Biology subjects

Song, J.

Publications and source records attributed to Song, J..

At least 163 records · Page 9Linked to original sources

bioRxiv2023

Phenotypic plasticity of antibiotic resistance, metabolism byproduct utilization and the evolution of mutually beneficial cooperation in Escherichia coli

Although tag-based donation and recognition have well explained how the cooperative individuals are positively assorted if the cooperative individuals possess some signals and are also able to detect such signals, an additional mechanism is required to explain why some individuals pay the costs of evolving such a tag that may not be rewarded subsequently, and how such tag-based cooperative individuals will meet other similar individuals with a very low mutation rate. Here, we show that many and even all Escherichia coli bacteria cells in the increased antibiotic concentration will plastically evolve to be antibiotic resistant individuals who could protect antibiotic sensitive strain from the attack of antibiotics, and the antibiotic resistant strain could reversibly evolve to be antibiotic sensitive in non-antibiotic supplement medium but in a harsher environment with low glucose. A further experiment showed that antibiotic-sensitive E. coli strain could in turn help reduce the concentration of indole produced by the resistant strain. This metabolic product is harmful to the growth of the antibiotic-resistant strain but benefits the antibiotic-sensitive strain by helping turn on the multi-drug exporter to discharge the antibiotic. The utilization of metabolism byproduct indole produced by antibiotic-resistant cells benefits antibiotic-sensitive cells, while the indole-absorbing service of antibiotic sensitive cells unconsciously help in nullifying the indole side effect on antibiotic resistant strain, and a mutual benefit cooperation could therefore evolve.

ecology↗

bioRxiv2023

ATP energy-independentlymodulates the folding equilibrium of ALScausing C71G-hPFN1

C71G is the most aggregation-prone and toxic mutant of 140-residue human profin-1 (hPFN1) that causes familial ALS by gain of toxicity, but its underlying mechanisms still remain unknown. C71G-hPFN1 exists in an equilibrium between folded and unfolded states, whose dynamic/thermodynamic properties and modulation are not yet defined. Here, we utilized NMR to quantify the populations to be 55.2% and 44.8% respectively for folded and unfolded states exchanging at 11.7 Hz. Intriguingly, even the folded state of C71G-hPFN1 has increased ps-ns flexibility and reduced thermodynamic stability, thus rationalizing its high aggregation-proneness. Strikingly, C71G-hPFN1 provides a unique model to unambiguously visualize the effects of ATP and 11 related molecules on its folding equilibrium by NMR. Unexpectedly, ATP completely converted C71G-hPFN1 into the folded state at 1:2, which is physiologically relevant in most living cells. By contrast, TMAO, a well-known protein-folding inducer, showed no detectable conversion even at 1:2000. Surprisingly, the inducing capacity of ATP comes from its triphosphate group, but free triphosphate strongly triggered aggregation. The inducing capacity was determined to rank as: ATP = ATPP = PPP > ADP = AMP-PNP = AMP-PCP = PP, while AMP, Adenosine, P and NaCl showed no detectable capacity. Mechanistically, ATP and triphosphate appear to enhance the intrinsic folding capacity encoded by the sequence. Therefore, by joining Adenosine and triphosphate ATP appears to integrate three abilities: inducing folding, inhibiting aggregation and increasing stability. Our study provide a mechanism for the finding that some single-cell organisms still use polyphosphates as primordial chaperones. Moreover, ATP continue to play foundational roles in modern cells, shedding light on the longstanding enigma of the age-related onset of FALS, which coincides with the ageing-dependent reductions in ATP concentrations.

biophysics↗

bioRxiv2023

Mapping Human Tissues with Highly Multiplexed RNA in situ Hybridization

In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. Recently there has been a surge of multiplexed RNA in situ techniques but their application to human tissues and clinical biopsies has been limited due to their large size, general lower tissue quality and high background autofluorescence. Here we report DART-FISH, a versatile padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections at cellular resolution. We introduced an omni-cell type cytoplasmic stain, dubbed RiboSoma that substantially improves the segmentation of cell bodies. We developed a computational decoding-by-deconvolution workflow to extract gene spots even in the presence of optical crowding. Our enzyme-free isothermal decoding procedure allowed us to image 121 genes in a large section from the human neocortex in less than 10 hours, where we successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. Additionally, we demonstrated the detection of transcripts as short as 461 nucleotides, including neuropeptides and discovered new cortical layer markers. We further performed in situ mapping of 300 genes on a diseased human kidney, profiled >20 healthy and pathological cell states, and identified diseased niches enriched in transcriptionally altered epithelial cells and myofibroblasts.

