs varied in their capacity to confer optimal protective function, and Treg suppressive capacity was in part determined by effector TCR affinity. Interestingly, when expressing the same TCR, Tregs showed higher Nur77-GFP expression than Teffs, suggesting Treg-intrinsic ability to compete for antigen. Our findings provide a new insight into TCR-dependent and independent mechanisms that regulate Treg function and indicate a TCR-intrinsic insufficiency in tissue-specific Tregs that may contribute to the pathogenesis of type 1 diabetes.

immunology↗

bioRxiv2023

Modeling the Effects of Transcranial Magnetic Stimulation on Spatial Attention

ObjectivesTranscranial magnetic stimulation (TMS) has been widely used to modulate brain activity in healthy and diseased brains, but the underlying mechanisms are not fully understood. Previous research leveraged biophysical modeling of the induced electric field (E-field) to map causal structure-function relationships in the primary motor cortex. This study aims at transferring this localization approach to spatial attention, which helps to understand the TMS effects on cognitive functions, and may ultimately optimize stimulation schemes. ApproachThirty right-handed healthy participants underwent a functional magnetic imaging (fMRI) experiment, and seventeen of them participated in a TMS experiment. The individual fMRI activation peak within the right inferior parietal lobule (rIPL) during a Posner-like attention task defined the center target for TMS. Thereafter, participants underwent 500 Posner task trials. During each trial, a 5-pulse burst of 10 Hz repetitive TMS (rTMS) was given over the rIPL to modulate attentional processing. The TMS-induced E-fields for every cortical target were correlated with the behavioral modulation to identify relevant cortical regions for attentional orientation and reorientation. Main resultsWe did not observe a robust correlation between E-field strength and behavioral outcomes, highlighting the challenges of transferring the localization method to cognitive functions with high neural response variability and complex network interactions. Nevertheless, TMS selectively inhibited attentional reorienting in five out of seventeen subjects, resulting in task-specific behavioral impairments. The BOLD-measured neuronal activity and TMS-evoked neuronal effects showed different patterns, which emphasizes the principal distinction between the neural activity being correlated with (or maybe even caused by) particular paradigms, and the activity of neural populations exerting a causal influence on the behavioral outcome. SignificanceThis study is the first to explore the mechanisms of TMS-induced attentional modulation through electrical field modeling. Our findings highlight the complexity of cognitive functions and provide a basis for optimizing attentional stimulation protocols.

neuroscience↗

bioRxiv2022

Chemoproteomics yields a selective molecular host for acetyl-CoA

Chemoproteomic profiling is a powerful approach to define the selectivity of small molecules and endogenous metabolites with the human proteome. In addition to mechanistic studies, proteome specificity profiling also has the potential to identify new scaffolds for biomolecular sensing. Here we report a chemoproteomics-inspired strategy for selective sensing of acetyl-CoA. First, we use chemoproteomic capture experiments to validate the N-terminal acetyltransferase NAA50 as a protein capable of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein retains this specificity and can be used to generate a bioluminescence resonance energy transfer (BRET) signal in the presence of a CoA-linked fluorophore. This enables the development of a ligand displacement assay in which CoA metabolites are detected via their ability to bind the Nanoluc-NAA50 protein host and compete binding of the CoA-linked fluorophore guest. We demonstrate that the specificity of ligand displacement reflects the molecular recognition of the NAA50 host, while the window of dynamic sensing can be controlled by tuning the binding affinity of the CoA-linked fluorophore guest. Finally, we show the methods specificity for acetyl-CoA can be harnessed for gain-of-signal optical detection of enzyme activity. Overall, our studies demonstrate the potential of harnessing insights from chemoproteomics for molecular sensing and provide a foundation for future applications in target engagement and selective metabolite detection.

biochemistry↗

bioRxiv2022

RNA sequencing analyses of gene expression by CRISPR/Cas9 knockout of CLL-1 gene in acute myeloid leukemia cells

