Coxiella burnetii is the causative agentof Qfever, for which there is yet to be an FDA-approved vaccine. Thisbacterial pathogen has both extra- and intracellular stages in itslife cycle, and therefore both a cell-mediated and humoral  immune response are necessary for effectiveeradication of this pathogen. However, most proposed vaccines elicitstrong responses to only one mechanism of adaptive immunity, and somecan either cause reactogenicity or lack sufficient immunogenicity.In this work, we aim to apply a nanoparticle-based platform towardproducing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugationof the immunodominant outer membrane protein antigen (CBU1910) tothe E2 nanoparticle to obtain a consistent antigen orientation: directgenetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-taggedCBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we foundthat the ST/SC approach yielded nanoparticles loaded with the highestnumber of antigens while maintaining stability, enabling formulationsthat could simultaneously co-deliver the protein antigen (CBU1910)and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Usingprotein microarray analyses, we found that after immunization, antigen-boundnanoparticle formulations elicited significantly higher antigen-specificIgG responses than soluble CBU1910 alone and produced more balancedIgG1/IgG2c ratios. Although T cell recall assays from these proteinantigen formulations did not show significant increases in antigen-specificIFN-γ production compared to soluble CBU1910 alone, nanoparticlesconjugated with a CD4 peptide epitope from CBU1910 generated elevatedT cell responses in mice to both the CBU1910 peptide epitope and wholeCBU1910 protein. These investigations highlight the feasibility ofconjugating antigens to nanoparticles for tuning and improving bothhumoral- and cell-mediated adaptive immunity against C. burnetii. Coxiella burnetii isthe Gram-negative intracellularbacterium that causes the life-threatening disease Q fever,3 and it has been classified by the US Center for Disease Controland Prevention as a potential bioterrorism agent due to its airbornetransmission, highly infectious nature, and extreme resistance toenvironmental conditions.5 Q fever has an almost global distribution and can be found in awide variety of animal reservoirs, with ruminants the most common.6 Human infections are often acquired from inhalationof contaminated aerosols resulting in an acute febrile illness, whichcan progress to pneumonia and hepatitis.7 In approximately 5% of cases, patients develop a potentially fatalchronic disease resulting in endocarditis, osteomyelitis, and chronicfatigue.8 In chronic forms of Q fever,that may arise weeks or years postinfection, long-term combinationtherapies are required to prevent death. Between 2007 and 2010, thelargest known outbreak of Q fever occurred in The Netherlands resultingin >4000 cases.9 Of those identifiedashaving chronic Q fever, mortality was 15.8%.1012 Unlike typical bacterial pathogens, C. burnetii exhibits a tropism for professional immune system phagocytes  and actively directs its own phagocytosis in order toreside within the terminal phagolysosomes of host cells in a favorablelow pH environment, enabling its long-term survival and persistence.14 For this reason, a T lymphocyteresponse, in addition to an adequate antibody response, is considerednecessary for eradication of the pathogen.17 In this investigation,we examine the ability to design and synthesize a C. burnetii vaccine using a protein nanoparticle (NP) platform to elicit bothstrong B and T cell responses. Although the advantages of NPs in vaccinedevelopment have been well-demonstrated,19 the design of antigen-conjugated nanoparticles for a Q fever vaccinehas not yet been reported.Despite its pathogenic potential, an FDA-approved vaccinefor thisinfectious agent is not yet available. A formalin-inactivated wholecell vaccine was previously licensed in Australia but was not approvedin the US, and was discontinued due to the costs of production andrequired associated screening to prevent severe side effects in patientswith previous exposure.Geobacillus stearothermophilus.21 E2 is a 60-subunit, self-assembling ∼25 nm dodecahedral scaffoldwith high stability that can be genetically engineered for precisechemical conjugation sites at the external surface and internal cavity.26 Our prior studies in developing cancer vaccines via a virus-mimeticstrategy have demonstrated the utility of this scaffold for both adjuvantand antigen delivery.31 However, the application of this E2-based strategy for protectionagainst bacterial pathogens has not yet been investigated. In thiswork, we utilize E2’s unique size, functional adaptability,and innate capability to elicit an antigen-specific immune responsetoward developing a prophylactic C. burnetii vaccine.The protein NP utilized in this researchis derived from the E2subunit (E2) of the multienzyme complex, pyruvate dehydrogenase, sourcedfrom C. burnetii.39 For this reason, CBU1910 was chosen as the antigen for this prophylacticvaccine formulation. Unlike cancers, which can utilize peptide neoantigensin a vaccine to produce the desired anti-epitope T cell responses,infectious disease vaccines typically require the use of whole proteinantigens to elicit both antibody and T cell responses.41 Protein antigens contain numerous immunogenic epitopes in nativestructural conformations, allowing for stronger antibody responsesand broader adaptive immune responses.42 Although immunogenicpeptide epitopes of C. burnetii have been identifiedand characterized for their potential use in vaccine development,application of these peptides in vaccines has not yet shown significantefficacy.46 More recently, vaccine formulationsusing C. burnetii protein antigens and triagonistadjuvants showed significant levels of protection for challenged animals,but to a lesser extent than the whole cell vaccine (which is not FDAapproved).47 Thus, there is still a needfor the development of a safer and efficacious prophylactic vaccinefor C. burnetii.Proteomics and antigen-specific serological assays have identifiedthe outer membrane protein CBU1910 as an immunodominant protein antigenof C. burnetii antigens onto thesurface of the E2 NP. It is known that B cellactivation and antibody responses are enhanced by a repetitive structuralarray on virus-like particles;49 however, there are currently limited options for conjugating proteinantigens onto a NP surface while maintaining this consistent geometricorientation. Here, we examined three bioconjugation strategies thatwould enable a desired consistent antigen configuration: (1) directrecombinant fusion, (2) high affinity tris-NTA-Ni conjugation to polyhistidine-tagged(His-tag) antigen, and (3) the SpyTag(ST)/SpyCatcher(SC) system  CBU1910antigen onto E2 NPs. To attach protein antigens, covalently and modularly,onto the surface of E2 NPs, the versatile protein–protein conjugationmethod, SpyTag/SpyCatcher, was implemented.62 The adaptive immune response to the most favorable NP construct was then examined to determinethe prophylactic potential of the vaccine formulation.In this study, we investigatedthe integration of ) system 1. DirectTable SI-1 liststhe descriptions of each of the components and the corresponding abbreviationsused in this work.We examined threestrategies to attach the CBU1910 protein antigen to protein nanoparticles,as summarized in 63 In our studies,we distill this protein down to its structural dodecahedral core forapplication as a nanoparticle scaffold;28 however, based on the native structure, we hypothesized that otherproteins with independent binding domains could be genetically fusedto its N-terminus. We created two E2 mutants with different N-terminallinker lengths and 60 internal cavity cysteines [E2_152(D381C) andE2_158(D381C)]. Relative to the truncated E2(D381C) mutant used previously,31 the E2_152and E2_158 mutants have an additional 20 and 14 amino acids of thewild-type protein sequence, respectively, added to their N-termini.65 Introduction of 60 internal cavity cysteines allows for adjuvantconjugation.66To investigate this loading strategy, we genetically fused CBU1910to the N-terminus of a truncated E2 monomer. The wild-type form ofthe core E2 nanoparticle (dihydrolipoyl transacetylase) includes,on its N-terminus, a lipoyl domain and a peripheral subunit-bindingdomain, which enables association with the E1 and E3 proteins in thecomplex.Table SI-2), and the fusion proteins were expressed in E. coli .CBU1910 protein antigen fused to an E2 monomer was strongly expressed.However, these fusion proteins aggregated as inclusion bodies andwere present only in the insoluble fraction, even under differentexpression conditions  . In contrast,the individual proteins  showedfractions which were soluble , with solubility linked to correct folding and nanoparticle assemblyin prior studies.23 Under the conditionstested, the fused proteins (CBU1910-E2) could not be expressed assoluble proteins, suggesting misfolding and/or misassembly of thecomplex. For this reason, the two subsequent loading strategies focusedon generating the two proteins separately, followed by conjugationtogether.The CBU1910 protein wasrecombinantly fused to E2 28 allows for conjugation of a synthesizedmaleimide-tris-NTA  linker,56 which enables a His-tagged protein to couple to the NP. This protocolwas performed for CBU1910 as shown in 6 to E2 yielded a mixture of single nanoparticlesand aggregates of nanoparticles. Optimization of conjugation conditions to yield non-aggregated nanoparticleswas required, and these conditions are summarized in Figure SI-2. HEPES buffer at pH 7.3 with 360 mM NaCl was foundto stabilize the nanoparticles and was used for subsequent purificationand characterization steps. Free CBU1910-(His)6 was separatedfrom E2-bound CBU1910 using size exclusion chromatography (SEC). Quantificationof the number of E2-attached CBU1910 was determined to be 6 ±3 per E2 nanoparticle.To conjugate CBU1910 onto the surface of the E2 protein NP, weused an affinity strategy that we had developed for conjugating greenfluorescent protein (GFP) and influenza hemagglutinin (HA).67 The small increase in diameterfor CBU1910-E2 was consistent with the relatively low number of CBU1910on the surface of the E2 nanoparticle. To dose an adequate amountof antigen for an in vivo vaccine study, a 10-foldincrease in the concentration was required; however, concentratingthe formulation to this extent led to significant protein aggregation.Although conjugation of the CBU1910 antigen to the protein nanoparticleusing a tNTA linker showed promise, this strategy showed limitationsfor this specific antigen that included low conjugation capacity andinconsistent physical stability. We note that this result is differentthan attachment of HA to E2, which had yielded reliable conjugationand stable, monodisperse particles, suggesting that these effectsare highly antigen-dependent. Furthermore, the use of the maleimide-tNTAfor surface conjugation via cysteine residues limits the use of cysteinesinternally for conjugation of immune-stimulating adjuvants.66Hydrodynamic diameters for E2, tNTA-E2, and CBU1910-E2NPs were27.3 ± 1.1 nm, 28.8 ± 2.2 nm, and 31.6 ± 4.0 nm, respectively,all of which fall in the size range shown to be beneficial for lymphaticsystem trafficking and antigen-presenting cell  engagement 2B.18,67 57 was used to attach CBU1910 to the E2 nanoparticle usingthe strategy outlined in The SpyTag/SpyCatcher systemFigure SI-3). The E2 mutantD381C possessed 60 internal cavity cysteines, which would enable conjugationof adjuvant.66 Because a high-resolution proteinstructure of CBU1910 has not yet been determined, we used the proteinfolding prediction tool Alphafold268 topredict the structure