Biology subjects

Sun, J.

Publications and source records attributed to Sun, J..

At least 343 records · Page 19Linked to original sources

bioRxiv2022

Structural basis of human LRRK2 membrane recruitment and activation

Mutations in LRRK2 are the most common genetic cause of late-onset Parkinsons disease (PD). LRRK2 encodes the leucine-rich repeat kinase 2 (LRRK2), whose kinase activity is regulated by Rab29, a membrane-anchored GTPase. However, molecular mechanisms underlying Rab29-dependent recruitment and activation of LRRK2 remain unclear. Here we report cryo-EM structures of LRRK2-Rab29 complexes in three oligomeric states, illustrating snapshots of key steps during LRRK2 membrane recruitment and activation. Rab29 binds to the ARM domain of LRRK2, and disruption at the interface abrogates LRRK2 kinase activity. Activation of LRRK2 is underpinned by the formation of an unexpected Rab29-induced super-assembly containing two central kinase-active and two peripheral kinase-inactive LRRK2 protomers. Central protomers undergo pronounced oligomerization-associated rearrangements and adopt an active conformation. Our work reveals the structural mechanism for LRRK2s spatial regulation controlled by Rab GTPases, provides mechanistic insights into pathogenic mutations and identifies new opportunities to design LRRK2 inhibitors for PD treatment.

biophysics↗

bioRxiv2022

Circular mRNA encoded PROTAC (RiboPROTAC) as a new platform for the degradation of intracellular therapeutic targets

Although proteolysis targeting chimera (PROTAC) technology that hijacking the ubiquitin-proteasome system (UPS) to artificially degrade protein is an emerging promising technology for many undruggable targets, it still faces several challenges, such as the difficulty to select high specificity molecule to protein of interest (POI), and only few of the E3-ligase are suitable for PROTAC mediated protein degradation. Protein-based PROTAC, termed BioPROTAC, owns the advantage of specificity but lacks membrane permeability. Here, we develop a new targeted protein degrading platform, which we termed RiboPROTAC, by encoding BioPROTAC protein degraders with circular mRNA (cmRNA) and delivering intracellularly. In this way, intracellular protein GFP and nuclear protein PCNA were successfully degraded. For PCNA targeting, not only the level of target protein is strongly decreased, but also the cell function related is strongly interfered, such as cell proliferation, apoptosis, as well as the cell cycle. What is more, for the first time, we observed that degrading PCNA with RiboPROTAC causes immunogenic cell death (ICD), although not strong, but was verified in vivo. Importantly, our in vivo results demonstrated that cmRNA encoded PCNA degrader exhibited significant tumor suppression effect, thus we provide the first proof of concept for the application of RiboPROTAC as potential mRNA therapeutic. We consider that RiboPROTAC is a new and superior PROTAC technology for targeting the undruggable targets.

molecular biology↗

bioRxiv2022

Persistent Memory as an Effective Alternative to Random Access Memory in Metagenome Assembly

The assembly of metagenomes decomposes members of complex microbe communities and allows the characterization of these genomes without laborious cultivation or single-cell metagenomics. Metagenome assembly is a process that is memory intensive and time consuming. Multi-terabyte sequences can become too large to be assembled on a single computer node, and there is no reliable method to predict the memory requirement due to data-specific memory consumption pattern. Currently, out-ofmemory (OOM) is one of the most prevalent factors that accounts for metagenome assembly failures. In this study, we explored the possibility of using Persistent Memory (PMem) as a less expensive substitute for dynamic random access memory (DRAM) to reduce OOM and increase the scalability of metagenome assemblers. We evaluated the execution time and memory usage of three popular metagenome assemblers (MetaSPAdes, MEGAHIT, and MetaHipMer2) in datasets up to one terabase. We found that PMem can enable metagenome assemblers on terabyte-sized datasets by partially or fully substituting DRAM at a cost of longer running times. In addition, different assemblers displayed distinct memory/speed trade-offs in the same hardware/software environment. Because PMem was provided directly without any application-specific code modification, these findings are likely to be generalized to other memory-intensive bioinformatics applications.

bioinformatics↗

bioRxiv2022

BIN2 phosphorylates the Thr280 of CO to restrict its function in promoting Arabidopsis flowering

