Q < 0.1) in 31% of patients during the first year of therapy, associated with adverse outcome. We also observe transcriptional downregulation of pathways mediating energy metabolism, cell cycle, and B cell receptor signaling. Known and previously undescribed mutations in BTK and PLCG2, or uncommonly, other candidate alterations are present in seventeen subjects at the time of progression. Thus, the frequently observed clonal shifts during the early treatment period and its potential association with adverse outcome may reflect greater evolutionary capacity, heralding the emergence of drug-resistant clones.Treatment of chronic lymphocytic leukemia (CLL) has shifted from chemo-immunotherapy to targeted agents. To define the evolutionary dynamics induced by targeted therapy in CLL, we perform serial exome and transcriptome sequencing for 61 ibrutinib-treated CLLs. Here, we report clonal shifts  treated with targeted agents, such as ibrutinib, drug resistant subclones emerge. Here, the authors report on transcriptional changes in CLL patients treated with ibrutinib and identify early clonal shifts associated with evolution of resistant clones. Indeed, in the context of standard frontline fludarabine-based chemo-immunotherapy for CLL, we have previously reported that tumor evolution following therapy is the rule rather than the exception6.Clonal evolution is a major driving force in the ability of malignancies to adapt to therapeutic bottlenecks, becoming more aggressive and resistant to therapy. Chronic lymphocytic leukemia (CLL), the most common leukemia in western countries, illustrates the challenge posed to modern oncology by cancer evolution: despite highly effective therapies, the leukemia invariably evolves and recurs7. Leading these is ibrutinib, a first-in-class Bruton tyrosine kinase (BTK) inhibitor, which blocks B cell receptor (BCR) signaling, a key pathway for CLL cell survival and proliferation. Ibrutinib has potent activity even in high-risk groups such as previously treated CLL or CLL with TP53 aberrations10. Approved for all CLL patients based on improved progression free survival in treatment-naive disease and a favorable safety profile13, ibrutinib is increasingly used as monotherapy or tested in combination regimens.Recently, the therapeutic landscape of CLL has been dramatically altered through the introduction of multiple highly effective targeted agentsBTK and in PLCG2 , as the most common adaptations to therapy17. In a limited series of CLL patients, we have also previously identified del(8p) as a putative resistance enabling driver emerging with disease progression on ibrutinib16. We further identified that the capacity for resistance  was already present at the time of study entry, thus emphasizing the role of ibrutinib in providing strong selection pressure for the emergence of resistant clones.Despite this high level of clinical activity, disease progression on ibrutinib has been increasingly appreciated, with mutations in n = 45) or ibrutinib in combination with rituximab (n = 16), examining early patterns of clonal growth prior to development of overt CLL relapse, dynamic transcriptional changes and the course of clonal evolution upon disease progression. Here, we demonstrate that early clonal shifts, detected in 31% of patients, are associated with disease progression and identify previously undescribed mutations in PLCG2 and ITPKB at the time of relapse. These data suggest that greater evolutionary capacity, as indicated by the presence of clonal shifts during the early treatment period, lead to the emergence of drug-resistant clones and an adverse clinical outcome.Collectively, these results suggest that a study of clonal evolution patterns based on dense serial sampling and the measurement of clone-specific growth with therapy can provide clues regarding the mechanisms of resistance to this targeted agent. We therefore performed a longitudinal study of 61 CLLs treated with ibrutinib monotherapy (n = 45) or ibrutinib with rituximab . The median age of the combined cohorts was 65 (range 33–85) years and the majority of patients had markers of poor prognosis such as deletion of chromosome 17p (del(17p)) by fluorescence in situ hybridization (FISH), unmutated IGHV (U-IGHV) status and relapsed or refractory disease (Supplementary Table P = 0.53). At a median follow-up of 48.5 months (range 5.8–58.5), 17 of 61 (28%) patients had evidence of progressive disease.We analyzed sequential samples from 61 patients enrolled in phase 2 clinical trials of single-agent ibrutinib  on a median of 3 (range 3–5) pre-relapse peripheral blood CLL samples with median depth of coverage of ×107  silent and non-silent somatic single nucleotide variants (sSNVs) and insertions and deletions (sIndels) per megabase across patients, with no significant differences between the two clinical cohorts (Mann–Whitney P = 0.156). Across the 61 pre-treatment samples, we observed 229 sSNVs and sIndels affecting 49 candidate CLL genes, and 108 CLL somatic copy number alterations (sCNAs)6. Of the 229 putative driver sSNVs/sIndels, 152 (66.4%) were observed at subclonal frequency6, while 33 of 108 (21.7%) driver sCNAs were subclonal in frequency , underlining the high-risk nature of CLL patients enrolled in these early clinical studies. We note that the presence of a TP53 alteration at baseline did not have a statistically significant association with adverse outcome . Other candidate drivers such as mutations in SF3B1 and ATM, del(13q) and del(11q) were found in a similar proportion of samples, compared with our previous report6.Consistent with previous characterizations of CLL20) and 14 (43.8%) of 32 had been previously reported in cancer . We did not observe the previously described ibrutinib resistance mutations in BTK or PLCG2, although we detected a clonal CARD11 L251P mutation reported to confer resistance to ibrutinib in diffuse large B cell lymphoma23. Despite the presence of baseline mutations in critical regulators downstream of BTK in more than a third of patients, the reduction in ALC during the first 6 months was not significantly different between patients with and without pathway mutations (rank sum P = 0.43). We noted only a trend towards higher risk of relapse (Fisher P = 0.091) and shorter time-to-progression  pathways are directly inhibited by ibrutinib, we examined whether the presence of mutations in these pathways prior to ibrutinib therapy was associated with inferior outcome. We identified 25 pre-study samples with 32 somatic mutations in the BCR and NF-κB pathways at both clonal and subclonal frequencies , across all samples. This procedure allows us to infer discrete clusters of somatic mutations that define each subclone, and to calculate the CCF of each subclone at each timepoint (Methods section).In addition to the baseline characterization, the frequent serial sequencing of early on-treatment (prior to relapse) samples enabled us to directly compare the relative diminution of different clonal subsets in response to ibrutinib therapy. To track clonal trajectories across serial samples, we first measured the variant allele fraction (VAF) of all sSNVs/sIndels identified in all available WES data  per patient. VAFs were transformed to cancer cell fractions (CCFs) using ABSOLUTEP value of <0.1 for change in CCF >0.1 in the largest rising or falling clone . We observed a wide range of CCF changes in these clones , where clonal dynamics may have resulted from a change in frequency between parent and progeny clones. Neither the number of serial samples per patient (rank sum P = 0.3), nor the time interval between the baseline and last sample (rank sum P = 0.055) differed significantly between CLLs with and without clonal shifts. Moreover, the propensity for clonal shifts was not impacted by the addition of rituximab treatment (Fisher P = 1.0), nor by ibrutinib-related lymphocytosis, which may persist for months after therapy initiation, but has not been associated with inferior clinical outcome11. Indeed, the change in ALC during the first 6 months of treatment was not different between CLLs with or without early clonal shifts (rank sum P = 0.49). A survey of additional baseline clinical characteristics, such as IGHV mutation status, prior treatment history, del(17p) or TP53 mutations, del(11q), and BCR or NF-κB pathway mutations, did not identify clear associations with early clonal shifts . Nineteen of 61 CLLs (31%) showed significant pre-relapse changes in CCF over time, defined as a FDR adjusted fts Fig. .Fig. 2EaATM-mutated clone in A18, Fig. t) of the most changing clones was 4.3 × 10−3 % per day (IQR 1.6–5.1 × 10−3 %) during the first month on ibrutinib, but 5-fold less at 8.6 × 10−4 % per day thereafter  showed a deceleration of –2.7 × 10−5 per day  nor with the addition of rituximab (ANOVA P = 0.3), but was consistent with exponential decline dynamics ) throughout treatment, we observed that clonal shifts were more pronounced during rather than after the first month on treatment . We examined the changes in CCF over time in those CLL drivers detected at subclonal frequency, focusing on the 14 candidate CLL genes and CNVs present in at least six cases across the 61 baseline samples. Overall, these recurrent subclonal drivers  showed clonal stability, without a clear trend towards clonal increase or clonal decrease , which have been associated with adverse outcome in the setting of conventional chemotherapy. Our finding of general stability of subclones over the early treatment period, even with TP53mut, is consistent with prior studies reporting comparable early response rates to ibrutinib for both TP53mut and TP53wt CLL25.CLLs that harbored a candidate CLL driverase Fig. . This inP = 0.032) and shorter time-to-progression , we reanalyzed the data with a less stringent criterion of CCF change greater than 0.05. While as expected, more CLLs showed clonal shifts that reached statistical significance (30/61 compared with 19/61), the shorter time-to-progression remains significant (hazard ratio 2.73 (95% CI 1.05–7.09), log-rank P = 0.049, Supplementary Fig. Despite the lack of detectable differential fitness across the individual genotypes, the presence of on-treatment clonal shifts during the first year of therapy was associated with a greater likelihood of disease progression . Overall, these data indicate a progressively stronger impact of therapy on the transcriptome of residual tumor cells in circulation over time.Comparing baseline to on-treatment samples, we identified 653 differentially expressed coding genes , of which 498 genes were downregulated and 155 were upregulated , and followed three main categories: (i) immune-receptor signaling, (ii) cytokine