Biology subjects

Zhou, C.

Publications and source records attributed to Zhou, C..

At least 235 records · Page 13Linked to original sources

bioRxiv2020

Identification of Lipid Droplets in Gut Microbiota

Recent research has accumulating evidence to support that gut microbiota can be a newly discovered organ/tissue for humans. Although the functions of gut microbiota have been linked to almost all aspects of human physiological, most findings so far are still either belonged to correlational research or at preliminary stage of causal study due to the complexity of gut microbiota as well as lack of proper tools and methods. Thus, development of new approaches is essential and necessary. Lipid droplet is a cellular organelle governing cell lipid homeostasis that is directly related to human metabolic syndromes. Previously we and other groups found lipid droplets in several bacterial strains. We wonder if gut bacteria have lipid droplets. If so, these bacteria may play key roles to regulate gut lipid content, remove unnecessary lipids and produce useful lipid molecules for host. Here we identified lipid droplet-like structures in freshly isolated bacteria from mouse small and large intestines, as well as mouse and human feces. We also purified and analyzed lipid droplets from a cloned human gut bacterium Streptomyces thermovulgaris. Together, we demonstrate the existence of lipid droplets in gut microbiota and established an approach to study them.

microbiology↗

bioRxiv2020

Rab18 Binds PLIN2 and ACSL3 to Mediate Lipid Droplet Dynamics

Rab18 has been linked to lipid metabolism and metabolic diseases in different model systems, but the mechanism of Rab18-mediated lipid droplet (LD) dynamics in muscle cells remains elusive. Here, we report that Rab18 plays an essential role in oleic acid (OA)-induced LD growth and formation in mouse myoblast cell line C2C12. Rab18 was translocated from endoplasmic reticulum (ER) to LDs during the LD growth induced by OA in C2C12 cells, which was directly regulated by perilipin 2 (PLIN2), a LD resident protein. LD-associated Rab18 bound with the C terminus of PLIN2, and the LD localization of Rab18 was diminished after PLIN2 deletion. Moreover, loss of function of Rab18 led to less triacylglycerol (TAG) accumulation and fewer but larger LD formation. In contrast, expression of wild type Rab18 and a constitutively active Rab18 (Q67L) mutant resulted in elevated TAG content and LD number. Furthermore, LD-associated Rab18 interacted with acyl-CoA synthetase long-chain family member 3 (ACSL3) and in turn, promoted the LD localization of ACSL3, which may play an important role in the accumulation of TAG induced by OA. These data showed that Rab18 was recruited to LD after OA treatment, and formed a complex with PLIN2 and ACSL3, which contributes to TAG accumulating and LD growth.

cell biology↗

bioRxiv2020

Longevity, clonal relationship and transcriptional program of celiac disease-specific plasma cells

Disease-specific plasma cells (PCs) reactive with transglutaminase 2 (TG2) or deamidated gluten peptides (DGP) are abundant in celiac disease (CeD) gut lesions. Their contribution toward CeD pathogenesis is unclear. We assessed expression of markers associated with PC longevity in 15 untreated and 26 treated CeD patients in addition to 13 non-CeD controls, and performed RNA-sequencing with clonal inference and transcriptomic analysis of 3251 single PCs. We observed antigen-dependent V-gene selection and stereotypic antibodies. Generation of recombinant DGP-specific antibodies revealed a key role of a heavy-chain residue that displays polymorphism, suggesting that immunoglobulin gene polymorphisms may influence CeD-specific antibody responses. We identified transcriptional differences between CeD-specific vs non-disease-specific PCs and between short-lived vs long-lived PCs. The short-lived CD19+CD45+ phenotype dominated in untreated and short-term-treated CeD, in particular among disease-specific PCs but also in the general PC population. Thus, the disease lesion of untreated CeD is characterized by massive accumulation of short-lived PCs that are not only directed against disease-specific antigens.