genomics↗

bioRxiv2023

NUCLEOPORIN1 mediates proteasome-based degradation of ABI5 to regulate Arabidopsis seed germination

NUCLEOPORIN1 (NUP1), a member of the Nuclear Pore Complex (NPC), is located on the inner side of the nuclear membrane. It is highly expressed in seeds; however, its role in seeds including germination has not been explored yet. Here, we identified an abscisic acid (ABA) hypersensitive phenotype of nup1 during germination. ABA treatment drastically changes the expression pattern of thousands of genes in nup1, including the major transcription factors (TFs) involved in germination, ABI3, ABI4, and ABI5. Double mutant analysis of NUP1 and these ABA-related genes showed that mutations in ABI5 can rescue the phenotype of nup1, suggesting that NUP1 acts upstream of ABI5 to regulate seed germination. ABI5, a key negative regulator of germination, is abundant in dry seeds and rapidly degrades during germination. However, its spatiotemporal regulation and interaction with other molecular players during degradation remained to be fully elucidated. We found that NUP1 is physically associated with ABI5 and the 26S proteasome. Mutation in NUP1 delayed ABI5 degradation through its post-translational retention in nucleolus under abiotic stress. Taken together, our findings suggest that NUP1 anchors the proteasome to NPC and modulates seed germination through proteasome-mediated degradation of ABI5 in the vicinity of NPC in the nucleoplasm.

plant biology↗

bioRxiv2023

Human receptive endometrial organoid for deciphering the implantation window

Human endometrial receptivity is a critical determinant of pregnancy success; however, in vivo studies of its features and regulation are particularly challenging due to ethical restriction. Recently, the development of human endometrial assembloids has provided a powerful model to investigate this intricate biological process. In this study, we established a specialized human window-of-implantation (WOI) endometrial assembloid system that mimics the in vivo receptive endometrium. It not only reproduces the structural attributes of pinopodes and cilia, but also molecular characteristics of mid-secretory endometrium. Furthermore, the WOI endometrial assembloid exhibits hormone responsiveness, an energy metabolism profile characterized by larger and functionally enhanced mitochondria, increased ciliary assembly and motility, and epithelial-mesenchymal transition (EMT), as well as promising potential for embryo implantation. As such, WOI assembloids hold great promise as a platform to unravel the intricate mechanisms governing the regulation of endometrial receptivity, maternal-fetal interactions, and associated pathologies, ultimately driving impactful advancements in the field.

cell biology↗

bioRxiv2023

Prominent contributions of selective neuronal subtypes to pediatric drug-resistant focal dysplasia

About 75% cases of epilepsy begin during childhood, and 10 - 30% of pediatric epilepsy patients are resistant to drug treatment. The predominant seizure type in children is focal cortical dysplasia (FCD) which is highly associated with drug-resistant epilepsy, but its underlying cause is poorly understood. We performed single-cell RNA sequencing and patch-clamp recording on fresh brain tissues obtained from pediatric FCD patients shortly after surgery to reveal critical factors contributing to FCD. We report that known epilepsy genes or significant transcriptomic alterations occur in only a few neuronal subgroups, suggesting that epilepsy-associated neurons cluster into only a few neuronal subtypes. These epilepsy-subtypes displayed significant epilepsy-related transcriptomic alterations, especially in the genes associated with excitation/inhibition balance and neuron functional signal pathways. Among eight epilepsy-subtypes, L2_4_CUX2_YWHAH and PVALB_RGS5 subtypes showed the most prominent alterations in FCD patient tissues. Supporting PV-neurons being important, recording from fast-spiking/parvalbumin-containing neurons in acute FCD patient brain slices revealed reduced excitatory synaptic inputs, implicating lower activity in these neurons and fewer synaptic inputs onto them. A higher percentage of mitochondria genes, and likely higher activity in the pathways associated with oxidative phosphorylation and ATP biosynthetic process, was observed in the epilepsy-subtypes, suggesting a high energy expenditure in them. Interestingly, the activity of the above two pathways in most epilepsy-subtypes was lower in the FCD patients, suggesting these subtypes may be more vulnerable to energy deficit in epilepsy. Altogether, transcriptomics is significantly altered in only a few neuronal subtypes in pediatric FCD patient brains. These selective epilepsy-subtypes may play prominent roles in the genesis of pediatric drug-resistant seizure and targeting them may provide new treatment options.

