Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder, which is linked to the contraction of the D4Z4 array at chromosome 4q35. Recent studies suggest that this shortening of the D4Z4 array leads to aberrant expression of double homeobox protein 4 (DUX4) and causes FSHD. In addition, misregulation of microRNAs (miRNAs) has been reported in muscular dystrophies including FSHD. In this study, we identified a miRNA that is differentially expressed in FSHD myoblasts and investigated its function.2C12 cells to determine the effect of miR-411 on myogenic markers.To identify misregulated miRNAs and their potential targets in FSHD myoblasts, we performed expression profiling of both miRNA and mRNA using TaqMan Human MicroRNA Arrays and Affymetrix Human Genome U133A plus 2.0 microarrays, respectively. In addition, we over-expressed miR-411 in C2C12 cells were transfected with miR-411 to determine whether miR-411 affects YAF2 expression in myoblasts. The results showed that over-expression of miR-411 reduced YAF2 mRNA expression. In addition, expression of myogenic markers including Myod, myogenin, and myosin heavy chain 1 (Myh1) were suppressed by miR-411.Using miRNA and mRNA expression profiling, we identified 8 miRNAs and 1,502 transcripts that were differentially expressed in FSHD myoblasts during cell proliferation. One of the 8 differentially expressed miRNAs, miR-411, was validated by quantitative RT-PCR in both primary  and immortalized  myoblasts. In situ hybridization showed cytoplasmic localization of miR-411 in FSHD myoblasts. By analyzing both miRNA and mRNA data using Partek Genomics Suite, we identified 4 mRNAs potentially regulated by miR-411 including YY1 associated factor 2 (YAF2). The down-regulation of YAF2 in immortalized myoblasts was validated by immunoblotting . CThe study demonstrated that miR-411 was differentially expressed in FSHD myoblasts and may play a role in regulating myogenesis. Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy with estimated prevalence of 1:20,000 . FSHD isDUX4) gene. Several DUX4 splice variants have been reported to be expressed in germ-line cells and myoblasts [DUX4 transcript (fl-DUX4) has been shown to be cytotoxic in vivo and ex vivo when ectopically expressed [DUX4 mRNA to be transcribed [DUX4 transcripts from the last D4Z4 repeat to be polyadenylated and therefore stable for protein translation. This combination of events leads to the aberrant expression of DUX4 and the downstream molecular changes involved in FSHD [Myod regulated pathways and inducing muscle atrophy pathways [Each D4Z4 repeat contains a double homeobox protein 4  are short (~22 nucleotides) non-coding RNAs which regulate gene expression by interfering translation or promoting degradation of target mRNAs ,36. A ma2. For in situ hybridization experiments, cells were seeded on poly-D-lysine/mouse laminin-coated coverslips .Primary myoblasts were obtained from EuroBioBank   with 10% heat-inactivated fetal bovine serum (Sigma). Myotube differentiation was induced by culturing the cells in differentiation medium consisting of DMEM with 2% heat-inactivated horse serum (Sigma) at 37°C, 5% CO2.C2. The culture dish was coated with 0.1% gelatin (Sigma-Aldrich).Immortalized human myoblasts were from the Boston Biomedical Research Institute and cultured as described in previously published protocol ,59. BrieMyoblasts purity was determined by performing immunofluorescent staining using anti-human desmin (Dako) antibody. Myoblasts that exhibited greater than 70% desmin-positive cells were utilized for expression profiling. Immunostaining was conducted as previously described . BrieflymirVana miRNA isolation kit (Life Technologies) according to manufacturer’s protocol. Following RNA isolation, RNA quality and concentration were determined by gel electrophoresis and NanoDrop (Thermo Fisher Scientific), respectively. The miRNA profile of each myoblasts was determined using TaqMan Human MicroRNA Array v2.0 (Human Array A) (Life Technologies) according to manufacturer’s protocol. Briefly, reverse transcription (RT) was performed with 100 ng of total RNA, Multiplex RT Human primer pools, and TaqMan MicroRNA Reverse Transcriptase Kit (Life Technologies). Real-time PCR was performed with TaqMan Universal PCR Master Mix, No AmpErase UNG (Life Technologies) using the Applied Biosystems 7900HT System. Ct values of all miRNAs were determined using RQ Manager 1.2 (Life Technologies) with a threshold of 0.1. Ct values were then imported into Partek Genomics Suite 6.5 and normalized to control, RNU48. ANOVA analysis was performed using Partek Genomics Suite 6.5 . No multiple testing corrections were performed.To identify differentially expressed miRNA in FSHD primary myoblasts, we performed miRNA expression profiling using proliferating primary FSHD and control myoblasts  following the manufacture’s protocol and as previously described ,61. AbsoT Method (ΔΔ CT Method) [Expression levels of miR-411 were analyzed by qRT-PCR using TaqMan MicroRNA assays according to the manufacturer’s instructions. RNU48 served as an endogenous control (Life Technologies). Briefly, RT reactions were performed with 10 ng of total RNA using TaqMan MicroRNA Reverse Transcriptase Kit (Life Technologies). Real-time PCR was performed using TaqMan Universal PCR Master Mix, No AmpErase UNG (Life Technologies) and Applied Biosystems 7900HT System. Relative fold changes were calculated using the comparative C Method) ,64. We cMyod, myogenin, myosin heavy chain 1 (Myh1), and YY1 associated factor 2 (Yaf2) were determined using qRT-PCR as previously described [Myod, myogenin and Myh1 primers were a gift from Dr. Tatiana V. Cohen [Myod, 5′- GGCTACGACACCGCCTACTA -3′; and 5′- GCTCCACTATGCTGGACAGG -3′. myogenin, 5′- GGGCAATGCACTGGAGTT -3′; and 5′- ATGGTTTCGTCTGGGAAGG -3′. Myh1, 5′- GCAAGAAGCAGATCCAGAAAC -3′; and 5′- CGGTCTTCCTCAGTTTGATAAG -3′. Yaf2, 5′- ATCAGGGTTAGCGCTGTTGT -3′; and 5′- TGGCAAGTTCTTTCCTGCTT -3′. All the Ct values were extracted with SDS (Life Technologies) and, after normalization using r18s, were converted into expression levels. Student t-tests were performed to determine the statistical significance of changes.Expression levels of escribed . 18s rRNV. Cohen . The pri′-digoxigenin (DIG) labeled miRCURY locked nucleic acid (LNA) detection probes, including hsa-miR-411 (38398–01), U6 (99002–01) and Scramble-miR (99004–01) (Exiqon) following the protocol from Exiqon and Roche with few modifications. Cultured cells were fixed with 4% paraformaldehyde (PFA)/PBS, rinsed with 100% methanol, then rehydrated and acetylated. Cells were then pre-hybridized in hybridization buffer at 37°C for 1 hour [In situ hybridization was performed using 5r 1 hour . The hybImmunoblotting was performed as described previously . Briefly3/cm2 C2C12 cells were plated in growth medium one day before the transfection. Cells were transfected when they reached 70% confluence. A final concentration of 30 nM oligos was used for the transfection. Differentiation was induced 6 hours after the pre-miR-411 transfection.Cells were transfected with 30 mM Ambion pre-miR-411 precursor oligos (Life Technologies) using Lipofectamine 2000 (Life Technologies) following the manufacture protocol. Briefly, 4×10MiRNAs have been shown to be misregulated in FSHD and other muscular dystrophies and were proposed to be involved in the pathological mechanisms of muscular dystrophies. To identify miRNAs misregulated in proliferating FSHD myoblasts and their potential mRNA targets, we performed miRNA and mRNA expression profiling using total RNA samples isolated from 3 sets of proliferating primary myoblasts. Using TaqMan Human MicroRNA Arrays, we identified 8 miRNAs differentially expressed in FSHD myoblasts comparing to controls  , calmodulin-like 4 (CALML4) , mitogen-activated protein kinase kinase 1 (MAP2K1) , and SH3 and multiple ankyrin repeat domains 2 (SHANK2) . Among the 4 genes, two genes, YAF2 and MAP2K1, are known to be involved in cell cycle regulation and early myogenesis. In addition, YAF2 negatively regulates myotube differentiation by inhibiting Yin Yang 1 (YY1) activity [To identify mRNA transcripts that can be potentially regulated by miR-411, mRNA profiles generated using the same total RNA samples used for miRNA profiling were analyzed to identify potential mRNA targets of miR-411. First, 1,502 mRNA transcripts were differentially expressed in the FSHD myoblasts comparing to the control samples during cell proliferation (p<0.05) . All five splice variants have one or two putative miR-411 binding site in their 3′ UTR , and Myh1 were examined by qRT-PCR. The results showed that Yaf2 expression level was significantly down-regulated  by miR-411 over-expression  and Myog  as well as a myotube differentiation marker, Myh1 , were also significantly down-regulated by miR-411 over-expression : locked nucleic acid; (PFA): paraformaldehyde; (AP): alkaline phosphatase; (BCIP): 5-bromo 4-chloro-3-indolyl phosphate; (NBT): nitroblue tetrazolium; (PVA): polyvinyl alcohol; (HRP): horseradish peroxidase; (CALML4): calmodulin-like 4; (MAP2K1): mitogen-activated protein kinase kinase 1; (SHANK2): SH3 and multiple ankyrin repeat domains 2; (YY1): Yin Yang 1; (Myog): myogenin; (PcG): Polycomb group; (Rb): retinoblastoma gene; (SRF): serum response factor.