Biology subjects

Zhong, X.

Publications and source records attributed to Zhong, X..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Multiplexed Short-wave Infrared Imaging Highlights Anatomical Structures in Mice

While multiplexed fluorescence imaging is frequently used for in vitro microscopy, extending the technique to whole animal imaging in vivo has remained challenging due to the attenuation and scattering of visible and traditional near infrared (NIR-I) wavelengths. Fluorescence imaging using short-wave infrared (SWIR, 1000 - 1700 nm, a.k.a. NIR-II) light enables deeper tissue penetration for preclinical imaging compared to previous methods due to reduced tissue scattering and minimal background autofluorescence in this optical window. Combining NIR-I excitation wavelengths with multiple distinct SWIR emission peaks presents a tremendous opportunity to distinguish multiple fluorophores with high precision for non-invasive, multiplexed anatomical imaging in small animal models. SWIR-emitting semiconductor quantum dots (QDs) with tunable emission peaks and optical stability have emerged as powerful contrast agents, but SWIR imaging demonstrations have yet to move beyond two-color imaging schemes. In this study, we engineered a set of three high quantum yield lead sulfide/cadmium sulfide (PbS/CdS) core/shell QDs with distinct SWIR emissions ranging from 1100 - 1550 nm and utilize these for simultaneous three-color imaging in mice. We first use QDs to non-invasively track lymphatic drainage, highlighting the detailed network of lymphatic vessels with high-resolution with a widefield imaging over a 2 hr period. We then perform multiplexed imaging with all three QDs to distinctly visualize the lymphatic system and spatially overlapping vasculature network. This work establishes optimized SWIR QDs for next-generation multiplexed preclinical imaging, moving beyond the capability of previous dual-labeling techniques. The capacity to discriminate several fluorescent labels through non-invasive NIR-I excitation and SWIR detection unlocks numerous opportunities for studies of disease progression, drug biodistribution, and cell trafficking dynamics in living organisms.

bioengineering↗

bioRxiv2024

Polaris Neurosense: A General-Purpose Non-invasive Neurotechnology Research Platform

This article describes our initial work toward a general-purpose platform for non-invasive neurotechnology research. This platform consists of a multi-modal wireless recording device, an associated software API, and full integration into BCI2000 software. The device is placed on the forehead and features two electroencephalographic (EEG) sensors, an inertial movement sensor (IMU), a photoplethysmogram (PPG) sensor, a microphone, and vibration-based feedback. Herein, we demonstrate different technical characteristics of our platform and its use in the context of sleep monitoring/modulation, simultaneous and synchronized recordings from different hardware, and evoked potentials. With further development and widespread dissemination, our platform could become an important tool for research into new non-invasive neurotechnology protocols in humans.

bioengineering↗

bioRxiv2023

De novo design of drug-binding proteins with predictable binding energy and specificity

The de novo design of small-molecule-binding proteins has seen exciting recent progress; however, the ability to achieve exquisite affinity for binding small molecules while tuning specificity has not yet been demonstrated directly from computation. Here, we develop a computational procedure that results in the highest affinity binders to date with predetermined relative affinities, targeting a series of PARP1 inhibitors. Two of four designed proteins bound with affinities ranging from < 5 nM to low M, in a predictable manner. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free-energy calculations performed directly on the designed models are in excellent agreement with the experimentally measured affinities, suggesting that the de novo design of small-molecule-binding proteins with tuned interaction energies is now feasible entirely from computation. We expect these methods to open many opportunities in biomedicine, including rapid sensor development, antidote design, and drug delivery vehicles. One Sentence SummaryWe use informatic sampling to design low nM drug-binding proteins, and physics-based calculations to accurately predict affinities.

biophysics↗

bioRxiv2023

Lrp10 suppresses IL7R limiting CD8 T cell homeostatic expansion and anti-tumor immunity.

