Membranes utilizing P-S, G-S, and EG-S were characterized in terms of the fouling amount on the membrane surface and fouling roughness. Electrostatic forces of EG-S provided 70% less and 90% smoother fouling on the membrane surface, leading to an only 14% less water flux reduction after 5 h of fouling. The importance of nanomaterial blending and polarization was successfully demonstrated herein. In membrane processes, a spacer is known to play a key role in the mitigation of membrane fouling. In this study, the effect of electric polarization on a graphene-blended polymer spacer , PLA) for organic fouling on membrane surfaces was investigated. A pristine PLA spacer (P-S), a graphene-blended spacer (G-S), and an electrically polarized graphene-blended spacer (EG-S) were successfully fabricated by 3D printing. Organic fouling tests were conducted by the 5-h filtration of CaCl Membrane treatment methods have been playing a crucial role in providing potable water . As a reVarious types of membrane fouling include crystalline fouling, organic fouling, particle fouling, colloidal fouling, and biofouling ,11. AmonFO membranes demonstrate immense potential and extensive applications to reduce the total energy consumption during desalination and waste water treatment. As a result of benefitting from the draw solution with a high osmotic concentration, FO systems can filter water with foulants without the use of any external pressure through high-pressure pumps . As FO iVarious studies on feed spacers have focused on the spacer shape ,20. Prev3 specific gravity, 26.4 MPa tensile strength, 2.3 GPa tensile modulus and elongation break at 4%, and graphene-blended PLA spacers were fabricated by using conductive graphene blended PLA filaments which has 1.11 gm/cm3 specific gravity, 53 MPa tensile strength, 2.9 GPa tensile modulus and elongation break at 5.1%  [Three fabricated spacers prepared by 3D printing after computer-aided design (CAD) modelling were used for experiments. One PLA and two graphene-blended PLA spacers were fabricated by using the same structural parameters  . All spaNY, USA) . Later, NY, USA) . In this2 was utilized in the continuous filtration mode at a cross flow rate of 200 cm3/s for the draw and feed sides  and 1 mM of CaCl2  were dissolved in DI water [2 was used as the Ca2+ source to bind alginate molecules. Filtration was conducted for 5 h after flux stabilization was performed for 1 h. Data obtained by the weight change in the feed solution in pure flux and fouling experiments, as well as feed conductivity for only pure flux experiments, were recorded every minute by a connected computer. From these recorded values, the water flux, Jw, and the reverse solute flux, Js, were calculated by using the following formulas [Flux measurements were conducted for pure water flux/reverse solute flux measurements and fouling-based flux reduction. For the performance investigation of the spacers, commercial membranes  were used, which were extracted from a commercial membrane module. An engineered osmosis system  with an ed sides . For theDI water . Here, Cformulas ,39:(1)Jww is the water flux, V is the volume of permeated water (L), Am is the effective membrane area (m2), and Δt is the permeation time (min). The reverse solute flux was calculated from the electrical feed conductivity per minute [t is the concentration (g/L) at time t, Vt is the volume (L) of the feed solution at time t, C0 is the initial concentration (g/L), and V0 the initial volume (L) of the feed solution. The resulting values were converted into Lm−2h−1 (LMH) for water flux and gm−2h−1 (gMH) for reverse solute flux.In the first formula, Jr minute . In the Surface roughness of fouled membranes, foulant volume on membranes, and visual topographic images of fouled membranes were obtained by a Surface Nano-Profiler  measurements. 3D measurements were performed with an x–y scanning distance of 500 µm and a scanning speed of 50 µm/s at 10 steps for each sample. These measurements were repeated 10 times for each sample. Notably, characterization was performed for surface areas that were not in contact with the spacers.The amounts of foulant adsorbed on the G-S and EG-S spacers were investigated by dipping the spacers in 200 mL of an alginate solution for 12 h (same solution as mentioned above) at room temperature. Spacer weights were measured before and after the dipping process (after drying in an oven at 40 °C for 2 h). Later, spacers were cleaned in an ultrasonicator  by dipping in DI water. After cleaning in an oven at 40 °C for 2 h, the spacer weight was measured again. Finally, the amounts of foulant adsorbed on G-S and EG-S spacers (before and after cleaning) were obtained. Notably, the resistance of spacers against heating and sonication was repetitively tested by using non-fouled spacers. G-S and EG-S spacers did not exhibit any deformation, while the P-S spacer exhibited weight loss and structural deformation. Therefore, the P-S spacer is not included in this investigation.P-S and two G-S spacers were successfully printed, and one of the G-S spacers was polarized to obtain an electrically polarized EG-S spacer. All three samples were examined for pure water flux and reverse solute flux measurements. Flux performances of all three spacers were relatively similar. More specifically, P-S, G-S, and EG-S exhibited water flux of 13 ± 0.85 LMH, 12.8 ± 0.55, and 14.4 ± 0.69 LMH, respectively . In addiAdditional tests were performed to investigate the organic fouling performance of P-S, G-S, and EG-S spacers. After 5 h of alginate-based fouling of membrane spacers, the fouled membranes with P-S or G-S exhibited a water flux reduction of 31% or 33%, respectively . This ra2 area, P-S, G-S, and EG-S membranes exhibited 12,685 ± 759 µm3, 12,391 ± 848 µm3, and 3570 ± 824 µm3 foulant, respectively, from an average of 10 measurements for each membrane  . In here2+ ions which are binding alginate molecules. Regarding to this, foulant adsorption test G-S and EG-S also showed that EG-S can collect 55% more foulant on itself than G-S, and it was also investigated that this additional amount of foulant collected by the effect of electric polarization is cleanable. However, this point should be extensively studied further in another research. For future studies, high-pressure membrane systems such as NF or RO also should be considered by utilizing various organic foulants such as humic acid or bovine serum albumin.In this article, the performance of electrically polarized graphene-blended poly(lactic acid) spacer (EG-S) was analyzed for alginate fouling. Compared to P-S and G-S, EG-S exhibited 70% less fouling on the membrane surface with a smoother fouling layer. Therefore, only 14% water flux reduction ratio was observed for the membrane of EG-S, while it was 31% or 33% for the membranes of P-S or G-S, respectively. This EG-S performance was attributed to the electrostatic manipulation of Ca-alginate bridges and Ca


KCNQ1 genetic variants associated with type 1 long QT or Romano-Ward syndrome, most frequently inherited in an autosomal dominant fashion, although recessive forms have been reported. Particularly in the case of missense variants, functional studies of mutants are of aid to establish variant pathogenicity and to understand the mechanistic basis of disease. Two compound heterozygous KCNQ1 mutations (p.A300T and p.P535T) were previously found in a child who suffered sudden death. To provide further insight into the clinical significance and basis for pathogenicity of these variants, different combinations of wildtype, A300T and P535T alleles were co-expressed with the accessory β-subunit minK in HEK293 cells, to analyze colocalization with the plasma membrane and some biophysical phenotypes of homo and heterotetrameric channels using the patch-clamp technique. A300T homotetrameric channels showed left-shifted activation V1/2 as previously observed in Xenopus oocytes, decreased maximum conductance density, slow rise-time300ms, and a characteristic use-dependent response. A300T slow rise-time300ms and use-dependent response behaved as dominant biophysical traits for all allele combinations. The P535T variant significantly decreased maximum conductance density and Kv7.1-minK-plasma membrane colocalization. P535T/A300T heterotetrameric channels showed decreased colocalization with plasma membrane, slow rise-time300ms and the A300T characteristic use-dependent response. While A300T left shifted activation voltage dependence behaved as a recessive trait when co-expressed with WT alleles, it was dominant when co-expressed with P535T alleles.Next Generation Sequencing has identified many Conclusions: The combination of P535T/A300T channel biophysical properties is compatible with recessive Romano Ward syndrome. Further analysis of other biophysical traits may identify other mechanisms involved in the pathophysiology of this disease. KCNQ1 gene and accessory β minK subunits encoded by KCNE1, with variable stoichiometry (1:4–4:4) . These patients are susceptible to malignant cardiac arrhythmia, which may cause syncope, seizures, and sudden death, frequently in young and/or apparently healthy individuals  largely contributes to shape the repolarization phase of the ventricular action potential. IKs results from the co-assembly of the Kv7.1 channel complex, consisting of four pore-forming α subunits encoded by the 1:4–4:4) . Heterozividuals . Homozygdeafness . Patientsubgroup .KCNQ1 genetic variants associated with type 1 LQTS. However, particularly in the case of missense variants, establishing pathogenicity remains challenging. The American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) published and recently updated guidelines to standardize the interpretation of genetic variants , suggesting recessive Romano-Ward syndrome  tagged with GFP and KCNE1 (NM_001127670) tagged with RFP plasmids were acquired from Origene . The A300T and P535T mutations were cloned in the KCNQ1 plasmid using QuickChange II XL Site-Directed Mutagenesis Kit  following manufacturer's instructions. Mutant primers were designed using QuikChange Primer Design tool: A300T-FW: 5′-ccaccacagcgcatccgtgtagctgccgaactc-3′; A300T-RV: 5′-gagttcggcagctacacggatgcgctgtggtgg-3′; P535T-FW: 5′-ccgcacatcgtaagtcttccgcgcttgct-3′, and P535T-RV: 5′-agcaagcgcggaagacttacgatgtgcgg-3′. The presence of the mutations was confirmed by Sanger sequencing.2 atmosphere at 37°C. Cells at 60–80% of confluence were used to transiently transfect both channel complex subunits (KCNQ1 and KCNE1) in a 1:1 ratio, using 1.5 μg of each construct. Transfection was made with Lipofectamine™ LTX Reagent with PLUS™ Reagent  according to manufacturer's instructions. Twenty-four hours after transfection, cells were seeded on poly-D-lysine-coated glass coverslips at 2 × 104 cells. Electrophysiological recordings were performed 2 h after seeded to ensure adhesion to coverslip.Human embryonic kidney cells (HEK293) were kindly provided by Dr. Ricardo Félix Grijalva. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS , 100 U/ml penicillin and 100 μg/ml streptomycin , in a humidified 5% CO2, 1 MgCl2, 0.7 NaH2PO4, and 10 HEPES; plus ~6.5 mM NaOH to bring pH to 7.4 and osmolality to ~295 mmol/kg. The pipette  solution was a conventional KCl solution: (in mM) 135 KCl, 7 NaCl, 0.1 CaCl2, 2 MgCl2, 3 Na2ATP, 10 EGTA, and 10 HEPES; plus ~33 mM KOH to bring pH to 7.3 and osmolality to ~300 mmol/kg. The patch clamp amplifier Multiclamp-700B and D-A/A-D converter Digidata 1550A  were controlled by pClamp 10.5 (Molecular Devices). Capacitive currents were electronically nulled. Series resistances ranged from 3 to 15 MΩ  and were compensated 76 ± 0.4% for a mean residual value of ~1.45 MΩ. Potentials were corrected for a liquid junction potential of −0.5 mV, calculated with JPCalc software  with 3–5 MΩ resistance in standard solutions. All reagents were purchased from Sigma Aldrich  unless otherwise indicated. In all experiments, the external solution was (in mM) 145 NaCl, 5 KCl, 1.3 CaClsoftware  as impleVoltage dependence of whole-cell currents was quantified by constructing activation (conductance density-voltage) curves from data collected using a voltage protocol consisting of an iterated series of 5.5 s test steps from a holding potential of −80.5 to 99.5 mV, that activates the slow outwardly rectifying K current (IKs). Steady-state conductances of IKs were calculated from tail currents at −40.5 mV, divided by driving force , mean across all cells, plotted against the test step voltage, and fitted with a Boltzmann function (Equation 1).1/2 is the voltage corresponding to half-maximal activation, and S is the voltage corresponding to an e-fold increase in G(V). Curve-fitting and statistical analyses were performed with OriginPro software . Parameters of curve fits  were compared for all experimental series, number of cells per group varied from 6 to 14. Gmax/Cm represents the maximum conductance density.where G(V) is conductance at voltage V, Gmin, and Gmax are minimum and maximum conductances, V300ms), within the physiological human ventricular action potential duration range.Rise time refers to the time required for a signal to change from a given low value to a given high value. Here, these values were 10 and 90% of the step height, as typically applied. To compare current activation time courses (kinetics) of different homo and heterotetrameric channels, rise time of currents activated from 0 to 100 mV steps was measured during the first 300 ms  following the manufacturer's protocol, and were observed with an LSM 780 NLO confocal microscope . All images are representative of at least three independent experiments.To investigate the colocalization of Kv7.1 WT, A300T, and P535T homo and heterotetrameric channels with minK and the plasma membrane, HEK293 cells transiently transfected with KCNQ1 and KCNE1 plasmids were tagged with GFP and RFP, respectively. Only cells expressing both GFP and RFP were selected for image analysis. Colocalization of the three signals  was quantified using the threshold overlap score (TOS), where TOS = 1 indicates colocalization, TOS = 0 indicates non-colocalization and TOS = −1 indicates anti-colocalization (https://imagej.net/Fiji) and EZColocalization plugin  was lower than WT channels , but not significantly different from P535T/WT channels  compared with WT homotetrameric  and A300T/WT heterotetrameric channels  (Voltage dependence of the A300T homotetrameric channel was significantly left-shifted (V7.64E-4) ; Table 17.64E-4) .1/2: 13.96 ± 2.31 mV, n = 14) compared with WT  and P535T channels ; but significantly more positive than A300T channels  (The activation of P535T/A300T channels was significantly left-shifted (V= 0.013) ; Table 1300ms of currents from A300T-containing channels were significantly slower than those from WT, P535T, and P535T/WT channels , followed by P535T/A300T  and by A300T/WT heterotetrameric currents ; however, only differences between A300T homotetrameric and A300T/WT heterotetrameric channels reached statistical significance  compared with WT, A300T, and A300T/WT channels. Second, for Kv7.1-plasma membrane (Kv7.1-Mem) colocalization, P535T channels clearly had the lowest score, being significantly lower than all other homo and heterotetrameric channels (p < 0.02). P535T/WT channels showed an intermediate colocalization score between WT and P535T channels, being significantly lower than the WT and A300T channels. Third, TOS comparisons of Kv7.1 and minK subunit colocalization (Kv7.1-minK) showed that all channels containing P535T monomers  were slightly lower than WT channels, however differences were not statistically significant. Finally, minK subunit-plasma membrane colocalization scores (minK-Mem) were highest for WT and A300T channels and lowest for P535T channels. Only comparisons between P535T and WT or A300T channels showed statistically significant differences (Our previous Kv7.1-A300T and -P535T protein model predicted defective trafficking of the Kv7.1-P535T potassium channel . We usedferences .KCNQ1 mutations (P535T/A300T). The P535T mutation was initially classified as of unknown clinical significance, and although the electrophysiological properties of A300T channels in Xenopus oocytes showed clear anomalies, interpretations of these anomalies and the clinical implications of the A300T mutation have been inconsistent  values. We thus further characterized the biophysical properties of A300T and P535T homo and heterotetrameric channels, analyzing prototypical activation curve parameters, current density, rise-time300ms, and use-dependent responses, in an effort to gain further insight into how these mutations affect the IKs and may cause LQTS.Remarkably, it has been observed that mutations that are dysfunctional at the molecular level may not cause clinical disease, and alternatively, some erations . In the KCNQ1-A300T mutation (rs120074187) is located at the pore domain, in the P-loop between S5 and S6 transmembrane domains, a topological location with high probability of pathogenicity . Post-translational processing, plasma membrane composition, and multimeric protein assembly in Xenopus oocytes can be different from mammalian cells . This analysis revealed an A300T dominant trait described here for the first time. We observed that currents from all A300T-containing channels had significantly slower activation than WT and P535T homo and heterotetrameric channels (A previous study in s faster . In the channels . Since schannels . The latchannels .https://gnomad.broadinstitute.org/).Summarizing, A300T causes a recessive, increased function biophysical trait (left-shifted activation voltage), concurrently with two dominant, decreased function biophysical traits (slow 300 ms activation and use-dependent response), likely resulting in a mild overall effect. It was first described as causal of a recessive form of Romano Ward in a homozygous child with normal hearing . To datein silico model predicted that P535T disrupts a calmodulin binding site by steric hindrance, most likely causing co-assembly and/or trafficking defects. P535T is localized within the C-terminus domain  (Because an undiagnosed child with sudden death was found to be compound heterozygous for T/A300T) , we func1/2 values, P535T/A300T channels showed intermediate activation V1/2 values, with a left-shift of 16.3 mV (p = 0.017) compared with P535T channels, and a right-shift of 13.8 mV compared with A300T (p = 0.013). Because P535T channels showed significantly lower colocalization scores with membrane than WT and A300T channels, it is likely that in a compound heterozygous state, a lower proportion of P535T and higher proportions of P535T/A300T and A300T channels will be assembled at the plasma membrane. This is compatible with the dominance of left-shifted activation voltage only in the compound heterozygous state. Finally, while P535T channels had significantly lower Gmax/Cm compared with WT channels, Gmax/Cm of P535T/A300T channels was also lower, although the difference did not reach statistical significance. These characteristics would most likely lead to abnormal repolarization and low possibilities of functional recovery since no Kv7.1-WT monomers can compensate the effects of the mutations.While A300T left shifted activation voltage dependence behaved as a recessive trait when co-expressed with WT alleles, it was dominant when co-expressed with P535T alleles. Although A300T/WT and WT channels showed similar activation VAlthough HEK293 cells have been widely used as host for heterologous expression and functional characterization of ion channels, they do not resemble the native cardiomyocyte environment where other ion channels and intracellular molecules interact to generate action potentials. For this reason, we cannot rule out that ion channels in their native environment are subject to regulation and interactions not considered in our model. Moreover, characterization of other yet unexplored biophysical traits such as response to PKA activators simulating adrenergic stimulation will provide further insight to better understand the mechanisms by which genetic mutations cause disease, and need to be performed in future studies.300ms, and a characteristic use-dependent response) that behaved as dominant traits. The A300T left-shifted activation V1/2 previously observed in Xenopus oocytes behaved as a recessive trait when co-expressed with WT alleles, but as a dominant trait when co-expressed with P535T alleles. The P535T variant significantly decreased maximum conductance density and Kv7.1-minK-plasma membrane colocalization, although how the P535T mutation affects trafficking remains to be elucidated. A lower density of P535T Kv7.1 monomers at the plasma membrane is compatible with the lower current density of P535T channels and with the dominance of A300T left shift voltage activation observed only when combined with P535T. The biophysical properties of P535T/A300T IKs are compatible with recessive Romano-Ward syndrome. Further analysis of other biophysical traits may identify other mechanisms involved in the pathophysiology of this disease.Co-expression of different combinations of WT, A300T, and P535T Kv7.1 with minK in mammalian cells revealed two previously undescribed biophysical phenotypes of the A300T mutation .The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: TV-M and AC conceptualized and designed the study. TV-M and AG-G designed the experimental approach and drafted the manuscript. AG-G performed all experiments and analyzed the data. MD-P performed confocal experiments. OL-R contributed to the data analysis and comments to the manuscript. LJ-A, JG-C, and OZ-G performed the experiments. MD-P, LJ-A, PI, and AC contributed to the manuscript. All authors contributed to the article and approved the submitted version.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.