of CBU1910 . Based on this predicted structure of N-terminal truncated CBU1910(to enable a soluble antigen),69 we decided to fuseSC to the N-terminus of CBU1910 .This ensured that when conjugated to the E2 nanoparticle, CBU1910would be oriented in the same direction as when it is displayed on C. burnetii, exposing more relevant B cell epitopes.The ST peptide was genetically fused to the N-terminusof E2 witha spacer sequence SI-3. TheFigure SI-3A). Therefore, we proceededwith purifying ST-E2(D381C) (henceforth referred to as ST-E2) andSC-CBU1910 for further characterization and studies. Both SDS-PAGEand mass spectrometry showed an expected molecular weight increaseof ∼2.2 kDa (ST and spacer) for ST-E2 monomers . The ST-E2 NP assembly yieldeda hydrodynamic diameter of 29.2 ± 0.5 nm, which is slightly largerthan the E2 diameter size of 27.8 ± 0.6 nm , as expected. This is approximately 1 nm larger,which is consistent with previous literature estimates of ST on virus-likeparticles (VLPs).58 For SC-CBU1910, weachieved a >95% purity and the average molecular weight of ∼40.8kDa, as determined by SDS-PAGE and mass spectrometry, which is consistentwith SC fused to CBU1910 with a linker .The attachment of ST and SC to E2 and CBU1910, respectively, didnot appear to decrease the expression levels or soluble protein amounts to aST-E2 monomer  can be found in Figure SI-5. From our investigation, we determinedfavorable reaction conditions to be a 1:0.5 molar ratio of ST-E2 (monomer):SC-CBU1910at room temperature for 20 h with the addition of 0.08–0.0875%(w/v) SLS; this resulted in stable, monodisperse nanoparticles  and the other CBU1910-CpG-E2 monomers  3B. QuantWe investigatedthe antibody responses of different vaccine formulations containingthe CBU1910 protein antigen, CpG1826, E2, and oil-in-water emulsionadjuvant, IVAX, after prime and boost immunization in mice 4A,B. The67 Nanoparticles between ∼20–50 nm in diameter with antigenvalences greater than ∼5 per nanoparticle and antigen spacinglarger than ∼25 nm are reported to obtain effective B cellengagement.18 The CBU-E2 formulations usedin this immunization study possess these characteristics and couldbe one explanation for obtaining >9 times increased IgG antibodyresponsetoward CBU by simply displaying the antigen on the nanoparticle platform(relative to unbound antigen). This also supports the premise thatB cell activation can be augmented by the decoration of nanoparticleswith repetitive epitopes; the repetitive geometry and spatial configurationof NP-attached antigens mimic natural pathogens such as viruses, whichcan yield strong innate adjuvating outcomes by improving uptake byantigen-presenting cells and enabling binding and simultaneous activationof multiple B cell receptors.77It has been previously described that nanoparticlesize and antigendisplay topography can play a crucial role in B cell engagement.78  or co-administered withthe E2 NP by mixing only (CBU-E2 + CpG) showed comparable resultson total IgG responses , monophosphoryl-lipidA (MPLA), and CpG1018, that has been shown to induce broadly reactiveresponses to influenza HA proteinsGroupf, 4D. Ourp83 Th1 responses are characterizedby the activation of antigen-presenting cells, stimulation of T cells,and induction of IgG2c antibodies.84 Thus, IgG1 and IgG2cproduction can be used as indicators of Th2 and Th1 responses, respectively.Profiles of the IgG1 and IgG2c antibody responses suggest modulationcapabilities that depend on the adjuvant used and whether it was loadedin the E2 nanoparticle . The additionof IVAX to this formulation significantly increased the level of IFN-γsecretion. This observation was not expected, given the similar levelsof antigen-specific IgG2c production after immunization with the CBU1910-CpG-E2nanoparticle, both with and without IVAX. The unexpectedly low IFN-γresponse from formulations that induced IgG2c/Th1 responses comparedto IVAX, such as CBU1910-CpG-E2, may have resulted from cytokinesother than IFN-γ  drivingthe IgG2c response, or a different temporal release of IFN-γ. Furtherexperiments would be necessary to test these hypotheses. Another reasonfor this discrepancy could stem from the TLR-4 agonist (MPLA) andTLR-9 agonist (CpG1018) present in IVAX. It has been shown that simultaneousstimulation of cell-surface and endosomal TLR receptors can causesynergistic increases in the activating/inflammatory immune response,and the soluble delivery of the adjuvants may cause a more systemicinitial innate immune response.86 This differs from thedelivery of CpG via a nanoparticle, which avoids non-specific systemicimmune system stimulation due to encapsulation and increases uptakeby antigen presenting cells .27 Furthermore, previous studies that co-delivered peptideantigens and adjuvant for cancer vaccines resulted in dosages of ∼2–5μg of CpG and ∼2–5 μg of T cell immunogenicpeptide epitopes;31 however, for this protein antigen vaccine, both theCpG and immunodominant T cell epitope dosage were nearly an orderof magnitude less than typically dosed for the peptide formulationsof the cancer studies.The more balanced IgG1/IgG2c antibody ratio elicited by CBU1910-CpG-E2suggests that T cell responses toward the CBU1910 antigen are likelyto be produced. However, when we examined the effector T cell response,we found that antigen-specific IFN-γ responses (which correspondsto an IgG2c/Th1 response) were low in mice that were administeredCBU1910-CpG-E2 alone coupled to the E2 NP could increase the cell-mediated response. Peptideantigens were conjugated to the NP using a linker and characterized,as described in the Supporting Information. Interestingly, we observed that immunizing with the CBU1910p-CpG-E2formulation, an E2 nanoparticle loaded with the immunodominant CD4T cell epitope peptide of CBU1910, HYLVNHPEVLVEASQ(CBU1910p), not only generated a higher frequency of CBU1910p-specificT cells, compared to soluble CBU1910p immunization, but also inducedstrong CBU1910-specific IFN-γ secretion . This confirms that this peptide is a bona fide epitope generatedby the natural processing of the whole CBU1910 antigen. This CBU1910pformulation allows for the delivery of ∼50-fold more of theimmunodominant T cell epitope and ∼6-fold more of CpG thanthe CBU1910-bound NP formulation . These data suggest that using this epitope may be sufficient toelicit a strong cellular immune response toward the immunodominantprotein antigen of C. burnetii, CBU1910. Given thestrong antibody and T cell responses observed from the CBU1910-basedand CBU1910p E2 NP formulations, respectively, a potential co-administrationof the two warrants further investigations.To determine if an E2 formulation was capable ofinducing a strongantigen-specific T cell response toward C. burnetii immunodominant protein antigen, CBU1910,to a protein nanoparticle:genetic fusion, tris-NTA-Ni linker, and ST/SC. We determined thatST/SC yielded vaccine nanoparticles with the highest antigen loading,had the capacity to internally encapsulate adjuvant, and could beformulated to yield stable, monodisperse particles. We engineeredthis platform, ST-E2(D381C), to allow for the simultaneous packingof Th1-skewing adjuvant, CpG, within the interior of the nanoparticleand displaying of protein antigen, CBU1910, on its surface. By displayingthe antigen on the nanoparticle only, i.e., in the absence of CpGadjuvant, significant increases in antigen-specific IgG antibodieswere observed in immunized mice compared to soluble antigen alone.The addition of an encapsulated or co-administered adjuvant balancedthe IgG1/IgG2c antibody profile, which suggests induction of bothTh1 (associated with cellular immunity) and Th2  responses.We investigated three methods of conjugatingthe C. burnetii infection.While antibodies play an important role during the early stages ofextracellular infection, T cell activation is vital for clearanceof intracellular bacteria.87 Of particularsignificance are Th1 cells, identified by class switched B cells toproduce IgG2c and IFN-γ positive CD4 T cells. Th1 cells contributetoward immunity by polarizing macrophages toward an M1 phenotype thatis less permissive for intracellular C. burnetii.88 Th1 cells also support the activation of cytotoxicT cells, which target infected cells and aid in the formation of protectivegranulomas that surround parasitized cells. Moreover, Th1 cells contributeto long-term immunity through the generation of memory responses.A balance between antibody mediated and Th1 mediated immunity is essentialfor effective protection against C. burnetii. Toconfirm the presence of cell-mediated immunity more directly, we showthat vaccination with a CBU1910 CD4 epitope peptide conjugated toE2 elicits robust Th1 responses, as measured via the T cell recallassay with IFN-γ ELISpot. This peptide-conjugation strategyhas clear potential to confer protection against a pathogen with bothextra- and intracellular stages to its life cycle, such as C. burnetii, which requires activation of both humoral andcellular arms of the immune system to grant protection.Both antibodies and T cells have been shownto contribute towardhost-mediated protection against E. coli wereused for general cloning and expression studies, respectively. DNAminipreps and gel extractions were performed with the QIAprep SpinMiniprep Kit (Qiagen) and GeneJET Gel Extraction Kit (Thermo FisherScientific), respectively. DNA primers were synthesized and orderedfrom Integrated DNA Technologies (IDT). CloneJET PCR cloning kit (ThermoFisher Scientific) was used for all polymerase chain reactions (PCRs).Plasmid pET11a was used as the expression vector for all protein constructs.All buffer and cloning reagents were purchasedfrom Fisher Scientific unless otherwise noted. All cloning enzymeswere purchased from New England Biolabs (NEB), unless otherwise noted.DH5α and BL21(DE3) 65 D381C is an E2 mutation thatintroduces 60 cysteines to the internal cavity of the nanoparticle,allowing for internal conjugation.27 To introducethe D381C mutation to E2_158 and E2_152 via site directed mutagenesis(SDM)90 the forward primer: 5′-/5Phos/GCCGATCGTTCGTTGCGGTGAAATCGTTGC-3′and reverse primer: 5′-/5Phos/TTTTCGGCTATACGACCAATACCCAG-3′were used. To introduce the DNA cut sites required for ligation tothe N-terminus of E2 mutants, Nde1 and Nhe1 cut sites were introducedto the N-terminus DNA coding region and C-terminus DNA coding region,respectively, of CBU1910 using the forward primer: 5′-CATATGCACCATCACCATCACCATCCGCAGCAAGTCAAAGACATTCAG-3′and reverse primer: 5′-GCTAGCTTAGCCGCCGGTTTCCGG-3′.The plasmid encoding the CBU1910 protein  was previously synthesizedby GenScript Biotech69 and was used as the DNA templatefor all genetic engineering of the protein antigen.Previouslyestablished E2 mutants E2_152 and E2_158 were used to engineer CBU1910-E2fusion constructs.Supporting Information.A standardPhusion High-Fidelity DNA polymerase protocol was used for PCRs. Thesereactions were performed in a thermal cycler using a 30 s denaturationstep at 98 °C, followed by 30 cycles of 15 s at 98 °C, 15s at 58 °C (E2 D381C mutation) or 53 °C (CBU1910), and 7min (E2 D381C mutation) or 45 s (CBU1910) at 72 °C, with a finalstep of 10 min at 72 °C. The CBU1910 gene was then ligated viathe Nde1/Nhe1 sites of a pET11a vector that contained the E2 genebetween Nhe1/BamH1. Sequencing was performed by GeneWiz/Azenta, andDNA and protein sequences are given in the 30 Expression studies were performed for each mutant and controls.Proteins were expressed in BL21(DE3) E. coli via 1 mM IPTG induction. After induction for 3 h at 37 °C,cells were pelleted and stored at −80 °C. Cells were thawedand lysed by vortexing with glass beads. Soluble and insoluble lysateswere centrifuged at 18000 × g for 15 min andanalyzed using SDS-PAGE for molecular weight and soluble:insolubleratios.TheCBU1910-E2 