Signals emanating from the T cell receptor (TCR), co-stimulatory receptors, and cytokine receptors each influence CD8 T cell fate. Understanding how these signals respond to homeostatic and microenvironmental cues can reveal new ways to therapeutically direct T cell function. Through forward genetic screening in mice, we discovered that loss-of-function mutations in LDL receptor related protein 10 (Lrp10) caused naive and central memory CD8 T cells to accumulate in peripheral lymphoid organs. Lrp10 encodes a conserved cell surface protein of unknown immunological function. Lrp10 was induced with T cell activation and its expression post-translationally suppressed IL7 receptor (IL7R) levels. Accordingly, Lrp10 deletion enhanced T cell homeostatic expansion through IL7R signaling. Lrp10-deficient mice were also intrinsically resistant to syngeneic tumors. This phenotype depended on dense tumor infiltration of CD8 T cells that displayed increased memory cell characteristics, reduced terminal exhaustion, and augmented responses to immune checkpoint inhibition. Here, we present Lrp10 as a new negative regulator of CD8 T cell homeostasis and a host factor that controls tumor resistance with implications for immunotherapy.

immunology↗

bioRxiv2023

CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provides molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. SignificanceCellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machineries. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

microbiology↗

bioRxiv2023

Coordination of focal adhesion nanostructure and mechano-signaling drives cardiomyocyte differentiation

Focal adhesion (FA) organization and signaling are essential for cell growth and differentiation. However, the molecular mechanism that coordinates the FA signaling with cardiomyocyte differentiation has not been fully understood. Here, we provide empirical evidence that BNIP-2, a BCH-domain-containing protein, is the organizer of FA nanostructure that potentiates FA signaling and cell traction force transmission. Mechanistically, BNIP-2 serves as a scaffold for focal adhesion kinase (FAK), paxillin and vinculin to control their molecular organization and assembly/disassembly within FAs. Constitutively active phosphomimetic mutant FAK Y397D shows enhanced binding to BNIP-2, whereas the depletion of BNIP-2 reduces FAK phosphorylation and interaction between FAK and paxillin. Using H9c2 myoblasts and human embryonic stem cells as model systems, we show that BNIP-2 depletion results in aberrant FA dynamics with impairment of traction force, and changes in signature target genes, hereby impeding cardiomyocyte differentiation. BNIP-2 regulation of FA organization and dynamic is therefore pivotal to the mechanotransduction in cardiomyocyte differentiation, shedding new light to how FA-transduced force modulates cell growth and differentiation.

cell biology↗

bioRxiv2023

Assessment of the macrovascular contribution to resting-state fMRI functional connectivity at 3 Tesla

In resting-state functional magnetic resonance imaging (rs-fMRI) functional connectivity (FC) mapping, temporal correlation are widely assumed to reflect synchronized neural-related activaty. Although a large number of studies have demonstrated the potential vascular effects on FC, little research has been conducted on FC resulting from macrovascular signal fluctuations. Previously, Tong et al. found (Tong et al., 2019b) a robust anti-correlation between the fMRI signals in the internal carotid artery and the internal jugular vein (and the sagittal sinus). The present study extends the previous study to include all detectable major veins and arteries in the brain in a systematic analysis of the macrovascular contribution to the functional connectivity of the whole-GM. This study demonstrates that: 1) The macrovasculature consistently exhibited strong correlational connectivity among itself, with the sign of the correlations varying between arterial and venous connectivity; 2) GM connectivity was found to have a strong macrovascular contribution, stronger from veins than arteries; 3) functional connectivity originating from the macrovasculature displayed disproportionately high spatial variability compared to across all GM voxels; 4) macrovascular contributions to connectivity were still evident well beyond the confines of the macrovascular space. These findings highlight the extensive contribution to rs-fMRI BOLD and FC predominantly by large veins, but also by large arteries. These findings pave the way for future studies aimed at more comprehensively modeling and thereby removing these macrovascular contributions.

