Biology subjects

Lou, C.

Publications and source records attributed to Lou, C..

8 recordsLinked to original sources

bioRxiv2026

Cellular context shapes the transcriptomic impact of somatic variants

Somatic SNVs are commonly prioritized by recurrence or sequence annotation, leaving the molecular consequences of private variants unresolved. We developed PrismSNV and Ligo-Seq, a single-cell full-length transcriptome sequencing platform, to quantify transcriptomic displacement associated with within-cell SNV-state toggling. Across 26 samples from three cancer types, more than 60% of candidate SNVs showed significant cell-type-specific displacement with over 30% variation in magnitude. Sequence-based deleteriousness was poorly aligned with cellular displacement, and fewer than 3% of top-displacement SNVs across six tumor types were catalogued in ClinVar. Displacement was strongest in transcriptionally plastic, less differentiated states. Analysis of 655,349 CD4+ T-cell transcriptomes further showed that SNV-gene associations changed in magnitude and sometimes direction during activation. Lack of recurrence or clinical annotation does not imply transcriptomic inertness; variant impact is shaped by cellular context rather than sequence alone, establishing the variant-cell pair as a critical unit for interpreting somatic variation.

genetics↗

bioRxiv2026

Intrinsic immune receptor triggering mechanism informs rational chimeric receptor design

The T cell receptor (TCR) couple antigen recognition to T-cell activation and immune homeostasis. As an eight-subunit assembly, TCR activation operates as a highly coordinated, multi-subunit signaling event. However, which subunit serves as the primary trigger upon ligand engagement still remains unresolved. we identify CD3{varepsilon} as a triggering subunit whose intracellular domain (ICD) is released from the plasma membrane during TCR activation. Elevated membrane tension facilitates this transition from a membrane-proximal to a membrane-distal conformation. The released ICD forms condensates through phase separation with free or co-receptor-associated Lck. These condensates recruit additional CD3{varepsilon} ICDs from neighboring TCRs, triggering resting receptors and promoting further recruitment of TCRs and Lck to amplify proximal signaling. The CD3{varepsilon} ICD alone support robust T cell activation independently of its own ITAM. We observe a similar triggering mechanism in several activating receptors in T and nature killer (NK) cell. These findings support a unifying model in which designated subunits initiate multi-subunit immune receptor activation through phase separation with key kinases. Guided by this model, we engineered triggering molecules (TRIG) that lack canonical intracellular signaling modules. These TRIG molecules recruit endogenous immune receptors in a ligand-dependent manner and efficiently activate both T cells and NK cells. This mechanism provides a basis for designing chimeric receptors that activate immune cells through endogenous receptor signaling.

immunology↗

bioRxiv2026

CD28 Co-stimulation Is Organized by Signaling Condensates and Counteracted by PD-1

T cell activation relies on the coordinated integration of TCR and co-signaling pathways, yet how these signals are physically organized remains unclear. Here, we demonstrate that CD28 co-stimulation is driven by a phase-separation mechanism in which the CD28 intracellular domain (ICD) forms signaling condensates with the Src-family kinase Lck. Ligand engagement promotes the CD28 ICD accessibility, and triggers rapid CD28/Lck condensation, enhancing CD28 phosphorylation, cytoskeletal polarization, and cytokine production. We further identify PD-1 as a graded regulator of this process: monophosphorylated PD-1 is recruited into CD28/Lck condensates, whereas di-phosphorylated PD-1 competitively binds the CD28 ICD and dissolves these assemblies. CD28 and PD-1 ICDs also undergo condensation directly, revealing a dynamic and compositionally heterogeneous signaling system. Guided by this competitive phase-separation mechanism, we engineer CD28 ICD mutants that preserve Lck condensation while resisting PD-1-mediated disruption. Incorporation of these condensation-selective mutants into CAR constructs confers resistance to inhibitory signaling and enhances antitumor efficacy in vivo. These findings establish phase separation as a central organizing principle of CD28 co-stimulation and provide a biophysical framework for engineering more resilient therapeutic T cells.

immunology↗

bioRxiv2024

Mechanical force regulates the inhibitory function of PD-1

The immune checkpoint molecule, programmed cell death 1 (PD-1), plays a pivotal role in regulating T-cell function. Upon binding to its ligands, PD-L1 or PD-L2, PD-1 suppresses T-cell receptor signaling, thereby preventing T-cell activation, making it a critical target in cancer immunotherapy. Although extensively studied, the molecular mechanism of PD-1s inhibitory function is still not fully understood, especially at the atomic level. Using the biomembrane force probe (BFP), we discovered that interactions between PD-1 and PD-L1/PD-L2 exhibit catch-slip bond behavior under force. At lower forces, catch bonds are observed, transitioning to slip bond as the force increases. Steered molecular dynamics (SMD) simulations revealed a force-induced bond state between PD-1 and its ligands, distinct from the force-free state observed in solved complex structures. Disrupting interactions that stabilize either state weakens the catch bond and diminishes PD-1s inhibitory function. Interestingly, soluble forms of PD-L1 and PD-L2 compete with their surface-bond counterparts, attenuating PD-1s suppression of T-cell activation. This suggests that soluble PD-1 ligands could potentially serve as anti-PD-1 drugs. Tumor growth studies in mice confirmed the anti-cancer activity of soluble PD-L1. Our findings highlight the critical role of mechanical force in PD-1s inhibitory function, suggesting that PD-1 may act as a mechanical sensor in suppressing T-cell activation. These insights indicate that mechanical regulation should be considered when designing PD-1 blocking inhibitors and other PD-1 related cancer immunotherapies. TeaserThis study shows that the interaction between PD-1 and its ligands is force-dependent, and soluble forms of PD-1 ligands can potentially serve as PD-1 blockers in cancer immunotherapy.