fusion protein was expressed in a similar fashion to previousmutants described.28 We have reported the generation of tNTA-E2 nanoparticles previously.56 Purified E2 (E279C) in 20 mM HEPES and 100 mMNaCl (pH 7.3) was incubated with an 8.5× molar excess of TCEP. A 10×molar excess of maleimido cyclic tris-NTA  (diluted to 4mg/mL in DMF) was added to the E2 and incubated at room temperaturefor 2 h and then at 4 °C overnight. Unreacted mal-tNTA, DMF,and TCEP were removed using Zeba spin desalting columns in 20 mM HEPESand 100 mM NaCl. Conjugation efficiency and characterization weredetermined using SDS-PAGE and mass spectrometry (Xevo G2-XS QTof). The hydrodynamic diameterof the purified constructs was analyzed by dynamic light scattering(DLS) .E2 (E297C) isan E2 mutant that displays 60 cysteines on its surface that can beused for thiol-based functionalization.6 to tNTA-E2 nanoparticlesfollowed similar procedures established using other protein-(His)n antigens.56 Briefly, a 10×molar excess of NiCl2 was incubated with tNTA-E2 for 2h at room temperature and subsequently purified from unchelated Niusing Zeba spin desalting columns into 20 mM HEPES + 360 mM NaCl bufferpH 7.3. To test optimal conjugation, varying molar ratios of (His)6-tagged CBU1910 (previously synthesized47) were added to the Ni-tNTA-E2 and incubated at room temperaturefor 2 h. The Ni-tNTA-E2 + CBU1910-(His)6 reaction requiredoptimization to yield unaggregated/precipitated constructs, the detailsof which are described in the Supporting Information. After conjugation, solutions were purified with size exclusionchromatography (SEC) using a Superose 6 Increase 10/300 GL column(Cytiva) on a FPLC . Fractions from the SEC were runon an SDS-PAGE gel and stained with a Pierce Silver Stain Kit (ThermoFisher Scientific) to determine the presence of CBU1910-E2, E2, andCBU1910, and conjugation efficiencies were estimated by evaluatingband intensities with standards. Fractions containing CBU1910-E2 werecombined and concentrated with a centrifuge concentrator . Protein concentration was measured via a bicinchoninicacid assay kit (Pierce). Nanoparticle size was assessed via dynamiclight scattering (DLS).Attachment of CBU1910-(His)Previously established mutants E2(D381C) and E2_152were used to engineer the SpyTag-E2 platforms. To introduce the D381Cmutation to E2_152 via site directed mutagenesis (SDM) the forwardprimer: 5′-/5Phos/GCCGATCGTTCGTTGCGGTGAAATCGTTGC-3′and reverse primer: 5′-/5Phos/TTTTCGGCTATACGACCAATACCCAG-3′were used. Introduction of the SpyTag to E2(D381C) and E2_152 wasdone using the forward primers: 5′-CATATGGCCCACATCGTTATGGTGGATGCCTACAAGCCAACTAAAGGTTCAGGAACAGCAGGTGGTGGGTCAGGTTCCCTGTCTGTTCCTGGTCCCGC-3′and 5′-CATATGGCCCACATCGTTATGGTGGATGCCTACAAGCCAACTAAAGCTAGCACCGGCAAAAATGGTCG-3′,respectively. E2 mutants used the same reverse primer: 5′-GGATCCTTAAGCTTCCATCAGCAGCAGTTCCGG-3′.69 The plasmidcontaining the SpyCatcher gene (pDEST14-SpyCatcher) was obtained fromAddgene. To introduce the endonuclease sites and GS-rich spacer onCBU1910 for fusion to SpyCatcher, the forward primer was 5′-GCTAGCGGTTCAGGAACAGCAGGTGGTGGGTCAGGTTCCCCGCAGCAAGTCAAAGACATTC-3′and the reverse primer was 5′-GGATCCTTATTTTTCGACACGGTCAATTTCTTTTTGCAGG-3′.To introduce the endonuclease sites on SpyCatcher, the forward primer5′-CATATGTCGTACTACCATCACCATCACCATCACG-3′and reverse primer 5′-GCTAGCAATATGAGCGTCACCTTTAGTTGCTTTGCC-3′were used. A standard Phusion High-Fidelity DNA polymerase protocolwas used for PCRs. These reactions were performed in a thermal cyclerusing a 30 s denaturation step at 98 °C, followed by 30 cyclesof 15 s at 98 °C, 15 s at 56 °C (SpyTag introduced to E2)or 55 °C (SpyCatcher) or 52 °C (CBU1910), and 45 s (SpyTagintroduced to E2) or 40 s (SpyCatcher) or 45 s (CBU1910) at 72 °C,with a final step of 10 min at 72 °C. Sequencing was performedby GeneWiz/Azenta, and DNA and protein sequences are given in Supporting Information.The plasmid encoding the truncated CBU1910 protein was previouslysynthesized by GenScript Biotech.30 Expression analysis of the ST-E2 mutants is describedin Supplementary Methods. Mutant ST-E2(D381C)was ultimately chosen for scale up expression. Briefly, a 1 L culturesupplemented with 100 μg/mL of ampicillin was inoculated withan overnight culture at 37 °C until an OD of 0.7–0.9 atwhich time it was induced by 1 mM IPTG and further incubated for 3h at 37 °C. Cells were pelleted and stored at −80 °Covernight before breaking. Cells were lysed using a lysing buffercontaining 3 mM PMSF and French Press (Thermo Fisher Scientific).Soluble cell lysates are heat shocked at 70 °C and ultracentrifugedto remove thermolabile containments. Subsequently, the lysates werepurified using a HiPrep Q Sepharose anion exchange column followed by a Superose 6 prep grade  size exclusioncolumn. The purified proteins were characterized by DLS , mass spectrometry (Xevo G2-XS QTof) and SDS-PAGE,and bicinchoninic acid assay (BCA) for size, molecular weight andpurity, and protein concentration, respectively.The new E2 protein mutants were prepared similarlyto previously described mutants.E. coli expression derived lipopolysaccharide (LPS)was removed following a previously described method.27 Briefly, Triton X-114 (Sigma) was added to the purifiedprotein at 1% (v/v), chilled to 4 °C, vortexed vigorously, andheated to 37 °C. The mixture was then centrifuged at 18000 × g and 37 °C for1 min, and the protein-containing aqueous phase was separated fromthe detergent phase. This total process was repeated 9 times. ResidualTriton was removed with detergent removal spin columns (Pierce). LPSlevels were tested to be below 0.1 EU per microgram of E2 protein.The residual E. coli via 1 mM IPTG induction. After inductionfor 3 h at 37 °C, the cells were pelleted and stored at −80°C before breaking. Cells were lysed via French Press and solubleprotein was purified using a HisPur Ni-NTA resin batch protocol (ThermoFisher Scientific). Briefly, soluble cell lysates were mixed withequal parts equilibration buffer and applied to a HisPur Ni-NTA affinityspin column using a packing ratio of 1.5 mL of resin per 10 mL oflysate slurry. The lysate was allowed to incubate with the resin for1 h at 4 °C. Wash buffers and elution buffer containing 75 and150 mM imidazole, and 250 mM imidazole, respectively, were used toattain pure SC-CBU1910. Pure protein fractions were collected anddialyzed into PBS to remove imidazole using 6–8 kDa MWCO dialysistubing. The purified protein was characterized by mass spectrometry(Xevo G2-XS QTof) and SDS-PAGE, and BCA for molecular weight and purity,and protein concentration, respectively.The SpyCatcher-CBU1910 fusion protein was expressed in a fashionsimilar to that of the E2 particles. Proteins were expressed in E. coli expression derived LPS was removed in a similarfashion to the E2 protein. Residual Triton was removed with detergentremoval spin columns (Pierce) or SM2 detergent removal beads (Bio-Rad).LPS levels were below 0.1 EU per microgram of SC-CBU1910 protein .Residual 27 In brief, the internal cavitycysteines of E2 were reduced with TCEP (Pierce) for 30 min, followedby incubation with the N- hydrazide (BMPH) linker (Pierce) for 2 h at room temperature(RT). Unreacted linker was removed using 40 kDa cutoff Zeba spin desaltingcolumns (Pierce). The aldehyde-modified CpG was subsequently addedand incubated overnight at RT. Unreacted CpG was removed by desaltingspin columns. Conjugation was estimated by SDS-PAGE and measured byband intensity analysis.27The oligodeoxynucleotide TLR-9 ligand CpG 1826 (5′-tccatgacgttcctgacgtt-3′)(CpG) was synthesized with a phosphorothioated backbone and 5′benzaldehyde modification by Integrated DNA Technologies (IDT). CpGwas conjugated to the internal cavity of the E2 nanoparticle as describedpreviously.Directlyincubating SpyCatcher-CBU1910 and SpyTag-E2 particles allowed forspontaneous isopeptide bond formation and conjugation. SC-CBU1910proteins were incubated with ST-E2 particles at a ∼0.5:1 (SC-CBU1910:ST-E2monomer) molar ratio, supplemented with 0.080–0.0875% (w/v)Sarkosyl (SLS), for 20 h at room temperature. SDS-PAGE densitometryanalysis with protein standards was used to quantify protein loadingonto the particles. DLS and transmission electron microscopy (TEM)were used to measure the size, assembly, and monodispersity of theparticles. Transmission electron micrographs of 2% uranyl acetate-stainednanoparticles on Cu 200 or 300 mesh carbon coated grids were obtainedon a JEM-2100F (JEOL) instrument with a Gatan OneView camera (Gatan).Supplementary Methods.Further details describing the optimization trials required todetermine the final formulation condition can be found in n = 5) were immunized subcutaneously at the left flank on Day 0 andfollowed by a booster on Day 14. Injections were 30 μL per mouseand contained definite amounts of CBU1910, E2, and CpG, based on theformulations investigated. In groups that used the adjuvant, IVAX,an equal volume of IVAX to formulation was supplemented . IVAX contains Addavax(InvivoGen), 1 nmol of CpG 1018, and 3 nmol of MPLA. Seven days afterthe last immunization, mice were sacrificed, blood was collected viacardiac puncture, and spleens were isolated.All animal studies were carriedout in accordance with protocols approved by the Institutional AnimalCare and Use Committee (IACUC) at the University of California, Irvine.Briefly, 6–8-week-old female C57BL/6 mice (For peptide (CBU1910p)formulations, the same prime boost immunization schedule was followedas described above. Each dosage of the peptide formulation contained10 μg of CBU1910p and 5 μg of CpG 1826 (when indicated).78 Briefly, CBU1910protein was diluted to a concentration of 0.1 mg/mL and printed ontonitrocellulose-coated glass Oncyte Avid slides (Grace Bio-Laboratories)using an Omni Grid 100 microarray printer (Genomic Solutions). Forprobing, mouse plasma samples were diluted 1:100 in protein arrayblocking buffer supplemented with 10 mg/mL E. coli lysate (GenScript) and His-tag containing peptide HHHHHHHHGGGG(Biomatik) to a concentration of 0.1 mg/mL to block anti-polyhistidineantibodies. Arrays were rehydrated with blocking buffer prior to additionof preincubated sera. Arrays were incubated overnight at 4 °Cwith gentle agitation. After overnight incubation, the slides werewashed with Tris-buffered saline (TBS) containing 0.05% Tween 20 (T-TBS)and incubated with biotinylated-SP-conjugated goat anti-mouse IgG,IgG1, or IgG2c (Jackson Immunoresearch). Arrays were washed with T-TBSand incubated with streptavidin conjugated Qdot-800 (ThermoFisher).Arrays were washed three times with T-TBS followed by TBS, dippedin water, and dried by centrifugation. Images were acquired usingthe ArrayCAM imaging system (Grace Bio-Laboratories). Spot and backgroundintensities were measured using an annotated grid  file. IgG1and IgG2c antibody subtype proportions were calculated using respectivesignal intensities: IgG1/(IgG1 + IgG2c) and IgG2c/(IgG1+IgG2c), respectively.47Protein microarrays were fabricatedas previously described.91 Antigens usedfor recall were C. burnetii CBU1910 and OVA as anirrelevant control antigen. Assays were performed in Roswell ParkMemorial Institute (RPMI) 1640, containing 5 × 10–5 M β-mercaptoethanol, 100 IU/mL penicillin, 100 μg/mLstreptomycin, and 10% heat-inactivated fetal bovine serum (completemedium). Briefly, erythrocyte-depleted splenocytes were incubatedat 5 × 105 cells per well in 96-well ELISpot plates,coated previously with IFN-γ capture antibody, and blocked incomplete medium, containing titrations of antigen ranging from 2.5to 10 μg/mL. Mice were assayed separately. Concanavalin A wasincluded as a viability control.Recall assays were performed usingIFN-γ ELISpot format and spleens collected on day 21 essentiallyas previously described.n ≥ 3), unless otherwise noted.Statistical analysis of immunization data was carried out by usingGraphPad Prism. Data are presented as mean ± standard error ofthe mean (S.E.M.) from at least five independent individuals (n ≥ 5). Statistical analysis was determined by aone-way or two-way ANOVA over all groups, followed by a Bonferronimultiple comparison test, unless otherwise noted. P-values less than 0.05 were considered significant.For nanoparticle characterization,including hydrodynamic diameter measurements, molecular weights determinedby mass spectrometry, and antigen/nanoparticle ratios, data are presentedas the mean ± standard deviation (S.D.) of at least three independentexperiments (