biophysics↗

bioRxiv2023

Adaptive Functions of Structural Variants in Human Brain Development

Quantifying the structural variants (SVs) in nonhuman primates could provide a niche to clarify the genetic backgrounds underlying human-specific traits, but such resource is largely lacking. Here, we report an accurate SV atlas in a population of 562 rhesus macaques, verified by two public SV benchmarks, an inhouse benchmark of eight macaque genomes with long-read sequencing and another inhouse benchmark of one macaque genome with whole-genome assembly. This accurate, quantitative SV map indicates stronger purifying selection on inversions, one type of poorly-clarified SVs to date, especially for those located on regulatory regions, suggesting a strategy for prioritizing inversions with the most important functions. Based on the distribution and the evolutionary features of these inversions in macaque population, we then identified 75 human-specific inversions, clarified their functional effects and prioritized them. Notably, the top-ranked inversions have substantially shaped the human transcriptome, through their dual-effects of reconfiguring the ancestral genomic architecture and introducing regional mutation hotspots at the inverted regions. As a proof-of-concept, we linked APCDD1, located on one of these inversions with the highest rank score and downregulated in human brains, to neuronal maturation. The accumulation of human-specific mutations on its promoter region, accelerated by the formation of the inversion, contributed to the decreased expression in humans. Notably, the overexpression of APCDD1 could accelerate the neuronal maturation, while its depletion in mice delays the neuronal maturation. This study thus highlights the contribution of SVs, especially the inversions, to the distinct features in human brain development.

evolutionary biology↗

bioRxiv2023

TRAF6 initiates inflammatory signaling via organizing membraneless cytoplasmic condensates

The tumor necrosis factor receptor-associated factor 6 (TRAF6) is a central molecule in multiple signaling pathways, i.e. the TNF receptor, the Toll-like receptors (TLRs), and interleukin-1 receptor (IL-1R) pathways. Upon pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) stimulations, TRAF6 activates downstream of NF-{kappa}B signaling. However, the precise mechanism of how TRAF6 activates downstream molecules remains unclear. Here, we demonstrate that TRAF6 acts as a sensor for upstream signals to initiate liquid-liquid phase separation (LLPS) of itself and downstream proteins, forming membraneless condensates. Subsequent recruitment, enrichment and activation of downstream effectors in the condensates lead to robust inflammatory signal transduction. The multivalent interactions mediated by its RING domain, zinc finger domain 1, and coiled-coil domain mediates the LLPS process. Forced phase separation of TRAF6 induced NF-{kappa}B activation. Disruption of TRAF6 phase separation abrogates activation of NF-{kappa}B signaling. Overall, we uncover the spatial organization of molecules by TRAF6 through phase separation as a subcellular platform to activate inflammatory signaling. Targeting TRAF6 phase separation hold promises for therapeutics aiming autoimmune diseases, inflammation and cancers.

immunology↗

bioRxiv2023

In vivo Ontogeny of human forebrain neural progenitor cell grafts in adult rats: an immunohistological study

A thorough understanding of the cell behaviors of the human neural grafts is fundamental to exploit them to achieve cell therapy for recovering brain functions. Here by using immunohistological staining, we trace the cell fate of the intrastriatal human neural progenitor cell (NPC) grafts up to 9 months in adult rats, with multiple examining time points to provide a unified working time frame for future transplantation study. Lots of Nestin+/Sox2+ human cells continuously migrate along the white matter tracts into distal brain parenchyma even long time after transplantation, providing a potential for curing diffuse brain damage. Further analysis reveals a significant heterogeneity of the long-term sustained neural stem cells (NSC)/NPCs that progressing throughout different stages, mimicking the neural development of human forebrain. More importantly, the initial GFAP expression in human grafts marks the NSC progression instead of terminal astrocyte differentiation. The distally migrating human cells continuously show the capability to produce new neurons, albeit at a low efficiency in the intact brain. Further investigations in neural disease models are needed. Such study would benefit neural cell therapy with regarding to the optimization of the transplantation strategy and choosing of acting mode by neural grafts (e.g. via cell replacement or ex vivo gene therapy).

cell biology↗

bioRxiv2023

Chemical screens in aging-relevant human motor neurons identify MAP4Ks as therapeutic targets for amyotrophic lateral sclerosis