biophysics↗

bioRxiv2023

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

Streptomyces has enormous potential to produce novel natural products (NPs) as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters (BGCs). However, the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery. Here, we developed an easy-to-use, highly flexible DNA assembly toolkit for gene cluster engineering. The DNA assembly toolkit are compatible with various DNA assembling approaches including Biobrick, Golden Gate, CATCH, yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly. This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters. To demonstrate the utility of this toolkit, we quantified a library of modular regulatory parts, and engineered a gene cluster (act) using characterized promoters that led to increased production. Overall, this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.

synthetic biology↗

bioRxiv2020

Fully Automated Detection of Paramagnetic Rims in Multiple Sclerosis Lesions on 3T Susceptibility-Based MR Imaging

Background and PurposeThe presence of a paramagnetic rim around a white matter lesion has recently been shown to be a hallmark of a particular pathological type of multiple sclerosis (MS) lesion. Increased prevalence of these paramagnetic rim lesions (PRLs) is associated with a more severe disease course in MS. The identification of these lesions is time-consuming to perform manually. We present a method to automatically detect PRLs on 3T T2*-phase images. MethodsT1-weighted, T2-FLAIR, and T2*-phase MRI of the brain were collected at 3T for 19 subjects with MS. The images were then processed with lesion segmentation, lesion center detection, lesion labelling, and lesion-level radiomic feature extraction. A total of 877 lesions were identified, 118 (13%) of which contained a paramagnetic rim. We divided our data into a training set (15 patients, 673 lesions) and a testing set (4 patients, 204 lesions). We fit a random forest classification model on the training set and assessed our ability to classify lesions as PRL on the test set. ResultsThe number of PRLs per subject identified via our automated lesion labelling method was highly correlated with the gold standard count of PRLs per subject, r = 0.91 (95% CI [0.79, 0.97]). The classification algorithm using radiomic features can classify a lesion as PRL or not with an area under the curve of 0.80 (95% CI [0.67, 0.86]). ConclusionThis study develops a fully automated technique for the detection of paramagnetic rim lesions using standard T1 and FLAIR sequences and a T2*phase sequence obtained on 3T MR images. HighlightsO_LIA fully automated method for both the identification and classification of paramagnetic rim lesions is proposed. C_LIO_LIRadiomic features in conjunction with machine learning algorithms can accurately classify paramagnetic rim lesions. C_LIO_LIChallenges for classification are largely driven by heterogeneity between lesions, including equivocal rim signatures and lesion location. C_LI

neuroscience↗

bioRxiv2020

Simple cloning of large natural product biosynthetic gene cluster by CRISPR/Cas12a-mediated fast direct capturing strategy

Directly cloning of biosynthetic gene clusters (BGCs) from microbial genomes has been revolutionizing the natural product-based drug discovery. However, it is still very challenging to efficiently clone, for example, large (> 80kb) and GC-rich (> 70%), streptomycete originating BGCs. In this study, we developed a simple, fast yet efficient and low-cost in vitro platform for direct cloning large BGCs from streptomycete genomic DNA, named as CAT-FISHING (CRISPR/Cas12a- and Agarose plug-based sysTem for Fast bIoSyntHetIc geNe cluster cloninG), by combining the advantages of CRISPR/Cas12a cleavage and bacterial artificial chromosome (BAC) library construction. CAT-FISHING was demonstrated by directly cloning large DNA fragments ranging from 47 to 139 kb with GC content of > 70% from the S. albus J1074 genome in a relatively efficient manner. Moreover, surugamides, encoded by a captured 87-kb BGC with GC content of 76%, was heterologously expressed in a Streptomyces chassis. These results indicate that CAT-FISHING is a powerful platform for BGCs batch cloning, which would be greatly beneficial to the natural products-based drug discovery. We believe that this system will lead a renaissance of interest in microorganisms as a source for drug development.

synthetic biology↗

bioRxiv2018

Systematically investigating the key features of the nuclease deactivated Cpf1 for tunable multiplex genetic regulation

With a unique crRNA processing capability, the CRISPR associated Cpf1 protein holds great potential for multiplex gene regulation. Unlike the well-studied Cas9 protein, however, conversion of Cpf1 to a transcription regulator and its related properties have not been systematically explored yet. In this study, we investigated the mutation schemes and crRNA requirements for the nuclease deactivated Cpf1 (dCpf1). By shortening the direct repeat sequence, we obtained genetically stable crRNA co-transcripts and improved gene repression with multiplex targeting. A screen of diversity-enriched PAM library was designed to investigate the PAM-dependency of gene regulation by dCpf1 from Francisella novicida and Lachnospiraceae bacterium. We found novel PAM patterns that elicited strong or medium gene repressions. Using a computational algorithm, we predicted regulatory outputs for all possible PAM sequences, which spanned a large dynamic range that could be leveraged for regulatory purposes. These newly identified features will facilitate the efficient design of CRISPR-dCpf1 based systems for tunable multiplex gene regulation.

synthetic biology↗