Histone H3 lysine 9 methylation (H3K9me), which is written by the Euchromatic Histone Lysine Methyltransferases EHMT1 and EHMT2 and read by the heterochromatin protein 1 (HP1) chromobox (CBX) protein family, is dysregulated in many types of cancers. Approaches to inhibit regulators of this pathway are currently being evaluated for therapeutic purposes. Thus, knowledge of the complexes supporting the function of these writers and readers during the process of cell proliferation is critical for our understanding of their role in carcinogenesis. Here, we immunopurified each of these proteins and used mass spectrometry to define their associated non-histone proteins, individually and at two different phases of the cell cycle, namely G1/S and G2/M. Our findings identify novel binding proteins for these writers and readers, as well as corroborate known interactors, to show the formation of distinct protein complex networks in a cell cycle phase-specific manner. Furthermore, there is an organizational switch between cell cycle phases for interactions among specific writer–reader pairs. Through a multi-tiered bioinformatics-based approach, we reveal that many interacting proteins exhibit histone mimicry, based on an H3K9-like linear motif. Gene ontology analyses, pathway enrichment, and network reconstruction inferred that these comprehensive EHMT and CBX-associated interacting protein networks participate in various functions, including transcription, DNA repair, splicing, and membrane disassembly. Combined, our data reveals novel complexes that provide insight into key functions of cell cycle-associated epigenomic processes that are highly relevant for better understanding these chromatin-modifying proteins during cell cycle and carcinogenesis. Emerging evidence implicates repressive H3K9 methylation as an important transcriptional regulatory and epigenetic pathway in tumorigenesis . H3K9 meDuring the cell cycle, a series of exquisitely coordinated events occur, which include DNA replication, histone synthesis, and duplication of chromatin structure during S phase and chromosome condensation and segregation for mitotic cell division during M phase. To ensure genomic and epigenomic integrity along with proper progression of the cell cycle, chromatin modifications and their regulators are an integral part of this process by regulating appropriate gene expression and chromatin structure . FurtherIn the present study, we used a multi-tiered approach to better define the interactomes of H3K9me writers (EHMT1 and EHMT2) and readers (CBX3 and CBX5), during the G1/S and G2/M cell cycle phases. Immunopurified complexes were identified by mass spectrometry, and these interacting protein networks were validated using several data science-based algorithms. Notably, our results reveal known and novel non-histone-related interactions, along with distinct cell cycle-specific protein networks. Given that EHMT1 and EHMT2 also methylate non-histone proteins, we analyzed the interacting proteins for the presence of an H3K9-like recognition domain, defining a list of novel candidate targets for histone mimicry. Combined, the present study extends the current paradigm on pathways that link, directly or indirectly, to these epigenetic regulators and offers unique insight into cell cycle-associated epigenomic processes that are highly relevant to human cancer.Following our prior publication, HeLa cells were used to obtain G1/S and G2/M lysates after double thymidine synchronization and release . Brieflym/z at resolution 70,000 (at 200 m/z), followed by HCD MS/MS scans on the top 15 ions at resolution 17,500. The MS1 AGC target is set to 1e6 and the MS2 target is set to 1e5 with max ion inject times of 50 and 100 ms, respectively. Only ions with charge states of +2, +3, and +4 were selected for MS2 fragmentation and dynamic exclusion placed the selected ions on an exclusion list for 30 s. Tandem mass spectra were extracted by msconvert version 3.0.9134. All MS/MS samples are analyzed using Mascot  and X! Tandem ). Mascot and X! Tandem were set up to search a combined Swissprot human database with reverse decoy (40620 entries), assuming the digestion enzyme strict trypsin and with a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 PPM. Glu->pyro-Glu of the n-terminus, ammonia-loss of the n-terminus, gln->pyro-Glu of the n-terminus, and oxidation of methionine were specified in X! Tandem as variable modifications and carbamidomethyl of cysteine was specified as a fixed modification. Oxidation of methionine and carbamidomethyl of cysteine were specified in Mascot as variable modifications. Scaffold  was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they were established at greater than 95.0% probability by the Scaffold Local FDR algorithm and contained at least two identified peptides. The decoy false discovery rate was less than 1%. Protein probabilities were assigned by the Protein Prophet algorithm [Antibodies to EHMT1 , EHMT2 , CBX3 , CBX5  or control IgG  were conjugated through disuccinimidyl suberate (DSS) cross-linking to Protein A/G Magnetic Beads (Thermo Fisher Scientific). Centrifuged pellets of cells in S phase or mitosis were lysed with IP Lysis/Wash Buffer (Thermo Fisher Scientific), and lysates were incubated with antibody conjugates overnight at 4°C. Immunoprecipitated complexes were washed and eluted to resolve on 4–15% Criterion™ Tris-HCl Protein Gels (Bio-Rad). Gels were subsequently visualized with BioSafe™ Coomassie Stain (Bio-Rad). Each gel lane was divided into eight sections, destained in 50% acetonitrile/50 mM Tris pH 8.1. Proteins were reduced with the addition of 50 mM TCEP/50 mM Tris pH 8.1 at 55°C for 30 min. Next, the samples were alkylated by adding 25 mM iodoacetamide/50 mM Tris pH 8.1 and incubated in the dark for 30 min at room temperature. In-gel digestion was carried out at 37°C overnight with 0.15 µg trypsin  in 25 mM Tris pH 8.1/0.0002% Zwittergent 3-16, followed by peptide extraction with 2% trifluoroacetic acid and acetonitrile. Extracts were pooled and subsequently used for LC/ESI-MS/MS analysis at the Mayo Clinic Proteomics Core. The pooled extracts were concentrated, and the proteins were identified by nano-flow liquid chromatography-electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS) using a Thermo Scientific Q-Exactive Plus Mass Spectrometer (Thermo Fisher Scientific) coupled to a Thermo Ultimate 3000 RSLCnano HPLC system. The extracts were then loaded onto a 250 nl OPTI-PAK trap (Optimize Technologies) custom packed with Michrom Magic C18 solid phase (Michrom Bioresources). Chromatography conditions were: solvent A water; 0.2% formic acid; 2% acetonitrile and solvent B water; 80% acetonitrile; 10% isopropanol. The Q-Exactive Plus mass spectrometer set up was a FT full scan from 340 to 1600 lgorithm . Proteinhttps://bioinformatics.psb.ugent.be/webtools/Venn/. Transcriptome microarray data for the HeLa cell synchronized cells was obtained from the Gene Expression Omnibus (GEO) database under accession number GSE27031. The data was accessed using the GEOquery package on R studio [Prior to data processing, proteins that contained less than four peptides were considered low enrichment proteins and removed from downstream analysis. Next, peptide counts which failed to reach a log2 foldchange threshold in the bait sample versus the IgG control sample were removed as potential contaminants to ensure that hits were specific to bait-protein interactions rather than non-specific binding . As a fiR studio . DiffereR studio . NuclearProtein interactions from the LC/ESI-MS/MS experiments fitting the present study’s criteria for novel histone mimicry were searched against the STRING database version 11.5 for protein–protein physical interactions . Edges wP-value cutoff = 0.05. Gene ontology for biological processes was visualized by heatmaps using ggplot2 [Biological processes GO enrichment analysis was carried out using the clusterProfiler and enrichGO R package. The interactions were annotated using org.Hs.eg.db with a  ggplot2 .Ten peptide sequences based on known EHMT2 lysine methylation targets  were aligned through Clustal Omega  and enteHigh-resolution (1.7 Å) crystal structure of a histone H3K9M mutant peptide (1–11)-bound form of human EHMT2 SET domain (PDB access code: 5T0K) was used in our study. This structure contains the natural cofactor S-adenosylmethionine (SAM) and the structural zinc ions. The H3K9M mutant peptide was replaced with the natural H3K9me1 peptide from PDB access code 3HNA and used as the WT peptide. For non-histone substrate complex modeling, amino acid substitutions were made within the Discovery Studio suite version 19.1 (Dassault Systèmes BIOVIA) by mutating the corresponding residues of the H3K9me1 peptide and selecting the side chain rotamer causing the least steric hindrance with the surrounding residues. These structures were subjected to the same energy minimization and molecular dynamics (MD) simulation protocol as the EHMT2 SET domain-histone H3K9me1 peptide complex.The energy minimized EHMT2 SET domain-histone H3K9me1 peptide complex was used to develop the pharmacophore model in Discovery Studio. The co-complexed peptide was extracted to create the model from the bioactive conformation. The maximum number of features considered was 6 within the minimum distance of 2.5 Å between features in the generated pharmacophore. The protocol also added excluded volumes  to indicate regions of inaccessibility by the bioactive conformation; however, these were concealed from view for better visualization of the model. The pharmacophore model with the highest selectivity score as predicted by a Genetic Function Approximation (GFA) model was selected .MD simulations were performed using the CHARMm36 all-atom-force-field  implemenThe current study was designed to identify cell cycle specific protein networks for both H3K9me writers (EHMT1 and EHMT2) and readers (CBX3 and CBX5). For this purpose, we immunopurified each of these four proteins from HeLa cells that were synchronized in G1/S and G2/M and performed mass spectrometry analyses, as previously described ,19, and Combined, the H3K9me writers and readers co-immunoprecipitated a total of 2218 unique proteins. From these interactions, we found that 674/2218 (30.39%) were shared by at least 2 of these epigenomic regulators D. WithinWe also found that the complete H3K9me writer–reader cellular interactome comprised 1282 (58%) nuclear and 935 (42%) non-nuclear interactors. Importantly, the group of nuclear interacting proteins formed more known interconnections than the non-nuclear group G–I. In fOntological analyses of the four Nuclear Interactomes demonstrated distinct and overlapping functional enrichments during the G1/S and G2/M phases A. The coBased on the themes that emerged from the above molecular function ontologies, we investigated whether the interactors of these writers and readers form specific multi-protein complexes B. This iSimilar to kinases, methylases recognize distinct Short Linear Motifs (SLiM), which are composed of short sequences recognized by other proteins for