CONSTANS (CO) is a central regulator of floral initiation in response to photoperiod. In this study, we show that the GSK3 kinase BIN2 physically interacts with CO and the gain-of-function mutant bin2-1 displays late flowering phenotype through down-regulation of FT transcription. Genetic analyses show that BIN2 genetically acts upstream of CO in regulating flowering time. Further, we illustrate that BIN2 phosphorylates the Thr280 residue of CO. Importantly, the BIN2 phosphorylation of Thr280 residue restricts the function of CO in promoting flowering. Moreover, we reveal that the N-terminal part of CO harboring the B-Box domain mediates the interaction of both CO-CO and BIN2-CO. We find that BIN2 inhibits the formation of CO dimer/oligomer. Taken together, this study reveals that BIN2 regulates flowering time through phosphorylating the Thr280 of CO and inhibiting the CO-CO interaction in Arabidopsis. HighlightBIN2 regulates flowering time through phosphorylating the Thr280 of CO in Arabidopsis.

molecular biology↗

bioRxiv2022

RNA helicase DDX5 mediates adaptive response to multi-kinase inhibitors in liver cancer

ObjectiveTo determine the role of RNA helicase DDX5 in sorafenib/multi-tyrosine kinase inhibitor (mTKI) response. Sorafenib and mTKIs downregulate DDX5 in vitro and preclinical hepatocellular carcinoma (HCC) models. In turn, sorafenib-mediated DDX5 downregulation activates Wnt/{beta}-catenin and non-canonical NF-{kappa}B signaling, resulting in ferroptosis escape and mTKI resistance. Design and ResultsMolecular, pharmacologic and bioinformatic approaches were employed in human HCC cell lines, preclinical HCC models, and HCCs from TCGA. Earlier studies linked sorafenib effectiveness to ferroptosis. Herein we demonstrate sorafenib/mTKIs downregulate DDX5 in vitro and in vivo. To understand the effect of DDX5 downregulation, we compared TCGA-derived HCCs expressing low vs. high DDX5 focusing on ferroptosis-related genes. Glutathione Peroxidase 4 (GPX4), a key ferroptosis regulator, was significantly overexpressed in DDX5LOW HCCs. Importantly, DDX5-knockdown (DDX5KD) HCC cell lines lacked lipid peroxidation by GPX4 inhibition, indicating DDX5 downregulation suppresses ferroptosis. RNAseq of wild type vs. DDX5KD cells untreated or treated with sorafenib, identified a unique set of genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps genes from Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways, including NF-{kappa}B inducing Kinase required for non-canonical NF-{kappa}B activation. Pharmacologic inhibition of these pathways in combination with sorafenib reduced DDX5KD cell viability. Mechanistically, sorafenib-mediated NIK expression induced NRF2 transcription, while DDX5KD extended NRF2 half/life by stabilizing p62/SQSTM1, enhancing GPX4 expression and ferroptosis escape. ConclusionSorafenib/mTKI-mediated DDX5 downregulation results in adaptive mTKI resistance by enhancing NRF2 expression, leading to ferroptosis escape. We propose inhibition of the pathways leading to NRF2 expression will enhance the therapeutic effectiveness of sorafenib/mTKIs. O_LIWhat is already known on this topic - In advanced HCC, mTKIs/sorafenib, offer limited survival benefits due to resistance. Combination of VEGF (bevacizumab) and PD-L1 antibody (atezolizumab) has led to its adoption as first line treatment; however, mTKIs are still widely used in patients with contra-indications to bevacizumab; also, in atezolizumab/bevacizumab failure, mTKIs are the gold standard in second or later-line systemic therapy, emphasizing the importance of delineating the mechanism of mTKI resistance. C_LIO_LIWhat this study adds - We show mTKIs and sorafenib downregulate the RNA helicase DDX5. This downregulation of DDX5 by sorafenib enables activation of Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways, leading to expression of genes that enable escape from ferroptotic cell death. C_LIO_LIHow this study might affect research, practice or policy The Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways activated by sorafenib/DDX5 downregulation can serve as druggable targets to enhance the anti-cancer effect of mTKIs by inducing ferroptosis, thereby opening-up new therapeutic directions against mTKI resistance. C_LI

cancer biology↗

bioRxiv2022

Mass red blood cell extravasation and tubular uptake results in toxic injury to the tubules during kidney ischemia from venous clamping

