Our study reveals a general mechanism found in the early secretory pathways of murine and human cells that apply to the quality control of MHC class I molecules, and it highlights the allotype-specific structural features of HLA-B*27:05 that can be associated with aberrant antigen presentation and that might contribute to the etiology of disease.HLA-B*27:05 is associated with the development of autoimmune spondyloarthropathies, but the precise causal relationship between the MHC haplotype and disease pathogenesis is yet to be elucidated. Studies focusing on the structure and cellular trafficking of HLA-B*27:05 implicate several links between the onset of inflammation and the unusual conformations of the molecule inside and at the surface of antigen presenting cells. Several lines of evidence emphasize the emergence of those unnatural protein conformations under conditions where peptide loading onto B*27:05 is impaired. To understand how cellular factors distinguish between poorly loaded molecules from the optimally loaded ones, we have investigated the intracellular transport, folding, and cell surface expression of this particular B27 subtype. Our findings show that B*27:05 is structurally unstable in the absence of peptide, and that an artificially introduced disulfide bond between residues 84 and 139 conferred enhanced conformational stability to the suboptimally loaded molecules. Empty or suboptimally loaded B*27:05 can escape intracellular retention and arrive at the cell surface leading to the appearance of increased number of  The binding of the peptide involves the concerted action of various proteins forming the peptide loading complex (PLC): the lectin chaperone calreticulin, binding to the N-linked glycans of the heavy chain; the disulfide isomerase ERp57, triggering disulfide bonding; the transporter associated with antigen processing (TAP), channeling the peptides generated in the cytosol into the ER lumen; and the class I-specific chaperones tapasin and TAPBPR, which are involved in peptide editing [β2m heterodimer is decisive for the further fate of the complex inside the cell, since suboptimally loaded class I molecules (bound to low-affinity peptides or possibly even devoid of any peptide) do not reach the cell surface under normal conditions but are retained in a cycle between the compartments of the early secretory pathway , and the cis-Golgi) instead [β2m, causing endocytic destruction of the heavy chain via its transport to degradative compartments [Major histocompatibility complex (MHC) class I molecules are transmembrane glycoproteins playing a key role in the anti-viral and anti-tumor immune response, by presenting antigenic peptides to cytotoxic T lymphocytes, which then kill aberrant cells. The assembly of the class I heavy and light chains  simulations on empty and peptide-bound class I molecules have shown an increased flexibility of the empty peptide binding domain, especially around the F pocket where the peptide C terminus binds ,10. Fluc peptide . Further peptide . In agres region . As a reThe human leukocyte antigen HLA-B27 is one of the most prominent and thoroughly studied MHC class I molecules due to the genetic association of some of its subtypes, such as B*27:05 and B*27:04 with ankylosing spondylitis (AS) and acute anterior uveitis –16. Whilin vitro analyses revealed increased molecular disorder in the alpha helices involved in the F pocket region in the 05 subtype compared to the 09, leading to a general instability of the heavy chains of B*27:05 [β2m affinity, and that it can reach the cell surface in the absence of optimal peptides. This confirms the importance of the F pocket for MHC class I quality control in cells and demonstrate that this region can artificially be stabilized not only in murine but also in human class I allotypes.We have previously attempted to understand the molecular features of B27 subtypes that confer susceptibility to AS by comparing disease-related subtype B*27:05 with the disease-unrelated B*27:09. MD simulations and  B*27:05 . In ordeTAP2 gene into the STF1 cells by viral transduction as described previously [β2m-specific antibody [Human TAP2-deficient STF1 cells ,20 were eviously . We usedeviously , BBM.1, antibody , and antantibody . SequencXhoI and BamHI and used in fluorescence microscopy experiments. The N-terminally HA-tagged HLA-B*27:05 expression construct (wild type and Y84C) were also cloned into puc2CL6IPwo via the XhoI and BamHI sites. Lentiviruses were produced and used for gene delivery as described previously [The A139C and Y84C mutations and the HA epitope tag (YPYDVPDYA) were sequentially introduced into the wild-type HLA-B*27:05 sequences by QuikChange site-directed mutagenesis (Agilent). Constructs were subcloned into pEGFP-N1 via eviously . In all Cells were harvested via trypsinization, fixed in sodium azide (0.02% in PBS), and stained with 12CA5 against the HA epitope tag or with W6/32 against HLA and were kept on ice. After washing, antibody-antigen complexes were labeled with secondary antibody conjugated with AlexaFluor488. Surface staining was recorded by a CyFlow®Space flow cytometer  and analyzed by Flowjo, LLC software. Presented histograms depict fluorescence measurements from intact cells that were gated according to their forward and side light scattering properties.35S-labeled methionine and -cysteine  as described previously [In pulse-chase and thermostability analyses, newly synthesized HLA-B*27:05 molecules were labeled with eviously . Brieflyeviously .β2m-bound B27 heavy chains in STF1 and STF1-TAP2 cells, we immunoprecipitated the heterodimeric complexes using mouse monoclonal anti-human β2m antibody BBM.1. Amount of B27 heavy chains that were readily associated and co-immunoprecipitated with β2m were analyzed in immunoblotting using anti-HA antibody. To prevent the dissociation of suboptimally loaded B27 molecules from β2m in STF cells, 20μM of IRAAPPPLF peptide was added to the cells immediately preceding the addition of lysis buffer. For specific peptide binding assay, we have added the IRAAPPPK(Biotin)F peptide to the post-nuclear supernatants of STF cells that were lysed with the native lysis buffer containing Triton X-100 (1%). Biotinylated proteins were recovered by neutravidin-agarose beads . Beads were boiled and associated proteins were resolved in SDS-PAGE. B27 heavy chains were detected using anti-HA antibody in immunoblotting.Cell lysates or immune complexes were resolved on 11% sodium dodecyl sulfate (SDS)- polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. Proteins were detected by incubating the membranes with primary and alkaline phosphatase-conjugated secondary antibodies, respectively. Protein-antibody complexes were visualized by 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitro blue tetrazolium (NBT) colorimetric method. For non-reducing gel electrophoresis, cells were incubated with 20 mM solution of the alkylating agent N-ethylmaleimide (NEM)  prior to and during lysis . Immunop35S-methionine and -cysteine for 7 min as described above and elsewhere. Post-nuclear supernatant (lysate) was kept either on ice or in a heat block at different temperatures  for 10 min. Folded HLA molecules were immunoprecipitated with W6/32. Next, HA-tagged HLA-B*27:05 molecules were isolated from the supernatant of boiled W6/32 conjugated Protein A beads as described above. The total amount of W6/32-reactive HLA-B*27:05 molecules in the heat-treated lysates was determined by quantifying the 35S signal from the individual samples run on SDS-polyacrylamide gels.Cells were labeled with + and Cl− ions and neutralized with eight counter-ions. The structures were then subjected to 1500 steps of steepest-descent (SD) energy minimization, positionally restrained , and heated from 100 to 300 K. The restraints were resolved in five steps, and then each complex was equilibrated for 1 ns and simulated for two independent 50 ns MD simulations. Short-range non-bonded interactions were taken into account up to a cut-off value of 9 Å. Long-range electrostatic interactions were treated with the particle mesh Ewald method [Coordinates of HLA-B*27:05 bound to the high affinity peptide IRAAPPPLF were taken from a 1.85 Å resolution X-ray crystal structure (PDB ID: 3BP4 ). The emd method .The Brefeldin A (BFA) decay was performed as described previously . BFA wasb via C84-C139 disulfide bond improves the conformational stability of the peptide-receptive class I-β2m heterodimer [b, we introduced two cysteines at positions 84 (Tyr) and 139 (Ala) of the HLA-B*27:05 coding sequence  in peptide-deficient STF1 cells , but within the next two hours (time points 120 and 240 min) the protein nearly disappeared, indicating rapid degradation  such that it could escape ER quality control [Human and mouse cells are capable of recognizing MHC class I molecules that have failed to load high-affinity peptides and of preventing them from reaching the cell surface in order to avoid binding of extracellular peptides. In search of a molecular explanation for the differences between class I molecules loaded with high-affinity peptides and suboptimally loaded class I molecules bound to low-affinity peptides, we have discovered a region in the class I heavy chain that is most likely targeted by the cellular quality control systems. This region, comprised of the amino acids that form the F pocket, becomes highly flexible when a peptide fails to interact with the heavy chain via its C terminus ,10. In t control . Since