signaling, and (iii) other general cellular processes  or to prolymphocytic leukemia . For 10 of 17 relapses, mutations in BTK and PLCG2 were tested by targeted sequencing of known hotspots , and detected in 6 of 10 cases  or that did not undergo targeted sequencing , which we previously linked to ibrutinib resistance16. This growing CLL clone yielded additional progeny clone(s) with a CCF of 0.12 at relapse, containing the canonical C481R BTK resistance variant, and two additional previously unreported PLCG2 variants . Although the resolution of our data led our clustering algorithm to assign these three mutations to a single subclone, these likely represent three distinct sibling subclones based on our previous single-cell sequencing analysis of a similar case -clone, containing a single Indel on the gene CDIPT ), consistent with previous estimates of relapse-driving clones in CLL33. As expected, the clone with ITPKB mutation in the Richter’s transformation (Patient A42) showed a much higher growth rate of 5.89% per day (95% CI: 5.86–5.93%). In some cases, the exponential growth dynamics of the likely resistant clone were insufficient to explain the resurgence of the CLL. For example, in Patient A43, in addition to the rise of the mutant PLCG2 clone, another large clone (BIRC3 mutant) that was previously declining in cell number, registered an increase in number in the peripheral blood upon relapse. This suggests that either additional genetic (not covered with WES) or non-genetic  factors are contributing to the relapse.From CCF information across serial measurements, we calculated clone-specific growth rates, based on exponential growth models, which are well supported by experimental CLL data9 circulating cells at treatment initiation (95% CI 2.8–5.5 × 109 cells), while the emerging progeny BTK/PLCG2 clones involved only 2.5 × 106 cells at baseline (95% CI: 0.26–19 × 106 cells). Likewise, the PLCG2 mutated clone in Patient A43 appeared to be present at the time of treatment initiation at a much smaller size with a predicted cell number of only 75 cells (95% CI 12–514). Similarly, the ITPKB mutated Richter’s transformation clone was estimated to involve a small proportion of circulating cells at treatment initiation, estimated at 10,700 cells .We back-extrapolated the calculated growth rates to estimate of the size of the resistant clone at treatment initiation. These analyses suggested that the del(8p) clone of Patient B06 involved 4 × 1019.The potent therapeutic efficacy of ibrutinib results in major clinical benefit for patients with CLL. However, this therapy also exerts strong selective pressure, which can promote the eventual outgrowth of resistant subclones. We therefore undertook an effort to perform unbiased sequencing of serially collected samples in order to define the evolutionary dynamics of ibrutinib-treated CLL, as we and others have shown that closely timed temporal sampling of tumor samples is a powerful approach to characterize such changesSF3B1mut or ATMmut) exhibited a clear trend of clonal expansion suggestive of selective fitness to ibrutinib. Furthermore, mutations in BCR signaling components, such as CARD11 L251P-conferring ibrutinib refractoriness in DLBCL, did not promote clonal fitness or preclude clinical response to ibrutinib in CLL. Likewise, in contrast to the patterns observed with fludarabine-based therapy, TP53mut subclones had similar likelihood to increase or decrease on treatment, arguing against a direct relationship between TP53mut and clonal fitness in the context of ibrutinib therapy. Instead, we speculate that the genomic instability in TP53mut cancer may potentially lead to greater clonal diversity and a higher chance of acquiring additional resistance mutations. This is consistent with the clinical observation that TP53mut CLLs show similar ibrutinib-response dynamics to TP53wt CLL9, but may be more likely to relapse. Thus, early clonal dynamics may serve as an indicator of the evolutionary capacity of the disease, which may underlie the potential association between early clonal dynamics and adverse outcome.Our data support the presence of marked clonal shifts within the early, pre-relapse treatment period in nearly a third of these high-risk ibrutinib-treated CLLs. While clonal dynamics were common, no single common CLL driver  Clinical Center (NCT01500733). Cohort B included 16 of 41 enrolled subjects on a phase 2 study of rituximab 375 mg/m2 on days 1, 8, 15, and 22, then once every 4 weeks during cycles 2–6 and ibrutinib 420 mg once daily starting on cycle 1 day 2 at the University of Texas MD Anderson Cancer Center . Inclusion criteria included diagnosis of high-risk CLL with either the presence of a 17p deletion or 11q deletion or TP53 mutation or previous treatment with up to three lines of prior therapy. Each clinical trial was approved by the NIH National Heart, Lung, and Blood and the MDACC institutional review board, respectively, and conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Guidelines for Good Clinical Practice. Written informed consent was obtained from all participants. Heparinized blood was collected before and after initiation of ibrutinib therapy; peripheral blood mononuclear cells from patient samples were isolated by Ficoll/Hypaque density-gradient centrifugation. Tumor cells were purified by CD19+ selection with MACS Cell Separation Columns  at 4 °C and stored as pellets at −80 °C. Of note, we have previously shown that during the first year of ibrutinib therapy the proportion of CD19 + non-malignant cells remains low (<5%)41, and have confirmed this by flow cytometry of the samples analyzed in the current work.Cohort A included 45 of 86 subjects enrolled on a phase 2 study of ibrutinib 420 mg once daily in patients with CLL, based on sample availability. Inclusion criteria included ≥ 65 years of age or CLL with del(17p) or IGHV gene in tumor cells. IGHV sequences were aligned to germline sequences in the international ImMunoGeneTics (IMGT) information system and database tools (IMGT/V-Quest)42. As per convention, the IGHV somatic mutation status was designated as unmutated if there was ≥98% homology; or as mutated if there was <98% homology to germline sequences43.Pretreatment evaluation included interphase FISH for common CLL chromosomal abnormalities with Vysis probes  and sequencing of the Genomic DNA from CLL cells and matched germline DNA  were extracted per manufacturer’s recommendations . The concentrations of tumor and normal DNA were measured using PicoGreen dsDNA Quantitation Reagent . A minimum DNA concentration of 60 ng/ml was required for sequencing and each Illumina sequencing library was created using the native DNA. Mass spectrometric fingerprint genotyping of 24 common SNPs  was used to confirm the identities of all tumor and normal DNA samples. Standard RNA extraction protocols  were used to extract RNA from CLL-B cells.http://broadinstitute.github.io/picard/) was used to generate a BAM file for each sample. Picard consists of four steps.Samples were sequenced using Agilent SureSelect capture kit and Illumina Rapid Capture Enrichment—37 Mb target kit on Illumina next-generation sequencers. The sequencing data processing pipeline known as “Picard”  are also included in the BAM file.The first step involves alignment to the genome. Here, BWA is used to align the sequence data to the NCBI Human Reference Genome GRCh37/hg19Q score that represents the probability that the base call is erroneous. The Q score represents −10*log (probability of error) and is rounded to an integer value. We also used GATK to empirically recalibrate the qualities according to the original Q score (generated by the Illumina software), the lane, the read cycle, the tile, the base in question and the preceding base45. The original quality scores are also represented by the read-level OQ tag within the BAM file.The second step involves base recalibration. Here, each base within a read sequence is assigned a Phred-like quality The third step involves aggregation of lane and library level data. Here, for each sample, multiple lanes and libraries were aggregated into a single BAM file. Lane-level BAM files were then combined into a single BAM file for each sample. The read group information within these BAM files represent the library and lane information. Information regarding the read groups appears in the BAM header.http://broadinstitute.github.io/picard/) is used here to flag molecular duplicate reads. Pairs of reads in which both ends map to the identical genomic position are considered as arising from the same DNA molecule and thus considered duplicate reads. Only one of these duplicate reads is retained in the BAM file.The fourth step involves marking of duplicated reads. The MarkDuplicates algorithm from Picard (http://archive.broadinstitute.org/cancer/cga/firehose) was used to analyze the whole-exome sequencing data. Firehose has been developed at the Broad Institute. All tumor-normal pairs were required to pass the Firehose QC pipeline. This QC pipeline involved testing for DNA contamination of a sample from other individuals using the ContEst algorithm, together with cross-checking lane fingerprints.Following the generation of BAM files using Picard, Firehose  were detected using Indelocator (http://archive.broadinstitute.org/cancer/cga/indelocator). All mutations were filtered using a panel of normals filter, which removes mutations commonly seen across a large number of sequenced normal (non-cancer) samples. All paired tumor-normal pairs were run through deTiN, a Bayesian method to estimate the contamination of tumor DNA in the normal sample, and keep mutations which would otherwise have been removed by germline filters  Furthermore, manual review of somatic mutations from their respective BAM files using the Integrative Genomics Viewer (IGV)48 was also used to filter mutations. For each patient, the union of all point mutations and indels from every sample was created. Then, the mutant and reference allele counts of every mutation in this union were measured in each sample using samtools in a process called force-calling49.Somatic mutations were identified in targeted exon data using the MuTect algorithm, version 1.1.650 was used to estimate the coverage profile for each tumor sample. In brief, this tool first normalizes read coverage for each target segment using the total number of aligned reads. The coverage for every segment is then normalized using tangent normalization against the coverage present across a Panel of Normals that have been sequenced using the same target regions. The circular binary segmentation algorithm51 is then used to merge target regions so as to form segments