Vascular congestion is common in ischemic acute kidney injury (AKI) and represents densely packed red blood cells (RBC) in the kidney circulation. In this study we tested the hypothesis that vascular congestion directly promotes tubular injury. Studies were performed in male and female Wistar-Kyoto rats. Vascular congestion and tubular injury were examined between renal venous clamping, arterial clamping and venous clamping of blood perfused and blood free kidneys. Vessels were occluded for either 15 or 45 minutes without reperfusion. We found that venous clamping resulted in greater vascular congestion than arterial clamping, particularly in the outer-medullary region (P<0.001). Venous clamping resulted in significant tubular injury, including cell swelling, tubular degeneration and luminal cast formation following as little as 15 minutes of occlusion. Tubular injury was significantly less following arterial clamping (P<0.001). Numerous red droplets were observed within tubular cells which were most prominent following venous clamping. Electron microscopy and immunohistochemistry identified these as derived from RBCs and indicated that RBCs from congested renal capillaries were extravasated and phagocytosed by tubular cells. CD235a staining confirmed tubular uptake and secretion of RBCs. Cast formation and tubular swelling were absent from blood free kidneys following venous clamping (P<0.001). Our data demonstrate that congestion of the kidney results in the rapid, mass extravasation and uptake of RBCs by tubular cells causing toxic injury to the tubules. Tubular toxicity from extravasation of RBCs appears to be a major component of tubular injury in ischemic AKI which has not previously been recognized.

physiology↗

bioRxiv2022

Hypoxia-induced tracheal elasticity in vector beetle facilitates the loading of pinewood nematode

Many pathogens rely on their insect vectors for transmission. Such pathogens are under selection to improve vector competence for their transmission by employing various tissue or cellular responses of vectors. However, whether pathogens can actively cause hypoxia in vectors and exploit hypoxia responses to promote their vector competence is still unknown. Fast dispersal of pinewood nematode (PWN), the causal agent for the destructive pine wilt disease and subsequent infection of pine trees is characterized by the high vector competence of pine sawyer beetles (Monochamus spp.), and a single beetle can harbor over 200,000 PWNs in its tracheal system. Here, we demonstrate that PWN loading activates hypoxia in tracheal system of the vector beetles. Both PWN loading and hypoxia enhanced tracheal elasticity and thickened the apical extracellular matrix (aECM) of the tracheal tubes while a notable upregulated expression of a resilin-like mucin protein Muc91C was observed at the aECM layer of PWN-loaded and hypoxic tracheal tubes. RNAi knockdown of Muc91C reduced tracheal elasticity and aECM thickness under hypoxia conditions and thus decreasing PWN loading. Our study suggests a crucial role of hypoxia-induced developmental responses in shaping vector tolerance to the pathogen and provides clues for potential molecular targets to control pathogen dissemination.

ecology↗

bioRxiv2022

Intestinal epithelial Axin1 deficiency protects against colitis via altered gut microbiota