CLL-1 has been revealed its potential role in acute myeloid leukemia (AML), however, the underlying mechanisms remain unclear. CRISPR/Cas9 strategy was employed to knock out CLL-1 gene in U937 cells and western-blot was used to validate the success of knock out. CCK8 and Transwell assays were used to detect cells viability and migration, respectively. RNA-sequencing was performed to profile mRNA expression in CLL-1 gene knock-out and wide type U937 cells. A cutoff of 1.5-fold change and false discovery rate (FDR) <0.05 was used to screen differentially expressed genes (DEGs), which were presented by volcano plots and hierarchical cluster heatmap. Protein-protein interaction (PPI) network was constructed by String database and Cytoscape software. Furthermore, hub genes were mined by CytoNCA and MCODE, which were subjected to functional enrichment using R package. Finally, the findings were validated using qRT-PCR and western-blot. The protein level of CLL-1 was significantly lowered, and cell viability and migration were suppressed in knock-out cells compared to wide type. Using RNA-sequencing and bioinformatics analysis, 452 DEGs (179 up-regulated and 273 down-regulated) were obtained, and several important hub genes (such as CCR2, FBXO21, UBB and UBE2C) were filtered out, which were enriched in 132 GO terms and 36 KEGG pathways such as chemokine signaling pathway and ubiquitin mediated proteolysis. A total of 8 representative genes mRNA expressions were validated by qRT-PCR, and the protein levels of 6 genes were confirmed by western-blot. CLL-1 gene might exert its role in AML through modulating genes enriched in multiple functions such as chemokine signaling and ubiquitination. Our results may give us new knowledge of CLL-1 in AML and provide a basis for mining novel targets.

genetics↗

bioRxiv2022

Single-nucleotide Differences and Cell Type Decide the Subcellular Localization of miRNA Isoforms (isomiRs), tRNA-derived Fragments (tRFs) and rRNA-derived Fragments (rRFs)

BackgroundMicroRNAs (miRNAs) and their isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent ~95% of all short RNAs found in cells. All three types modulate mRNA and protein abundance and are dysregulated in diseases. Experimental studies to date assumed that the subcellular localization of these molecules is well understood and constant across cell types. ResultsWe investigated the localization of isomiRs, tRFs, and rRFs in biological replicates from three frequently-used model cell lines. In each case, we analyzed the contents of the nucleus, cytoplasm, whole mitochondrion, mitoplast, and the whole cell. We used a rigorous mathematical model to account for cross-fraction contamination and technical errors and adjusted abundances accordingly. We found that isomiRs, tRFs, and rRFs exhibit complex and unexpected patterns of subcellular localization. These patterns depend on the type of the RNA molecule, its exact sequence, and the cell type. Even for "sibling" RNAs from the same parental RNA whose sequences differ by only a few nucleotides, their subcellular localization depends on each siblings exact sequence and the cell type. ConclusionsPrevious studies of isomiRs, tRFs, and rRFs that used ectopic expression without accounting for isoforms may need to be re-evaluated. Future experiments with these molecules will need to distinguish among the multiple isoforms and account for the fact that each isoforms abundance and destination depend on its exact sequence and cell type. The findings additionally suggest the existence of an intracellular trafficking program for isomiRs, tRFs, and rRFs and, by extension, expanded roles for these molecules - both dimensions await characterization. To help design future experiments, we compiled a first-of-its-kind Atlas to catalogue the subcellular localization and abundance of 5,898 isomiRs, tRFs, and rRFs across three model cell lines. Results SummaryO_LIWe analyzed the distribution of microRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) in the{whitebullet} nucleus {whitebullet}cytoplasm {whitebullet}mitochondrion, and {whitebullet}mitoplast of biological replicates from three cell lines from the same tissue. C_LIO_LIWe corrected the measured abundances by accounting for cross-fraction contamination and technical errors through a rigorous mathematical model. C_LIO_LIOur analysis revealed complex localization patterns involving numerous isomiRs, tRFs, and rRFs. C_LIO_LIThe subcellular localization of these RNAs depends on their exact sequence and differs even for molecules that arise from the same parental miRNA, tRNA, or rRNA. C_LIO_LIFor a given RNA, its subcellular localization additionally depends on cell type. C_LIO_LIThe findings have implications for previous and future molecular studies of the function of isomiRs, tRFs, and rRFs. C_LIO_LIThe findings suggest the existence of a complex subcellular trafficking program, and hint at expanded functions for these RNA molecules that differ by compartment. C_LIO_LITo assist with the design of future experiments, we created a first-of-its-kind Atlas that catalogues the subcellular distribution and abundance of 5,898 isomiRs, tRFs, and rRFs across three cell lines. C_LI

molecular biology↗

bioRxiv2022

Development of Equine Polyclonal Antibodies as a Broad-Spectrum Therapy Against SARS-CoV-2 Variants

