Biology subjects

Shi, J.

Publications and source records attributed to Shi, J..

At least 217 records · Page 12Linked to original sources

bioRxiv2022

ABL1 Kinase plays an important role in spontaneous and chemotherapy-induced genomic instability in multiple myeloma

Genomic instability contributes to cancer progression and is at least partly due to dysregulated homologous recombination. Here, we show that an elevated level of ABL1 kinase overactivates the HR pathway and causes genomic instability in multiple myeloma (MM) cells. Inhibiting ABL1 with either shRNA or a pharmacological inhibitor (nilotinib) inhibits HR activity, reduces genomic instability, and slows MM cell growth. Moreover, inhibiting ABL1 rescues the HR dysregulation and genomic instability caused by melphalan, a chemotherapeutic agent used in MM treatment, and increases melphalans efficacy and cytotoxicity in vivo in a subcutaneous tumor model. In these tumors, nilotinib inhibits endogenous as well as melphalan-induced HR activity. These data demonstrate that inhibiting ABL1 using the clinically approved drug nilotinib reduces MM cell growth, promotes genome stability, increases the cytotoxicity of melphalan (and similar chemotherapeutic agents), and can potentially prevent or delay progression in MM patients.

cancer biology↗

bioRxiv2022

Apurinic/apyrimidinic nuclease 1 drives genomic evolution contributing to chemoresistance and tumorigenesis in solid tumor

Genomic instability fuels genomic alterations that befit cancer cells with necessary adaptations to keep proliferating and overcome the impact of host anti-tumor immunity and cytotoxic therapy. Since DNA breaks are required for genomic rearrangements to take place, we hypothesized that dysregulated nuclease activity mediates genomic instability in cancer. Using an integrated genomics protocol, we identified a four gene deoxyribonuclease signature correlating with genomic instability in six human cancers which included adenocarcinomas of esophagus (EAC), lung, prostate, stomach, pancreas and triple negative breast cancer. Functional screens confirmed the role of these nucleases in genomic instability and growth of cancer cells. Apurinic/apyrimidinic nuclease 1 (APE1), identified as top nuclease in functional screen, was further investigated in five cell lines representing four solid tumors (EAC, lung, prostate and breast cancer). We demonstrate that chemical as well as transgenic suppression of APE1 impaired growth/colony formation and increased cytotoxicity of chemotherapeutic agent, whereas inhibited spontaneous as well as chemotherapy-induced DNA breaks, homologous recombination (HR) activity and genomic instability in all cancer cell types tested. Treatment with APE1 inhibitor also impaired tumor growth and significantly increased efficacy of a chemotherapeutic agent in a subcutaneous mouse model of EAC. Overexpression of APE1 in normal esophageal epithelial cells increased DNA breaks and HR activity, leading to massive mutational, copy number as well as karyotypic instability. Evaluation of by whole genome sequencing identified HR as the top mutational process activated by APE1. Normal cells overexpressing APE1 grew as tumors in mice and tumors removed from mice displayed additional karyotypic changes, providing evidence of genomic instability in vivo. Overall, our data demonstrate that elevated APE1 dysregulates HR activity, G2/M checkpoint and genome stability thus contributing to tumorigenesis and chemoresistance in cancer. Therefore, inhibitors of APE1 have potential to inhibit growth and increase cytotoxicity of chemotherapeutic agents while minimizing spontaneous as well as chemotherapy-induced genomic damage and instability in EAC and other solid tumors.

cancer biology↗

bioRxiv2022

Self-avoidance alone does not explain the function of Dscam1 in mushroom body axonal wiring

Alternative splicing of Drosophila Dscam1 into 38,016 isoforms provides neurons with a unique molecular code for self-recognition and self-avoidance. A canonical model suggests that homophilic binding of identical Dscam1 isoforms on sister branches of mushroom body (MB) axons supports segregation with high fidelity, even when only a single isoform is expressed. Here we generated a series of mutant flies with a single exon 4, 6, or 9 variant, encoding 1,584, 396, or 576 potential isoforms, respectively. Surprisingly, most of the mutants in the latter two groups exhibited obvious defects in the growth, branching, and segregation of MB axonal sister branches. This demonstrates that repertoires of 396 and 576 Dscam1 isoforms were not sufficient for normal patterning of axonal branches. Moreover, reducing Dscam1 levels largely reversed the defects caused by reduced isoform diversity, suggesting a functional link between Dscam1 expression levels and isoform diversity. Taken together, these results indicate that canonical self-avoidance alone does not explain the function of Dscam1 in MB axonal wiring.