Background and AimsIntestinal homeostasis is maintained by specialized host cells and the gut microbiota. Wnt/{beta}-catenin signaling is essential for gastrointestinal development and homeostasis, and its dysregulation has been implicated in inflammation and colorectal cancer. Axin1 negatively regulates activated Wnt/{beta}-catenin signaling, but little is known regarding its role in regulating host-microbial interactions in health and disease. Here, we aim to demonstrate that intestinal Axin1 determines gut homeostasis and host response to inflammation. MethodsThe expression of Axin1 was analyzed in human IBD datasets. To explore the effects and mechanism of intestinal Axin1 in regulating intestinal homeostasis and colitis, we generated mouse models with Axin1 conditional knockout in intestinal epithelial (Axin1{Delta}IEC) and Paneth cells (Axin1{Delta}PC) to compare with control (Axin1LoxP) mice. ResultsWe found increased Axin1 expression in the colonic epithelium of human IBD. Axin1{Delta}IEC mice exhibited altered goblet cell spatial distribution, Paneth cell morphology, reduced lysozyme expression, and enriched Akkermansia muciniphila. Absence of intestinal epithelial and Paneth cell Axin1 decreased susceptibility to DSS-induced colitis in vivo. Axin1{Delta}IEC and Axin1{Delta}PC became more susceptible to DSS-colitis after cohousing with control mice, suggesting the non-colitogenic effect is driven by the gut microbiota. ConclusionWe found loss of intestinal Axin1 protects against colitis, which is likely driven through Paneth cell Axin1 and the microbiota. Our study demonstrates a novel role of Axin1 in mediating intestinal homeostasis and the microbiota. Further mechanistic studies using specific Axin1 mutations elucidating how Axin1 modulates microbiome and host inflammatory response, will provide new therapeutic strategies for human IBD. What you Need to Know1. Background and ContextWnt/beta-catenin is a fundamental molecular pathway that affects intestinal proliferation and differentiation. Axin1 negatively regulates activated Wnt/{beta}-catenin signaling, but little is known regarding its role in the microbiome. Dysfunction of Wnt/beta-catenin was reported in human inflammatory bowel disease (IBD) and Axin1 serum level was elevated in patients with UC. 2. New FindingsWe found increased Axin1 expression at both the mRNA and protein level in human IBD. Specifically, we identified increased Axin1 expression positive correlated with pro-inflammatory cytokines IL-6 and TNF- in CD. Our study, for the first time, identifies links between the gut microbiota and intestinal Axin1 in intestinal inflammation through utilization of innovative deletion mouse models in intestinal epithelium and Paneth cells. Loss of intestinal Axin1 plays a novel role in intestinal inflammation by altering the Paneth cells and microbiome (e.g., enriched Akkermansia mucinlphila). Our study has provided insights into the molecular mechanism that might contribute to IBD, especially the novel role of Paneth cell Axin1 in colitis. 3. LimitationsThere are no human or mice studies assessing the role of intestinal epithelial and Paneth cell Axin1 in inflammation and the microbiome. 4. ImpactFurther explorations of the gut microbiota and Axin1 interaction as we report will provide novel mechanistic strategies for therapeutic approaches for human IBD by targeting intestinal Axin1 and Axin1-associated microbiome.

pathology↗

bioRxiv2022

Evidence for the role of music on the growth and signal response in duckweed

Sound vibration, an external mechanical force, has been proved to modulate plant growth and development like rain, wind, and vibration. However, the role of music on plants, especially on signal response, has been usually neglected in research. Herein, we investigated the growth state, gene expression, and signal response in duckweed treated with soft music. The protein content in duckweed after music treatment for 7 days was about 1.6 times that in duckweed without music treatment. Additionally, Fv/Fm in duckweed treated with music was 0.78, which was significantly higher in comparison with the control group (P < 0.01). Interestingly, music promoted the Glu and Ca signaling response. To further explore the global molecular mechanism, we performed transcriptome analysis. A total of 1296 DEGs were found for all these investigated genes in duckweed treated with music compared to the control group. Among these, up-regulation of the expression of metabolism-related genes related to glycolysis, cell wall biosynthesis, oxidative phosphorylation, and pentose phosphate pathways were found. Overall, these results provided a molecular basis to music-triggered signal response, transcriptomic, and growth changes in duckweed, which also highlighted the potential of music as an environmentally friendly stimulus to promote improved protein production in duckweed.

plant biology↗

bioRxiv2022

Alveolar regeneration following viral infection is independent of tuft cells

Severe injuries following viral infection cause lung epithelial destruction with the presence of ectopic basal progenitor cells (EBCs), although the exact function of EBCs remains controversial. We and others previously showed the presence of ectopic tuft cells in the disrupted alveolar region following severe influenza infection. Here, we further revealed that the ectopic tuft cells are derived from EBCs. This process is amplified by Wnt signaling inhibition but suppressed by Notch inhibition. Further analysis revealed that p63-CreER labeled population de novo arising during regeneration includes alveolar epithelial cells when Tamoxifen was administrated after viral infection. The generation of the p63-CreER labeled alveolar cells is independent of tuft cells, demonstrating segregated differentiation paths of EBCs in lung repair. EBCs and ectopic tuft cells can also be found in the lung parenchyma post SARS-CoV-2 infection, suggesting a similar response to severe injuries in humans.

