Biology subjects

Jin, A.

Publications and source records attributed to Jin, A..

9 recordsLinked to original sources

bioRxiv2024

Synthetic genomes unveil the effects of synonymous recoding

Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers1-4. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement--recoding--is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a synthetic Escherichia coli genome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organisms genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.

synthetic biology↗

bioRxiv2024

Accelerated protein retention expansion microscopy using microwave radiation

Protein retention expansion microscopy (ExM) retains genetically encoded fluorescent proteins or antibody-conjugated fluorescent probes in fixed tissue and isotropically expands the tissue through a swellable polymer network to allow nanoscale (<70 nm) resolution on diffraction-limited confocal microscopes. Despite numerous advantages ExM brings to biological studies, the full protocol is time-consuming and can take multiple days to complete. Here, we adapted the ExM protocol to the vibratome-sectioned brain tissue of Xenopus laevis tadpoles and implemented a microwave-assisted protocol to reduce the workflow from days to hours. In addition to the significantly accelerated processing time, our microwave-assisted ExM (M/WExM) protocol maintains the superior resolution and signal-to-noise ratio of the original ExM protocol. Furthermore, the M/WExM protocol yields higher magnitude of expansion, suggesting that in addition to accelerating the process through increased diffusion rate of reagents, microwave radiation may also facilitate the expansion process. To demonstrate the applicability of this method to other specimens and protocols, we adapted the microwave-accelerated protocol to whole mount adult brain tissue of Drosophila melanogaster fruit flies, and successfully reduced the total processing time of a widely-used Drosophila IHC-ExM protocol from 6 days to 2 days. Our results demonstrate that with appropriate adjustment of the microwave parameters (wattage, pulse duration, interval, and number of cycles), this protocol can be readily adapted to different model organisms and tissue types to greatly increase the efficiency of ExM experiments.

neuroscience↗

bioRxiv2023

Endodermal BRD4 mediates epithelial-mesenchymal crosstalk during lung development

Lung morphogenesis relies on diverse cell intrinsic and extrinsic mechanisms to ensure proper cellular differentiation and compartmentalization. Using genetic mouse models, tissue explants, and transcriptomic analysis, we demonstrate that the epigenetic reader Bromodomain Containing Protein 4 (BRD4) is required for lung morphogenesis and perinatal survival. Endodermal BRD4 deletion impairs epithelial-mesenchymal crosstalk, leading to disrupted proximal-distal patterning and branching morphogenesis. These early defects result in dilated airways and the formation of cystic distal airway structures, containing both airway and alveolar features. Moreover, BRD4 deficient lungs exhibit abnormal airway and alveolar epithelial cell lineage allocation and differentiation. Restoration of SHH signaling partially rescues defects due to loss of BRD4, suggesting a role for BRD4 in regulating the SHH-FGF signaling axis. Together, these data identify the essential role of Brd4 in lung development to ensure proper intercellular crosstalk to enable proper lineage specification, identity, and maturation.

developmental biology↗

bioRxiv2022

A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA.1 and BA.2

Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC50) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 g of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.

immunology↗

bioRxiv2021

Mismatch repair deficiency is not sufficient to increase tumor immunogenicity

DNA mismatch repair deficiency (MMRd) in human cancer is associated with high tumor mutational burden (TMB), frameshift mutation-derived neoantigens, increased T cell infiltration, and remarkable responsiveness to immune checkpoint blockade (ICB) therapy. Nevertheless, about half of MMRd tumors do not respond to ICB for unclear reasons. While tumor cell line transplant models of MMRd have reinforced the importance of TMB in immune response, critical questions remain regarding the role of immunosurveillance in the evolution of MMRd tumors induced in vivo. Here, we developed autochthonous mouse models of lung and colon cancer with highly efficient ablation of MMR genes via in vivo CRISPR/Cas9 targeting. Surprisingly, MMRd in these models did not result in increased immunogenicity or response to ICB. Mechanistically, we showed this lack of immunogenicity to be driven by profound intratumoral heterogeneity (ITH). Studies in animals depleted of T cells further demonstrated that immunosurveillance in MMRd tumors has no impact on TMB but shapes the clonal architecture of neoantigens by exacerbating ITH. These results provide important context for understanding immune evasion in cancers with high TMB and have major implications for therapies aimed at increasing TMB.

cancer biology↗

bioRxiv2021

Ultrapotent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

immunology↗

bioRxiv2020

Identification of Cross-Reactive CD8+ T Cell Receptors with High Functional Avidity to a SARS-CoV-2 Immunodominant Epitope and Its Natural Mutant Variants

Despite the growing knowledge of T cell responses and their epitopes in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Using a peptide library predicted with HLA class I-restriction, specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients. Among the 15 SARS-CoV-2 epitopes identified from the S and N proteins, N361-369 (KTFPPTEPK) was the most dominant epitope. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity, and they exhibited complementary cross-reactivity to reported N361-369 mutant variants. In dendritic cells (DCs) and the lung organoid model, we found that the N361-369 epitope could be processed and endogenously presented to elicit the activation and cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, illuminating natural ways of viral clearance with high relevancy in the vaccine development.

immunology↗

bioRxiv2020

A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD

The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies1-3. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic4,5. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S6. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.

immunology↗

bioRxiv2020

A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

Neutralizing antibodies (Abs) have been considered as promising therapeutics for the prevention and treatment of pathogens. After the outbreak of COVID-19, potent neutralizing Abs to SARS-CoV-2 were promptly developed, and a few of those neutralizing Abs are being tested in clinical studies. However, there were few methodologies detailly reported on how to rapidly and efficiently generate neutralizing Abs of interest. Here, we present a strategically optimized method for precisive screening of neutralizing monoclonal antibodies (mAbs), which enabled us to identify SARS-CoV-2 receptor-binding domain (RBD) specific Abs within 4 days, followed by another 2 days for neutralization activity evaluation. By applying the screening system, we obtained 198 Abs against the RBD of SARS-CoV-2. Excitingly, we found that approximately 50% (96/198) of them were candidate neutralizing Abs in a preliminary screening of SARS-CoV-2 pseudovirus and 20 of these 96 neutralizing Abs were confirmed with high potency. Furthermore, 2 mAbs with the highest neutralizing potency were identified to block authentic SARS-CoV-2 with the half-maximal inhibitory concentration (IC50) at concentrations of 9.88 ng/ml and 11.13 ng/ml. In this report, we demonstrated that the optimized neutralizing Abs screening system is useful for the rapid and efficient discovery of potent neutralizing Abs against SARS-CoV-2. Our study provides a methodology for the generation of preventive and therapeutic antibody drugs for emerging infectious diseases.

immunology↗