参考文献

Chlamydia infection and disease are endemic in free-ranging koalas. Antibiotics remain the front line treatment for Chlamydia in koalas,despite their rates of treatment failure and adverse gut dysbiosis outcomes. A Chlamydia vaccine for koalas has shown promise for replacing antibiotic treatment in mild ocular Chlamydia disease. In more severe disease presentations that require antibiotic intervention,the effect of vaccinating during antibiotic use is not currently known. This study investigated whether a productive immune response could be induced by vaccinating koalas during antibiotic treatment for Chlamydia-induced cystitis. Plasma IgG antibody levels against the C. pecorum major outer membrane protein (MOMP) dropped during antibiotic treatment in both vaccinated and unvaccinated koalas. Post-treatment,IgG levels recovered. The IgG antibodies from naturally-infected,vaccinated koalas recognised a greater proportion of the MOMP protein compared to their naturally-infected,unvaccinated counterparts. Furthermore,peripheral blood mononuclear cell gene expression revealed an up-regulation in genes related to neutrophil degranulation in vaccinated koalas during the first month post-vaccination. These findings show that vaccination of koalas while they are being treated with antibiotics for cystitis can result in the generation of a productive immune response,in the form of increased and expanded IgG production and host response through neutrophil degranulation. View Publication

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders,resulting in early-onset,therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy,severe developmental delay,progressive microcephaly,visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent,homozygous variant (chr2:64083454A {\textgreater} G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform,which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells,leading to altered glycogen metabolism,upregulated unfolded protein response and premature neuronal differentiation,as modeled during pluripotent stem cell differentiation in vitro. In contrast,the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly,it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease,even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies. View Publication

Cerebrospinal fluid (CSF) is a vital liquid,providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP),a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here,we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally,the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes. View Publication

Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore,the objective was to study the effects of SCFAs on biomarkers of ISC activity,differentiation,barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET),propionate (PROP),or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments,but increased with ACET. As a result of BUT and PROP treatments,there was an increase in differentiation markers for enterocyte,Paneth,goblet,and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3$\beta$,Reg3$\gamma$,and Defb1 were stimulated by BUT and PROP,but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures,although tight junctions were influenced by BUT and PROP,but not ACET in monolayer format. Furthermore,BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall,individual SCFAs are potent stimulators of cellular gene expression,however,PROP and especially BUT show great efficacy for driving cell differentiation and gene expression. View Publication

Site-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells,via single-cell RNA-Seq and protein mass spectrometry,demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C-expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro,recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells,with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells,S1P ablation results in an increased production of granulocyte-macrophage progenitors,the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche. View Publication

Oncolytic virus therapy leads to immunogenic death of virus-infected tumor cells and this has been shown in preclinical models to enhance the cytotoxic T-lymphocyte response against tumor-associated antigens (TAAs),leading to killing of uninfected tumor cells. To investigate whether oncolytic virotherapy can increase immune responses to tumor antigens in human subjects,we studied T-cell responses against a panel of known myeloma TAAs using PBMC samples obtained from ten myeloma patients before and after systemic administration of an oncolytic measles virus encoding sodium iodide symporter (MV-NIS). Despite their prior exposures to multiple immunosuppressive antimyeloma treatment regimens,T-cell responses to some of the TAAs were detectable even before measles virotherapy. Measurable baseline T-cell responses against MAGE-C1 and hTERT were present. Furthermore,MV-NIS treatment significantly (P {\textless} 0.05) increased T-cell responses against MAGE-C1 and MAGE-A3. Interestingly,one patient who achieved complete remission after MV-NIS therapy had strong baseline T-cell responses both to measles virus proteins and to eight of the ten tested TAAs. Our data demonstrate that oncolytic virotherapy can function as an antigen agnostic vaccine,increasing cytotoxic T-lymphocyte responses against TAAs in patients with multiple myeloma,providing a basis for continued exploration of this modality in combination with immune checkpoint blockade. View Publication

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however,the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here,we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins,including PDGFR$\alpha$,Sca1,and CD81. Notably,CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with $\alpha$V/$\beta$1 and $\alpha$V/$\beta$5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly,CD81 loss causes diet-induced obesity,insulin resistance,and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis. View Publication

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis,regeneration and cancer,little is known about the post-translational regulation of LGR5. Here,we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation,increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically,we find that both NEDD4 and NEDD4L negatively regulate Wnt/$\beta$-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers,which subsequently enhances tumour predisposition and progression. View Publication

The network topology of a protein interactome is shaped by the function of each protein,making it a resource of functional knowledge in tissues and in single cells. Today,this resource is underused,as complete network topology characterization has proved difficult for large protein interactomes. We apply a matrix visualization and decoding approach to a physical protein interactome of a dendritic cell,thereby characterizing its topology with no prior assumptions of structure. We discover 294 proteins,each forming topological motifs called bow-ties" that tie together the majority of observed protein complexes. The central proteins of these bow-ties have unique network properties display multifunctional capabilities are enriched for essential proteins and are widely expressed in other cells and tissues. Collectively the bow-tie motifs are a pervasive and previously unnoted topological trend in cellular interactomes. As such these results provide fundamental knowledge on how intracellular protein connectivity is organized and operates." View Publication

Severe acute respiratory syndrome (SARS) coronavirus (CoV) envelope (E) protein is a transmembrane protein. Several subcellular locations and topological conformations of E protein have been proposed. To identify the correct ones,polyclonal and monoclonal antibodies specific for the amino or the carboxy terminus of E protein,respectively,were generated. E protein was mainly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of cells transfected with a plasmid encoding E protein or infected with SARS-CoV. No evidence of E protein presence in the plasma membrane was found by using immunofluorescence,immunoelectron microscopy and cell surface protein labeling. In addition,measurement of plasma membrane voltage gated ion channel activity by whole-cell patch clamp suggested that E protein was not present in the plasma membrane. A topological conformation in which SARS-CoV E protein amino terminus is oriented towards the lumen of intracellular membranes and carboxy terminus faces cell cytoplasm is proposed. View Publication

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation,here,we compared,in multiple cell and in vivo models,mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1m$\Psi$),synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1m$\Psi$ made by the modified process,while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA. View Publication

Integrating nutrient sensing with the synthesis of complex molecules is a central feature of metabolism. Yet the regulatory mechanisms underlying such integration are often unknown. Here,we establish that the transcription regulators Rtg1/3 are key determinants of sphingolipid homeostasis in the human fungal pathogen Candida albicans. Quantitative analysis of the C. albicans lipidome reveals Rtg1/3-dependent alterations in all complex sphingolipids and their precursors,ceramides. Mutations in the regulators render the fungus susceptible to myriocin,a sphingolipid synthesis inhibitor. Rtg1/3 exert control on the expression of several enzymes involved in the synthesis of sphingolipids' building blocks,and the regulators are activated upon engulfment of C. albicans cells by human neutrophils. We demonstrate that Rtg1p and Rtg3p are regulated at two levels,one in response to sphingolipids and the other by the nutrient sensor TOR. Our findings,therefore,indicate that the Rtg1/3 system integrates nutrient sensing into the synthesis of complex lipids. View Publication