mediating protein binding and posttranslational modifications. For instance, the ARKS sequence within histones was the first SLiM identified to undergo targeting by EHMT1 and EHMT2 for methylation . HoweverP≤0.05) complexes were CtBP, PAF1, Nup107-160, and the Spliceosome. In summary, the multi-tiered analyses applied here to our interactomes expand the repertoire of non-histone candidates that can be regulated by enzymes initially believed to only function as histone methylases.Consequently, we investigated whether these H3K9-mimetic candidates cluster into distinct multiprotein complexes. First, among the 171 H3K9-mimetic proteins identified in both G1/S and G2/M, 75 form physical connections at high confidence C. Second5T0K). We identified principal chemical features involved in this receptor–ligand complex. The lysine (K9) substrate occupies the hydrophobic channel that crosses the core of the EHMT2 catalytic domain and links to the co-factor SAM (S-adenosyl-methionine) binding site .nd D1088 C. Theref10) (10). By comparing the effect of our controls, we can reinforce the critical role of the ‘RK’ motif during interaction with EHMT2, responsible for loss of 46% of interaction energy when compared with the H3-A10  and co-activators  ,74–78. T10) D. Our ree H3-A10 D. The ene H3-A10 D. IndeedPrevious studies from our laboratories have focused on understanding how the histone code functions in human disease, documenting the importance of the H3K9 methylation pathway and its associated epigenetic regulators in monogenic disorders ,81 and cEHMT1 mutations. Like many monogenic diseases with developmental delay, it is believed that alterations in the asymmetric cell division and migration of neuroblasts play a role in disease presentation [Cell cycle progression contributes to pathobiological mechanisms underlying Kleefstra syndrome, which is a congenital disease caused by entation . Alteratentation . For exaentation . In addientation . Moreoveentation . Thus, tConsidering these four H3K9me regulators at the level of systems biology, we have considered its emergent properties outside the canonical H3K9 methylation pathway. First, we find that while the EHMT1 and EHMT2 writers do not directly interact with the CBX3 and CBX5 readers in G1/S, intriguingly, these regulatory systems organize as two distinct reader–writer pairs, namely EHMT1–CBX5 and EHMT2–CBX3 through their shared interactomes. The observed organization of overlapping interactomes between these reader-writer pairs has functional implications for their roles in distinct processes in this cell cycle phase, as the cell either prepares or proceeds to copy its DNA. For example, EHMT2 and CBX3 exhibited associations with proteins relevant to replication fork biology and regulation of DNA methylation through interactions with DNMT1, UHRF1, and USP7, facilitated by histone mimicry mechanisms . AlternaAn important emergent property of the identified interactomes, independent of the cell cycle, is its link with complexes that regulate other pathways important for the epigenome. For instance, EHMT2 interacts with the ALL-1 Supercomplex (ALL-1), which forms a membrane-less nuclear aggregation of more than 20 proteins that are part of the Sin3-HDAC, the NuRD histone deacetylase (Nucleosome remodeling and deacetylation), and SWI/SNF (SWItch/Sucrose Non-Fermentable) protein complexes . This noIn summary, the present study is the first comprehensive characterization of the repertoire of proteins that interact with the H3K9me writers (EHMT1 and EHMT2) and readers (CBX3 and CBX5), which reveal systems-level organization across cell cycle phases. We deconvoluted the cellular interactomes into cytoplasmic and nuclear proteins to develop specific G1/S and G2/M pathways and networks. In addition to examining these protein–protein interactions, we investigated the extensive repertoire of proteins that can be potentially methylated and/or read by these writers and readers. While the H3K9me mark is consistently present in both G1/S and G2/M , the emeClick here for additional data file.Click here for additional data file.


In addition, 1,25(OH)2D3 treatment promoted cell viability and invasion and inhibited apoptosis of H/R-exposed trophoblast cells. Notably, overexpression of LAMP3 abrogated the protective effect of 1,25(OH)2D3 on H/R-exposed trophoblast cells. Collectively, we demonstrated trophoblast cytoprotection by vitamin D, a process mediated via LAMP3.Inadequate invasion and excessive apoptosis of trophoblast cells are associated with the development of preeclampsia. Vitamin D deficiency in pregnant women may lead to an increased risk of preeclampsia. However, the underlying mechanisms by which vitamin D is effective in preventing preeclampsia are not fully understood. The objectives of this study were to investigate the role of lysosome-associated membrane glycoprotein 3 (LAMP3) in the pathogenesis of preeclampsia and to evaluate whether vitamin D supplementation would protect against the development of preeclampsia by regulating LAMP3 expression. Firstly, the mRNA and protein levels of LAMP3 were significantly upregulated in the placentas of preeclampsia patients compared to normal placentas, especially in trophoblast cells (a key component of the human placenta). In the hypoxia/reoxygenation (H/R)-exposed HTR-8/Svneo trophoblast cells, LAMP3 expression was also upregulated. H/R exposure repressed cell viability and invasion and increased apoptosis of trophoblast cells. siRNA-mediated knockdown of LAMP3 increased cell viability and invasion and suppressed apoptosis of H/R-exposed trophoblast cells. We further found that 1,25(OH) It is tta in PE ,8. ThereLysosome-associated membrane glycoprotein 3 (LAMP3) is a member of the lysosome-associated membrane glycoprotein family , which p2D3], exerts essential functions in regulating cell proliferation and differentiation ntiation ,16. A clntiation . Low vitntiation . Moreoventiation . Vitaminin vitro . Our prein vitro . In the A previous study demonstrated that LAMP3 expression is downregulated by vitamin D in dendritic cells . NeverthPlacenta samples were obtained from 23 women: 10 women with PE defined by hypertension (systolic blood pressure ≥140 and/or diastolic blood pressure ≥90 mmHg) and proteinuria (0.3 g/24 h)  supplemented with 10% fetal bovine serum  at 37°C in a humidified atmosphere with 5% CO2 concentration of 2% for 8 h and then for an additional 16 h to an ambient O2 concentration of 20%.H/R was achieved by exposing HTR-8/Svneo cells to an ambient OSmall interfering RNA targeting LAMP3 (siLAMP3), LAMP3 overexpression plasmid (oe-LAMP3), and their corresponding negative controls (siNC and vector) were transfected into HTR-8/Svneo cells using Lipofectamine 3000  according to manufacturer's instruction. After transfection for 24 h, cells were cultivated under H/R condition for another 24 h.2D3 treatment, H/R-induced HTR-8/Svneo cells were treated with 100 nM 1,25(OH)2D3 for 24 h.For 1,25(OH)TM 96 real-time quantitative thermal block . GAPDH was used as an internal control. The primers were as follows: LAMP3 F: CTCGGAGATACTTCAACAT; LAMP3 R: GAGACGGTCAAATAGGC.Total RNA was extracted from placenta cells using TRIpure lysate . RNA was reverse-transcribed with BeyoRT™ II M-MLV reverse transcriptase . The reaction was followed by a melting curve protocol according to the specifications of ExicyclerTotal proteins were extracted using RIPA buffer (Solarbio). Sample proteins were separated by SDS gels and transferred to PVDF membranes . The membranes were probed with primary antibodies by LAMP3 antibody , Bcl2 antibody , Bcl-xL antibody , Bax antibody . GAPDH  was used as an internal control. Chemiluminescence detection was performed using goat anti-rabbit IgG/HRP antibody  or goat anti-mouse IgG/HRP antibody . The immunoreactive protein bands were visualized using ECL reagent (Solarbio).Frozen sections of placenta tissue were washed three times with phosphate buffered saline (PBS). Slides were blocked using 5% BSA  for 15 min and then incubated overnight at 4°C with both LAMP3 antibody  and CK7 antibody , respectively. Slides were then incubated with secondary antibody including goat anti-rabbit IgG H&L (FITC)  and goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody  at 37°C for 90 min. The nuclei were stained with DAPI, and the staining effect was viewed by a microscope  at 400× magnification.Cell viability was monitored using MTT cell proliferation and a cytotoxicity detection kit  according to the manufacturer's instructions. The absorbance value was detected at 490 nm under a microplate reader .4/well) were cultured into the upper well. The medium with 10% fetal bovine serum was added to the lower chamber. Incubation was performed at 37°C for 48 h, and cells were stained with crystal violet . The number of invaded cells was measured using a microscope (Olympus) at 200× magnification.The matrigel invasion assay was performed using a transwell chamber  precoated with matrigel . The cells 2 and MMP9 were tested using commercial ELISA kits  according to the manufacturer's instructions.in situ cell death detection kit . Briefly, sections were incubated with TUNEL solution for 60 min, and then stained with DAPI for 5 min. The images were observed under a fluorescence microscope (Olympus) at 400× magnification.TUNEL was done using the t-test was used to compare the differences between two groups, and one-way analysis of variance  was used to analyze the differences among multiple groups.Data analysis was performed using GraphPad Prism 8.0 software (USA). Experiments were repeated three times and data are reported as means±SD. Unpaired The mRNA and protein levels of LAMP3 in preeclampsia placenta were upregulated compared with normal samples . In doubin vitro, we established a H/R model of trophoblast cell line HTR-8/SVneo. The flow diagram is shown in To mimic preeclampsia To explore the function of LAMP3 in cell viability, invasion, and apoptosis of trophoblast cells, we used siRNA to knockdown LAMP3 . MTT ass2D3 was involved in H/R-induced trophoblast cell function, HTR-8/SVneo cells were stimulated with H/R followed by 1,25(OH)2D3 treatment reatment . MTT assreatment . 1,252D3-treated HTR-8/SVneo cells under H/R stimulation 2D3-mediated promotion of invasion and inhibition of apoptosis under H/R stimulation mulation . The effmulation . The MTTondition . Comparemulation . Therefoin vitro results corroborated the role of LAMP3 as a pathogenic factor regulating cell viability, invasion, and apoptosis of trophoblast cells, LAMP3 is a lysosomal membrane glycoprotein with complex functions and diverse effects, therefore, in vivo evidence is needed to support the inference that LAMP3 contributes to disease progression in PE.PE has been the focus of research due to its adverse effects on pregnant women and perinatal babies ,26. As fExcessive trophoblast cell apoptosis and senescence may contribute to the progression of PE . The incAccumulating studies have confirmed that deficient trophoblast cell migration and invasion may result in PE -32. Tro2D3 diminishes the oxidative stress-induced microparticle release from placental trophoblasts in PE  in placental trophoblasts, thereby increasing amino acid transport across the placenta and decreasing the risk of fetal growth restriction in PE . WestwooIn summary, our study clarified the important role of LAMP3 in the dysfunction of trophoblast cells. In particular, we provided evidence that vitamin D supplementation during pregnancy may be beneficial for decreasing trophoblast cell dysfunction by downregulating LAMP3 expression. These findings may provide a theoretical basis for the use of vitamin D in clinical prevention and treatment of PE and propose a novel therapeutic strategy by targeting LAMP3 in PE treatment.