bioinformatics↗

bioRxiv2023

Manipulation of Bacterial ROS Production Leads to Self-escalating DNA Damage and Resistance-resistant Lethality for Intracellular Mycobacteria

The high prevalence of drug resistance in mycobacteria calls for antimicrobial mechanisms that suppresses the development of resistance. As a structurally conserved multi-site bio-macromolecule, DNA is presumed to be an ideal candidate for such resistance-resistant drug target. However, survey of marketed and investigational DNA interactors indicates that they are not immune to resistance development. Here, we report our strategy to achieve real resistance-resistant DNA targeting by incurring "catastrophic" DNA damage with an organoruthenium-natural product hybrid. The dual-mode DNA damage, in the form of strong tri-valent binding and concomitant oxidative modification, is achieved by manipulating of bacterias native endogenous ROS production mechanism upon lethal stress (such as DNA binding). Such self-escalating DNA damage, together with precise targeting of intracellular bacteria via vacuole fusion, thus endows the hybrids resistance-resistant lethality against mycobacteria and in vivo efficacy in animal models.

microbiology↗

bioRxiv2023

Dendrimeric DNA Coordinate Barcoding Design for Spatial RNA Sequencing

Spatially resolved transcriptomic technologies show promise in revealing complex pathophysiological processes, but developing sensitive, high-resolution, and cost-effective methodology is challenging. Here, we report a dendrimeric DNA coordinate barcoding design for spatial RNA sequencing (Decoder-seq). This technology combined dendrimeric nano-substrates with microfluidic coordinate barcoding to generate high-density spatial DNA arrays with deterministically combinatorial barcodes in a resolution-flexible and cost-effective manner ([~]$0.5/mm2). Decoder-seq achieved high RNA capture efficiency, [~]68.9% that of in situ sequencing, and enhanced the detection of lowly expressed genes by [~]five-fold compared to 10x Visium. Decoder-seq visualized a spatial single-cell atlas of mouse hippocampus at near-cellular resolution (15 m) and revealed dendrite-enriched mRNAs. Application to renal cancers dissected the heterogeneous tumor microenvironment of two subtypes, and identified spatial gradient expressed genes with the potential in predicting tumor prognosis and progression. Decoder-seq is compatible with sensitivity, resolution, and cost, making spatial transcriptomic analysis accessible to wider biomedical applications and researchers.

genomics↗

bioRxiv2023

Multi-omics data and analysis reveal the formation of key pathways of different colors in Torenia fournieri flowers

AbstractTorenia fournieri Lind. is an ornamental plant, popular for its numerous flowers and variety of colors. However, its genomic evolution, as well as the genetic and metabolic basis of flower color formation, remain poorly understood. Here we report a chromosome-level reference genome of T. fournieri comprising 164.4 Mb. Phylogenetic analysis revealed the phylogenetic placement of the species, and comparative genomics analysis indicated that T. fournieri shared a whole genome duplication (WGD) event with Antirrhinum majus. Through joint transcriptomics and metabolomics analyses, we characterized the differential genes and metabolites in the anthocyanin synthesis pathway in five T. fournieri varieties. We identified many metabolites related to pelargonidin, peonidin, and naringenin in Rose (R) color samples. On the other hand, the blue (B) and blue-violet (D) color samples contained many metabolites related to petunidin, cyanidin, quercetin, and malvidin. The formation of different flower colors in T. fournieri involves multiple genes and metabolites. We analyzed the results and obtained significantly different genes and metabolites related to the biosynthesis of flavonoids and anthocyanins, which are key metabolites in the formation of different flower colors. Our T. fournieri genome data provide a basis for studying the differentiation of this species and provide a valuable model genome enabling genetic studies and genomics-assisted breeding of T. fournieri. HighlightThe genome of Torenia fournieri was reported for the first time, and the formation mechanism of different colors in Torenia fournieri flowers was analyzed by genomics, transcriptomics and metabolomics.