t control  and tend control .First, we used comparative MD simulations of HLA-B*27:05, we showed reduced flexibility of the residues in the F pocket for the empty Y84C mutant similar to that of the peptide-bound HLA-B*27:05. Due to high computational demand, MD simulations are limited in predicting and analyzing conformational changes in proteins only within a nanosecond-timescale, which does not reflect all possible alterations that are taking place in class I molecules, which have lost their peptide ligands. Nevertheless, our results are consistent with previous MD simulations  of human and murine allotypes –40 and sFig 2). The steady state levels were restored by the introduction of the TAP2 gene (STF1-TAP2 cells), indicating that the reduced cell surface expression of the wild type protein is due to the lack of high-affinity peptides in the ER. The disulfide mutant B*27:05-Y84C, however, was readily detected at the surface of STF1 cells, indicating the ability of this molecule to bypass intracellular retention mechanisms that apply to empty or suboptimally loaded MHC class I molecules. This observation reinforces the suggestion that a general mechanism exists in cells that surveys the flexibility of the helices in the peptide binding domain of class I heavy chains as an indicator of optimal peptide selection. Similar levels of wild type B*27:05 and B*27:05-Y84C at the surface of STF1-TAP2 cells  and pull-down experiment using B*27:05-specific peptide  imply that the disulfide mutant can bind peptides and can present them at the cell surface. Despite its ability to bind high-affinity peptide , B*27:05-Y84C disappeared more quickly from the surface of TAP2-proficient cells than the wild type B*27:05 . Loss of the conformation recognized by W6/32 antibody in B*27:05-Y84C despite the presence of optimal peptides is supported by our observation that lesser amounts of β2m-bound Y84C heavy chains can be recovered from TAP-proficient cells ( compare lanes 4 and 6). This contrasts with our previous biochemical and cell biological analyses that show that the disulfide mutant of H-2Kb is equally or only slightly less stable than the wild type in peptide-bound form [β2m affinity and ability to bind peptides, Figs S2D), it can be speculated that the mutant attains a different (and probably much more stable) conformation in the ER when it is devoid of peptide. This is supported by our finding that the B*27:05-Y84C mutant, when newly synthesized, is more resistant to thermal denaturation (loss of W6/32 epitope) than wild type B*27:05  and that it continues to gain increasing recognition by W6/32 during its folding in STF1 cells .Next, we showed that cell surface expression of HA-tagged wild type HLA-B*27:05 is dependent on peptides delivered to the ER via the TAP transporter since it is intracellulary retained in TAP2-deficient STF1 cells . In the same cells, B*27:05-Y84C is increasingly recognized by W6/32 within the first hour of chase, suggesting also slow folding but more efficient ER exit. The slight decrease of W6/32-reactive amounts of B*27:05-Y84C after two hours of chase  are possibly due to the endocytic degradation of the molecules that have reached the cell surface. The drastic reduction in the amount of W6/32-reactive wild type B*27:05 after two hours of chase in TAP2-deficient STF1 cells is on the other hand most likely due to ER-associated degradation (ERAD) in the absence of peptide [S2C Fig) also confirms the requirement of cell surface transport of B*27:05 for the generation of β2m-free heavy chains and disulfide linked structures arising from them [Surprisingly, the improvements in the binding strength between the heavy chain and  for β2m . HLA-B*2 for β2m –45. Our  peptide . The lacrom them ,37.β2m, rather than the occupancy of class I molecules with peptides. Indeed, surface lifetimes of both wild type and disulfide mutant of B*27:05 were restored in STF1 cells when the BFA decay was performed at 25°C . To our surprise, the enhanced structural stability of the peptide-receptive heterodimers of B*27:05-Y84C  is insufficient in rescuing the mutant from endocytic degradation. This suggests that the Y84C-heterodimers dissociate shortly after their arrival at the cell surface. Supporting this assumption, we observe elevated levels of HC-10 positive heavy chains of B*27:05-Y84C at the surface of both STF1 cells . The behaviour of B*27:05-Y84C in TAP-deficient cells might be considered comparable to the phenotype of AS-related B*27:05 subtypes in conjunction with non-synonymous ERAP1 polymorphisms [ERAP1-silenced model system [β2m-free B27 heavy chains [β2m-free B*27:05 in ERAP1 knock-down cell models, while surface HC-10 levels of other tested HLA-B allotypes remained unchanged [In cells that lack components of the peptide loading complex, class I molecules are retained in the early secretory pathway , and loworphisms –51. Whill system , underlil system . Severall system –56. Not y chains –52. Thisnchanged ,55. Thosnchanged  and promnchanged . Empty Bin vitro T-cell activation.Our study confirms the presence of a quality control mechanism in the early secretory pathway that surveys the flexibility of the F pocket of class I molecules in general. The artificially stabilized mutants of human and murine MHC molecules are useful tools to delineate protein folding and monitoring in cells. It is also noteworthy to mention that the disulfide mutants of class I are potentially powerful tools for diagnostic and therapeutic approaches involving MHC-multimers and S1 Fig(A) TAP2 expression is restored in STF-TAP2 cells. The TAP2 gene was introduced into STF1 cells by viral transduction (generating STF1-TAP2 cells), and the expression of TAP2 was verified western blotting. Lysates of STF1 and STF-TAP2 cells were probed for TAP2 with the monoclonal antibody 429.3. Full length TAP2 could only be detected in STF-TAP2 cells migrating slightly more slowly than the 72 kDa band of the molecular marker. Calnexin was used as a loading control (upper band at 90 kDa). The asterisk marks an unspecific band detected in both lysates.(B) TAP2 is fully functional in STF1-TAP2 cells. TAP function was confirmed by measuring the surface levels of endogenous HLA levels by using the monoclonal anti-HLA antibody, W6/32 and anti-mouse IgG conjugated to AlexaFluo-488 in flow cytometry. In peptide-deficient STF1 cells, only low cell surface expression of endogenous HLA molecules could be detected (dashed line), in peptide-proficient STF1-TAP2 cells, however, the surface expression was strongly enhanced (solid line) confirming functionality of the reconstituted TAP transporter.(C) Cell surface expression of B*27:05 and B*27:05-Y84C. Cells were stained with W6/32 and anti-mouse IgG conjugated with AlexaFluor-488 and subjected to flow cytometry. Surface signal intensities from B*27:05 (blue) and B*27:05-Y84C (orange) are displayed as histograms. Grey lines indicate cells that were stained only with the secondary antibody.(D) The scatter plot  shows individual cell surface W6/32 measurements in STF1 and STF-TAP2 cells (black dots). In TAP2-deficient STF1 cells, surface expression of B*27:05-Y84C was about three times higher than for the wild type construct (left) whereas in TAP2-proficient cells, both constructs showed comparable cell surface expression (right).(TIF)Click here for additional data file.S2 Fig(A) Wild type B*27:05 reaches the cell surface of TAP2-deficient cells at 25°C. Peptide-deficient STF1 cells expressing wild type B*27:05 were kept at 25 and 37°C, respectively, stained with anti-HA and anti-mouse IgG conjugated with AlexaFluor-488, and subjected to flow cytometry. Wild type B*27:05 shows a much higher cell surface expression at 25 (blue line) than at 37°C (orange line). The grey curve in both histograms shows the background signal without primary antibody. Quantification of surface signals obtained at 25°C (blue) and 37°C  revealed a 4-fold increase in surface levels of wild type B*27:05 .(B) Averaged BFA decay from the cell surface at 25°C. STF1 cells were kept at 25°C and surface levels of B*27:05 and B*27:05-Y84C were detected by staining STF1 cells with anti-HA. Cells were harvested and stained at the times indicated representing the duration of treatment with Brefeldin A. The graph shows the cell surface levels normalized to the values detected at time point zero , which was set to 100% with the following values depicted as its percentage. Both constructs show similar residence times at the cell surface when incubated at 25°C.(C) B*27:05 free heavy chains on the surface of TAP-deficient cells. Scatter plot  shows the levels of class I free heavy chains detected by HC-10 antibody at the surface of STF1, STF1-B*27:05 and STF1-B*27:05-Y84C cells at 37°C, respectively. Acquired staining intensities from individual experiments were normalized to wild type B*27:05 levels. B*27:05-Y84C reveals approximately 4-fold higher more free heavy chains that the wild type protein.μM of the B*27:05-specific peptide IRAAPPPLF overnight (black bars). Amount of B*27:05 molecules were detected with anti-HA antibody and displayed in comparison with the samples without peptide addition (grey bars). IRAAPPPLF can bind and stabilize B*27:05-Y84C molecules that have reached cell surface whereas surface levels of B*27:05-WT cannot be improved by the peptide.(D) Peptide binding to B*27:05 at the cell surface. STF1 cells expressing either B*27:05-WT or B*27:05-Y84C were incubated with 20 (TIF)Click here for additional data file.