Pneumococcal infections caused by non-vaccine serotypes, including serotype 15B and 15C, emerged after the introduction of pneumococcal conjugate vaccines (PCVs). Serotypes 15B and 15C have been added to new different pneumococcal-conjugate vaccines . We sought to derive a serum anti-15B antibody level that would be a correlate of protection (COP) against acute otitis media (AOM) and nasopharyngeal colonization and assess functional cross-reactivity against serotype 15B and 15C in children following natural immunization.Method: IgG antibody to serotype15B polysaccharide was measured by ELISA in 402 sera from 6-36 month olds collected before, at the time of, and after pneumococcal culture-confirmed AOM and colonization caused by serotypes 15B and 15C. 153 serum samples from 6-36 month olds where no colonization with either 15B or 15C was observed were included as controls. A two-step method was used for the construction of COP models: a generalized estimating equation followed by logistic-regression. Opsonophagocytic (OPA) assays were used to assess functional cross-reactivity between serotypes 15B and 15C.The COP for prevention of colonization was 1.41µg/ml for 15B and 0.63µg/ml for 15C with a predictive probability of 80%. Significantly higher anti-15B antibody levels were measured in children who had AOM caused by serotype 15B compared to control children and compared to children who had AOM caused by serotype 15C (p< 0.03). Consequently, a COP for serotypes 15B and 15C could not be calculated for AOM. ELISA-measured antibody levels did not correlate with OPA titers. Thirty percent of child samples, with varying moderate to high amounts of ELISA-measured antibody, showed no OPA titer against either serotype 15B or 15C. For the remaining samples, very low or no functional cross-reactivity between serotypes 15B and 15C was measured.Conclusions: A COP for prevention of colonization in young children based on naturally-induced antibody levels was derived for serotype 15B and 15C that differed. A COP for AOM could not be derived. ELISA-measured antibody levels correlated poorly with OPA titers and low functional cross-reactivity between serotypes 15B and 15C in child sera was observed.All Authors: No reported disclosures


The glycosylation of viral envelope proteins can play important roles in virus biology and immune evasion. The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) includes 22 N-linked glycosylation sequons and 17 O-linked glycosites. Here, we investigated the effect of individual glycosylation sites on SARS-CoV-2 S function in pseudotyped virus infection assays and on sensitivity to monoclonal and polyclonal neutralizing antibodies. In most cases, removal of individual glycosylation sites decreased the infectiousness of the pseudotyped virus. For glycosylation mutants in the N-terminal domain (NTD) and the receptor binding domain (RBD), reduction in pseudotype infectivity was predicted by a commensurate reduction in the level of virion-incorporated spike protein. Notably, the presence of a glycan at position N343 within the RBD had diverse effects on neutralization by RBD-specific monoclonal antibodies (mAbs) cloned from convalescent individuals. The N343 glycan reduced overall sensitivity to polyclonal antibodies in plasma from COVID-19 convalescent individuals, suggesting a role for SARS-CoV-2 spike glycosylation in immune evasion. However, vaccination of convalescent individuals produced neutralizing activity that was resilient to the inhibitory effect of the N343 glycan. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 disease and has caused a devastating pandemic –6. The SGlycosylation of viral envelope proteins can play an important role in virus-host interactions . In the Little is known about how SARS-CoV-2 S glycosylation might affect immune surveillance. It is conceivable that glycans sterically shield underlying epitopes from recognition by antibodies, as is the case in HIV-1 . Many SACo-expression of SARS-CoV-2 S with envelope-deficient viruses such as HIV-1 (human immunodeficiency virus-1) produces pseudotyped viruses capable of infecting ACE2-expressing cells and is widely used as a surrogate to study viral entry and neutralization by antibodies , 28. To Δ19), along with an envelope-deficient HIV-1 proviral plasmid encoding NanoLuc. The number of SARS-CoV-2 spikes incorporated into HIV-1 pseudotype virions were estimated using purified recombinant purified S protein . Co-transfection with as little as 2 ng of S expression plasmid produced virions that induced 1000x the level of luciferase observed with ‘bald virions’, indicating that small amounts of S protein (0.15 ng/ml or a mean of ~1–2 S trimers per virion) were sufficient to mediate viral entry  and 1C. To investigate the contribution of glycosylation to S protein function, we generated 22 spike substitution mutants, each containing a single Asn to Asp (N to D) substitution at one of the 22 N-glycosylation sequons. Additionally, we generated a mutant with Ala replacements at potential O-linked sites S323 and T325 in the receptor binding domain (RBD) . None ofhe RBD is the major target of neutralizing antibodies. To determine the effect of glycan removal on sensitivity to neutralizing antibodies, we focused on glycosylation sites in the RBD (N331 and N343), and one site adjacent to the RBD (N282). Because these glycosylation sites affected the level of spike incorporation into virions, we first tested the effect of SARS-CoV-2 spike density on neutralization sensitivity, as this parameter could be a potential confounder. We harvested pseudotyped virions from cells transfected with varying amounts (from 2 ng to 1 μg) of wild-type S expression plasmid and tested their sensitivity to C144, a potent class 2 neutralizing human monoclonal antibody cloned from a convalescent individual . Notably50) of C144 was reduced from 1.8 to 3.4 ng/ml (against the WT pseudotype) to 0.45 ng/ml (against the N343D pseudotype), while the C121 IC50 was reduced from 2.3 to 2.9 ng/ml (against the WT pseudotype) to 0.21 ng/ml (against the N343D pseudotype).We next tested the neutralization sensitivity of the RBD glycosylation site mutants bearing Asn to Asp mutation at N-linked sites (N331D and N343D) or proximal (N282D) to the RBD or alanine substitutions at O-linked sites S323 and T325. To provide matched control viruses with approximately similar numbers of S trimers and similar levels of infectivity, neutralization of the glycosylation site mutants was compared to virions generated with an appropriate level of the WT spike protein. We compared the neutralization sensitivity of WT and mutant virions to C144 and another potent class 2 neutralizing antibody, C121, both of which target the ACE2 binding site. While the N282D, S323/T325A, and N331D mutant pseudotypes exhibited neutralization sensitivities that were similar to the WT pseudotype, the N343D substitution conferred significantly increased neutralization sensitivity to both C144 and C121 . SpecifiTo determine the effects of glycosylation more broadly on SARS-CoV-2 sensitivity to neutralizing antibodies, we used pseudotyped viruses bearing spike proteins with R683G substitution, which ablates the furin cleavage site. This substitution does not affect S incorporation into virions  but enha50 =21.3 ng/ml against WT (10ng) or 24.5 ng/ml against WT (3ng) and its activity was resilient to many naturally occurring mutations in the RBD (50 was increased to 176.9 ng/ml (50 was increased from 41.1 ng/ml against WT (10ng) or 54.9 ng/ml against WT (3ng) to 467.1 ng/ml against N343D  to 2.6 ng/ml (against the N343D), while the C144 IC50 was reduced from 5.7 to 6.1 ng/ml (against the WT) to 1.7 ng/ml (against the N343D)  .50 (IC50= 8.3–11.5ng/ml), was able to achieve almost complete neutralization of the N343D pseudotypes at high concentrations  did not alter the neutralization sensitivity to most monoclonal antibodies tested herein , Fig. S5Given that the N343D substitution exhibited different effects on sensitivity to neutralizing monoclonal antibodies, we next asked whether this substitution affected neutralization by polyclonal antibodies present in convalescent plasma. As with the monoclonal antibodies, we compared the neutralization sensitivity of N343D pseudotyped particles with that of WT pseudotyped particles containing same amount of WT spike protein or showing the same infectivity as the N343D pseudotype, to convalescent plasma from 15 patients collected early in the COVID19 pandemic (at 1.3 months after infection) and from the same individuals ~1 year later following subsequent vaccination . The N34While SARS-CoV-2 spike pseudotyped viruses have been widely used as a surrogate to study S-mediated viral entry  little iIn this study, we found that glycosylation at several sites in the spike NTD and RBD, including N61, N122, N165, N331, N343 and S323 T325 are required for full pseudotyped virus infectivity. Substitution of these glycosylation sites resulted in a reduction in pseudotype infectivity of about 10-fold or more and correlated with a reduction in spike incorporation into virions. While it is unclear precisely how glycosylation on these sites drives S incorporation into virions, glycans could in principle affect protein stability and trafficking through the secretory pathway . We noteThe N343 glycan also affected neutralization by RBD-targeted monoclonal antibodies, and the effect was largely dependent on the class to which antibodies belong. The N343 glycan is positioned on the RBD distal to the ACE2 and class 1 antibody (C098) binding site, but increased sensitivity to class 1 antibodies , which bind only to “up” RBDs , suggestThe N343 glycan is proximal to the binding sites of class 3 antibodies, and the N343D substitution had a range of effects on sensitivity to class 3 antibodies. Three clonally related antibodies, C032, C080 and C952, were unaffected by the N343D substitution, while substantial but opposing effects were seen for two others, C135 and C581. In the case of C135, removal of the glycan reduced the fraction of virions that resisted neutralization at high antibody concentration. A possible explanation for this phenomenon is that the N343 site is heterogeneously glycosylated, and some subfraction of the glycans occlude the C135 binding site. For C581, removal of the N343 glycan had the opposite effect, reducing neutralization sensitivity. In this case it seems likely C581 mimics the properties of two previously described cross-reactive class 3 antibodies, S309 and SW186, for which the N343 glycan constitutes part of the antibody binding site  26). Fi. Fi26). The N343 glycan reduced neutralization by convalescent plasma collected at 1.3 months after infection, echoing the effects on sensitivity to the C121, C144 (class 2) and C135 (class 3) antibodies. Conversely, neutralization by convalescent plasma from the same individuals (collected at 12 months following subsequent vaccination) was unaffected by the N343 glycan. That neutralizing antibodies are relatively sensitive to glycan-mediated protection early after infection may be a reflection of the fact that the initial neutralizing response is based to a large extent on class 2 antibodies that are easily escaped , 34. SubAntibodies used here are anti-SARS CoV-2 spike antibody [1A9]  and anti-HIV capsid p24 . Secondary antibodies included goat anti-mouse conjugated to IRDye 800CW or IRDye 680 for Western blot analysis. The recombinant HIV p24 protein was purchased from abcam (ab43037).Δ19) has been previously described  and the derivative expressing ACE2, ie 293T/ACE2.cl22, were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Sigma F8067) and gentamycin (Gibco). All cell lines used in this study were monitored periodically to ensure the absence of retroviral contamination and mycoplasma.Δ19). In transfection experiment to generate viruses bearing R683G substitution, 1 ng, 3 ng, 10 ng, 30 ng, 100 ng, and 300 ng of expression plasmid were used instead. The total amount of DNA was held constant by supplementing the transfection with empty expression vector. Cells were harvested at 48 hr post transfection and subjected to Western blot analysis. Virus-containing supernatant was filtered (0.2 μm), and, to remove cell debris, clarified by Lenti-X (TaKaRa). Particle infectivity was measured as previously described  of WT or 1000 ng of glycosylation site mutant SARS-CoV-2 expression plasmids (pSARS-CoV-2escribed . BrieflyCell lysates were separated on NuPage Novex 4–12% Bis-Tris Mini Gels (Invitrogen). Proteins were blotted onto nitrocellulose membranes. Thereafter, the blots were probed with primary antibodies and followed by secondary antibodies conjugated to IRDye 800CW or IRDye 680. Fluorescent signals were detected and quantitated using an Odyssey scanner (LI-COR Biosciences).To express S-6P-NanoLuc proteins, Expi293 cells were transfected with S-6P-NanoLuc expression plasmids using ExpiFectamine 293 (ThermoFisher Scientific). Four days later, the supernatant was harvested and loaded on Ni-NTA agarose and, after thorough wash, S-6P-NanoLuc proteins were released after HRV 3C protease treatment.To measure neutralizing activity of monoclonal antibodies, serial dilutions of antibodies beginning at 3 μg/ml were four-fold serially diluted in 96-well plates over seven dilutions. To determine the neutralizing activity in convalescent plasma, the initial dilution started at a 1:30 (for plasma at 1.3 months) or a 1:150 (for plasma at 12 months). Thereafter, SARS-CoV-2 spike pseudotyped viruses were incubated with monoclonal antibodies or the convalescent plasma for 1 hr at 37°C in 96-well plates. The mixture was then added to 293T/ACE2cl.22 target cells seeded one day prior to infection so the final starting dilutions were 1.5 μg/ml for monoclonal antibodies and 1:60 or 1:300 for plasma. Cells were then harvested 48 hours post infection for NanoLuc luciferase assays.Monoclonal antibodies C022, C032, C080, C098, C099, C118, C121, C135, C144, C581, C936, and C952 used in this study were previously reported , 34–37. Supplement 1