neuroscience↗

bioRxiv2022

CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever

Dengue virus infection can cause dengue hemorrhagic fever (DHF). Dengue NS1 is multifunctional: the intracellular dimeric NS1 (iNS1) forms part of the viral replication complex, the extracellular multi-oligomeric secreted NS1 (sNS1) is a major factor contributing to DHF. The structure of the iNS1 is well studied but not sNS1. Here we show the tetrameric (stable and loose conformation) and hexameric structures of sNS1. Stability of the stable and loose tetramers is determined by the conformation of their N-terminal domain - elongated {beta}-sheet or {beta}-roll. Binding of an anti-NS1 Fab breaks the loose tetrameric and hexameric sNS1 into dimers, whereas the stable tetramer remains largely unbound. Our results show detailed quaternary organization of different oligomeric states of sNS1 and will contribute towards the design of dengue therapeutics.

molecular biology↗

bioRxiv2022

Long-range inhibition synchronizes and updates prefrontal task activity

Changes in patterns of activity within the medial prefrontal cortex enable rodents, non-human primates, and humans to update their behavior to adapt to changes in the environment, e.g., during cognitive tasks1-5. Within medial prefrontal cortex, inhibitory neurons expressing parvalbumin are important for updating strategies in a rule shift task6-8. Nevertheless, causal mechanisms through which parvalbumin neurons recruit specific circuits to produce prefrontal network dynamics that switch from maintaining to updating task-related patterns of activity remain unknown. Here we identify a new long-range inhibitory projection from prefrontal parvalbumin neurons to the contralateral cortex that mediates this process. Whereas nonspecifically inhibiting all callosal projections does not prevent mice from learning rule shifts, selectively inhibiting these callosal parvalbumin projections disrupts rule shift learning, desynchronizes gamma-frequency activity that is necessary for learning8, and suppresses changes in prefrontal activity patterns that normally accompany rule shifts. Thus, callosal parvalbumin projections switch prefrontal circuits from maintaining to updating behavioral strategies by synchronizing callosal communication and preventing it from inappropriately maintaining outdated neural representations. These findings may explain how deficits in prefrontal parvalbumin neurons and gamma synchrony cause impaired behavioral flexibility in schizophrenia9-10, and identify long-range projections from prefrontal parvalbumin neurons as a key circuit locus for understanding and treating these deficits.

neuroscience↗

bioRxiv2022

The addition of antibiotics to embryo culture media caused altered expression of genes in pathways governing DNA integrity in mouse blastocysts

Antibiotics are common components of embryo culture media and minimize the risk of microbial contamination and infection during assisted reproductive technology procedures (ART). The effects of two aminoglycoside antibiotics (gentamicin, streptomycin) and penicillin on the global profiles of gene expression (DE) were assessed by RNA-seq of individual mouse blastocysts. Zygotes were cultured in an optimized defined medium formulation (KSOM) to which a dose range of each antibiotic was added. A dose-dependent retardation of the rate of zygote development to morphologically normal blastocyst was observed and this was accompanied by a reduction in the number of cells present within the resulting blastocysts. The lowest dose of each antibiotic tested (similar to the concentrations used in clinical grade media) caused significant differential expression of approximately 1800 genes. In most cases antibiotic treatment caused a reduction in gene expression and gene ontology analysis showed that down regulated genes were enriched for several biological processes related to the maintenance of genomic integrity. All three antibiotics caused the downregulation of Brca2, Blm, Rad51c and Rad54l, genes involved DNA homologous recombination pathways and also several p53-dependent genes. Immunolocalization studies showed that each antibiotic also reduced level of BRCA2 and RAD51C detected within blastocysts. The present study shows that the supplementing embryo culture media with antibiotics is associated with wide ranging alterations in gene expression in a manner that could potentially compromise the genomic integrity of the resulting embryos.

developmental biology↗

bioRxiv2022

Photoacoustic imaging reveals mechanisms of rapid-acting insulin formulations dynamics at the injection site

