The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers. Protein amphitropism is a phenomenon where soluble proteins, usually globular, reversibly interact with the cellular plasma membrane. The reversibility is caused by ligand binding, post-translational modification , or locaIntrinsic disorder is also overrepresented in proteins that form in vivo aggregates of different types, including amorphous aggregates and b-sheet-rich amyloid fibrils . The dis31P NMR, triple-resonance NMR, conformational clustering, and molecular dynamics (MD). We comment critically on the suitability of the model system and our approaches.Here, we investigate the chimeric A-Cage-C polypeptide, one of the artificially designed recombinant peptides previously investigated by Strømland et al. . A-Cage-To initiate the study, we performed in silico assessments of the A-Cage-C sequence. We calculated its hydropathy index and secondary structure propensity using the approach of Tanford and Protparam, respectively ,38. Then13C and 15N labelled A-Cage-C, and a suitable membrane mimicking system with a low proton background. To that end, double labelled A-Cage-C was expressed and purified by adapting a protocol published earlier [NMR is one of the most powerful methods available to resolve individual atoms and their chemical environment in solution . However earlier , as descq, as well as solvent conditions and interacting polypeptides [q values < 2) are isotropically fast-tumbling membrane-mimicking systems and are suitable for studying many aspects of membrane–protein interactions [q = 0.4 from deuterated D22-DHPC, deuterated D54-DMPC, DHPS and DMPS as described in Materials and Methods and [31P NMR on the bicelles in the presence and absence of A-Cage-C  and non-binding (pH 7.0) conditions. 1H-15N HSQC spectra of A-Cage-C acquired at pH 4.5 in the absence of bicelles and the presence of bicelles, respectively. Data acquired at pH 7.0 is presented in Earlier work showed that the A-Cage-C bound to acidic lipid membranes at pH 4.5 and that at pH 7.0 this binding was practically absent . In addiThere may also be efficient dipole–dipole relaxation from the remaining proton background of the partially deuterated bicelles . The resAlthough the assignment was incomplete, the determined Cα and Cβ chemical shifts can be used to investigate the secondary structure propensity of A-Cage-C in its different states using the Secondary Structure Propensity (SSP) program . The SSPThe incomplete sequential assignment and a paucity of interresidual Nuclear Overhauser Effects (NOEs) precluded traditional structural assessment using distance constraints. Therefore, an alternative approach using the calculation of structural ensembles was undertaken. Briefly, structural representations of A-Cage-C in both its free and bicelle-associated forms were produced based on SSP and TALOS N dihedral angle data. These were then incorporated into the Flexible-Meccano software . For eacThe representative conformations of the two clusters (centroids) obtained for the free peptide are presented in Based on incomplete experimental data, the cluster analysis provided us only with quite tentative models for states that may be relevant for A-Cage-C binding. In an attempt at complementing these models with an independent approach, we performed MD simulations of A-Cage-C in the presence of lipid bilayers. To find a representative A-Cage-C starting conformer, preparative simulations were performed on two A-Cage-C representations picked from an ensemble of 10,000 structures generated using Flexible-Meccano. The most compact and the most extended structures were treated in silico as described in Materials and Methods and simulated in explicit water for up to 520 ns . The DSSThe two simulations represent extremes within a structurally diverse ensemble, and because of this, they did not converge within the simulation time . InsteadThe previous study using CD and fluorescence support that A-Cage-C is somewhat helical in solution at both pHs. When bound to bilayers, this helicity increases, and there is evidence of coils but no β-sheets . In siliq-values have been criticized for being fluctuating and not always retaining a disc-like morphology [q-value in the upper range of what could be considered small bicelles, and at least some evidence for disc-like morphologies could be found in the TEM micrographs . D-13C-Glucose Monohydrate was obtained from Cambridge Isotope Laboratories . NMR-tubes  were obtained from Shigemi Inc . Tobacco Etch Virus (TEV) protease was expressed and purified according to [vide infra.Egg yolk phosphatidylcholine  and porcine brain phosphatidylserine  lipids were obtained from Avanti Polar Lipids , Inc. Buffer components, as well as Drding to . Other mIn silico analyses were performed using the A-Cage-C sequence “GSEQLTKAEVFRELKDLNLYIQWLKDGGPSSGRPPPSFLDDDLTDDIMAVKKILDKVG” as input. Three different metrics for protein disorder were assessed by DisEMBL , using default settings. These metrics were COILS as defined by DSSP, and HOTLOOPS and REMARK465, which compare the query sequence with sequences in the Protein Databank with high B factors and missing coordinates in X-ray structures, respectively. These metrics are described in [http://aias.biol.uoa.gr/AMYLPRED2 (accessed on 7 April 2018).ribed in , and refribed in , using aribed in . The onlribed in . AMYLPREribed in , Amyloidribed in , Amyloidribed in , Averageribed in , b-stranribed in , Hexapepribed in , NetCSSPribed in , Pafig [ribed in , SecStr ribed in , and TANribed in ,81. EachE. coli BL21 Star™ (DE3) (Invitrogen™) using M9 minimal medium supplemented with 13C-glucose, 15N-ammonium chloride and 100 ug/mL ampicillin. Cultures were grown at 37 °C until OD595 reached 0.6, and protein expression was induced by 1 mM Isopropyl β-D-1-thiogalactopyranoside, IPTG. Expression was maintained at 26 °C for 3 h, and then the cells were pelleted by centrifugation . The pellet was resuspended in lysis buffer , supplemented with Complete EDTA free protease inhibitor cocktail, 1 tablet per 50 mL  and chicken egg white lysozyme (1 mg/mL). The resuspended pellet was kept on ice for 20 min and sonicated for 2 min with 15-s intervals. The cell lysate was centrifuged  and purified using a Ni-NTA column  following manufacturers recommendations. Eluted A-Cage-C was buffer exchanged using PD-10 Desalting columns  to TEV cleavage buffer and the His-tag was cleaved off with TEV protease overnight at 4 °C on a rotating wheel (TEV: polypeptide 1:30 molar ratio). To remove the affinity tag and TEV protease, a second Ni-NTA purification was performed, and the flow through was collected and concentrated. Finally, size exclusion chromatography was performed on a ÆKTA Purifier fitted with a Superdex 75 16/60 HiLoad prep grade  equilibrated with 50 mM Tris-HCl, 300 mM NaCl, at pH 8.0.Expression was performed in q-value, a measure of the disk elongation [2O 10% (v/v), pH 4.5) was added. The glass tube containing the bicelles was placed in an ultrasonic bath for 20 min, resuspending the lipid film. The lipid mixture was then subjected to 10 cycles of freezing in liquid nitrogen, thawing in 60 °C water bath and vortexing at 1800 rpm for 40 s. The lipid mixture was further subjected to three cycles of 10 min incubation at 37 °C at 350 rpm and 10 min of incubation on ice. The final lipid concentrations of the bicelles corresponded to 130 mM D22-DHPC, 46 mM D54-DMPC, 12 mM DHPS and 12 mM DMPS.Bicelles of at a total lipid concentration 200 mM were prepared using deuterated and non-deuterated lipids for the lamellar and hexagonal part of the disk, D22-DHPC and D54-DMPC, and DHPS and DMPS. The bicelle ongation , was setBicelles were characterized using Transmission Electron Microscopy (TEM). Samples were prepared on carbon mesh copper grids and stained with Uranyl Acetate (UA) (negative stain contrast for imaging). The TEM grid was placed in sample solution for 60 s and dipped in separate drops of water, then in 1% of UA for 60 s and then back in Milli-Q. Excess fluid was removed by touching a filter paper to the side of the grid. Prepared grids were left in a petri dish on a filter paper to dry. TEM imaging was performed on a Jeol Jem-1011, equipped with a tungsten filament, operating at an acceleration voltage of 80 kV. Images were captured by a MORADA camera, with optical image stabilization data system, and processed using iTEM. ImageJ was utilized for post-processing and analysis of the TEM images.31P-NMR experiments were carried out on an AV500 (Bruker) instrument, with a field strength of 11.7 T and fitted with a 5 mm Broadband Observe (BBO) probe. The carrier frequencies for the respective nuclei channels were 500.1 MHz and 202.2 MHz for 1H and 31P, respectively. Bicelle-containing samples were prepared by diluting a set amount of bicelles in NMR buffer at pH 4.5  D2O) to a volume of 500 µL, so that a final lipid concentration of 45 mM was achieved. The samples were transferred to a 528-PP-7 NMR tube and subjected to standard 1D proton-decoupled 31P-NMR experiments, using a pulse width of 13.3 µs, 64 scans, a relaxation delay of 4 s, and spectral width of 20 ppm. Inverse gated proton decoupling was achieved using a Waltz16 pulse-train with a 100 µs duration. The FID treated with a 1.2 Hz line broadening window function prior to Fourier transformation. Further information on the NMR acquisition parameters can be found in All NMR-experiments were acquired at 298 K and processed using TopSpin 2.1. The 1H-15N HSQC and triple-resonance NMR experiments were acquired on a Bruker Avance spectrometer operating at a proton frequency of 600.13 MHz (14.4 T) fitted with a cryogenically cooled 5 mm-TCI probe with pulse field gradients along the z-axis. The NMR sample used for resonance assignment contained 1.6 mM 13C-15N uniformly labelled A-Cage-C in citrate buffer at pH 4.5 or pH 7.0, containing 50 mM NaCl, 1 mM NaZ and 10% (v/v) D2O. To characterize the protein in the presence of bicelles, 1H-15N HSQC spectra of 1.1 mM A-Cage-C in the presence of DMPC: DMPS (q = 0.4) bicelles at a final lipid concentration of 100 mM were acquired. The following spectra were acquired for the purpose of resonance backbone assignment: HNCA, HNCO, CBCA(CO)NH, CBCANH, Sequence-specific backbone resonance assignment was performed using CARA (Computer Aided Resonance Assignment) version 1.8.4.2 [ 1.8.4.2 . Based o 1.8.4.2 . For morEnsembles of 10,000 A-Cage-C structural conformers were calculated in the Flexible-Meccano program using the amino acid sequence and SSP scores as input parameters . In the + counterions to obtain a neutral net charge. Each system was subjected to the following regime: (i) 5000-step minimization ; (ii) 5 ps heating from 0 to 100 K at constant volume; (iii) 100 ps heating from 100 K to 310 K at constant pressure; (iv) unrestrained simulation for about 500 ns at 310 K and constant pressure. Ten kcal mol−1Å−2 restraints were applied to the peptide backbone during both heating steps.Two conformations, the ones with the longest and shortest centre-of-mass distance from the first to the last residue, were selected from the Flexible Meccano ensemble of A-Cage-C and subjected to MD simulations in solution. In each case, the peptide was assigned ff14SB parameters  and plac+ counterions for neutralization of the net charge. The simulated volume was large enough to allow full peptide unfolding with a minimum of 20 Å between any solute atom and the edge of the octahedral water box. The same minimization/simulation regime as described above was performed, with two notable differences: semi-isotropic pressure coupling was used in step (iii) and (iv) while isotropic pressure was applied for A-Cage-C in solution, and each of the bilayer-containing systems were simulated for 800 ns in step (iv).To find a reasonable starting conformation for simulation in the presence of DOPC and DOPC:DOPS lipid bilayers, we applied the following strategy. The two simulations of A-Cage-C in solution were combined and subjected to the same clustering approach as described above for Flexible-Meccano output, from which a representative frame from the largest cluster was extracted . The resulting A-Cage-C conformation was placed in the aqueous phase of two bilayer systems containing: (A) 232 DOPC lipids  and (B) −1 collision frequency, and pressure was maintained at a reference value of 1 bar and a relaxation time of 1.0 ps by the Berendsen barostat [All simulations were run with the GPU-accelerated version of AMBER18 ,95,96,97barostat .


