Our results reveal interplay between A- and B-type lamins on radial locus positioning, suggesting complementary contributions to large-scale genome architecture. The data also unveil a hitherto unsuspected impact of cytotoxic drugs on genome conformation.Abbreviations: ChIP-seq: chromatin immunoprecipitation sequencing; CsA: cyclosporin A; FISH; fluorescence in situ hybridization; ICMT: isoprenylcysteine methyltransferase; LAD: lamina-associated domain; TAD: topologically-associated domainImmunosuppressive drugs such as cyclosporin A (CsA) can elicit hepatotoxicity by affecting gene expression. Here, we address the link between CsA and large-scale chromatin organization in HepG2 hepatocarcinoma cells. We show the existence of lamina-associated domains (LADs) interacting with lamin A, lamin B, or both. These ‘A-B’, ‘A-only’ and ‘B-only’ LADs display distinct fates after CsA treatment: A-B LADs remain constitutive or lose A, A-only LADs mainly lose A or switch to B, and B-only LADs remain B-only or acquire A. LAD rearrangement is overall uncoupled from changes in gene expression. Three-dimensional (3D) genome modeling predicts changes in radial positioning of LADs as LADs switch identities, which are corroborated by fluorescence  Drug-induced hepatotoxicity, a common cause of clinical trial failure, has led to the use of cellular models such as HepG2 hepatocarcinoma cells for drug testing ,2. At miThe mammalian genome is organized into compartments of active and inactive chromatin  and withLamin-chromatin interactions can be altered during differentiation ,15,18–20in situ hybridization (FISH). The data suggest an A- and B-type lamin interplay in radial genome conformation and reveal unsuspected effects of cytotoxic compounds such as CsA on nuclear organization.In light of these observations, to address the relationship between CsA exposure and genome organization, we examined here the effect of CsA on the association of chromatin with nuclear lamina. We report an impact of CsA on the dynamics of interactions of lamins A and B with the genome. Such interactions can be gained, lost or interchanged in a manner uncoupled from gene expression changes. They also correlate with distinct patterns of radial repositioning of loci predicted from 3D genome models and confirmed by fluorescence −5], paired t-tests relative to controls; LMNA or LMNB1 transcript levels  and lamin A LADs (‘A-LADs’) by chromatin immunoprecipitation-sequencing (ChIP-seq) of lamin B1 and lamin A/C, respectively. Of note, the anti-lamin A/C ChIP antibody (Santa-Cruz sc7292x) immunoprecipitated not only lamin A/C but also pre-lamin A ), rulingde novo A-LADs with pre-existing B-LADs.We therefore examined the overlap of A- and B-LADs in control and CsA-treated cells by computing Jaccard indices (Ji) of their genome coverage ). We finTo further investigate the relationship between A-LADs and B-LADs, we divided these into LAD classes associated with lamin A only, lamin B only and both lamins A and B  and analyzed the properties of these LADs in control and CsA-treated cells (Supplementary Table S3). We find that (i) genome coverage by B-only LADs slightly increases ) due to de novo acquisition of lamin A on B LADs inferred from Jaccard indices . These −3; unpaired t-tests; –8 respectively, unpaired t-tests; We next determined whether LAD dynamics was associated with changes in gene expression. Genes in A-B, A-only and B-only LADs are not significantly differently expressed after CsA treatment , P > 0.01, unpaired t-tests) although trends are detectable. We thus asked whether genes in each LAD class in control cells showed altered expression as a function of LAD class fate after CsA treatment. Due to the low number of genes in LAD classes of CsA-treated cells, many LAD class changes were not associated with significant expression changes ). Howeve−16, unpaired t-tests; To assess whether a gain or loss of LAD or a change in LAD identity would coincide with changes in the radial positioning of these domains, we generated 3-dimensional (3D) models of the HepG2 genome and analyzed properties of the models. We used Chrom3D, a 3D genome modeling platform that integrates TAD-TAD interactions from Hi-C data, and genome interactions with the nuclear periphery from lamin ChIP-seq data ,28. Our −16). Moreover, retention of a LAD class does not result in significant repositioning. Significantly however, loss of lamin B from an A-B LAD (pair 2) or a switch from a B-only to an A-only LAD (pair 10) correlates with repositioning towards the center (P < 2.2 × 10−16). In contrast, gain of lamin B resulting from an A-only to B-only switch (pair 7), a de novo gain of A-B (pair 13) or a de novo gain of B-only (pair 15), correlates with repositioning towards the periphery (P < 2.2 × 10−16). Lastly, gain of lamin A does not correlate with significant repositioning ; however the impact of lamin A loss on radial placement depends on whether lamin B is bound in the region . Analysis of 3D genome models therefore predicts a dominant impact of lamin B over lamin A on radial positioning of the genome.We next estimated the radial repositioning of each LAD class according to their fate in CsA-treated cells ). Loss o−16, t-tests) . Thus, FISH data validate predictions from the 3D models and confirm that lamin B association influences the radial distribution of loci more prominently than lamin A.To independently validate Chrom3D model predictions on locus repositioning, we designed FISH probes to loci found in specific LAD classes in the ChIP-seq data (Supplementary Table S5). We also positioned in Chrom3D models the loci targeted by each FISH probe and measured their distance from the nucleus center ), consisper se. To address this, we expressed a Flag-tag wild-type (wt) pre-lamin A construct. Since, as we have shown earlier, wt pre-lamin A is fully processed into mature lamin A27, we also expressed a lamin A mutant (L647R) exclusively localized at the nuclear periphery as unprocessed pre- lamin A27 by retention of its pre-lamin A farnesyl group [Pre-lamin A accumulation following CsA exposure see ) could iyl group . We thenper se. Repositioning is probably independent of pre-lamin A interaction with chromatin; it is however likely dependent on lamin B, as discussed above.The data strikingly reveal no significant repositioning of the three loci in lamin A L647R vs. wt cells . Thus, overexpression of pre-lamin A (as lamin A L647R) does not recapitulate locus repositioning detected in CsA-treated cells (which show some pre-lamin A expression). This suggests that relocalization observed after CsA treatment is a CsA-linked phenotype and not a pre-lamin A expression phenotype We report a marked effect of CsA on the association of chromatin with A- and/or B-type lamins. We notably reveal distinct patterns of lamin A and B interaction with chromatin, resulting in variable LAD identities and correlated with a radial repositioning of loci concordant with predictions from 3D genome models. An account of redistribution of lamin A and B (A-B) LADs, A-only and B-only LADs between two cellular states provides biochemical evidence for previous microscopy-based observations of distinct lamin A and B micro-domains . Our resStudies of drug hepatotoxicity have revealed an accumulation of pre-lamin A in HepG2 cells exposed to an inhibitor of isoprenylcysteine carboxylmethyltransferases (ICMTs) . Pre-lamat the nuclear periphery at the single-cell level. In addition, lamin B-chromatin interactions detected by ChIP from millions of cells do not imply peripheral association of loci in all cells. Our results extend previous findings [12 [A key question in understanding spatial genome architecture is whether changes in lamin-chromatin interactions reported here occur at the nuclear periphery or in the nuclear interior. Pre-lamin A is detected at the nuclear envelope but could also be nucleoplasmic (if not farnesylated ), suggesfindings ,32,33 anfindings . They alings [12 ,18,35,36ings [12  or euchrings [12 .Lamins A and B are important regulators of spatial genome organization ,22,38, a21 but also a more local depletion promotes a restricted conformation change manifested by increased chromatin mobility. It will be relevant to investigate the impact of a local loss of LAD on the biophysical properties of chromatin in the vicinity.Global loss of lamin A results in increased chromatin mobility in the nuclear interior . A loss de novo, we predict and observe locus repositioning towards the periphery. In contrast, in the local absence of lamin B, A-LAD loss correlates with repositioning towards the center. This suggests that lamin B is an important factor in the directional radial organization of chromatin. The surrounding LAD neighborhood could also influence LAD behavior: LAD repositioning may not only depend on the LAD class per se, but also on LADs in the vicinity. For instance, absence of repositioning of a B-only LAD towards the nuclear center after losing lamin B, as seen by FISH, could be due to an A-B LAD in the vicinity. Thus, radial genome positioning may be influenced by a combination of both local and medium-range contributions of the linearly nearest region. This idea extends our previous observations that a lamin A neighborhood affects expression of genes in that neighborhood even though they are not bound by lamin A18.Notwithstanding, repositioning of A-LADs upon loss of lamin A depends on whether lamin B is in the