Aedes aegypti. One mutation, V1016G, confers sodium channel resistance to pyrethroids, but a different substitution in the same position V1016I alone had no effect. In pyrethroid-resistant Ae. aegypti populations, V1016I is often linked to another mutation, F1534C, which confers sodium channel resistance only to Type I pyrethroids including permethrin (PMT), but not to Type II pyrethroids including deltamethrin (DMT). Mosquitoes carrying both V1016G and F1534C exhibited a greater level of pyrethroid resistance than those carrying F1534C alone. More recently, a new mutation T1520I co-existing with F1534C was detected in India. However, whether V1016I or T1520I enhances pyrethroid resistance of sodium channels carrying F1534C remains unknown.Multiple mutations in the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides in a major human disease vector v1-1, a sodium channel from Aedes aegypti. The mutant channels were then expressed in Xenopus oocytes and examined for channel properties and sensitivity to pyrethroids using the two-electrode voltage clamping technique. The results showed that V1016I or T1520I alone did not alter the AaNav1-1 sensitivity to PMT or DMT. However, the double mutant T1520I+F1534C was more resistant to PMT than F1534C, but remained sensitive to DMT. In contrast, the double mutant V1016I+F1534C was resistant to DMT and more resistant to PMT than F1534C. Furthermore, V1016I/G and F1534C channels, but not T1520I, were resistant to dichlorodiphenyltrichloroethane (DDT). Cryo-EM structures of sodium channels suggest that T1520I allosterically deforms geometry of the pyrethroid receptor site PyR1 in AaNav1-1. The small deformation does not affect binding of DDT, PMT or DMT, but in combination with F1534C it increases the channel resistance to PMT and DDT.V1016I, V1016G, T1520I and F1534C substitutions were introduced alone and in various combinations into AaNaAe. aegypti. We proposed that mutation F1534C first emerged in response to DDT/pyrethroids providing a platform for subsequent selection of mutations V1016I and T1520I that confer greater and broader spectrum of pyrethroid resistance.Our data corroborated the previously proposed sequential selection of kdr mutations in  Aedes aegypti. Among the mutations, the V1016I and T1520I substitutions were found to be associated with F1534C but rarely found alone. F1534C confers sodium channel resistance to Type I pyrethroids including permethrin (PMT). However, whether V1016I or T1520I enhances the F1534C-mediated sodium channel resistance remain unknown. In this study, our electrophysiological results confirmed their involvement in kdr and corroborate the previously proposed sequential selection of kdr mutations in Ae. aegypti: F1534C likely emerged first in response to DDT and/or pyrethroids, whereas V1016I and T1520I appeared later under more intensive selection from pyrethroid use.Intensive use of pyrethroids has led to the selection of resistance in mosquitoes, and knockdown resistance (kdr) is one of the major mechanisms of pyrethroid resistance. So far, eleven kdr mutations were identified to be associated with pyrethroid resistance in  Chrysanthemum spp. [Pyrethroid insecticides are synthetic analogs of naturally occurring pyrethrins from mum spp. . Due to mum spp. .Pyrethroids target voltage-gated sodium channels in insects. The pore-forming α1 subunit of sodium channels has four homologous repeat domains (I-IV), each containing six transmembrane segments, S1-S6 Figs  and 2A. Aedes aegypti, a vector of viruses causing dengue fever, chikungunya, Zika fever, yellow fever and other diseases.Pyrethroids, as well as DDT which was used intensively before 1960s, prolong the opening of sodium channels by inhibiting their inactivation and deactivation [Ae. aegypti. Co-occurrence of multiple mutations appears to be a common phenomenon in populations of pyrethroid-resistant Ae. aegypti [Eleven sodium channel mutations, V410L , L982W , S989P , V1016G/ aegypti . Example aegypti , 20, 21, aegypti  and V101 aegypti , 23. AmiAe. aegypti from Indonesia, Vietnam and Thailand [Ae. aegypti populations in Latin America [Ae. aegypti populations in Singapore [Ae. aegypti populations in Thailand, Vietnam [Mutation V1016G located in IIS6 was first identified in PMT- and DDT-resistant Thailand . V1016G Thailand , MalaysiThailand , Saudi AThailand  and othe America . V1016I  America , 26, Bra America , 27, Mex America , 29, and America . In contingapore , they alingapore . Interes Vietnam  and Vene Vietnam . More re Vietnam .Ae. aegypti sodium channel, AaNav1-1, and the cockroach sodium channel, BgNav1-1a, to Type I pyrethroids  in the Xenopus oocyte expression system [Previous studies demonstrated that mutation F1534C reduced sensitivity of the n system –34. V101n system , 33. Howv1-1 channel, expressed the mutant channels in Xenopus oocytes, and examined their gating properties, pyrethroid and DDT sensitivity. We found that (i) like V1016I [v1-1 channels to PMT or DMT, (ii) the T1520I+F1534C channel was resistant to PMT, but sensitive to DMT, (iii) the V1016I+F1534C channel was resistant to DMT and more resistant to PMT than the F1534C channel, and (iv) V1016I/G and F1534C channels, but not T1520I, were resistant to DDT.In this study, we introduced V1016I, F1534C and T1520I alone and in various combinations in the AaNae V1016I , T1520I v1-1 allosterically distorts the pyrethroid receptor site PyR1. The small distortion per se does not affect action of insecticides, which we studied here, but in combination with F1534C it affects binding of PMT and DDT.Cryo-EM structures of sodium channels suggest that T1520I in AaNaAe. aegypti: F1534C emerged first in response to DDT and/or pyrethroids, whereas V1016I and T1520I appeared later under more intensive selection from pyrethroid use.Our results corroborated sequential selection of kdr mutations in -deltamethrin and isomer-mixed cypermethrin were purchased from Sigma-Aldrich . (1R)-cis-permethrin and bifenthrin were purchased from Chem Service . β-cyfluthrin was purchased from Fluka . (1R)-cis-NRDC 157, which is structurally similar to deltamethrin, but lacks the α-cyano group next to the phenylbenzylalcohol moiety, was a gift from Bhupinder Khambay . The purities of these compounds were above 98%. Stock solutions of the compounds (100 mM) were dissolved in dimethyl sulfoxide (DMSO). The working solution was prepared in ND96 recording solution immediately prior to experiments. The concentration of DMSO in the final solution (< 0.5%) had no effect on the function of sodium channels in the experiments.1016I, V1016G and F1534C channel constructs in the background of AaNav1-1, a pyrethroid-sensitive sodium channel from Ae. aegypti, were available from a previous study [1016I+F1534C by introducing V1016I into the F1534C construct. We introduced T1520I into AaNav1-1 or F1534C to generate T1520I and T1520I+F1534C mutant channels, respectively. Site-directed mutagenesis was performed by PCR using Phusion High-Fidelity DNA Polymerase . The sequence of the primers used in mutagenesis to introduce T1520I mutation were “CAGCCGATTCGCGAGATCAACATCTACATGTACC” (forward primer) and “GGTACATGTAGATGTTGATCTCGCGAATCGGCTGC” (reverse primer). The mutant clones were verified by DNA sequencing.Vus study . In thisv1-1 and mutants were expressed in the Xenopus oocytes, Ovaries from oocyte-positive female Xenopus laevis purchased from Xenopus 1 . The procedures for oocyte preparation, cRNA synthesis and injection were identical to those described previously [v1-1 and mutant channels, their cRNAs were co-injected into oocytes with Ae. aegypti tipE cRNA in the 1:1 molar ratio [AaNaeviously . cRNA waar ratio , 37.Sodium currents were recorded by using the oocyte clamp instrument OC-725C , Digidata 1200A, and pCLAMP 6 software interface . Methods for electrophysiological recording and data analysis were similar to those described previously . The pearev), where V is the test potential and Vrev is the reversal potential for sodium ions. The peak conductance values were normalized to the maximal peak conductance (Gmax) and fitted with a two-state Boltzmann equation:1/2 is the voltage of half-maximal activation, and k is the slope factor.The voltage dependence of sodium channel conductance (G) was calculated by measuring the peak current at test potentials ranging from -80 to +65 mV in 5 mV increments and divided by  and Type II pyrethroids (cypermethrin and β-cyfluthrin). β-cyfluthrin, cypermethrin and bifenthrin are commonly used in mosquito control. Consistent with data on PMT and DMT, T1520I+F1534C channel was resistant to Type I, but not Type II pyrethroids.Previously the Fethroids , 28. We v1-1 sensitivity to DDT by measuring DDT-induced non-inactivating current. v1-1 and mutants V1016G, V1016I, F1534C, T1520I, V1016I+F1534C and T1520I+F1534C. DDT caused smaller inhibition of fast inactivation in V1016G and V1016I than in the wild-type AaNav1-1, indicating that the mutants were resistant to DDT. The V1016G channel was more resistant to DDT than the V1016I channel. In contrast, the T1520I channel was not resistant to DDT   in the extracellular loop