immunology↗

bioRxiv2020

Adiposome Targeting and Enzymatic Activity of Lipid Droplet-Specific Proteins

New strategies to decode the specific protein targeting mechanism on lipid droplet (LD) are urgently needed. Using adiposome, the LD binding of perilipin 2 (PLIN2), perilipin 3 (PLIN3), and adipose triglyceride lipase (ATGL) were studied. Scatchard analysis found that the binding of PLIN2 to the adiposome surface was saturable, pointing to a specific membrane binding partner. Phosphatidylinositol (PI) was found to inhibit PLIN2 binding while it did not impede PLIN3. Structural analysis combined with mutagenesis revealed that the 73rd glutamic acid of PLIN2 is significant for the effect of PI on the protein binding. The presence of PI significantly stimulated the activity of ATGL in vitro. The phosphorylation site mutants of ATGL were found reducing the lipase activity in the adiposome system. Our study demonstrates the utility of adiposome as a powerful, manipulatable model system for the characterization of LD binding and enzymatic activity of LD proteins in vitro.

biochemistry↗

bioRxiv2020

METTL5, an 18S rRNA-specific m6A methyltransferase, modulates expression of stress response genes

RNA N6-methyladenosine (m6A) modification is present in different RNA molecules, including protein-coding mRNAs and non-coding RNAs such as ribosomal RNAs (rRNAs). Previous studies identified m6A in both the 18S and 28S rRNAs, but the roles of these methylation events are poorly understood due to the lack of knowledge of the responsible methyltransferases. Here, we report that mammalian METTL5, a member of a highly conserved methyltransferase family, specifically methylates adenosine 1832 (A1832) in the 18S rRNA in vivo and in vitro. We identify TRMT112 as a near stoichiometric partner of METTL5 important for the enzymatic activity of METTL5. By mapping the positions of translating ribosomes (Ribo-seq), we found translation of multiple stress response-related mRNAs, including Atf4 mRNA, is selectively reduced in the Mettl5 knockout (KO) mouse B16 melanoma cells. Atf4 is a key transcription factor that mediates the Integrated Stress Response (ISR), as exemplified by the Endoplasmic Reticulum (ER) stress. Consistently, transcription of ISR effector genes is reduced in Mettl5 KO cells during ER stress, suggesting a compromised ISR. Our findings reveal a new mechanism that regulates expression of stress response genes and suggest that chemical modifications of ribosomal RNAs may play a key role in selectively impacting translation and possibly ISR.

biochemistry↗

bioRxiv2020

Cryo-EM structure of C9ORF72-SMCR8-WDR41 reveals the role as a GAP for Rab8a and Rab11a

A massive intronic hexanucleotide repeat (GGGGCC) expansion in C9ORF72 is a genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently, C9ORF72, together with SMCR8 and WDR41, has been shown to regulate autophagy and function as Rab GEF. However, the precise function of C9ORF72 remains unclear. Here, we report the cryo-EM structure of the human C9ORF72-SMCR8-WDR41 complex at a resolution of 3.2 [A]. The structure reveals the dimeric assembly of a heterotrimer of C9ORF72-SMCR8-WDR41. Notably, the C-terminal tail of C9ORF72 and the DENN domain of SMCR8 play critical roles in the dimerization of the two protomers of the C9ORF72-SMCR8-WDR41 complex. In the protomer, C9ORF72 and WDR41 are joined by SMCR8 without direct interaction. WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix. Interestingly, the prominent structural feature of C9ORF72-SMCR8 resembles that of the FLNC-FNIP2 complex, the GTPase activating protein (GAP) of RagC/D. Structural comparison and sequence alignment revealed that Arg147 of SMCR8 is conserved and corresponds to the arginine finger of FLCN, and biochemical analysis indicated that the Arg147 of SMCR8 is critical to the stimulatory effect of the C9ORF72-SMCR8 complex on Rab8a and Rab11a. Our study not only illustrates the basis of C9ORF72-SMCR8-WDR41 complex assembly but also reveals the GAP activity of the C9ORF72-SMCR8 complex. Significance StatementC9ORF72, together with SMCR8 and WDR41, has been shown to form a stable complex that participates in the regulation of membrane trafficking. We report the cryo-EM structure of the C9ORF72-SMCR8-WDR41 complex at atomic resolution. Notably, the stoichiometry of the three subunits in the C9ORF72-SMCR8-WDR41 complex is 2:2:2. Interestingly, the C-termini of C9ORF72 and the DENN domain of SMCR8 mediate the dimerization of the two C9ORF72-SMCR8-WDR41 protomers in the complex. Moreover, WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix without direct contact with C9ORF72. Most importantly, the C9ORF72-SMCR8 complex works as a GAP for Rab8a and Rab11a in vitro, and the Arg147 of SMCR8 is the arginine finger.