neighborhood. When a lamin B LAD appears These findings raise the question of which factors may regulate the radial positioning of chromatin. Nuclear envelope proteins , histoneIt is becoming increasingly clear that patterns and mechanisms of nuclear lamin association with the genome are more complex and dynamic than previously understood. Functional and live-cell imaging studies are needed in order to fully understand the mechanisms by which lamins contribute to organizing the 3D conformation of chromatin in the nucleus.HepG2 cells were cultured in DMEM/F12 (Life Technologies) with 10% fetal calf serum and 1% penicillin-streptomycin. Cells were also cultured with 10 µM CsA  for 72 h before harvest.HepG2 cells were transiently transfected with pCMV-Flag-preLA-WT (lamin A wt) and pCMV-Flag-preLA-L647R (pre-lamin A L647R) plasmids  using LiProteins were separated by 10% SDS-PAGE, transferred onto an Immobilon-FL membrane (Millipore) and membranes blocked with Odyssey blocking buffer (LI-COR). Membranes were incubated in Odyssey blocking buffer with antibodies against lamin A/C Cells cultured on coverslips were fixed with 3% paraformaldehyde and permeabilized for 10 min in PBS/0.1% Triton X-100. Cells were blocked with 2% BSA in PBS/0.1% Tween (PBST-BSA) for 30 min and incubated with primary antibodies in PBST-BSA overnight at 4°C. Cells were incubated with secondary antibodies in PBST-BSA for 45 min and washed in PBST-BSA before mounting with DAPI . Antibodies used were mouse anti-lamin A/C , goat anti-pre-lamin A , anti-mouse Alexa Fluor® 594 and anti-goat Alexa Fluor® 488 .oC with Avidin-Alexa Fluor 488 . Slides were washed in 4x SSC/0.1% Tween 20 and incubated with Biotinylated Anti-Avidin D  for 45 min at 37°C. Slides were washed and incubated with Avidin-Alexa Fluor 488 as above and mounted with DAPI in Dako Fluorescent Mounting Medium.FISH was done as described . Cells wBased on ChIP-seq profiles, FISH probes were designed for regions changing LAD class after CsA (Supplementary Table S5). Since HepG2 cells harbor copy number variations, probes were designed outside these areas. 100 FISH images  were analyzed using FISHfinder  to calcuImmuno-FISH was done as described . Transfe6 cells per ChIP) were cross-linked with 1% formaldehyde and lysed in RIPA buffer . To generate 200–500 bp DNA fragments, cells were sonicated 4 × 10 min in a Bioruptor (Diagenode). After sedimentation, the supernatant was diluted 10x in RIPA and incubated with anti-lamin A/C  or lamin B1  antibodies coupled to Dynabeads Protein G (Invitrogen). ChIP samples were washed 3x in ice-cold-RIPA. Samples were incubated for 6 h at 68°C in elution buffer  and 40 ng proteinase K for crosslink reversal and DNA elution. DNA was extracted and libraries prepared and sequenced on an Illumina HiSeq2500.ChIP was done as described . Cells (ENCODE ChIP input data for HepG2 cells were downloaded from NCBI GEO accession No. GSE29611. Lamin A and B ChIP-seq reads and input reads were mapped to hg19 using Bowtie v2.25.0  with defHi-C data of HepG2 cells were downloaded from ENCODE (GEO accession No. GSM2825569)  and procWe applied Chrom3D to generate 3D genome models from Hi-C and lamin B1 ChIP-seq data ,28. We gTotal RNA was isolated from two CsA exposure experiments using the RNeasy Mini Kit (Qiagen). Libraries were sequenced on an Illumina HiSeq2500. RNA-seq reads were processed with Tuxedo . TopHat FPKM levels from RNA-seq data were compared using unpaired t-tests. For LAD size comparisons, a 2-way ANOVA was first applied to determine the global significance of CsA treatment vs. control. A Tukey honest significant difference (HSD) test was then applied to determine the significance of any difference between LAD classes. Comparisons of normalized LAD distances to the nuclear center in Chrom3D models and in FISH analyses were done using unpaired t-tests.Our ChIP-seq and RNA-seq data are available under GEO accession No. GSE119631. ChIP input data are available under GEO accession No. GSE29611. Hi-C data for HepG2 cells  are avai


Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids. In plants, it is largely accepted that import pathways that target proteins into and across the plastid envelope are critical for plant development by regulating the response to physiological and metabolic changes within the cell –4. On th–6–in vitro import assays  and outside plastids (implying that plastid import did not occur). Here, we show that the calmodulin isoform CaM5A plastid protein, termed ceQORH AT4G13010), originally predicted as a quinone oxido-reductase homolog, was recently shown to reduce long-chain, stress-related oxidized lipids that are spontaneously produced in the chloroplast envelope from the unstable allene oxide formed in the biochemical pathway leading to 12-oxo-phytodienoic acid, a precursor of the defense hormone jasmonate 010, orig. Targete16–t assays –21. Howeplastids  but was A). Then we compared the CaM-binding properties of the recombinant ceQORH protein produced in Escherichia coli with those of the endogenous protein ecQOR (as a negative control), the closest bacterial homolog of plant ceQORH (B). As demonstrated in Fig. 1C, although the plant ceQORH is detected in the elution fraction of CaM affinity chromatography, its bacterial homolog is only detected in the pass-through fraction, indicating that it does not bind CaM.In the present study, we provide several lines of evidence demonstrating the specific CaM-binding property of ceQORH. First, the natural plant ceQORH was enriched in the fraction eluted from a calmodulin-affinity resin when compared with its level in crude cell extracts A. Then wt ceQORH B. As demA) and expressed these constructs in E. coli (B). Using these experiments, we were able to demonstrate that this CaM-binding domain is located in the C terminus of ceQORH (B and C) between residues 229 and 280 of the protein. In this region, we searched for a putative CaM-binding site within this domain using the CaM prediction program  the following candidate motif: 255AMWTYAVKKITMSKKQLVPLLL277. CaM-binding peptides have the potential to fold into a basic, amphiphilic α-helix  within the helix or adjacent to this helix according to the recently established 3D structure of ceQORH . As deduced from CaM overlay experiments performed on purified recombinant proteins , mutagenesis of only three residues abolishes the CaM-binding properties of ceQORH. This mutated form, termed Mut2-ceQORH, was thus considered as the best tool for further approaches.To establish the location of the CaM-binding domain of ceQORH, we first performed successive deletions of the ceQORH protein A and exp E. coli B. Using f ceQORH , B and C program , which isee Ref.  in whichf ceQORH . Three oArabidopsis transformants expressing one truncated form  and one mutated form  of ceQORH and examined the subplastidial location of these mutated forms. Note that to limit artifacts resulting from overexpression of the various ceQORH constructs, transgenic plants were selected for expression levels of recombinant proteins similar to endogenous ceQORH level . Strikingly, whereas the full-length ceQORH is enriched in purified chloroplast envelope fractions (A), ceQORH lacking its C terminus also accumulates in thylakoid membranes at a similar level (C). The simplest interpretation is that the C terminus of ceQORH (which contains its CaM-binding domain) also promotes selective location at the envelope membrane. However, Mut2-ceQORH only lacking CaM-binding properties, is also targeted to the chloroplast and only detected in the purified envelope fraction and not in thylakoids (B), similarly to the endogenous ceQORH protein. This clearly demonstrates that the CaM-binding domain of ceQORH is not essential, in planta, for its targeting to the chloroplast or for its specific association to the chloroplast envelope.To determine whether the CaM-binding properties of ceQORH are responsible for the targeting of this protein to the chloroplast envelope, we established stable ractions A, ceQORHar level C. The siylakoids B, similaD). Furthermore, a function of CaM associated to ceQORH independently of the envelope membrane was further provided through quantification of CaM within purified envelope fractions. Indeed, through dilution and Western blotting analyses, we observed that although representing ∼1/2000 (∼10 ng CaM in 20 μg of crude leaf extract) of all leaf proteins , CaM should represent less than 1/600,000 (less than 0.1 ng of CaM in 60 μg of envelope proteins) of all envelope proteins. Knowing that ceQORH represents 2.5% of envelope proteins  CaM isoforms and tens of CaM-like proteins  than classical (shorter) CaM isoforms (i.e. 16 kDa). This high-molecular-mass signal was also enriched in the membrane fraction of epidermal cells , whereas the soluble fraction contained CaM isoforms of lower molecular mass. Finally, this high-molecular-mass CaM signal was 10–20 times more enriched in epidermal cells  compared with crude leaf extract  . In screening databases for a membrane-bound CaM-like isoform of higher molecular mass than classical (short) CaM isoforms, we identified CaM53 from petunia (CALM3_PETHY), a CaM isoform that contains an additional C-terminal sequence compared with short CaM isoforms  . A close homolog of this CaM53 protein from petunia is present in Arabidopsis, and this isoform is named CaM5 (AT2G27030.3). Compared with other CaM isoforms, this CaM5 isoform contains an additional C terminus sequence that is highly conserved  with CaM53 from petunia (CALM3_PETHY). Notably, using AT2G27030 as a query in the Uniprot database, only a classical (short) version  of CaM5 is referenced (Q682T9 or TCH1 or CALM5_ARATH). However, in the TAIR database, a query using the term AT2G27030 results in the gene model AT2G27030.3, which encodes a 181-amino acid protein with a molecular mass of 20,575.9 Da (i.e. similar to CaM53 from petunia).Having previously noted that ceQORH was mainly present at the periphery of plant cells in epidermal tissues , we deciFig. 