IIIP2-S6 is far from F3i13 and V2i18 and cannot directly interact with pyrethroids bound to the latter residues. In NavPaS, T3(i-1) donates an H-bond to the backbone carbonyl of D3p62 in the AID motif at the C-terminus of helix IIIP2 (v1-1 has the same-length loop IIIP2-S6 and the same AID motif (3(i-1)I eliminates the H-bond, thus destabilizing the loop conformation. The loop contains isoleucine I3i1, which forms tight inter-repeat contacts with residues L2p37 and H2p38 in the P-loop helix IIP1. AaNav1-1 has the same residues I3i1, L2p37 and H2p38 (3(i-1)I would cause small shifts of IIIS6, IIP1, as well as IIS5 and IIS6, which form tight contacts with IIP1. Possible consequences of this changes are discussed in a later section.In NavPaS, Tix IIIP2 . This H-ID motif . Mutationd H2p38 , which aAe. aegypti field populations [Ae. aegypti kdr mutation, F1534C. Specifically, V1016I+F1534C caused a high resistance to both a Type I pyrethroid, PMT, and a Type II pyrethroid, DMT, whereas T1520I+F1534C increased resistance to PMT, but not to DMT.Mutations V1016I and T1520I alone with F1534C cause high levels of pyrethroid resistance in ulations , 17, 49,Xenopus oocytes established that F1534C confers sodium channel resistance only to Type I pyrethroids, but not to Type II pyrethroids including DMT, cypermethrin and cyfluthrin [Ae. aegypti populations carrying the F1534C mutation [1520I+F1534C channel is not resistant to Type II pyrethroids, populations in which T1520I+F1534C were detected were also resistant to DMT [Ae. aegypti additional kdr mutation(s) in the sodium channel or other pyrethroid-resistance mechanisms, such as enhanced metabolic detoxification, contribute to resistance to DMT.Our electrophysiological analysis in fluthrin , 34. Themutation , 50, 51.mutation . Populatmutation . Similart to DMT . It is pAe. aegypti collected in Mexico from 2000 to 2012. It is also consistent with the observation that in natural populations frequencies of F1534C are higher and increase more rapidly than frequencies of V1016I [1534C channel is resistant to only Type I pyrethroids, V1016I+F1534C is highly resistant to both Type I and Type II pyrethroids. Thus, V1016I enhanced F1534C-mediated pyrethroid resistance to both Type I and Type II pyrethroids. We suggest a similar evolution path I may augment pyrethroid resistance of channel F1534/3i13C? In NavPaS T1520/3i(-1) is far above F1534/3i13 (1520/3i(-1)I alone does not affect action of pyrethroids or DDT (1520/3i(-1)I increases the channel resistance to PMT, which is caused by F1534/3i13C (1520/3i(-1)I allosterically induces a small deformation of PyR1.How mutation T534/3i13  and canns or DDT . The fac34/3i13C , indicat1520/3(i-1)I+F1534/3i13C was much more resistant to PMT than F1534/3i13C, but remained sensitive to DMT I+F1534/4i13C increase the channel resistance to DDT (1520/3(i-1)I does not affect the channel resistance to DDT (1520/3(i-1)I+F1534/3i13C as it does in case of PMT. Thus, the experimental data of this study can be explained in view of our previous models of mosquito sodium channel and recent cryo-EM structures of eukaryotic sodium channels. Furthermore, the latter structures suggest an allosteric mechanism by which mutation T1520/3(i-1)I induces a small change of the PyR1 site geometry.In our Kv1.2-based model of mosquito sodium channel , DDT dire to DDT . Furthere to DDT  is consiAe. aegypti: F1534C (and probably V1016I) appeared first in response to DDT, while T1520I emerged later under the continued pyrethroid selection. Both V1016I and T1520I can be established in kdr populations that possess the F1534C mutation.In conclusion, our functional characterization revealed common and unique effects of V1016I and T1520I mutations on the channel sensitivity to DDT and Type I and Type II pyrethroids. Our results supported the hypothesis on sequential selection of kdr mutations in


Poly I:C significantly promoted more IL-2 and IFN γ production as well as higher lytic activity of CAR-T cells. Upon systemic administration in vivo, CAR-T cells obviously suppressed tumor growth, and poly I:C significantly enhanced the suppression. Further study showed that poly I:C exerted antitumor effect dependent on type I IFNs. In addition, poly I:C decreased myeloid-derived suppressor cells (MDSC) number in peripheral blood and spleen, and attenuated the immunosuppressive activity of MDSC on proliferation and cytolytic function of CAR-T. Depletion of MDSC with anti-Gr1 Ab further increased the antitumor effect of CAR-T cells plus poly I:C treatment. In conclusion, CAR-T treatment combined with intratumoral delivery of poly I:C resulted in synergistic antitumor activity. We thus provide a rationale to translate this immunotherapeutic strategy to solid tumors.Chimeric antigen receptor modified T cells (CAR-T) therapy is an emerging immunotherapy against malignancies. However, only limited success was obtained in solid tumors. Polyinosinic-polycytidylic acid (poly I:C), ligand of TLR3, mediates innate immune and adaptive immune and shows broad antitumor effect on many types of cancer. In the present study, we combined EGFRvIII-targeted CAR-T cells with poly I:C treatment and evaluated the synergic antitumor effect  Adoptive T cell immunotherapy has been demonstrated to be a new way to fight malignancies. In particular, T lymphocytes engineered to express chimeric antigen receptor (CAR) have shown great promise in treating hematological malignancies . CD19-taCAR-T therapy is also a novel approach to treat other malignant tumors. Currently, Glypian-3 (GPC3), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor2 (HER2), carcinoembryonic antigen (CEA), disialoganglioside 2 (GD2), mesothelin, prostate-specific membrane antigen (PSMA), and interleukin-13Ra2 (IL13Ra2) have been tested as targets of CAR-T cells in solid tumor. However, CAR-T therapy for solid tumors has not been as efficient as those targeting hematologic malignancies –7.The reasons for the limited success of CAR T cells in solid tumor are being actively investigated, and are likely multifactorial. One major reason is the immunosuppressive microenvironment that may impede the function and persistence of CAR T cells. This includes the presence of regulatory T cells, tumor-associated macrophages, myeloid-derived suppressor cells (MDSC), and cancer-associated fibroblasts, which promote higher levels of inhibitory ligands and cytokines . FurtherPolyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA), is recognized by toll-like receptor 3 (TLR3), dsRNA-activated protein kinase (PKR), retinoic acid-inducible gene I protein (RIG-I) and melanoma differentiation- associated protein 5 (MDA5) . The appIn this study, we explore the potential of using poly I:C to increase the antitumor activities of CAR-T cells and the underlying mechanism.NEG) was maintained in our laboratory. Human embryonic kidney cell line 293T was purchased from the American Type Culture Collection . Both cells were grown as monolayer in DMEM culture medium (Gibco) containing 10% fetal bovine serum  and 1% L-glutamine. Murine breast cancer cell line E0771  was cultured in RPMI 1,640 medium (Gibco) supplemented with 10% FBS and 10 mmol/L HEPES. All cells were routinely maintained at 37°C in a 5% CO2 atmosphere incubator. These cell lines were tested on a regular basis for mycoplasma and were negative.The murine colon carcinoma line CT26 . Activated T cells were seeded in 96-well plates with poly I:C concentrations of 10 and 50 μg/mL. Cell proliferation was analyzed by CCK8 method at 24, 48, and 72 h, respectively.Tumor cells were plated at 10The recombinant murine EGFRvIII specific CAR retrovirus was generated as follows. 806 scFv  was inseT cell transduction was measured using EGFP/Biotin labeled EGFRvIII protein generated in our laboratory, followed by PE-streptavidin when needed. T cell phenotypes were identified using the following antibodies: mouse CD3e  conjugated to FITC and mouse CD8α  conjugated to PE.To detect MDSC from blood, spleen and tumors of mice, tumor-bearing mice were euthanized 24 h after last poly I:C treatment. Fifty microliter of peripheral blood drawn from the cheek vein was incubated with antibodies to mouse CD11b  and Gr1  followed by red blood cell removal and then was analyzed by flow cytometry. Single cell suspensions of spleen were obtained by mechanical disruption through a 70 μm cellular sieve with a plunger, and red cells were then removed. Tumor cell suspensions were obtained by digesting dissected tumor tissues with tissue dissociation kit (Miltenyi). All antibodies were used according to manufacturer's recommendations. Live cells were gated by forward scatter/side scatter (FSC/SSC). Analysis was performed using a FACSCelesta flow cytometer (BD Biosciences) and FlowJo software (TreeStar).in vitro was evaluated using lactate dehydrogenase (LDH) release assay . Target cells were incubated with CAR transduced effector T cells at varying effector-to-target (E:T) ratios. After 18 h of co-incubation at 37°C, supernatant was transferred to a new microplate and the LDH released into the supernatant was assessed using a Microplate Reader. Specific