genomics↗

bioRxiv2023

CyVerse: Cyberinfrastructure for Open Science

CyVerse, the largest publicly-funded open-source research cyberinfrastructure for life sciences, has played a crucial role in advancing data-driven research since the 2010s. As the technology landscape evolved with the emergence of cloud computing platforms, machine learning and artificial intelligence (AI) applications, CyVerse has enabled access by providing interfaces, Software as a Service (SaaS), and cloud-native Infrastructure as Code (IaC) to leverage new technologies. CyVerse services enable researchers to integrate institutional and private computational resources, custom software, perform analyses, and publish data in accordance with open science principles. Over the past 13 years, CyVerse has registered more than 110,000 verified accounts from 160 countries and was used for over 1,600 peer-reviewed publications. Since 2011, 45,000 students and researchers have been trained to use CyVerse. The platform has been replicated and deployed in two countries outside the US, with additional private deployments on commercial clouds for US government agencies and multinational corporations. In this manuscript, we present a strategic blueprint for creating and managing SaaS cyberinfrastructure and IaC as free and open-source software.

scientific communication and education↗

bioRxiv2023

TPR domain assigns versatility of BcTir/Tpr system against viral infection

NAD+-derived signal produced by TIR domain triggered the host immune responses. The ubiquitous TPR domain involved in signal recognition and effector activation were found widely assembled with the TIR domain. However, the immune roles of these assemblies remain elusive. Here, a two-gene operon, one containing a TIR domain, designated as BcTir, and the other, BcTpr, from Bacillus cereus, exhibited anti-phage immunity. BcTpr, but not BcTir, exhibited NADase activity to produce the cyclic ADPR (cADPR) isomer and mediate NAD+ depletion. Noticeably, the truncated N terminus of BcTpr only depleted NAD+ unless at the presence of TPR domain to generate cADPR isomer unveiling its role played for glycosite selection. In addition, the BcTir/Tpr system significantly repressed viral proliferation and increased oxidation resistance by scavenging excessive reactive oxygen species (ROS) upon phage infection. These findings unraveled a multifunctional role of the BcTir/Tpr system during immune responses. In BriefThe bacterial BcTir/Tpr system was identified with the ability to protect against phage infection via NAD+ depletion, viral replication repression, and ROS homeostasis, in which BcTpr played a dual role in NAD+-derived signal production and NAD+ depletion. HighlightsO_LIThe BcTir/Tpr system works as a BcTir-BcTpr complex against phage infection. C_LIO_LIBcTpr, instead of BcTir, generates the NAD+-derived cADPR isomer through its glycosidase domain at N terminus. C_LIO_LIThe amount of cADPR isomer production is regulated by the helix numbers of the TPR domain at C terminus of BcTpr. C_LIO_LIThe BcTir/Tpr system can depress phage proliferation and decrease ROS production upon phage infection. C_LI

microbiology↗

bioRxiv2023

ATP and Zn2+ induce folding of ALS-associated hSOD1 with fundamentally distinctive mechanisms