5i.e. where ceQORH is mostly located at the periphery of the cells), we were encouraged to pursue its analysis using confocal imaging. First, ceQORH and CaM5 were colocalized at the periphery of plant cells in epidermal cells  (A). Because of its lack of CaM-binding properties, the Mut2-ceQORH mutant was used as a negative control during these experiments . Strikingly, although both ceQORH and Mut2-ceQORH accumulate at similar levels in plant cells , BiFC was only detected using the WT version of ceQORH (i.e. containing the CaM-binding domain) and mostly at the periphery of leaf cells  as a candidate for the most abundant (and high-molecular-mass CaM) isoform detected in epidermal cells  A. Becausi.e. ceQORH lacking its CaM-binding domain) in planta and analyzed its subcellular localization compared with that of the WT form of ceQORH. Again, to limit artifacts resulting from overexpression of the various GFP fusions, the selected transgenic plants were the above-used ones , in which the expression levels of GFP fusions are similar to endogenous ceQORH level. Interestingly, whereas ceQORH-GFP is never detected in plastids in epidermal cells , Mut2-ceQORH accumulates at the periphery of plastids , thus most likely in their envelope membrane, in agreement with Fig. 4B. The simplest interpretation is that, in planta, the CaM-binding domain of ceQORH promotes an alternative subcellular location of ceQORH via its retention in the cytosol by CaM5.We then expressed Mut2-ceQORH , in contrast to the location pattern (exclusively at the periphery of the cells) observed above in control WT cells. In agreement with its lost CaM-binding properties, Mut2-ceQORH was also detected in plastids in both WT plant and cam5 mutant . This result confirms that CaM5 is indeed a crucial actor of the observed alternative location of ceQORH. However, other actors may also contribute to this regulatory process as suggested by the observation that a certain level of ceQORH is still observed at the cell periphery for ceQORH in the cam5 mutant and Mut2-ceQORH in both WT and cam5 backgrounds .As stated above, plant cells contain seven classical (short) CaM isoforms and tens of CaM-like proteins , 26. It n et al. , which win planta, Tic32 was also detected at the periphery of epidermal cells and in a few dotted structures distinct from plastids (A), as observed for ceQORH (i.e. its CaM-binding domain) was mostly detected in plastids (B). Thus, these results indicate that, like ceQORH, the alternative subcellular location of the chloroplast envelope protein Tic32 is also CaM-dependent.To examine whether this calmodulin-dependent mechanism plays a more global role, we used Tic32, an inner envelope membrane protein, whose CaM-binding capacity was previously demonstrated and whose CaM-binding domain was previously localized in its C terminus . More replastids A, as obsr ceQORH –9. Similplastids B. Thus, in vitro import experiments using isolated chloroplasts. We propose here that, when tightly bound to CaM5, ceQORH cannot reach plastids. The direct role of a calmodulin-like protein in controlling ceQORH location is shown by the fact that the loss of the CaM-binding properties of ceQORH (i.e. Mut2-ceQORH) greatly abolishes retention outside of the plastids and instead leads to a plastid location of this protein. The expression of ceQORH in the cam5 mutant proves that CaM5, a calmodulin-like protein, is a crucial actor involved in this phenomenon.During evolution, plants evolved complementary subsets of strategies for dual targeting of proteins. This work documents a novel strategy allowing a dual plastid–cytosol location of plant proteins. This evidence is achieved despite the fact that ceQORH has been shown to be imported into plastids in It should be mentioned here that CAM5 binding to ceQORH does not prevent its import into the chloroplast through interaction with a canonical chloroplast transit peptide because ceQORH is devoid of such an N-terminal extension and is known to be imported via a “noncanonical” pathway . The latin planta . The latter shows the closest similarity to the 1-16 class of CaM-binding domains . The positions of the residues, whose mutagenesis abolished CaM-binding capacity of ceQORH, demonstrate that not only this amphiphilic α-helix structure but also adjacent basic residues are essential for this interaction.Interestingly, the bacterial homolog of ceQORH does not appear to bind calmodulin  and 3, dn planta . Thus, t domains , 30 whic domains . This Ca domains  of the ci.e. cam5 mutant) or when the CaM-binding site of ceQORH is lost (i.e. Mut2-ceQORH mutant). However, this phenomenon also exists in mesophyll cells where, however, the balance between plastid and nonplastid locations is in favor of the former (and therefore relocation is not that easy to monitor).Another condition for CaM5 to trap ceQORH outside plastids is that it must be sufficiently abundant. This is the case in epidermal cells . EpidermWe also showed that this mechanism also influence the subcellular location of another “noncanonical” plastid protein, namely Tic32, which also lacks a cleavable chloroplast transit peptide . However16–Arabidopsis and spinach chloroplast subfractions , amino acid synthesis metabolism , lipid and hormone metabolism , protein targeting and folding , DNA transcription (e.g. PTAC17), thylakoid biogenesis (e.g. ViPP1), etc. Furthermore, some of these proteins showed CaM-binding properties that are either dependent or independent of the presence of calcium , were germinated in Petri dishes containing solidified medium  sucrose, and 1% (w/v) agarose) for 2 weeks before being transferred to soil. Plants were then grown in growth rooms at 23 °C (12-h light cycle) with a light intensity of 100 μmol·m−2·s−1.TM/EMBL databases under the following accession numbers: ceQORH (At4g13010), CaM5 (AT2G27030.3), and Tic32 (At4g23430).The sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBankArabidopsis homozygous mutant (SALK_027181) affected in the cam5 gene (AT2G27030.3) is a T-DNA insertion mutant termed cam5-4 , and 1% (w/v) SDS. The crude extract was centrifuged for 5 min at 13,200 rpm and 4 °C. Because of the presence of a high concentration of detergent, the supernatant resulting from this centrifugation step contains whole-cell proteins (including highly hydrophobic membrane proteins). Purification of epidermal tissues from Arabidopsis leaves was performed by peeling the epidermis of Arabidopsis leaves using a thin forceps used for plant crosses. Crude epidermal extracts were obtained using the procedure described above and the same extraction buffer. Membrane and soluble fractions of crude cell extracts were obtained as previously described (Whole-cell proteins from escribed .Arabidopsis cDNA coding for the CAM1 protein was performed by RT-PCR using pFU ultra high-fidelity DNA polymerase (Stratagene) starting from a homemade Arabidopsis cDNA library. The primer GAAGAACATATGGCGGATCAACTC was designed to introduce an NdeI restriction site (underlined residues) at the 5′ end of the DNA fragment coding for CAM1. The primer ATCACCTGGATCCAATCACTTAGC was designed to introduce a BamHI restriction site (underlined residues) at the 3′ end of the DNA fragment coding for CaM1. The amplified fragments were cloned in pBluescriptSK−. The insert was then digested with NdeI and BamHI and inserted into the expression vector pET-15b (Novagen). The resulting expression plasmids allowed the expression of N-terminal His-tagged versions of the CaM1 protein. The purified protein was desalted  and stored at −80 °C. The purified recombinant His-CaM1 was used as a control (from 100 to 0.1 ng) to estimate the content of the natural CaM proteins in crude leaf extracts, epidermal fractions, or purified chloroplast envelope membranes. Two independent rabbit polyclonal antibodies (including CaM-767) raised against the Arabidopsis recombinant CaM1 protein were obtained from the Elevage Scientifique des Dombes .The CaM1 isoform (AT5G37780) was selected because of its high sequence conservation with other CaM isoforms . Cloning of the Arabidopsis ceQORH produced in E. coli  ethanol). The membrane was then washed for 5 min in TBST and incubated for 1 h in TBST containing 5% (w/v) defatted milk. Incubation with the primary antibody at a 1/1500 dilution requires an overnight incubation at 4 °C. The membrane was then washed three times for 10 min in TBST and incubated for 1 h in TBST containing the secondary antibody  at a 1/10,000 dilution. The membrane was then washed three times for 10 min in TBST before the proteins were detected using ECL. Notably, because of the 97% sequence identity between the two isoforms, the rabbit polyclonal antibody CaM-767 raised against the Arabidopsis CaM1 isoform cross-reacts with all Arabidopsis CaM isoforms, including CaM5.SDS-PAGE analyses were performed as described by Chua  using