that correspond to the same copy number event. The allelic copy ratio in each tumor sample was then estimated by measuring the allelic fraction of germline heterozygous SNPs. These allelic copy ratio estimates were then combined with the observed copy ratio of each segment.The ReCapSeq tool (version 34)24 was performed to assess purity and overall ploidy of each sample, as well as the cancer cell fraction (CCF) of each mutation (percentage of tumor cells harboring mutation in the sample). The ABSOLUTE algorithm requires two inputs, an input maf (mutation annotation file), and segmented allelic specific copy ratio file . The allelic seg file was produced as described in the previous section. The input maf file passed in to ABSOLUTE for each sample contained all of the force-called mutation calls. This made it possible to estimate the CCF of mutations which were not originally detected in a given sample, but detected in other samples belonging to that patient. ABSOLUTE produces an array of possible purity/ploidy combinations, ordered by their relative likelihoods. The final purity and ploidy of each sample were chosen by manual review of these possible solutions.ABSOLUTE52, and improved and extended the method to incorporate analysis of a large number of samples from a single patient. In brief, the method uses a Bayesian clustering framework based on a Dirichlet Process53, where the parameter defining the number of clusters is varied during the process and inferred over many MCMC iterations.To estimate the number of clusters and subsequent clonal structure in each individual, we further developed an approach previously published by our groupn = 61), the two clusters with maximal negative and positive change between baseline and latest post-treatment samples were identified. For each of these clusters the posterior probability density of the two-dimensional cluster CCF distribution lying within ±10% of the diagonal was quantified to evaluate the null assumption that the cluster CCF was similar between timepoints. These probabilities were submitted to a Benjamini–Hochberg FDR procedure to account for multiple hypotheses testing, and the null was rejected if the adjusted P value was <0.1.For each individual in the cohort  parent-progeny relationships were assigned only if the parent clone had a higher cancer cell fraction than the progeny clone in all samples, and sibling relationships were enforced by the constraint of sibling clones having a summed cancer cell fraction of no more than 1 at any timepoint. When more than one phylogeny resulted from the application of these constraints, the phylogenetic relationship that maximized the goodness of fit for the exponential growth model of each clone was chosen.An MCMC (Monte-Carlo Markov Chain) algorithm was run on the final clustering results of the four individuals shown in Figs. BTK and exons 19, 20, and 24 of PLCG2 were amplified using custom oligonucleotides and analyzed by bidirectional Sanger sequencing. To increase the limit of detection for hotspot mutations, mutant alleles were preferentially amplified by wild-type blocking polymerase chain reactions followed by hybrid-capture based next-generation sequencing with custom SureSelect QXT Target Enrichment  or Nextera Rapid Capture  panels that include BTK and PLCG2 genes .Exon 15 of 2 transformed counts data were used for downstream analysis. Subject effect and time effect of treatment of the samples were qualitatively assessed by the first few principal components of the data. A two-way ANOVA model was then used to account for subject effect and time point effect for each gene. Genes with 2 fold-change in expression on treatment compared to baseline and with a false-discovery rate less than 10% were considered differentially expressed and selected for further investigation. Overrepresentation of experimentally derived gene sets in the list of selected genes was estimated by the P values  calculated from hypergeometric distribution. Under-enrichment or over-enrichment scores were calculated based on the cumulative distribution function of the hypergeometric distribution. The Graeber Lab CDF calculator54 , JMP 12  and R software55 were used in data analysis. Gene sets with a minimum of five differentially expressed genes, P < 0.05 and a FDR q < 0.1 were considered overrepresented. The mean change in gene expression was calculated by averaging the change across all differentially expressed genes per patient at each time point for each overrepresented gene set.A cDNA library was prepared from poly-A selected RNA and sequenced on an Illumina platform. The total counts of reads across samples were normalized, and logt test, while associations across gene sets overtime was determined by paired Student's t test.Associations between clonal evolution, progressive disease and clinical features were assessed by the Wilcoxon rank-sum test or Fisher exact test, as appropriate. The Kaplan–Meier method was used to estimate time-to-progression and differences between groups were compared using the log-rank test. Associations between patient groups were determined by unpaired Student's The sequence data have been deposited in dbGaP under the accession code 26784.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6


Escherichia coli, Salmonella enterica subsp. serovar Typhymurium, Staphylococcus aureus and Listeria monocytogenes.In food microbiology, novel strategies to fight foodborne pathogens are certainly welcome. In this data report, cationic nanostructures built from combinations of nanoparticles, antimicrobial peptide and cationic lipid are evaluated against important foodborne pathogens such as The cationic lipid dioctadecyldimethylammonium bromide (DODAB), the antimicrobial peptide gramicidin D (Gr), the antimicrobial cationic polymer poly  (PDDA) and the biocompatible polymer poly (methyl methacrylate) (PMMA) can be combined to yield a variety of antimicrobial cationic nanostructures as previously described by our group The cationic lipid (DODAB) and the cationic polymer PDDA bear quaternary antimicrobial nitrogens and form a variety of cationic nanostructures as the closed or open bilayers; the hybrid polymeric nanoparticles NPs and the DODAB/Gr combinations. Schemes, physical properties and antimicrobial activity for the cationic assemblies against the foodborne pathogens are on the data set  and NaCl were from Sigma-Aldrich . DODAB LV were obtained by hydrating and vortexing the DODAB powder in 1 mM NaCl aqueous solution, at 60°C at 2 mM DODAB  in TFE was added to previously prepared LV or BF at a 1:10 Gr:DODAB molar ratio. DODAB/Gr dispersions were prepared from DODAB LV incubated for 1 h/60°C with Gr , zeta-potential (ζ) and polydispersity (P) using a Zeta Plus-Zeta Potential Analyzer  equipped with a laser (677 nm) for DLS with measurements at 90°  had some colonies transferred to a 1 mM NaCl solution and turbidity was adjusted to 0.5 McFarland   were Gr insertion in DODAB LV bilayer reduced Dz and increased the positive ζ-potential. Gr tryptophans anchoring the peptide at the bilayer-water interface sterically stabilized DODAB/Gr. Disrupting DODAB LV/Gr led to cationic bilayers with Gr molecules inserted as dimeric channels so that the packing of the cationic lipids in the bilayer and the ζ-potentials increased. Other assemblies also tested in this work against the food pathogens had DODAB embedded in PMMA or PDDA making an outer layer (shell) in core-shell PMMA/PDDA positively charged NPs.S. enterica. Mostly DODAB BF was more efficacious against the bacteria than LV. The Gr peptide in DODAB BF reduced MBC values against three bacteria strains  or as cells survival in log (CFU/mL) (MBC). Here MBC determinations and reduction in log(CFU/mL) at MBC properly quantified the antimicrobial effect of the cationic nanostructures Table . As concμM Table  and eukaDODAB and DODAB/Gr interacted with bacteria driven by the electrostatic attraction and their mechanism of action involved lysis of the bacteria with leakage of intracellular compounds to the external medium and distortions in cell morphology .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.


XRCC2 gene on the risk of head and neck cancer (HNC) in 400 patients and 400 controls. Five polymorphisms of XRCC2 gene G4234C (rs3218384), G4088T (rs3218373), G3063A (rs2040639), R188H (rs3218536) and rs7802034 were analyzed using Allele- specific polymerase chain reaction (ARMS-PCR) followed by sequence analysis. For rs3218373, the GG genotype indicated a statistically significant 3-fold increased risk of HNC (P < 0.001) after multivariate adjustment. For rs7802034, the GG genotype suggested statistically significant 2-fold increased risk of HNC (P < 0.001). For SNP of rs3218536, the AA genotype indicated a significant 3-fold increased risk of HNC (P < 0.001). Additionally, haplotype analysis revealed that TACAG, TGGAG, TACGG and TAGGA haplotypes of XRCC2 polymorphisms are associated with HNC risk. Two SNPs in XRCC2 (rs2040639 and rs3218384) were found increased in strong linkage disequilibrium. Furthermore, joint effect model showed 20 fold  increased HNC risk in patients carrying four homozygous risk alleles of selected polymorphisms. These results show that allele distributions and genotypes of XRCC2 SNPs are significantly associated with increased HNC risk and could be a genetic adjuster for the said disease.We aimed to investigate the effect of hotspot variations of  Accumulation of these unrepaired DSBs can cause cell death and initiate malignancies2. There are several mechanisms which repair these DSB. Homologous recombination repair (HRR) is the key pathway for this DNA repair, functioning in S phase of somatic mammalian cell cycle2. Defective HRR has been reported to be closely related to different human cancers3. A wide range of crucial molecules have been identified to participate in HRR process such as RAD51 paralogs  and can serve as central proteins during HRR process5. X-ray repair cross complementing group 2 (XRCC2) gene, XRCC2 protein, together with other proteins of RAD516, forms a complex which plays a critical role in chromosomal segregation and apoptotic response to DSBs7. This crucial function of the XRCC2 protein for the HRR process has been demonstrated in earlier studies where over 100 fold HRR reduction in XRCC2 deficient hamster cells was observed compared to parental cells8.Various damaging agents such as chemicals, radiations and some endogenous elements affect DNA integrity which ultimately result in single strand breaks (SSBs). Unrepaired SSBs may lead to double strand breaks (DSBs) during the S phase of the cell cycle9. Common genetic variants in XRCC2, particularly a coding SNP in exon 3 , have been identified as potential cancer susceptibility loci recently, though the association results were controversial10. The XRCC2 R188H polymorphism has been proposed to be a genetic modifier for smoking related pancreatic cancer10, pharyngeal cancer11, oral cancer12 and ovarian cancer13, though validation studies could not provide confirmation15. Some of the studies have also implicated XRCC2 R188H in breast cancer19, however, the Breast Cancer Association Consortium20 and other subsequent studies found no association between R188H and breast cancer risk22, or evidence of a modest protective association24.Many earlier studies have found that single nucleotide polymorphisms (SNPs) in the DNA repair gene might modify DNA repair capacity and subsequently influence the susceptibility of cancerXRCC2 genomic sequence is highly polymorphic, it is of interest to identify genetic defects which have a functional potential to affect the final repairing efficiency of XRCC2, and subsequently the development of head and neck cancer. On the basis of these observations, it was planned to study the role of XRCC2 gene as a candidate involved in the underlying cause of head and neck carcinogenesis.Since The subjects included in this study consisted of 400 diagnosed head and neck cancer patients and an equal number of age- and sex-matched controls. These subjects were collected during 2011 to 2015. The diagnosis of the head and neck cancer patients was made histologically at Nuclear Oncology Radiation Institute (NORI) Islamabad and Pakistan institute of medicine Sciences (PIMS). Controls were selected from individuals receiving routine medical examinations in these hospitals. The selection procedure for patients included confirmed histological diagnosis of HNC, no preoperative therapy and availability of complete follow-up data. The inclusion criterion for the controls was age and sex matched healthy individuals with absence of prior history of cancerous or precancerous lesions. Patients and controls suffering from any other familial disease  were excluded from this study. A written informed consent was obtained from all subjects. Additionally, a structured questionnaire, including information about demographic factors, smoking habits and dietary habits was also used to interview all subjects who provided written informed consent. Peripheral blood samples were collected from all study subjects. This study was approved by the institutional Ethical Review Boards of COMSATS Institute of Information Technology, Islamabad and both collaborating hospitals. Additionally, all experiments performed were in accordance with relevant guidelines and regulations.25. The extracted DNA was quantified by 2% ethidium bromide gels and spectrophotometrically using Nano Drop  and stored at −20 °C until used.Approximately 3–4 ml blood sample was collected in vacutainer tubes from enrolled subjects in this study. DNA was extracted from whole blood by Phenol chloroform method with some modificationsXRCC2 were selected using a set of web-based SNP selection tools (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm). Potential functional SNPs were included to meet the following criteria: (1) Validated SNPs with minor allele frequency > 5% in the Asian population; (2) SNPs present in the promoter of XRCC2 gene such as G4234C (rs3218384), G4088T (rs3218373) G3063A (rs2040639); (3) SNPs present in exonic region of XRCC2 gene such as R188H (rs3218536); (4) SNPs present in intronic region of XRCC2 gene such as (rs7802034).Five functional polymorphisms in DNA repair gene 26. Primers specific for each polymorphism are given in Supplemetary Table Genotyping was performed by Allele- specific polymerase chain reaction (ARMS-PCR). Primers for PCR amplification were designed by WASP Statistical analysis was performed using GraphPad prism software v 6.0. The chi-squared test and one sample t-test was performed to assess difference of collected data of age, gender, family history, smoking status, histological type of HNC and different treatment modalities for HNC between the control and case group. Hardy-Weinberg equilibrium test was used to compare the actual genotypes with the expected number based on Hardy-Weinberg equilibrium theory  in controls. The difference in allele frequencies and genotypes between the control and case group was analyzed by Chi-squared tests. Logistic regression, with the adjustment of age and gender, was applied to calculate the odds ratios (ORs) and 95% confidence intervals (CIs). Three logistic regression models  were also used to analyze the SNPs. For SNP-SNP interactions, we used a adjusted logistic regression model to estimate the multiplicative interaction effect of the SNPs. P < 0.05 was considered to be statistically significant.Haplotypes were generated from the genotyped data. The linkage disequilibrium (LD) and haplotype analysis were performed using Haploview 4.2, which uses the expectation maximization (EM) algorithm. Bonferroni correction was used to account for multiple testing and a two-tailed p value < 0.01 (=0.05/5 SNPs) was considered statistically significant.XRCC2 gene . Demographic data of these head and neck cancer patients and control individuals is given in Table 400 head and neck cancer patients and 400 control subjects were tested for five selected SNPs of XRCC2 and head and neck cancer. In case of first selected SNP (rs3218373) of XRCC2 gene, frequency of heterozygous mutant (TG) and homozygous mutant (GG) genotypes was observed significantly higher in HNC patients compared to healthy controls . Frequency of G allele of respective polymorphism (rs3218373) was also found statistically higher in the patient group . Genotyping of second selected SNP (rs2040639) of XRCC2 showed that the frequency of G allele of respective SNP was significantly higher in the control group  compared to patients as shown in Table The distribution of the genotypes and the allele frequencies of all of the studied polymorphisms are shown in Table  SNP rs328373 of XXRCC21 revealed that the frequencies of heterozygous mutant (GC)  and homozygous mutant (CC)  genotype along with C allele frequency  were significantly higher in the control group compared to patients. In case of fourth selected SNP (rs7802034), frequency of heterozygous mutant (AG) genotype was significantly higher in control group compared to patient group . However, the frequency of homozygous mutant (GG) genotype was statistically significantly higher in patients compared to control group . Genotyping of fifth SNP (rs3218536) showed that frequency of heterozygous mutant (GA)  and homozygous mutant (AA) genotypes  was significantly higher in patients compared with the controls. Moreover, the frequency of the A allele of respective SNP (rs3218536) was also statistically higher in the patients  as shown in Table Genotyping of third selected SNP rs3218384) of 8384 of XIn genetic association studies, statistical power to detect disease susceptibility loci depended on the genetic models tested. Therefore, the genotype frequencies were further analyzed by three genetic models: additive, dominant, and recessive model. For rs3218373, a significant association between this polymorphism and increased HNC risk was found in dominant model , recessive model  and additive model . Similarly, an increased risk of HNC risk was also found in recessive model  of polymorphism rs7802034. Moreover, significant positive correlations between rs3218536 and HNC risk were also identified in dominant , recessive  and additive model  as shown in Table Frequencies of selected polymorphisms were calculated in different histological subtypes of head and neck cancer such as oral, nasal, pharyngeal and laryngeal carcinoma. In case of oral carcinoma, mutant genotype of polymorphism rs3218373 , rs2040639 , rs3218384  and rs3218536  were observed significantly associated with oral carcinoma. In case of nasal carcinoma, mutant genotype of polymorphisms rs3218373 , rs7802034  and rs3218536  were observed significantly associated with the said histological type. For pharyngeal carcinoma, mutant genotype of polymorphisms rs3218373 , rs7802034  and rs3218536  were observed significantly associated with the pharyngeal carcinoma. For laryngeal carcinoma, mutant genotype of polymorphism rs3218373  and rs3218536  were observed significantly associated with said histological type of head and neck cancer, as shown in Table In case of genotype frequency correlation and smoking status of head and neck cancer, logistic regression model analysis was conducted using SNPs genotypes as a dependent variable and demographic parameters such as age, sex and smoking status as independent variable Table . The resIt was also investigated whether the five SNPs were in linkage disequilibrium. Any common haplotypes associated with the disease and rare haplotypes (with frequency < 5%) were excluded from the association analysis. The most common haplotypes of the five polymorphisms, calculated by Haploview 4.2, are summarized in Table XRCC2 intragenic SNPs  among the HNC cases and controls, and thirty different haplotypes were generated which accounted for most of the haplotypes in the cancer patients and control groups (with frequency < 5%). For commonly observed haplotypes, GAGAA haplotype (P = 1.6e-007), GAGAG haplotype (P = 2.7e-010), GAGGG haplotype (P = 0.02), GGCGA haplotype (P = 0.04), TAGAA haplotype (P = 0.03), TAGGA haplotype (P = 8.10e-009), TGCAA haplotype (P = 0.01), TGCAG haplotype (P = 0.01) and TGCGA haplotype (P = 0.03) were found linked with significant increase in head and neck cancer risk while GGGGG (P = 0.0004), TGGAA (P = 0.004), TGGAG (P = 4.06e-014) and TGGGG (P = 0.001) were observed associated with a significant reduction in head and cancer risk. The other seventeen common haplotypes including GACAG, GACGA, GACGG, GGCAA, GGCAG, GGCGG, GGGAG, TACAA, TACAG, TACGA, TACGG, TAGAG, TAGGG,TGCGG, GAGGA, GGGAA and TGGGA were observed not associated with the risk of head and neck cancer as shown in Table XRCC2 (rs2040639 and rs3218384) were in strong LD  in XRCC2 gene are associated with head and neck cancer. In this study, we successfully genotyped a total of five SNPs in different regions of XRCC2 gene such as promoter region, exonic region and intronic region and examined their possible association with head and neck cancer risk. We also investigated that whether or not these five polymorphisms are in linkage disequilibrium and common haplotypes of these SNPs are associated with head and neck carcinogenesis. Finally, we estimated the association among the combined genotypes of five selected SNPs and overall risk of head and neck cancer.Many earlier studies have reported