biochemistry↗

bioRxiv2020

A loss-of-function mutation in IL-17F enhances susceptibility of mice to oropharyngeal candidiasis

Oropharyngeal candidiasis (OPC) is an opportunistic infection of the oral mucosa caused by the commensal fungus C. albicans. IL-17 receptor signaling is essential to prevent OPC in mice and humans, but the individual roles of its ligands, IL-17A, IL-17F and IL-17AF, are less clear. A homozygous IL-17F deficiency in mice does not cause OPC susceptibility, whereas mice lacking IL-17A are moderately susceptible. In humans, a rare heterozygous mutation in IL-17F (IL-17F.S65L) was identified that causes chronic mucocutaneous candidiasis, suggesting the existence of essential antifungal pathways mediated by IL-17F and/or IL-17AF. To investigate the role of IL-17F and IL-17AF in more detail, we exploited this experiment of nature by creating a mouse line bearing the homologous mutation in IL-17F (Ser65Leu) by CRISPR/Cas9.The resulting Il17fS65L/S65L mice showed increased susceptibility to OPC, but only in homozygous, not heterozygous, mutant mice. The mutation was linked to impaired CXC chemokine expression and neutrophil recruitment to the infected tongue but not to alterations in antimicrobial peptide expression. These findings suggest mechanisms by which the enigmatic cytokine IL-17F contributes to host defense against fungi.

immunology↗

bioRxiv2020

Single-cell RNA sequencing deconvolutes the in vivo heterogeneity of human bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells, which have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat found in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte cells that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of transcriptomes suggest that osteoblast precursors may induce angiogenesis coupled with osteogenesis, and chondrocyte precursors may have the potential to differentiate into myocytes. We discovered transcripts for several cluster of differentiation (CD) markers that were highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs and could be novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing insight into the extent of their cellular heterogeneity and bone homeostasis.

cell biology↗

bioRxiv2020

CasRx-mediated RNA targeting prevents choroidal neovascularization in a mouse model of age-related macular degeneration

The smallest Cas13 family protein, CasRx, has a high cleavage activity and targeting specificity, offering attractive opportunity for therapeutic applications. Here we report that delivery of CasRx by adeno-associated virus via intravitreal injection could efficiently knockdown Vegfa transcripts and significantly reduce the area of laser-induced choroidal neovascularization in a mouse model of age-related macular degeneration. Thus, RNA-targeting CRISPR system could be used for in vivo gene therapy.

genetics↗

bioRxiv2020

High-fidelity base editor with no detectable genome-wide off-target effects

Base editors hold promise for correcting pathogenic mutations, while substantial single nucleotide variations (SNVs) on both DNA and RNA were generated by cytosine base editors (CBEs). Here we examined possibilities to reduce off-target effects by engineering cytosine deaminases. By screening 24 CBEs harboring various rAPOBEC1 (BE3) or human APOBEC3A (BE3-hA3A) mutations on the ssDNA or RNA binding domain, we found 8 CBE variations could maintain high on-target editing efficiency. Using Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) method and RNA sequencing analysis, we found DNA off-target SNVs induced by BE3 could be completely eliminated in BE3R126E but the off-target RNA SNVs was only slightly reduced. By contrast, BE3-hA3AY130F abolished the RNA off-target effects while could not reduce the DNA off-target effects. Notably, BE3R132E, BE3W90Y+R126E and BE3W90F+R126E achieved the elimination of off-target SNVs on both DNA and RNA, suggesting the feasibility of engineering base editors for high fidelity deaminases.