Ras proteins are membrane-bound small GTPases that promote cell proliferation, differentiation, and apoptosis. Consistent with this key regulatory role, activating mutations of Ras are present in ∼19% of new cancer cases in the United States per year. K-Ras is one of the three ubiquitously expressed isoforms in mammalian cells, and oncogenic mutations in this isoform account for ∼75% of Ras-driven cancers. Therefore, pharmacological agents that block oncogenic K-Ras activity would have great clinical utility. Most efforts to block oncogenic Ras activity have focused on Ras downstream effectors, but these inhibitors only show limited clinical benefits in Ras-driven cancers due to the highly divergent signals arising from Ras activation. Currently, four major approaches are being extensively studied to target K-Ras–driven cancers. One strategy is to block K-Ras binding to the plasma membrane (PM) since K-Ras requires the PM binding for its signal transduction. Here, we summarize recently identified molecular mechanisms that regulate K-Ras–PM interaction. Perturbing these mechanisms using pharmacological agents blocks K-Ras–PM binding and inhibits K-Ras signaling and growth of K-Ras–driven cancer cells. Together, these studies propose that blocking K-Ras–PM binding is a tractable strategy for developing anti–K-Ras therapies. RAS genes were initially identified as the viral oncogenes of acute transforming retroviruses, and it was designated as a mammalian proto-oncogene when mutated RAS genes were discovered in human cancer cells , 1 (1p22-p32), and 12 (12p12.1-pter), respectively , where the AAX tripeptide is removed by the Ras and a-factor–converting enzyme (Rce1). The now C-terminal cysteine is methylated by isoprenylcysteine carboxyl methyltransferase (Icmt)  of each isoform, which accounts for <15% homology being shared between any two isoforms . These He (Icmt) . The CAAe (Icmt) .via the classical secretory pathway to the PM (1/2 of <20 min), the depalmitoylation/repalmitoylation machinery is important for delivering consistent H- and N-Ras distribution between the Golgi and the PM at a steady state  . While tIn addition to K-RasG12C–specific direct inhibitors, there are three other approaches that are currently being investigated for blocking all oncogenic mutant K-Ras activity. They are 1) blocking K-Ras interaction with the PM, 2) inhibiting K-Ras downstream effectors, and 3) dysregulating cell energy metabolism. This review will focus on mechanisms that regulate the PM localization of K-Ras, which could be tractable targets for developing new anti–K-Ras therapeutics.in vivo and in vitro models, which allowed phase I studies on FTIs in 1999, with some progressing to phase III clinical trials in 2002  for redelivery to the PM via vesicular transport  is an anionic phospholipid synthesized from phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEth) by PtdSer synthase 1 and 2, respectively, in mammalian cells. While PtdSer is found in the ER and mitochondria, it is concentrated in the inner PM ucidated . PM PtdSucidated . The aniucidated . K-Ras bucidated . Recent 2 P2 level by overexpressing PI4P 5-kinase (PIP5K) β reduces PM PI4P levels. In both cases, the exchange of ER PtdSer for PM PI4P is perturbed, resulting in PtdSer reduction in the inner PM leaflet  K-Ras in cohorts of pancreatic cancer, NSCLC, and 33 types of cancer in the TCGA (the Cancer Genome Atlas) database (Phosphatidylinositol (PI) is phosphorylated to PI 4-phosphate (PI4P) by four PI 4-kinases in mammalian cells: PI4K IIα and β (PI4K2A and 2B) and PI4K IIIα and β (PI4KA and PI4KB) . PI4KA a2 . Several leaflet . Also, t leaflet . Ras pro leaflet . PM PI4P leaflet . Consist leaflet . ORP5 ovdatabase . Furtherdatabase .In addition to PM PI4P, a recent study has demonstrated that Golgi PI4P is involved in the PM localization of PtdSer and K-Ras. Chemical inhibition of PI4KB, which depletes PI4P at the Golgi complex, but not the PM, translocates K-Ras and PtdSer from the PM to the mitochondria and endomembrane, respectively . Supplemvia the classical vesicular trafficking. Once PM PtdSer is endocytosed, it enters the sorting endosomes, where it either returns to the PM via the RE or is transported to lysosomes for its degradation by phospholipases  inhibitor, staurosporine, and its analogs accumulate PtdSer internalized from the PM in the RE, resulting in PM PtdSer depletion in a PKC-independent manner . These cSMPD1 gene–encoding ASM has inactivating and partial loss-of-function mutations, respectively -induced multi-vulva phenotype (Recent studies have demonstrated that perturbing the enzymes involved in sphingomyelin (SM) metabolism depletes the PM localization of PtdSer and K-Ras, and blocks oncogenic K-Ras signaling. Ceramide, which is synthesized in the ER, trafficks to the Golgi complex, where it is converted to SM. SM is further transported to the PM and lysosomes, where it is reverted to ceramide by sphingomyelinases . Severalectively . These iectively . Supplemectively . In addiectively . They fuectively . In a suhenotype .Acacia victoriae, have proapoptotic, anti-inflammatory, and anticancer activities . Although combination therapy with K-RasG12C inhibitors and other anticancer approaches is promising, it is limited to K-RasG12C–specific cancers, which accounts for ∼20% of K-Ras–driven cancers. Therefore, it would be worthwhile to examine the effects of combining pharmacological agents that can block all oncogenic mutant K-Ras by dissociating it from the PM with drugs developed for targeting the other approaches.Cancer chemotherapy is most effective when a combination of drugs targeting different molecular mechanisms are applied. There are four major approaches that are currently being perused for developing anti–K-Ras therapies, and any one approach alone may not be sufficient to completely block oncogenic K-Ras signaling due to high cytotoxicity and/or nonspecificity. A recent study has demonstrated that a K-RasG12C inhibitor potentiates the anticancer effect of the MEK, mTOR, and insulin-like growth factor 1 receptor (IFG1R) inhibitors in NSCLC cells. While combined mTOR, IGF1R, and MEK inhibition shows significant tumor regression in K-RasG12C–driven lung cancer mouse models, replacing the MEK inhibitor with a K-RasG12C inhibitor in combination demonstrates greater efficacy, specificity, and tolerability . Moreove


Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete  Escherichia coli and Salmonella enterica serovar Typhimurium share a remarkably high degree of similarity with respect to overall structure, as the key components are conserved among the external flagella and the periplasmic flagella of spirochetes  strain (bb0259 mutant and complemented strains were constructed as described below. All B. burgdorferi strains were grown in Barbour–Stoenner–Kelly II (BSK-II) liquid medium or on soft agarose plates at 35°C in the presence of 2.5% CO2 (Escherichia coli strains were cultured in Luria-Bertani (LB) liquid medium or on LB agar plates  strain . The iso2.5% CO2 . Antibior plates , 2004. WBasic local alignment search tool (BLAST) was used to determine protein homologs from the sequence database , 1997. FB. burgdorferi BB0259 with variable lengths using short-affinity tags (His6 or Strep) failed, as all these recombinant BB0259 proteins with short affinity tags formed inclusion bodies in the E. coli host. In order to produce soluble recombinant BB0259 protein, a DNA fragment harboring the BB0259 open reading frame (ORF) without signal peptide (1-49 aa) was polymerase chain reaction (PCR)-amplified from chromosomal DNA of B. burgdorferi B31-A using the primers PF rBB0259-BamHI (GGATCC TGGTTATGGAATTTTGATTATAC) and PR rBB0259-PstI  and cloned into the pMAL c5x (NEB Inc.) through BamHI and PstI restriction sites to produce maltose binding protein (MBP)-tagged BB0259. Similarly, the 1xFLAG (DYKDDDDK) tagged FlgJ with gene locus number  was constructed for far-western or affinity blotting. A DNA fragment of full-length flgJ fused with 1xFLAG tag coding sequence (GACTACAAAGACGATGACGACAAG) at C-terminus was amplified by PCR with primers PF MBPFlgJBamHI (CGTCGACGGATCCGAAACCAAAATTAAT TCACAAAATC) and PR MBPFlgJPstI (TAATTACCTGCAG TTACTTGTCGTCATCGTCTTTGTAGTCTTTACTTTTTTGTA ATTGATTGTA) and cloned into the pMAL c5x using BamHI and PstI restriction sites. MBP tagged FlaB (BB0147), FlgE (BB0283), FlgD (BB0284), FlgG (BB0774), and FlgL (BB0182) were also prepared to determine protein–protein interactions. The coding regions of FlaB, FlgE, FlgG, and FlgL were amplified by PCR with primers PF MBPflaB_NotI (GTCCATGGGCGG CCGCATGATTATCAATCATAATACATC) and PR MBPflaB_BamHI (GGAATTCGGATCCTTATCTAAGCAATGACAAAA CATATTG), PF MBPflgE_NotI (GTCCATGGGCGGCCGC ATGAT GAGGTCTTTATATTCTGG) and PR MBPflgE_BamHI (GGAATTCGGATCCTTAATTTTTCAATCTTACAAGTTCTTG), PF MBPflgG_NotI (GTCCA TGGGCG GCCGCATGATGAGA GCATTATGGACAGC) and PR MBPflgG_BamHI (GGAATTC GGATCC TTATTGCCTTTTTAAGTTATTTGC), PF MBPflgL_NotI (GTCCATGGGCGGCCGCATGATAAATAGAGTAAGTCA TCC) and PR MBPflgL_BamHI (GGAATTCGGATCCTTATTT TATAAAATCTAATAAAGTCG), respectively, and cloned into the pMAL c5x (NEB Inc.) using NotI and BamHI restriction sites. Recombinant FlgD construct was similarly prepared using NdeI-NcoI restriction enzymes with primers PF MBPflgD_NdeI (GCTGGACGACATATGAATAAAATAAACGGTGTTGAAAATG) and PR MBPflgD_NcoI (TGCTGGGCACCATGG TTATTCC TCCAAACCTACCGATAAT). All E. coli DH5α strains carrying the pMAL c5x constructs for expressing BB0259, FlaB, FlgE, FlgG, FlgL, FlgD, 1xFLAG tagged FlgJ were induced with 0.25mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at room temperature, and purifications of recombinant proteins were performed with amylose resin (NEB Inc.) according to the manufacturer’s protocol.Multiple attempts to express soluble B. burgdorferi cells were harvested and washed with phosphate-buffered saline (PBS) and resuspended in the same buffer to process the preparation of cell lysate for SDS-PAGE. Immunoblotting was performed with B. burgdorferi FlaB, FlgE, MotB, FliL, and DnaK-specific antibodies (™ ECL or ECL 2 substrate (Thermo Fisher Scientific Inc.). Concentration of the proteins was determined using a Bio-Rad protein assay kit with bovine serum albumin (BSA) as the standard. Unless specified, approximately 10μg of cell lysates was subjected to SDS-PAGE.Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as mentioned previously . Exponentibodies  and Pier® M2 antibody (Sigma-Aldrich Co. LLC) followed by ECL 2 detection, as mentioned above. The X-ray films were exposed to the membranes for less than a minute.Far-western or affinity blotting assay with recombinant proteins was performed as described previously . Brieflybb0259 and bb0531 inactivation plasmids, electroporation, and plating of B. burgdorferi were as described earlier (bb0259 was inactivated by flgBP-kanamycin resistance cassette (flgB-aph1P), which possessed the flgB promoter driving kanamycin resistance gene  using primers BB0259-KO-F (GTTGAGTATATTGACGAGAAG) and BB0259-KO-R (TCCCAACAA CTCCGGTAACA), which was further TA cloned into pGEM-T Easy (Promega Inc.), yielding pGEM-T Easy::bb0259. flgB-KanP was introduced into pGEM-T Easy::bb0259 through the first and last HindIII sites located within bb0259 gene, creating pGEM-T Easy::flgB-Kanbb0259KO-P, of which the direction of transcription of flgB-KanP was confirmed to be the same as that of bb0259. Plasmid pGEM-T Easy::flgB-Kanbb0259KO-P was linearized by NotI restriction digestion and electroporated into competent B. burgdorferi B31-A cells to create ∆bb0259 mutant cells. bb0531 mutant was similarly prepared using a promoter-less kanamycin cassette (Pl-Kan), as described  and BB0259 Comp R (CTGCAGTTAATTT TTGGGGAATTCGCCC) and ligated into pGEM-T Easy, yielding plasmid bb0259ORF-Easy. DNA fragment containing bb0259 ORF was then released using NdeI restriction digestion from bb0259ORF-Easy, and further ligated into NdeI-treated PflgB-motB-Easy vector  and bb0259-D606N (D aspartate to N asparagine) point-mutated strains were constructed through allelic exchange mutagenesis (bb0259 gene), promoter-less kanamycin cassette , and right arm  were PCR amplified, respectively, using primer pairs P1F BB0259PointMuKOplKan (CCTAACGTAAGCGGAGAATACA AGAGTCTTTTGCATTCTG) and P1R BB0259Point MuKO plKan (TAAAATTGCTTTTAACTATTAATTTTTGGGGAATTC GCCC), P2F BB0259PointMuKOplKan (GGGCGAATTCCCC AAAAATTAATAGTTAAAAGCAATTTTA) and P2R BB0259 PointMuKOplKan (CTTTTCATACAAAGCATCATTTAGAAA AACTCATCGAGC), P3F BB0259PointMuKOplKan (GCTCGA TGAGTTTTTCTAAATGATGCTTTGTATGAAAAG) and P3R BB0259PointMuKOplKan (CCCAAGCCTTGCATCAGCCCCA TAAAAATTCCTGCTAAC). Through overlapping PCR, Pl-Kan was inserted between the left arm and right arm, and further cloned into pGEM-T Easy, creating vector pGEM-T Easy:: bb0259PointMuKO-Pl-Kan. Using pGEM-T Easy::bb0259 PointMuKO-Pl-Kan as the template, point mutations of BB0 259E580Q were introduced by QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies Inc.) using the primers PF BB0259E580Q (CTTTAAT AAAAGCACAA AGTAG CTTTG AAAAAAATG) and PR BB0259E580Q , to create pGEM-T Easy::bb0259E580QKO-Pl-Kan. Similarly, through site-directed mutagenesis pGEM-T Easy::bb0259D606NKO-Pl-Kan was generated with primers PF BB0259D606N (GCCATCAACAGC AAATAATATTTCTAAA GAACTTAAG) and PR BB0259D606N (CTTAAGTTCTTTA GAAATATTATTTGC TGTTGATGGC). These two vectors were linearized by NotI digestion and electroporated into the competent B. burgdorferi B31 A cells to generate bb0259-E590Q and bb0259-D606N mutant strains, of which the point mutations of bb0259 were further verified by sequencing.agenesis . Simply,B. burgdorferi cells were observed using a Zeiss Axio Imager M1 dark-field microscope to determine bacterial morphology and motility. To evaluate the motility of B. burgdorferi cells, swarm plate assays were performed following our well-established protocol  for 25s. The grids were blotted with filter paper for 3 to 5s and rapidly frozen in liquid ethane, using a homemade plunger apparatus as described previously  equipped with a field emission gun and a 4,096-by-4,096 charge-coupled device (16-megapixel) camera . The microscope was operated at 300kV with a magnification of ×31,000, resulting in an effective pixel size of 5.7Å. Using the FEI batch tomography program, low-dose single-axis tilt series were collected from each bacterium at ~8μm defocus with a cumulative dose of ~100 e−/Å2 distributed over 65 images. In total, 80 tomographic reconstructions of Δbb0259 cell tips and 10 tomographic reconstructions of complemented bb0259 cells were generated.The Δbb0259 point mutants (bb0259-E580Q and bb0259-D606N) were imaged with Krios electron microscope (Thermo Fisher) with a field emission gun, a volta phase plate, and a direct electron detector (Gatan K3 Summit). SerialEM was used to collect tilt series at focus. A total dose of 55 e−/Å2 is distributed among 35 tilt images covering angles from −51° to +51° at tilt steps of 3°. For every single tilt series collection, dose-fractionated mode was used to generate 11 frames per projection image. Collected dose-fractionated data were first subjected to the MotionCor2 to generate drift-corrected files . BB0531 shares only 15% amino acid sequence identity with S. enterica FlgJ and alignment of amino acid sequences of S. enterica FlgJ and BB0531 from FFAS shows that the catalytic residues of FlgJ are not well conserved in BB0531 leading us to speculate that BB0531 is not involved in peptidoglycan hydrolyzing activity for flagellar penetration (not shown).The single-domain FlgJ of enterica , as flgJructures . Given tB. burgdorferi lacks a significant homolog of S. enterica FlgJ, we used the model organism’s SLT domain as a query in the BLAST search and identified BB0259 as a homolog in B. burgdorferi. BB0259 shares 35–41% amino acid sequence identity with significant E-value at the SLT domain  cells observed under a microscope as well as in swarm plate motility assays indicated morphology and motility phenotypes restored to wild-type level . As described above, while the full-length bb0259 was able to complement the Δbb0259 mutant and therefore restored the morphology and motility phenotypes (bb0259Δsec was unable to restore any of the phenotypes (data not shown). These results suggest that the signal sequence is essential for BB0259 proteins to be secreted in the periplasm.Completion of the appropriately assembled flagellar rod, hook, and filament is critical for the spirochete motility and morphology . If the  Δbb0531 , and S5.e plates . Furtherd-shaped . To demoe vector . Complempe level ,B. Furthpe level . Consistpe level . To deteenotypes ,B, the SB. burgdorferi cells lacking the gene-encoding filament FlaB or hook FlgE were reported to be non-motile and rod-shaped , we created a bb0259-D606N B. burgdorferi point mutant to determine whether this non-conserved D606 residue codes for any enzyme function , and the hooks of the mutant cells look to be underneath the peptidoglycan layer . As the C-terminal domain possesses the enzymatic motif, we speculate that the N-terminal domain is involved in binding to FlgJ, as LTaseBb secretes into the periplasm using its Sec-signaling sequence. Since LTaseBb appears to possess peptidoglycan lytic activities and FlgG and FlgJ are required for proper rod/hook/filament assembly  also assemble the rod, P-ring, and hook but are unable to synthesize/assemble the filament . This result also enforces the notion that FlgJ and LTaseBb are crucial for hook/ filament assembly and/or for hook-to-filament transition  and consistent with the previously reported hook length . Part of cryo-ET data were collected at Yale CryoEM resource that is funded in part by the NIH grant 1S10OD023603-01A1.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Foot-and-mouth disease (FMD) vaccine efficacy is mainly determined by the content of intact virions (146S) and empty capsids (75S). Both particles may dissociate into 12S subunits upon vaccine manufacturing, formulation, and storage, reducing vaccine potency. We report the isolation of capsid-specific llama single-domain antibodies (VHHs) with broad strain recognition that can be used to quantify intact capsids in FMD vaccines by double antibody sandwich (DAS) ELISA. One capsid-specific VHH displayed remarkably broad strain reactivity, recognizing 14 strains representing the 13 most important lineages of serotype A, and two VHHs cross-reacted with other serotypes. We additionally show that the newly isolated VHHs, as well as previously characterized VHHs, can be used to identify antigenic differences between authentic 146S and 75S capsids, as well as corresponding genetically engineered virus-like particles (VLPs). Our work underscores that VHHs are excellent tools for monitoring the quantity and stability of intact capsids during vaccine manufacturing, formulation, and storage, and additionally shows that VHHs can be used to predict the native-like structure of VLPs. Picornaviridae. The RNA genome of FMDV is surrounded by a capsid assembled from 60 protomers, each comprising one copy of the four capsid proteins VP1, VP2, VP3, and VP4. The latter protein is located on the inside of the particle. The capsid proteins adopt a T = 3 pseudo-icosahedral structure with VP1 located close to the fivefold axes of symmetry and VP2 and VP3 alternating around the threefold axes [Foot-and-mouth disease (FMD) affects cloven-hoofed animals, such as cattle, pigs, and sheep. Its ability to spread rapidly across geographic areas and cause large epizootics poses a continuous threat to the livestock industry, especially in developing countries. FMD is caused by FMD virus (FMDV), an aphthovirus of the family FMDV exists as seven serotypes: A, O, C, Asia1, SAT1, SAT2, and SAT3. The most prevalent serotypes in the field are O and A, followed by SAT2 and Asia1. FMD can be controlled effectively through vaccination. However, due to antigenic variation, vaccines do not elicit cross-protection. Conventional FMD vaccines are mostly multivalent and are based on chemically inactivated FMDV strains formulated with an adjuvant. Apart from these classical vaccines, next-generation vaccines based on FMD virus-like particles (VLPs) are being developed that mimic the native structure of the FMDV particle, produced with mammalian-, insect- or bacterial-cell expression systems ,5. FMDV virions sediment at 146S in sucrose density gradients (SDG), whereas natural empty capsids lack the viral genome and VLPs sediment at 75S. Incubation of intact capsids at high temperatures or low pH leads to irreversible dissociation into 12S subunits, resulting in a strongly reduced immunogenicity . ConsequSeveral methods have been developed to quantify FMDV 146S antigens for use in vaccines. SDG fractionation and 146S quantification by short-wavelength (254–260 nm) UV absorbance to measure RNA content is the traditional and most commonly used method. Such SDG is not suitable for quantification of VLPs and natural 75S capsids since these are not detected by 254–260 nm absorbance. It is also not suitable for high throughput applications and relies on delicate manual operation, making it difficult to standardize . Novel mThe primary objective of the present work was to isolate 146S-specific VHHs against FMDV serotype A, which is antigenically the most heterogeneous serotype ,26. In aEarlier isolated VHHs used are shown in 2 roller bottles. FMDV present in the clarified supernatant was inactivated with 10 mM binary ethylenimine and concentrated using polyethylene glycol-6000 precipitation, resulting in crude antigen. The FMDV strains AF72, A/HuBWH/CHA/2009, A/GDMM/CHA/2013, and O/BY/CHA/2010 were obtained from the OIE/National Foot-and-Mouth Disease Reference Laboratory of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China. Antigens