ObjectiveUltra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog(R) and Lyumjev(R) formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. MethodsWe employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. ResultsThrough experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. ConclusionsThis study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and the formulation excipients, which could be useful to make faster and better controlled insulin formulations in the future. HighlightsO_LIHexamer dissociation is not the absorption rate-limiting step for insulin lispro C_LIO_LILyumjev excipients enhance insulin microvascular absorption and diffusion C_LIO_LIVascular endothelial transit determines the overall absorption for insulin lispr C_LIO_LIInsulin diffusion studied for the first time at the injection site of live animals C_LIO_LIIn vivo imaging is a powerful tool to study injection site dynamics C_LI

systems biology↗

bioRxiv2022

The Receptor Kinase FER Mediates Phase Separation of Glycine-Rich RNA-Binding Protein 7 to Confer Temperature Resilience in Arabidopsis

Temperature fluctuations repress plant growth. Although glycine-rich RNA-binding proteins (GRPs) and cold shock proteins (CSPs) have been implicated in cold adaptation, their physiological roles in the response to temperature fluctuations are largely unknown. The receptor kinase FERONIA (FER), a master regulator of cell growth, phosphorylates GRP7 within its intrinsically disordered region to modulate mRNA alternative splicing in the nucleus. Here we show that natural variations at a GRP7 residue phosphorylated by FER influences GRP7 liquid-liquid phase separation (LLPS), aiding Arabidopsis to grow over a wider temperature range. LLPS of GRP7 in the cytoplasm leads to the formation of stress granules that recruits RNAs, along with the translation machinery component eIF4E1 and mRNA chaperones, CSP1 and CSP3, to inhibit translation. Mutations in FER and the GRP7-LLPS-recruited components attenuate root growth under temperature shift conditions. Our findings illustrate the roles of GRP7 LLPS in improving plant root capacity to withstand temperature fluctuations.

plant biology↗

bioRxiv2022

Design of the mammalian cone photoreceptor to Off bipolar cell synapse

Graded synapses in sensory systems reliably transmit small signals in the presence of continuous quantal noise. To understand how signaling is optimized during graded transmission, we counted the number of vesicles released by a mammalian cone terminal and compared it to the simultaneous responses in each Off bipolar cell type. Off bipolar cells contacting the terminal base comprised two groups depending on how they sampled transmitter release. In both groups, responses initially grew non-linearly with the number of released vesicles implicating a role for cooperativity during sparse release. One group sampled release from most of a cones [~]20 ribbons and can exploit averaging to improve signal reliability. The other, less-sensitive group made 1-3 contacts at the terminal center and responded to pooled transmitter, a consequence of membrane depolarization, using an insensitive kainate receptor. Off bipolar cells use different strategies to minimize transmission noise and encode cone output over different ranges.

neuroscience↗

bioRxiv2022

Estimating the overall fraction of phenotypic variance attributed to high-dimensional predictors measured with error

In prospective genomic studies (e.g., DNA methylation, metagenomics, and transcriptomics), it is crucial to estimate the overall fraction of phenotypic variance (OFPV) attributed to the high-dimensional genomic variables, a concept similar to heritability analyses in genome-wide association studies (GWAS). Unlike genetic variants in GWAS, these genomic variables are typically measured with error due to technical limitation and temporal instability. While the existing methods developed for GWAS can be used, ignoring measurement error may severely underestimate OFPV and mislead the design of future studies. Assuming that measurement error variances are distributed similarly between causal and noncausal variables, we show that the asymptotic attenuation factor equals to the average intraclass correlation coefficients of all genomic variables, which can be estimated based on a pilot study with repeated measurements. We illustrate the method by estimating the contribution of microbiome taxa to body mass index and multiple allergy traits in the American Gut Project. Finally, we show that measurement error does not cause meaningful bias when estimating the correlation of effect sizes for two traits.

genetics↗

bioRxiv2022

Structure of IgG-Fc hexamer reveals a mutual lock-and-key mode of Fc-Fc interaction

IgG exists mainly as monomer, but recent studies suggest that IgG forms hexamer to mediate antibody functions. Although many structures of IgG monomer and its fragments are determined, there is no IgG hexamer structure at atomic level due to the weak Fc-Fc interactions. Here we engineered a hexameric IgG with IgM tailpiece fusion and determined the structure by cryo-EM. IgG-Fc hexamer forms hexagon symmetry with the six Fcs lie in a plane with Fc-Fc interaction in a mutual lock-and-key mode. This structure provides structural insights into Fc-Fc interaction of IgG and reveals molecular basis for its function.

biophysics↗

bioRxiv2022

An allosteric agonist activates BK channels by perturbing coupling between Ca2+ binding and pore opening