RANKL gene leads to increased bone resorption and can influence the incidence of osteoporosis. Although much has been learned about the intracellular signals activated by RANKL/RANK complex, significantly less is known about the molecular mechanisms of regulation of RANKL expression. Here, we report on the structure of an unprecedented DNA G-quadruplex, well-known secondary structure-mediated gene expression regulator, formed by a G-rich sequence found in the regulatory region of a RANKL gene. Solution-state NMR structural study reveals the formation of a three-layered parallel-type G-quadruplex characterized by an unique features, including a G-A bulge. Although a guanine within a G-tract occupies syn glycosidic conformation, bulge-forming residues arrange in a pseudo-loop conformation to facilitate partial 5/6-ring stacking, typical of G-quadruplex structures with parallel G-tracts orientation. Such distinctive structural features protruding from the core of the structure can represent a novel platform for design of highly specific ligands with anti-osteoporotic function. Additionally, our study suggests that the expression of RANKL gene may be regulated by putative folding of its G-rich region into non-B-DNA structure(s).Bone remodeling is a fine-tuned process principally regulated by a cascade triggered by interaction of receptor activator of NF-κB (RANK) and RANK ligand (RANKL). Excessive activity of the  DNA can adopt various structures outside the double stranded margins of classical Watson-Crick geometries, which can interactively regulate biological processes such as replication, transcription, translation, and splicing ,3,4,5,6.Pu24 structure from the human c-myc promoter, where a discontinuous G-strand is formed by a guanine from the 3′-end and two guanines from one of the middle G-tracts [c-kit87up sequence from the human c-kit promoter, where two guanines from the 3′-G-tract connect into a discontinuous G-strand with an isolated non-G-tract guanine from the middle of the sequence [The core of the G-quadruplex structure is composed of stacked G-quartets, Hoogsteen hydrogen-bonded planes of four guanines, which are connected by short random loop-forming sequences in different orientations. Due to various possible options of G-quartet geometries and several loop combinations, G-quadruplexes are considered to be a very polymorphic structural family ,22,23,24G-tracts . Similarsequence . Additiosequence ,28,29,30sequence . Howeversequence ,28,29,30sequence ,28, cytoRANKL activity plays an important role in combat against osteoporosis. Thus, next to direct inhibition of RANKL/RANK interaction, influencing the RANKL expression could represent a promising way in a treatment of osteoporosis. One of the recently introduced ways to reduce the amount of RANKL protein is siRNA-mediated knockdown. Injections of RANKL siRNA-loaded virus-like-particles (VLPs) demonstrated a successful decrease in RANKL mRNA and protein levels in rats [Pathogenic conditions that affect the human skeleton, such as osteoporosis, disrupt balanced bone remodeling by accelerating differentiation and maturation of osteoclasts. The key regulator of osteoclast’s activity is receptor activator of NF-κB (RANK) that interacts with RANK ligand (RANKL) formed by osteoblasts ,32,33,34 in rats ,39. A 35RANKL gene (designated RANwt), characterized by three GGG tracts and one G-tract with four guanines [RANKL gene.Additionally, gene expression can be notably influenced by the formation of G-quadruplexes in the regulatory regions of a gene ,16,17,18guanines . RANwt iguanines . UnpreceRANKL gene expression. It has been shown before that different G-quadruplex structures can affect the gene transcription differently, revealing the importance of individual conformation for function [pseudo-loop arrangement, while adjacent guanine G16 resides in syn glycosidic conformation. RAN1* structure highlights the potential role of non-G-tract guanines on G-quadruplex folding, structure, and, furthermore, function. At the same time, it offers novel insights into exciting structural elements that can be used for the development of highly selective ligands that may serve as an innovative approach in osteoporosis treatment in the future.The ability of RANwt to form other G-quadruplexes, different from RAN4, indicates possible topology-dependent regulation of function . Since sfunction ,42. Ther1H NMR spectrum of RANwt shows several weak, overlapping signals, indicating formation of multiple G-quadruplex structures in the presence of KCl  indicates that the latter belongs to the unfolded form of the investigated oligonucleotide.Although a single set of 12 signals in the imino region of 1D bout 40% b. Conseqbout 40% . By intrbout 40% . The redectively b. Additi species . A17-to- species . Notewor species . Preserv15N-edited HSQC experiments acquired on residue-specific 6% 15N/13C-isotopically labeled oligonucleotides . Concurrent observation of these connectivities suggest that H8 and H1′ of G16 are both oriented towards sugar moiety of G13, which is only possible if G16 is in syn glycosidic conformation  topology ,23 Figuc. Althouguanines ,23, inten of G16 . This obormation .High-resolution structure of RAN1* revealed unprecedented structural details of the G-quadruplex with a G-A bulge. Structural calculations were conducted based on 630 NOE-derived distance restraints, along with 24 hydrogen bond, 37 torsion angle, and 36 planarity restraints using restrained simulated annealing protocol . Resultsanti glycosidic conformation, while G16 is in syn conformation , one wide (P-P distance 19.4 Å) and two medium grooves -3′ alone, allows for formation of the G-quadruplex core similar to RAN1* , no signals characteristic for RAN1* were observed. This indicates that ∆G14A15 folds into G-quadruplex structures distinct from topology of the parent RAN1*. Moreover, the detrimental effects of G14 and/or A15 deletions on the RAN1* G-quadruplex structure suggest that pseudo-loop arrangement can only connect G13 and G16 in syn glycosidic conformation if the bulge is at least two residues long. Intrigued by this observation, we designed a construct with inserted thymine residue between G14 and A15 to test the ability of a G-quadruplex formation with a longer bulge. Similar distribution of 12 signals in the imino region of 1D 1H NMR spectrum of G14A15insT in comparison to RAN1* corroborates the formation of a G-quadruplex with prolonged G-T-A bulge.On the other hand, more than 20 signals in the imino region of the 1D RANKL gene, RANwt, encompasses three G-tracts with three guanines and one G-tract with four guanine residues. Four successive guanines in the first G-tract of the sequence (G1 to G4) are most probably the cause of severe structural polymorphism of RANwt  hybrid G-quadruplex scaffold, which exhibits an A-bulge [anti glycosidic conformations if G-strands are oriented parallel to each other [syn guanines in PARP1 (3 + 1) hybrid topology [anti guanines from the G-quadruplex core of RAN1*, G16 occupies syn glycosidic torsion angle. G-A bulge in the G-quadruplex structure of RAN1* thus connects two guanines with different glycosidic conformations. This significantly influences the progression of the backbone of the bulge-containing G-strand  hybrid structure is defined by one narrow, one wide, and two medium grooves [Detailed comparison of RAN1* G-quadruplex to previously described structures containing bulges revealed significant structural differences including the progression of the backbone and related glycosidic conformations of residues next to the bulge . In the topology ,28. Hightopology . Additio A-bulge . Regardlch other ,28,29 ortopology . In contG-strand a. In ordbackbone . Structu grooves ,28,29,30RANKL gene raises several exciting points. First of all, RAN1* structure together with antiparallel-stranded RAN4 [RANKL gene. Furthermore, special structural features of RAN1*, such as pseudo-loop, can represent unique platform for binding to protein partners. Interestingly, some of the G-quadruplex-binding proteins preferentially recognize the phosphate backbone of the loop, regardless of the exact G-quadruplex topology or even DNA sequence or simultaneously interact with G-quartet and loop [Characterization of parallel-stranded RAN1* structure with a G-A bulge as one of the possible G-quadruplexes that are able to form by a G-rich sequence from regulatory region of ded RAN4  represenand loop ,47. On tand loop ,49,50. Dand loop ,50,51.pseudo-loops, can provide a rationale for development of specific ligands, which would allow for explicit preference for one G-quadruplex over another. Stacking of A15 on G-quartet III and dynamic residues A9 and G14 can hinder the access and stacking of the potential ligand onto the 3′-G-quartet  in the regulatory region of the RANKL gene can be correlated with its lower expression is yet to be determined.Last but not least, unprecedented high-resolution structure of RAN1* G-quadruplex and detailed description of et al. in Nucleic Acids Research [Note added in proof: During the revision process of our work, an article related to the topic of unusual G-quadruplex structures was published by Ngoc Nguyen Research . The aut15N/13C-labeled RAN1 and RAN1* oligonucleotides were synthesized on K&A Laborgeraete GbR DNA/RNA Synthesizer H-8 using standard phosphoramidite chemistry in DMT-on mode. Oligonucleotides were cleaved from the solid support by treatment with concentrated aqueous ammonia at 55 °C overnight and purified using reverse-phase HPLC chromatography, followed by removal of DMT group with reaction in 80% AcOH for 30 min. Oligonucleotides were then extracted with pure ethanol and 8 M LiCl. Samples were after rinsing with 70% ethanol dissolved in pure water. The pH value of samples was adjusted to around 7 using LiOH. Samples were subsequently desalted using ultrafiltration cell with 1 kDa cellulose membrane. Concentration of desalted samples was determined by UV absorption at 260 nm using UV/VIS Spectrophotometer Varian CARY-100 BIO UV–VIS. Extinction coefficients were determined by the nearest neighbor method. 10% of 2H2O, 50 mM KCl, and 10mM potassium phosphate buffer with pH 7.0 were added to the samples. The pH of the oligonucleotide samples was adjusted to 7.0 using HCl or LiOH. The samples were heated up to 90 °C for 5 min and then slowly cooled down to room temperature.All isotopically unlabeled and residue-specific Circular dichroism (CD) spectra were recorded on an Applied Photophysics Chirascan CD spectrometer at 25 °C using a quartz cuvette with a 1.0 mm path length. The wavelength range was from 220 to 320 nm. CD samples were prepared at 30 μM oligonucleotide concentrations in 50 mM KCl and 10 mM potassium phosphate buffer. A blank sample containing only 50 mM KCl and 10 mM potassium phosphate buffer was used for baseline correction. Three scans were averaged to obtain each CD spectrum.