were produced as earlier described [The FMDV strains A/IRN/2/87, A/TUR/20/2006, A/MAU/1/2006, A/ETH/4/2007, A/KEN/12/2005, A/ETH/13/2005, A/SUD/2/84, A/ERI/2/98, and A10/HOL/1/42, were propagated at Wageningen Bioveterinary Research . A/TUR/1escribed . BrieflyE. coli using purified small ubiquitin-like modifier (SUMO) protein-tagged recombinant proteins and self-assembly in vitro as previously described [VLPs of FMDV strain A/GDMM/CHA/2013 were generated by expression in VLPs of strains O1/Manisa/TUR/69, A22/IRQ/24/64, Asia1/Shamir/ISR/89, and SAT2/ZIM/7/83 were produced by a vaccinia virus expression system at The Pirbright Institute. They were produced as both wt and either one or more stabilizing mutations at VP2 residue 93 from a serine  or a histidine (A22/IRQ/24/64) to either cysteine (93C), phenylalanine (93F) or tyrosine (93Y), as described earlier, for serotype O, A and SAT2 . The VLPg. The gradients were fractionated into twenty 0.60 mL aliquots, and the absorbance at 254 nm was determined to identify the 146S peak. The 146S concentration in μg/mL was then calculated by multiplying the absorbance at 254 nm by 126.7 [FMDV particles (inactivated authentic virus) were fractionated using 10–40% sucrose density gradients (SDG) and centrifuged for 2 h at 10 °C and 200,000× by 126.7 . We prevby 126.7 . The conImmunizations of llamas with various FMDV strains are summarized in 7 unique clones were generated, and phages were rescued. Phage display selections were performed by two consecutive rounds of biopanning in polystyrene 96-well plates , using 100 µL/well for each incubation. SDG-purified 146S from strains A/TUR/14/98, A24/Cruzeiro/BRA/55, A22/IRQ/24/64, O/TAW/3/97, and Asia1/Shamir/ISR/89 was used for phage display selections. These FMDV authentic 146S particles were immobilized using a specific capturing VHH to prevent capsid denaturation due to passive adsorption during the biopanning procedures. Each FMDV 146S particle was used in two separate biopanning procedures using either the broadly reactive capturing VHH M8F or using one of the serotype/strain-specific VHHs M326F, M321F, M23F, or M98F  supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK− mK−)) cells. In each selection round, a phage ELISA was performed, simultaneously with phage display selection. For this purpose, a duplex plate containing similar concentrations and types of FMDV 146S particles and phage was incubated with a peroxidase-conjugated mAb against M13 phage instead of incubation with trypsin. The amount of bound 146S particle-specific phage was then measured by phage ELISA.Libraries consisting of at least 10 or M98F . The useE. coli TG1 cells, and individual colonies were picked. The expression of the VHH genes was induced by adding 1 mM isopropyl β-d-thiogalactopyranoside (IPTG). Soluble recombinant VHHs, directed to the periplasm, were tested for binding to FMDV authentic particles as described below in Section ELISAs.After the second round of panning, phages were transduced to ® 3130 Genetic Analyzer and the BigDye Terminator V1.1 Cycle Sequencing kit (ThermoFisher Scientific). The SeqMan Pro program of the Lasergene suite was used to align and merge the sequence reads. The deduced VHH amino acid sequences were aligned according to the IMGT system [For sequencing the P1 region of the various FMDV strains, cDNA was synthesized using superscript II reverse transcriptase (ThermoFisher Scientific) and primer FMDV-116 (5′-GACATGTCCTCCTGCATCTG-3′). A 3.5 kB PCR fragment was generated using the Expand Long Template PCR system  and primer FMDV-83 (5′-CCCCCCCCCCCCCCCCCCCCTAGGT-3′) in combination with either FMDV-116 or FMDV-115 (5′-GACATGTCCTCCTGCATCTGGTTGAT-3′). PCR products purified from agarose gel as well as individual VHHs were sequenced using both strands by fluorescent dideoxy DNA sequencing using an ABI PrismVHHs fused to the natural llama heavy-chain antibody long hinge region, and a hexahistidine (his6) tag was produced in baker’s yeast using vector pRL188 and purified from culture supernatant using immobilized-metal affinity chromatography as described earlier . VHHs prVHHs were subjected to reducing SDS-PAGE using precast gels , and stained using GelCode Blue reagent (ThermoFisher Scientific). VHHs were digested with endoglycosidase H (Roche Applied Science) according to the manufacturer’s instructions. 2PO4; 8.1 mM Na2HPO4; pH 7.4) or PBSTM for 45 min at RT. Plates were next incubated with either phage-displayed VHH, E. coli-produced VHH, or yeast-produced biotinylated VHH, and subsequently with a suitable specific peroxidase conjugate, using the same buffer as used for incubation with FMDV particles (ELISA buffer or PBSTM). The peroxidase conjugate was subsequently detected by staining with 3,3′,5,5′-tetramethylbenzidine. After stopping the reaction by the addition of 0.5 M H2SO4 (50 µL per well), the absorbance at 450 nm was measured using a Multiskan Ascent spectrophotometer . Three different ELISA procedures were used. We first describe the basic ELISA procedure that was used in all three procedures. ELISAs were performed by coating high-binding polystyrene 96-well plates (Greiner) with 0.1–1 μg/mL of unlabeled VHH in 50 mM carbonate/bicarbonate buffer, pH 9.6 (coating buffer), overnight at 4 °C. Coating and subsequent incubations were performed using 100 µL per well. After washing, the coated plates were incubated with FMDV particles in ELISA buffer  and FMDV 146S particles (1 μg/mL) as described before . Bound pE. coli-produced soluble VHH, plates were preferably coated with 1 μg/mL yeast-produced VHH and then subsequently incubated with 1 μg/mL FMDV authentic particles in ELISA buffer. Bound FMDV authentic particles were detected by incubation with 10-fold diluted E. coli-produced soluble VHH, which contains a myc-tag, and a peroxidase-conjugated mAb against the c-myc tag . Four ELISAs with different capturing VHH/FMDV authentic particles combinations were conducted to evaluate the 146S specificity of the E. coli-produced VHH, with two parallel comparisons done in each ELISA. One comparison used the M326F-, M321F-, M98F- or M23F-captured authentic particles versus M3F-captured 12S, and the other comparison used M8F for comparing authentic particles with 12S.For ELISA analysis of Plates were coated with 0.5 μg/mL of unlabeled yeast-produced VHH to determine FMDV particle concentrations by double antibody sandwich (DAS) ELISA. These plates were then incubated with serial 2-fold dilution series of FMDV particle preparations in ELISA buffer. Normally, standards of untreated FMDV particles were included in the ELISAs for the quantification of FMDV particles. Standards were serially 2-fold diluted using both 1 μg/mL and 0.66 μg/mL as starting concentrations. Plates were next incubated with 0.25 μg/mL of biotinylated VHH. The different DAS-ELISAs employed the same VHH for coating in unlabeled form and for detection in biotinylated form. Bound biotinylated VHH was detected with 1 μg/mL peroxidase-conjugated streptavidin . Absorbance data were evaluated using an Excel spreadsheet template . A four-parameter logistic curve was fitted to absorbance and FMDV particle concentrations of standards by non-linear least squares using the Excel solver tool. The FMDV particle concentration in unknown samples was then determined by interpolation. For the determination of the binding of VHHs to particular FMDV particles, the particles were titrated in two or three-fold dilution series. The effective particle concentration (EC) required to reach an absorbance value of 1 was then interpolated after four-parameter logistic curve fitting of absorbance and particle concentrations. The LOD of ELISAs was measured by titrating FMDV particles in triplicate and interpolating the FMDV particle concentration required to reach the average absorbance value of background plus 3 times the standard deviation.Phagemid libraries from four llamas previously immunized and two llamas immunized in this study  were useIn total, 26 novel VHHs belonging to 20 CDR3 groups were produced in yeast for further study . They weAll 26 yeast-produced VHHs migrated as diffuse bands in SDS-PAGE, suggestive of O-glycosylation, with a molecular weight of about 24 kDa , as expeThe yeast-produced VHHs were analyzed for FMDV particle specificity in a DAS-ELISA setup using the same VHH for coating in an unlabeled form and detection of FMDV particles in biotinylated form. Furthermore, to determine the FMDV strain/serotype specificity of the novel VHHs, 21 vaccine strains were used as FMDV particles in this DAS-ELISA . Three bVHHs from the same CDR3 group generally show similar particle- and strain-specificity. Overall, for serotype A, VHHs M659F, M702F, M691F, M703F, M686F, M688F, M669F, M676F, M677F, M678F, which encompass six CDR3 groups, bound to untreated particles of strain A/TUR/14/98 and a variable number of additional serotype A strains while not binding to heated (12S) particles of any of these strains . These 1Among the 146S-specific VHHs, M691F from the green group displayed the broadest strain recognition while showing high 146S specificity. This VHH binds to all 14 tested serotype A strains, except FMDV strain AF72, and did not react with 12S from any of these strains. The other VHH from the green group, M703F, recognized 6 out of 15 serotype A strains in a 146S specific manner. It especially did not bind A/Africa topotype strains. The four VHHs from the purple CDR3 group similarly bound to most of the A/Asia topotype strains but, in addition, bound one A/Africa topotype strain. They did not bind A/Euro SA topotype strains except M652F, which bound A24/Cruzeiro/BRA/55. Of note, strain AF72, which was not recognized by M691F, could be recognized by M659F. Only VHHs M659F and M702F from the purple group showed a consistent 146S-specific binding to all strains recognized. M643F and M652F from the purple group, in general, showed much lower 146S specificity for most strains but were shown to be 146S-specific for strain A/GDMM/CHA/2013. Surprisingly, two VHHs exhibited both 146S specificity and cross serotype reactivity. M652F was shown to cross-react with C1/Detmold/FRG/60 with a high 146S specificity. Similarly, M661F, which did not show considerable specificity for 146S particles of the 15 tested serotype A strains, displayed 146S specificity to Asia1/Shamir/ISR/89. The additional color-coded 146S-specific VHHs were found to be highly strain-specific. Among these, VHHs of the orange, gray, and magenta groups bound two strains, A/TUR/14/98 and A/IRN/2/87, whereas the red group VHHs only bound A/TUR/14/98. M651F also showed some 146S specificity for some serotype A strains. The remaining 11 novel VHHs were not 146S specific and sometimes even specific for 12S of certain strains. Among them, five VHHs were serotype/strain-specific without 146S specificity. M655F, M662F, and M679F