(FSHD): Facioscapulohumeral muscular dystrophy; (DUX4): double homeobox protein 4; (fl-DUX4): full-length DUX4 transcript; (miRNA): microRNA; (pri-RNA): primary-miRNA; (RISC): RNA-induced silencing complex; (Mef2): myocyte enhancing factor 2; (PBS): phosphate buffer saline; (RT): reverse transcription; (qRT-PCR): quantitative reverse transcription polymerase chain reaction; (Myh1): myosin heavy chain 1; (YAF2): YY1 associated factor 2; (DIG): 5The authors declare no conflicts of interests.PS and MCW provided the primary myoblasts and participated in manuscript preparation. NH performed the experiments and prepared the manuscript. Y-WC designed and supervised the experiments and prepared the manuscript. All authors read and approved the final manuscript.Human primary myoblasts used for miRNA and mRNA profiling (the top 6 samples) and validation (the bottom 8 samples).Click here for filemiRNAs differentially expressed in proliferating FSHD myoblasts (p<0.05).Click here for filemRNAs differentially expressed in proliferating FSHD myoblasts (p<0.05).Click here for file′ UTR of 5 splice variants of YAF2. The seed region of miR-411 is underlined. The complimentary bases are highlighted in yellow. The variant 2 has been reported to be expressed in skeletal muscles.Putative miR-411 binding sites in the 3Click here for file


Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca2+]i. We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca2+ dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover.The calpains are physiologically important Ca 2+ or mediate dimerization; however, typical and atypical calpains, which lack EF hand motifs, are both present in mammals. Hence, any therapeutic intervention designed to suppress degenerative conditions, particularly those caused by elevated Ca2+ levels, should also consider the potential involvement of atypical calpains. We have taken advantage of the model organism C. elegans, which only encodes atypical calpain proteins, to gain an understanding of the evolution and activities of these proteins. We show that the CLP-1 atypical calpain is normally expressed in muscle and localizes to sarcomeric sub-structures. We find that CLP-1 contributes to the muscle degeneration observed in a model of Duchenne muscular dystrophy. Our studies also highlight the importance of calcium dysregulation in promoting CLP-1 activity and muscle degeneration.Calpains are calcium activated non-lysosomal proteases that cleave proteins with exquisite selectivity. Proteins can be activated by calpain cleavage, because they are released from inhibitory constraints, or they can be targeted for further degradation to facilitate their normal physiological turnover or to promote cellular remodelling. Inappropriate calpain activity can lead to degenerative pathologies and cancers. Our understanding of calpain function is based primarily on typical calpains, which carry EF hand motifs that bind Ca This absence of EF hands was also responsible for the initial belief that atypical calpains were insensitive to CaC. elegans encodes multiple atypical calpains, but no typical calpains. Given the paucity of typical calpains in non-mammals, phylogenetic arguments have been presented to suggest that the EF hand motifs of typical calpains were late evolutionary additions C. elegans and DrosophilaGenes encoding predicted calpain proteins have been identified in many organisms ranging from single-celled yeasts to higher vertebrates through the sequencing of whole genomes. Mammalian genomes encode nine typical and five atypical calpains CAPN3) CAPN10) CAPN8/9) Mammalian calpains participate in many cellular processes, including, but not limited to, cytoskeletal remodelling Despite their involvement in cell maintenance and disease, it has been difficult to predict the targets of calpain proteolysis with any precision, because substrate specificity is only weakly determined by primary sequence C. elegans as a model for exploring the function of atypical calpains. Previous work in C. elegans has highlighted the importance of the tra-3/clp-5 calpain gene in sex determination C. elegans clp-1 and tra-3/clp-5 genes are involved in neurodegeneration and necrosis of a subset of vulval cells and in the intestine C. elegans development. To gain information about the potential site of action of a subset of atypical calpain genes, we have examined their in vivo expression patterns. Given the association between calpain activity and degenerative pathologies, we have investigated factors that influence the activity of clp-1 in a C. elegans model of muscular dystrophy and reveal the importance of both sustained calpain expression and intracellular Ca2+ levels ([Ca2+]i). Taken together, we hypothesize that the muscle degenerative phenotype caused by ectopic clp-1 stems from a conserved physiological role for calpain in the turnover of sarcomeric muscle proteins.By contrast to the many studies focused on the activity of the typical calpains, it is clear that the specific involvement of atypical calpains in development and disease pathologies remains underexplored. We are using C. elegans genome sequence using the typical human CAPN1 sequence and identified 14 atypical calpain-like sequences. Seven of these genes had previously been named clp-1 to clp-7, so we named the remaining seven genes clp-8 to clp-10 and clpr-1 to clpr-4 (for clp-related) for reasons explained below  and CLP-10 (W05G11.4) proteins, which are homologous to C. briggsae Cbr-G19393; the proteins in this cluster are each predicted to have a SolH domain. The second cluster includes clp-8 (F44F1.3) and four predicted clpr genes, which are missing a critical cysteine residue of the catalytic triad; together these genes are paralogous to Cbr-G04776 and Cbr-G00485, which encode proteins with an intact catalytic triad. The existence of clpr genes raises the possibility that these predicted calpains are inactive, or that they have possibly gained novel non-proteolytic activities  with human CAPN7, CAPN5/CAPN6 and CAPN15, respectively . Application of these profiles to other typical calpain proteins showed that the calpain from Trichoplax is predicted to have a penta EF hand whereas the sponge has only four predicted motifs  domains using existing models d motifs .C. elegans calpain-like proteins are predicted to lack proteolytic activity because the catalytic triad has not been conserved. Such inactive calpains have also been identified in protozoa C. elegans, CLP-3 and CLPR-1 to CLPR-4, Drosophila CALPC and mammalian CAPN6 proteins, which have incomplete catalytic triads. Surprisingly, putative inactive calpain proteins were conserved across the plant, animal and fungus kingdoms  clp-3 (RNAi) treated animals, except for clp-10 (ok2713) mutants in which the average brood size was reduced by approximately 50%  without a corresponding increase in embryonic lethality. Embryonic lethality was slightly elevated in mutants carrying deletions in calpain genes, but gross developmental, mobility or morphological defects were not observed. For reasons discussed below, we also stained the clp-1, -4, -6 and -7 deletion mutants with phalloidin, but failed to detect disruptions to the sarcomeric structure of adult body wall muscle ::nls::mRFP transcriptional reporters were co-expressed in animals carrying one of five different tissue-specific GFP reporters , vulva  and hypodermis (tra-3/clp-6 and clp-7)  . The expC. elegans, we next investigated the consequences of increasing calpain activity. This was first achieved by ectopically expressing full-length clp-1, -2, -4, -7 and tra-3/clp-5 cDNAs under the control of the inducible hsp-16.41 promoter, which is activated by heat shock in almost all tissues, except the germ line Increased calpain activity is associated with degenerative pathologies, such as cataract, neuronal degeneration and muscular dystrophy unc-54 promoter was used to drive ectopic expression of calpain cDNAs in body wall muscle unc-54p::clp-1::myc transgene crEx325 caused 1.5%±0.4% of adult animals to develop paralysis (n>1000); similar results were obtained for two other independent transgenic lines (data not shown). Affected animals displayed an uncoordinated (Unc) phenotype at the L4/early adult stage, which progressed to paralysis and finally to premature death as animals matured to day 2 adults  transgene crEx336 [unc-54p::clp-1(C371A)::mrfp] animals (n>1200), whereas paralysis developed in 1.9%±0.6% (n>300) of crEx335 [unc-54p::clp-1::mrfp] animals. We also observed that the level of CLP-1::mRFP expressed from crEx336, as measured by western blot analysis, was not reduced when compared to crEx335, and so could not account for the difference in their activities  