cell biology↗

bioRxiv2022

Impaired peri-olfactory Cerebrospinal Fluid clearance mediated cognitive decline after dyssomnia

Animal experiments have demonstrated the dependency of CSF clearance function on age and sleep, which partially underlay the cognitive decline in the elderly. However, evidence is lacking in humans mainly due to limited method to assess CSF clearance function. We aimed to image CSF clearance pathways in human brain by dynamic MRI with intrathecal contrast agent as a CSF tracer. We performed T1-weighted and T2-fluid attenuated inversion recovery imaging with equal sequence parameters before and at multiple time points after intrathecal injection of contrast agent to visualize the putative meningeal lymphatic pathway, peri-olfactory nerve pathway and peri-optic nerve pathway. We defined the CSF clearance function as the percentage change in signal unit ratio of critical locations in these pathways from baseline to 39 hours after intrathecal injection. CSF clearance through the putative meningeal lymphatic and peri-neural pathways were clearly visualized in all 85 patients. The CSF clearance function of meningeal lymphatic and peri-neural pathways were reduced with aging (all P < 0.05). The CSF clearance function through peri-olfactory nerve pathway was positive correlated with sleep quality and cognitive function (both P < 0.05). Moreover, the CSF clearance function through peri-olfactory nerve pathway mediated the association of sleep quality with cognitive function (percent change in {beta} [bootstrap 95% confidence interval]: 35% [-0.220, -0.003]). The study shows promise for dynamic MRI with intrathecal injection of contrast agent as a method to assess CSF clearance function through putative peri-neural pathways, and interprets the impaired clearance through putative peri-olfactory nerve pathway may explain the cognitive decline in patients with dyssomnia.

neuroscience↗

bioRxiv2022

Balancing activation and costimulation of CAR tunes signaling dynamics and enhances therapeutic potency

BackgroundPrimary human T cells engineered with chimeric antigen receptors (CARs) ex vivo can be adoptively transferred to treat cancer. CD19-targeting CAR with CD28 costimulatory domain and CD3{zeta} activation domain have been approved by the US FDA for treating B cell malignancies. MethodsHere we generated mutation of immunorecpetor tyrosine-based activation motifs (ITAMs) in CD3{zeta}, namely 1XX CAR, which altered the balance of activation and costimulation. Next we investigated whether 1XX design could enhance therapeutic potency against solid tumors. We constructed both CD19- and AXL-specific 1XX CARs and compared their in vitro and in vivo functions with their WT counterparts. ResultsEven though 1XX CARs decreased cytotoxicity against tumor cells in vitro, they showed better anti-tumor efficacy in both pancreatic and melanoma mouse models. Detailed analysis revealed that 1XX CAR-T cells proliferated more in response to antigen stimulation in vitro, persisted longer in vivo and had higher percentage of central memory cells. As 1XX modification directly calibrates CAR activation potential, we utilized fluorescence resonance energy transfer (FRET)-based biosensor to monitor signaling dynamics downstream of CARs. Decreased ITAM numbers in 1XX resulted in similar ZAP70 activation, while 1XX induced higher Ca2+ elevation and faster Erk activation than WT CAR, which may contribute to the better therapeutic potency of 1XX. ConclusionsOur results established the surpiosity of 1XX against two targets in different solid tumor models and shed light on the underlying molecular mechanism of CAR signaling, paving the way for the clinical application of 1XX CARs against solid tumors.

immunology↗

bioRxiv2022

The chemokine receptor CXCR3 promotes CD8+ T cell-dependent lung pathology during influenza pathogenesis

While the role of CD8+ T cells in influenza clearance is established, their contribution to pathological lung injury is increasingly appreciated. To explore if protective versus pathological functions can be linked to CD8+ T cell subpopulations, we dissected their responses in influenza-infected murine lungs. Our single-cell RNASeq (scRNAseq) analysis revealed significant diversity in CD8+ T cell subpopulations during peak viral load vs. infection-resolved state. While enrichment of Cxcr3hi CD8+ T effector (Teff) subset was associated with a more robust cytotoxic response, both CD8+ Teff and CD8+ T central memory (TCM) exhibited equally potent effector potential. The scRNAseq analysis identified unique regulons regulating the cytotoxic response in CD8+ T cells. The neutralization of CXCR3 mitigated lung injury without affecting viral clearance. IFN-{gamma} was dispensable to regulate the cytotoxic response of Cxcr3hi CD8+ T cells. Collectively, our data imply that CXCR3 interception could have a therapeutic effect in preventing influenza-linked lung injury. TEASERThe CXCR3 expressing CD8+ T cell subset causes severe lung pathology and exacerbates disease severity without affecting viral clearance during influenza infection

immunology↗

bioRxiv2022

Emodin Combined with Multiple Low-frequency Low-intensity Ultrasound to Relieve Osteomyelitis Through Sonoantimicrobial Chemotherapy