bound several (2–3) serotype A strains, and M675 bound to most of the serotype A strains. In addition, M685F bound to both 146S and 12S of Asia1/Shamir/ISR/89. However, the other six VHHs, M684F, M642F, M665F, M664F, M663F, M680F, reacted broadly with various strains representing several (3–4) serotypes. Especially M663F and M680F bound to all strains analyzed, similar to M3F, and displayed 12S specificity for some strains. In summary, the set of 10 VHHs that showed high 146S specificity comprised several VHHs with limited strain recognition but also included four VHHs from two CDR3 groups with much broader strain recognition. Together, this set of VHHs covers all 15 serotype A strains analyzed. Although an additional five serotype A vaccine strains were not used in ELISA, they showed a close phylogenetic relationship to other serotype A strains that were tested . It suggTo further investigate the absolute particle specificity of the novel isolated VHHs, 146S, 75S, and 12S, particles of four serotype A strains and one Asia1 strain were separated by SDG. The number of FMDV particles in 20 fractions collected from top to bottom was quantified by DAS-ELISA with the novel isolated VHHs as well as the control VHHs M8F and M3F, using standards of crude antigens that were either heated  or untreated (rest of the ELISAs). The presence of 12S and 75S in the five crude antigens was ignored when using them as standards in this ELISA, which can explain the differences in the FMDV particle concentrations determined by ELISAs or A254. All FMDV particle preparations contained a clear 12S peak in fractions 1–5 as detected by M3F ELISA and 146S peak in fractions 13–16 as detected by A254 a,d,g,m,pY-axis  did not interfere with intact capsid quantification by M691F or M702F, whereas M652F and M661F were affected by such spiking with 12S, but only at lower intact capsid concentrations , consistAfter demonstrating antigenic differences between 75S and 146S particles, we further explored the capsid specificity of VHHs using vaccinia virus-produced wt and stabilized mutant VLPs from four different FMDV serotypes. We also used 146S-specific VHHs binding serotypes O and SAT2 strains and some additional control VHHs recognizing both 12S and 146S . We usedM691F did not bind both wt- and 93C VLPs of A22/IRQ/24/64 c,e, showWe here report the isolation and characterization of 26 novel VHHs, 13 of which display capsid specificity. We earlier ,24 referE. coli or vaccinia virus by the novel capsid-specific VHHs as well as earlier isolated ones, which is summarized in Most previously published DAS-ELISAs for quality control of conventional vaccines were used to detect 146S particles but not 75S or VLPs ,21,24,34Vaccinia virus-produced VLPs of serotypes O, A, Asia 1, and SAT2 comprised both wt VLPs as well as corresponding particles with an amino acid substitution of VP2 residue 93 to F, Y, or C resulting in the stabilization of VLPs against dissociation into 12S particles upon heating for 2 h at 56 °C, although the 93Y VLP of O1/Manisa/TUR/69 showed only partial stabilization . The wt Recently, the Cryo-EM structure of the capsid-specific VHH M170F with 146S particles was elucidated . M170F wIrrespective of the molecular basis of differences in antigenicity, which is one of the focus points of our current research, our capsid-specific VHHs prove useful tools not only for VLP quantification but also for qualitative assessment of their suitability for vaccine use. In this respect, the demonstration that several stabilized mutant VLPs of serotypes A, O, and SAT2  show morE. coli, vaccinia virus, or baculovirus. In addition to their practical use for vaccine quality control, the VHHs can be used to gain more insight into the structure-function relationship of FMDV capsids.The newly-developed VHHs presented here are promising tools for the quality control of vaccines based on 146S, 75S, or (stabilized) VLPs produced by


The substrates and received products were investigated by TEM, XRD, Raman and IR spectroscopies, XPS, XAES and REELS. MWT assisted reduction using different agents resulted in rGOs of a large number of vacancy defects, smaller than at GO surface C sp3 defects, oxygen groups and interstitial water, interlayer distance and diameter of stacking nanostructures (flakes). The average number of flake layers obtained from XRD and REELS was consistent, being the smallest for CH2O and then increasing for HCO2H and N2H4. The number of layers in rGOs increases with decreasing content of vacancy, C sp3 defects, oxygen groups, water and flake diameter. MWT conditions facilitate formation of vacancies and additional hydroxyl, carbonyl and carboxyl groups at these vacancies, provide no remarkable modification of flake diameter, what results in more competitive penetration of reducing agent between the interstitial sites than via vacancies. MWT reduction of GO using a weak reducing agent (CH2O) provided rGO of 8 layers thickness. Reduced graphene oxide (rGO) was prepared by chemical reduction of graphene oxide (GO) (with a modified Hummers method) in aqueous solutions of hydrazine (N Nowadays, a large number of methods of graphene production using “top-to-bottom” methods  and “bottom-to-top” methods  are being applied ,2,3. HowThe mass production used for separating graphene layers from graphite leads to defected graphite of uncontrolled structural parameters , where c4 higher times than that of GO paper [2) reported the wide range of values for C/O ratio, Raman D to G modes ratio and conductivity typical for graphene [The quick (3 min) synthesis of exfoliated graphite materials has been prepared by 1 min microwave irradiation using different weight ratios of natural graphite and nitric acid to potassium permanganate, resulting in various expanded volume and bulk density values . The graGO paper . SimultaGO paper . The micGO paper . The graGO paper . A mild GO paper . ApplicaGO paper . Compilagraphene . The effect of a microwave treatment (MWT) at 250 °C in a high pressure microwave reactor (HPMWR) at 55 bar and microwave assisted (MW at RT) conditions reduction of GO to rGO applying different reducing agents is examined. The chemical and structural properties were investigated using transmission electron microscopy (TEM), X-ray diffraction (XRD), Raman spectroscopy (Raman), infrared spectroscopy (IR) and electron spectroscopic methods like X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy (AES) and reflection electron energy loss spectroscopy (REELS). UHV, i.e., GO paste dried in an ultra-high vacuum (UHV) and GO-exp-foil. The graphene oxide, denoted as GO, was prepared from commercially available 99.0% purity graphite, denoted as Gr, from AcrosOrganics, USA, 325 mesh and expanded graphite, denoted as Gr-exp, by using a modified Hummers method . These G2O) and formic acid (HCO2H), whereas for hydrazine (N2H4) reduction with 50 mL of 50% solution of hydrazine hydrate in conditions of a microwave heating at 250 °C (MWT) in a high pressure (55 bar) microwave reactor (HPMWR) for 30 min . The reaction mixtures of rGO samples prepared under heating conditions were finally cooled to room temperature (RT), washed in deionized water till the pH of filtrate stabilized to ca. 7–8 and then dried.The reduction of GO was carried out in 100 mL water suspension of GO (0.6 wt%) mixed with 50 mL 1M water solution of formaldehyde , GO suspension treated for 15 min in microwaves at power at 400 W (rGO-N2H4-MW) [2O boiled under reflux for 30 min (rGO-CH2O), Gr and Gr-exp standards [The samples prepared under MWT conditions were compared to samples already reported elsewhere, i.e., GO and GO-exp reduced using hydrazine in 100 mL water suspension of GO (0.6 wt%) mixed with 50 mL of 50% water solution of hydrazine hydrate boiled under reflux for 30 min (rGO-NN2H4-MW) , GO redutandards .A high resolution transmission electron microscopy (HR-TEM) images were measured using microscope Tecnai 20F X-Twin, equipped wth an electron source, a cathode with field emission gun (FEG), EHT = 200 keV, a camera Orius, a high-angle annular dark-field (HAADF) detector, spectrometer for energy dispersive X-ray spectroscopy (EDX) with energy resolution of 134 eV (EDAX RTEM SN9755+) and parallel electron energy loss spectroscopy (PEELS) with the energy resolution of 0.8 eV. EDX quantification was performed according to the modified standardless/thin foil method. The samples for TEM measurements were prepared by proceeding with sonication for 5 s (a few milligrams of sample in 99.8% anhydrous ethanol using ultrasound), then applying a drop (5 μL) on a carbon coated copper mesh with holes  and evaporating the solvent at room temperature. Then, the remaining dried powder stuck on copper mesh was examined. The X-ray diffraction (XRD) spectra were recorded using an X’Pert Pro diffractometer .−1.Raman spectra were recorded using Raman micro-spectrometer  equipped with a 532 nm laser using laser power of 2 mW, acquisition time of 360 s, number of scans 2 and in the spectra range of 800–3000 cmp = 10−1 Pa, 70–4000 cm−1 range) with a resolution of 4 cm−1, number of scans 100, in the 750–1900 cm−1 range. Prior the measurements a “vacuum spectrum” was recorded to be subtracted automatically as a background from the investigated samples spectra. The samples were prepared by mixing with KBr at a ratio of 1/300 mg, then compressing at 7 MPacm−2 to form a pellet. The IR transmission spectra were recorded in a vacuum spectrometer  and an Ar+ ion source of AG21 (VG Scientific) [The electron spectroscopic (XPS and REELS) measurements were performed in UHV chamber of the home-made ESA-31 electron spectrometer. The ESA-31 spectrometer was equipped with a hemispherical electron energy analyzer of a high energy resolution, an electron gun (LEG62-VG Microtech), a home-made X-ray excitation source (Al Kα X-rays hentific) . The XPS2O [2O-MWT indicate disordered, thin transparent structures of various transparency, where for GO-CH2O-MWT the diameter size of stacking nanostructures (flakes) is larger and crystallinity smaller than those for GO-CH2O  and then the average number of layers in graphene flakes, n. The (10) reflex was applied for evaluating the average diameter of graphene flakes, D. The evaluated parameters are shown in d, H and D resulting from a few peaks are listed in The XRD diffractograms for the investigated samples indicating (002) and (10) reflexes, notation according to Warren , are shoUHV and the smallest for GO-exp. The remaining rGOs show values of d between 0.35 nm and 0.383 nm. The d values in rGOs obtained using the MWT procedure depend on the reducing agent, i.e., 0.35 nm (rGO-CH2O) and 0.375 nm (rGO-CH2O-MWT), whereas 0.383 (rGO-N2H4) and 0.37 (rGO-N2H4-MWT)  . Reducti2H4-MWT) .UHV < GO-foil < GO-exp, whereas in rGOs in the order: In GOs, the average number of layers increases in the following order: GO-dried2H4 < CH2O-MWT ≈ N2H4-MWT < HCO2H-MWT < CH2O < exp-N2H4.ND (~1320–1350 cm−1), G (~1570–1605 cm−1), D‘ (~1620 cm−1), D + D‘ (~2900 cm−1) modes and the second order 2D mode (2640–2680 cm−1) [D mode occuring due to in-plane breathing vibration of carbon rings reflects disorder due to destroyed carbon hexagons present in amorphous structures. The D mode ntensity and FWHM is a measure of structural disorder. The G mode results from the in-plane stretching vibration of carbon atom pairs and is observed for all carbon structures containing sp2 bonds, both aromatic carbon and other sp2 structures. For an ideal single layer graphene, 2D allowed mode is the most intense, whereas for graphite a low intensity, D mode is accompanied by a presence of 2D mode. Modification of D and D’ modes results from introducing defects such as (i) vacancy defects (deformation of the carbon lattice bond), (ii) on-site defects (sp3 hybridizations) describing atoms bonded to carbon and (iii) boundary or edge defects [The Raman spectra for the investigated nanomaterials show the first order modes such as 80 cm−1) ,28,29,30 defects ,32.aL, the distance between defects, DL and density of defects, Dn, where aL~DL and DL~1/√Dn, requires the spectra classification according to three stages amorphization trajectory [2 phase, (ii) bond disorder, (iii) the presence of sp2 rings or chains and (iv) the sp2/sp3 ratio. Raman spectra of stage 1  exhibit a high intensity, small FWHM of G mode and the smallest I(D)/I(G) values (D to G mode intensities is expressed by equation: λ is Raman wavelength and C (515.5 nm) = 4.4 nm [Quantification of structural parameters of carbon nanomaterials from Raman spectra, i.e., evaluation of the effective crystallite size in the direction of the graphite plane or graphitic cluster, ajectory , conside) values , indicat= 4.4 nm .D and G modes, very weak intensity of 2D mode and I(D)/I(G) values close to 1. Their characteristic features like positions, FWHM and intensity ratios of D, G and 2D modes ~0.55 nm−2. The average distance between defects, DL and density of defects, Dn, can be also observed in I(D)/I(G) ratios since these values increase up to DL~4 nm (stage 2) and then decrease for DL > 4 nm (stage 1) [D and G mode in GOs and rGOs indicate the distance between defects smaller than 3 nm [D mode and decreasing frequency of G mode accompanied by decreasing FWHM of D and G bands   exhibit high intensities and FWHMs of 2D modes  reveal callinity , Table 3stage 1)  in agreestage 1) . The FWHhan 3 nm . Generalhan 3 nm , increas G bands  indicatetion (1)  in the r−1 are shown in −1 and 1300–950 cm−1 represent C=O and C-O moieties, respectively [−1 is ascribed to the stretching vibration of C=O in carboxylic (conjugated and/or non-conjugated) or carbonate systems . The overlapping modes at 1635 cm−1 and 1580 cm−1 can be attributed to olefinic and aromatic carbon-carbon bonds (sp2), carbonyl moieties in various chemical surroundings , carbon-oxygen ion-radical structures and conjugated systems . The absorption band at 1385 cm–1 is present due to aliphatic carbon-carbon bonds (sp3 C-H bend) [−1, deformation vibration band δ(HOH) can be assigned to adsorbed/intercalated water. The overlapping absorption bands in the 1300–950 cm−1 region can be assigned to C-O moieties existing in different structural environment. In this spectral region, the followings can be observed: the presence of the C-O-C symmetric stretching vibration bands from epoxide group at 1290 cm−1, ether-, ester- lactone-, pyrone-, furane-like structure at 1225–1100 cm–1, C-OH vibration of phenol and hydroxyl molecular groups at 1070 cm−1 and alkene sp2 C-H bend at about 980 cm–1.The IR spectra in the region of 800–1800 cmectively ,36. The -H bend) . At abou2) at 1635 cm−1 and 1580 cm−1 and C=O moieties at 1720 cm−1. The contribution of aliphatic carbon-carbon bonds and C-O moieties is less remarkable than in case of GO samples. The spectra from graphite samples indicate the predominant contribution of absorption band from olefinic and aromatic carbon-carbon bonds (sp2O) a relative decrease of the intensity of the mode attributed to carboxylic moieties (1720 cm−1) and a relative increase of the intensity of the mode associated with the presence of hydroxyl groups (near 1620 cm−1) take place [2O microwave heating assisted (rGO-CH2O-MWT) leads to decrease of the relative intensity of the peak ascribed to hydroxyl groups with increasing intensity of peaks in C-O-C region .The GO reduction procedures decrease the contribution of C=O moieties and aromatic carbon-carbon bonds, what is accompanied by increasing content of C-O moieties depending on the agent. This reduction provides different modifications in IR spectra recorded for the obtained samples. After the reduction with formaldehyde  [n, from REELS the values of interlayer distance, d, evaluated from XRD  , the valcosαout) . When defrom XRD  were con and XRD  are list and XRD  and REEL and XRD  are consUHV < GO-foil < GO-exp. The rGOs resulting from MWT exhibit the smallest number of flake layers for reducing conditions: CH2O-MWT < HCO2H-MWT < N2H4-MWT. The N2H4 reducing agent accompanied by MW and MWT procedures leads to increasing number of flake layers with no remarkable difference between MW and MWT treatments. In contrast, the CH2O reducing agent MWT procedure provides smaller number of flake layers in comparison to only CH2O reducing agent. Reduction using N2H4 of GO-exp provides larger number of flake layers in comparison to reduction of GO prepared from graphite.The GOs show the least number of flake layers, i.e., GO-driedXPS analyzes the intensity and binding energy (BE) values of photoelectrons from different atoms and their orbitals, where the intensity and BE are attributed to surface atomic concentration and chemical state of atom at the surface, respectively. The atomic content of elements present at the surface  was quantified from the survey XPS spectra accounting for an area under C 1s, O 1s, N, 1s, Na 1s, Si 2p, S 2p and Mn 2p photoelectron peaks after Tougaard background subtraction  applyingThe carbon and oxygen chemical forms and their content were investigated from fitting of C 1s and O 1s spectra after Tougaard background subtraction  using XP2 bonds, vacancy and C sp3 defects and carbon oxygen groups like hydroxyl (C-OH), epoxy (C-O-C), carbonyl (C=O) and carboxyl (C-OOH), whereas in O 1s spectra fitting, C-OH, C-O-C, C=O, C-OOH and adsorbed water. The values of BE in the fitting of C 1s and O 1s spectra were considered according to the literature data [The following chemical forms of C atom in C 1s spectrum were assumed in the fitting: C spure data ,53,54,55UHV  [MED) in XPS and AES and mean penetration depth (MPD) in REELS, are defined as an average depth normal to the surface from which the specified particles escape and for assumed single scattering of electrons can be evaluated from equations reported previously [ID (99%), 2.17 nm (MPD), REELS-bulk (2000 eV)—6.53 nm (ID (99%), 1.42 nm (MPD), C 1s-EELS XPS—9.72 nm (ID (99%), 2.11 nm (MED) and C KLL AES—3.32 nm (ID (99%), 0.72 nm (MED). Therefore, ID increases in the following order:Evaluation of the content of C speviously ,49. A mesly [ID) ,42. The eviously ,42. Accoeviously  and appl2/sp3 hybridized atoms are listed in 2/sp3 contributions evaluated from C 1s spectra in 3 content at the surface sensitivity of C KLL spectra is evaluated from parameter D, i.e., the energy distance between maximum and minimum intensity values of the first derivative of C KLL spectra, (2 (graphite) and C sp3 (diamond) standards from [REELS-surface < C KLL AES < REELS-bulk (2000 eV) < REELS-bulk (4000 eV) ≈ C 1s-EELS XPS, where the REELS-surface component provides information from the first outer layer of graphene. The percent contribution of REELS bulk and surface components of C spspectra, , Table 4rds from  as propords from .3 content at various surface information depths is compared in MED for C 1s-EELS XPS and REELS-bulk (4000 eV) is about 3–7 graphene layers, for C KLL spectra—1–2 graphene layers, respectively, whereas REELS-surface component reflects information from the outer layer. Discrepancies between C sp3 for first graphene layers (REELS-surface component and C KLL AES) and further layers (C 1s-EELS XPS and REELS-bulk component) result from non-homogeneous distribution of C sp3 and oxygen groups of the largest content at the flakes plane  analyses  GO samples prepared using a modified Hummers method  and (ii) rGOs prepared in aqueous solutions of N3 defect, oxygen groups and adsorbed water. The GO dried in UHV resulted in a smaller number of layers, larger number of vacancy and C sp3 defects and a smaller number of oxygen groups (except C-O-C) and adsorbed water than GO dried in atmosphere. GO-exp and GO-foil show larger number of flake layers, smaller number of defects and larger content of adsorbed water than GO-driedUHV. This would suggest that in UHV the interstitial water and weakly bonded C-OH, C-OOH and C=O groups are being removed, creating more defects than in GO-foil dried in the atmosphere, where water can dissociate forming additional oxygen groups. Samples showed different interlayer distance, flake diameter, number of vacancy and C sp2O, lead to increasing content of oxygen groups, resulting probably from water dissociation and/or removal of oxygen from epoxy group, leading to a smaller number of flake layers. In the case of a strong N2H4 reducing agent accompanied by microwaves , the content of oxygen groups is not modified due to microwave treatment; however, these procedures result in a larger number of flake layers. The rGOs prepared using MWT procedure showed the average number of layers in the following order: CH2O-MWT < HCO2H-MWT < N2H4-MWT. Number of layers in rGOs increases with decreasing content of vacancy, C sp3 defects, oxygen groups, water and flake diameter. This would suggest that MWT facilitates formation of oxygen groups at vacancies and provides more competitive penetration of reducing agent between the interstitial sites than via vacancies. The smaller number of flake layers show rGOs, where reduction of GOs oxygen groups between the interstitial sites is faster than those in the flake planes. This process depends on reducing agent, its reducing ability and dynamic interaction of MWT conditions with the treated material. Different reducing agents at MWT conditions resulted in various in-depth distribution of C sp3 at the surface and in the bulk, what depends on MW kinetic and thermal effect on the treated material modifying the rates of oxygen group decomposition.The MWT conditions result in formation of larger amount of vacancy defects and in the case of weaker reducing agent like CHThe microwave assisted methods of GO reduction, being more efficient, less energy and time consuming, may be utilized for a large scale graphene production.