genomics↗

bioRxiv2020

Prediction of meiosis-essential genes based upon the dynamic proteomes responsive to spermatogenesis

Mammalian meiosis is a specific cell division process during sexual reproduction, whereas a comprehensive proteome of the different meiotic stages has not been systematically investigated. Here, we isolated different types of spermatocytes from the testes of spermatogenesis-synchronized mice and quantified the corresponding proteomes with high-resolution mass spectrometry. A total of 8,002 proteins were identified in nine types of germ cells, and the protein signatures of spermatogenesis were characterized using the dynamic proteomes. A supervised machine learning package, FuncProFinder, was developed to predict meiosis-essential candidate genes based on changes in their protein abundance. Of the candidates without functional annotation, four of the ten genes with the highest prediction scores, Zcwpw1, Tesmin, 1700102P08Rik, and Kctd19, were validated as meiosis-essential genes using knockout mouse models. The proteomic analysis of spermatogenic cells provides a solid foundation for studying the mechanism of mammalian meiosis.

developmental biology↗

bioRxiv2020

Dynamic Laser Speckle Imaging meets Machine Learning to enable Rapid Antibacterial Susceptibility Testing (DyRAST)

Rapid antibacterial susceptibility testing (RAST) methods which measure change of a bacterial phenotype in response to a given treatment are of significant importance in healthcare, as they can assist care-givers in timely administration of the right treatment. Various RAST techniques have been reported for tracking bacterial phenotypes, such as size, shape, motion, and metabolic activity. However, they still require bulky and expensive instruments (which hinders their application in resource-limited environments) and/or utilize labeling reagents (which can interfere with antibiotics and add to cost). Furthermore, the existing ultra-rapid methods do not address possible adaptation of gradual adaptation of bacteria to antibiotics, which can lead to false interpretation of resistance when using ultra-rapid methods. In this work, we present a RAST approach leveraging machine learning analysis of time-resolved dynamic laser speckle imaging (DLSI) results to accurately predict the minimum inhibitory concentration (MIC) of a model strain of Escherichia coli in 60 minutes, compared to 6 hours using the currently FDA-approved phenotype-based RAST technique. To demonstrate the DLSI performance, we studied the effect of a {beta}-lactam ampicillin and an aminoglycoside gentamicin on Escherichia coli strain K-12. DLSI captures change of bacterial motion/division in response to treatment. The machine learning algorithm was trained and validated using the overnight results of gold standard, broth microdilution method. Empowered by machine learning, DyRAST can predict MIC with high accuracy comparable to gold standard methods through a voting strategy.

microbiology↗

bioRxiv2020

Non-invasive red-light optogenetic control of Drosophila cardiac function

Drosophila is a powerful genetic model system for cardiovascular studies. Recently, optogenetic pacing tools have been developed to control Drosophila heart rhythm noninvasively with blue light, which has a limited penetration depth. Here we developed both a red-light sensitive opsin expressing Drosophila system and an integrated red-light stimulation and optical coherence microscopy (OCM) imaging system. We demonstrated noninvasive control of Drosophila cardiac rhythms, including simulated tachycardia in ReaChR-expressing flies and bradycardia and cardiac arrest in halorhodopsin (NpHR)-expressing flies at multiple developmental stages. By using red excitation light, we were able to pace flies at higher efficiency and with lower power than with equivalent blue light excitation systems. The recovery dynamics after red-light stimulation of NpHR flies were observed and quantified. The combination of red-light stimulation, OCM imaging, and transgenic Drosophila systems provides a promising and easily manipulated research platform for noninvasive cardiac optogenetic studies.

bioengineering↗

bioRxiv2019

CSF1R inhibition by small molecule inhibitor affects hematopoiesis and the function of macrophages.