that the genes involved in DNA repair and in the maintenance of genome integrity plays a crucial role in protection against mutations. Although single nucleotide polymorphisms have been identified in these DNA repair genes, such as XRCC2 rs3218536 SNP. In present study five polymorphisms, present in different regions of XRCC2 gene, such as promoter region, exonic region and intronic region were screened in HNC cancer patients and controls. Among selected promoter polymorphisms, frequency of risk allele of first promoter polymorphism rs3218373 was observed significantly higher in patients compared to controls. However, in the case of other two promoter polymorphisms (rs2040639 and rs3218384), frequency of risk allele was observed significantly higher in controls compared to patients. Similar results have earlier been reported where significant association was observed between XRCC2 promoter polymorphism and oral cancer risk32, breast cancer risk33, thyroid cancer risk34 and bladder cancer risk35. Although the functional consequences of these polymorphisms are unknown, their location in important domain(s) of XRCC2 may control translation and mRNA decay and are also sites for RNA interference34.The majority of earlier studies on cancer susceptibility have focused only on XRCC2 gene, rs7802034, was located in the non-coding region and mutant allele frequency was observed associated with increased risk of HNC. Even though intronic SNPs are unlikely to have a direct functional role, still several studies have provided evidence that SNPs located in non-coding DNA, especially in intronic gene regions near the exon/intron boundaries, can inactivate pre-mRNA splice sites consequently affecting gene expression37, or can activate cryptic splice sites leading to exonization38. Furthermore, the presence of SNPs in 3‘-UTR of selected genes can modify the binding with specific microRNAs (miRNAs)39. For fifth selected polymorphism of XRCC2 gene, rs3218536, frequency of risk allele was observed significantly higher in patients compared to controls. Similar results have earlier been reported where significant association was observed between XRCC2 polymorphism rs3218536 with laryngeal and pharyngeal cancer risk40, thyroid cancer risk41, ovarian cancer42, gastric cancer risk43, oral cancer44 and head and neck cancer45. Nevertheless, some of the studies have also reported that there is no significant association between XRCC2- rs3218536 polymorphism and thyroid cancer47. In some of the studies rs3218536 polymorphism has been considered a genetic adjuster for ovarian and colorectal cancer patients49. The influence of these specific genetic variants on repair phenotype and cancer risk is yet not clear. However, amino acid 188 is conserved in humans, mice and rat XRCC2 proteins as well as human RAD51C, suggesting a potential functional role in DNA repair activity50. Romanowicz-Makowska et al., (2016) reported that rs3218536 polymorphism has shown a functional significance and may be responsible for a low DNA repair capacity phenotype characteristic of cancer patients including larynx carcinoma31. Future determination of functional and active sites in human XRCC2 protein may clarify the biological importance of this amino acid residue.In this study, fourth selected polymorphism of 19. In second step of study, we successfully established haplotypes for the XRCC2 gene from different combinations of five SNPs. GAGAA, GAGAG, GAGGG, GGCGA, TAGAA, TAGGA, TGCAA, TGCAG and TGCGA haplotypes of selected polymorphism were observed linked with significant increase in head and neck cancer risk. These haplotypes possess risky alleles which are consistently over represented in head and neck cancer patients relative to healthy controls, suggesting a leading role of selected polymorphisms in risk determination. To produce more information, linkage disequilibrium was calculated for five SNPs of XRCC2 gene and rs2040639 was found in complete linkage disequilibrium with rs3218384. It is possible that SNPs of this gene may have a collective effect on DNA repair outcomes. It has earlier been reported that interaction of polymorphisms, of the same gene or other genes, by linkage disequilibrium may be important in modulating the overall repair activity. This might explain the influence of genetic variations in the carcinogenic process51.It is believed that haplotype analysis can provide more information than single-locus analysisXRCC2 gene may contribute to decreased or lost DNA repair capacity46. Furthermore, SNPs of XRCC2 may also increase the risk of several types of cancer including thyroid, brain and breast cancer53. In previously reported studies, cumulative meta-analyses have suggested no such significant association. Theoretically, genetic variants in XRCC2 gene can change the regular function of this gene, disturb the DNA repair and subsequently increase the cancer risk54. Nevertheless, some of the previous studies have reported that the variant alleles of this polymorphism can increase resistance to DNA damage induced by cisplatin55 which enlightens protective function of this polymorphism under certain conditions.After this step, SNP-SNP interaction was calculated which showed that rs3218373-rs2040639 and rs2040639-rs3218536 combinations were associated with an increased head and neck cancer risk. Similar results have earlier been reported where it was suggested that mutations in XRCC2 polymorphisms are directly associated with HNC risk. Nevertheless, several potential limitations of this study need to be considered before making a final conclusion. Firstly, studies should incorporate a larger sample size with various ethnic groups to further confirm the association between SNPs of XRCC2 and head and neck susceptibility. Secondly, HNC is a polygenic disease, therefore other genetic and environmental factors should also be assessed. Thirdly, few well-known risk factors for HNC such as human papilloma virus (HPV) has been discovered. In such cases correlation between sexual behavior of the subjects and head and neck carcinogenesis should be considered. Fourthly, subjects in this case-control study came from two hospitals and this may cause selection bias that can have substantial impact on the overall conclusions. As a result, large-scale studies adjusting for a wide range of factors are recommended to validate these findings. In conclusion, our results indicate that these five polymorphisms of XRCC2 gene may be related to individual susceptibility to head and neck risk in Pakistani population.In conclusion, current evidence suggest that analyzed supplementary Tables


Saccharomyces cerevisiae rescues toxicity associated with the strong [PSI+] prion. This counterintuitive finding can be explained by changes in chaperone balance and distribution whereby the folding of the prion protein is improved and the prion is rendered nontoxic. In particular, the ribosome-associated Hsp70 Ssb is redistributed away from Sup35 prion aggregates to the nascent chains, leading to an array of aggregation phenotypes that can mimic both overexpression and deletion of Ssb. This toxicity rescue demonstrates that chaperone modification can block key steps of the prion life cycle and has exciting implications for potential treatment of many human protein conformational disorders.The nascent polypeptide-associated complex (NAC) is a highly conserved but poorly characterized triad of proteins that bind near the ribosome exit tunnel. The NAC is the first cotranslational factor to bind to polypeptides and assist with their proper folding. Surprisingly, we found that deletion of NAC subunits in  Misfolded proteins can be toxic to cells, causing pathologies such as Alzheimer’s disease, Parkinson’s disease, prion diseases, and ALS. One mechanism by which organisms combat protein misfolding involves molecular chaperones, proteins that help other proteins fold correctly. Here, we describe a novel role for a family of chaperones called the nascent polypeptide-associated complex (NAC). The NAC is a group of proteins that exist in all multicellular organisms, yet we do not fully understand its function. Using yeast as a model system, we have found that deletion of NAC subunits can reduce the toxicity associated with misfolded proteins. This work has implications for human protein misfolding diseases, as modulation of the NAC may present a viable therapeutic avenue by which to slow the progression of neurodegeneration and other protein conformational disorders. Protein synthesis is an essential process undertaken by all organisms, but its necessity also presents cells with a myriad of challenges. An extensive network of molecular machines is active throughout translation, folding, and degradation in order to preserve protein homeostasis (proteostasis). Perturbations to that machinery can have ripple effects that impact many cellular systems.Misfolded proteins are one such challenge to proteostasis. Improperly folded proteins are generally non-functional; thus, the correct folding and trafficking of polypeptides is essential to the maintenance of cellular viability . ProteinPSI+] prion, which is cytoplasmically inherited via amyloid seeds [PSI+] is toxic under certain circumstances, including Sup35 overexpression, due to severe disruption of proteostasis as a consequence of excessive aggregation of Sup35  prion as a model for protein misfolding and a reporter for the activities of the chaperone network. We screened for factors that, when disrupted, rescued the prion-dependent toxicity and restored viability. Surprisingly, disruption of β-NAC, a subunit of the NAC chaperone complex, was identified as one of the rescuing mutants in our screen. This counterintuitive result suggests that depletion of chaperones can, in some cases, rescue defects associated with misfolded proteins.Here, we describe a surprising mechanism for preventing aggregation-related cytotoxicity by manipulating cotranslational folding pathways. We utilized the  prion. Though [PSI+] is generally well-tolerated by cells, the overexpression of Sup35 in [PSI+] cells is cytotoxic [PSI+]  [PSI+] NAC deletion strains onto rich media (YPD) and SD-Ade plates to assess their nonsense suppression phenotypes. All NAC deletion strains formed white colonies and grew strongly on SD-Ade  . We spotn SD-Ade , indicatPSI+] control . We found that the overall distribution of SDS-sensitive population of Sup35 aggregates did not change following growth in media without copper , confirmPSI+]-related Sup35 overexpression toxicity without observable changes to the prion or to nonsense suppression. To further investigate the reduction of Sup35 toxicity, we examined the aggregation and solubility of the Sup35 