from an tivities . Attemptclp-1 activity in neurons. Previous studies have shown that RNAi inactivation of clp-1 partially suppressed the degeneration of touch receptor neurons in animals carrying gain-of-function mutations in the mec-4 or deg-1 Na+ channel subunits clp-1 was overexpressed in touch receptor neurons unc-119p::clp-1 or unc-47::clp-1 reporter was expressed in other neurons.We next examined crEx325 [unc-54p::clp-1] array to generate crIs4, in order to circumvent potential problems associated with mosaic expression crIs4 array retained the same phenotypic characteristics as crEx325; 1.69±0.34 (n = 352) of crIs4 animals displayed paralysis. We next synchronized the growth of crIs4 worms and separated crIs4 adults into three distinct classes: 1) phenotypically wildtype with normal sinusoidal movement 2) Unc and 3) paralyzed, and examined the integrity of body wall muscles by staining actin thin filaments with phalloidin. We found that phenotypically wildtype crIs4 animals exhibited only occasional muscle cell abnormalities ; hlh-1(cc561ts) animals C. elegans, a null mutation in the only dystrophin-like protein gene, dys-1(cx18), caused only occasional muscle degeneration hlh-1(cc561ts), sensitized dys-1(cx18); hlh-1(cc561ts) mutants to become uncoordinated (Unc), but not paralyzed, and to display increased muscle degeneration In mammals, the absence of the large structural muscle protein dystrophin underlies the muscle degenerative disorder Duchenne muscular dystrophy (DMD) C. elegans DMD by constructing the dys-1(cx18); hlh-1(cc561ts); clp-1(tm690) strain. The clp-1(tm690) mutation deletes a 624 bp region of the clp-1 gene and introduces a translational frame-shift that is predicted to produce a 493 amino acid truncated protein, which lacks two of the three critical catalytic residues required for proteolytic activity. clp-1 (tm690) mutants are phenotypically wild-type and do not display any obvious defects in their muscle structure (dys-1(cx18); hlh-1(cc561ts); clp-1(tm690) was compared to that found in dys-1(cx18); hlh-1(cc561ts) animals after phalloidin staining, we found that the absence of clp-1 reduced the number of abnormal muscle cells by almost 50% (p<0.001). A body wall muscle cell was scored as abnormal when: 1) the classic striated pattern of actin filaments was disrupted 2) actin bundles were visible as puncta or 3) muscle cells were missing due to cell death. Thus, clp-1 is normally active in muscle and contributes to the muscle degeneration observed in dys-1(cx18); hlh-1(cc561ts) animals , itr-1(sy290gf), slo-1(js379) and unc-24(e138) , unc-24(e138) or slo-1(js379) (egl-19(ad695gf) mutation produced the most dramatic increase in paralysis (p<0.001), whereas the unc-24(e138) mutation caused a mild, but significant increase (p = 0.043) (slo-1(js379) mutation also increased the level of crIs4 paralysis, this effect could be attributed to either its role in Ca2+ regulation or to its participation in the Dystrophin Associated Protein Complex (DAPC), discussed below itr-1(sy290gf) mutation did not significantly affect the number of paralyzed crIs4 animals; however, ITR-1 expression has not been detected in muscle We found that the number of animals displaying paralysis was significantly increased when 1(js379) . Inclusi= 0.043) . AlthougcrIs4 and members of a second group of allelic modifiers that form the Dystrophin Associated Protein Complex (DAPC): dys-1(cx18), snf-6(ok720), dyb-1(cx36) and stn-1(ok292), which encode a dystrophin-like protein, acetylcholine transporter, dystrobrevin and syntrophin, respectively slo-1(js379) share a similar phenotype marked by hyperactivity and exaggerated head bending hlh-1(cc561ts) temperature sensitive allele of a MyoD homolog was also examined because this mutation sensitizes dystrophin dys-1(cx18) mutants to muscle damage We next constructed a set of double mutants between clp-1 provided by crIs4 (hlh-1(cc561ts) allele substantially increased the proportion of paralyzed crIs4 animals. We next tested whether the effects of crIs4 could be further enhanced in a dys-1(cx18); egl-19(ad695gf) genetic background by increasing [Ca2+]i. Adult dys-1(cx18); egl-19(ad695gf) double mutants in the absence of crIs4 are Unc, but not paralyzed, and exhibit moderate muscle degeneration dys-1(cx18); egl-19(ad695gf); crIs4 adults developing paralysis was vastly elevated (56.3±6.4%) when compared to animals expressing crIs4 in combination with a mutation in either dys-1(cx18) or egl-19(ad695gf) alone (dys-1(cx18); egl-19(ad695gf); crIs4 animals was dramatically reduced and embryonic lethality elevated  alone . Moreoveelevated .dys-1(cx18); egl-19(ad695gf) mutants to crIs4 induced paralysis led us to investigate whether dys-1(cx18); egl-19(ad695gf) mutants might also be sensitized to the effects of ectopic clp-2, -4, -7 or tra-3/clp-5 expression in muscle under control of the unc-54 promoter. We examined over 1000 animals from at least three independent transgenic lines for each calpain, however, we failed to detect any exacerbated defects in movement aside from those already reported for dys-1(cx18); egl-19(ad695gf) mutants clp-1 was found to induce paralysis when overexpressed in muscle under conditions of elevated [Ca2+]i.The heightened sensitivity of 2+ channel is located along the basal membrane of muscle. To demonstrate that egl-19(gf) was exerting an effect on clp-1 activity specifically by altering [Ca2+]I, we asked if the small molecule antagonist, nemadipine-A, could suppress CLP-1 mediated muscle degeneration; nemadipine-A has been shown to be a specific and highly effective inhibitor of EGL-19 egl-19(ad695); crIs4 mutants (hlh-1(cc561ts); crIs4 mutants with nemadipine-A and found that paralysis was also reduced by over 50% compared to untreated animals (hlh-1(cc561ts) mutation is likely to sensitize muscle cells to the effects of crIs4 indirectly by causing an increase in [Ca2+]I, which can be suppressed by inhibiting the activity of the EGL-19 Ca2+ channel.The EGL-19 L-type voltage gated Ca mutants . We next animals . This reasp-1, asp-3 and asp-4, have previously been shown to be constituents of a neural degenerative pathway involving the calpains clp-1 and tra-3/clp-5asp genes also promote muscle degeneration, we performed RNAi with the genes asp-1 to asp-6 on hlh-1(cc561ts); crIs4 animals. The effectiveness of RNAi knockdown was measured by quantitative PCR (qPCR) (asp-3(RNAi) reduced paralysis levels by over 40% and that asp-2(RNAi) and asp-4(RNAi) each reduced paralysis by ∼15%, whereas asp-1(RNAi) had no measurable effect in muscle. We further showed that clp-1(RNAi) almost completely abolished crIs4 induced paralysis in hlh-1(cc561ts); crIs4 animals, as might be expected  . We founexpected .C. elegans and the presence of both typical and atypical calpain genes in Schistosomes and Drosophila further suggests that C. elegans is likely to have undergone a lineage-specific loss of typical calpain genes; it is noteworthy that Drosophila and C. elegans have both been assigned to the clade Ecdysozoa CAPN1 and CAPN2 are still referred to as the main or major calpains despite the rich variety and abundance of genes encoding atypical calpain proteins . HoweverC. elegans presents an ideal model in which to examine the function of atypical calpains, because their genome encodes a range of variants that are representative of those found across phyla, including ancient metazoan lineages, and because of the availability of mutant alleles (clp-10(ok2713) mutants have a reduced brood size, but that the remaining mutants, with the exception of tra-3/clp-5, do not display obvious phenotypes affecting viability, motility or fertility clp-1 suppressed muscle degeneration in dys-1(cx18); hlh-1(cc561ts) mutants  DMD, which is based on a mouse model of myopathy involving the combined mutation of MyoD and dystrophin  allele of a MyoD homolog, sensitizes C. elegans to the effects of crIs4, as measured by the increase in paralysis, and that treatment of these worms with nemadine-A suppressed paralysis (2+ channel egl-19(gf); crIs4 mutants. Based on these results, we propose that the hlh-1(cc561ts) mutation creates a sensitized background for calpain activity by indirectly elevating [Ca2+]I, although it remains unclear if EGL-19 activity is disrupted. Following this logic, we speculate that inappropriate CLP-1 activity in a sensitized background could generate a positive feedback loop whereby calpain disrupts Ca2+ channel activity, leading to increased [Ca2+]i, and further activation of CLP-1. In support of this model, several studies have shown that typical calpains can cleave Ca2+ channels and disrupt their activities We have also shown that the aralysis . As nemaclp-1 and clp-4 genes appear to be expressed in muscle ; crIs4 mutants and to perform a chemical screen to identify compounds that ameliorate muscle damage. Such a screen could potentially identify calcium channel blockers or specific inhibitors of atypical calpains. Given that atypical calpain genes