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a method for microglia depletion, with the assumption that it does not affect peripheral immune cells. Here, we show that CSF1R inhibition by PLX5622 indeed affects the myeloid and lymphoid compartments, causes long-term changes in bone marrow-derived macrophages by suppressing their IL-1{beta}, CD68 and phagocytosis, but not CD208, following exposure to endotoxin, and also reduces the population of tissue resident macrophages of peritoneum, lung, liver, but not spleen. Thus, small molecule CSF1R inhibition is not restricted to microglia only, but rather causes strong effects on circulating and tissue macrophages that perdure long after cessation of the treatment. Given that peripheral monocytes repopulate the CNS after CSF1R inhibition, these changes may have practical implications on relevant experimental data.

neuroscience↗

bioRxiv2019

Mitochondrial Electron Transport Chain Inhibition Suppresses LPS-Induced Inflammatory Responses via TREM1/STAT3 Pathway in BV2 Microglia

Mitochondrial damage and neuroinflammation belong to two of the most important pathological factors in multiple neurological disorders. However, the effect of mitochondrial damage of microglia on microglial activation under pathological conditions has remained unclear. In our current study, we used BV2 microglia as a cellular model to determine the effects of mitochondrial electron transport chain (ETC) inhibitors on LPS-induced inflammatory responses of microglia. We found that all of the three mitochondrial ETC inhibitors, including rotenone, sodium azide and antimycin A, significantly inhibited LPS-induced inflammatory responses of the microglia, assessed by determinations of the protein or mRNA levels of IL-1{beta}, IL-6, TNF-, iNOS and COX2. Nuclear translocation of NF-{kappa}B p65 subunit does not appear to play an important role in the mitochondrial ETC inhibition-produced suppression of microglial activation. Instead, our study found that the mitochondrial ETC inhibitors significantly attenuated not only the LPS-induced increase in the TREM1 levels - an amplifier of inflammatory process, but also the LPS-induced increase in the ratio of phosphorylated STAT3 / STAT3. In summary, our study has suggested that mitochondrial ETC inhibition of microglia can lead to suppression of LPS-induced microglial activation, which may be mediated by the inhibitory effects of mitochondrial ETC inhibition on the LPS-induced increases in the level of TREM1 and the ratio of p-STAT3 / STAT3. These findings have provided valuable information for elucidating the relationships between mitochondrial damage and neuroinflammation in multiple neurological diseases.

immunology↗

bioRxiv2019

Out of the Qinghai-Tibetan Plateau and get flourishing - the evolution of Neodon voles (Rodentia: Cricetidae) revealed by systematic sampling and low coverage whole genome sequencing

Neodon, genus of a short time evolutionary history, was reported to be diverged from its relatives in early stage of Pleistocene. Only 4 species were well documented in Neodon for a long period of time until last years when a systematic work described and added three new species, adjusted three species used to belong to Lasiopodomys, Phaiomys, Microtus to Neodon and removed one species (Neodon juldaschi) to genus Blanfordimys, leading to a total of eight species recorded in Neodon. To gain a better insight into the phylogeny and ecology of Neodon, we have systematically sampled Neodon species along the whole Hengduan and Himalayan Mountains in the last 20 years. In addition to morphological identification, we generated 1x - 15x whole genome sequencing (WGS) data and achieved the mitochondrial genomes and an average of 5,382 nuclear genes for each morpho-species. Both morphology and phylogeny results supported an extra six new species in Neodon (nominated Neodon shergylaensis sp. nov., N. namchabarwaensis sp. nov., N. liaoruii sp. nov., N. chayuensis sp. nov., N. bomiensis sp. nov., and N. bershulaensis sp. nov.). This is the first study that included Neodon samples covering its entire distribution area in China and this systematic sampling also revealed a long-time underestimation of Neodons diversity, and suggested its speciation events linked highly to founder event via dispersal (from Plateau to surrounding mountains). The results also revealed that the Qinghai-Tibetan Plateau is the center of origin of Neodon, and the impetus of speciation include climate change, isolation of rivers and mountains.