prion aggregates in all NAC deletion strains. We sought to do so by a method that would allow observation of intact aggregates in cells, rather than simply the SDS-resistant aggregates detected by SDD-AGE (Sup35GFP) into the WT and NAC deletion strains. We induced Sup35-GFP expression by the addition of small amounts (Vf = 50μM) of CuSO4 to the culture media and monitored GFP localization by fluorescence microscopy. Sup35-GFP exhibited a diffuse pattern of fluorescence in [psi-] cells  ,21,25. Sild type .We theorized that NAC deletion may increase the role of Ssb-RAC in cotranslational folding, as this protein complex is next in line to receive nascent chains following the NAC. This increased folding pressure upon Ssb-RAC as a result of NAC deletion might also localize a larger fraction of Ssb to the ribosome and nascent chains while titrating Ssb away from the available cytosolic pool. The titration would reduce the overall availability of cytosolic Ssb relative to Ssa, thereby creating a chaperone imbalance.PSI+] strain with endogenous Ssa expression and found that the cells grew as expected, with a moderate toxicity phenotype and consistent Sup35 expression  led to poor growth, and severely imbalanced Hsp70s (ssb1Δ with Ssa1OE) led to pronounced toxicity , as it is known that ssb1Δ yeast are sensitive to the fungicide , and its curing ability is influenced by the Hsp70s. Ssb overexpression in conjunction with Hsp104 overexpression promotes loss of [PSI+], while Ssa1 overexpression prevents Hsp104-mediated curing  prion, an effect that is inhibited by simultaneous Ssa overexpression. We wondered whether the Hsp70 imbalance in the NAC deletion strains would mimic this phenotype. We transformed each of the NAC deletion and WT strains with a plasmid that constitutively overexpresses Hsp104 and verified that Hsp104 levels were increased while not altering the growth of the cells or amount of Sup35 expressed  were resistant to Hsp104-mediated curing of [PSI+], as a significant proportion of their colonies remained white on rich media on first pass , a strong inhibitor of Hsp104 , which cGiven the toxicity rescue phenotype resulting from loss of NAC subunits and altered chaperone activity, we considered that NAC deletion may bring about a broader modification to proteostasis and the cellular response to protein misfolding. First, we probed the ability of the NAC deletion strains to manage global protein misfolding. We challenged cells with canavanine, an arginine analog that induces misfolding , and fouTo determine how NAC deletion strains tolerate elevated protein misfolding, we asked whether misfolded proteins were differentially acted upon by protein quality control machinery in the NAC deletion strains. We questioned whether the NAC deletion strains resist canavanine-induced misfolding due to increased activity of the ubiquitin-proteasome system (UPS). We assessed the presence of ubiquitinated species in the NAC deletion strains in the presence and absence of canavanine and found no differences between the WT and the most stabilized NAC deletion strains . AdditioWe considered the possibility that NAC deletion strains may package misfolded proteins into insoluble aggregates, rendering them nonfunctional but nontoxic. We performed solubility assays to visualize total protein aggregation in the NAC deletion strains, and observed no changes relative to the wild type , S9C FigPSI+] prion with other chaperones, including Hsp104, which is less able to efficiently cure [PSI+] in the NAC deletion backgrounds. The deletion of the NAC does not impact expression levels for any of the proteins examined in this study  are the first fully-functional chaperones that interact with nascent polypeptides. Nascent polypeptides encountering the RAC-Ssb system in the NAC deletion strains are presumably less protected or folded than in WT cells. The RAC-Ssb system, compensating for loss of NAC subunits, might retain Ssb on nascent polypeptides, thereby reducing free Ssb elsewhere in the cell.PSI+]-related Sup35 overexpression toxicity. Deletion of the entire NAC may exacerbate the chaperone imbalance in such a way that leads to a harmful depletion of the cytosolic pool of Ssb. However, in other cases, the triple NAC deletion did mimic phenotypes associated with the double deletions. For example, NAC deletion confers resistance to hygromycin B and increases resistance to Hsp104-mediated curing. This leads us to hypothesize that there is a function for each of the NAC subunits that can persist independent of the complex. For example, hygromycin B resistance is demonstrated by every strain that has a deletion of EGD1, indicating that this subunit may be particularly important in modulating the interaction between nascent chains and molecular chaperones. Further, in the NAC deletion strains, any subunits that remain in the cell might act independently on nascent chains or upon misfolded cytosolic proteins. The precise function of all of the NAC subunits remains unclear and we look forward to future studies that will shed light on this dynamic complex.Interestingly, deletion of the whole NAC did not recapitulate all of the phenotypes associated with the double deletions , and observed with an Olympus FV1200 laser scanning confocal microscope fitted with a 100x oil immersion objective.Cells expressing pCUP1-RNQ+] into [rnq-][psi-] strains  with acid-washed glass beads (Sigma) for 2x3 minutes in a multi-tube vortexer (Scientific Industries). Lysates were incubated with 1μl Sup35 antibody at 4°C for 2 hours, and 40ul of a 50% slurry of Protein G sepharose beads (GE) and lysis buffer was added and tubes were incubated at 4°C overnight. An unbound fraction was retained and beads were washed 3X in lysis buffer and resuspended in SDS-PAGE sample buffer as the bound fraction. Fractions were boiled in sample buffer for 5 minutes before SDS-PAGE (10% polyacrylamide) and Western blotting with enhanced chemiluminescence (G-Biosciences) and film (GeneMate). Bands were quantified with ImageJ and normalized to immunoprecipitated Sup35.Yeast were cultured in rich media under homeostatic conditions. Cells were lysed in buffer  with glass beads as described above. A “total” fraction was retained, and then lysates were centrifuged at 250,000xg in a TLA100 rotor in an Optima TLX Ultracentrifuge (Beckman Coulter). The supernatant was retained as the “soluble” fraction, and then the pellets were washed in lysis buffer and centrifuged again. Supernatant was discarded and the pellets were resuspended in a 1:1 ratio of lysis buffer to RIPA buffer . Following SDS-PAGE, gels were stained with coomassie blue to visualize the total protein content of each fraction.S1 FigPSI+] yeast containing pRS315CUP SUP35 for the copper-inducible expression of Sup35. This strain harbors the strong [PSI+] prion variant. Prion variants  result from particular amyloid structures propagated by the prion-forming protein -associated toxicity when Sup35 is overexpressed. The egd2Δbtt1Δ and btt1Δ strains show poor growth on selective media containing 350μM CuSO4.(A) We performed a transposon screen with a mini-transposon (3XHA/lacZ URA3 (mTn3)) mutagenized library . The lib protein . Transfo(TIF)Click here for additional data file.S2 FigEGD1 and EGD2 genes (“clone 2”) displays a similar pattern of Sup35 aggregation as is shown in 4 to the culture medium was consistent between strains. Note the proportion of Sup35-GFP present in the “uninduced” lane, consistent with leaky expression from the CUP1 promoter [RNQ+] [PSI+] was performed as previously described [egd1Δ deletion with a plasmid expressing Egd1 gene rescues [PSI+] induction to WT levels. Data are represented as mean ± SEM.(A) A second disruption of the promoter . (D) WT r [RNQ+]  and tranescribed . At leas(TIF)Click here for additional data file.S3 Figssb1Δ strains from (A) Wild type and NAC deletion strains were grown overnight in YPD before lysis, SDS-PAGE, and Western blotting for the specified proteins. Expression levels of chaperone  and prion-forming  proteins is not changed as a result of NAC deletion. The vertical white bar in the Hsp104 blot indicates non-contiguous lanes of the same blot. (B) Sup35 expression was analyzed by Western blot of lysates of the WT and (TIF)Click here for additional data file.S4 FigRNQ+] and the strong [PSI+] variant; none of the strains are overexpressing Sup35.WT and NAC deletion strains were transformed with p415GPD-SSA1 for the overexpression of Ssa1. Overexpression of Ssa1 is not toxic without the concurrent overexpression of Sup35. All strains contain [(TIF)Click here for additional data file.S5 FigRNQ+] and the strong [PSI+] variant, and the strains on selective media are overexpressing Sup35.Spottings performed as in  demonstr(TIF)Click here for additional data file.S6 FigRNQ+] and strong [PSI+]) were grown in YPD at 30C and separated into 500μl fractions in glass culture tubes. Culture tubes were “pretreated” for 30 minutes at 37°C prior to heatshock to promote the induction of heat-responsive elements. A non-pretreated control (-P) was incubated at 30°C. Cultures were heat shocked at 50 degrees for the indicated number of minutes. NS = no shock. No differences were observed between the WT and deletion strains.Strains Click here for additional data file.S7 FigThe WT and NAC deletion strains were transformed with a plasmid to overexpress Hsp104, as described in (TIF)Click here for additional data file.S8 FigPSI+] WT and NAC deletion strains were spotted onto plates containing variable levels of GdnHCl (5mM plates are shown), which inactivates Hsp104. All strains demonstrated equal curability, as demonstrated by their red coloration.(A) As in (TIF)Click here for additional data file.S9 FigRNQ+] and strong [PSI+]) were spotted onto plates containing the proteasome inhibitor MG132. The strains were not differentially affected by the stressor. (B) The induction of the unfolded protein response (UPR) was measured at baseline (uninduced) and upon addition of tunicamycin to culture medium (activated UPR). The NAC deletion strains did not show a differential ability to induce the UPR in response to misfolding stress. (C) The solubility assay from (A) WT and NAC deletion strains Click here for additional data file.S1 Tableegd1Δegd2Δ and egd1Δbtt1Δ strains were able to rescue prion–associated toxicity and had the most severe phenotypes in the remaining assays. Strains that showed variable phenotypes (e.g. egd2Δ could resist canavanine but not HygB) may harbor a slight chaperone imbalance that could not produce a detectable readout in our assays.The (PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 References(PDF)Click here for additional data file.