pr E. coli  at a 1/5E. coli were previously described (TAAGAGCGTTGGATC) and ceQORH 1–175 reverse (GATCCAACGCTCTTACAGCTGGACTGCATA). Construct 8, Δ(229–329)ceQORH-GFP (ceQORH lacking 100 residues in the N terminus fused to GFP), was amplified using the following primers: ceQORH 1–229 (GTGGTCCATTGTGCATAAGAGCGTTGGATC) and ceQORH1–229 reverse (GATCCAACGCTCTTATGCACAATGGACCAC). Construct 9, Δ(175–329)ceQORH-GFP (ceQORH lacking 146 residues in the N terminus fused to GFP), was amplified using the following primers: ceQORH1–280 (CTCTTGTTGATCCCAAAATAAGAGCGTTGGATC) and ceQORH1–280 reverse (GATCCAACGCTCTTATTTTGGGATCAACAAGAG). For targeted mutagenesis and the creation of construct 10 (Mut1), construct 11 (Mut2), and construct 12 (Mut3), vectors were created from targeted mutagenesis of available vectors using the QuikChange® site-directed mutagenesis kit from Stratagene. Mut1-ceQORH was obtained using the following primers: mut1qorBamHI (GGGCCTAATGCAATGGGATCCTATGCGGTTGCTGCAATAACCATGTCAAAG) and mut1qorBamHIreverse (CTTTGACATGGTTATTGCAGCAACCGCATAGGATCCCATTGCATTAGGCCC). Mut2-ceQORH was obtained using the following primers: mut2qorBamHI (GTTAAGAAAATAACCGGATCCGCAGCTCAGTTAGTGCCACTC) and mut2qorBamHIreverse (GAGTGGCACTAACTGAGCTGCGGATCCGGTTATTTTCTTAAC). Mut3-ceQORH was obtained using the following primers: mut3qorBamHI (GGGCCTAATGCAATGGGATCCTATGCGGTTGCTGCAATAACCGGGTCAGCAGCTCAGTTAGTGCCACTC) and mut3qorBamHIreverse (GAGTGGCACTAACTGAGCTGCTGACCCGGTTATTGCAGCAACCGCATAGGATCCCATTGCATTAGGCCC).Several constructs used to express ceQORH and several truncated forms in escribed , 19: conE. coli were previously described  were solubilized in 100 μl of 10 mm Tris-HCl, pH 6.8. Because GFP was present in all protein fusions, it was possible to estimate the relative expression levels (using Western blotting and GFP antibody detection) obtained for all recombinant proteins. Variation in expression levels was thus controlled before CaM overlay experiments. As a negative control, the bacterial ecQOR protein (from E. coli K12) was also produced and tested.Several constructs used to express full-length ceQORH and truncated forms in escribed , 19. TheArabidopsis ceQORH from crude cell extracts was performed on crude solubilized plant proteins diluted in CaM-binding buffer  containing 0.1% (w/v) Nonidet P-40. CaM-binding proteins were purified using CaM affinity resin (Stratagene) and the small-scale quick batch method according to the manufacturer's instructions with minor modifications. Purification from bacteria started with bacterial pellets  that were solubilized in 5 ml of CaM-binding buffer. After three rounds (for 2 min) of sonication, cell extracts were centrifuged for 20 min at 5500 rpm at 4 °C. Then Nonidet P-40 (0.1% (w/v) final concentration) was added to the resulting supernatant (5 ml). CaM affinity resin  was then added to the solution before incubation (stirring wheel) for 1.5 h at 4 °C. The resin was then washed with 5 ml of CaM-binding buffer, and bound proteins were eluted in 250 μl of elution buffer  Nonidet P-40). Purified recombinant proteins were then quantified using the Bio-Rad protein assay reagent  gelatin, 50 mm Tris-HCl, pH 7.5, 50 mm MgCl2, 150 mm NaCl, 5 mm CaCl2, and 0.1% (w/v) Tween 20) + 0.1 μg/ml biotinylated CaM (Sigma). Detection of the bound CaM protein was then performed using a streptavidin–HRP conjugate (Sigma) and ECL using the manufacturer's instructions. Alternatively, after transfer, washing, and saturation, the membrane was incubated in the hybridization solution containing 0.3 μg/ml of a homemade CaM–HRP conjugate (the recombinant Arabidopsis CaM1 (AT5G37780) was covalently coupled to HRP). Detection of the bound CaM protein was then performed using ECL according to the manufacturer's instructions.After SDS-PAGE and transfer, the resulting membrane was first dried and further incubated for 45 min in TBS buffer containing 0.1% (w/v) Triton X-100. The membrane was then incubated for 1 h in TBS buffer containing 0.1% (w/v) Triton X-100 and 5% (w/v) defatted milk followed by 1 h in saturation buffer containing 3% (w/v) gelatin, 50 mArabidopsis plants (ecotype WS) were transformed by dipping the floral buds of 4-week-old plants into an Agrobacterium tumefaciens (C58 strain) solution containing a surfactant (Silwett L-77), according to Clough and Bent  starting from a homemade Arabidopsis cDNA library. Correct orientation and sequence of the inserted fragment were controlled. Plasmids used for A. tumefaciens transformation were prepared using the QIAfilter plasmid midi kit (Qiagen).To construct vectors for the expression in GGATCCATGGCTGGAAAACTCATG) and ceQORHSacI–C-ter (ACAGAGCTCTTATGGCTCGACAATGATCTTC), and the PCR product was cloned into the pBluescript SK− vector (Stratagene) to create the ceQORH-pKS vector. The BamHI–SacI fragment cleaved from this plasmid was inserted into the BamHI–SacI–digested pEL103 binary vector (kanamycin resistance to transform WT plants), resulting in a vector allowing stable expression of ceQORH-GFP.The coding region of Arabidopsis ceQORH (AT4G13010) was PCR-amplified using the two flanking primers ceQORHBamHI–N-ter (CCTGGATCCGCAGCTCAGTTAGTGCCACTC) and mut2qorBamHIrevers (GAGTGGCACTAACTGAGCTGCGGATCCGGTTATTTTCTTAAC).Mut2-ceQORH was produced using targeted mutagenesis of pre-existing constructs using the following primers: mut2qorBamHI (GTTAAGAAAATAACCArabidopsis Tic32 (At4g23430) cDNA was PCR-amplified using the two flanking primers Tic32SalI–N-ter (GCTAGTCGACATATGTGGTTTTTTGGATC) and Tic32NcoI–C-ter (TCCTCCATGGAACTGCTTTCTCCTGATTG). This fragment was cloned into the pKS vector (Stratagene) and then transferred to pUC-GFP .Tic32 lacking its C terminus (TCTAGAATGGCTGGAAAACTC for ceQORH and Mut2-ceQORH or TCATCTAGAATGGCAGATCAGCTC for CaM5) and SalI–C-ter (CCAGTCGACTGGCTCGACAATGATC for ceQORH and Mut2-ceQORH or CCAGTCGACGAGAATACGGCAGTG for CaM5). The PCR products were cloned into the pBlueScript SK− vector. XbaI–SalI fragments cleaved from this plasmid were inserted into the XbaI–SalI–digested YFP C-terminal or YFP N-terminal reporter plasmid pUC-SPYCE and pUC-SPYNE (A. tumefaciens strain C58 and used to transform tobacco leaves using agroinfiltration. Plasmids used for A. tumefaciens transformation were prepared using the NucleoSpin plasmid kit (Macherey–Nagel). Localization of the GFP and GFP fusions in tobacco leaves was analyzed by confocal fluorescence microscopy 3–4 days after agroinfiltration.Construction of the plasmids for the expression of ceQORH, Mut2-ceQORH and CaM5 proteins fused to the YFP C terminus or N terminus was performed as follows. The coding regions of candidate proteins were PCR-amplified using two flanking primers, XbaI–N-ter .L. M., D. S., I. B., S. M., L. P., M. K., and N. R. conceptualization; L. M., D. S., I. B., and N. R. resources; L. M., D. S., I. B., D. S.-B., M. K., and N. R. data curation; L. M., D. S.-B., M. K., and N. R. formal analysis; L. M., D. S., I. B., and N. R. supervision; L. M., M. K., and N. R. funding acquisition; L. M., I. B., S. M., M. K., and N. R. validation; L. M., D. S., I. B., S. M., L. P., D. S.-B., M. K., and N. R. investigation; L. M., D. S., I. B., S. M., and N. R. visualization; L. M., D. S., I. B., S. M., L. P., D. S.-B., M. K., and N. R. methodology; L. M., D. S., I. B., M. K., and N. R. writing-original draft; L. M., M. K., and N. R. writing-review and editing.


T2), which renders it 4-hydroxytamoxifen (4-OHT) inducible for the access of genome, and a nuclear export signal (NES), which lowers the background activity. Tight and efficient drug-inducible genome editing was achieved across several human cell types, including embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), upon vigorous optimization. Optimized terminal device, which we named hybrid drug inducible CRISPR/Cas9 technology (HIT-Cas9), delivered advantageous performances over several existing designs. Such architecture was also successfully applied to an orthogonal Cas9. The HIT-Cas9 system developed in this study will find broad utility in controlled editing of potentially any genomic loci.The CRISPR/Cas9 enabled efficient gene editing in an easy and programmable manner. Controlling its activity in greater precision is desired for biomedical research and potential therapeutic translation. Here, we engrafted the CRISPR/Cas9 system with a mutated human estrogen receptor (ER  In the CRISPR/Cas9 system, guide RNA (gRNA) directs Cas9 protein to its complementary DNA adjacent to a protospacer-adjacent motif (PAM), a species specific requirement for Cas9 recognition.5T2, which contains three mutations, G400V, M543A, and L544A, selectively responds to a synthetic ligand 4-hydroxytamoxifen (4-OHT) over β-estradiol, the endogenous ER ligand, a property crucial for tight drug control.Drug control of genetic events allows dissection of gene functions in greater precision, which also reduces undesired off-target events, thus providing potential avenues toward safer gene therapies. One of the most widely used drug-inducible systems in genetic research coupled Cre recombinase with estrogen receptor (ER).T2, it is tempting to harnessing the power of the CRISPR/Cas9 with drug control by coupling it with ERT2. Here, we engineered and optimized such a system, which we named hybrid inducible CRISPR/Cas9 technology (HIT-Cas9). Tight and efficient genome editing was accomplished in a 4-OHT-inducible fashion across multiple human cell types, including ESCs and MSCs with clinical utilities, through