zoology↗

bioRxiv2019

Annexin A2 depletion exacerbates the intracerebral microhemorrhage induced by acute rickettsia and Ebola virus infections

Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infections induced by some intracellular pathogens, including rickettsia, can result in CMHs. Annexin a2 (ANXA2) has been documented to play a functional role during intracellular bacterial adhesion. Here we report that ANXA2-knockout (KO) mice are more susceptible to CMHs in response to rickettsia and Ebola virus infections, suggesting an essential role of ANXA2 in protecting vascular integrity during these intracellular pathogen infections. Proteomic analysis via mass spectrometry of whole brain lysates and brain-derived endosomes from ANXA2-KO and wild-type (WT) mice post-infection with R. australis revealed that a variety of significant proteins were differentially expressed, and the follow-up function enrichment analysis had identified several relevant cell-cell junction functions. Immunohistology study confirmed that both infected WT and infected ANXA2-KO mice were subjected to adherens junctional protein (VE-cadherin) damages. However, key blood-brain barrier (BBB) components, tight junctional proteins ZO-1 and occludin, were disorganized in the brains from R. australis-infected ANXA2-KO mice, but not those of infected WT mice. Similar ANXA2-KO dependent CMHs and fragments of ZO-1 and occludin were also observed in Ebola virus-infected ANXA2-KO mice, but not found in infected WT mice. Overall, our study revealed a novel role of ANXA2 in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated tight junctions and its role in stabilizing the BBB in these deadly infections. Author SummaryTraditionally, spontaneous intracerebral microhemorrhages (CMHs) were defined as small foci of intracerebral hemorrhages. Such atraumatic CMHs are due to the rupture of a weak blood vessel wall. Infections complicating cerebrovascular accidents have been extensively investigated. However, the role of CMHs complicating infections, in particularly acute systemic infections, has been poorly explored. Population-based retrospective cohort studies suggest there are potentially more undiscovered cases of CMHs accompanying acute systemic infections. Given both the lack of an animal model and cellular/molecular pathophysiology of CMHs following acute systemic infections, there is an urgent need to increase our comprehensive understanding of acute infection-induced CMHs. Overall, our study revealed a novel role of annexin a2 (ANXA2) in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated endothelial tight junctions and its role in stabilizing the blood-brain barrier in these deadly infections.

pathology↗

bioRxiv2019

Obesity Potentiates TH2 Immunopathology via Dysregulation of PPAR

How obesity affects immune function is not well understood. Clinically, obesity is strongly associated with severe TH2 immunopathology1-3, though the physiological, cellular, and molecular underpinnings of this association remain obscure. Here, we demonstrate that obese mice are susceptible to severe atopic dermatitis (AD), a major manifestation of TH2 immunopathology and disease burden in humans4,5. Mechanistically, we show that dysregulation of the nuclear hormone receptor (NHR) PPAR{gamma} (peroxisome proliferator-activated receptor gamma) in T cells is a causal link between obesity and the increased TH2 immunopathology. We find that PPAR{gamma} oversees a cellular metabolic transcriptional program that restrains nuclear gene expression of the chief TH2 priming and effector cytokine interleukin-4 (IL-4). Accordingly, thiazolidinediones (TZDs), potent PPAR{gamma} agonists, robustly protect obese mice from TH2 immunopathology. Collectively, these findings establish PPAR{gamma} as a molecular link between obesity and TH2 immune homeostasis and identify TZDs as novel therapeutic candidates for TH2 immunopathology. Fundamentally, these findings demonstrate that shifting physiologic metabolic states can shape the tone of adaptive immune responses to modulate differential disease susceptibility.

immunology↗