153-residue copper-zinc superoxide dismutase 1 (hSOD1) is the first gene whose mutation was linked to FALS, while wild-type hSOD1 aggregation is associated with SALS. So far >180 ALS-causing mutations have been identified within hSOD1, but the underlying mechanism still remains enigmatic. Mature hSOD1 is extremely stable constrained by a disulfide bridge to adopt a Greek-key {beta}-barrel fold that houses copper and zinc cofactors. Conversely, nascent hSOD1 is unfolded and prone to aggregation, requiring Zn2+ to initiate folding to a coexistence of folded and unfolded states. Recent studies demonstrate mutations disrupting Zn2+-binding correlate with their capacity to form toxic aggregates. Therefore, to decode the role of cations in hSOD1 folding provide not only mechanistic insights, but also therapeutic prospects for hSOD1-linked ALS. Here by NMR, we visualized the effect of 12 cations, encompassing 8 essential for humans (Na+, K+, Ca2+, Zn2+, Mg2+, Mn2+, Cu2+, Fe2+), 3 mimicking zinc (Ni2+, Cd2+, Co2+), and environmentally abundant Al3+. Surprisingly, most cations, including Zn2+-mimics, exhibited negligible binding or induction for folding of nascent hSOD1. Cu2+ displayed extensive binding to the unfolded state but induced severe aggregation. Unexpectedly, here for the first time Fe2+ was deciphered to have Zn2+-like folding-inducing ability. Surprisingly, Zn2+ failed to induce folding of H80S/D83S-hSOD1, while Fe2+ could. By contrast, Zn2+ could trigger folding of G93A-hSOD1, but Fe2+ failed. Notably, pre-existing Fe2+ disrupted the Zn2+-induced folding of G93A-hSOD1. Comparing with ATP-induced folded state, our results delineate that hSOD1 maturation requires: 1) intrinsic folding capacity encoded by the sequence; 2) specific Zn2+-coordination; 3) disulfide formation and Cu-load catalyzed by hCCS. This study decodes a previously-unknown interplay of cations in controlling the initial folding of hSOD1, thus not only underscoring the critical role of Zn2+ in hSOD1-associated ALS, but also suggesting novel hSOD1-dependent mechanisms for Cu2+/Fe2+-induced cytotoxicity likely relevant to other diseases and aging.

biophysics↗

bioRxiv2023

mitoSpliter: a mitochondrial RNA variants-based method for single-cell RNA-seq analysis of multiplexed samples

Single-cell RNA-seq (scRNA-seq) analysis of multiple samples separately can be costly and lead to batch effects. Exogenous barcodes or genome-wide RNA mutations can be used to demultiplex pooled scRNA-seq data, but they are experimentally or computationally challenging and limited in scope. Mitochondrial genomes are small but diverse, providing concise genotype information. We developed "mitoSplitter", an algorithm that demultiplexes samples using mitochondrial RNA (mtRNA) variants, and demonstrated that mtRNA variants can be used to demultiplex large-scale scRNA-seq data. Using affordable computational resources, mitoSplitter can accurately analyze 10 samples and 60,000 cells in 6 hours. To avoid the batch effects from separated experiments, we applied mitoSplitter to analyze the responses of five non-small cell lung cancer (NSCLC) cell lines to BET chemical degradation in a multiplexed fashion. We found the synthetic lethality of TOP2A inhibition and BET chemical degradation in BET inhibitor-resistant cells. The result indicates that mitoSplitter can accelerate the application of scRNA-seq assays in biomedical research.

bioinformatics↗

bioRxiv2023

Activation of peroxisome proliferator-activated receptor-gamma prevents monocrotaline-induced pulmonary arterial hypertension by suppressing of nuclear factor-kappa B mediated autophagy

To investigate the molecular mechanisms underlying autophagy inducing pulmonary vascular remodeling and rosiglitazone inhibiting pulmonary arterial hypertension (PAH). Monocrotaline (MCT) was intraperitoneally injection to induce the rat PAH model. The right ventricular hypertrophy index (RVHI), right ventricle systolic pressure (RVSP), percentage of medial wall thickness (%MT) and histomorphologic analyses were performed to evaluate the development of PAH. The translocation of nuclear factor-kappa B (NF-{kappa}B) p65 subunit from cytosol to nucleus, the protein expression levels of LC3B, Beclin 1 and RND3 were determined by western blot. Furthermore, NF-{kappa}B inhibitor, pyrrolidine dithiocarbamate (PDTC) and peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) activator, rosiglitazone, were used to inhibit the activation of NF-{kappa}B and activate PPAR{gamma} signaling, respectively. MCT injection dramatically induced PAH models in rats as manifested by the increased RVSP, RVHI, and %MT. In addition, the activation of NF-{kappa}B and autophagy were significantly enhanced and the RND3 were markedly decreased in MCT-induced PAH in rat. However, these effects could be significantly suppressed either by the supplementation of PDTC or rosiglitazone. NF-{kappa}B promotes the development of PAH by activation of autophagy and consequent down-regulation of RND3 expression. Activation of PPAR{gamma} suppresses autophagy by inhibiting NF-{kappa}B in MCT-induced PAH. Our results not only uncovered the mechanisms of PPAR{gamma} activator in the protection of PAH but also provided potential therapeutic target for PAH treatment.