combinatory engineering of Cas9, ERT2, and nuclear export signal (NES). In head-to-head comparisons, our terminal HIT-Cas9 system delivered better performances than several designs published previously, including an ERT2-based iCas system reported lately, while this study was in progress.Encouraged by the wide utility of CreERT2 (T2 domains to Cre appeared to exert additive effect for lowering background activity.T2 domains to either the N or C terminus of Cas9 (T2 fusion to the C terminus of Cas9 (CE and C2E) less compromised its nuclease activity  introduced lower background in the absence of 4-OHT in comparison with that fused with one ERT2 (CE) , a cell-surface protein highly expressed in HEK293T cells, and examined NHEJ-induced gene knockout using flow cytometry . A populRT2 (CE) B, consisTo examine both NHEJ and HDR events induced by C2E, we next employed a traffic-light reporter (TLR) assay A.19 We uT2 (Cas9-NES-2ERT2/CN2E) (T2 domains (C2N2E). This indeed further lowered background activity while retaining drug inducible action, though compromised, in the CD201 knockout assay  . ConsistT2/CN2E) . On the T2/CN2E)  and S5. T2/CN2E) . Howeverut assay B. Considut assay , we adoput assay C. This apromised D. Taken promised E.Figure Considering that such drug-inducible systems would find broad utility in targeting various cell types, among which human stem cells with clinical utilities would be of particular interest,So far, we have used several assays, including the FCR assay, the TLR assay, the CD201 knockout assay, and the plasmid SSA (pSSA) assay, in optimization toward a tight and effective drug-inducible Cas9 construct. The higher sensitivity of the FCR assay than the TLR assay likely reflects higher HDR efficiency in point mutagenesis (FCR) than in longer DNA fragment exchange (TLR). In addition, mCherry signal in the TLR reporter that probes NHEJ events displayed a high background noise, consistent with its previous use after prior purification to remove mCherry-positive cells.T2 domains to Cas9 (iCas), two to each terminus, resulted in 4-OHT-inducible genome-editing activity. It was reported that this construct introduced a comparable background activity with intein and split systems but higher efficiency upon drug induction and a more rapid response to drug induction than Tet-on, intein, and split systems.In pursuit of CRISPR/Cas9 technologies in greater precision, the field has reported several designs of drug-inducible systems previously.https://clontech.com/US/Products/Inducible_Systems/Tetracycline-Inducible_Expression/Tet-On_3G)  A. Among t-On_3G) A, in whit-On_3G) A. Interet-On_3G) A.Figure To be less biased, we next further interrogated HIT-Cas9 in comparison with other systems by probing NHEJ events in Surveyor assays. All systems except the one utilizing the intein mutant (G521R) displayed similar on-target indel frequencies at an EMX1 site upon drug treatment B,11, 15 T2 regulation and it was reported to elicit a faster response than Tet-on, intein, and split systems.In addition to the tightness and efficiency, an advantageous drug-inducible system should also be sensitive, selective, and rapid in response. We next further characterized HIT-Cas9 based on these additional criteria. In doing so, we used intein systems as comparison given that the rationale to utilize intein mutant is to increase selectivity of 4-OHT over β-estradiol.We first examined dose dependent response of the optimized terminal HIT-Cas9 system to 4-OHT and β-estradiol respectively. Sensitive and selective response of HIT-Cas9 to 4-OHT over β-estradiol was observed in the FCR assay A and S10iCas was reported to act at a higher speed than intein, split, and Tet-on constructs in response to drug induction.Streptococcus pyogenes (SpCas9). Orthogonal Cas9 species have distinct PAM requirements, thus expanding target coverage in the genome.Staphylococcus aureus Cas9 (SaCas9) has been of considerable interest as an orthogonal Cas9 species due to high activity in mammalian cells, the high incidence of its PAM sequence (NNGRR), and its smaller protein size, a crucial property for the development of gene therapy because of restrictive cargo sizes for certain delivery vehicles such as adeno-associated virus (AAV).T2 construct for drug-inducible genome editing. When delivered with a sgRNA targeting the same region on BFP and compatible with SaCas9, this construct managed to convert the fluorescence to GFP. It showed robust drug-inducible regulation dependent on a specific sgRNA, albeit higher background activity than that of SpCas9, a feature that warrants further optimization , compromised activity was observed upon drug induction for multiple drug-inducible CRISPR/Cas9 systems in our hands and others .11, 13, Above all, continuous development toward greater precision in using drugs to control biological events is always desired for biomedical research and potential clinical applications in a safer and more effective manner. Temporal control and dose-dependent control of the CRISPR/Cas9 machinery using HIT-Cas9, together with other HIT devices we have reported recently for transcriptional programming based on CRISPR/Cas9 and those based on transcription activator-like (TAL) effectors,T2 were cloned from the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid http://crispr.mit.edu/)To identify effective sgRNA candidates, we employed the genetic perturbation platform (GPP) web portal  medium supplemented with 10% FBS at 37°C and with 5% CO2. ESC line H9 was purchased from Wicell Research Institute and maintained in a 6-well plate coated with Matrigel (BD Biosciences) in Essential 8 medium (Thermo Fisher).HEK293T and HepG2 cells (American Type Culture Collection [ATCC]) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM GlutaMAX (Thermo Fisher), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and with 5% COHIT-Cas9 and EMX1 sgRNA were cloned in lentiviral expression plasmids, packaged in lentiviral particles, and delivered to H9 cells. The cells were then treated with 2.5 μg/mL of Zeocin (InvivoGen), whose drug resistance cassette was co-expressed with HIT-Cas9, and 0.5 μg/mL Puromycin (InvivoGen), whose drug resistance cassette was co-expressed with sgRNA, to enrich cells that were successfully infected by both constructs. An unrelevant sgRNA was used as a negative control. 3 days post-antibiotic selection, 4-OHT was added into the medium to the working concentration at 200 nM. Genomic DNA was isolated and applied for surveyor assay and TIDE analyses 4 days upon 4-OHT treatment.As for MSCs, HIT-Cas9 and AAVS1 sgRNA constructs were packed in polyethyleneimine (PEI)-based lipid particles and transfected following standard protocol. A plasmid that constitutively expresses GFP was co-transfected into the MSCs. Two days after 4-OHT induction, GFP-positive cells were harvested by fluorescence-activated cell sorting (FACS) and genomic DNA was isolated for surveyor assay and TIDE analyses.TLR and FCR reporter constructs was integrated into the genome via lentiviral infection. Monoclonal cell lines were obtained respectively. Transfections were conducted with Biotool DNA transfection reagent (Biotool) following standard protocol. To ensure proper controlled comparison, the molar amount of Cas9, sgRNA, and donor as well as the total weight of transfected DNA were matched for each well in all experiments. Unless stated otherwise, 125 nM of 4-OHT or a matched volume of ethanol was added to culture medium 5 hr after transfection, and treatments lasted for 48 hr.X3 Multilabel Plate Reader (PerkinElmer) according to the manufacturer’s recommended protocol.pSSA reporter, sgRNA, and Cas9 constructs and an internal control construct that constitutively expresses firefly luciferase were co-transfected into HEK293T cells. Medium was collected 48 hr after transfection and gaussia luciferase substrate (New England Biolabs) was added. Cells were lysed and firefly luciferase substrate (Promega) was added. Luciferase readouts were measured using VICTOR TLR reporter cell line were pre-seeded and transfected with various Cas9 constructs and sgRNA with or without GFP donor template . Flow cytometry analysis was conducted using the CytoFLEX (Beckman Coulter).FCR stable cell line was co-transfected with various Cas9 constructs and BFP sgRNA and single strand DNA (ssDNA) donor template. The sequence of donor was 5′-GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACGTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGA-3′GFP or 3×Flag tagged Cas9 constructs were transfected into HEK293T Standard immunofluorescence staining protocol was followed to label 3×Flag epitopes. Images were acquired and quantitatively analyzed using Operetta High Content Screening system (PerkinElmer) after cells being fixed by 4% (w/v) paraformaldehyde and stained with Hochest 33342 (Thermo Fisher).T2 fusion constructs and sgRNAs targeting either the EMX1 gene or AAVS1 site (https://tide.nki.nl/).Genomic DNA from various types of cells delivered with different Cas9-ERVS1 site  was extrY.W. conceived and supervised the study. C.Z., Y. Zhao, J.Z., J.L., L.C., Y. Zhang, Y.Y., and Y.W. designed experiments. C.Z., Y. Zhao, J.Z., J.L., L.C., Y. Zhang, Y.Y., and S.W. performed experiments. C.Z., Y. Zhao, J.Z., J.L., L.C., Y. Zhang, Y.Y., J.X., and Y.W. analyzed data. C.Z., Y. Zhao, J.Z., J.L., L.C., and Y.W. wrote the manuscript.Patents covering the novel designs in this work have been filed.


Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections in children and immunocompromised individuals. A multi-center surveillance of the epidemiologic and molecular characteristics of RSV circulating in Lebanon was performed. The attachment (G) and fusion (F) glycoproteins were analyzed and compared to those reported regionally and globally. 16% (83/519) of the nasopharyngeal swabs collected during the 2016/17 season tested positive for RSV; 50% (27/54) were RSV-A and 50% (27/54) were RSV-B. Phylogenetic analysis of the G glycoprotein revealed predominance of the RSVA ON1 genotype, in addition to two novel Lebanese genotype variants, hereby named LBA1 and LBA2, which descended from the ON1 and NA2 RSV-A genotypes, respectively. RSV-B strains belonged to BA9 genotype except for one BA10. Deduced amino acid sequences depicted several unique substitutions, alteration of glycosylation patterns and the emergence of palivizumab resistance among the Lebanese viruses. The emergence of ON1 and other novel genotypes that are resistant to palivizumab highlights the importance of monitoring RSV globally. Respiratory syncytial virus (RSV) is a leading cause of morbidity and mortality in infants, young children and immunocompromised patients . Nearly Paramyxoviridae, order Mononegavirales. It is an enveloped virus containing a 15.2 kb linear, negative-sense single-stranded RNA genome that encodes 11 proteins. RSV is divided into two antigenic subgroups, A and B, based on the reactivity of the virus with monoclonal antibodies mainly against the attachment (G) and fusion (F) glycoproteins ) of the prototype strain A2 G protein (M11486) and to globally reported strains including an ON1 (JN257693) and an NA2 (AB470479) reference strain . Specimens belonging to the LBA1 genotype (represented by 16LB03/2016) had 100% homology and shared six substitutions: G284S, E295K, Y304H, S314P (S290P-d), T319I (T295I-d) and P320K (P296K-d). Specimens of the novel LBA2 cluster had 100% homology and shared two substitutions: L258Y and R297I. This cluster carried the unique R297I mutation recently reported in two specimens (02-281/2014 and 52-151/2013) from France.To explain the distinct clustering of the novel RSV-A LBA1 and LBA2 strains, deduced amino acid sequences of representative ON1, LBA1 and LBA2 Lebanese specimens were aligned and examined relative to the region spanning residues 221–321  and to globally reported strains including a BA9  and a BA10  reference strain . Three uTo examine the susceptibility of our RSV-A specimens to palivizumab, the derived amino acid sequences (n = 54) were aligned relative to the region spanning residues 241–286 of the prototype RSV-A F protein  . RepreseTo further characterize the effect of the observed substitutions in the F glycoprotein on palivizumab binding, we analyzed the interactions between palivizumab and F glycoprotein carrying the mutations of interest . The obsPredicted RSV-A F glycoprotein structure carrying the N276S substitution bound to palivizumab revealed no structural changes but showed the loss of a 2.3 Å polar bond with Tyr100 of the palivizumab heavy chain while maintaining the 2.9 Å polar bond with Tyr94, although both asparagine and serine belong to the same class of polar amino acids . Amino aTwo N-glycosylation sites (Asn-Xaa-Ser/Thr where Xaa is any amino acid except proline) and 8 O-glycosylation sites (Ser or Thr) were predicted in the partial G protein sequence of the prototype strain A2 . ON1 genIn order to determine whether characteristic O-glycosylation patterns exist for the novel LBA1 and LBA2 genotypes, sites of glycosylation in LBA1 were surveyed in all reported ON1 G sequences  while sites of glycosylation in LBA2 were checked against NA2 G sequences (n = 795) . All posRegarding RSV-B, 1 N-glycosylation site and 20 O-glycosylation sites were predicted in the G protein of the prototype strain BA4128/99B . BA9 genFor the F protein amino acid sequences, two sites at positions 244 and 245 were predicted to be O-glycosylated in the RSV-A sequences whereas no N-glycosylation sites were predicted . The preet al. screened children in Northern Lebanon reporting an RSV incidence of 26.7% with a peak in January 2008 [et al. reported an incidence of 19% with a peak in December 2013 [In this study, RSV incidence among patients with acute respiratory infections during the 2016/17 season was 16% with a peak in December. Two epidemiologic studies of RSV were previously conducted in Lebanon; however, none attempted to genetically characterize the virus , 34. Hamary 2008 , while Fber 2013 . TherefoOur findings demonstrate that during the 2016/17 season, two distinct lineages of RSV were co-circulating, ON1 and BA9, with the temporal disappearance of NA2 and BA10 genotypes. Retrospective screening of RSV specimens from the previous seasons, revealed that ON1 and BA9 circulated in Lebanon as early as the 2014/2015 season. The novel LBA2 genotype was detected during the 2014/2015 season but was replaced in the subsequent seasons by the fast-spreading ON1 genotype. Nonetheless, the detection of LBA2 in France suggests a broad geographic spread and warrants further analysis.The RSV-A ON1 genotype has a 72-nucleotide duplication that was identified initially in Canada and spread worldwide thereafter likely due to the fitness advantage of this genotype . This trLBA1 is characterized by six amino acid substitutions and possesses an additional O-glycosylation site (G284S) compared to reported ON1 viruses. While, LBA2, a descendant of NA2 genotype, was characterized by two amino acid substitutions and an additional O-glycosylation site (S292) compared to NA2.The G glycoprotein is a heavily glycosylated (30–40 O- and 4–5 N-linked glycans) 80 kDa protein with a 289–299 amino acid backbone (32–33 kDa) . The putImmunocompromised individuals are at greater risk of severe RSV infections. Palivizumab, a high-affinity F-specific humanized murine monoclonal antibody, is effective at preventing the debilitating consequences of the viral infection . The F pCurrently, there is no vaccine available for prevention of RSV infections and candidate vaccines are in various stages of clinical evaluation . It is iIn this study, we characterized multiple co-circulating RSV genotypes in Lebanon and identified two novel genotypes, LBA1 and LBA2. Substantial genetic diversity was observed within RSV-A and RSV-B. The increased use of palivizumab along with the universal spread of the N276S mutation represent alarming concerns for the emergence of mutant, resistant viruses and a need for the development of new antiviral drugs and vaccines.S1 Fig(a) Predicted interactions within the RSV-A F protein (PDB: 3RRT). (b) Predicted interactions within the palivizumab antibody (PDB: 2HWZ). (c) Predicted interactions between palivizumab and the RSV-A F protein. (d) Predicted interactions between palivizumab and RSV-A F protein carrying the N276S mutation. (e) Predicted interactions between palivizumab and RSV-A F protein carrying the N276S and A241V mutations. (f) Predicted interactions between palivizumab and RSV-A F protein carrying the N276S and V247I mutations. (g) Predicted interactions between palivizumab and the RSV-B F protein. (h) Predicted interactions between palivizumab and RSV-B F protein carrying the S276N and Q279H mutations.(PPTX)Click here for additional data file.


Triticum aestivum L) arose by two polyploidisation events from three diploid species with homoeologous genomes. Nullisomic-tetrasomic (nulli-tetra or NT) lines are aneuploid wheat plants lacking two and adding two of six homoeologous chromosomes. These plants can grow normally, but with significantly morphological variations because the adding two chromosomes or the remaining four chromosomes compensate for those absent. Despite these interesting phenomena, detailed molecular mechanisms underlying dosage deletion and compensation in these useful genetic materials have not been determined.Hexaploid bread wheat  occurred throughout the whole genome with a subgenome and chromosome preference. The deletion effect of nulli-chromosomes was compensated partly by the tetra-chromosomes via the dose level of expressed genes, according to the types of homoeologous genes. The functions of differentially regulated genes primarily focused on carbon metabolic process, photosynthesis process, hormone metabolism, and responding to stimulus, and etc., which might be related to the defective phenotypes that included reductions in plant height, flag leaf length, spikelet number, and kernels per spike.The perturbation of the expression levels of transcriptional genes among the NT stocks for homoeologous group 7 demonstrated the gene dosage effect of the subgenome at the genome-wide level. The gene dosage deletion and compensation can be used as a model to elucidate the functions of the subgenomes in modern polyploid plants.The online version of this article (10.1186/s12864-018-5421-3) contains supplementary material, which is available to authorized users. Arabidopsis thaliana [Chromosomes are partitioned equally during cell division for most eukaryotic organisms . Howeverthaliana . The defthaliana . In hexathaliana ., Triticum urartu  and Aegilops speltoides , forming the allotetraploid wheat, Triticum turgidum . In the second hybridisation event, another diploid grass species, Aegilops tauschii , was introduced into the allotetraploid wheat, producing the ancestral allohexaploid Triticum aestivum  [, subgenome A, subgenome B and subgenome D. Unlike the paleopolyploid maize and soybean, no transcriptional dominance was observed among the three sets of subgenomes at the genome-wide level in the modern wheat [Allohexaploid wheat arose by two polyploidisation events from three diploid species with homoeologous genomes. The first hybridisation event occurred in the genomes of two diploid species, i.e.6x = 42) , 8, whicrn wheat , 10. Basrn wheat , 10. WitAccording to the characteristics of chromosomes, these stocks are currently primarily used for localisation of genes on chromosomes. Although one study  showed thttp://www.k-state.edu/wgrc/) provided the seeds of the NT stocks for group 7: N7AT7B, N7AT7D, N7BT7A, N7BT7D, N7DT7A and N7DT7B. We planted these seeds in the field and only harvested those spikes bagged before flowering. Then, according to published methods [The Wheat Genetics Resource Center  were downloaded from the IWGSC website. Reads were then mapped to reference sequences without mismatch using the Tophat2 program . To prec were dowP-value , and the FDR correction of the Benjamini and Hochberg multiple hypothesis testing method was used to reduce the false negatives with FDR < 0.05 [The GO (Gene Ontology) term annotation of wheat genes is available in the IWGSC website. The functional enrichment of significantly expressed genes was performed by the BINGO program with reference to the CS genome . The hypR < 0.05 .To compare the phenotypic traits between the NT stocks for group 7 and euploid CS, several phenotypical characters were investigated Fig. a-e. ReduDifferent types of chromosome karyotypes exhibited differently phenotypic traits, which should be defined as the expressed gene dosage effect. Thus, to investigate the differences in the gene expression profiles among the NT stocks for group 7 and euploid CS, seven RNA-seq libraries were constructed from the leaves of two-week-old seedlings and