We found that NR supplementation had no influence on body or organ weight, glucose metabolism or hepatic lipid accumulation, energy expenditure, or metabolic flexibility but increased mitochondrial respiration in soleus muscle in both mouse strains. Strain-dependent differences were detected for body and fat depot weight, fasting blood glucose, hepatic lipid accumulation, and energy expenditure. We conclude that, in mild obesity, NR supplementation does not alter metabolic phenotype in two commonly used laboratory mouse strains.Supplementation with precursors of NAD has been shown to prevent and reverse insulin resistance, mitochondrial dysfunction, and liver damage in mouse models of diet-induced obesity. We asked whether the beneficial effects of supplementation with the NAD precursor nicotinamide riboside (NR) are dependent on mouse strain. We compared the effects of NR supplementation on whole-body energy metabolism and mitochondrial function in mildly obese C57BL/6N and C57BL/6J mice, two commonly used strains to investigate metabolism. Male C57BL/6N and C57BL/6J mice were fed a high-fat diet (HFD) or standard chow with or without NR supplementation for 8 weeks. Body and organ weights, glucose tolerance, and metabolic parameters as well as mitochondrial O Repletion of NAD with vitamin B3 precursors or metabolites, mainly nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) has been used widely in experimental animal studies to counteract negative consequences of metabolic and mitochondrial disorders. Beneficial effects on energy metabolism and mitochondrial function were found in animal models of diet-induced obesity , mice were fed either a standard chow  or a high-fat diet  for 8 weeks. For NR supplementation, 3.0 mg/mL NR chloride (Chromadex Inc.) was dissolved in drinking water in light-protected bottles and changed every 3 days since this regimen was shown to lead to a stable increase in hepatic NAD  and MRC Harwell, UK (B6N Taconic). Mice were group-housed in a standard temperature (22°C) and humidity-controlled environment with 12 h light:12 h darkness cycle, except for indirect calorimetry experiments, where animals were single-housed for 72 h. Nesting material was provided and mice had 2 or used for mitochondrial respiration measurements. Genotyping was performed according to Mice were weighed weekly. Mice were sacrificed using cervical dislocation after exsanguination under isofluorane anesthesia. Liver, inguinal, and epididymal fat as well as skeletal muscle , soleus) were immediately frozen in liquid NExperiments were performed following a 5 h fast starting at ~08:00 h. Blood glucose was monitored using the Contour XT glucometer (Bayer) from samples collected at the distal tail vein. Following an initial blood glucose measurement, glucose (2 g/kg body weight) was injected intraperitoneally. Blood glucose was measured after 15, 30, 60, 90, and 120 min.Quantification of liver triglycerides was performed using a triglyceride quantification kit  according to the manufacturer’s instructions. One lobe (lobus hepatis sinister) was fixed in 4% paraformaldehyde and paraffin-embedded. Sections were stained with hematoxylin and images were taken at 20× magnification.n = 10 for each group) and chow-fed (n = 4 four for each group) were measured in a Phenomaster System . Briefly, animals were group-acclimatized for 24 h before isolation. On day 2 at 12:00 h, mice were removed from the home cages, weighed and their overall condition was checked, then placed into new cages in isolation. On the morning of day 3, mice were visually checked for signs of stress at 09:00 h and food/drink consumption was recorded. If mice were not eating and/or drinking (determined as less than 1 g of food or 1 mL of water), they were removed from the experiment (n = 1 B6N mouse). For the data collection phase, mice were then left undisturbed until 13:00 h on day 5, where they were removed. Food/drink consumption data were checked again and mice were weighed. If the mice had not eaten or drank and/or had lost more than 20% body weight since day 1, the data were excluded from the experiment (n = 2 B6N mice). The last 48 h were used for data analysis. ∆RER was calculated by subtracting the mean of the five highest RER values during the dark phase from the mean of the five lowest RER values during the light phase. Caloric consumption was calculated using the following values: HFD 5.24 kcal/g, chow 3.18 kcal/g.Energy expenditure, respiratory exchange ratio (RER), food and water intake of HFD-fed  or protein concentration (liver mitochondria).Mitochondrial respiration was measured by high-resolution respirometry . Liver mitochondria were isolated according toCitrate synthase (CS) activity in liver mitochondria and homogenized muscle samples was measured in a microplate format according toStrategy: our main aim was to check whether NR supplementation has a differential effect on B6J and B6N mice during HFD feeding. We first analyzed the effect of NR supplementation with supplementation (NR vs no NR) and mouse strain (B6J vs B6N) as factors in HFD-fed and chow-fed mice separately. Since we unexpectedly saw no effect of NR supplementation, but a difference in response to HFD feeding between mouse strains, we then tested for differences using mouse strain (B6J vs B6N) and diet (HFD vs chow) as factors and included mouse data regardless of whether or not mice were supplemented with NR. Tests for statistical significance were done using GraphPad Prism version 9  with ordinary two-way ANOVA followed by Tukey’s multiple comparisons test, except for the time course of body weight data , which wt-tests with Holm–Sidak correction were used for analyzing differences in time courses for energy expenditure and respiratory exchange ratio (RER). Energy expenditure and ∆RER were analyzed using linear regression and analysis of covariance (ANCOVA) between average absolute daily energy expenditure/∆RER and body weight  differences were marked by + for comparisons between chow and HFD, by * for comparisons between B6J and B6N mouse strains, and by # for comparisons between NR vs no NR supplementation.Multiple +P  < 0.05 B6J HFD vs chow-fed mice, *P  < 0.05 chow-fed B6J vs B6N). The liver weight of B6J chow-fed mice was higher than B6J HFD-fed mice (+P  < 0.05 B6J HFD vs chow-fed mice).Previous studies reported that NR supplementation counteracted negative effects of HFD-induced obesity, decreased fat depot size in HFD-fed mice  nor affe+P  < 0.05 HFD vs chow-fed mice). After 8 weeks, HFD-fed mice of both strains had higher fasting blood glucose levels than mice on chow feed, while HFD-fed B6J mice displayed increased fasting blood glucose levels compared to HFD-fed B6N . HFD feeding for 8 weeks , B and C+P  < 0.05 B6N HFD vs chow; *P  < 0.05 HFD-fed B6J vs B6N).HFD-induced obesity is associated with increased hepatic lipid accumulation. NR supplementation did not decrease lipid accumulation in any experimental group. B6N mice on HFD stored significantly more hepatic lipids than chow-fed B6N mice or HFD-fed B6J, while there was no significant difference between HFD and chow-fed B6J mice . Chow-fed B6J mice had higher body weights than B6N on the chow diet as opposed to HFD-fed B6J mice, which had lower body weights than HFD-fed B6N mice . The RER was higher in chow-fed than HFD-fed mice and indicated a preferential use of lipids after 8 weeks of HFD , C and D+P  < 0.05 for HFD vs chow-fed) and were more active as indicated by feeding frequency (+P  < 0.05 for HFD vs chow-fed). B6J mice drank significantly more water compared to B6N both on chow and on HFD during the night (*P  < 0.05 for B6J vs B6N) and also were more active (+P  < 0.05 for HFD vs chow-fed). Except for a borderline significantly higher caloric consumption in chow-fed vs HFD-fed B6J mice , there w#P  < 0.05 for NR vs no NR) or when respiration was uncoupled (#P  < 0.05 for NR vs no NR). Mitochondrial respiration of tibialis anterior (TA) muscle fibers  was found by indirect calorimetry  and subsequently increase mitochondrial respiration (In our hands, the beneficial effects of NR supplementation reported for HFD-induced obesity mouse models were not apparent. We only detected slight mouse-strain-dependent variation in the response to NR supplementation. In line with this, a recent study examining indirect calorimetry results from two large mouse projects revealed the institutional site as causing the biggest experimental variance (The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.Wellcome Trusthttp://dx.doi.org/10.13039/100010269 Senior Fellowship, grant number GGL-104612/Z/14/Z, an Early Career Grant from the Society for Endocrinologyhttp://dx.doi.org/10.13039/501100000382 and received support from the Research and Development Fund of the College of Medical and Dental Sciences, University of Birmingham, UK.This work was funded by the European Union, grant number 705869, a Conceptualization, A G, D J H and G G L; methodology, A G, D M C, L O, R S F, C L D, S H, D P L, D N, D J H.; formal analysis, A G; investigation, D M C, L O, R S F, C L D, S H, D P L; C E B, S R H; resources, G G L; writing – original draft preparation, A G; writing – review and editing, A G; visualization, A G; supervision, D J H and G G L; project administration, A G and G G L; funding acquisition, A G and G G L.


Microarray and qRT-PCR assays demonstrated that miRNA-205 was upregulated in tamoxifen resistance MCF-7/TAMR-1 (M/T) cells and M/T cell-derived exosomes (M/T-Exo). The M/T-Exo was internalized by human BC cells (BCCs), causing increased expression of miRNA-205 in BCCs. Coculturing with M/T-Exo promoted tamoxifen resistance, proliferation, migration, and invasion while suppressed apoptosis in recipient BCCs, which were associated with activating the caspase pathway and phosphorylating Akt. Luciferase reporter assays showed that miRNA-205 directly targeted E2F Transcription Factor 1 (E2F1) in BCCs. Furthermore, knockdown of miRNA-205 or overexpression of E2F1 reversed the roles of M/T-Exo in BCCs. In vivo experiments showed that the intratumoral injection of M/T-Exo caused greater tamoxifen resistance and larger tumor size relative to mice treated with miRNA-205-knockdown or E2F1-overexpressing BCCs. Together, the results suggest that exosomal miRNA-205 may promote tamoxifen resistance and tumorigenesis in BC through targeting E2F1 in vivo and in vitro.Breast cancer (BC) is a common malignant tumor in females. The challenge in treating BC is overcoming chemoresistance. Exosome-mediated transfer of miRNAs is a molecule-shuttle in intercellular communication. Thus, we aimed to investigate whether exosomal miRNA-205 could affect chemoresistance and tumorigenesis in recipient tumor cells and to elucidate the underlying mechanism  Breast cancer (BC) is the most common invasive malignancy among women worldwide, accounting for 31% of all female cancer types . CurrentAlthough the initial treatment has been demonstrated to be effective for most patients with BC, eventually, more aggressive tumors develop due to the resistance of tumor cells from radiotherapy or chemotherapy , 6. ThusExosomes are a group of extracellular vesicles (30–150 nm in size) , 11. GroMicroRNAs (miRNAs) are a class of small (21–24 nucleotides in length) non-coding RNAs . The miRE2F Transcription Factor 1 (E2F1), a transcription activator, can bind to DNA with dimerization partner (DP) proteins through the recognition site of E2 . It has in vitro and in vivo.Therefore, this study aimed to determine whether miRNA-205 can be transmitted via exosomes derived from chemoresistant breast cancer cells (BCCs) and the roles of exosomal miRNA-205 in the regulation of tamoxifen chemoresistance and tumorigenesis in BC Exosomes were first isolated from BCCs and M/T cells. The morphology of exosomes was identified by the transmission electron microscope which showed round-shape exosomes with bilayer membranes . Also, tThe microarray assay was used to determine the miRNA expression profile in M/T cells. As shown in Thus, to further investigate the molecular mechanism underlying exosomal miRNA-205-associated tamoxifen resistance in BCCs, bioinformatics tools, TargetScan  and StarTo investigate the functional correlation between miRNA-205 and E2F1 in tamoxifen resistance of BCCs, the BCCs were transfected with the miRNA-205 negative control inhibitor (NCi), miRNA-205 inhibitor (205i), or the combination of 205i and E2F1-expressing vector (E2F1). Next, the M/T-Exo with their respective treatments were added into the BCCs medium. The results showed that the mRNA and protein expressions of E2F1 decreased in M/T-Exo-treated BCCs transfected with NCi but was not affected in the other treatment groups  and 3E. Next, M/T-Exo was applied to treat BCCs that were treated with NCi, 205i, or the combination of 205i and E2F1. Next, the apoptotic level was assessed by flow cytometry assays which demonstrated that BCCs treated with NCi and M/T-Exo exhibited lower apoptotic levels compared to the other groups , along wBCCs treated with NCi and M/T-Exo showed higher levels of proliferation, migration, and invasion  and 5C, in vivo, nude mice were subcutaneously injected by BCCs treated with NCi, 