Effective therapeutics is much needed for amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease mainly affecting motor neurons. By screening chemical compounds in human patient-derived and aging-relevant motor neurons, we identify a neuroprotective compound and show that MAP4Ks may serve as therapeutic targets for treating ALS. The lead compound broadly improves survival and function of motor neurons directly converted from human ALS patients. Mechanistically, it works as an inhibitor of MAP4Ks, regulates the MAP4Ks-HDAC6-TUBA4A-RANGAP1 pathway, and normalizes subcellular distribution of RANGAP1 and TDP-43. Finally, in an ALS mouse model we show that inhibiting MAP4Ks preserves motor neurons and significantly extends animal lifespan.

neuroscience↗

bioRxiv2023

Gemcitabine plus nab-paclitaxel preserves skeletal and cardiac mass and function in a murine model of pancreatic cancer cachexia

More than 85% of patients with pancreatic ductal adenocarcinoma (PDAC) suffer from cachexia, a debilitating syndrome characterized by the loss of muscle and fat and remains an unmet medical need. While chemotherapy remains an effective treatment option, it can also induce weight and muscle loss in patients with cancer. Gemcitabine combined with nab paclitaxel (GnP) is a first line treatment option for patients with PDAC but GnPs effect on cachexia has not been comprehensively investigated. We interrogated the effects of GnP in a murine model of pancreatic cancer cachexia. Mice were orthotopically implanted with the cachexia inducing pancreatic cell line (KPC) and were administered GnP or vehicle. The controls underwent sham surgery. We defined GnP effects on cachexia and tumor burden by evaluating muscle and cardiac mass and function, fat mass, bone morphometry, and hematology measurements. We completed RNA sequencing and deep proteome profiling in skeletal and cardiac muscle. KPC+GnP reduced tumor burden over 50% and increased survival compared to KPC. KPC vehicle group had more than 15% muscle mass loss and decreased left ventricular mass, this was not present in KPC+GnP when compared to controls. RNA Seq and deep proteomics analyses suggested that muscle and cardiac dysfunction pathways activated in KPC group were either reversed or decreased in KPC+GnP. In all, our data suggests that GnP protects against muscle and cardiac wasting in an experimental model of PDAC cachexia. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/536434v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1d394f7org.highwire.dtl.DTLVardef@1d8fdaforg.highwire.dtl.DTLVardef@f709d4org.highwire.dtl.DTLVardef@a668e0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cancer biology↗

bioRxiv2023

An automated proximity proteomics pipeline for subcellular proteome and protein interaction mapping

Proximity labeling (PL) through biotinylation coupled with mass spectrometry (MS) has emerged as a powerful technique for capturing spatial proteomes within living cells. Large-scale sample processing for proximity proteomics requires a workflow that minimizes hands-on time while enhancing quantitative reproducibility. Here, we present a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. By combining this pipeline with an optimized quantitative MS acquisition method based on data-independent acquisition (DIA), we not only significantly increased sample throughput but also improved the reproducibility of protein identification and quantification. We applied this pipeline to delineate subcellular proteomes across various cellular compartments, including endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, employing 5HT2A serotonin receptor as a model, we investigated temporal changes of proximal interaction networks induced by the receptors activation with serotonin. Finally, to demonstrate the applicability of our PL pipeline across multiple experimental conditions, we further modified the PL pipeline for reduced sample input amounts to accommodate CRISPR-based gene knockout, and assessed the dynamics of the 5HT2A network in response to the perturbation of selected proximal interactors. Importantly, the presented PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.

systems biology↗

bioRxiv2023

SFyNCS detects oncogenic fusions involving non-coding sequences in cancer

Fusion genes are well-known cancer drivers. However, very few known oncogenic fusions involve non-coding sequences. We develop SFyNCS with superior performance to detect fusions of both protein-coding genes and non-coding sequences from transcriptomic sequencing data. We validate fusions using somatic structural variations detected from the genomes. This allows us to comprehensively evaluate various fusion detection and filtering strategies and parameters. We detect 165,139 fusions in 9,565 tumor samples across 33 tumor types in the Cancer Genome Atlas cohort. Among them, 72% of the fusions involve non-coding sequences and many are recurrent. We discover two long non-coding RNAs recurrently fused with various partner genes in 32% of dedifferentiated liposarcomas and experimentally validated the oncogenic functions in mouse model.