sequenced. Approximately 11–12 million reads were generated by RNA sequencing, and ~ 8–9 million reads were perfectly mapped to the reference genomes of CS (Table 2(NT/CS)) distribution of gene expression for each category and found that the medium and high expression genes contributed substantially to up-regulation in the NT stocks compared with CS , mediumly expressed genes , and highly expressed genes . Totally, 17–24, 12–14, and more than two thousand genes were identified at the low, medium, and high expression level, respectively  in genome-wide level, we defined them in the NT stocks, relative to those in CS, using the DEGseq program with an FDR less than 0.001. A total of ~ 4000–5000 genes were SDEGs , almost all of them were HEGs followed by MEGs and only a few of them were LEGs Fig. e-f. HoweArabidopsis, the pattern of high expression level of genes affects similar and conserved cellular pathways. Genes involved in a stress response are consistently up-regulated, whereas those genes associated with the cell cycle and cell proliferation are down-regulated [For aneuploid organisms, including fungi such as yeast, animals such as mouse and human, and plants such as egulated . To undeThen, using the common SDEGs, we performed GO enrichment analysis as shown in Fig. In NT stocks, there also were many NT-specific SDEGs in each nullisomic stocks  families have 31 homoeologous copies totally in hexaploid wheat. Of these copies, 27 are distributed in the subgenomes A and B and only four are located in subgenome D. TaGH3.1 has two alleles on chromosome 3A, with none in subgenomes B and D. It showed asymmetrical distribution in the three subgenomes on common wheat [A polyploidisation event occurs at least once in the evolution history of most angiosperm species . The genica rapa , synthetbidopsis , and thebidopsis , 13, 24.bidopsis . With apon wheat .Fig. 7ThHKT1;5  increases the salt tolerance of hexaploid wheat [WSEP) and the homologs of OsMADS45  in rice showed similar genomic structures and expression profiles. By contrast, the three homoeologs of wheat LEAFY HULL STERILE1 (WLHS1) and the homologs of OsMAD1 in rice showed genetic and epigenetic alterations. WLHS1-A, because of structural alteration, had no apparent function; WLHS-B, because of cytosine methylation, was also silenced; only WLHS-D had functions in hexaploid wheat [Q gene, a member of the AP2 transcription factors, also with three homoallele copies, only the 5AQ as the dominant allele with hyper-functionalisation played a primary role in conferring domestication-related traits [In the hexaploid wheat, the homoeoalleles globally showed additive expression but with local nonadditive asymmetric expression. However, the local asymmetric expression may contribute to excellent agronomic traits. For example, change in the expression pattern of the D homoeoallele id wheat . For theid wheat . For thed traits , 30. ThuTaSus2 (Sucrose synthase 2), TaGW2, TaTEF (transcript elongation factor) and TaGS5. TaSus2 could be active in endosperm development. In the wheat accession, two haplotypes, Hap-H and Hap-L, in TaSus2B and the Hap-A of TaSus2A were significantly associated with the thousand kernel weight (TKW), whereas no diversity was found in TaSus2D [TaGW2-6B has a stronger influence than TaGW2-6A on TKW (Thousand Kernel Weight) [TaTEF-7A, as a functional regulatory factor for grain number in per spike, had a higher level of expression than that of TaTEF-7B and TaTEF-7D [TaGS5-3A and TaGS5-3A-T with higher enzymatic activity than that of TaGS5-3A-G were significantly correlated with larger grain size and higher TKW [Additionally, strong positive selection fixed many genes in the early breeding in the domestication of wheat . As exam TaSus2D . TaGW2-6 Weight) , and TaTTaTEF-7D . The twogher TKW .WRKY, NAC , MYB, ARF (Auxin Response Factors), AP2/ERF (AP2-like ethylene-responsive transcription factor), GATA, NFYA (Nuclear transcription factor Y subunit A), SPB (Squamosa promoter binding like protein), WD, and MADS-box, and some of these might be singleton or functionalisation diversity homoeologs. For these deleted genes involved in the interaction of multiple genes, dysregulation of one gene will affect the entire balance of pathway regulation  and Oligo-pSc119.2 (green) as probes. A-F represented the N7AT7B, N7AT7D, N7BT7A, N7BT7D, N7DT7A, and N7DT7B, respectively. (PDF 143 kb)Additional file 2:Figure S2. The flow chart of RNA-seq data analysis on the NT stocks referred to CS genome. (PDF 21 kb)Additional file 3:Table S1. The mapping information of the transcriptomes with only one hit loci referred to the CS genome in the NT stocks for group 7 and CS. (XLS 20 kb)Additional file 4:Table S2. The expressed genes in the NT stocks for group 7 and CS. (XLS 25 kb)Additional file 5:Significantly differentially expressed genes with FDR less than 0.001 in N7AT7B stock compared with CS. (XLSX 416 kb)Additional file 6:Significantly differentially expressed genes with FDR less than 0.001 in N7AT7D stock compared with CS. (XLSX 500 kb)Additional file 7:Significantly differentially expressed genes with FDR less than 0.001 in N7BT7A stock compared with CS. (XLSX 416 kb)Additional file 8:Significantly differentially expressed genes with FDR less than 0.001 in N7BT7D stock compared with CS. (XLSX 412 kb)Additional file 9:Significantly differentially expressed genes with FDR less than 0.001 in N7DT7A stock compared with CS. (XLSX 475 kb)Additional file 10:Significantly differentially expressed genes with FDR less than 0.001 in N7DT7B stock compared with CS. (XLSX 451 kb)Additional file 11:Figure S3. The number of the up- and down-regulated genes for the lowly (A), mediumly (B), and highly (C) expressed genes. (D-F) The distribution of SDEGs in the chromosome 1–6 and chromosome 7 for the lowly (D), mediumly (E) and highly (F) expressed genes. (G) The preference of proportion distribution of SDEGs on the chromosome 2D and 3D in the NT stocks compared with that in CS. (PDF 51 kb)Additional file 12:Table S3. The significantly differentially expressed genes in the NT stocks for group 7 compared with CS. Chrs, chromosomes. (XLS 24 kb)Additional file 13:Figure S4. The distribution of significantly differentially expressed genes along the chromosomes. (PDF 57 kb)Additional file 14:Figure S5. The overlapped up- and down-regulated genes in N7A (N7AT7B ∩ N7AT7D), N7B (N7BT7A ∩ N7BT7D), and N7D (N7DT7A ∩ N7DT7B) stocks. (PDF 105 kb)Additional file 15:Figure S6. The GO enrichment analysis for the up-regulated genes in N7D (N7DT7A ∩ N7DT7B) stocks referred to CS. (PDF 52 kb)Additional file 16:The GO enrichment for the N7AT7B-specific down- or up-regulated expressed genes in N7A stocks. (XLS 40 kb)Additional file 17:The GO enrichment for the N7AT7D-specific down- or up-regulated expressed genes in N7A stocks. (XLS 250 kb)Additional file 18:The GO enrichment for the N7BT7A-specific down- or up-regulated expressed genes in N7B stocks. (XLS 66 kb)Additional file 19:The GO enrichment for the N7BT7D-specific down- or up-regulated expressed genes in N7B stocks. (XLS 52 kb)Additional file 20:The GO enrichment for the N7DT7A-specific down- or up-regulated expressed genes in N7D stocks. (XLS 87 kb)Additional file 21:The GO enrichment for the N7DT7B-specific down- or up-regulated expressed genes in N7D stocks. (XLS 57 kb)


Background: Immunosenescence contributes to reduced vaccine response in elderly persons, and is worsened by deficiencies in nutrients such as Vitamin (Vit-D). The immune system is a well-known target of Vit-D, which can both potentiate the innate immune response and inhibit the adaptive system, and so modulate vaccination response.Objective: This randomized placebo-controlled double-blind trial investigated whether Vit-D supplementation in deficient elderly persons could improve influenza seroprotection and immune response.Design: Deficient volunteers (Vit-D serum <30 ng/mL) were assigned (V1) to receive either 100,000 IU/15 days of cholecalciferol , or a placebo , over a 3 month period. Influenza vaccination was performed at the end of this period (V2), and the vaccine response was evaluated 28 days later (V3). At each visit, serum cathelicidin, immune response to vaccination, plasma cytokines, lymphocyte phenotyping, and phagocyte ROS production were assessed.Results: Levels of serum 25-(OH)D increased after supplementation . No difference was observed for serum cathelicidin levels, antibody titers, and ROS production in D vs. P groups at V3. Lower plasma levels of TNFα (p = 0.040) and IL-6 (p = 0.046), and higher ones for TFGβ (p = 0.0028) were observed at V3. The Th1/Th2 ratio was lower in the D group at V2 .Conclusions: Vit-D supplementation promotes a higher TGFβ plasma level in response to influenza vaccination without improving antibody production. This supplementation seems to direct the lymphocyte polarization toward a tolerogenic immune response. A deeper characterization of metabolic and molecular pathways of these observations will aid in the understanding of Vit-D's effects on cell-mediated immunity in aging. This clinical trial was registered at clinicaltrials.gov as NCT01893385. Influenza infection occurs in people of all ages, but complications are more frequent in elderly persons , 2. This2D (Recent studies have demonstrated an expression of the Vit-D receptor (VDR) in almost all immune cells, suggesting that Vit-D has anti-infectious and immunomodulatory effects . These c2D , 11. Amo2D . Catheli2D , 13. Mor2D , 15. Tri2D , 17. Rec2D . A furth2D , 20.in vitro the regulatory T cells (Treg) differentiation via an indoleamine 2,3-dioxygenase (IDO)-dependent pathway (Vit-D is known to shift the T-cell response from a T helper 1 (Th1) to a Th2-mediated cell response, and thereby reduce inflammation and promote an immunosuppressive state –23. More pathway , 25. Thu pathway , 27.The public health strategy for influenza is to reduce severe outcomes such as hospitalization and death by recommending annual vaccinations, particularly for people over 65 years old , 29. HowConsidering these data, we assessed the impact of Vit-D supplementation on the immune response to influenza vaccination in Vit-D-deficient elderly volunteers by evaluating (i) cathelicidin status, and (ii) antibody response to vaccine, cytokine production, IDO activity, lymphocyte polarization and ROS production.Eligible volunteers were over 65 years old and accepted Vit-D or placebo supplementation and influenza vaccination. Exclusion criteria included prior hypersensitivity to Vit-D (in the previous year), ongoing Vit-D supplementation, previous side effects, and complications after vaccination, hypercalcemia (>2.6 mmol/L), dysparathyroidism, renal impairment, and long-term treatment with bisphosphonates, corticosteroids, or fibrates.Volunteers were randomly assigned to blocks of four by sex and age using a computerized random-sequence-generation program run by an independent researcher who was not involved in the data collection, analysis, or reporting. For the supplementation, placebo and Vit-D doses were identical in appearance to maintain blinding, and all participants, investigators, and outcome assessors remained blinded until after all of the data was inputted.This randomized double-blind controlled trial was authorized by the ethics committee  and the French state authority . It was registered on EudraCT under ref. 2012-005658-52 and on clinicaltrials.gov as