205i, and E2F1, respectively. The results showed that the tumor of mice treated with NCi grew larger and faster than those of mice treated with 205i and E2F1, respectively  [In this study, exosomes secreted from tamoxifen resistance M/T cells expressed higher levels of miRNA-205. Exosomes are extracellular vesicles (30 to 100 nm in diameter) and are secreted by various cell types through fusion of vesicular bodies with plasma membranes . Exosomeg (PTEN) . Thus, eIn addition to chemoresistance, exosomes-based communications have been demonstrated to be an effective way to regulate cancer development and progression. Exosomal miRNA-1246 enhances cell proliferation and invasion in BC through cyclin-G2 (CCNG2) signaling . Long noAs a multifunctional factor, miRNA-205 has been reported to be involved in a variety of physiological and pathological processes, such as cell proliferation , angiogeTo further determine the mechanism underlying exosomal miRNA-205-induced chemoresistance in BCCs, both mechanical and functional experiments were performed to investigate the target gene of miRNA-205. The results revealed that miRNA-205 directly binds to E2F1 and overexpression of E2F1 could reverse the effect of miRNA-205 on chemoresistance and tumorigenesis in BCCs. In addition, E2F1, a cellular transcription, belongs to the E2F family . Also, EAll subjects recruited in this study were informed before inclusion and written consents were given. This study was reviewed and approved by the Ethics Committee of Cangzhou Central Hospital (2017R-K1174). All animal involved experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of Cangzhou Central Hospital (CZ-208-R8743) and were performed based on the guidelines and regulation of the Management of Laboratory Animals published by the Ministry of Science and Technology of the People’s Republic of China.Breast tumor samples were collected from two patients who were diagnosed with BC and undergone BC surgical resection at the Cangzhou Central Hospital in April 2017 (35 and 46 years old). The detailed information of patients was summarized in 6) were placed in the medium supplemented with 10% exosome-free FBS for 48 hours at 37°C in a humidified atmosphere with 5% CO2. Next, to collect the exosomes, 40 ml of conditioned mediums were taken from each cell line and the ExoQuick-TC Kit  was used. The morphology and diameter size of the exosomes were assessed by Morgagni 268D transmission electron microscopy  according to a protocol previously described [Exosome-free fetal bovine serum (FBS)  was collected through 120,000 × g ultracentrifugation at 4°C for 6 hours. The BCCs and M/T cells  corrected p <0.05 and |log2 fold-change (FC)|>2.Total RNA was extracted from BCCs and M/T cells using the TRIzol reagent . The concentration and quality of total RNA were examined by NanoDrop™ 2000 . The miRNA microarray and data analysis were performed as previously described , 67. Brilization . The avelization . The t-tThe coculture system of the exosome donor and recipient cells were modified as previously described . The M/T6/well) were transfected with 100 nM of 205i or NCi using the Lipofectamine™ 3000 Reagent  according to the manufacturer’s instructions. Small interfering RNA (siRNA) specific for E2F1 (siE2F1)  and E2F1-expressing plasmids  were obtained from GenePharma Co., Ltd . Co-transfection of 50 nM 205i and 50 nM siE2F1 was performed according to the manufacturer’s instructions using the Lipofectamine™ 3000 Reagent . After 24 hours of transfection, transfected cells were used for subsequent experiments.The miRNA-205 inhibitor (205i) and the corresponding negative control (NCi) were obtained from GenePharma Co., Ltd . The BCCs  according to the manufacturer’s instructions. After 24 hours of transfection, relative luciferase activities were quantified using the Luciferase Reporter Assay Substrate Kit .−ΔΔCT method [Total RNA was extracted from exosomes, cells, and tumor tissues of xenograft mice using the TRIzol reagent . The qRT-PCR reactions were performed as previously described . The relT method . Table 2Total protein isolation of exosomes, cells, and tumor tissues of xenograft mice and western blots were performed as previously described . The priThe Annexin V-APC Assay Kit  and flow cytometry assay were used to quantify cell apoptosis according to the manufacturer’s instructions.Evaluation for cell proliferation, colony formation, migration, and invasion were performed as previously described . Cell pr6) in 100 μl PBS and Matrigel were subcutaneously injected into each mouse. Mice were randomly divided into four groups (n = 3): mice in group 1 and 2 were injected with NCi-treated cells, mice in group 3 were injected with 205i-treated cells, and mice in group 4 were injected with E2F1-treated cells. Tumor growth was monitored daily and tumor volume (V) was evaluated by measuring the length (L) and width (W) with a caliper and calculated using the formula: V = (L × W2) × 0.5. When the tumor tissue volume was around 50 mm3, the mice in group 2, 3, and 4 were intratumorally treated with M/T-Exo (40 μg) three times a week. Once the tumor tissue volume was around 100 mm3, all mice were treated with tamoxifen (3 mg/kg) through the tail vein three times a week for two weeks and then were sacrificed. The tumor tissues were isolated and immediately stored in –80°C for subsequent experiments.Twelve male BALB/c nude mice (6 weeks old) were obtained from Jackson Labs . Transfected BCCs  with post-hoc test was applied to analyze statistical differences between three or more experimental groups. P < 0.05 was considered statistically significant.In this study, data were presented as means ± SD from at least 3 independent replicates. The statistical analyses were completed using the SPSS 17.0 software . Two-tailed Student’s in vivo and in vitro and that exosomes-mediated transfer of functioning molecules may provide novel insight into developing therapeutic strategies for BC.In conclusion, the results suggest that exosomal miRNA-205 could promote tamoxifen resistance and tumorigenesis in BC through targeting E2F1


The major histocompatibility complex (MHC) in humans includes three classical class I loci , which are important biomarkers for the transplantation of organs and hematopoietic stem cells. In the MHC, polymorphism is known to be extremely high while interlocus recombination is rare. We report a rare interlocus recombination between HLA-A and HLA-H, which was analyzed using next generation sequencing and nanopore sequencing.In the sample, the genotypes of HLA-A, B, C, DRB1, and DQB1 were firstly determined using the methods of sequence-specific primer, sequence-specific oligonucleotide, Sanger’s sequencing, and NGS; however, HLA-A could not be phased. Nanopore sequencing was finally utilized to distinguish the sequence of the novel allele.Finally, the novel HLA-A*11:335 allele was identified as an interlocus recombination involving HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles; this was mainly achieved by nanopore sequencing.The identification of the interlocus recombination indicated that nanopore sequencing can be helpful in the characterization of novel alleles with complex rearrangements. Interlocus recombination has been identified as one of the mechanisms involved in the generation of novel HLA alleles.The online version contains supplementary material available at 10.1186/s12920-022-01176-1. HLA-A*11 allele, A*11:335, which was identified as an interlocus recombination involving the HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles in a Chinese bone marrow donor and analyzed the consequences of this recombination. This interlocus recombination was mainly characterized by nanopore sequencing.The main function of human classical class I loci  is to display intracellularly digested foreign peptides (at antigen recognition site) to CD8 T cells . For claA total of 2964 specimens were sampled (approximately 2%) from the database of recruited volunteers of the China Marrow Donor Program in 2017 and subsequently genotyped for HLA-A, B, C, DRB1, and DQB1. The DNA sample 17ZZ2298 was originally extracted from a volunteer’s peripheral blood by the BGI laboratory, which was a cooperating partner of the China Marrow Donor Program. HLA typing for A, B, C, DRB1, and DQB1 was firstly performed with the BGI Next Generation Sequencing Typing method-RCHSBT  [HLA typing of the sample 17ZZ2298 was performed for a second time using the Sanger’s sequencing method  to examine the results acquired from RCHSBT. Because the result of HLA-A typing was A*01:01/11:126, which was different from that obtained by RCHSBT (A*01:01/11:01), the sample was typed for a third time using the sequence-specific oligonucleotide (SSO method) . The result obtained with the SSO method was A*01:01/11:01, which was the same as that obtained using the RCHSBT method. The sample was further typed using Miseq based sequencing (Onelambda) and the assignment was A*01:01/11:126, with the following system comments: “Warning: mismatch in an intron, two or more variants cannot be phased, Locus has a high background position in exon.” The sample was further typed by next generation sequencing (NGS) using commercially available reagents  and a MiniSeq system , and MinION based nanopore sequencing . Data were analyzed using the NGSengine software program (GenDX).The sequence of the novel allele (mismatched area) was blasted in the IMGT/HLA database using the “BlastN” tool.http://bioinf.cs.ucl.ac.uk/psipred/). The amino acid sequences of exon 5 of HLA-A*11:335 and HLA-A*11:01:01:01 were entered into the online tool and analyzed.The effects of the six missense mutations in exon 5 on the function of the transmembrane domain were analyzed and predicted with the PSIPRED online tool  the assignment for HLA-A was A*01:01/11:126. The sample was then reanalyzed by the SSO method and Miseq sequencing-based typing (Onelambda). The assignment determined by these methods was HLA-A*01:01/11:01 and HLA-A*01:01/11:126, respectively. However, the Miseq assignment had the following system comments: “Warning: mismatch in an intron, two or more variants cannot be phased.” Indicating the possibility that HLA-A*01:01/11:126 was not the correct assignment. The only difference between HLA-A*11:01:01:01 and HLA-A*11:126 was at c.874A > G in exon 4 . The data showed that there was a new allele, but exon 3 and exon 4 could not be phased with the MiSeq data. Therefore, the Miseq reads were further analyzed together with a low number of MiniON reads. The recommended genotype was HLA-A*01:01:01:01/A*11:126 . It was suggested that the sample contained a new HLA-A*11 allele (HLA-A*11:335), which was the result of the interlocus genomic exchange of HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18. The distance between HLA-H and HLA-A on chromosome 6 is approximately 50 kb. The alignment of the genomic sequence of HLA-A*11:01:01:01 with A*11:126, A*11:335, H*02:07, H*02:14, and H*02:18 is shown in Fig. The sequence of region 1824–2437  was then blasted in the IMGT/HLA database. As shown in Table HLA-A*11:335 differs from HLA-A*11:01:01:01 by 10 nucleotide substitutions, which resulted in three synonymous mutations and six missense mutations, mainly in exon 5. Exon 5 encodes the transmembrane domain of HLA-A. We analyzed the effects of the six missense mutations on the property of the transmembrane domain using the PSIPRED online tool (http://bioinf.cs.ucl.ac.uk/psipred/). The results showed that although six missense (three in the transmembrane domain) mutations were produced as a result of interlocus recombination between HLA-A and HLA-H, these mutations did not create destructive effects on the helix structure of the transmembrane domain  and to the IPD-IMGT/HLA Database [A number of authors , 11 haveA*11 was common (frequency: 23.203%) in Chinese volunteers [A*11:335) was determined.Based on our latest data, 191 alleles of the A locus were identified and lunteers . In thisThe sequence of region 1824–2437 was then blasted in the IMGT/HLA database and the results showed that 612/612 bases matched exactly with HLA-H*02:07/14/18  in A*11:01:01:01/126 before 2763 and a mutation (T) in H*02:07/14/18 after 2783, respectively, compared with A*11:335. The upper crossover region was not easily determined; several possibilities for this may exist. It was certain that the crossover point was after 1810. Four possible allele pairs and their upper crossover regions were: (a) HLA-A*11:01:01:01 versus HLA-H*02:07/14 in 1811–2148; (b) HLA-A*11:126 versus HLA-H*02:07/14 in 1811–2211; (c) HLA-A*11:01:01:01 versus HLA-H*02:18 in 1896–2148; (d) HLA-A*11:126 versus HLA-H*02:18 in 1896–2211. The exact panel of the interlocus recombination may be resolved by sequencing of the HLA region of the donor’s parents. Detailed information is listed in the Additional file A*11:335 differed from HLA-A*11:01:01:01 by 10 nucleotide substitutions, resulting in three synonymous mutations and six missense mutations in exon 5  was the common sequence of the aligned alleles, which could cover the total length of all the sequences. “Red nucleotide” means the nucleotide was a mutation at this position in the aligned sequences.