−1 from 10 °C to 90 °C and absorbance at 295 nm was measured at 0.5 °C steps. Mineral oil was used to prevent evaporation and sample loss due to high temperatures. A stream of nitrogen was applied throughout the measurements to prevent condensation at lower temperatures. Tm was determined from the normalized A295 versus temperature plot.UV melting experiments of RAN1 and RAN1* were performed on a Varian CARY-100 BIO UV–VIS spectrophotometer with the Cary Win UV Thermal program using a 1.0 cm pathlength cells. Samples for UV measurements were prepared at 30 μM oligonucleotide concentrations in 50 mM KCl and 10 mM potassium phosphate buffer. Samples were heated at 0.1 °C min15N, 13C-labeled samples of RAN1* was performed by 1D 15N-edited HSQC NMR experiments. Exchangeable and non-exchangeable proton resonances of RAN1* were assigned using 2D NOESY spectra  recorded in 90% H2O/10% 2H2O. The 2D DQF-COSY and TOCSY (mixing time of 40 ms) spectra were used to estimate sugar conformations. All spectra were processed in VNMRJ program (Agilent Technologies). Resonance assignment and integration were achieved using SPARKY software [anti glycosidic conformations, we could reference the averaged volume to the value of 3.9 Å. With the help of this reference, we classified the remaining signals as strong (1.8–3.6 Å), medium (2.6–5.0 Å), and weak (3.5–6.5 Å).NMR experiments were recorded on Agilent-Varian VNMRS 600 MHz and 800 MHz spectrometers at 25 °C. Double-pulsed field gradient spin echo (DPFGSE) pulse sequence was used to suppress the water signal. Identification of guanine imino protons in residue-specific 6% anti and between 25 and 95° for syn (G16) guanine residues. Torsion angle χ restrains for all adenine residues were set between 200 and 280° and between 170 and 310° for thymine and cytosine residues describing their anti glycosidic conformation. Torsion angle ν2 was used to restrain sugar pucker to C2′-endo conformation for all residues except G10, G14 and G16, which were left unrestrained.NOE distance restraints for exchangeable protons were obtained from 2D NOESY spectrum recorded at 25 °C with a mixing time of 300 ms. Cross-peaks were classified as medium (2.6–5.0 Å) or weak (3.5–6.5 Å) based on their intensities. Torsion angle χ along glycosidic bond was restrained to a range between 200 and 280° for −1 A−2) and hydrogen bond distance restraints , torsion angle χ and ν2 restraints  and planarity restraints . Planarity restraints were excluded in the last 25 ps of SA. A family of 10 structures was selected based on the lowest energy and subjected to energy minimization with a maximum of 20,000 steps. Figures displaying structures and their details were visualized and prepared with UCSF Chimera software [Structure calculations were performed with AMBER 14 software  using pasoftware . The cooRANKL gene arrange in a pseudo-loop to allow proper partial 5/6-ring stacking of G-quartets and maintain parallel orientation of G-tracts. Unique structural features, such as pseudo-loop and alterations of groove widths along the G-core progression, may serve as targets for highly selective structure-based drug design. Structural insights presented in this study may, in the future, represent the basis for the development of novel therapeutic approaches against osteoporosis. However, the biggest question still remains the correlation between G-quadruplex(es) formation and expression levels of RANKL gene.In this study, we determined an unprecedented three-layered G-quadruplex structure with a G-A bulge. Bulge-forming residues G14 and A15 of a 20 nt G-rich sequence found in the regulatory region of an osteoporosis-related


DYRK1A haploinsufficiency is associated with a recognizable developmental syndrome and variable clinical features. Here, we present a patient with DYRK1A haploinsufficiency syndrome, including facial dysmorphism, delayed motor development, cardiovascular system defects, and brain atrophy. Exome sequencing identified a novel de novo heterozygous mutation of the human DYRK1A gene (c.1185dup), which generated a translational termination codon and resulted in a C-terminally truncated protein (DYRK1A-E396ter). To study the molecular effect of this truncation, we generated mammalian cell and Drosophila models that recapitulated the DYRK1A protein truncation. Analysis of the structure and deformation energy of the mutant protein predicted a reduction in protein stability. Experimentally, the mutant protein was efficiently degraded by the ubiquitin-dependent proteasome pathway and was barely detectable in mammalian cells. More importantly, the mutant kinase was intrinsically inactive and had little negative impact on the wild-type protein. Similarly, the mutant protein had a minimal effect on Drosophila phenotypes, confirming its loss-of-function in vivo. Together, our results suggest that the novel heterozygous mutation of DYRK1A resulted in loss-of-function of the kinase activity of DYRK1A and may contribute to the developmental delay observed in the patient.Dual-specificity tyrosine phosphorylation-regulated kinase 1 A (DYRK1A) is essential for human development, and  Due to the variety of proteins that DYRK1A phosphorylates, it plays essential roles in a wide range of cellular signalling pathways and processes, including neurogenesis, neuronal differentiation and proliferation3, synaptic transmission4, cell cycle5, apoptosis6, splicing7, and RNA transcription8. Many of these pathways and processes regulate neurological function, and, indeed, DYRK1A overexpression or overactivity is associated with many neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, Huntington’s, and Pick’s diseases, and even certain types of cancers 14. DYRK1A is also thought to contribute to several of the phenotypic and neurocognitive features of Down Syndrome (DS), because individuals with DS carry an extra copy of DYRK1A17. Decreased DYRK1A expression or activity is also pathogenic. For example, mice hemizygous for DYRK1A have substantial phenotypic defects, including smaller body size, microcephaly, reduced numbers of neurons, abnormal motor function, gait disturbances, and impaired cognitive function19. Human DYRK1A haploinsufficiency is generated by a variety of mutations and is a potential cause of a recognizable developmental syndrome that is characterized by variable clinical features, including intellectual disability, developmental delay, microcephaly, dysmorphic facial features, speech delay, autism, febrile seizures, and ocular malformations 21. Individuals with this syndrome were first identified with partial monosomies of chromosome 21 on routine karyotypes that encompassed the DYRK1A gene (21q22.13)22. More recently, the diagnosis of numerous de novo mutations in DYRK1A has been achieved by next generation sequencing, which has facilitated and broadened the clinical characterization of DYRK1A disruptions. To date, many mutations associated with DYRK1A have been identified and include gross deletions, small deletions, point mutations, complex rearrangements, small indels, and splice-site mutations . Many of these mutations result in truncated proteins that partially or completely lack the DYRK1A kinase domain and thereby lose their catalytic activity.Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a member of the highly conserved DYRK kinase family that belongs to the CMGC kinase superfamily. DYRK1A phosphorylates serine and threonine residues on its target substrates and autophosphorylates its own tyrosine residuesde novo DYRK1A mutation occurring in the β-sheet of the CMGC insert, which is located in the C-terminal end of the kinase domain. This nonsense mutation led to the production of a C-terminally truncated kinase domain protein (DYRK1A-E396ter). The resulting mutant protein was not only efficiently degraded by the proteasome but was also catalytically inactive in mammalian cell and fly models, indicating complete loss-of-function of DYRK1A.Here, we report a novel The study was approved by the Institutional Review Board of Pusan National University Yangsan Hospital  and adhered to the tenets of the Declaration of Helsinki involving ethical principles for medical research with human subjects. Informed consent was obtained from the child’s parents.23. Captured targeted regions were sequenced using the Hiseq 2500 Sequencing System (Illumina Inc.) following the manufacturer’s instructions. Alignment and variant calling was done automatically by on-instrument tools. Imported sequence data was filtered for specified genes and converted into a customized report using the VariantStudio software. Pathogenic variants were evaluated by the practical statement released by the American College of Medical Genetics and Genomics24.Written informed consent was obtained from all participants before blood was drawn. Genomic DNA was isolated using the QIAamp DNA Blood Midi kit  from participants’ leukocytes in the peripheral blood, according to the manufacturer’s standard protocols. The extracted gDNA was evaluated using the TruSight One Sequencing Panel  as described previouslyNheI/HindIII sites, and the open reading frame of human DYRK1A (NM_001396.4) was cloned into a pcDNA3.1(+) vector at HindIII/XbaI sites. Plasmids expressing FLAG-DYRK1A-E396ter and FLAG-DYRK1A-K188R were generated by mutating the original sequence with a QuikChange II Site-Directed Mutagenesis Kit , according to the manufacturer’s method. The following primers that are specific to each mutant were used: 5ʹ-CAAAAGCAAGAAAGTTCTTTTGAGAAGTTGCCAGATG-3’ (forward) and 5ʹ-CATCTGGCAACTTCTCAAAAGAACTTTCTTGCTTTTG-3ʹ (reverse) for FLAG-DYRK1A-E396ter; 5ʹ-CAAGAATGGGTTGCCATTAGAATAATAAAGAACAAGAAG-3ʹ (forward) and 5ʹ-CTTCTTGTTCTTTATTATTCTAATGGCAACCCATTCTTG-3ʹ (reverse) for FLAG-DYRK1A-K188R.To construct plasmids expressing FLAG-DYRK1A proteins, the DNA fragment encoding FLAG (DYKDDDDK) was inserted into a pcDNA3.1(+) vector at 2. When the cells reached 60–80% confluency, they were transfected with plasmids using the XtremeGene Transfection Reagent , according to the manufacturer’s instructions. Transfected cells were incubated at 37 °C for 24 h prior to harvest or analysis.Human embryonic kidney 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% foetal bovine serum  supplemented with 1% streptomycin and penicillin. The cells were seeded at approximately 50% confluency into cell culture plates and were maintained overnight at 37 °C under 5% CO4Cl , the calpain inhibitor calpeptin , and