cytolysis was calculated by using the formula: (Test–effector control–target control/Tmax control–target control) × 100, in which “Tmax control” was the value obtained from supernatant of target cells exposed to 1% Triton-X 100, “effector control” was the spontaneous LDH release value of CAR-T alone, and “target control” was the spontaneous LDH release value of target cells alone; background control  was subtracted from each value before the calculation.The activity of CAR-T cells to kill target cells Murine CAR-T cells were tested for antigen-specific activity in cytokine release assay using tumor cells. In these experiments, effector cells were co-cultured with an equal number of target cells in complete RPMI 1,640 medium in a final volume of 0.2 ml in triplicate wells of a 96-well microplate. Culture supernatants were harvested 24 h after the initiation of co-culture and assayed for IL-2 and IFN γ by ELISA (Multisciences). Serum IFN γ level of treated tumor-bearing mice was detected by ELISA (Multisciences).5 C26-EGFRvIII cells suspended in 200 μl PBS were injected s.c. into the flank of mice. To establish orthotopic breast tumors, we injected 5 × 105 E0771-EGFRvIII cells into the 4th inguinal mammary fat pads of 6–7 weeks old female C57BL/6 mice. Tumor bearing mice were treated with 5 × 106 CAR-T cells by tail vein injection. Mice of control groups were injected with same amount of untransduced T (UT) cells. Fifty micrograms high molecular weight poly I:C  was injected intratumorally 3 and 6 days after CAR-T infusion. Mice intratumorally injected with the same volume of saline were used as control. Tumor volume was estimated using the digital caliper measurement (0.5 × length × width × width) twice a week. Mice were euthanatized when the tumor volume reached 2,000 mm3.All animal procedures were approved by the Shanghai Cancer Institute Committee for the Use and Care of animals and performed in accordance to the protocol. For tumor induction, 6–7 weeks old female Balb/c mice were irradiated with 3 Gy whole-body irradiation and then 3 × 10Tumor-bearing mice were injected i.t. with 50 μg poly I:C. Peripheral blood was collected from cheek veins at the indicated times. Saline was used as control. IFN-β level in sera was quantified with ELISA. To interfere type I IFN signaling, 50 μg anti-IFNAR1 mAb  was intratumorally injected on day 0 and 2 after poly I:C or saline treatment .To determine the copy number of integrated CAR in genetically modified T cells, 100 ng DNA genomic DNA was extracted from the tumors of treated mice with the QIAamp DNA Mini Kit (Qiagen). DNA was amplified in triplicate with primers and TaqMan probes specific for the CAR transgenes, using the 7,500 Real Time PCR System (Applied Biosystems) in a PCR reaction . To generate DNA standards, we established serial dilution of DNA plasmids encoding the specific cassette. The primers used were as follows: Primer-F: 5′- GACGTTGGGTTACCTTCTG C-3′), Primer-R: 5′- TTCCCAGGTCACGATGTAGG−3′ and probe: 5′-(FAM)-ATGGCCGCGA GACGGCACCT-(BHQ1)-3′.MDSCs were isolated via Ly6G magnetic selection from spleens of tumor-bearing mice 24 h after poly I:C or saline treatment. Isolated MDSCs were titrated into CAR-T-cell cultures at varying MDSC: CAR-T cell ratios and incubated overnight. For flow cytometry-based proliferation assays, mouse T cells were pre-labeled with CSFE (ThermoFisher Scientific). Anti-CD3 and anti-C28 Abs coated dynabeads were added at initial time. After 72 h, cells were collected and CFSE expression was detected by flow cytometry.To evaluate the MDSC suppression on CAR-T lytic activity, CAR-T cells and target cells were co-cultured at an E:T ratio of 0.3:1. Isolated MDSC were added at varying MDSC: CAR-T-cell ratios. Cytotoxicity efficiency was calculated after 18 h of co-incubation.in vivo MDSC, mice were intraperitoneally injected with anti-Gr1 mAb  24 h before poly I:C initially and then three times per week for 2 weeks . Data were presented as the mean values ± SEM. In all cases, p-values < 0.05 were considered statistically significant.In all experiments, statistical significance was evaluated with unpaired We generated murine EGFRvIII specific CAR-T cells by retroviral transduction of splenocytes derived from Balb/c or C57 mice. High transduction efficiency of 80% was obtained as detected by flow cytometry . T cell in vitro activity of CAR-T cells, poly I:C was added to the co-culture of effector cells and target cells. In the cytotoxicity assay, murine CAR-T cells showed significant specific cytolysis activity against CT26-EGFRvIII and E0771-EGFRvIII tumor cells but not CT26 and E0771 cells (To determine the impact on 71 cells . However71 cells .in vitro killing activity of CAR-T against both CT26-EGFRvIII and E0771-EGFRvIII tumor cells.To further assess the function of poly I:C on CAR-T cells upon antigen-specific stimulation, CAR-T cells were co-cultured with EGFRvIII- negative or positive tumor cells. Supernatant was harvested, and IL-2 and IFN γ levels were measured by ELISA. Upon antigen stimulation for 24 h, we found that CAR-T cells had high level of IL-2 and IFN γ secretion. Moreover, CAR-T cells treated by poly I:C secreted higher level of IL-2 and IFNγ, when compared to the untreated CAR-T cells . HoweverTo address whether poly I:C could increase the antitumor activities of CAR-T cells, mice bearing CT26-EGFRvIII or E0771-EGFRvIII tumor xenografts were treated by EGFR-targeted CAR-T cells, or their combination with poly I:C. Interestingly, we found that intratumoral injections of poly I:C significantly enhance the tumor-suppression activity of CAR-T cells, as showed by the delayed tumor growth  and smalin vivo, body weight of mice was monitored. Results showed that no significant difference in body weight was observed between poly I:C treatment and corresponding control group in both animal models (In addition, to explore whether poly I:C injection caused severe toxicity l models .in vivo, mice bearing CT26-EGFRvIII or E0771-EGFRvIII tumors were treated with intratumoral injections of IFNAR-blocking Ab on days 0 and 2 after injection with poly I:C or saline (in vitro (Poly I:C is a potent activators of systemic type I IFN responses . Upon por saline . Type I r saline . Similarr saline . These rin vitro .Previous reports showed that tumor growth induces expansion of MDSC in peripheral blood. In turn, these immunosuppressive MDSC contributed to tumor growth . Indeed,To examine whether poly I:C affects the suppressive function of MDSC, we isolated MDSC from the spleen of tumor-bearing mice treated by poly I:C or saline, and co-cultured these cells with splenic T cells of healthy mice. We found that MDSC from poly I:C treated CT26-EGFRvIII and E0771-EGFRvIII tumor-bearing mice were significantly less suppressive on the beads-activated T cell proliferation than that isolated from mice treated with saline, as manifested by CFSE expression  and cellTo determine whether the decline of MDSC suppression activity following poly I:C treatment could also be observed in cytotoxicity, we examined the specific lytic function of CAR-T cells on target cells with the presence of MDSC. As shown in These data suggested that MDSCs from poly I:C treated tumor-bearing mice showed diminished suppressive potency compared with those from saline treated mice.As shown above, poly I:C treatment reduced MDSC quantity and diminished MDSC suppression on T cell proliferation and CAR-T lytic activity. Furtherly, we wanted to know whether the enhanced function of poly I:C was mainly due to the inhibition of MDSC. Mice bearing subcutaneous CT26-EGFRvIII tumor were i.p. injected with MDSC depletion agent one day prior to CAR-T cells injection. The depletion of CD11b+ Gr1+ MDSC in peripheral blood was verified by flow cytometry . TreatmeImmunosuppressive microenvironment has been regarded as the major issue for the limited success of CAR-T therapy on solid tumors. Combination of CAR-T therapy with immune checkpoint molecule inhibitors has been regarded as a promising strategy. For example, a patient with DLBCL and progressive lymphoma was first infused with CAR-modified T cells directed against CD19, and then was administered with anti-PD-1 antibody. Subsequently, the patient had a clinically significant antitumor response, an expansion of CAR-T cells, and decreased coexpression of PD-1 on CAR-T cells . BesidesSystemic administration of poly I:C, including intravenous and intraperitoneal injection, caused significant reduction of body weight and pulmonary thrombosis as well as hepatic necrosis of mice. This toxicity was related to dose and injection schedule. Moreover, weight loss showed a tendency to return to baseline levels with time . In the in vitro and in vivo. This was in accordance with previous report that poly I:C cells increased cytokines secretion induced by TCR-dependent and -independent stimulation, but did not affect their proliferation or specific cytolytic activity within short time (To explore the mechanism through which poly I:C enhances CAR-T therapy, we firstly investigated whether poly I:C exert direct impact on tumor and T cells. Previous studies