Treatment of osteomyelitis is still challenging as conventional antibiotic therapy is limited by the emergence of resistant strains and the formation of biofilms. Sonoantimicrobial chemotherapy (SACT) is a novel therapy of low-frequency and low-intensity ultrasound (LFLIU) combined with sonosensitizer. Therefore, in our study, a sonosensitizer named emodin (EM) was proposed to be combined with LFLIU to relieve acute osteomyelitis caused by Methicillin-resistant Staphylococcus aureus (MRSA) through synergistic antibacterial and anti-biofilm effects. The efficiency of different intensities of ultrasound, single (S-LFLIU, 15 min) and multiple ultrasound (M-LFLIU, 5 min every 4 h, three times) against bacteria and biofilm was compared, contributing to develop the best treatment regimen. Our results demonstrated that EM plus S-LFLIU or M-LFLIU (EM+S-LFLIU or EM+M-LFLIU) have significant synergetic bactericidal and anti-biofilm effects and EM+M-LFLIU exhibits superior performance in anti-biofilm. Furthermore, it was suggested that EM+M-LFLIU could produce a large amount of reactive oxygen species (ROS), destroy the integrity of bacterial membrane and wall, down-regulate the expression of oxidative stress, membrane wall synthesis, bacterial virulence and other related genes (agrB, PBP3, sgtB, GMK, zwf, msrA). In vivo study, micro-CT, H&E staining, ELISA assay and bacterial quantification of bone tissue indicated that EM+M-LFLIU could also relieve osteomyelitis of MRSA infection. Our work proffers an original treating bacterial osteomyelitis approach that weakens drug-resistant bacterial and suppresses biofilm formation through SACT, which may provide new prospects for clinical treatment.

microbiology↗

bioRxiv2022

PIMT suppresses endothelial activation and vascular inflammation through methylation of TRAF6

As a repair enzyme, protein L-isoaspartyl O-methyltransferase (PIMT) methylates and converts isoaspartyl residues (isoAsp) back to conventional forms to avoid protein damage during aging and stress responses. This study aims to investigate the pathological significance of PIMT in vascular inflammation. Herein, we show that PIMT is highly expressed in lung endothelial cells (ECs), and its reduction significantly exacerbates pulmonary inflammation in a murine model of acute lung injury (ALI). Mechanistically, we identify tumor necrosis factor receptor-associated factor 6 (TRAF6) as a substrate of PIMT. PIMT-mediated methylation of TRAF6 inhibits TRAF6 oligomerization, autoubiquitination, and LPS-induced NF-{kappa}B transactivation in ECs. Importantly, in addition to transcriptionally attenuating expression of adhesion molecules, PIMT post-translationally impedes site specific N-glycosylation of intercellular adhesion molecule-1 (ICAM-1), which leads to an increased protein degradation of ICAM-1 and a subsequent inhibition of EC interaction with immune cells during inflammatory process. Our results for the first time identify PIMT-mediated protein O-methylation as a key post-translational mechanism in controlling vascular inflammation, and suggest that PIMT may represent a novel therapeutic target in inflammatory vascular diseases.

physiology↗

bioRxiv2022

Dietary butyrate treatment enhances healthy metabolites by longitudinal untargeted metabolomic analysis in amyotrophic lateral sclerosis mice