the autophagy inhibitor 3-methyladenine (Sigma-Aldrich) for protein degradation pathway analyses. All chemicals were dissolved in dimethyl sulfoxide (DMSO) prior to treatment, and the cells were treated with 10 μM of each compound for 15 h after plasmid transfection.We used the proteasome inhibitor MG132 , the lysosomal inhibitor NH25. The following antibodies were used: anti-FLAG M2 antibody , anti-HA antibody , anti-hnRNP A1 antibody , anti-Tau antibody , and anti-phospho-Tau (at residue Thr-212) antibody . Signals were detected on a LAS-4000 image analyser using a ClarityTM Western ECL Substrate  and then quantitatively analysed using the NIH ImageJ software .Quantitative western blot analysis was performed as described previouslyThe 293T cells were seeded in 6-well cell culture plates and transfected with FLAG-tagged wild-type or mutant DYRK1A plasmids for 24 h. TRIzol reagent  was used for total RNA extraction, and the Omniscript RT Kit  was used to synthesize first-strand cDNA from 1 μg of total RNA, which was primed with oligo-dT primers. qRT-PCR was performed using the TOPreal SYBR Green PCR Kit  in triplicate with the following primer sets: 5ʹ-TAGCATGGATTACAAGGATGACGATG-3ʹ (FLAG-DYRK1A forward), 5ʹ- ACATAAGTGACCAACAGGTTTCTGC-3ʹ (FLAG-DYRK1A reverse), 5ʹ-AGAGCTACGAGCTGCCTGAC-3ʹ (Human β-actin forward), and 5ʹ-AGCACTGTGTTGGCGTACAG-3ʹ (Human β-actin reverse).26. Briefly, the 293 T cells were co-transfected with FLAG-DYRK1A-E396ter and HA-ubiquitin plasmids using the XtremeGene Transfection Reagent (Roche) for 24 h. Cells were then treated with 10 μM MG132 for 11 h, harvested, and lysed in an immunoprecipitation (IP) buffer . The cell lysates were mixed with anti-FLAG M2 antibody-conjugated protein A/G-agarose beads (Santa Cruz Biotechnology) in 0.2% NP-40-containing IP buffer using a tubing-rotator at 4 °C for 2 h. The bound beads were washed three times with 0.2% NP-40-containing IP buffer and were solubilized with 2X sample buffer for western blot analysis.Polyubiquitination assays were performed as described previouslyDrosophila melanogaster were cultured at 25 °C on standard cornmeal media. GMR-gal4 (#9146), MS1096-gal4 (#8860), Mhc-gal4 (#55133), UAS-Tau 27, and all other stocks and balancers were obtained from the Bloomington Stock Centre . UAS-mnb transgenic flies were described in our previous report28. Based on amino acid sequence alignment, the human DYRK1A E396 residue corresponded to the Drosophila mnb D401 residue. Both wild-type full-length mnb and truncated mutant (mnb-D401ter) cDNAs were generated by PCR using Drosophila S2 cell cDNA as a template. Phusion Pfu PCR polymerase (Invitrogen) was used with the following primer pairs: 5ʹ-GAATTCATGTATAGATTAGAGGATACGA-3ʹ , 5ʹ-TCTAGACTAATGTATAACTACAGGATTC-3ʹ (reverse for mnb-WT), and 5ʹ-TCTAGATCAGAAGAACTTGCGGGTCTTG-3ʹ (reverse for mnb-D401ter). The cDNAs of the full-length and truncated forms of mnb were ligated to the pUAST-FLAG vector digested with EcoRI/XbaI restriction enzymes to place the transgenes under the control of the UAS promoter. UAS-mnb-WT and UAS-mnb-D401ter transgenic flies were obtained by P-element-mediated germline transformation29. To analyse the eye phenotype, newly eclosed flies were collected and allowed to mate for 2–3 days. Wings from adult flies were dissected in 100% ethanol and mounted in Canada Balsam mounting medium (Gary’s magic mountant). The eye and wing phenotypes were photographed using a SZ60 binocular microscope equipped with an eXcope K5 CCD system . Fly tissue sizes were measured on multiple samples (n > 10) from each genotype using NIH ImageJ software. The average eye size was presented as a normalized percentage of the control eye size. For adult lethality assessments, at least 100 flies were included in each individual experiment. For statistical analyses, two-tailed unpaired Student’s t-tests were used.30. Briefly, fixed larval preparations were washed in phosphate buffered saline with Tween-20 (PBST) three times (10 min each), blocked in PBST with 5% normal goat serum for 30 min, and incubated with primary antibody at room temperature for 2 h or at 4 °C overnight. Fluorescein isothiocyanate-labelled goat anti-horseradish peroxidase  was used to detect the synaptic bouton in larval tissue. All images were collected using a FV1000 confocal microscope (Olympus) and processed using NIH ImageJ software. Neuromuscular junction (NMJ) quantifications were conducted based on published procedures31. The filopodial NMJ structure was processed from complete z-stacks using the entire NMJ of the A4 abdominal segment. All statistical comparisons were performed using Prism software . P-values were calculated using two-tailed Student’s t-tests.Immunostaining of larval samples was performed as previously describedth percentile), her weight was 5.8 kg (10th percentile), and her head circumference was 48.5 cm (25th percentile). Based on these clinical manifestations, the patient was initially suspected to have either Ohdo or Hallermann-Streiff syndrome. However, we discovered a novel heterozygous DYRK1A variant  on exon 8 by exome sequencing. The novel variant was only detected in the patient sample and was not present in the control population . We confirmed the variant by Sanger sequencing , the patient demonstrated delayed global developmental milestones and growth parameters. At 21 months of age, the patient developed seizures with generalized stiffness.A 5-month-old female was referred to our hospital for evaluation of delayed development and facial dysmorphism. The patient was born after 39 weeks and 4 days of gestation by vaginal delivery after an uneventful pregnancy and weighed 2.89 kg. She was the third child of healthy Korean parents  identified in this study was generated by the insertion of a single T at nucleotide position 1,185 of NM_001396.3. This insertion altered the translational frame, generated a premature translation termination codon (TGAG) at the E396 (GAG) position, and produced a truncated protein of 395 amino acids (DYRK1A-E396ter)  and substrate peptide  Fig. . To gainide Fig. . Structuide Fig. . As a reide Fig. .Figure 24Cl, calpeptin, and 3-methyladenine were used to inhibit proteasome-, lysosome-, calpain-, and autophagy-mediated protein degradation pathways, respectively. Western blotting with an anti-FLAG antibody revealed a prominent FLAG-DYRK1A-E396ter protein (~45 kDa) band only after treatment with MG132  expression clone and then introduced the mutation identified in the patient to produce a truncated protein of 395 amino acids (FLAG-DYRK1A-E396ter). In contrast to FLAG-DYRK1A-WT protein, FLAG-DYRK1A-E396ter protein was barely detectable by western blotting with an anti-FLAG antibody when overexpressed in 293 T cells Fig. . Since q132 Fig.  and not 132 Fig. . Togethe33. Phosphorylation of Tau at the T212 residue is highly dependent on DYRK1A and was chosen as a marker of DYRK1A kinase activity34. Tau was transiently co-expressed with each FLAG-DYRK1A-WT, FLAG-DYRK1A-E396ter, and FLAG-DYRK1A-K188R1  in 293 T cells. Phosphorylation of Tau at T212 was detected by western blotting with a phosphorylation-specific antibody. As previously reported, the co-expression of Tau and wild-type DYRK1A induced remarkable phosphorylation of Tau  gene is 82% identical to its human homolog, DYRK1A37. Loss-of-function mutations in mnb result in reduced brain size with abnormal visual and olfactory behaviour due to defects in neurogenesis and brain development38, which recapitulates the microcephaly phenotype of DYRK1A haploinsufficiency in human patients. In addition, the tissue-specific overexpression of mnb also induces various phenotypic and neurological defects in central nervous system structure, consistent with various phenotypes of DS patients39.Because basic biological and neurological properties are highly conserved between human and in vivo, we generated transgenic Drosophila harbouring wild-type mnb (UAS-mnb-WT) or truncated mnb (UAS-mnb-D401ter). Based on sequence comparison, Drosophila D401 was equivalent to human E396   Fig. . Similarer) Fig. , the D40mnb-D401ter flies with those of mnb-WT flies. Overexpression of mnb-WT in wing tissue using the MS1096-Gal4 driver showed shortening of the L5 vein in the adult wing, which we previously identified as one of the most prominent phenotypic defects27. In contrast, there was no recognizable phenotypic defect when the D401ter protein was overexpressed in the wing  or pan-neuronal  expression of mnb-WT caused severe embryonic lethality of 80–100%, whereas mnb-D401ter expression barely affected fly viability or any other phenotypes  morphology of mnb Fig. . Comparepes Fig. . Collectde novo heterozygous mutation of the DYRK1A gene. This novel mutation generated a translational termination codon and produced a C-terminally truncated protein (DYRK1A-E396ter)  Fig. . Structuity Fig. . AccordiDrosophila as an in vivo model. Transgenic mnb-D401ter flies, which have the Drosophila mutation equivalent to human DYRK1A-E396ter, had no recognizable phenotypic defects in the wings, eyes, and NMJs, and no severe embryonic lethality, whereas mnb-WT transgenic flies had severe phenotypic defects and lethality (Fig. More importantly, we further revealed that the mutant protein did not have a dominant-negative effect on the wild-type protein. Tau phosphorylation induced by the expression of the wild-type protein was barely affected by the additional expression of the mutant protein, even though the level of the mutant protein was recovered to the level of the wild-type protein by treatment with MG132 Fig. . In the ity Fig. . Collect44. Thus, loss of this region in the DYRK1A-E396ter mutant may directly affect protein stability. Indeed, we observed that the mutant protein was efficiently degraded by the ubiquitin-mediated proteasomal pathway and was consequently undetected in mammalian cells (Fig. βTrCP to the N-terminus of DYRK1A45. In addition, the CDC37/HSP90 chaperone has been suggested to regulate DYRK1A protein stability through interaction with its N-terminal lobe46. Whether these two mechanisms are associated with the degradation of the DYRK1A-E396ter protein needs further experimental validation.According to previous reports, the C-terminal end of the DYRK1A kinase domain is required for  protein stability as well as kinase activitylls Fig. . It has de novo DYRK1A nonsense mutation in a patient with DYRK1A haploinsufficiency syndrome. The mutation generates a C-terminally truncated protein at the β-sheet of the CMGC insert within the kinase domain that behaves as a loss-of-function mutant in both mammalian cell and Drosophila models.In conclusion, we have identified a novel Supplementary Information.


Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease. Multiple sclerosis (MS) is a polyphasic immune-mediated disorder characterized by multifocal inflammatory infiltrates  within the CNS, with concomitant degradation of the myelin sheath, oligodendrocytes, and axons, along with reactive astrogliosis and activated microglia .Multiple areas of myelin loss within the CNS called “plaques” or “lesions” are the pathologic hallmark of MS. It is evident that MS lesions evolve differently during the early/acute versus the chronic phase of the disease, and within each phase, different plaque types occur in particular stages of activity . FurtherMS-related damage to CNS tissues has been found to include two main pathological processes: inflammatory myelin destruction (demyelination) and progressive irreversible axonal loss (neurodegeneration). The underlying pathology of the inflammatory component is generally believed to be associated with an autoimmune attack upon myelin antigens. However, extensive studies have not yet established the predominant target antigenic structures involved in the autoimmune response most relevant for MS background . Both prSphingolipids (SLs), as the major component of CNS myelin sheaths, seem to be potential biomarker candidates in MS . They paComprehensive profiling of CNS lipids in MS lesions might provide a better insight into their role in the pathogenesis of the disease, including an attempt to define the metabolic pathways leading to autoimmune demyelination and/or neurodegeneration. Such findings might contribute to evaluate the usefulness of SLs as biomarkers of various phases of MS or even as potential targets for therapeutic interventions. Myelin lipids in MS have already been investigated in some studies, but few of them analyzed their distribution in brain MS tissues , 26, 27 In this study, we performed a targeted sphingolipidomic analysis of post mortem brain tissues in patients with MS as well as subjects with the CNS affected by other neurological diseases (ONDs) or with substantially normal CNS (nCNS). The comparative analyses were further conducted for particular types of MS lesions, namely chronic active and chronic inactive. We have observed the distinctive pattern of SL metabolism in chronic inactive MS (In-MS) lesions in comparison to the nCNS. Based on our findings, we propose Cer 1-phosphate (C1P) to be a potential new biomarker of the MS progressive phase.Fresh frozen brain tissues were obtained from the Human Brain and Spinal Resource Center, Veterans Affairs West Los Angeles Healthcare Center, 11301 Wilshire Blvd., Los Angeles, CA 90073, which is sponsored by the National Institutes of Health, the National MS Society, and the US Department of Veterans Affairs. Brain tissue specimens were derived from autopsy of patients with clinically diagnosed and neuropathologically confirmed MS (n = 13) and ONDs (n = 15) as well as from three controls who had been diagnosed with diseases without CNS involvement, with essentially normal brain confirmed on autopsy findings, nCNS. OND samples were further subdivided into inflammatory OND  and noninflammatory OND  reference subgroups. Patients’ clinical and autopsy characteristics are provided in Normal-appearing and pathological tissues were selected by gross examination and verified by microscopic examination. To assess the presence of myelin and identify areas of demyelinated plaques and normal-appearing white matter (NAWM), frozen 4 μm-thick cryostat tissues sections were stained with Luxol fast blue and H&E myelin stains. Neuropathological evaluation also comprised staining for axons (Bielschowsy’s silver impregnation). This allowed identification of lymphocytes and plasma cells as well as foamy macrophages containing myelin degradation products. The stage of lesional development was determined immunochemically as well. Anti-CD68 antibody was used to identify macrophages (data not shown). MS lesions were classified as either chronic active MS (Ac-MS) (profound inflammation with macrophages present) or In-MS .Part of each frozen human brain tissue (100 mg) was cut and homogenized with 2 ml of tissue homogenization buffer containing 0.25 M sucrose, 25 mM KCl, 50 mM Tris-HCl, and 0.5 mM EDTA (pH 7.4). Homogenization was performed on ice using a Polytron electric homogenizer until no solid pieces were observed. Next, 100 μl of the tissue homogenate was diluted 1:10 with the homogenization buffer and aliquots of 10 μl were taken for protein determination assay using a Pierce BCA protein assay kit. Aliquots corresponding to 1 mg of protein were transferred to 15 ml Falcon tubes and subjected to lipid extraction.All solvents were analytical grade from Fisher Scientific . SL standards were from the Medical University of South Carolina Lipidomics Shared Resource or from a commercially available source  with purity of ≥98%.erythro-sphingosine (17C/Sph), 17C base D-erythro-S1P (17C/S1P), 17C base D-erythro-dihydrosphingosine (17C/dhSph), D-erythro-N-palmitoyl-13C-D-erythro-sphingosine (13C/C16-Cer), N-heptadecanoyl-D-erythro-sphingosine (18C/C17-Cer), N-heptadecanoyl-D-erythro-dihydrosphingosine (18C/C17-dhCer), D-erythro-N-palmitoyl-17C-D-erythro-sphingosine (17C/C16-Cer), D-erythro-N-nervonoyl-17C-D-erythro-sphingosine (17C/C24:1-Cer)]; hexosylceramide (HexCer)/lactosylceramide (LacCer) ISs ; C1P ISs ; and SM ISs .Homogenates of tissues (1 mg per protein) were fortified with 50 μl of the appropriate internal standards (ISs), specifically: sphingosine (Sph)/Cer/dihydroceramide (dhCer) ISs  as well as dhCer, Cer, SM, HexCer, LacCer, and C1P species. HPLC-tandem mass spectrometry analysis was performed on a Thermo Fisher TSQ Quantum or SCIEX Q-Trap triple-stage quadrupole mass spectrometer, operating in a multiple reaction monitoring positive ionization mode, as previously described , 33. ChrPeaks corresponding to the target analytes of SLs and ISs were collected and processed using the Xcalibur™ software system (Fisher Scientific). Quantitative analysis was based on calibration curves using a linear regression model as described previously . SLs witP < 0.05 being considered as statistically significant.SL levels for each sample were calculated by summing up the total number of all SL subspecies measured expressed in picomoles per milligram of protein and then normalizing that total to 100%. Because of the uneven distribution of our data, a nonparametric test was used for each two group comparison. Statistical differences between groups were determined by Mann-Whitney test using GraphPad PRISM 7.01, with Histopathological analysis in clinically diagnosed cases of MS revealed features typical for this disease such as demyelination, oligodendrocyte and axonal loss, inflammation with evidence of monocyte infiltrates present, and some degree of gliosis. MS lesions were classified depending on their activity, examples of which are shown in supplemental Fig. S1A). The next largest group was comprised of Cer species, which was composed of C18-Cer (52.0 ± 1.9%), C18:1-Cer (16.0 ± 2.7%) and C24:1-Cer (14.2 ± 0.2%) . Contrary to SM precursors of Cer, precursors derived from de novo Cer synthesis, i.e., dhCer species, constitute a very low amount of SLs. The major dhCer species were dhC18-Cer (39.6 ± 1.1%), dhC24:1-Cer (26.1 ± 3.0%), and dhC24:0-Cer (6.8 ± 0.9%) . Similar abundance to Cer subspecies represents HexCer subspecies that included: C18-HexCer (42.0 ± 2.6%), C24:1-HexCer (30.5 ± 0.9%), and C24:0-HexCer (7.4 ± 1.1%) . Comparatively, SLs such as LacCer subspecies: C18-LacCer (53.4 ± 3.2%); C16-LacCer (32.8 ± 4.0%); C24:1-LacCer (9.5 ± 2.4%) , as well as C1P subspecies: C18:0-C1P (40.3 ± 7.0%); C24-C1P (15.8 ± 1.7%), and C24:1-C1P (15.8 ± 1.7%)  made up a very small proportion of the entire sphingolipidome . The detailed molecular distribution of the individual lipid subspecies measured are summarized in supplemental Figs. S1 and S2.Compositional analysis of total SLs indicated that SM was the dominant species of the NAWM of the nCNS . The moslipidome . Sphingolipidome , were alOverall, whereas the lipid profile of Ac-MS lesions  seemed tTo determine the relationships between the active/inactive type of MS-related chronic brain damage and bioactive lipids such as Cer and its main precursors derived from de novo Cer synthesis as well as SM hydrolysis, we determined the level of Cer, dhCer, and SM subspecies in chronic Ac- and In-MS lesions. Besides the NAWM from the nCNS, two types of reference groups, I-OND and NI-OND, were included. There was a clear decrease in total Cer level in In-MS plaques in comparison to the NAWM from the nCNS as well as the I-OND reference group , C18:0-CThe overall Cer level was increased in Ac-MS plaques, mostly due to their major dhCer precursors, i.e., C18:0 , and C24As shown in It has recently been reported that glycosphingolipids may participate in CNS chronic inflammation , and theNext, we investigated whether the MS activity dependent on altered HexCer level  in the sInterestingly, some LacCer species, i.e., C24-LacCer  and C24:We next assessed whether C1P subtypes are involved in disease activity , C18-C1PTo further understand the mechanism of MS course, sphingoids and their derivatives  were quantified in particular phases of the disease . ElevatiSLs belong to one of the several families of bioactive lipids that activate specific G protein-coupled receptors, thereby acting in both stages involved in MS background: inflammation  and neurOur data suggest different pathological scenarios for Ac- and In-MS-related damage differentiated mainly by a Cer source. Cer is the central hub of the SL pathway, which includes dhCer, SM, HexCer , LacCer and sphingoid bases (Sph and dhSph) and sphingoid base 1-phosphates (dhS1P and S1P), and other SLs. In order to test the association between the features of chronic MS-related brain damage and SL levels, we applied sphingolipidomics to quantify bioactive SL mediators in post mortem tissues obtained from subjects with advanced stages of MS. There are two main pathways of Cer production: de novo biosynthesis and endocytic recycling. Which of these pathways dominates for supplying Cer depends on the cell type and specific conditions and remains to be elucidated. Although it was originally thought that SMase was the key enzyme responsible for Cer generation, our ex vivo studies implicate de novo SL biosynthesis, as indicated by elevated levels of C18:0-dhCer , C24:0-dCer is the structural backbone of SLs and a precursor of complex SLs. Consequently, further perturbation in its metabolism may have an important implication for disease progression. First, our data indicate that Cer could be metabolized to its glycosylated derivatives, as suggested by the significant increase of all HexCer species examined, e.g., C16:0-HexCer , C18:0-H2+ ion-dependent enzyme has been reported to be highly active in brain tissue , which is the rate-limiting releaser of arachidonic acid used for production of pro-inflammatory eicosanoids  released in different types of cells, and this chemokine was revealed to be a major mediator of C1P-stimulated cell migration events , 55. HowIn conclusion, our investigation of chronic MS lesions in the brain revealed different SL molecules supposed to differentiate inflammatory and neurodegenerative processes underlying MS pathology. These guardian SL molecules and their corresponding pathologic pathways could be potentially exploited in both Ac-MS and In-MS forms. More neuropathological research is needed in order to define the relationship between the accumulation of these particular SLs and MS activity or progression. All of these SL molecules might serve as relevant biomarkers and, hopefully, also platforms for novel therapies.The data supporting this study are available in the article and are available from the corresponding author upon reasonable request.