The Coronavirus disease 19 (COVID-19) pandemic has accumulated over 550 million confirmed cases and more than 6.34 million deaths worldwide. Although vaccinations has largely protected the population through the last two years, the effect of vaccination has been increasingly challenged by the emerging SARS-CoV-2 variants. Although several therapeutics including both monoclonal antibodies and small molecule drugs have been used clinically, high cost, viral escape mutations, and potential side effects have reduced their efficacy. There is an urgent need to develop a low cost treatment with wide-spectrum effect against the novel variants of SARS-CoV-2. Here we report a product of equine polyclonal antibodies that showed potential broad spectrum neutralization effect against the major variants of SARS-CoV-2. The equine polyclonal antibodies were generated by horse immunization with the receptor binding domain (RBD) of SARS-CoV-2 spike protein and purified from equine serum. A high binding affinity between the generated equine antibodies and the RBD was observed. Although designed against the RBD of the early wild type strain sequenced in 2020, the equine antibodies also showed a highly efficient neutralization capacity against the major variants of SARS-CoV-2, including the recent BA.2 Omicron variant (IC50 =1.867g/ml) in viral neutralization assay in Vero E6 cells using live virus cultured. The broad-spectrum neutralization capacity of the equine antibodies was further confirmed using pseudovirus neutralization assay covering the major SARS-CoV-2 variants including wild type, alpha, beta, delta, and omicron, showing effective neutralization against all the tested strains. Ex vivo reconstructed human respiratory organoids representing nasal, bronchial, and lung epitheliums were employed to test the treatment efficacy of the equine antibodies. Antibody treatment protected the human nasal, bronchial, and lung epithelial organoids against infection of the novel SARS-CoV-2 variants challenging public health, the Delta and Omicron BA.2 isolates, by reducing >95% of the viral load. The equine antibodies were further tested for potential side effects in a mouse model by inhalation and no significant pathological feature was observed. Equine antibodies, as a mature medical product, have been widely applied in the treatment of infectious diseases for more than a century, which limits the potential side effects and are capable of large scale production at a low cost. A cost-effective, wide-spectrum equine antibody therapy effective against the major SARS-CoV-2 variants can contribute as an affordable therapy to cover a large portion of the world population, and thus potentially reduce the transmission and mutation of SARS-CoV-2.

immunology↗

bioRxiv2022

BIN2 phosphorylates the Thr280 of CO to restrict its function in promoting Arabidopsis flowering

CONSTANS (CO) is a central regulator of floral initiation in response to photoperiod. In this study, we show that the GSK3 kinase BIN2 physically interacts with CO and the gain-of-function mutant bin2-1 displays late flowering phenotype through down-regulation of FT transcription. Genetic analyses show that BIN2 genetically acts upstream of CO in regulating flowering time. Further, we illustrate that BIN2 phosphorylates the Thr280 residue of CO. Importantly, the BIN2 phosphorylation of Thr280 residue restricts the function of CO in promoting flowering. Moreover, we reveal that the N-terminal part of CO harboring the B-Box domain mediates the interaction of both CO-CO and BIN2-CO. We find that BIN2 inhibits the formation of CO dimer/oligomer. Taken together, this study reveals that BIN2 regulates flowering time through phosphorylating the Thr280 of CO and inhibiting the CO-CO interaction in Arabidopsis. HighlightBIN2 regulates flowering time through phosphorylating the Thr280 of CO in Arabidopsis.

molecular biology↗

bioRxiv2022

Maintaining hypoxia environment of subchondral bone alleviates osteoarthritis progression

Abnormal subchondral bone remodeling featured by over-activated osteoclastogenesis leads to articular cartilage degeneration and osteoarthritis (OA) progression, but the mechanism is still unclear. In this study, we used lymphocyte cytosolic protein 1 (Lcp1) knock-out mice to suppress subchondral osteoclast formation in mice OA model with anterior cruciate ligament transection (ACLT) and Lcp1-/- mice showed decreased bone remodeling and sensory innervation in subchondral bone accompanied by retarded cartilage degeneration. For mechanisms, in wildtype mice with ACLT the activated osteoclasts in subchondral bone induced type-H vessels and elevated oxygen concentration which ubiquitylated hypoxia-inducible factor 1 (HIF-1), vital for maintaining chondrocyte homeostasis in articular chondrocytes and led to cartilage degeneration. Deletion of Lcp1 impeded osteoclast-mediated angiogenesis, which maintained the low levels of oxygen partial pressure (pO2) in subchondral bone as well as the whole joint and delayed the OA progression. Stabilization of HIF-1 delayed cartilage degeneration and knockdown of Hif1a abolished the protective effects of Lcp1 knockout. Notably, we identified a novel subgroup of hypertrophic chondrocytes highly associated with OA by single cell sequencing analysis of human articular chondrocytes. Lastly, we showed that Oroxylin A, an Lcp1-encoded protein L-plastin (LPL) inhibitor, could alleviate OA progression. In conclusion, maintaining hypoxic environment in subchondral bone is an attractive strategy for OA treatment. TeaserInhibiting subchondral osteoclastogenesis alleviates OA progression via maintaining joint hypoxia environment.

biochemistry↗