are also present in mammals, our results emphasize the importance of investigating the contribution of atypical calpains to degenerative disorders, especially under conditions associated with inappropriately elevated [Ca]I, such as muscular dystrophies, neurodegeneration and cataract.Finally, the ability of nemadipine-A to reduce the level of paralysis in activity  [96]. ThSee Protocol S1 for sequence accession numbers and details about sequence similarity searches and protein alignments.C. elegans atypical calpains were shown to be true negatives. An E-value cutoff level of 0.01 was used.A custom profile hidden Markov model was created for each of the five EF-hand motifs of the penta EF hand, using the following proteins as a training set: HsCAPN1 (AAH75862.1), HsCAPN2 (NP_001739.2), HsCAPN3/p94 (AAI46650.1), HsCAPN8 (NP_001137434.1), HsCAPN9 (NP_006606.1), HsCAPN11 (EAX04252.1), DmCALPA (NP_001097378.1), DmCALPB (NP_524016.4), DmCALPC (AAF48591.2) and HsGRAN (P28676) hlh-1(cc561ts) II, which were grown at 15°C ccIs4215 [myo-3::gfp::nls] I, LS292: dys-1(cx18) I, clp-8 [F44F1.3] (ok1878) I, clp-9 [T11A5.6] (ok1866) I, clpr-1 [W04A4.4] (ok2601) I, LS505: dyb-1(cx36) I, LS721: stn-1(ok292) I, LS587: dys-1(cx18) I; hlh-1(cc561ts) II, LS706: dys-1(cx18) I; egl-19(ad695gf) IV, NL2099: rrf-3(pk1426) II, PD4605: hlh-1(cc561ts) II, clp-1(tm690) III, clp-2 (pk323) III, clp-4 (ok2808) III, clp-10 [W05G11.4] (ok2713) III, JR667: unc-119 (e2498::Tc1) III, VC591: okIs53 snf-6(ok720) III, dpy-20(e1282) IV, DA695: egl-19(ad695gf) IV, clp-6 (ok1779) IV, clp-7 (ok2750) IV, itr-1(sy290) dpy-20(e1282) IV, unc-24(e138) IV, NM1968: slo-1(js379) V, EG1285: lin-15(n765); oxIs12 [unc-47::gfp+lin-15(+)] X, IM19: urIs13 [unc-119::gfp ], NK358: unc-119(ed4) III; qyIs43, UG756: bgIs312 (pes-6::gfp), wIs51 [scm::gfp (seam cell)+unc-119(+)]. The clp-1(tm690) III allele (kindly provided by Shohei Mitani) was sequenced using primers GGATGAGCTCTTCTATCGTG and GTCTGACCATGGTCCATTCC to confirm the presence of a 624 bp deletion, which is predicted to create a frame-shift at A(460), and produce a truncated protein of 493 amino acids.Worms were grown and maintained at 20°C as described egl-19 encodes an L-type voltage gated Ca2+ channel; an egl-19(ad695gf) gain-of-function mutation delays the inactivation of the EGL-19 L-type voltage gated Ca2+ channel, and hence leads to increased [Ca2+]iitr-1 gene encodes an inositol 1,4,5-triphosphate receptor (IP3R) homolog that resides on the endoplasmic reticulum (ER) and releases Ca2+ into the cytoplasm in response to IP3 signalling itr-1(sy290gf) allele is a gain-of-function mutation that increases [Ca2+]i. Because the itr-1(sy290gf) mutant carries a point mutation and does not display an obvious phenotype, the dpy-20(e1282) mutation, which is closely linked to the itr-1 gene, was included to facilitate the identification of itr-1 homozygotes. To account for potential marker effects, paralysis was also scored in a dpy-20(e1282); crIs4 genetic background. slo-1(js379) is a loss-of-function allele of a gene that encodes a Ca2+ activated potassium BK channel 2+]i, which leads to plasma membrane hyperpolarization. In turn, [Ca2+]i is reduced by the inactivation of L-type Ca2+ channels, such as EGL-19 unc-24(e138) is a loss-of-function allele of a gene encoding a protein containing stomatin-like and lipid transfer domains that indirectly regulates Na+ channels, and hence, similar to slo-1, unc-24 affects [Ca2+]i through plasma membrane hyperpolarisation Primer sequences are available in asp-1 to asp-6 were obtained from a library RNAi was performed as described by cloning PCR amplified products into the L4440 RNAi feeding vector ama-1 as an endogenous reference gene relative to N2 wildtype animals ama-1 (PK1084/PK1085); clp-1 (PK1062/PK1063); clp-2 (PK1113/PK1114); clp-3 (PK1066/PK1067); clp-4 (clp-4_f2/clp-4_r2); clp-6 (clp-6_f2/clp-6_r2); clp-7 (PK1074/PK1087); clp-8 (clp-8_f2/clp-8_r2); clp-9 (clp-9_f2/clp-9_r2); clp-10 (clp-10_f2/clp-10_r2); clpr-1 (clpr1_f2/clpr1_r2); asp-1 (PK1095/PK1096); asp-2 (PK1097/PK1098); asp-3 (PK1099/PK1100); asp-4 (PK1101/PK1102); asp-5 (PK1103/PK1104); asp-6 (PK1105/PK1106).Animals were harvested and RNA was extracted as described by Hope (1999). cDNA was amplified from 1.5 µg of DNAse treated mRNA using Taqman Reverse Transcription Reagents (Applied Biosystems), as directed by the manufacturer. qPCR was performed in triplicate in 20 µl reactions using Fast SYBR Green Master Mix (Applied Biosystems) and analysed using Fast System SDS software (Applied Biosystems); PCR products were verified by agarose gel electrophoresis. Gene expression data were analysed using the ΔΔCT method and normalized using rol-6 (su1006) or 50 µg/ml of TG96 sur-5p::gfp. At least three independent transgenic strains were generated and examined for each construct, but only those displayed in this manuscript are listed, as they are representative of the patterns observed for a given construct. The following extrachromosomal arrays were generated with the pRF4 co-transformation marker: crEx65 (clp-1p::nls::mrfp), crEx70 (clp-2p::nls::mrfp), crEx72 (clp-3p::nls::mrfp), crEx74 (clp-4::nls::mrfp), crEx78 (tra-3p::nls::mrfp), crEx83 (clp-6p::nls::mrfp), crEx79 (clp-7::nls::mrfp), crEx202 (clp-1p::gfp), crEx110 (hsp-16.64p::clp-1), crEx86 (hsp-16.64p::clp-2), crEx88 (hsp-16.64p::clp-4), crEx8 (hsp-16.64p::tra-3), crEx96 (hsp-16.64p::clp-7) and crEx333 (unc-54p::clp-1::mrfp). The following extrachromosomal arrays were generated with the TG96 co-transformation marker: crEx325 (unc-54p::clp-1), crEx141 (unc-54p::clp-2), crEx147 (unc-54p::clp-4), crEx263 (unc-54p::tra-3), crEx258 (unc-54p::clp-7), crEx241 (unc-47p::clp-1), crEx190 (unc-119p::clp-1), crEx319 (unc-54p::clp-1(C371A)), crEx335 (unc-54p::clp-1::mrfp) and crEx336 (unc-54p::clp-1(C371A)::mrfp).Worms were transformed by germ line microinjection unc-54p::clp-1(crEx325) extrachromosomal array, crEx325 was chromosomally integrated by gamma irradiation using a 137Cs source . The integrated strain, crIs4 (unc-54p::clp-1) was outcrossed five times with N2 prior to study.The unc-119::gfp, a pan-neuronal marker unc-47::gfp, which is specifically expressed in GABAergic neurons of the ventral nerve cord myo-3::gfp::nls, which is expressed in all muscle cells except those of the pharynx scm::gfp, a seam cell marker pes-6::gfp, a excretory cell GFP reporter.Tissue-specific GFP reporters used to identify tissues expressing calpain reporters include: Brood size and embryonic lethality was scored by placing individual L4 staged hermaphrodites on NGM plates and transferring them daily to a fresh plate until egg laying had ceased. The number of dead eggs  and live animals (brood size) were scored two days after transfer of the mother.The percentage of animals displaying paralysis was scored on day 2 of adulthood after first obtaining synchronized populations of animals of the specified genotype by bleaching gravid adults with alkaline hypochlorite 2, 0.004% (w/v) SDS) containing 2 U Alexa Fluor 594 phalloidin, incubated for 1 hour in the dark and washed twice with PBS-Tween 20 (0.5%) (Sigma) before viewing.Animals were stained with Alexa Fluor 594 phalloidin (Invitrogen). Briefly, day 2 adults displaying wildtype, Unc or paralyzed phenotypes were lyophilized in an Automatic Environmental Speedvac (Savant) prior to fixation in ice-cold acetone. Animals were resuspended in 20 µl S-Mix (0.2 M Na phosphate (pH 7.5), 1 mM MgClEighty day 2 adult animals were placed in 20 µl of SDS protein sample buffer, electrophoresed on 10% or 4–12% gradient SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Blots were incubated with primary antibodies at the following concentrations: anti-mRFP/GFP primary antibody (Invitrogen), 1∶1000; anti-myc 9E10, 1∶1000; anti-α tubulin (Abcam), 1∶4000; anti-actin (Sigma), 1∶1000. Protein was visualized using anti-mouse or anti-rabbit horseradish peroxidase (HRP) linked antibodies (Amersham) at 1∶5000 dilution and Western Lightning™ chemiluminescent substrate (Perkin Elmer). Nitrocellulose membranes were stripped using Restore Plus stripping buffer (Thermo Scientific). Protein levels were quantified using ChemiDoc-It imaging system (UVP).hlh-1(cc561ts) mutants, which were raised at 15°C. Phenotypic scoring is described above.Worms were grown in wells of 24-well plates containing 1 ml of MYOB agar Animals were immobilized using 10 mM sodium azide. Differential interference contrast (DIC) and fluorescent images were captured with a Zeiss Axioskop 2 fitted with an ORCA-ER (Hamamatsu) digital camera driven by Openlab 4 software (Improvision), or a Zeiss LSM 710 confocal driven by Zeiss Zen software.Statistical analysis was performed using Student's T-test.Figure S12+-activated calpain proteases. Atypical and typical calpains share a conserved catalytic DII, which is further separated into Ca2+ binding sites, IIa and IIb. Residues of the catalytic triad are highlighted with arrows; missing catalytic residues are marked with an X. Many