Modifications are differentially accepted at multiple positions, but are all tolerated in the flanking regions. Introduction of Aib and Daa in the binding core had the most deleterious effect on binding to HLA-DR molecules and T-cell activation. Their introduction at the positions close to the P1 anchor residue abolished T-cell priming, suggesting they might be sufficient to dampen peptide immunogenicity. Other modifications led to variable effects on binding to HLA-DR molecules and T-cell reactivity, but none exhibited an increased ability to stimulate T cells. Altogether, non-natural modifications appear generally to diminish binding to HLA-DR molecules and hence T-cell stimulation. These data might guide the design of therapeutic peptides to make them less immunogenic.Non-natural modifications are widely introduced into peptides to improve their therapeutic efficacy, but their impact on immunogenicity remains largely unknown. As the CD4 T-cell response is a key factor in triggering immunogenicity, we investigated the effect of introducing D-amino acids (Daa), amino isobutyric acid (Aib), N-methylation, C Therapeutic peptides represent approximately one-tenth of marketed drugs, owing to their remarkable target selectivity, the high flexibility of their sequence and their low toxicity . As therα-methylation (Cm), amino isobutyric acids (Aib), Ψ(CH2NH) reduced amide bonds (Rd), and peptoids (Pp). Daa are the enantiomeric forms of L-amino acids. Incorporation of Daa into peptides has been associated with low immunogenicity in mice (2NH) Rd, and Pp are modifications introduced in the peptide bond. Studies on their immunogenicity have been limited to assessing their effects on binding to MHC class II molecules , N-methylation (Nm), C in mice , 7, 8 an in mice . Aib con in mice , 16. Nm,olecules , 14, 17 olecules  and T-ceolecules , 18, 19.We therefore addressed this issue by evaluating the effects of introducing six frequent non-natural modifications into an immunogenic peptide on the ability to bind to HLA class II molecules and to activate human CD4 T cells. A wide range of T-cell reactivity and HLA binding activity was found for the 69 investigated analogs, many of which resulted in substantial loss of activity.306−318 and the 69 analogs (The influenza hemagglutinin (HA) peptide HA analogs  were froPeripheral blood mononuclear cells (PBMCs) were obtained from blood cells collected at the Etablissement Français du Sang  as buffy coat preparations from anonymous healthy donors who gave informed consent, in accordance with EFS guidelines. PBMCs were isolated by Ficoll-Paque PLUS density gradient centrifugation . Monocyte-derived DCs were generated from plastic-adherent PBMCs after 4 or 5 days of culture in AIM-V medium  supplemented with 1,000 U/mL of recombinant human IL-4  and of rh-GM-CSF (R&D systems). Monocyte-derived DCs were matured with 1 μg/mL of lipopolysaccharide  for 2 days at 37°C. CD4 T cells were isolated from autologous PBMCs by positive selection using an anti-CD4 monoclonal antibody coupled to magnetic microbeads  and magnetic cell sorting, as recommended by the manufacturer. The HLA-DR genotypes were determined using the Gold SSP DRB1 typing kit (Invitrogen) after DNA extraction from PBMCs with the NucleoSpin Blood L Kit .PBMCs were seeded at 5 M/mL with the peptide HA (10 μg/mL) in RPMI 1640 medium supplemented with 5% SAB, 10 U/mL IL-2 (R&D systems) and 1 μg/mL anti-CD28 (Miltenyi). The culture medium was changed every 2–3 days. Cells were harvested at day 10 and submitted to interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay with HA or analogs as described below. This assay was carried out with 4 different healthy donors.+ T cells in 200 μL of Iscove's modified Dulbecco medium supplemented by 10% human AB serum , 1,000 U/mL rh-IL-6 (R&D systems) and 10 ng/mL rh-IL-12 (R&D systems). Co-cultures were incubated at 37°C in 5% CO2 for 21 days. The CD4+ T cells were restimulated on days 7 and 14 with fresh autologous peptide-loaded DCs, 10 U/mL IL-2 (R&D systems) and 5 ng/mL IL-7 (R&D systems). The specificity of each cell culture replicate (CD4+ T-cell line) was assessed by IFN-γ ELISPOT assay using HA or selected analogs at day 21. T cell lines were generated from 7 healthy donors, different from the previous donors.Mature dendritic cells (mDCs) were loaded for 4 h at 37°C with either the peptide HA or analogs (3 μM). After washings, loaded DCs were added (10 replicates per peptide) at 20,000 cells/well to round-bottom microwells with 200,000 autologous CD4Fifty thousand cells from the short-term culture or a mix of 50,000 cells from T-cell lines and 50,000 autologous PBMCs were incubated with HA or analogs (10 μg/mL) in Multiscreen 96-well plates  previously coated with 2.5 μg/mL anti-human IFN-γ monoclonal antibody . After overnight incubation, spots were revealed using 0.25 μg/mL biotinylated anti-human IFN-γ mAb  in phosphate-buffered saline/1% bovine serum albumin, extravidin-phosphatase , and NBT/BCIP (Sigma-Aldrich). Spot number was determined by the AID ELISPOT Reader System . PHA was introduced in the assay as positive control. T-cell responses was considered as specific when a spot count was 2-fold higher in the presence of the peptide than in its absence, with a minimal difference of 25 spots.*01:01, 04:01, 07:01, 11:01, and DRB5*01:01) was assessed by competitive ELISA (50) were calculated. IC50 values of HA for each HLA class II molecule were 0.8 nM (DRB1*0101), 37 nM (DRB1*0401), 29 nM (DRB1*0701), 20 nM (DRB1*1101), and 8 nM (DRB5*0101). Data were reported as relative affinity corresponding to the ratio of the IC50 of the tested analog to that of HA. Means were calculated from at least two independent experiments.Human leukocyte antigen-DR molecules were immunopurified from homozygous EBV B lymphoblastoid cells as previously reported . HLA-DR ve ELISA , 21. Bin306−318 peptide (HA) as a T-cell epitope model. HA binds to multiple HLA-DR molecules  within the peptide core gave rise to weak T-cell activation, while substitution of Nm, Cm, Pp, and Rd at 3–6 core positions induced 50% T-cell activation. Introduction of a single non-natural amino acid into the HA peptide could therefore dramatically alter its T-cell antigenicity, depending on the nature of the modifications, and the position in the peptide sequence.To evaluate the impact of non-natural modifications on T-cell response, we selected the HAolecules  and partolecules . We firsolecules . HA inte regions , 25. As *01:01 allele, while one was typed HLA-DRB1*04:01 and 11:01. These three allotypes are known to bind the HA peptide as well as HLA-DRB1*07:01 and DRB5*01:01  was found between T-cell priming and HLA binding, suggesting that the loss of priming activity resulted from the binding loss , deeply affected the affinity for HLA-DR molecules. Binding to HLA-DR molecules is the result of interactions with peptide side chains, which are accommodated in five specificity pockets  and 15 hydrogen bonds between mainly conserved residues of the HLA-DR molecules and the main chain of the peptide  , 41, 42,f HLA-DR  and consf HLA-DR , 44. Subolecules , 44. P1-ition 86 . Other m (*15:01  and DRB1 (*03:01  and at p (*13:01  stronglyIn conclusion, we have comprehensively analyzed the impact on T-cell activation of six non-natural modifications widely used to design therapeutic peptides. None of the modifications increased T-cell activation or generated strongly immunogenic peptides. In contrast, they mainly diminished binding to HLA molecules and hence T-cell stimulation. Our data provide important clues to the appropriate use of non-natural modifications to minimize the immunogenicity of therapeutic peptides.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.AA designed and performed the research, analyzed the data, and wrote the paper. SM, SI, OD, and CP designed the research and analyzed the data. BM designed the research, analyzed data, and wrote the paper. All authors contributed to the article and approved the submitted version.AA, SM, SI, OD, and CP were employed by Sanofi. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Influenza virus infection is a global public health threat. Current seasonal influenza vaccines are efficacious only when vaccine strains are matched with circulating strains. There is a critical need for developing “universal” vaccines that protect against all influenza viruses. HA stem is a promising target for developing broad-spectrum influenza vaccines due to its relatively conserved feature. However, HA stem is weakly immunogenic when administered alone in a soluble form. Several approaches have been employed to improve the immunogenicity of HA stem, including conjugation of HA stem with a highly immunogenic carrier protein or displaying HA stem on a nanoparticle scaffold. Converting a weakly immunologic protein into a multimer through aggregation can significantly enhance its immunogenicity, with some multimeric protein aggregates previously shown to be more immunogenic than their soluble counterparts in animal models. Here, we show that a chemically coupling a peptide derived from the head domain of PR8 HA (P35) with the poorly immunogenic HA stem protein results in aggregation of the HA stem which significantly increases stem-specific B cell responses following vaccination. Importantly, vaccination with this conjugate in the absence of adjuvant still induced robust B cell responses against stem in vivo. Improving HA stem immunogenicity by aggregation provides an alternative avenue to conjugation with exotic carrier proteins or nanoparticle formulation. Seasonal influenza