Microbial metabolites affect the neuron system and muscle cell functions. Amyotrophic Lateral Sclerosis (ALS) is a multifactorial neuromuscular disease. Our previous study has demonstrated elevated intestinal inflammation and dysfunctional microbiome in ALS patients and an ALS mouse model (human-SOD1G93A transgenic mice). However, the metabolites in ALS progression are unknown. Using an unbiased global metabolomic measurement and targeted measurement, we investigated the longitudinal changes of fecal metabolites in the SOD1G93A mice over the course of 13 weeks. We compared the changes of metabolites and inflammatory response in age-matched WT and SOD1G93A mice treated with bacterial product butyrate. We found changes in carbohydrate levels, amino acid metabolism, and formation of gamma-glutamyl amino acids. Shifts in several microbially-contributed catabolites of aromatic amino acids agree with butyrate-induced changes in composition of gut microbiome. Declines in gamma-glutamyl amino acids in feces may stem from differential expression of GGT in response to butyrate administration. Due to signaling nature of amino acid-derived metabolites, these changes indicate changes in inflammation (e.g. histamine) and contribute to differences in systemic levels of neurotransmitters (e.g. GABA, glutamate). Butyrate treatment was able to restore some of the healthy metabolites in ALS mice. Moreover, microglia in the spinal cord were measured by the IBA1 staining. Butyrate treatment significantly suppressed the IBA1 level in the SOD1G93A mice. The serum IL-17 and LPS were significantly reduced in the butyrate treated SOD1G93A mice. We have demonstrated an inter-organ communications link among metabolites, inflammation, and ALS progression, suggesting the potential to use metabolites as ALS hallmarks and for treatment. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/476456v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@16d3e4dorg.highwire.dtl.DTLVardef@1155c05org.highwire.dtl.DTLVardef@14c6155org.highwire.dtl.DTLVardef@18a30d6_HPS_FORMAT_FIGEXP M_FIG We compared the changes of metabolites and inflammatory response in age-matched WT and SOD1G93A mice treated with bacterial product butyrate. Butyrate treatment was able to restore some of the healthy metabolites in ALS mice. Due to signaling nature of amino acid-derived metabolites, these changes indicate changes in inflammation and contribute to differences in systemic levels of neurotransmitters (e.g. GABA, glutamate). Moreover, butyrate treatment significantly suppressed the microglia IBA1 level and aggregated SOD1G93A in the SOD1G93A mice. The inflammatory cytokine, e.g serum IL-17, was significantly reduced in the butyrate treated SOD1G93A mice. We have demonstrated an inter-organ communications link among metabolites, inflammation, and ALS progression, suggesting the potential to use metabolites as ALS hallmarks and for treatment. C_FIG

physiology↗

bioRxiv2022

Comparison of double-stranded and single-stranded cfDNA library construction methods for targeted genome and methylation sequencing

Cell-free DNA (cfDNA) profiling by next generation sequencing (NGS) has wide applications in cancer diagnosis, prognosis, and therapy response monitoring. One key step of cfDNA deep sequencing workflow is NGS library construction, whose efficiency determines effective sequencing depth, sequencing quality, and accuracy. In this study, we compared two different cfDNA library construction methods for the applications of mutation detection and methylation profiling: the conventional method which captures double-stranded DNA (dsDNA) molecules, namely the dsLib workflow, and an alternative method which captures single-stranded DNA (ssDNA), namely the ssLib workflow. Our results suggest that the dsLib method was preferrable for mutation detection while the ssLib method proved more efficient for methylation analysis. Our findings could help researchers choose more appropriate library construction method for corresponding downstream sequencing applications.

molecular biology↗

bioRxiv2021

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron

Genomic analyses have revealed how the gut microbiota impacts human health. However, knowledge about the physiology of most gut commensals is largely lacking. Here, we sorted cells from a pooled library to construct an ordered collection of transposon-insertion mutants in the model commensal Bacteroides thetaiotaomicron. We applied a pooling strategy with barcode sequencing to locate mutants and created a condensed collection with single insertions in 2,565 genes. This effort enabled the development of an accurate model for progenitor-collection assembly, which identified strain-abundance biases and multi-insertion strains as important factors that limit coverage. To demonstrate the potential for phenotypic screening, we analyzed growth dynamics and morphology of the condensed collection and identified growth defects and altered cell shape in the sphingolipid-synthesis gene BT0870 and the thiamine scavenging gene BT2397. Analyses of this collection and utilization of the platform described herein to construct future ordered libraries will increase understanding of gut commensal physiology and colonization strategies.

microbiology↗