NCT01893385. At the inclusion visit the volunteers gave fully informed written consent, and then blood samples were taken to determine serum Vit-D levels and the biological parameters required to validate eligibility criteria: blood cell count, and usual plasma and urinary levels of calcium, phosphorus, creatinine, liver enzymes , glucose, and total proteins. Based on serum Vit-D data, the volunteers were grouped as follows: (i) persons with a serum Vit-D level greater than or equal to 30 ng/mL: these individuals were excluded, and advised to accept an influenza vaccine in autumn; (ii) persons with a Vit-D level below 30 ng/mL: these individuals were randomly assigned to one of two groups: (1) a supplemented group (D) receiving six Vit-D doses  over 3 months, followed by an influenza vaccination; (2) a control group (P) receiving a placebo  over 3 months, followed by an influenza vaccination. The participants' compliance was verified by restitution of all empty vials at each visit.Influenza vaccination was carried out using the IM vaccine Vaxigrip® (Sanofi Pasteur), which provides seroprotection against all seasonal influenza strains, namely A/California/7/2009 , A/Texas/50/2012 (H3N2), and B/Massachusetts/2/2012 (Yamagata lineage). The volunteers committed not to change their eating habits, and were assessed at three different stages: at inclusion (V1), after 3 months of supplementation (V2), and 1 month after vaccination (V3). A survey of side effects and complications was performed at each visit and by telephone interview. For the D group, serum Vit-D concentration, calcemia, and calciuria were monitored after 2 months of supplementation. The biological parameters discussed in the following paragraphs were measured at each visit .Serum 25-(OH)D was measured by chemiluminescence immunoassay . Serum cathelicidin (LL-37 protein) was quantified using a double-sandwich ELISA, following the manufacturer's guidelines (Hycult Biotechnology—HK321).Vaccine response was assessed at two points (V2 and V3) by measuring hemagglutination inhibition (HAI) antibody (Ab) titers against the influenza vaccine antigens. The HAI test was performed in microplates by incubating serum with the 2013-2014 influenza reference strains , following the WHO procedure. Inter assay quality control was performed with reference antisera as positive controls. Erythrocyte controls allowed adjustments in incubation time and were performed on each plate. Each field isolate antigen and the control antigens have been tested with a negative serum control. The HAI Ab titer was defined as the highest dilution of serum inhibiting the agglutination of guinea pig erythrocytes (Charles River Lab.). In accordance with the European Agency for the Evaluation of Medicinal Products' guidelines , data waThe concentrations of plasma cytokines were quantified using a multiplex assay , following the manufacturer's instructions: IL-5, IL-6, IL-10, IL-13, IL-17A, IFNγ, TNFα (Hcytomag-60K-7plex). For IL-23 and TGFβ, a singleplex assay was used (Tgfbmag-64K-01-1plex).n = 10; D group, n = 9), and estimated by the ratio of Kyn to Trp serum concentrations as described previously  as described previously . LymphocROS production of polymorphonuclear cells (PMN) was quantified from hemolyzed blood by an intracellular fluorescent probe  using flow cytometry, as described previously .The primary outcome of the trial was the difference in serum cathelicidin levels between the placebo and Vit-D study arms after 3 months of supplementation. To detect this significant difference, the calculation was based on the hypothesis that the mean ± SD baseline serum cathelicidin concentration was 13.3 ± 1.8 ng/ml and that the Vit-D supplementation would cause a difference of 1.26 ± 2.1 ng/ml in cathelicidin between the 2 arms (80% power with α = 0.05). Taking these assumptions into account, we calculated a group size of 42 participants per arm.post hoc test. Differences within groups were determined by a paired Student t-test or a Wilcoxon matched-pairs signed-ranks test. Differences between groups were tested by an independent Student t-test or a Mann-Whitney U-test. Differences were considered statistically significant at p < 0.05. The relationship between serum Vit-D and cathelicidin data was assessed using a Pearson correlation (significant threshold: p < 0.05).Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism® 5.03 for Windows . Vit-D supplementation and period effects were analyzed by two-way ANOVA followed by a Bonferroni n = 47) was half the original intended sample size (n = 84). An intermediary analysis was conducted, and showed a discrepancy between the initial hypothesis and the results obtained for cathelicidin. For these reasons, and because the vaccine strains were set to change the following year (2015), we decided to end recruitment.The volunteers were recruited over 2 years (2013 and 2014) on a similar schedule: the first visit (V1) in June, the second in October at the end of supplementation and for vaccination (V2), and the third in November, 28 days after vaccination (V3). Because of recruitment difficulties, the number of volunteers selected (n = 19) and Vit-D (n = 19) groups; the causes of drop-out are indicated in the flow chart , and for all subjects, 25-(OH)D concentration was >30 ng/mL at V2. The highest serum 25-(OH)D level was 58 ng/mL with the 600,000 IU cumulative Vit-D dose. This supplementation was demonstrated to be safe: no variation in plasma or urinary calcium levels and no clinically relevant adverse effects were observed. One month after the end of the supplementation (V3), no significant decrease in serum Vit-D level was observed in the D group (V3: 36.5 ± 6.3 vs. V2: 44.3 ± 8.6 ng/mL).At inclusion, no statistical difference in serum Vit-D level was observed between the groups , no statistical difference between groups was observed in serum cathelicidin levels . For the pre-vaccination Ab titers, there was no significant difference between the groups for any strain (p4). Nor was there any significant difference for post-vaccination Ab titers, except for the H3N2 strain which was significantly lower in the D than the P group (p5). No significant differences were observed after vaccination between P and D groups in either seroconversion (p < 0.05). This data needed to be stratified with regards to volunteers' serologic status before vaccination.Ab titers to inactivated influenza virus strains are presented in y strain ; p4. Nor P group ; p5. No nversion  or seropnversion  rates, ep < 0.05) after vaccination in both P and D groups, with a significantly lower level of H3N2 in the D than in the P group  after vaccination in the P group, although in the D group only the H3N2 strain increased significantly : Th1 , Th2 , Th17 , and Treg (TGFβ) . No datap = 0.0478, U-test) and IL-6  were observed in the D than in the P group, with no significant variation in TNFα/IL-6 ratio .The IFNγ and IL-5 plasma levels were similar irrespective of group or period, with no variation in the IFNγ /IL-5 ratio . Significantly lower levels of TNFα  and in V2 than V3 in the D group .No change was observed in the Th17-cytokine response (IL-17A and IL-23) either between groups or over different periods. The level of TFGβ was significantly higher in the D than in the P group after vaccination  , Th2 (CrTh2), Th17 (CCR6), and Treg (CD125 CD127−) . PercentThe PMN basal ROS production, expressed in fluorescence arbitrary unit, did not vary over periods or between groups .By analyzing several immune biomarkers, this trial assessed the effects of Vit-D supplementation on the response to influenza vaccination in Vit-D-deficient elderly persons.The study carries limitations that warrant consideration. Firstly, the differences in volunteers' vaccine status before vaccination may have limited the ability to observe the effects of Vit-D supplementation on influenza vaccination response. Secondly, the small sample size, which could mean the study was underpowered to detect changes in serum cathelicidin levels despite volunteers' well-defined Vit-D deficiency status and the significant (2-fold) increase in Vit-D level in the supplemented group. The lack of a reference analytical method and physiological ranges for serum cathelicidin may further compound this issue. Consequently, the study power calculated on cathelicidin variations with Vit-D supplementation was reduced from 80 to 47%.At the end of Vit-D supplementation , the mean change in serum Vit-D level ranged from +16.7 to +37.1 ng/mL, corresponding to 0.23–0.52 ng/mL for 100 IU. This is in line with data from Schleck et al. , who repThe antimicrobial properties of Vit-D have been extensively studied with respect to tuberculosis, where Vit-D enhances cathelicidin production and autophagy . These eThe effect of 25-(OH)D supplementation on the humoral immune response to influenza vaccination was evaluated. It showed no effect on Ab production in either seroprotection or seroconversion. This finding is consistent with two randomized controlled trials of Vit-D supplementation in influenza-vaccinated healthy adults  and adolThe volunteers' seroprotection (11–84%) and seroconversion rates (10–42%) were in line with data from the CDC, suggesting a clinical efficacy of 17–53% in elderly persons . The preConsidering the innate immune response, Vit-D supplementation induced a shift to a Th2- cytokine response as previously described  , 22. In 2D can inhibit Th1 differentiation (via expression of IFNγ) and increase the Th2 response by stimulating IL-5 production. The IFN-γ/IL-5 ratio is of interest when evaluating the Th1/Th2 balance (After Vit-D supplementation we noted a significant decrease in the Th1/Th2 ratio in link with TNFα and IL-6 reduced levels. This is in accordance with Penna's data  showing  balance , 54.Interestingly, the Vit-D supplementation was associated with an increase in TGFβ plasma levels after influenza vaccination, while no change in the Treg cell sub-population was observed. Likewise, previous studies showed that neuraminidase from influenza vaccine strains directly activates TGFβ production , which c2D strongly up-regulates the cathelicidin gene and protein expression NOX2-dependently, and induces antibacterial activity by NADPH oxidase pathway in phagocytes (Previous results  demonstragocytes . In our Our data demonstrate for the first time that Vit-D supplementation in deficient elderly persons promotes a higher TGFβ plasma level in response to influenza vaccination without improving antibody production. The Vit-D supplementation seems to direct the lymphocyte polarization toward a tolerogenic immune response as suggested by the lower Th1/Th2 ratio compared to controls. Taken together, our results suggest that vitamin D supplementation in deficient elderly persons is not an effective way to improve their antibody response to influenza vaccine. A deeper characterization of metabolic and molecular pathways of these observations will aid in the understanding of vitamin D's effects on cell-mediated immunity in deficient elderly persons.NG-M, AG, BE, HL, and M-PV designed the protocol. NG-M, JT, PB, and MP-V performed the experiments, analyzed the data and wrote the manuscript. CD and VC conducted the medical visits. GM and VS participated to the biological analysis and serum vit D quantification. All authors read and approved 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.