biochemistry↗

bioRxiv2023

Two haplotype-resolved genomes of highly heterozygous AAB allotriploid bananas provide insights into subgenome asymmetric evolution and banana wilt control

Bananas (Musa spp.) are one of the most important tropical fruits and staple food, which are of great significance to human societies. Plantain and Silk are two important banana subgroups, which are both triploid hybrids (AAB) between the wild diploid Musa acuminata and M. balbisiana. In this study, we reported the first haplotype-resolved genome assembly of Plantain and Silk bananas with genome size of approximately 1.4 Gb. We discovered widespread asymmetric evolution in the subgenomes of Plantain and Silk, which could be linked to frequent homologous exchanges (HEs) events. This is the first study to uncover the genetic makeup of triploid banana and verify that subgenome B harbors a rich source of resistance genes. Of the 88,078 and 94,988 annotated genes in Plantain and Silk, only 58.5% and 59.4% were present in all three subgenomes, with >50% genes containing differently expressed alleles in different haplotypes. We also found that Plantain is more resistant to banana Fusarium wilt, exhibiting a much faster defense response after pathogenic fungi infection. Many differentially expressed genes in abscisic acid, ethylene, jasmonic acid and salicylic acid pathways were identified in Plantain. Our analysis revealed that MpMYB36 promotes the biosynthesis of secondary cell wall and deposition of lignin by directly binding to the promoter of MpPAL and MpHCT, which allows Plantain to inhibit the penetration of early infection. Moreover, the insertion of the key carotenoid synthesis gene (CRTISO) may be the potential genetic basis for the richness of carotenoids in Plantain. Our study provides an unprecedented genomic basis for basic research and the development of elite germplasm in cultivated bananas.

plant biology↗

bioRxiv2023

Targeted engagement of β-catenin-Ikaros complexes in refractory B-cell malignancies

In most cell types, nuclear {beta}-catenin functions as prominent oncogenic driver and pairs with TCF7-family factors for transcriptional activation of MYC. Surprisingly, B-lymphoid malignancies not only lacked expression and activating lesions of {beta}-catenin but critically depended on GSK3{beta} for effective {beta}-catenin degradation. Our interactome studies in B-lymphoid tumors revealed that {beta}-catenin formed repressive complexes with lymphoid-specific Ikaros factors at the expense of TCF7. Instead of MYC-activation, {beta}-catenin was essential to enable Ikaros-mediated recruitment of nucleosome remodeling and deacetylation (NuRD) complexes for transcriptional repression of MYC. To leverage this previously unrecognized vulnerability of B-cell-specific repressive {beta}-catenin-Ikaros-complexes in refractory B-cell malignancies, we examined GSK3{beta} small molecule inhibitors to subvert {beta}-catenin degradation. Clinically approved GSK3{beta}-inhibitors that achieved favorable safety prof les at micromolar concentrations in clinical trials for neurological disorders and solid tumors were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of {beta}-catenin, repression of MYC and acute cell death. Preclinical in vivo treatment experiments in patient-derived xenografts validated small molecule GSK3{beta}-inhibitors for targeted engagement of lymphoid-specific {beta}-catenin-Ikaros complexes as a novel strategy to overcome conventional mechanisms of drug-resistance in refractory malignancies. HIGHLIGHTSO_LIUnlike other cell lineages, B-cells express nuclear {beta}-catenin protein at low baseline levels and depend on GSK3{beta} for its degradation. C_LIO_LIIn B-cells, {beta}-catenin forms unique complexes with lymphoid-specific Ikaros factors and is required for Ikaros-mediated tumor suppression and assembly of repressive NuRD complexes. C_LIO_LICRISPR-based knockin mutation of a single Ikaros-binding motif in a lymphoid MYC superenhancer region reversed {beta}-catenin-dependent Myc repression and induction of cell death. C_LIO_LIThe discovery of GSK3{beta}-dependent degradation of {beta}-catenin as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3{beta}-inhibitors for the treatment of refractory B-cell malignancies. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/532152v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1b70e4dorg.highwire.dtl.DTLVardef@10bfcc8org.highwire.dtl.DTLVardef@b10eorg.highwire.dtl.DTLVardef@1683a3e_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIAbundant nuclear {beta}-catenin C_LIO_LI{beta}-catenin pairs with TCF7 factors for transcriptional activation of MYC C_LIO_LIB-cells rely on efficient degradation of {beta}-catenin by GSK3{beta} C_LIO_LIB-cell-specific expression of Ikaros factors C_LI Unique vulnerability in B-cell tumors: O_LIGSK3{beta}-inhibitors induce nuclear accumulation of {beta}-catenin. C_LIO_LI{beta}-catenin pairs with B-cell-specific Ikaros factors for transcriptional repression of MYC C_LI