BK type Ca2+-activated K+ channels activate in response to both the membrane voltage and intracellular Ca2+ with distinct mechanisms. Ca2+ binds to the cytosolic domain (CTD) to open the pore across the membrane, but the mechanism that couples Ca2+ binding to pore opening is not clear. Here we show that a compound, BC5, identified using in silico screening, interacts with BK channels at the interface between the CTD and the transmembrane voltage sensing domain (VSD) and enhances channel activity by specifically affecting the Ca2+ dependent mechanism. BC5 activates the channel in the absence of Ca2+ binding but Ca2+ binding inhibits BC5 effects. Thus, BC5 perturbs the pathway that couples Ca2+ binding to pore opening to allosterically affect both, which is supported by atomistic simulations and mutagensis. The results suggest that the CTD- VSD interaction makes a major contribution to the mechanism of Ca2+ dependent activation and is an important site for allosteric agonists to modulate BK channel activation.

biophysics↗

bioRxiv2022

A Genome-wide CRISPR Screen Identifies WDFY3 as a Novel Regulator of Macrophage Efferocytosis

AbstractsPhagocytic clearance of dying cells, termed efferocytosis, must occur efficiently to maintain homeostasis and prevent disease. Yet, our understanding of this important biological process remains incomplete. To search for novel regulators of efferocytosis, we performed a FACS-based genome-wide CRISPR knockout screen in primary murine macrophages. We identified a novel role for WDFY3 in efferocytosis by macrophages. WDFY3 deficiency in macrophages specifically impaired uptake, not binding, of apoptotic cells due to defective actin depolymerization. We further revealed that WDFY3 directly interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Although the C-terminus of WDFY3 was sufficient to rescue impaired degradation, full-length WDFY3 is still required for regulating uptake. Finally, WDFY3 is required for efficient efferocytosis in vivo in mice and in primary human macrophages. The work expands our knowledge of the mechanisms of macrophage efferocytosis, and more broadly, provides a general strategy for genome-wide CRISPR screen to interrogate complex functional phenotypes in primary macrophages. HighlightsO_LIFunctional readout for pooled genome-wide CRISPR screen in primary macrophages. C_LIO_LIWDFY3 is discovered as a regulator of macrophage efferocytosis in vitro and in vivo. C_LIO_LIWDFY3 deficiency led to impaired uptake, as opposed to binding, of apoptotic cells due to defective actin depolymerization. C_LIO_LIWDFY3 directly interacts with GABARAP, facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. C_LIO_LIC-terminal WDFY3 is sufficient to regulate the degradation of engulfed apoptotic cells while full-length WDFY is required for regulating uptake. C_LI

cell biology↗

bioRxiv2021

Assessment of chimeric antigen receptor T (CAR-T) cytotoxicity by droplet microfluidics in vitro

Chimeric antigen receptor T (CAR-T) cells are cytotoxic T cells engineered to specifically kill cancer cells expressing specific target receptor(s). Prior CAR-T efficacy tests include CAR expression analysis by qPCR or ELISA, in vitro measurement of interferon-{gamma} (IFN{gamma}) or interleukin-2 (IL-2), and xenograft models. However, the in vitro measurements did not reflect CAR-T cytotoxicity, whereas xenograft models are low throughput and costly. Here we presented a robust in vitro droplet microfluidic assay for CAR-T cytotoxicity assessment. This method not only enabled assessment of CAR-T cytotoxic activity under different fluid viscosity conditions, but also facilitated measurement of CAR-T expansion and dissection of mechanism of action via phenotype analysis in vitro. Furthermore, our data suggested that label-free cytotoxicity analysis is feasible by acquiring data before and after treatment. Hence, this study presented a novel in vitro method for assessment of cellular cytotoxicity that could potentially be applied to any cell-kill-cell experiment with varying solvent composition.

cancer biology↗

bioRxiv2021

Modeling of microRNA-derived disease network repurposes methotrexate for the prevention and therapy of abdominal aortic aneurysm in mice