report that poly I:C could directly induce proapoptotic and antiproliferation of many cancer cells , 30. Howort time .in vitro. Thus, we speculated that that IFN β-mediated therapeutic effect requires adaptive immunity, rather than direct killing of tumor cells. This may be due to that IFN β directly targets intratumoral dendritic cells, which reactivate CTL by increasing antigen cross-presentation within the tumor microenvironment (One of the main consequences of poly I:C administration is the secretion of type I IFNs. Indeed, we observed obviously elevated IFN β release after poly I:C treatment. Type I IFN signaling pathway blockade almost offsetted the enhanced antitumor activity mediated by poly I:C after CAR-T infusion. Reports demonstrated that type I IFNs can directly and potently induce apoptosis of tumor cells for tumor regression . But ourironment . In addiironment . What's ironment .MDSCs have emerged as important contributors to solid tumor immune evasion . These cin vivo, implicating the immunosuppressive solid tumor microenvironment as a modulator of CAR-T cell efficacy (Tumor-induced MDSC were associated with poor CAR-T cells efficacy against xenografts efficacy . After pefficacy .Poly I:C also inhibited the suppressive activity of MDSC on T cell proliferation and abrogated the immunosuppressive function of MDSC, concomitant with an increase in local T cell response of the immune system in a murine model of breast cancer . Our furIn summary, our study for the first time shows that administration of poly I:C can significantly enhance the therapeutic efficacy of CAR T-cell therapy in solid tumors. We also demonstrate the need for mobilizing host immune systems and limiting microenvironmental immunosuppressive activity of solid tumors. The combination of these two modalities is likely to have a significant impact on increasing the effectiveness of immunotherapy against a number of solid tumors, thus provide a new perspective to adoptive immunotherapy of cancer.in vitro and in vivo experiments. MZ and ZP took part in in vivo analysis. RS took part in the cell culture. MC helped a lot in the vector construction. HJ and BS helped a lot in the spleen T cell isolation and stimulation. HL took part in the retrovirus production and flow cytometry. ZL is the principle investigator of our research group.SD took part in the vector construction, ZL was employed by company CARsgen Therapeutics and has ownership interests of combined therapy of CAR-T cells with poly I:C. The remaining 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.


To do so, β-clamp inhibits the strand displacement activity of Pol I by interfering with the interaction between the finger subdomain of Pol I and the downstream primer-template junction. Conversely, β-clamp stimulates the 5’ exonuclease property of Pol I to cleave single nucleotides or shorter oligonucleotide flaps. This single nucleotide flap removal at high frequency increases the probability of ligation between the upstream and downstream DNA strands at an early phase, terminating nick translation. Besides β-clamp-mediated ligation helps DNA ligase to seal the nick promptly during the maturation of Okazaki fragments.The nick translation property of DNA polymerase I (Pol I) ensures the maturation of Okazaki fragments by removing primer RNAs and facilitating ligation. However, prolonged nick translation traversing downstream DNA is an energy wasting futile process, as Pol I simultaneously polymerizes and depolymerizes at the nick sites utilizing energy-rich dNTPs. Using an  E. coli cells, DNA polymerase III (Pol III) holoenzyme is the major replicative DNA polymerase [1τ2δδ‘χψ polypeptides) and β-sliding clamps at the primer-template junctions of a replication fork [E. coli [In replicating lymerase . Three Pion fork . The dimion fork –12. Suchion fork ,14. Immeion fork –17. The [E. coli –25. Thes[E. coli –25. Furt[E. coli . TherefoSynthesis of the leading strand DNA presumably requires a few priming events –30, whilE. coli and Bacillus subtilis [in vitro studies in the past have been done to illuminate the different functions of Pol I, as described above [in vitro experiments to assess the influence of β-clamp on the functions of Pol I and DNA ligase in the nick translation process. Our approach unraveled a hitherto unknown mechanism of clamp-assisted DNA gap repair in E. coli.After synthesizing an Okazaki fragment, the replisome dissociates from the β-clamp. As a result, the “left over” β-clamp remains attached at the 3’-side of the ssDNA gap or nick –41. Conssubtilis ,43. Pressubtilis ,44,45. Aed above –37,46, nth to 67th positions while traversing 28 bases downstream RNA oligonucleotide , as described in Materials and Methods section. Thus, as depicted in the We conducted the control experiments to show that the clamp loader could efficiently load β-clamp on the above-mentioned specific template types (see the detail in polA (the Pol I encoding gene) by site directed mutagenesis to disrupt the 3’ and 5’ exonuclease activities. The purified 3’-exonuclease-free (3’-exo-) and 5’-exonuclease-free (5’-exo-) Pol I were then used to dissect the role of 3’ and 5’ exonuclease activities of Pol I in the nick translation process. The nick translation mediated by the 3’-exo- Pol I , which lacks both 3’ and 5’ exonuclease functions, in the assay. The exo- Klenow exhibited a trail of scattered pause complexes, as judged by the band intensities between the 39th to 67th nucleotide positions  but retains both of the domains. Using the exo- Pol I, we show that the pattern of paused complexes  encoding β-clamp and minimal clamp loader complex  [NdeI and EcoRI sites of pET21a (+) vector and expressed as tag-less native proteins. The overexpressed proteins were purified using standard protocols [- Klenow, E. coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB). Furthermore, to get full length hexa-histidine-tagged Pol I, PCR amplified E. coli polA gene was cloned at the NdeI and HindIII sites of pET28a (+) vector. Site directed mutagenesis was performed on the pET28a-polA vector using Q5 Site-Directed Mutagenesis Kit from NEB to get 3’-exo- Pol I , 5’-exo- Pol I , and exo- Pol I  [+) was purchased from Sigma-Aldrich. The hexa-histidine-tagged β-clamp was purified by cloning and overexpressing dnaN gene as described in GGATCCCACAC TCATTAAAATTAGTCGCTAATGCATTCTAAAAGCATTCGCAACGAGAAGATAGA GG), 19 bases (CCTCTATCTTCTCGTTGC G), 28 bases (GCGACTAATTTTAATGAGTG TGGGATCC and GCGACUAAUUUUAAUGAGUGUGGGAUCC), and 38 bases (AGAA CTTGACGCGACTAATTTTAATGAGT GTGGGATCC) in lengths. The 5’-end of 28 bases primer was phosphorylated. The biotinylated template made for the control experiments are described in PCR of the genes ( and δ‘)  were per and δ‘) . The PCRrotocols ,52. Pol , E357A) ,53. The 32P-γATP. The radiolabeled oligonucleotides were purified using BioGel P-6 columns  to remove the salts and free radioactivity. Templates were prepared as described previously [+) to catalyze the ligation process. The final reaction products were separated by urea denaturing PAGE and were exposed to the phosphor-imager screen. A Fujifilm-9000 phosphor-image scanner was used to visualize the bands. Counts of the bands, or the desired stretches of the lanes, were recorded for further analyses. A minimum of three experiments were performed for each reaction to get the average counts and standard deviations. The relative ligation frequency (RLF), i.e. fraction of nick translating products that take part in ligation at 30 seconds time-point, was calculated, as defined below. If, X = average counts of the 67-nucleotide long products generated in the presence of ligase, Y = average counts of the 67-nucleotide long products generated in the absence of ligase, Z = average counts of the products between 39th and 67th nucleotide positions, then RLF = (X-Y)/Z. Nick translation, single nucleotide and 10 nucleotides cleavage rates were calculated from the initial slopes of the fitted curves, as described in the 5’-end radiolabeling of the desired oligonucleotides were performed using T4 polynucleotide kinase (NEB) and eviously ,54 usingThe assay was designed with modifications to the protocol mentioned . BrieflyGeobacillus stearothermophilus. Klenow complexed with 9 base pairs of duplex DNA (PDB ID 1L3S). The additional upstream and downstream DNA segments were built using COOT , 28 nucleotides and 67 nucleotides (lane 3) long oligonucleotides. When they mixed in an annealing reaction, the individual oligonucleotides disappeared from their original position and gave rise to a higher band (Lane 4), suggesting that annealing was 100% efficient. However, non-complementary oligonucleotides (19 and 28 bases) did not found to be annealed (lane 5). On the other hand, the 67 bases long oligonucleotide, which has complementary regions for both 19 and 28 bases long oligonucleotides, has also completely annealed to these 19 and 28 bases oligonucleotides .