We developed human monoclonal antibodies that selectively bind the myostatin and GDF11 precursor forms, including a subset that inhibit myostatin proteolytic activation and prevent muscle atrophy in vivo. The most potent myostatin activation-blocking antibodies promoted robust muscle growth and resulted in significant gains in muscle performance in healthy mice. Altogether, we show that blocking the extracellular activation of growth factors is a potent method for preventing signaling, serving as proof of concept for a novel therapeutic strategy that can be applied to other members of the TGFβ family of growth factors.Many growth factors are intimately bound to the extracellular matrix, with regulated processing and release leading to cellular stimulation. Myostatin and GDF11 are closely related members of the TGFβ family whose activation requires two proteolytic cleavages to release the growth factor from the prodomain. Specific modulation of myostatin and GDF11 activity by targeting growth factor-receptor interactions has traditionally been challenging. Here we demonstrate that a novel strategy for blocking myostatin and GDF11, inhibition of growth factor release, specifically and potently inhibits signaling both  Muscle atrophy is associated with a variety of clinical conditions, including diseases of denervation, genetic disorders, cachexia syndromes, and disuse. In these patients, reduced muscle strength and function are highly disabling and lack adequate treatments, often undermining successful management of the primary condition. Loss of muscle strength and mass also occurs as a natural process of aging and in severe cases is categorized as sarcopenia. While varied in their cause, these indications lead to significant disability, lengthy physical rehabilitation and recovery times, and quality of life impairment.1 and cardiac2 tissues. Naturally occurring and engineered mutations that reduce myostatin signaling lead to a hypermuscular phenotype8. Conversely, myostatin overexpression in rodents induces a cachexia-like syndrome with muscle wasting and fat loss9. Because of the well-established impact of myostatin signaling on muscle mass, a number of agents that target the myostatin signaling pathway have entered the clinic for indications involving muscle atrophy.Despite intense interest, pharmacological interventions for preventing or reversing muscle atrophy remain an unmet medical need. Therapeutics for muscle atrophy currently in preclinical or clinical development include inhibitors of myostatin , a secreted growth factor that negatively regulates muscle mass. Myostatin’s expression is mainly restricted to skeletal muscle, with low levels of mRNA reported in adipose1. To date, all of the anti-myostatin drug candidates in early-stage clinical studies block the interaction between mature myostatin and its receptors through antibodies, ligand traps, or overexpression of natural inhibitors such as Follistatin26. However, because the receptor recognition surfaces of mature myostatin and other Transforming Growth Factor β (TGFβ) family members share a high degree of similarity29, many of these myostatin-targeted biologics cross-react with other members of the TGFβ family (most commonly GDF11 or Activin A). In all of these cases, while promising results have been reported, it is too early to fully appreciate both their efficacy in patients and potential clinical toxicities associated with their lack of target specificity.Myostatin signals by binding a cell surface receptor complex consisting of a type I receptor (ALK4/5) and a type II receptor (ACTRIIa/b)32. This cleavage produces an inactive latent complex, here termed latent myostatin or latent GDF11, with two prodomains remaining associated with the growth factor dimer, preventing receptor binding.Here we demonstrate the efficacy of a novel alternative therapeutic approach to preventing growth factor engagement with cell surface receptors: blocking growth factor maturation from its autoinhibited precursor forms. Like other TGFβ family members, myostatin and its close homolog GDF11 are synthesized as inactive precursor polypeptides (here termed proMyostatin and proGDF11). Mature growth factor release from the unprocessed precursor is regulated through two discrete protease cleavage events Fig. . ProMyos33  or Bone Morphogenetic Protein 1 (BMP1)33 Fig. . This adIn vivo, myostatin is detected both in circulation, where it exists predominantly in its latent form38, and in muscle where it resides primarily as unprocessed proMyostatin39. Notably, myostatin precursor forms are significantly more abundant in these compartments than the active mature growth factor40. Here we show that pro- and latent myostatin reside predominantly in the extracellular space within the muscle fiber, in close proximity to the cell surface. In addition, we show that myostatin precursors alter their distribution during glucocorticoid-induced atrophy, suggesting that myostatin processing plays a biologically important role in muscle atrophy.in vitro. In vivo, myostatin-specific activation-blocking antibodies were able to prevent atrophy in a corticosteroid atrophy model, and also increased muscle mass and function in healthy animals. Taken together, these data suggest that inhibition of extracellular activation is a potent and highly selective method for inhibiting TGFβ family signaling.We also demonstrate that highly specific TGFβ family growth factor regulation can be achieved by modulating extracellular activation, a fundamentally distinct approach from blocking receptor/growth factor interactions. We developed fully human monoclonal anti-proMyostatin and anti-proGDF11 antibodies that potently inhibit their target by blocking growth factor release from the prodomain and, consequently, signaling. Because the prodomains share a much lower degree of conservation (47% identity between myostatin and GDF11 prodomains vs. 90% in the mature domains), this approach allows greater control of antibody specificity. These precursor-directed antibodies were specific for their intended targets Most growth factor-targeted therapeutic antibodies block the growth factor-receptor interaction. However, release of mature growth factor from the prodomain is tightly regulated in many TGFβ family members, offering an alternative strategy to modulate signaling. We therefore developed a novel phage display approach, using recombinant proMyostatin, latent myostatin and proGDF11 as antigens to screen for antibodies against the pro- and latent forms of myostatin and GDF11. Antibodies were then counterscreened against the mature growth factors to identify fully human monoclonal antibodies that specifically recognize the pro- and latent forms of myostatin or GDF11 without recognizing the mature growth factors. Table To evaluate whether the antibodies inhibit proMyostatin or proGDF11 activation, we developed an assay in which recombinant proMyostatin or proGDF11 were incubated with a tolloid protease (mTLL2 or BMP-1) and a proprotein convertase (Furin or PCSK5) Fig. .. FollowWe identified multiple antibodies that specifically blocked proteolytic activation of either proMyostatin or proGDF11 Fig. , Table 1in vitro and in vivo. We chose ten antibodies with a wide range of binding profiles for this experiment as assessed by epitope binning and affinity measurements towards murine GDF8 proteins (Table We employed an expanded panel of myostatin precursor-specific antibodies (identified in our screen) to ask whether inhibition of myostatin activation is necessary and sufficient to inhibit myostatin signaling ns Table . These ans Table  and S2.Tin vitro.Three antibodies in the panel  , Krueppl-like factor 15 (KLF15), and the E3 ubiquitin ligases Murf1/TRIM63 and F-box only protein 32 (FBXO32/Mafbx/atrogin-1)49. Importantly, myostatin is thought to be a key mediator of catabolic effects of steroid hormones through glucocorticoid responsive elements in its promoter53.For this experiment we chose a dexamethasone-induced muscle atrophy model due to its relevance to myostatin signaling and human disease. Elevated circulating glucocorticoids are associated with a number of muscle wasting diseasesMuscle atrophy in male C57BL/6 mice was induced by two weeks of dexamethasone treatment. Starting on day 1 of the study, mice were given either normal drinking water (vehicle) or water containing dexamethasone. Concurrent with dexamethasone treatment, mice were administered anti-Myostatin antibodies Fig.  and a nein vitro, GDF8_021, showed no significant effect on atrophy. Similar results were observed with rectus femoris muscle weights and total body lean mass  compared to controls that did not receive dexamethasone (No Dex) Fig. . In contin vivo. Furthermore, the equivalent potency of SRK-015 and GDF8-C5 suggest that blocking myostatin proteolytic cleavage is as effective as conventional blockade of myostatin receptor/ligand binding.These data confirm that pro- and latent myostatin-specific antibodies that prevent growth factor release can inhibit myostatin signaling 54, recognized the latent and mature forms of myostatin. The second control antibody, GDF8-C355, displayed a broader recognition profile, binding to not only GDF11 and myostatin, but also to Activin A and BMP10, demonstrating the lack of specificity which arises from targeting the mature growth factor.As shown in Table We next explored the mechanism of SRK-015 and SRK-015P action in more detail. Importantly, SRK-015P inhibited activation of both latent myostatin and proMyostatin, suggesting that the furin cleavage step is not significantly affected by antibody binding Figs  and 3a. 39. We next performed immunoprecipitation experiments using non-reducing SDS-PAGE to separate precursor myostatin forms. Figure As SRK-015 binds both the pro and latent forms of myostatin, we investigated the abundance of these proforms in mouse serum and skeletal muscle. Our goal was to establish whether these forms of myostatin are present in compartments where they can be bound and inhibited by the antibody. First we confirmed by western blot that the predominant form of myostatin in serum is latent, while proMyostatin is the primary form in muscle , signifying that these mice had undergone muscle atrophy, an effect that was even more pronounced at day 15  at frequencies greater than 60 Hz (main effect P = 0.003)  Fig. . While t03) Fig. . Because03) Fig. , there a03) Fig. , suggestin vitro to confirm direct effects of myostatin blockade on muscle, independent of nerve function and blood supply. muSRK-015P treatment resulted in a 20–29% increase in maximal force at frequencies greater than 60 hz Fig. , which w011)Fig.  or cross011)Fig. . To our activation of the mature growth factor rather than by blocking receptor-ligand interactions. This is important, as the high homology of the mature ligands has confounded discovery of truly selective antibodies. Indeed, selective pharmacological modulation of highly related mature myostatin and GDF11 growth factors has proven to be technically challenging. Divergent results have been reported regarding the biological functions of GDF11, with much of the controversy stemming from a lack of reagent specificity60. We show in this manuscript that by targeting precursor forms of myostatin and GDF11 we were able to generate several highly specific and pharmacologically effective antibodies towards these two homologs. Encouragingly, SRK-015 limited dexamethasone-induced muscle atrophy as effectively as a clinically vetted mature myostatin-targeting antibody  are naturally occurring inhibitors of myostatin and GDF11, these appear to bind a small fraction of mature myostatin in circulation58, our data highlight the importance of understanding myostatin processing and signaling in the muscle as well as in serum, as circulating myostatin levels may not adequately reflect the extent of myostatin signaling in the muscle itself. Quantitation of myostatin by ELISA or immunoaffinity mass spectrometry has been reported40, but these measurements do not distinguish between free mature myostatin and mature myostatin in latent complexes (bound to the propeptide or other inhibitory proteins). Reagents with specificity for the various forms of myostatin, such as the antibodies described in this study, could be useful in developing truly specific quantitative assays. This, coupled with additional studies to understand myostatin forms in patient samples and preclinical models, will help elucidate the broader role of extracellular processing in myostatin regulation and help to identify patient populations responsive to anti-myostatin therapy.While circulating myostatin has been examined as a biomarker of muscle atrophy and dysfunctionWhile our results highlight the therapeutic potential of inhibiting myostatin activation in muscle wasting diseases, an important step towards clinical development is identification of patient populations that would benefit the most from such a therapy. Evaluation in additional disease models, as well as more detailed analysis of the pharmacokinetic and pharmacodynamic properties of SRK-015 is ongoing. In addition, the panel of antibodies that we have developed also affords the opportunity to measure the distribution and dynamics of myostatin precursor forms in healthy and diseased serum and muscle. Such translational insights, complemented by biomarker data previously generated using other assay systems, can further inform the selection of clinical indications in which blockade of extracellular myostatin activation may bring greatest benefit to patients. Based on the highly regulated release of these growth factors in tissue, the novel mechanism of SRK-015 may afford improved safety and/or efficacy profiles compared to antibodies targeting the mature growth factor domain.Finally, the finding that prodomain-targeted antibodies can modulate signaling of two independent growth factors, myostatin and GDF11, suggests the broader applicability of our approach. We note that these proteins are closely related, and additional investigation is necessary to understand whether our approach can be extended to other TGFβ family members or, more broadly, to prodomains of other signaling molecules. Nevertheless, the data presented here clearly demonstrate the efficacy of our novel approach to inhibit signaling by blocking maturation of a proprotein to its active form, and suggest a potential to selectively modulate a range of growth factor signaling pathways by targeting extracellular activation.TM T-REXTM 293 cells  and expressed according to manufacturer’s instructions . Fully human anti-myostatin and anti-GDF11 antibodies were identified using a phage library displaying scFvs (single chain variable fragments)64. Separate antibody discovery campaigns were run using recombinant human proMyostatin, latent Myostatin, and proGDF11 for both positive and negative selection as appropriate to obtain antibodies with the desired binding profile. Antibodies were cloned and expressed using standard techniques, and binding profiles assessed on a FortéBio Octet QKe in various formats according to manufacturers directions (Supplementary methods). To potentially reduce immunogenicity, SRK-015 was generated from its parental version, SRK-015P, by introduction of five mutations in the variable domain. These antibodies are identical in the complementarity determining regions (CDRs) and have similar affinities and functional activity both in vitro and in vivo. muSRK-015P is identical to SRK-015P but contains a murine IgG1 constant region instead of a human IgG4 constant region.Full-length human and murine Myostatin and GDF11 proteins were stably integrated into FLP-INTM reagent  according to manufacturer’s instructions.Fully matured growth factors were purchased from R&D systems. Samples of human or murine proMyostatin  or human/murine proGDF11 (50 nM) were incubated with supernatants (either singly or in combination as appropriate) collected from stably integrated 293 cells overexpressing a pro-protein convertase (either Furin/PACE3 or PCSK5) and/or a Tolloid protease (either BMP-1 or mTLL2) at 37 °C for 16 hrs. After the proteolysis reaction, Myostatin or GDF11 activity was assessed by adding samples to 293 T cells containing a stably integrated pGL4 plasmid  with a promoter comprising SMAD- responsive CAGA sequences. Cells were incubated at 37 °C for 6 hours before detection of luciferase expression using BRIGHT-GLOResults were used to calculate the percent inhibition for each condition (% inhibition = 100 × ((Reading with test antibody but in the presence of protease – reading without tolloid protease)/(reading without antibody but in the presence of protease-reading without tolloid protease)). EC50 values were calculated in GraphPad Prism 7.0.1 from a four parameter logistic curve. Activation assay data are representative of at least 3 independent experiments, and means and standard deviations of three technical replicates are shown.In vivo dexamethasone studies were conducted at the Covance Laboratories Inc. Greenfield, Indiana. The Institutional Animal care and Use Committee approved experiments and procedures conformed to their guidelines. Animals were randomly assigned to treatment groups based on body weight at study initiation. Sample sizes (n = 8–10) are standard for detecting statistically different changes in muscle weights of the muscles examined. All data from animals that perished due to dexamethasone toxicity prior to study completion were removed from the analysis. Dexamethasone data are representative of four separate studies for SRK-015 and two studies for SRK-015P. All other antibodies were evaluated once in this model.2 overdose and blood was collected via cardiac puncture for serum preparation. Additionally, upon study termination, gastrocnemius and rectus femoris muscles were dissected from both the right and left legs of study mice. All tissues were weighed and then snap frozen for storage at −80 C.Ten male mice  were enrolled in the studies at 13–14 weeks of age. Starting on day 1 of the study, mice were given either normal drinking water (vehicle) or water containing dexamethasone  treatment for two weeks. In studies with antibody treatments, test articles were administered by intraperitoneal (IP) injection (20 mg/kg) once per week Fig.  or once For analysis, weights of the individual muscles from both legs were combined and the average muscle weight in grams was calculated, the average weights were normalized by initial body weight and the percent difference in muscle weight was calculated relative to the mean of the IgG vehicle control group (no Dex). In all experiments, outliers were identified and removed in GraphPad Prism 7.0.1 using the robust regression and outlier removal (ROUT) method (Q = 1%).Using standard procedures, clarified supernatants from homogenized muscle tissues or murine serum were analyzed by SDS-PAGE followed by western blot with the following primary antibodies: anti-myostatin prodomain AF1539 (R&D Systems); anti-mature Myostatin ab124621 (Abcam). Immunoprecipitations with SRK-015, IgG control, or GDF8-C1 antibodies from homogenized clarified muscle lysate and murine serum were carried out using the Thermo Scientific Pierce™ Co-Immunoprecipitation Kit according to the manufacturer’s specifications.For quantitative fluorescent western blots, samples were loaded onto Any kD Mini-PROTEAN TGX Stain-Free Gels (Bio-Rad) and signal was detected by near-IR imaging  and quantified using the western blot analysis tool in AzureSpot software (Azure Biosystems). Band intensity (number of positive pixels per unit area) from pro and latent Myostatin was measured and normalized to total loaded protein per lane. For all data presented, a minimum of three biological replicates were measured to generate the presented average values, and error bars on all graphs represent standard deviations. Statistical significance was determined by t-test .The Institutional Animal Care and Use Committee of the Office of Animal Welfare Assurance at the University of Maryland School of Medicine approved muscle performance studies. Experiments conformed to all Association for Assessment and Accreditation of Laboratory Animal Care guidelines. Muscle physiology experiments were conducted once.in vivo, followed by in vitro functional testing of the extensor digitorum longus (EDL).Nine-week old male and female C57BL/6 J mice (n = 10) were acclimated for one week and randomized based on body weight, then assigned to treatment groups with approximately equal body weights across all groups. The treatment or controls were administered I.P. via tuberculin syringe weekly. Following 4 weeks of treatment, plantarflexor function was tested in vivo with a 305 C muscle lever system . Anesthesia was accomplished via inhalation , and maintained via nose-cone . The knee was isolated using a pin partially inserted into the tibial head and the foot firmly fixed to a footplate on the motor shaft. Contractions were elicited by percutaneous electrical stimulation of the sciatic nerve. Optimal isometric twitch torque was determined by increasing the current with a minimum of 30 seconds between each contraction to avoid fatigue. A series of stimulations were then performed at increasing frequency of stimulation : 1, 10, 20, 40, 60, 80, 100, 150 Hz, followed by a final stimulation at 1 Hz. Following the measurements, the foot was released and the pin removed and the mouse allowed to recover in a separate clean cage prior to returning to its living chamber.Muscle performance was measured in vitro with the 305 C muscle lever system adapted with a horizontal perfusion bath. Briefly, the skin on the lower limb was degloved half-length to the knee and the tibialis anterior muscle carefully dissected free from the underlying EDL. A silk suture (4-0) was tied to the distal tendon of the EDL and the tendon severed. The muscle was then carefully dissected free from the tibia and adjacent muscles, the proximal tendon visualized and severed. The muscle was placed in an ice-cold physiological buffered solution and a silk suture tied to the proximal tendon. The muscle was placed in the horizontal bath of the 305 C muscle lever system and perfused with physiological buffer oxygenated with 95% O2/5% CO2 and kept at 37 °C. The sutures were tied to a fixed post on one side, and the lever arm on the other. A series of 1 Hz and 100 Hz field stimulations  at 0.01 Hz frequency were delivered via platinum electrodes flanking the muscle to ensure that the sutures are tight and that the maximal developed force was stable. Once stable, a series of stimulations were performed at increasing frequency of stimulation : 1, 10, 20, 40, 60, 80, 100, 150 Hz, followed by a final stimulation at 1 Hz.EDL muscle performance was measured Data were analyzed using Aurora Scientific 615 A Dynamic Muscle Analysis Software Suite in high-throughput mode. Each data file was manually inspected to ensure that cursors and fits were assigned properly and corrected when necessary. Data were then grouped and means and standard errors calculated. Force was normalized to limb length defined as the distance from the knee to the bottom of the ankle. Data were compared over time by calculating the % change using standard equations. Statistical analysis was performed using SigmaPlot v11. Data were analyzed using either a Student’s t-test with Mann-Whitney test, a One-way ANOVA or Two-way ANOVA with Holm-Sidak post-hoc when appropriate with an α = 0.05.The plantarflexor muscle group, soleus, gastrocnemius and tibialis anterior muscles frozen for histology were sectioned and stained according to standard protocols. For quantization using fluorescence microscopy, sections were stained with wheat germ agglutinin to determine cross sectional area or antibodies against MyHC-I, MyHC-IIa, or MyHC-IIb  for fiber-type determination. For proMyostatin localization studies, sections were stained with rabbit anti-laminin (Abcam) and GDF8_086 (Scholar Rock). H&E staining was performed according to Treat-NMD protocol MDC1a_M1.2.004.Correspondence and requests for reagents described in this manuscript (available via MTA) can be directed at Adriana Donovan, adonovan@scholarrock.com.Supplementary Information