calpains have a short non-conserved DI sequence, which are subject to autolysis, and a DIII that carries a C2 Ca2+-binding domain. DIV is exclusive to typical calpains and is distinguished by the presence of a penta EF-hand domain; the fifth EF-hand motif mediates heterodimerization with a small regulatory subunit CAPNS1 (not shown). A number of additional domains and motifs are also associated with calpain proteins: G and G E, regions rich in glycine or glycine and glutamate, respectively; SEEL, a potential C-terminal ER target sequence; PBH, PalB homology domain with some domain III homology; T, C2 domain originally identified in TRA-3 2+, zinc finger motif-containing; SolH, small optic lobes (SOL) homology domain; and MIT 1, microtubule interacting and transport domain.The modular arrangement of Ca(TIF)Click here for additional data file.Figure S2C. elegans calpains  and human calpains (CAPN1 and CAPN2). Domains are highlighted with coloured lines as follows: DI (green), DII (red) and DIII (purple). The catalytic residues C, H and N are highlighted with red boxes. Conserved residues involved in coordinating Ca2+ within DII are highlighted yellow, and residues R and E which coordinate the Ca2+ induced conformational change between domain IIa and IIb in human CAPN2 are highlighted yellow and indicated with arrows Sequence alignments of calpain proteins. Protein sequence alignment of fourteen (DOC)Click here for additional data file.Figure S3Porphyromonas gingivalis (Pg). Prefixes used to identify species include: C. elegans (Cel), C. briggsae (Cbr), D. melanogaster (Dm) and H. sapiens (Hs). GenBank accession numbers are provided in the Methods section.Phylogenetic relationships between calpain proteins. Cladogram of calpain and calpain-like proteins from worm, fly and human, which is rooted using the prokaryote calpain-related protein, TPR-1, from (TIF)Click here for additional data file.Figure S4Identification of proteolytically inactive calpain-like proteins. (A) Inactive calpain proteins (344) that are missing key residues of the catalytic triad were identified across all eukaryotic kingdoms. (B) Inactive calpain proteins (100) were found distributed across animal phyla, ranging from ancient Placozoa and Cnidaria to Chordata.(TIF)Click here for additional data file.Figure S5clp-1 (tm690), (B) clp-4 (ok2808), (C) clp-6 (ok1779), and (D) clp-7 (ok2750) deletion mutants at day 3 of adulthood. Following phalloidin staining, the 20 most central muscle cells from each of the two most visible body wall muscle quadrants were scored , as described in Gieseler et al. (2000). Abnormal body wall muscle cells were not seen in any of the four clp mutants from 3 independent experiments involving at least 30 animals per experiment. Scale bar is 20 µm.Calpain deletion mutants do not display defects in body wall muscle. Representative images of phalloidin stained body wall muscle from (A) (TIF)Click here for additional data file.Figure S6C. elegans atypical calpain transcriptional reporters. (A) Basic construction of nls::mrfp expression reporters driven from calpain promoters. (B–F) Nuclear localized expression patterns of calpain reporters in adult hermaphrodites. (B) clp-1p::nls::mrfp (crEx65) (C) clp-2p::nls::mrfp (crEx70) (D) clp-4p::nls::mrfp (crEx74) (E) tra-3p::nls::mrfp (crEx78) (F) clp-7p::nls::mrfp (crEx79). Each micrograph is typical of the pattern observed with at least two other independent transgenic strains generated with the same reporter construct. Nomarski DIC micrograph, left; mRFP fluorescence micrograph, right. A montage of overlapping images captured in the same focal plane was created to show the entire worm. Scale bar, 50 µM.Expression patterns of five (TIF)Click here for additional data file.Figure S7C. elegans atypical calpain transcriptional reporters. (A–E) Nuclear localized expression patterns of calpain reporters at different larval stages. (A) L2 larvae expressing clp-1p::nls::mrfp (crEx65), (B) L3 larvae expressing clp-2p::nls::mrfp (crEx70), (C) L4 larvae expressing clp-4p::nls::mrfp (crEx74), (D) L2 larvae expressing tra-3p::nls::mrfp (crEx78), (E) L1 larvae expressing clp-7p::nls::mrfp (crEx79). Nomarski DIC micrograph, left; mRFP fluorescent micrograph, right. Scale bar, 10 µM.Larval stage expression patterns of five (TIF)Click here for additional data file.Figure S8nls::mrfp transcriptional reporters and the GABA-ergic unc-47::gfp reporter. (A) clp-1p::nls::mrfp (crEx65) (B) clp-4p::nls::mrfp (crEx74), and (C) clp-7p::nls::mrfp (crEx79) co-localize with the unc-47::gfp reporter. unc-47::gfp reporter, left (green); calpain promoter driven nls::mrfp expression, middle (red); and co-localization, right (yellow). The tra-3/clp-5::mrfp reporter fails to co-localize with unc-47::gfp (data not shown). Each micrograph is typical of the pattern observed with at least two other independent transgenic strains generated with the same reporter construct. Scale bar, 10 µM.Co-localization of calpain (TIF)Click here for additional data file.Figure S9clp-2p::nls::mrfp (crEx70), (B) tra-3p::nls::mrfp (crEx78) and (C) clp-7p::nls::mrfp (crEx79). tra-3p::nls::mrfp (crEx78) is expressed in (D) the vulva and (E) the hypodermis. clp-7p::nls::mrfp (crEx79) is expressed in (F) the vulva and (G) the hypodermis. Vulval and hypodermal expression are indicated with a (v) and arrowheads, respectively. Nomarski DIC micrographs were overlaid with false colored mRFP fluorescence micrographs, and the entire image was converted to greyscale. Each micrograph is typical of the pattern observed with at least two other independent transgenic strains generated with the same reporter construct. Scale bar, 10 µm.Atypical calpain expression is associated with the intestine, vulva and hypodermis. Intestinal expression, indicated with arrows, is detected in (A) (TIF)Click here for additional data file.Figure S10unc-54p::clp-1. Animals were mechanically prodded (arrowhead) at t = 0 and examined for their ability to move away from a stimulus over a 10 s interval. Unc animals (top panel) are able to change body position, but have impaired mobility. Paralyzed animals (middle panel) show marginal movement of head and/or tail and an absence of mobility. The behavior of a wildtype adult (bottom panel) is shown by comparison. The time elapsed after the application of mechanical stimulus is indicated. Scale bars, 500 µm.Test to discriminate between phenotypically Unc and paralyzed adult animals expressing (TIF)Click here for additional data file.Figure S11unc-54p::clp-1::myc (crEx325) and hsp16-41p::clp-1::myc (crEx329) animals without (−HS) and four hours after heat shock (+HS). α-tubulin (TBA) provides a protein loading reference and CLP-1::myc expression levels are presented as a percentage of unc-54p::clp-1::myc protein levels and normalized to TBA. (B) CLP-1::myc expression levels in N2 wildtype and in hsp16-41p::clp-1::myc (crEx329) animals taken at intervals following heat shock. TBA provides a protein loading reference. CLP-1::myc expression levels are presented as a percentage of hsp16-41p::clp-1::myc protein levels 8 hours post heat shock and normalized to TBA. (C) CLP-1::mRFP protein expression levels in wildtype unc-54p::clp-1::mrfp (crEx335) and catalytically inactive unc-54p::clp-1(C371A)::mrfp (crEx336) animals. Actin was used as a protein loading reference. CLP-1::mRFP expression levels are presented as a percentage of unc-54p::clp-1::mRFP protein levels and normalized to actin. Values represent the average from two independent experiments.Detection and comparison of transgenic CLP-1::mRFP protein levels. (A) CLP-1::myc levels in N2 wildtype, (TIF)Click here for additional data file.Figure S12clp-1::gfp. (A) clp-1::gfp (crEx202) is expressed throughout the animal. Scale bar, 50 µM. (B–F) clp-1::gfp (crEx202) is expressed in many somatic tissues, including: (B) the head – pharyngeal muscles and the nerve ring, (C) body wall muscle, (D) the ventral nerve cord and muscle arms (white arrows), (E) vulva, (F) intestine. Each micrograph is typical of the pattern observed with at least two other independent transgenic clp-1::gfp strains. Scale bar, 10 µM.Translational expression pattern of (TIF)Click here for additional data file.Protocol S1Protein blast and phylogenetic analyses.(DOC)Click here for additional data file.Protocol S2Oligonucleotide sequences.(DOC)Click here for additional data file.Protocol S3Molecular biology and gene cloning methods.(DOC)Click here for additional data file.Table S1Eukaryotic catalytically inactive calpains.(DOC)Click here for additional data file.Table S2Effects of calpain mutants and feeding RNAi on brood size and embryonic lethality.(DOC)Click here for additional data file.Table S3clp-1 expression on brood size and embryonic lethality.Effects of ectopic (DOC)Click here for additional data file.Table S4asp-1 to asp-6 (RNAi).qPCR analysis of (DOC)Click here for additional data file.Text S1Profile hidden Markov models detecting EF hand motifs.(TXT)Click here for additional data file.