Abdominal aortic aneurysm (AAA) is a highly lethal vascular disease characterized by permanent dilatation of the abdominal aorta. The main purpose of the current study is to search for noninvasive medical therapies for abdominal aortic aneurysm (AAA), for which there is currently no effective drug therapy. Network medicine represents a cutting-edge technology, as analysis and modeling of disease networks can provide critical clues regarding the etiology of specific diseases and which therapeutics may be effective. Here, we proposed a novel algorithm to quantify disease relations based on a large accumulated microRNA-disease association dataset and then built a disease network that covered 15 disease classes and included 304 diseases. Analysis revealed a number of patterns for these diseases; for example, diseases tended to be clustered and coherent in the network. Surprisingly, we found that AAA showed the strongest similarity with rheumatoid arthritis and systemic lupus erythematosus, both of which are autoimmune diseases, suggesting that AAA could be one type of autoimmune disease in etiology. Based on this observation, we further hypothesized that drugs for autoimmune disease could be repurposed for the prevention and therapy of AAA. Finally, animal experiments confirmed that methotrexate, a drug for autoimmune disease, was able to prevent the formation and inhibit the development of AAA.

bioinformatics↗

bioRxiv2021

CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

It has recently been shown that CFIm25, a canonical mRNA 3 processing factor, could play a variety of physiological roles through its molecular function in the regulation of mRNA alternative polyadenylation (APA). Here, we used CRISPR/Cas9-mediated gene editing approach in human embryonic stem cells (hESCs) for CFIm25, and obtained three gene knockdown/mutant cell lines. CFIm25 gene editing resulted in higher proliferation rate and impaired differentiation potential for hESCs, with these effects likely to be directly regulated by the target genes, including the pluripotency factor rex1. Mechanistically, we unexpected found that perturbation in CFIm25 gene expression did not significantly affect cellular mRNA 3 processing efficiency and APA profile. Rather, we provided evidences that CFIm25 may impact RNA polymerase II (RNAPII) occupancy at the body of transcribed genes, and promote the expression level of a group of transcripts associated with cellular proliferation and/or differentiation. Further study indicated that CFIm25 association with LEO1, an RNAPII associated factor, might contribute to the effect. Taken together, these results reveal novel mechanisms underlying CFIm25s modulation in determination of cell fate, and provide evidence that the process of mammalian gene transcription may be regulated by an mRNA 3 processing factor.

molecular biology↗

bioRxiv2021

Rhythmic auditory stimulation rescues cognitive flexibility in mutant mice with impaired gamma synchrony

Neural synchronization at gamma ([~]40 Hz) frequencies is believed to contribute to brain function and be deficient in disorders including Alzheimers disease and schizophrenia. Gamma-frequency sensory stimulation has been proposed as a non-invasive treatment for deficient gamma synchrony and associated cognitive deficits, and has been shown to be effective in mouse models of Alzheimers disease. However, both the mechanism and applicability of this approach remain unclear. Here we tested this approach using mutant (Dlx5/6+/-) mice which have deficits in gamma synchrony and the ability to learn to shift between rules which use different types of cues to indicate reward locations. 40 Hz auditory stimulation rescues rule shifting in Dlx5/6+/- mice. However, this improvement does not outlast the period of stimulation, and is not associated with normalized gamma synchrony, measured using genetically encoded voltage indicators. These results show how gamma-frequency sensory stimulation may improve behavior without fully restoring normal circuit function.

neuroscience↗

bioRxiv2021

Fast anther dehiscence state recognition system establishing by deep learning to screen heat tolerant cotton

Cotton is one of the most economically important crops in the world. The fertility of male reproductive organs is a key determinant of cotton yield. The anther dehiscence or indehiscence directly determine the probability of fertilization in cotton. Thus, the rapid and accurate identification of cotton anther dehiscence status is important for judging anther growth status and promoting genetic breeding research. The development of computer vision technology and the advent of big data have prompted the application of deep learning techniques to agricultural phenotype research. Therefore, two deep learning models (Faster R-CNN and YOLOv5) were proposed to detect the number and dehiscence status of anthers. The single-stage model based on YOLOv5 has higher recognition efficiency and the ability to deploy to the mobile end. Breeding researchers can apply this model to terminals to achieve a more intuitive understanding of cotton anther dehiscence status. Moreover, three improvement strategies of Faster R-CNN model were proposed, the improved model has higher detection accuracy than YOLOv5 model. In addition, the percentage of dehiscent anther of randomly selected 30 cotton varieties were observed from cotton population under normal temperature and high temperature (HT) conditions through the integrated Faster R-CNN model and manual observation. The result showed HT varying decreased the percentage of dehiscent anther in different cotton lines, consistent with the manual method. Thus, this system can help us to rapid and accurate identification of HT-tolerant cotton. One sentence summaryThe deep learning technique was applied to identify the anther dehiscence state for the first time to quickly screen heat tolerant cotton varieties and help to explore key genetic improvement genes.

bioengineering↗