Cancers originate from a single cell according to Nowell’s theory of clonal evolution. The enrichment of the most aggressive clones has been developed and the heterogeneity arises for genomic instability and environmental selection. Multiple myeloma (MM) is a multiple relapse plasma cell cancer generated from bone marrow. Although there were accumulating researches in multiple myeloma pathogenesis, the heterogeneity remains poorly understood. The participants enrolled in this study were 4 EMP+  and 2 EMP- primarily untreated MM patients. Single cell RNA sequencing and analysis were conducted for the single cell suspension, which was sorted by flow cytometry from peripheral blood mononuclear cells or bone marrow cells. In our research, the results of single cell RNA sequencing show that FAM46C determines MM tumor heterogeneity predicting extramedullary metastasis by influencing RNA stability. Further, we integrated and analyzed 2280 multiple myeloma samples from 7 independent datasets, which uncover that FAM46C mediated tumor heterogeneity predicts poorer survival in multiple myeloma. Multiple myeloma (MM) is a multiple relapse plasma cell malignancy in the bone marrow, which is characterized by clonal proliferation of plasma cells in the bone marrow and vast secretion of monoclonal immunoglobulin , 2. In rFAM46C, a member of nucleotidyltransferase superfamily –16, encoCancers originate from a single cell according to Nowell’s theory of clonal evolution . The enrP = 5.3E-30, one-way analysis of variance), while IGHV4-28 is specifically expressed in P21 multiple myeloma patients . IGKV3D-7 and IGHA1 were specially expressed in P20 and P19 MM patients, respectively . IGKC was expressed in several MM patients, including P19, P20, and P23 . The specifically expressed immunoglobulin genes in each multiple myeloma patient with an extremely significant P-value suggest that we have obtained the right sample and our method of single-cell RNA sequencing is robust.Intriguingly, 3 immunoglobulin genes also were expressed in various MM patients. IGKV3D-20 and IGKV4-1 were specifically expressed in both P20 and P21 multiple myeloma patients .To determine the differentially expressed genes in CPCs compared with bone marrow mononuclear cells (BMMCs), we performed differential expression analysis in CPCs and BMMCs isolated from same EMP-positive patient (P17 and P20). A total of 95 genes were differentially expressed , of thesP = 0.0003; P20: P < 0.0001; Kolmogorov-Smirnov test), indicating that decreased level of FAM46C reduces mRNA stability in MM.FAM46C acts as an active non-canonical poly (A) polymerase which enhances mRNA stability. To explore whether the decreased FAM46C level influences mRNA stability in MM, we analyzed the expression level of the whole transcriptome in both CPCs and BMMCs. As compared to BMMCs, the significantly lower level of the whole transcriptome was identified in CPCs isolated from P17 and P20 patient  of BMMCs were divided through the silhouette method according to the biggest average silhouette width , 3B. TheP < 0.05, unpaired t test, two sided). Immunoglobulin gene IGLV3-1 decreased in the C1 compared with C2 in P19 BMMCs . In P17 BMMCs, C1 + C3 was low-FAM46C group and C2 was high-FAM46C group . A total of 11 immunoglobulin genes were downregulated in the low-FAM46C group compared with high-FAM46C group . In P23 BMMCs, the C1 trended to be FAM46C high group and C2 trended to be FAM46C low group . Immunoglobulin genes such as IGKV1-33, IGKV1D-33 decreased in the C1 compared with C2 in P23 BMMCs . Collectively, these results further demonstrate that FAM46C mediated tumor heterogeneity in MM.According to silhouette method, the BMMCs of P20, P21, and P23 were also divided into 2 clusters  and 6B. 6C group . ImmunogDue to having identified that FAM46C-mediated tumor heterogeneity of MM and lower expression of FAM46C in CPCs compared with BMMCs, we constructed a single sell model to show the contribution of FAM46C-meditated tumor heterogeneity for extramedullary metastasis. In P17 patient, the low-FAM46C group (C1+C3) provided 83.3% of the contribution for extramedullary metastasis, whereas the high-FAM46C group (C2) provided only 16.7% . Then, wFAM46C mediated tumor heterogeneity contributes to extramedullary metastasis of multiple myeloma. To further explore whether FAM46C could predict poor survival of MM patients at the population level, we integrated 7 independent datasets, a total of 2280 MM samples (2072 patients), and subsequently analyzed the relationship between FAM46C gene expression and clinical characteristics of MM.P = 0.0078; Kruskal-Wallis test). After combining with molecular subtype, similar results were also identified in immunoglobulin G subtype of MM . In another independent dataset with 293 multiple myeloma samples, FAM46C was lower expressed in ISS stage III compared with ISS stage I and II . FAM46C was lower expressed in FISH 1q21 amplification samples (≥4 copies) compared with 2 and 3 copies . FAM46C was lower expressed in proliferation and nuclear factor kB (NFKB) molecular subtypes .We analyzed the FAM46C expression in different ISS stages and molecular subtypes. FAM46C was lower expressed in ISS stage II compared with ISS stage I, and was no difference between ISS stage III and ISS stage II/I . For stage II and III MM patients with lower FAM46C expression, an extremely poorer survival can be observed in these patients . The 559 multiple myeloma patients can be divided into FAM46C-low group (57 patients) and FAM46C-high group (502 patients)  , 12.We compared the whole transcriptome expression level between the low-FAM46C and the high-FAM46C group. Lower transcriptome expression level was identified in the low-FAM46C group compared with the high-FAM46C group in 559 MM patients .Moreover, we further investigated whether clinical outcomes were different between the low-FAM46C and the high-FAM46C group. For 295 ISS stage I MM patients, Kaplan-Meier survival curves indicated that patients with lower FAM46C expression had poorer clinical outcomes (shorter OS and EFS time) . SimilarP = 0.016, P < 0.001, P = 0.045, unpaired t test, two sided), especially LDH, were lower in high-FAM46C group. Among them, LDH is the most significantly different clinical characteristic . LDH is active in the anaerobic glycolysis, which seems closely related to FAM46C. So, we compared the expression of hypoxia-related genes, including HIF1A, HIF1AN, and HIF3A, between low-FAM46C and high-FAM46C group, but none of them were significantly expressed . Interestingly, 2 other hypoxia related genes (CITED2 and TAZ) were significantly expressed in FAM46C-low vs. FAM46C-high group . CITED2 was the top 1 down regulated gene in FAM46C-low vs. FAM46C-high group .Further analysis of clinical characteristics between low-FAM46C and high-FAM46C group revealed that they were similar in age, sex, race, etc. However, several blood indexes  or TT3 treatment (incorporating bortezomib up-front into a tandem transplant regimen) . This sueline MM .P = 3.2E-4, ANOVA test) and 120 h .The best FAM46C-siRNA was selected based on the results of western blot . To idenCancers originate from a single cell according to Nowell’s theory of clonal evolution . The enrBy analyzing the single cell RNA sequencing data of BMMCs from 6 multiple myeloma patients, we found the BMMCs from 3 patients could be divided into a significantly FAM46C-low and a FAM46C-high group. The other 3 patients trended to be one of the FAM46C-low and FAM46C-high group. There are 4 points that support the heterogeneity in BMMCs: (a) Unsupervised clustering of BMMCs of patients were conducted, which supports that FAM46C is a statistically dominate factor in distinguishing between clusters of BMMCs and the heterogeneity in BMMCs. (b) The immunoglobulin genes which are known as the hallmarks of clonal plasma cells were differently expressed among clusters of BMMCs. (c) Low expression of transcriptome level was observed in the FAM46C-low cluster which supports a functional cluster mediated by FAM46C. (d) The single cell metastasis model showed the high metastasis contribution of FAM46C low expression group in BMMCs. Thus, the tumor heterogeneity of BMMCs mediated by FAM46C exits in the multiple myeloma.FAM46C-meditated tumor heterogeneity in BMMCs is functional. The following 3 points support the functional FAM46C-meditated tumor heterogeneity: (a) FAM46C low expression cluster contribute much more than FAM46C high expression cluster. (b) Low expression of FAM46C is closely related to the poorer survival and therapeutic response in multiple myeloma. (c) FAM46C is one of the highest frequently mutated genes with many frameshift and nonsense mutations , 29. ThuWhat is the role of FAM46C in the FAM46C-meditated heterogeneity cluster? In our research, we can uncover the role of FAM46C layer-by-layer. (a) FAM46C-meditated RNA stability is a distinct role of FAM46C in the heterogeneity cluster. Low expression of transcriptome was observed in the BMMCs vs. CPCs comparison (single cell), FAM46C-low cluster vs. FAM46C-high cluster comparison (single cell), FAM46C low group vs. FAM46C high group comparison (population comparison). (b) FAM46C is closely related to the LDH level of multiple myeloma patients. (c) CITED2 is the top 1 down regulated gene in FAM46C-low vs. FAM46C-high group (population comparison). CITED2 is an efficient brake hypoxic response . Low expThe participants enrolled in this study were 4 EMP+  and 2 EMP- primarily untreated MM patients at the Peking University Third Hospital and Beijing Chao-Yang Hospital during 2014-10 to 2015-06 . All the4 ml peripheral blood and bone marrow were drawn from untreated MM patients. Peripheral blood mononuclear cells (PBMCs) or bone marrow cells (BMCs) for Single cell RNA sequencing were isolated and purified through Ficoll-Paque Plus  and human CD138 MACS Microbeads  according to the manufacturer's instructions. Single cell suspension was then analyzed or sorted by flow cytometry with antibodies   through UltraComp beads , Aria II SORP flow cytometer .Single cell libraries were constructed according to Smartseq2 protocol  and sequP-value < 1E-6 was defined as specifically expressed immunoglobulin genes in each patient at the single cell transcriptome level.Immunoglobulin genes of 6 patients were used for One-way analysis of variance. The t test. The different expressed genes (DEGs) were defined using the criterion: (a) P < 0.05 in unpaired t test; (b) foldchange  > 1 or < −1; (c) The criterion of (a and b) should be meet in both P17 and P20 patient. These DEGs may be related to extramedullary metastasis in multiple myeloma.The transcriptome of clonal circulating plasma cells (CPCs) versus myeloma cells in bone marrow (BMMCs) in P17 and P20 patient were compared using unpaired Unsupervised clustering of BMMCs single cells was done using the DEGs and immunoglobulin genes related genes. Silhouette method was used for cluster selecting. Average silhouette width was calculated for one to ten cluster. The cluster number with the biggest average silhouette width was chosen.P < 0.05 in unpaired t test and foldchange  > 1 or < −1 was used to defined the different expressed genes if no special designation. For C1 cluster and C2 cluster of P14 BMMCs comparison, P < 0.0001 in unpaired t test and foldchange  > 1 or < −1 was used to defined the different expressed genes.The different expressed genes were used for the whole transcriptome level comparison. + patient P17 and P20 at single cell transcriptome level was conducted and showed using Sankey diagram. The Pearson correlations were calculated between each CPCs and BMMCs. Only the BMMC with the biggest Pearson correlation value was chosen for a certain CPC. And this BMMC was defined as a possible contributor for a certain CPC. So, a BMMC can contribute 0, 1 or more CPCs. The metastasis rate means a BMMC contributes to how many CPCs. The metastasis rate (Mj) was calculated using the method in (1).The model of extramedullary metastasis in EMPj indicates the metastasis rate for the jth BMMC.Where Mj indicates the number of CPC contributed with the jth BMMC.Where Cp indicates the number of CPC analyzed for the certain patient. 