Pseudomonas aeruginosa immune-dominant outer membrane protein F (OprF) epitope 8 (Epi8) in five distinct sites of the Ad5 fiber, loops CD (AdZ.F(CD)Epi8), DE (AdZ.F(DE)Epi8), FG (AdZ.F(FG)Epi8), HI (AdZ.F(HI)Epi8) and C terminus (AdZ.F(CT)Epi8), or the hexon HVR5 loop (AdZ.HxEpi8) were compared in their capacity to elicit anti-P. aeruginosa immunity to AdOprF, an Ad expressing the entire OprF protein. Intramuscular immunization of BALB/c mice with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 elicited higher anti-OprF humoral and cellular CD4 and CD8 responses as well as enhanced protection against respiratory infection with P. aeruginosa compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8. Importantly, repeat administration of the fiber- and hexon-modified Ad vectors boosted the OprF-specific humoral immune response in contrast to immunization with AdOprF. Strikingly, following three doses of AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 anti-OprF immunity surpassed that induced by AdOprF. Furthermore, in the presence of anti-Ad5 immunity, immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8, but not with AdOprF, induced protective immunity against P. aeruginosa. This suggests that incorporation of epitopes into distinct sites of the Ad fiber is a promising vaccine strategy.Adenovirus (Ad) vector-based vaccines displaying pathogen-derived epitopes on Ad capsid proteins can elicit anti-pathogen immunity. This approach seems to be particularly efficient with epitopes incorporated into the Ad fiber protein. Here, we explore epitope insertion into various sites of the Ad fiber to elicit epitope-specific immunity. Ad vectors expressing the 14-mer  Pseudomonas aeruginosa is one of the leading nosocomial bacterial pathogens worldwide and can cause serious infections of the respiratory tract. A vaccine against P. aeruginosa would be useful as treatment is often challenged by antibiotic resistance of the organism. No efficient and marketable vaccine is yet available P. aeruginosa outer membrane protein F (OprF) is one of the promising vaccine antigens. OprF is surface exposed, antigenically conserved in wild-type strains of P. aeruginosa and elicits cross-reactive, opsonizing and protective antibodies in various animal models and humans P. aeruginosa. Human Ad serotype 5 (Ad5) or non-human primate Ad serotype C7 (AdC7) expressing OprF induced robust protective immunity against pulmonary infections with P. aeruginosa in mice Adenovirus (Ad) vectors are attractive delivery vehicles for genetic vaccines due to their ability to act as immune system adjuvants and to rapidly evoke robust immune responses against the transgene product and viral capsid proteins One of the limitations of Ad as vaccine carrier is that anti-Ad immunity elicited by the initial immunization usually prevents productive infection with subsequent immunizations, critical to achieve boosting of the anti-transgene immunity All animal studies were conducted in accordance to the protocols reviewed and approved by the Weill Cornell Institutional Animal Care and Use Committee (Permit Number 0703-594A). All efforts were made to minimize suffering to the animals.AdEasy™ adenoviral vector system  was used to construct the replication-defective recombinant human Ad5 vectors. The vectors expressed either β-galactosidase, referred to as “Z” in the vector (AdZ), or no transgene (AdNull) 10 pu/animal diluted in PBS to the left thigh muscle using a 0.5 ml insulin syringe .Female BALB/c mice were obtained from Taconic Farms. The animals were housed under specific pathogen–free conditions and were used at 6–8 weeks of age. Mice were immunized by injection of 50 µl of the Ad vectors AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF, AdZ or AdNull at a dose of 1010 virus particles) were denatured (95°C for 5 min) in NuPAGE sample buffer  and separated by 4 to 12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Following transfer to a polyvinylidene difluoride membrane  equal loading was confirmed by GelCode silver staining . The membrane was exposed to blocking solution  in PBS for 1 h and then incubated with either polyclonal anti-OprF serum, obtained from C57Bl6 mice immunized with recombinant OprF protein (1∶500) To evaluate the presence of the Epi8 epitope on the Ad fiber protein, purified Ad vectors  or dendritic cells  was analyzed. A549 cells , maintained in complete Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum, 100 U of penicillin/ml, 100 mg of streptomycin/ml , were infected with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8 or AdZ  in low-serum medium  for 2 h, washed, and then maintained in complete medium for 24 h. Bone marrow-derived DC were generated from bone marrow precursors as described previously To evaluate if incorporation of Epi8 in the different fiber sites interferes with the coxsackie-adenovirus receptor (CAR)-dependent or -independent infection 10 pu/animal and boosted twice with the same virus after 2 and 5 wk. Serum was collected after 2, 5 and 8 wk of initial immunization. Lung bronchioalveolar lavage fluid was collected by intratracheal instillation and aspiration of 0.5 ml PBS, pH 7.4., which was centrifuged at 6000 rpm at 4°C for 10 min and the supernatant was stored at −80°C. Anti-OprF, anti-Ad and anti-β-galactosidase IgG were assessed by ELISA using flat bottomed 96-well EIA/RIA plates  coated with recombinant OprF (0.5 µg/well) 9 pu/well) or β-galactosidase (0.1 ug/well) in 0.05 M carbonate buffer, pH 7.4. The plates were blocked with 5% dry milk in PBS for 1 h at 25°C and two-fold serial serum dilutions were added to each well and incubated for 1 h at 25°C. Following three washes with PBS containing 0.05% Tween (PBS-Tween) a peroxidase-conjugated sheep anti-mouse IgG (Sigma-Aldrich), diluted 1∶10,000 in PBS containing 1% dry milk, was added and incubated for 1 h at 25°C. Absorbance at 415 nm was measured with a microplate reader (Bio-Rad Laboratories) and the antibody titers were calculated with a log (OD)–log (dilution) interpolation model and a cutoff value equal to 2-fold the absorbance of the background.Mice were immunized intramuscularly with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF or AdZ at a dose of 1010 pu/well). Disruption of Ad5 vector was carried out in 0.5% sodium dodecyl sulfate  To assess surface exposure of Epi8 on the capsid-modified Ad vectors, ELISA plates were coated with intact or disrupted AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8 or AdZ (1010 pu of AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF or AdZ and boosted after 2 and 5 wk with the same vector. The frequency of OprF-specific CD4 and CD8 T lymphocytes was determined with a IL-4- and/or interferon-γ (IFN-γ)-specific enzyme-linked immunospot (ELISPOT) assay (R&D Systems) 7 days following the last immunization. Splenic CD4 or CD8 T cells were purified by positive selection with CD4 (L3T4) or CD8 (Ly-2) MACS microbeads . The purity of the T cells was more than 95%. To serve as antigen-presenting cells, splenic DCs were purified from syngeneic naive animals by positive selection with CD11c MACS beads (Miltenyi Biotec) and two consecutive purifications over MACS LS columns (Miltenyi Biotec). The purity of the DC was more than 90%. DC (5×106/ml) were incubated for 2 h with purified recombinant OprF protein (100 µg/ml) in RPMI medium supplemented with 2% fetal bovine serum , 10 mM HEPES , and 105 µM β-mercaptoethanol (Sigma-Aldrich). CD4 or CD8 T cells (2×105) were incubated with splenic DC with or without recombinant OprF protein at a ratio of 4∶1 in IL-4 and/or IFN-γ plates (R&D Systems) for 48 h. Following washing, biotinylated anti-IFN-γ or anti-IL-4 (both from R&D Systems) antibodies were added and incubated overnight at 4°C. For final spot detection a streptavidin-alkaline phosphatase conjugate followed by 3-amino-9-ethylcarbazole substrate (both R&D Systems) was added. The spots were counted by computer-assisted ELISPOT image analysis .To evaluate OprF-specific cellular immune responses induced by the capsid-modified Ad vectors, BALB/c mice were immunized intramuscularly with 10P. aeruginosa strain PAO1 was used to assess protective immunity. PAO1-containing agar beads were prepared based on the method of Starke et al. P. aeruginosa, BALB/c mice were immunized intramuscularly with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF or AdZ  followed by boost immunizations after 2 and 5 wk. Eight weeks following initial immunization, the mice were challenged intranasally with PAO1 (4×106 cfu in 50 ul) encapsulated in agar beads. Mice were sacrificed 24 h post challenge and lung homogenates were plated on McConkey agar plates. The numbers of colonies were quantified after 48 h.The Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using Two-Way ANOVA and statistical significance was determined at p<0.05.The Ad vectors with Epi8 incorporated in the fiber loops CD (AdZ.F(CD)Epi8), DE (AdZ.F(DE)Epi8), FG (AdZ.F(FG)Epi8), HI (AdZ.F(HI)Epi8) or C terminal (AdZ.F(CT)Epi8) were generated in the HEK 293-derived cell line that constitutively expresses the Ad5 fiber protein (293F) and subsequently propagated in regular HEK 293 cells as described in material and methods section. The presence of Epi8 epitope in the intact fiber protein on each of the purified vectors was assessed by Western analysis with serum from mice that had been immunized with OprF  transgene expression was assessed following infection of A549 cells or bone marrow-derived DC. In A549 cells, highest transgene expression was seen with AdZ and AdZ.HxEpi8 . An anti-Ad antibody was used to assess if even amounts of disrupted and intact vectors were plated (not shown). Overall, these results indicate that Epi8 inserted into the FG or HI is favorably exposed compared to insertion into the CD, DE loops, CT or the loop 1 of HVR5 in hexon.Epitopes inserted in capsid protein could be hidden by protein folding and so affect immune recognition. To investigate surface exposure and accessibility of the Epi8 epitope on the capsid-modified Ad vectors to antibody binding, intact or disrupted Ad vectors were probed with anti-OprF antibody in an ELISA plate. Intact AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 interacted strongly with anti-OprF compared to intact AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8 . At 2 weeks, anti-OprF titers were higher in animals immunized with AdOprF compared to animals immunized with capsid-modified vectors. Immunization with AdZ.F(FG)Epi8 and AdZ.F(HI)Epi8 elicited comparable titers (p>0.05) that were higher compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8 Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF or AdZ Epi8, AdZ.F(DE)Epi8, AdZ.F(FG)Epi8, AdZ.F(HI)Epi8, AdZ.F(CT)Epi8, AdZ.HxEpi8, AdOprF or AdZ and boosted twice with the same vector. The frequencies of OprF-specific splenic CD4 and CD8 T cells stimulated by syngeneic DC pulsed with recombinant OprF protein were analyzed by ELISPOT. Immunization with AdOprF induced