bioinformatics↗

bioRxiv2022

Comparative genomic analysis between Leonurus japonicus and Leonurus sibiricus

Leonurus japonicus Houtt. is an important medicinal plant in East Asia and is now widely recognized for its role in treating cerebral apoplexy and lowering blood lipids. Here, we report two sets of chromosome-level genome sequences for leonurine-producing Leonurus japonicus and for its closely related species leonurine-free Leonurus sibiricus, where 99.78% of 518.19 Mb of L. japonicus was assembled into ten pseudochromosomes with a contig N50 of 17.62 Mb and 99.33% of 472.29 Mb of L. sibiricus was assembled into nine pseudochromosomes with a contig N50 of 13.29 MB. The reference genomes of Leonurus will accelerate the decoding of novel bioactive molecules in medicinal plants, especially in the Lamiaceae family.

plant biology↗

bioRxiv2022

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic--Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans. One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

systems biology↗

bioRxiv2022

Disulfide stabilization reveals conserved dynamic features between SARS-CoV-1 and SARS-CoV-2 spikes.

SARS-CoV-2 spike protein (S) is structurally dynamic and has been observed by cryo-EM to adopt a variety of prefusion conformations that can be categorized as locked, closed and open. The locked conformations feature tightly packed trimers with structural elements incompatible with RBD in "up" position. For SARS-CoV-2 S, it has been shown that the locked conformations are transient under neutral pH. Probably due to their transience, locked conformations remain largely uncharacterized for SARS-CoV-1 S. Intriguingly, locked conformations were the only conformations captured for S proteins of bat and pangolin origin SARS-related coronaviruses. In this study, we introduced x1, x2, and x3 disulfides into SARS-CoV-1 S. Some of these disulfides have been shown to preserve rare locked conformations when introduced to SARS-CoV-2 S. Introduction of these disulfides allowed us to image a variety of locked and other rare conformations for SARS-CoV-1 S by cryo-EM. We identified bound cofactors and structural features that are associated with SARS-CoV-1 S locked conformations. We compare newly determined structures to other available spike structures of Sarbecoviruses to identify conserved features and discuss their possible functions.

biochemistry↗

bioRxiv2022

Brevicidine, a bacterial non-ribosomally produced cyclic antimicrobial lipopeptide with a unique modus operandi

Due to the accelerated appearance of antibiotic-resistant (AMR) pathogens in clinical infections, new first-in-class antibiotics, operating via novel modes of action, are desperately-needed. Brevicidine, a bacterial non-ribosomally produced cyclic lipopeptide, has shown potent and selective antimicrobial activity against Gram-negative pathogens. However, before our investigations, little was known about how brevicidine exerts its potent bactericidal effect against Gram-negative pathogens. In this study, we find that brevicidine has potent antimicrobial activity against AMR Enterobacteriaceae pathogens, with a MIC value ranging between 0.5M (0.8mg/L) and 2M (3.0mg/L). In addition, brevicidine showed potent anti-biofilm activity against the Enterobacteriaceae pathogens, with same 100% inhibition and 100% eradication concentration of 4M (6.1mg/L). Further mechanistic studies showed that brevicidine exerts its potent bactericidal activity via interacting with lipopolysaccharide in the outer membrane, targeting phosphatidylglycerol and cardiolipin in the inner membrane, and dissipating the proton motive force of bacteria. This results in metabolic perturbation, including inhibition of adenosine triphosphate synthesis, inhibits the dehydrogenation of nicotinamide adenine dinucleotides, accumulation of reactive oxygen species in bacteria, and inhibition of protein synthesis. Lastly, brevicidine showed a good therapeutic effect in a mouse peritonitis-sepsis model. Our findings pave the way for further research on clinical applications of brevicidine, to combat the prevalent infections caused by AMR Gram-negative pathogens worldwide.

microbiology↗