viruses circulate in all geographical regions of the world and cause recurrent disease in humans. Worldwide, these annual epidemics are estimated to cause 1 billion infections, with 3–5 million cases of severe illness and 290,000–650,000 deaths . There iHA is the most important surface glycoprotein which mediates viral entry into host cells , 6. InflImmunogen multimerization is an effective strategy for enhancing the immunogenicity of protein antigens . MultimeProtein aggregates are high molecular weight proteins composed of multimers of native or denatured monomers . AlthougMouse studies and related experimental procedures in this study were approved by the University of Melbourne Animal Ethics Committee (#1914874). Female C57BL/6 mice (6–8 weeks old) were anesthetized by isoflurane inhalation prior to infection or immunization. For intranasal influenza infections, mice were instilled with 50μL of 50 TCID50 of A/Puerto Rico/8/34 (PR8) as described previously . For intDraining lymph nodes from 5 PR8-infected mice were mashed into single cell suspensions, pooled and cultured in RPMI 1640 supplemented with 10% fetal calf serum and penicillin/streptomycin (RF10). To identify antigen-specific Tfh cells using peptide stimulation , cell suPeptide 11-cysteine  or peptide 35-cysteine  were conjugated with PR8 HA stem protein , HIV gp1ex vivo, freshly isolated LN cell suspensions from individual mice were stained with the following panel: Live/dead Red (Thermo), B220 BV605 , CD3 BV510 , CD4 BUV737 , CXCR5 BV421 , PD-1 BV786 . For Ag-specific Tfh identification [For detection of antigen-specific GC B cells, freshly isolated LN cell suspensions from individual mice were stained with Aqua Viability Dye (Thermo) and Fc blocked with an anti-CD16/32 antibody . Cells were then surface stained with the relevant probes   and the fication , cells w2SO4 and read at 450nm. Endpoint titers were calculated as the reciprocal serum dilution giving signal 2x background using a fitted curve (4-parameter log regression).Stem-specific IgG titre of serum from PR8 HA stem or peptide-stem conjugates immunized mice were detected using sandwich ELISA as previously described . Briefly2SO4 and read at 450 nm.The antigenicity of PR8 HA stem and P35C-stem conjugate was assessed with a panel of anti-HA stem monoclonal antibodies  and a negative control antibody VRC01 (produced in-house) as preciously described . BrieflyDynamic light scattering and zeta potential measurements were performed on a Zetasizer Nano ZS  instrument and analysed with Zetasizer software. Samples kept on ice were allowed to equilibrate to room temperature prior to analysis, with all measurements taking place at 25°C. Concentration of all samples was 0.38 mg/mL in PBS for light scattering experiments, and in Milli-Q water (pH 7) for zeta potential experiments to limit salt interference with measurements. Data represent mean values plus/minus standard deviation of three measurements each consisting of 12 runs.Data is presented as mean ± SEM. Statistical significance was assessed by one-way ANOVA and performed using GraphPad Prism version 7 (GraphPad Software). P values less than 0.05 were considered statistically significant and marked with one asterisk, P values less than 0.01 were marked with two asterisks, P values less than 0.001 were marked with three asterisks.The HA stem is relatively conserved and an attractive target for improved influenza vaccines. However, the HA stem is relatively poorly immunogenic. To study whether the immunogenicity of the HA stem could be improved, we conjugated peptides (P35 and P11) from HA head domain with HA stem proteins and evaluated the immune responses induced by various peptide-stem conjugates in mice. The P35 or P11 peptides and HA stem proteins were coupled with a crosslinker, Sulfo-SMCC. Sulfo-SMCC is a heterobifunctional crosslinker that contains N-hydroxysuccinimide (NHS) ester and maleimide group. The NHS ester reacts with primary amine from N-termimal or lysine of the protein and maleimide group reacts with sulfhydryl from cysteine of the peptide. Therefore, we added a cysteine at the C-terminal of the peptide during peptide synthesis (to produce P11C or P35C peptides). Antigenicity of the stem on peptide-stem conjugate is not altered, which is demonstrated by the similar binding activity of HA stem and P35C-stem with anti-HA stem monoclonal antibodies , P11C-stem with AddaVax (P11C-stem Ad+), P35C-stem without AddaVax (P35C-stem Ad-) and P35C-stem with AddaVax (P35C-stem Ad+) on day 0. Draining lymph nodes (dLNs) and blood were collected on day 14 post immunization for FACS and ELISA analyses. As expected, P11C-stem elicited little stem-specific B cell responses regardless of the presence of AddaVax . In contWe found that P35 is not a CD4 T cell epitope , and thuWe next performed biophysical characterisations of gel-filtrated P35C-stem and unfiltered P35C-stem through dynamic light scattering (DLS) and zeta potential experiments. The hydrodynamic size of gel-filtrated P35C-stem, as determined by the most frequent particle population in solution (hydrodynamic size by Number), was determined to be approximately 9.2 nm, similar to stem alone (10.2 nm); in contrast, the hydrodynamic size of unfiltered P35C-stem was >10,000 nm, outside the limit of distribution analysis, and so population peaks could not be resolved . Both saThis characterization suggests P35C self-aggregates after conjugation with the HA stem protein to form larger multimers which elicited superior immunity compared to soluble stem proteins.The unfiltered P35C-stem was highly immunogenic and we hypothesised that this phenomenon might also be true when other immunogens were conjugated to P35. To assess the generalizability of the application of P35C, we tested another immunogen, hen egg lysozyme (HEL), for conjugation with P35C and mouse immunization. Three groups of C57BL/6 mice were immunized: with HEL protein alone, with the P11C-HEL conjugate or with the P35C-HEL conjugate. Similar to results obtained with the HA stem protein alone, HEL alone elicited little or no HEL-specific B cell responses . The P35To further probe the ability of P35C conjugation to improve humoral immunity, we selected another well-studied and immunogenic model antigen, OVA, for conjugation with P35C and mouse immunization. C57BL/6 mice were immunized with OVA protein alone or P11C-OVA conjugate or P35C-OVA conjugate. Compared with the HA stem and HEL proteins, OVA was more immunogenic and elicited a strong OVA-specific B cell responses in the absence of peptide conjugation . After cNext, we selected another immunogen, the HIV-1 Env monomer gp120, for conjugation with P35C and mouse immunization. C57BL/6 mice were immunized with gp120 protein alone or P11C-gp120 conjugate or P35C-gp120 conjugate. gp120 alone elicited reasonable gp120-specific B cell responses . SurprisSynthetic oligopeptides have displayed significant adjuvant potential in animal studies recent years –28. HereTan et al. from our group recently demonstrated that HA stem was a poor immunogen which elicited little or no stem-specific antibody response in C57BL/6 mice . In the To investigate whether the enhanced immunogenicity of stem protein by P35C conjugation is generalizable, we tested another immunogen, HEL, for conjugation with P35C and mouse immunization. Our data in groups of 5 mice showed that P35C-HEL conjugates could increase the frequency of HEL-specific GC B cells and HEL-specific serum IgG titre. We also conjugated P35C with HIV gp120 protein and OVA protein and elevated their immune immunogenicity in mice. We found that P35C-gp120 conjugates or P35C-OVA conjugates did not increase gp120-specific or OVA-specific antibody responses compared with soluble gp120 protein or OVA protein. Possible reasons are: 1) since peptide and protein conjugation using Sulfo-SMCC is not site-specific, P35C might block some of the original B cell epitopes of gp120 or OVA protein; 2) protein aggregation can cover some of the original B cell epitopes of gp120 or OVA protein. Either scenario could limit gp120-specific or OVA-specific B cell responses and offset the enhancement effect of P35C induced aggregation. This is consistent with previous studies that, in some cases, aggregation of a protein antigen did not increase, or even reduced, immunogenicity due to loss of immunogenic epitopes .Conjugation of a highly immunogenic carrier protein with a poor immunogen is an effective strategy to rescue its immunogenicity . HoweverS1 FigBinding activity of anti-HA stem monoclonal antibodies CR9114 (A), TN1F11 (B), TN1G09 (C) and control anti-HIV-1 gp120 antibody VRC01 (D) with PR8 HA stem (red line) or P35C-stem (blue line) measured by ELISA. Titration curves were generated using sigmoid dose-response of nonlinear fit with GraphPad Prism.(TIF)Click here for additional data file.S2 Fig(A) Gating strategy to identify CD4 T cells in the mLN. Lymphocytes were identified by forward scatter area (FSC-A) and side-scatter area (SSC-A). Doublets were excluded by gating on single cells as determined by FSC-A versus FSC-H, and live cells were identified by viability dye exclusion. T cells were identified as CD3+B220- and CD4 T cells were identified as CD3+CD4+. For each step, the parental population is indicated above the plot. (B) Representative flow cytometric plots of CD154 expression on CD4 T cells after DMSO, P11, P35, or Con A stimulation for 18 h. (C) Representative flow cytometric plots of CD25/OX40 expression on CD4 T cells after DMSO, P11, P35, or Con A stimulation for 18 h.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.