the highest OprF-specific IFN-γ CD4 Epi8, AdZ.F(HI)Epi8, AdOprF or AdZ, boosted twice with the same vector and challenged by intranasal administration of agar-encapsulated PAO1 three weeks after the last vector administration. Mice immunized with AdZ.F(FG)Epi8, AdZ.F(HI)Epi8 and AdOprF showed reduction in the P. aeruginosa colony count compared to the AdZ group Epi8 was administered in the presence or absence of anti-Ad immunity Epi8 or AdOprF. In the presence of pre-existing anti-Ad immunity, AdOprF inoculated mice showed a marked reduction in anti-OprF titers that were close to basal levels  influenced the epitope-specific immune responses. Consistent with our results, upon second administration a stronger humoral response was elicited when ovalbumin epitope was inserted into fiber protein compared to hexon One attractive feature of Ad-based vaccines is the feasibility to modify the Ad capsid to enhance immune responses or change the Ad tropism P. aeruginosa is mainly dependent on sufficient levels of humoral immunity induced by the vaccine. T cell-mediated immunity has received less attention in the development of a vaccine against P. aeruginosa but is a part of the response against natural infection with the organism P. aeruginosa challenge is not clear.Protective immunity against extracellular bacteria such as in vitro infectivity and its capacity to elicit anti-peptide immunity. It would be interesting to assess infectivity and immunogenicity of a bispecific Ad vector with peptides at both FG and HI loops.The HI loop in the fiber knob has been the usual choice for incorporation of antigenic epitopes or targeting moieties Anti-Ad immune responses impair efficacy of Ad vectors of the same serotype, as pre-existing neutralizing antibodies against Ad prevent the cellular uptake of Ad and expression of the transgene in a previously immunized host P. aeruginosa immunity even in the presence of high levels of pre-existing anti-Ad immunity. This suggests that pre-existing Ad vector immunity can be effectively circumvented by the incorporation of antigenic epitopes into the fiber protein. Taken together, incorporation of antigenic peptides on the FG or HI loop of Ad fiber knob is an efficient strategy to generate protective immunity that can be boosted by repeated administration and is effective in the presence of pre-existing anti-Ad immunity. The display of antigenic epitopes on the Ad fiber should also be valuable in the development of Ad-based vaccines against other pathogens. The robust protective immunity induced by AdZ.F(FG)Epi8 and AdZ.F(HI)Epi8 make both of these sites attractive for the insertion of epitopes as a general vaccine strategy.The prevalence of pre-existing vector immunity in humans may limit the utility of human Ad serotype vaccines. Notably, in sharp contrast to immunization with AdOprF, AdZ.F(FG)Epi8 induced protective anti-


By contrast with P. falciparum msp-7 loci and ancestral P. vivax msp-7 genes, specie-specific duplicates of the latter specie display high genetic variability, generated by single nucleotide polymorphisms, repeat regions, and recombination. At least three major allele types are present in Pvmsp-7C, Pvmsp-7H and Pvmsp-7I and positive selection seems to be operating on the central region of these msp-7 genes. Although this region has high genetic polymorphism, the C-terminus (Pfam domain ID: PF12948) is conserved and could be an important candidate when designing a subunit-based antimalarial vaccine.A completely effective vaccine for malaria (one of the major infectious diseases worldwide) is not yet available; different membrane proteins involved in parasite-host interactions have been proposed as candidates for designing it. It has been found that proteins encoded by the merozoite surface protein ( Plasmodium vivax malaria should not be considered a benign disease anymore due to its wide geographical distribution P. vivax, relatively few vaccine candidate molecules have been thoroughly characterized P. falciparum merozoite surface msp-1 and msp-6 are both single copy genes while msp-7 has a large number of genes which seem not to have diverged functionally. Furthermore, the Plasmodium genus has dissimilar copy numbers of this gene msp-7 genes have a single exon and have been named in alphabetical order according to their position regarding the PVX_082640 flanking gene msp-7 genes are expressed simultaneously in the schizont stage of Plasmodium species MSP-1, MSP-6 and MSP-7 form the most abundant protein complex on the P. falciparum has eight msp-7 genes and P. vivax eleven, P. vivax msp-7A (GenBank ID: XM_001614080.1) and msp-7K (GenBank ID: XM_001614090.1) genes P. vivax specie-specific duplicates MSP-7C (GenBank ID: XM_001614082.1) and MSP-7H (GenBank ID: XM_001614087.1) are recognized by IgG antibodies from P. vivax-infected patient sera P. vivax msp-1 allele infections were considered for PCR amplification even though amplicons were not detected in a few samples .Forty-eight parasite samples were collected from symptomatic patients living in representative regions of Colombia for this study: Amazonian , Andean , Caribbean , Orinoquia  and Pacific  . Forty-tPvmsp-7C, 1,232-bp Pvmsp-7H and 1,182-bp Pvmsp-7I gene fragments were amplified and direct sequenced. Nucleotide sequence data here reported are available in GenBank: accession numbers JQ423957-JQ424037. Twenty-three haplotypes were found for Pvmsp-7C while twenty-eight haplotypes were found for both Pvmsp-7H and Pvmsp-7I. Haplotype diversity (Hd) was lower in the Pvmsp-7C gene than in Pvmsp-7H and Pvmsp-7I  was higher than that previously reported for other P. vivax msp-7 family members (Pvmsp-7A π = 0 and Pvmsp-7K π = 0.0022) msp-7 genes from this study were among the most polymorphic P. vivax genes reported to date  in the different Colombian regions, the Pacific area was the most diverse followed by the Andean, Caribbean and Orinoco regions while the Amazonian was the least polymorphic GT[V/E] repeat region from residue 195 to 209   and 3′-ends   to the Colombian  and F*) ; these v values) .S) and non-synonymous substitutions per non-synonymous site (dN) was estimated to determine whether natural selection was affecting the msp-7 loci. Although dN was higher than dS in Pvmsp-7C and Pvmsp-7I, dN was lower than dS in the Pvmsp-7H gene even though these values were not statistically significant (N/dS) , central  and 3′-end    showed td 1,188) ; dN and ee genes .Pvmsp-7C had 19 sites under positive selection and 21 negatively selected sites according to SCAL, FEL, IFEL and REL methods. Positive selection signatures were found for Pvmsp-7H in 50 sites while another 32 sites were negatively selected and Pvmsp-7I had 10 sites under positive selection and 30 negatively selected sites  for whole data revealed no statistically significant values for the three msp-7 genes (2 (linkage disequilibrium) with physical distance in regression analysis showed that LD declined as nucleotide distance increased  are particularly interesting since they have expanded differentially P. vivax msp-7 gene family has 11 functional genes P. vivax schizont stage At least eleven MSPs have been reported in msp-7 gene family members, at least in the two main human malaria species. By contrast with the very little polymorphism displayed by Pfmsp-7 genes Pvmsp-7 genes  have high genetic diversity. It is worth noting, however, that not all the Pvmsp-7 genes display a similar pattern. Pvmsp-7A and Pvmsp-7K, like Pfmsp-7A, Pfmsp-7B, Pfmsp-7C, Pfmsp-7D, Pfmsp-7E, Pfmsp-7F and Pfmsp-7H (GenBank ID numbers: XM_001350038.1–44.1) display very low polymorphism msp-7 genes might be exposed to different selective pressure (such as that exerted by the immune system) or have different biological constraints. Although low genetic polymorphism does not hold for Pvmsp-7C, Pvmsp-7H and Pvmsp-7I, vaccine development using these proteins could be focused on their conserved C-terminal domain (Pfam ID number PF12948) due to the high level of conservation it displays and, like in P. falciparum, the C-terminal domain could be involved in interaction with erythrocytes Besides being recognized by the immune system, vaccine candidates must have limited polymorphism. Results found in this study highlighted differences in allele diversity among P. vivax genes described so far .The genetic diversity found in these three genes places them among the most diverse d so far . DifferePvmsp-7C and Pvmsp-7I genes appear to be under balancing selection. It has been suggested that this type of selection increases variability within a population The Pvmsp-7C and Pvmsp-7I results discussed above, Pvmsp-7H appeared to be under a standard neutral model of molecular evolution since there were no significant values in neutrality tests and no difference between dN and dS was found; consequently, high polymorphism would be expected in regions lacking functional constraints N average from the whole gene is significantly greater than dS (the opposite occurs for negative selection). The sliding window test  for different selective pressures appeared to be acting throughout Pvmsp-7 gene sequences, as has been described for others proteins N and dS rates at the 5′- and 3′-ends and in the central region of the three genes. The central region was under positive selection while the 5′- and 3′-ends seemed to be under negative selection . Similar results were observed when using a second approach in which several maximum likelihood methods were performed to determine positively and negatively selected codons. The central region of the Pvmsp-7C, Pvmsp-7H and Pvmsp-7I genes had codons under positive selection while sites under negative selection were preferentially located at the 5′- and 3′-ends which were relatively fully conserved in Pvmsp-7 genes.By contrast with the msp-7 genes. These events affect the accuracy of detecting selected sites, increasing type I errors N substitutions (and/or positive selection) would have been randomly found across the genes; instead, these sites were just observed in the msp-7 central region and the non-synonymous substitutions in the positively selected sites were found as parsimonious sites and not as singleton sites. Recombination and selection thus seem to drive Pvmsp-7 genes’ antigenic variation, similar to what occurs in other pathogen antigens E. coli fimA gene Pvmsp-7 genes  possibly due to the presence of a functional domain (Pfam ID number PF12948) within this region. Therefore, the central region (the most diverse) could have been under selective pressure exerted by the immune system; consequently, the intragenic recombination and, to a lesser extent, the positively selected sites increased genetic diversity, generating different allele variants as an evasion mechanism.On the other hand, multigene families might be evolving by recombination among paralogous genes, thereby contributing to allele diversity or the homogenization of the multigene family in the event of unequal crossover or gene conversion P. vivax gene