30 CPCs for P17 patient and 47 CPCs for P20 patient.Where Ct test and a P-value of < 0.05 was considered to be statistically significantly. Only genes with foldchange (log2) > 1 or < −1 were defined as different expression genes.To further analysis the gene expression profiling of multiple myeloma and the relationship between FAM46C expression and clinical characteristics of multiple myeloma, we integrated 7 independent datasets including totally 2280 samples (2072 patients) from the NCBI GEO database. Probeset measures of all the arrays were calculated by RMA (robust multiarray averaging) method. Relative RNA expression value was log-transformed using log2. Data were analyzed with the unpaired GSE24080 with 559 samples (559 patients) were retrieved from the NCBI GEO database. The gene expression array was Affymetrix Human Genome U133 Plus 2.0 Array . The relGSE9782 with 238 samples (238 patients) were retrieved from the NCBI GEO database. The 238 samples were tested by Affymetrix Human Genome U133A Array and Affymetrix Human Genome U133B Array respectively  . FAM46C GSE19784 with 308 samples (308 patients) were retrieved from the NCBI GEO database. The 308 samples were tested by Affymetrix Human Genome U133 Plus 2.0 Array . The relGSE39754 with 136 samples (136 patients) were retrieved from the NCBI GEO database [GSE82307 with 66 samples (33 patients) were retrieved from the NCBI GEO database . The 66 st  paired samples in GSE19554 dataset  were analyzed.GSE19554 with 36 samples (18 patients) were retrieved from the NCBI GEO database . The 33 GSE31161 with 937 samples (780 patients) were retrieved from the NCBI GEO database. The 937 samples were tested by Affymetrix Human Genome U133 Plus 2.0 Array , 43. FAMThe best FAM46C-siRNA was selected based on the results of western blot. siRNA was purchased from RiboBio (China). Antibody against FAM46C was purchased from Abcam (UK) and were used at a dilution of 1:1000.The proliferation ability of H929 cells was examined by CCK8 assay. H929 cells of different groups were seeded into 96-well plates, and the optical density (OD) value at 450 nm was determined at gradient times  via using an automatic microplate reader.P < 0.05 was used.R software v3.1.3  was used in this study. A statistical significance level of Supplementary FiguresSupplementary Tables


Marinobacter nanhaiticus D15-8W is known for its ability to metabolize polycyclic aromatic hydrocarbons. Here, we report the complete circular genome sequence of this strain to be 5,336,660 bp  with one plasmid . Marinobacter nanhaiticus D15-8W is a slightly halophilic bacilliform Gram-negative bacteria that is facultatively anaerobic and was previously isolated from a South China Sea sediment  (PET), characterized as PETase type IIa  and harvested at an OD600 of around 0.3. The genomic DNA was extracted using the Genomic-tip 20/G system (Qiagen). A genomic DNA library for sequencing was prepared and multiplexed using the rapid barcoding kit . The library was sequenced with FLO-MIN111 flow cell (Oxford Nanopore Technologies) on a GridION X5 device , base-called, demultiplexed, and adapter-trimmed by guppy version 6.3.8 (Super-Accurate Mode). Illumina sequencing was performed for error correction using the HyperPlus library preparation kit (Kapa Biosystems) and a MiSeq sequencer with Reagent Kit v3 600 cycles as 300 bp paired ends (Illumina). All nanopore reads  were used for the de novo assembly using Canu version 2.2 (A0A0K8P6T7 PETase (e = 3e−75) (M. nanhaiticus D15-8W decomposes organic pollutants in marine environments.The assembly completeness was assessed with CheckM (DFAST), resulting in 99.61% completeness with the Alteromonadaceae marker lineage , which w


Identifying the subcellular localization of a protein within a cell is often an essential step in understanding its function. The main objective of this report was to determine the presence of the P2X7 receptor (P2X7R) in healthy human cells of skeletal system, specifically osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells. This receptor is a member of the ATP-gated ion channel family, known to be a main sensor of extracellular ATP, the prototype of the danger signal released at sites of tissue damage, and a ubiquitous player in inflammation and cancer, including bone and cartilaginous tissues. Despite overwhelming data supporting a role in immune cell responses and tumor growth and progression, a complete picture of the pathophysiological functions of P2X7R, especially when expressed by non-immune cells, is lacking. Here we show that human wild-type P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. By fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3 cell types, especially IVD cells and OBs. P2X7R mitochondrial immunoreactivity was predominantly detected in OBs and IVD cells, but not in Chs. Evidence of subcellular localization of P2X7R may help to i. understand the participation of P2X7R in as yet unidentified signaling pathways in the joint and bone microenvironment, ii. identify pathologies associated with P2X7R mislocalization and iii. design specific targeted therapies. It is well known that function and stability of intracellular proteins are critically determined by their subcellular localization. Each cell compartment offers different biochemical environments, is exposed to different mechanical triggers and allows interaction with different partners, which altogether contribute to a fine-tuned mechanism of control of intracellular protein localization .+, Ca2+ influx, and K+ efflux)  . Dependi efflux) . The pro efflux) . No doub efflux) . Finally efflux) . Data on efflux) . During To date, a rigorous investigation of P2X7R subcellular distribution is still hampered by the lack of highly specific anti-P2X7R antibodies and by the need of well controlled protocols to obtain highly purified subcellular fractions.Nevertheless, a detailed “subcellular anatomy” of the P2X7R is strongly needed, also in view of the many P2X7R-associated responses that may not directly (or entirely) depend on its channel function, such as changes in plasma membrane lipid symmetry, microparticle shedding, activation of lipases, kinases, and transcription factors, as well as cytokine release and apoptosis . These mEx vivo human primary osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells from surgical biopsies were used as experimental model. Our data show that the human wild-type full length P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. Through fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3 cell types, in particular IVD cells and OBs. Mitochondrial P2X7R immunoreactivity was predominantly detected in OBs and in IVD cells, but not in Chs.Studies that have so far investigated P2X7R subcellular localization are few. To date, there is evidence suggesting the presence of the P2X7R in the endoplasmic reticulum, lysosomes, and phagosomes and in the mitochondria . Some ofHuman samples were collected after written informed consent provided by the participants and approval of the Ethics Committee of the University of Ferrara and S. Anna Hospital (protocol no. 160998). IVD cells were isolated from human lumbar disc tissues of patients undergoing spinal surgery for herniation and subjected to mild digestion  as previously described . Human o2. The cells were then expanded until confluent , harvested and used for the experiments here described (passage 2 to passage 4). The morphology and phenotypic characterization of the cells used is reported in the Cells  that were released from the dissected tissue were cultured in standard medium (50% DMEM (Dulbecco’s Modified Eagle Medium) high-glucose/50% DMEM F-12/10% fetal calf serum)  supplemented with antibiotics (penicillin 100 mg/mL and streptomycin 10 mg/mL), at 37 °C in a humidified atmosphere of 5% COP2X7R negative HEK293 (wild type) and HEK293 stably expressing P2X7A  were grown in standard culture medium as above reported .See Total cell lysates were prepared in lysis buffer , 0.1% SDS (Sodium Dodecyl Sulfate) and 1 mM NaF) supplemented with protease inhibitors (Sigma-Aldrich). Proteins were quantified using the Bradford protein assay  . Thirty See 4) were seeded on glass coverslips put into 24 well plates and fixed in 4% paraformaldehyde for 2 min at 37 °C. After washes, the cells were permeabilized using 0.05% Triton X-100 and then blocked with 2% BSA (Bovine Serum Albumin)/0.05% Triton X-100/PBS (Phosphate Buffered Solution). After that, cells were incubated overnight at 4 °C with the primary antibodies: anti-P2X7R  and anti-TOM20 . P2X7R blocking peptide  was added to the primary antibody at a 1:1 ratio (http://fiji.sc/Fiji).Cells (2 × 10:1 ratio . Appropr6) were harvested by trypsinization. The cell suspension was fixed in 2% paraformaldehyde/PBS for 1 h, permeabilized with 0.1% Triton X-100 and blocked with PBS/2% BSA (http://fiji.sc/Fiji).Cells . All the results were expressed as means ± SD from triplicate measurements performed in at least 3 independent experiments.The expression of P2X7R was investigated by Western blotting analysis. As shown in We then analyzed the 3 cell types by confocal microscopy. All cell types were diffusely stained by the anti-C-terminus antibody, with high intensity in the nucleus. Overall, the signal was higher in OBs and IVD cells than in Chs .To analyze the P2X7R subcellular localization we immunolocalized the P2X7R by either fluorescence microscopy (LM) or immunogold transmission electron microscopy (TEM). Given structural and functional changes observed during osteogenic and chondrogenic differentiation in the mitochondria and the nucleus , we focuCo-labeling of cells with anti-P2X7R C-tail antibody and DAPI for nuclear staining  confirmeHigh-resolution visualization of P2X7R distribution by TEM revealed P2X7R immunoreactivity  modulate its activation state and stability- B) promote its degradation, turnover or recycling via different pathways2+ signals) (- C) integrate the P2X7R in different intracellular circuitries, intracellular organelle exchange, or specific metabolic pathways or intracellular second messenger systems  - D) support non-canonical nuclear ATP-generating systems - E) support nuclear mechano-transduction .It is conceivable that P2X7R uses some alternative pathways based on molecular adaptors or protein scaffolds capable of modulating the localization of the receptor and consequently its function, all aspects that certainly deserve to be investigated in detail. It is important to highlight these issues because the design of novel P2X7R-targeted therapies will have to take them into account . The unoThese tips might help to resolve many controversial aspects regarding the role of P2X7R in the cartilage and bone tissues. A large scientific literature has demonstrated that the protection of articular cartilage and the maintenance of joint extracellular matrix homeostasis, as well as the fine-tuning, initiation and termination of balanced bone remodeling are strongly dependent on the purinergic signaling, but many aspects remain to be understood . Importa