cancer biology↗

bioRxiv2023

Debottlenecking and reformulating feed media for improved CHO cell growth and titer by data-driven and model-guided analyses

Designing and selecting cell culture media and feed are a key strategy to maximize culture performance in industrial biopharmaceutical processes. However, this is a major challenge for therapeutic proteins production since mammalian cells are very sensitive to their culture environment and require specific nutritional needs to grow and produce high-quality proteins such as antibodies. In this regard, in our previous study, we developed data-driven and in-silico model-guided systematic framework to investigate the effect of growth media on Chinese hamster ovary (CHO) cell culture performance, allowing us to design a new media formulation. To expand our exploration to feed, in this study, we evaluated two chemically defined feed media, A and B, in Ambr15 bioreactor runs using a monoclonal antibody-producing CHO K1 cell line. The feeds had a significant impact on cell growth, longevity, viability, and productivity and toxic metabolites production. Specifically, concentrated feed A was not sufficient to support prolonged cell culture and high titer compared to feed B. The framework systematically characterized the major metabolic bottlenecks in the TCA cycle and its related amino acid transferase reactions, and identified key design components, such as asparagine, aspartate, and glutamate, needed for highly productive cell cultures. From our results, we designed three new feeds by adjusting the levels of those amino acids and successfully validated their effectiveness in promoting cell growth and/or titer.

bioengineering↗

bioRxiv2023

Analysis of the SARS-CoV-2 spike protein revealed that blocked receptor-binding domain antigenicity decreases the production of neutralizing antibodies in vivo

The identification of SARS-CoV-2 spike protein distribution and function in target cells has raised concerns about its possible impacts on vaccine efficacy and pathogenic effect in host cells. Thus, a better understanding of such consequences is necessary. In this study, we studied the biological characteristics of six variants of SARS-CoV-2 in A549 and HEK293 cells using four different technologies. The results showed that compared to the other fragments, the full-length spike protein exhibited the highest expression on the cell surface and was detectable in the cell supernatant, cytoplasm, and nucleus. Except for the cell surface, the S1 subunit generally expressed higher than the full-length spike protein. RBD and S2 subunits were expressed in the cytoskeleton. The SS-RBD peptide, which consists of a 19-amino acid signal peptide sequence (SS)-linked RBD, exhibited the highest expression in the cell supernatant among all other studied peptides. The SS positively enhanced the expression, migration, and secretion of SS-RBD from the cytoskeleton to the supernatant. Importantly, the FACS assay results showed that neutralizing antibodies (NAbs) could recognize SS-RBD but not RBD in the transfected cells, suggesting that RBD was tightly bound by ACE2 in HEK293 cells. In contrast, the antigenicity of the RBD in the spike protein was revealed and efficiently monitored only by 6-His-tag mAbs. Thus, our findings demonstrated that ACE2 blocks crucial immunogenic epitopes of the RBD, and the full-length spike protein mainly induces non-neutralizing antibodies in vivo. Therefore, we suggest that reducing ACE2 binding affinity and exposing the immunogenicity of the RBD on the spike protein is imperative for improving vaccine efficacy and generating new SARS-CoV-2 mRNA vaccines.

microbiology↗