flow in Colombian regions. This result agreed with previous reports of other highly polymorphic P. vivax genes not involved in geographical clustering Phylogenetic analysis showed that, regardless of an isolate’s origin, sequences tended to cluster without having clear geographical distribution. This pattern might indicate a constant Previous studies have shown the potential role of MSP-7 in parasite invasion of erythrocytes P. vivax-infected patients who provided us with the blood samples signed an informed consent and the purpose of the study was carefully explained to them. All procedures carried out in this study were approved by our institute’s ethics committee.All P. vivax malaria by microscope examination were collected from geographical regions of Colombia from 2007 to 2010. DNA was obtained using a Wizard Genomic DNA Purification kit (Promega) following manufacturer’s instructions.Peripheral blood samples from patients proving positive for Pvmsp-1 gene’s blocks 6, 7 and 8 as previously described P. vivax msp-1 allele infection.All parasite samples were genotyped by PCR-RFLP of the Pvmsp-7C, Pvmsp-7H and Pvmsp-7I DNA fragments based on the Sal-I reference sequences . The DNA fragment from Pvmsp-7C was amplified with 7Cdto 5′ ACCACAAAGATGAATAAAACG 3′ and 7Crev 5′ CACCTCAATCGTGTTCAGC 3′ primers. Pvmsp-7H was amplified by using 7Hdto 5′ GTGTGCATCAGTATAGCGAC 3′ and 7Hrev 5′ AAGAAGGTTAGCCATAAATGC 3′ primers and Pvmsp-7I was amplified with 7Idto 5′ ACAATGAGGGGCAAGTACG 3′ and 7Irev 5′ TTCATTCGTTGCTCACTTCG 3′ primers. All PCR reactions were performed using KAPA HiFi HotStart Readymix containing 0.3 µM of each primer in a final 25 µL volume. Thermal conditions were set as follows: one cycle of 5 min at 95°C, 25 cycles of 20 sec at 98°C, 15 sec at 62°C for Pvmsp-7C and Pvmsp-7I and 60°C for Pvmsp-7H, 30 sec at 72°C, followed by a 5 min final extension at 72°C. PCR products were purified using the Wizard PCR preps kit (Promega), and then sequenced with a BigDye Terminator kit  in both directions. Internal primers were used for sequencing . Two PCR products obtained from independent PCR amplifications were sequenced per isolate to discard errors.Primers were designed to amplify msp-7 amino acid sequences were detected by using the T-REKS algorithm P. vivax msp-7C, msp-7H and msp-7I sequences were compared against the previously described Sal-I strain sequences available in GenBank .Electropherograms were assembled using CLC DNA workbench 6  and Clustal W N) and synonymous (dS) substitutions. Differences between dN and dS were assessed by applying the Z-test using MEGA software v.5 N/dS ratio) at every codon in the alignment. A ≤0.1 p-value was considered significant for IFEL, FEL and SLAC methods and ≥50 Bayes factor for REL. Only Colombian sequences were considered for all analyses performed; positions containing gaps or repeats in the alignment were not taken into account  and nucleotide distances was performed to evaluate whether recombination was taking place in Pvmsp-7 genes. Recombination events were assessed using DnaSP v.5 software msp-7 genes. This tool looks for evidence of recombination among aligned sequences by examining all possible triplet combinations following a scanning approach with a range of different recombination detection algorithms. Additionally, the algorithm developed by Betrán et al. (1997) Linkage disequilibrium (LD) was evaluated by calculating the ZPvmsp-7C, Pvmsp-7H and Pvmsp-7I sequences were aligned and trees were constructed by using ML with the TN93+G model selected by the ModelTest algorithm Maximum Likelihood (ML) trees describing the phylogenetic consequences of the recombination events were constructed using RDP3 v3.4 software Figure S1Geographical location of the study regions within Colombia. Amazonian region , Andean region (samples from Apartadó and El Bagre in the Antioquia department), Caribbean region (samples from Tierra Alta and Puerto Libertador in the Córdoba department), Orinoco region (samples from Tauramena in the Casanare department and Mapiripán in the Meta department), and Pacific region (samples from Istmina in the Chocó department and Tumaco in the Nariño department). Black dots on the map represent the areas from which patients came who donated the infected blood samples. 1: Puerto Libertador, 2: Tierra Alta, 3: Istmina, 4: Tumaco, 5: Apartado, 6: El Bagre, 7: Tauramena, 8: Mapiripan, 9: Calamar.(TIF)Click here for additional data file.Figure S2Sliding window analysis of Tajima D and Fu & Li D* and F* statistics along the Pvmsp-7C (A), Pvmsp-7H (B) and Pvmsp-7I (C) genes. Bars  below each figure represent the regions where the tests showed a significant deviation from the neutral expectation. 5′-end , central  and 3′-end .(PDF)Click here for additional data file.Figure S3N/dS) throughout the Sliding window analysis for ω rates (dPvmsp-7C (Blue), Pvmsp-7H (Red) and Pvmsp-7I (Green) genes. Discontinuity of the curves is due to gaps within the alignments which were not considered for the analysis. 5′-end , central  and 3′-end .(PDF)Click here for additional data file.Figure S4The linkage disequilibrium (LD) plot for P. vivax Pvmsp-7C (A), Pvmsp-7H (B) and Pvmsp-7I (C). Trace line represents the regression line which declined as nucleotide distance increased suggesting that intragenic recombination was taking place in msp-7 genes.(PDF)Click here for additional data file.Figure S5ML trees describing the phylogenetic consequences of the intragenic recombination in Pvmsp-7C. A topology is inferred for each recombinant fragment, (A) from nucleotides 685 to 855, (B) from nucleotides 1,060 to 1,167  and from 18 to 266, and (C) from nucleotides 62 to 266. Isolates clustered without a clear geographical distribution. Sal-I: Salvador strain, AMA: Amazon, AND: Andean, CAR: Caribbean, ORI: Orinoco, PAC: Pacific.(TIF)Click here for additional data file.Figure S6ML trees describing the phylogenetic consequences of the intragenic recombination in Pvmsp-7H. A topology is inferred for each recombinant fragment, (A) nucleotides 189 to 543 (excluding nucleotides 436 to 450 and nucleotides 484 to 486), (B) nucleotides 353 to 661 (excluding nucleotides 436 to 450 and 568 to 627), (C) nucleotides 425 to 655 , (D) nucleotides 500 to 1,057 (excluding nucleotides 568 to 627 and 772 to 774), (E) nucleotides 628 to 977 (excluding nucleotides 772 to 774), (F) nucleotides 630 to 997, (G) nucleotides 416 to 590 (excluding nucleotides 436 to 450 and 484 to 486), (H) nucleotides 472 to 531 (excluding nucleotides 484 to 486), and (I) nucleotides 482 to 646 (excluding nucleotides 568 to 627). Isolates clustered without a clear geographical distribution. Sal-I: Salvador strain, AMA: Amazon, AND: Andean, CAR: Caribbean, ORI: Orinoco, PAC: Pacific.(TIF)Click here for additional data file.Figure S7ML trees describing the phylogenetic consequences of the intragenic recombination in Pvmsp-7I. A topology is inferred for each recombinant fragment identified, (A) nucleotides 19 to 187 and nucleotides 683 to 1,188, (B) nucleotides 683 to 849, (C) nucleotides 684 to 797, (D) nucleotides 19 to 358 and nucleotides 796 to 1,188, (E) nucleotides 798 to 1,105, (F) nucleotides 868 to 1,188, (G) nucleotides 920 to 1,188, (H) nucleotides 733 to 1,044, (I) nucleotides 688 to 797, and (J) nucleotides 683 to 849. Isolates clustered without a clear geographical distribution. SAL-I: Salvador strain, AMA: Amazon, AND: Andean, CAR: Caribbean, ORI: Orinoco, PAC: Pacific.(TIF)Click here for additional data file.Figure S8Pvmsp-7 sequences based on the TN93+G model, ignoring recombination. Three monophyletic groups are shown; the first groups clustered Pvmsp-7H (red) sequences, the second group clustered Pvmsp-7I (green) sequences and the third group clustered Pvmsp-7C (blue) sequences. (B-E) Trees describing phylogenetic consequences of some gene conversion tracks identified. (B) nucleotides 71 to 265 (amino acids 24 to 89), (C) nucleotides 913 to 1,090 (amino acids 305 to 390), (D) nucleotides 913 to 1,090 (amino acids 305 to 354), (E) nucleotides 913 to 1,175 (amino acids 305 to 392). Positions are numbered according to the alignment in Pvmsp-7C, Pvmsp-7H and Pvmsp-7I genes did not evolve independently. Numbers represent bootstrap values with 1,000 replicates.(A) Phylogenetic tree obtained by ML for (PDF)Click here for additional data file.Figure S9Schematic representation of gene conversion tracks identified by DnaSP and GENECONV for Pvmsp-7C (blue), Pvmsp-7H (red) and Pvmsp-7I (green) as a combined data set. Rectangles in a different color are graphical representations of sequence fragments that have potentially been originated by gene conversion and are localized at the conserved 5′- and 3′-ends. The black bars delimit the 5′-end, the central region and the 3′-end, .(PDF)Click here for additional data file.Figure S10Alignment of deduced PvMSP-7C amino acid sequences.(PDF)Click here for additional data file.Figure S11Alignment of deduced PvMSP-7H amino acid sequences.(TIF)Click here for additional data file.Figure S12Alignment of deduced PvMSP-7C amino acid sequences.(TIF)Click here for additional data file.Figure S13Alignment of the paralogous PvMSP-7deduced amino acid sequences.(PDF)Click here for additional data file.Table S1Nucleotide diversity for P. vivax antigens. n: number of isolates, π: nucleotide diversity.(PDF)Click here for additional data file.Table S2Nucleotide diversity (π) values for subpopulations within Colombia.(PDF)Click here for additional data file.Table S3Nucleotide and amino acid positions within the 5′- end, central region and 3′-end.(PDF)Click here for additional data file.Table S4Positively selected sites detected for Pvmsp-7 genes without taking recombination into account. Numbers according to the reference Sal-I protein sequence Pvmsp-7C: XP_001614132.1, Pvmsp-7H: XP_001614137.1 and Pvmsp-7I: XP_001614138.1.(PDF)Click here for additional data file.Table S5Negatively selected sites detected for Pvmsp-7 genes without taking recombination into account. Numbers according to the reference Sal-I protein sequence Pvmsp-7C: XP_001614132.1, Pvmsp-7H: XP_001614137.1 and Pvmsp-7I: XP_001614138.1.(PDF)Click here for additional data file.Table S6Positively selected sites detected for Pvmsp-7 genes taking recombination into account. Numbers according to the reference Sal-I protein sequence Pvmsp-7C: XP_001614132.1, Pvmsp-7H: XP_001614137.1 and Pvmsp-7I: XP_001614138.1.(PDF)Click here for additional data file.Table S7Negatively selected sites detected for Pvmsp-7 genes taking recombination into account. Numbers according to the reference Sal-I protein sequence Pvmsp-7C: XP_001614132.1, Pvmsp-7H: XP_001614137.1 and Pvmsp-7I: XP_001614138.1.(PDF)Click here for additional data file.Table S8Conversion tracks identified by DnaSP and GENECONV, between Pvmsp-7 genes. Nucleotides and amino acids based in alignment of the (PDF)Click here for additional data file.Table S9Nucleotides and amino acid positions excluded from the analysis.(PDF)Click here for additional data file.