(TIF)Click here for additional data file.S2 FigThe fraction of the initial counts that appeared at 67 nucleotide position were plotted. The rate of the nick translation through RNA substrate was calculated from the fitted curve shown above also see . The neg(TIF)Click here for additional data file.S3 Fig(A) The lanes 2–5 are showing purified individual proteins that are used in the pull down assay. The streptavidin-bound primed biotinylated template could pull-down β-clamp (marked by white arrow), while clamp loader proteins washed away . (B) The his-tag β clamp pull down primed template, as indicated by the pulled down radiolabeled 19nt long oligonucleotide, which was the integral part of the template.(TIF)Click here for additional data file.S4 FigThe urea denaturing gel indicating that washing the streptavidin-bound reaction products, which washes out the clamp loader protein, but retains β clamp , generat(TIF)Click here for additional data file.S5 Fig(A) The autoradiogram represents nick translation product formation at three different time points by Pol I and 3’-exo- Pol I. (B) The rate of nick translation was calculated as described in - Pol I.(TIF)Click here for additional data file.S6 Fig(A). A new template was assembled using 67 bases, 5’-phosphorylated 38 bases and 5’-radiolabelled (asterisk) 19 bases oligonucleotides, so that a 10 nucleotide long 3’ overhang was generated as shown. (B) The autoradiogram represents Pol I mediated nick translation-coupled ligation in the presence or absence of β-clamp. Visibly, 77 nucleotide ligation product that appears at lower time points, degraded quickly due to 3’ exonuclease activity of intact Pol I.  The representative autoradiograms show that 3’-exo- Pol I and exo- Pol I produced stable nick translation-coupled ligation products in the presence or absence of β-clamp in three different time points. (E) RLFs calculated at different time points in the presence or absence of β-clamp from panels (TIF)Click here for additional data file.S7 Fig- Pol I and dNTPs exhibited pausing at the 39th and downstream locations at two different time points. Addition of the Pol I and ligase efficiently produces 67nt products, suggesting that in the absence of clamp loader complex the nick translation coupled ligation reactions are similar as demonstrated in The nick translation coupled ligation assay was performed on streptavidin beads to remove clamp loader proteins after loading of the β-clamp. Addition of 3’ exo(TIF)Click here for additional data file.S8 Fig(A). Single nucleotide cleavage was shown in (B) Similarly, flap cleavage was shown in (TIF)Click here for additional data file.S9 Fig(A) Cartoon representation of the docked model of Klenow (cyan) /β-clamp (gray and blue) protein complex. The possible protein-protein interaction interfaces are encircled in red and blue. (B) Molecular dynamics simulations for the docked Klenow/β-clamp protein-protein complex. (C) Surface representation of the modelled Klenow (grey) /β-clamp (yellow and blue)/ DNA (orange and green) ternary complex.(TIF)Click here for additional data file.S10 FigE. coli small domain of Pol I (light pink) was built using Prime module in the Schrodinger software suite using 1BGX as the template. The models were manually placed near the Klenow/nicked DNA (A) and Klenow/ β-clamp/nicked DNA (B) models using PyMOL. The predicted flexible linker region connecting small domain and Klenow fragment has been shown by undulating lines in different grey shades. The conformational space available in β-clamp-bound and β-clamp free Klenow/nicked DNA structures are shown by multiple copies of small domains. From this model, we hypothesize that the presence of β- clamp could sterically restrict (shown by red T blocker) the conformational freedom of small domain hence increasing the chance of accessing 5’ exonuclease site.The structural model of (TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Citrobacter rodentium infection. A single oral dose of retinoic acid (RA) at d7 post-infection was sufficient to induce clearance of the pathogen in A– mice. RA treatment of A– mice induced il17 expression in the colon. In A– mice, colonic IL-17 was primarily produced by CD11b+ cells; however, in A+ mice, the major source of colonic IL-17 was CD4+ T cells. To determine the cellular targets of vitamin A required for host resistance to C. rodentium, mice that express a dominant negative (dn) retinoic acid receptor (RAR) in T cells (T-dnRAR) or macrophage/neutrophils (LysM-dnRAR) were used. T-dnRAR mice had T cells that produced a robust intestinal IL-17 response and for 40% of the mice was enough to clear the infection. The remainder of the T-dnRAR mice developed a chronic infection. A– LysM-dnRAR mice developed early lethal infections with surviving mice becoming chronically infected. RA treatment of A– LysM-dnRAR mice was ineffective for inducing colonic IL-17 or clearing C. rodentium. Retinoid signaling is required in T cells and CD11b+ cells for complete elimination of enteric pathogens.Vitamin A deficiency affects over 250 million preschool-age children worldwide and is associated with increased childhood mortality and risk of developing enteric infections. Vitamin A deficient (A–) mice developed chronic  Citrobacter rodentium, a gram negative murine enteric pathogen, developed a severe infection, including premature mortality of 40% of the A– mice  that produce IL-22 and IL-17 (ILC3) and clearance of infection requires robust Th17 cell responses  is an important public health problem in resource limited countries including areas in sub-Saharan Africa and southeastern Asia. The most recent World Health Organization studies estimate that 250 million pre-school age children worldwide are vitamin A deficient and these children have increased rates of enteric infection , 2. Vitaesponses , 7. The esponses . IL-17 e WT mice . Host rein vitro (C. rodentium infection and the reduced colonic inflammation corresponded with lower IL-17 expression (Toxoplasma gondii had lower IL-17 responses and RA treatments in this model induced more T cells that produced IL-17 (Vitamin A is a well described regulator of immune cells. Retinoic acid (RA) has been reported to suppress IFN-γ and IL-17 production from T cells in vitro , 11. Thein vitro . RA induin vitro , 13. RA pression –16. Paraed IL-17 , 17.C. rodentium in the A–host were determined. A single dose of RA was sufficient to promote host survival and clearance of C. rodentium infection in A– mice. Although RA was protective, retinoid repletion of the A– host resulted in an immunologically and metabolically distinct phenotype, different from both A+ and A– mice. RA mediated protection from C. rodentium was associated with an increase in colonic IL-17 production. Sixty percent of mice that expressed a dominant negative (dn) retinoic acid receptor (RAR) in T cells (T-dnRAR), developed a chronic C. rodentium infection. Infected A+ T-dnRAR mice had robust IL-17 production by T cells in the colon that for some mice (forty percent), was effective for inducing clearance of the infection. RA treatments of the T-dnRAR was ineffective for clearing C. rodentium. IL-17 production in the A– host was derived primarily from CD11b+ cells rather than T cells. RA treatment of A– LysM-dnRAR mice was ineffective for induction of CD11b+ mediated IL-17 or for clearance of the infection. The effects of retinoids in A– mice are distinct from those in A+ mice, and retinoid regulation of CD11b+ and T cell function contributes to effective protection of the host from C. rodentium infection.We hypothesized that the effects of RA depended on the vitamin A status of the host. Here, the mechanisms and targets underlying RA-mediated clearance of −/− (WT littermates) and dnRAR fl/fl cre +/– (T-dnRAR or LysM-dnRAR) were used for experiments. All mice were bred at the Pennsylvania State University  according to university and IACUC guidelines. A+ and A– mice were generated by breeding C57BL/6J WT animals on lab prepared purified diets as described previously with and without retinyl acetate (25 μg/d) . The dnRAR fl/fl mice  were a g25 μg/d) . A– miceSerum samples were saponified and quantified for total retinol concentration by ultra-performance liquid chromatography (UPLC) . BrieflyC. rodentium strain ICC169  was a kind gift from Gad Frankel . Bacteria were grown and cultured in Difco Luria-Bertani broth and agar  containing 50 μg/mL nalidixic acid . Overnight cultures containing log phase bacteria were used to prepare inoculums. Individually housed mice were fasted overnight and then orally gavaged with 5 × 109 CFU C. rodentium in 100 μl of sterile saline. Feces and organs were collected, homogenized, and plated in serial dilutions on LB agar plates containing nalidixic acid to quantify bacterial burdens.6 colonic lymphocytes for 4 h with phorbol 12-myristate 13-acetate , and ionomycin (6 μg/mL) in the presence of brefeldin A  St. Louis, MO). Cells were stained with fluorescein isothiocyanate (FITC) CD8β, FITC GL3 (γδ TCR), phycoerythrin PE GL3, PE-CF594 CD4, PE-CF594 GR-1, PE-Cy 5 T cell receptor β (TCRβ) , PE-Cy5 CD11b , PE-Cy7 F4/80, PE- PE-Cy7 CCR9, or PE-Cy7 CD8α . Intracellular cytokine staining was performed using PE IL-17 and the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Single positive and fluorescence minus one (FMO) controls were used for gating purposes. Cells were analyzed on an FC500 benchtop cytometer , and data was analyzed using FlowJo 7.6.5 software .Single cell suspensions of spleen, MLN, thymus, small intestinal (SI) intraepithelial lymphocytes (IEL) and colonic lamina propria (LP) cells were isolated as described previously –24. PeyeWhole tissues were snap frozen in liquid nitrogen and stored until processing. RNA was isolated using TriZOL  as described in the manufacturer's protocol. Complementary DNA (cDNA) was created by reverse transcribing 4–5 μg of RNA using the TaqMan reverse transcription reagents kit . RT-PCR was performed using SYBR green mix  and the MyiQ Single-Color Real Time PCR machine (BioRad). Standards were prepared by serially diluting DNA products for each gene of interest. Gene expression levels were then normalized to one or more housekeeping genes (HPRT and/or GAPDH). Fold change values were reported relative to untreated or un-infected control tissues which were set at 1. Primers for RT-PCR are listed Supplementary Table 1H NMR spectra were recorded at 298 K on a Bruker Avance III 600 MHz spectrometer equipped with an inverse cryogenic probe . NMR spectra of liver samples were acquired for each employing the first increment of NOESY pulse sequence (NOESYPR1D) with the recycle delay (RD) of 2 s and mixing time (tm) of 100 ms. NMR spectra of samples were acquired using the Carr-Purcell-Meiboom-Gill sequence [RD-90°-(τ-180°-τ) n-acquisition]. The 90° pulse length was adjusted to about 10 μs for each sample and 64 transients were collected into 32 k data points for each spectrum with spectral width of 20 ppm. The chemical shift of 1H NMR spectra were referenced to TSP. Each bucketed region (0.004 ppm) was then normalized to the total sum of the spectral integrals prior to statistical data analysis. Principal component analysis (PCA) and orthogonal projection to latent structure-discriminant analysis (OPLS-DA) were carried out using the SIMCA-P+ software .The liver samples for NMR was prepared as previously described . 1H NMR t-tests were used to compare normally distributed data with only two groups. A student's t-test with Welch's correction was also used to compare data sets with unequal variances. Two-way ANOVA with Bonferroni's post-hoc test was used to compare fecal shedding curves and mRNA abundance through time and by treatment. A P ≤ 0.05 was used as the cut off for a significant change.Statistical analyses were performed using GraphPad Prism . Two-tailed student's C. rodentium and left untreated (A–) or dosed with RA starting on the day of infection (RA d0) or at d7 post-infection (RA d7) and the RA dosing was continued throughout the experiment. RA dosing beginning at either d0 or d7 induced clearance of the C. rodentium infection  revealed that A+ and A– mice had unique liver Figure  metaboliC. rodentium infected A– mice were untreated or dosed with RA 1x (d7) or RA 2x (d7 and d9) and sacrificed on d8 or d10 post-infection . The shedding kinetics of C. rodentium in the WT littermates  out to d48 post-infection  and macrophages (P < 0.05) that produced IL-17 increased in the RA 2x treated mice compared to the A– controls  littermates resembled those in Figure C. rodentium by d29 post-infection  of The Pennsylvania State University, University Park, PA, USA. The protocol was approved by the IACUC committee of The Pennsylvania State University, University Park, PA, USA.MC, LS, KM, and YT conceptualized and designed the experimental studies. LS, KM, and YT performed the experiments and acquired, and analyzed the data. C-HW quantified serum retinol levels and MK scored histology slides. LS drafted the manuscript with the help of MC, YT, and C-HW. MK, AP, AR, and MC critically revised the manuscript. All authors approved the publication of the manuscript.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.


PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients.Parkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic   •Patient-derived DA neurons recapitulate several PD-related disease phenotypes•Establishment of a system for drug screening against PD using patient-derived cells•Calcium channel antagonists suppress rotenone-induced apoptosis in PARK2 DA neurons•The involvement of dysregulated T-type calcium channels in the progression of PD Our study demonstrate the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD patient-derived DA neurons, which are further prevented by T-type calcium channel antagonists. These findings suggest that calcium homeostasis in DA neurons would be a useful target for developing new drugs for PD patients. PINK1 (PARK6) and PARKIN (PARK2) are involved in mitochondrial homeostasis and stress responses (Parkinson disease (PD) is one of the most common neurodegenerative disorders, and PD patients display progressive motor dysfunction such as tremor, bradykinesia, rigidity and postural instability due to a preferential loss of dopaminergic (DA) neurons in the substantia nigra (SN) . While tesponses . PARK1/4esponses , and PAResponses . TherefoThe recent progress of human induced pluripotent stem cell (iPSC)-assisted technology presents new opportunities for disease modeling based on precise cellular contexts, which had previously been difficult to obtain, especially in neurodegenerative disorders . In the PARK2 null (PARK2−/−) iPSC-derived DA neurons  for in vitro disease modeling and found that these neurons exhibited neurite abnormalities, elevated oxidative stress, and apoptosis. We also performed a phenotypic screening to identify neuroprotective compounds and identified benidipine, a voltage-gated calcium channel antagonist, as a potential chemical targeting PD. Importantly, we found that the selective vulnerability of DA neurons to rotenone-induced stress in PARK2 was attributable to the dysregulation of intracellular calcium homeostasis via T-type calcium channels. In summary, we have established a robust platform to model PD in a dish and revealed an additional layer of the pathogenesis of PD, offering a potential therapeutic target.Here, we developed an efficient directed differentiation protocol to generate DA neurons with less variation of efficacy than previous protocols . We usedin vitro disease model using PD patient-specific iPSC-derived DA neurons (PARK2−/− iPSC line (B7PA21) were also generated to evaluate the disease-relevant phenotypes in the same genetic background + cells, indicating that astrocytic differentiation was negligible. Importantly, tyrosine hydroxylase (TH)+ neurons constituted more than 40% of the βIII-tubulin+ neurons , engrailed-1 (EN1), nur-related factor 1 (NURR1), and G-protein-activated inward rectifier potassium channel 2 (GIRK2)  , further (GIRK2) D. Becaus (GIRK2) A–S2E, we (GIRK2) E. Furthe (GIRK2) F. Taken PARK2−/− iPSC line , which labels apoptotic cells . In addition, we observed significantly increased intracellular calcium levels in the rotenone-treated PARK2 iPSC lines  in the rotenone-exposed cell-based assay was found to be similar to the therapeutic range of this drug in clinical use (3.7–7.4 μM as an antihypertensive) , an uncoupler that is known to dissipate mitochondrial membrane potential and induced entry of extracellular calcium through L/N-type calcium channels  , this cotensive) . Althougchannels . Similarnidipine . TherefoBecause dysregulated mitochondrial function occurs ubiquitously in the PD brain, additional cell-intrinsic circumstances in midbrain DA neurons might determine cell-type-specific vulnerability, triggering selective death of DA neurons in PD patients. For example, transcriptional regulation of T-type calcium channels in DA neurons is of interest based on our findings that the PARK2-DA neurons expressed more T-type calcium channels  and S5. in vitro disease model that recapitulates several PD-related disease phenotypes; this model can be used to investigate the pathogenesis of the disease and search for potential therapeutic targets for PD.In summary, our study indicates that PARK2-DA neurons provide an A detailed description of the experimental procedures is given in the The iPSC line 201B7 , PARK2 iWe used an FDA-approved drug library consisting of 1,165 biologically active compounds . The PAR50 was also calculated using GraphPad Prism.Statistical analysis was performed using GraphPad Prism 7.0 . The data are described as the means ± standard error of the mean (SEM). Unpaired t tests were used for pairwise comparisons between the groups. One-way analysis of variance (ANOVA) followed by Dunnett's test and two-way ANOVA followed by either Tukey's test or a t test with Sidak's correction were applied for multiple comparisons. The results were considered statistically significant when p < 0.05. The ICAll the experimental procedures were approved by the Keio University School of Medicine Ethics committee .Study concept and design: Y.T., J.K., and H.O.; data acquisition and analysis: Y.T., Y.I., M.S., T.A.-N., S.B., M.C., T.S., K.Y., H.S., and N.H.; drafting the manuscript and figures: Y.T., M.I., K.T., J.K., and H.O.; and reviewing and editing the manuscript: all authors.