ICOS encodes the Inducible T-cell Co-Stimulator (ICOS). Deficiency of this receptor in humans causes a common variable immunodeficiency (CVID) characterised by an absence of class-switched memory B cells and hypogammaglobulinemia. Three pathogenic mutations in ICOS have been described to date in a total of 13 cases. Here we report a novel homozygous 10 base pair frameshift deletion in exon 2 discovered by whole exome sequencing of two siblings from a family of Pakistani origin. Both patients presented in early childhood with diarrhea, colitis and transaminitis and one showed defective handling of human herpesvirus 6. Activated patient CD3+CD4+ T lymphocytes demonstrated a complete absence of ICOS expression and, consistent with previous reports, we detected a reduction in circulating T follicular helper cells. Findings in this kindred emphasise the phenotypic variability of ICOS deficiency and, in particular, the variably impaired antiviral immunity that is a poorly understood facet of this rare disorder.The online version of this article (doi:10.1007/s10875-015-0193-x) contains supplementary material, which is available to authorized users. The Inducible T-cell Co-Stimulator (ICOS) is a receptor structurally related to CD28. ICOS is expressed by T cells following activation while its ligand (ICOS-L) is expressed on antigen presenting cells including B cells . Co-stimICOS was reported in 2003 as the cause of adult-onset autosomal recessive common variable immunodeficiency in multiple patients from the Danube region [A homozygous 1815 base-pair deletion in the ICOS-encoding gene e region , 7. The e region .ICOS.We investigated two siblings who presented in early childhood with persistent pathogen-negative diarrhea and identified a novel homozygous 10 base-pair deletion in exon 2 of Cryptosporidium.The parents of the patients are from the same ethnic background and are not, to their knowledge, related Fig. a. From tLiver and gut biopsies showed mild chronic hepatitis and severe active chronic panenteritis, respectively. Samples from sigmoid colon, duodenum and liver were PCR positive for human herpesvirus 6 (HHV6). Treatment with intravenous ganciclovir and then oral valganciclovir together with nitazoxanide resulted in some improvement of the hepatitis: liver and duodenal samples became negative for HHV6 but sigmoid biopsy remained positive. However, the patient continued to have clinically severe colitis with persistent diarrhea and abdominal pain which limited her school attendance. By analogy with other combined immunodeficiencies we were concerned that this situation would only deteriorate over time. Following extensive discussion among the team and with family, it was therefore decided to attempt curative treatment with hematopoietic stem cell transplantation (HSCT). She received an unrelated 11/12 HLA matched transplant following reduced intensity conditioning. Unfortunately she developed capillary leak syndrome on day 5 with respiratory distress followed by toxic epidermal necrolysis and died from these complications.The younger brother of the proband (patient 2) presented similarly at age two years with an episode of diarrhea. Investigations again revealed low immunoglobulins, absent class-switched memory B-cells and raised liver enzymes . Filtering out common variants left a homozygous 10 base-pair deletion in To confirm ICOS deficiency at protein level, cryopreserved frozen aliquots of peripheral blood mononuclear cells from patient 2 were analyzed by flow cytometry after stimulation with PHA. This demonstrated complete absence of ICOS expression  peripheral CD4+ T cell population [loCXCR5hiCD4+ T cells, and the overall proportion of CD4+ lying in this region was lower than the normal range , and a previously reported reference range for the patient’s age group (approximately 15–65 %) [Previous reports have concluded that there is an association between ICOS deficiency and a reduction in the circulating pool of Tfh, as represented by the CXCR5pulation , 15. Defpulation , 16. In 15–65 %) .Crytosporidium, while the Kuwaiti patients suffered from Pneumocystis jirovecii pneumonia (PJP) and cytomegalovirus viremia [Pneumocystis or Cryptosporidium infections [This report clearly demonstrates that ICOS-deficiency can be associated with clinical features of cellular as well as humoral immunodeficiency. The most common presentation in previous cases was pneumonia, which could be mechanistically explained by defective antibody production . In cont viremia . Thus thfections .Although a less prominent feature, earlier reports of ICOS deficiency also included evidence of impaired viral immunity. Patients from the Danube cohort have been reported to experience recurrent HSV infection and one individual developed HPV-driven vulval carcinoma, while one of the Japanese siblings is described as having prolonged viral infections in infancy , 9. The Patient 2 lacked a discrete Tfh population, but the size of this compartment varied markedly in healthy controls. This finding is consistent with data from the Danube cohort where average Tfh proportions were markedly lower in the patient group compared to controls, but outliers from the two groups had overlapping values . This su+ T cells are activated before being evaluated for expression of ICOS and activation markers including CD69 and CD25 or HLA-DR. Inclusion of these markers is important because there is a danger that non-damaging variants in ICOS could be incorrectly designated as pathogenic if T cell activation is defective for another reason, resulting in a failure of ICOS induction. Evaluation of Tfh numbers might also contribute diagnostically although, as we show, these may overlap normal values.ICOS deficiency should be considered as part of the differential diagnosis in patients with antibody deficiency or combined immunodeficiency. An early clue is the complete lack of class switched memory B cells, which is a consistent feature of the disorder. In the absence of another molecular diagnosis we suggest that such patients should undergo an assay in which CD4. was successfully transplanted with improvement in his colitis [Haematopoietic stem cell transplantation offers patients with ICOS deficiency a chance of cure should their disease be clinically severe. Although transplantation was sadly unsuccessful in our patient, one of the Kuwaiti siblings reported by Chou et al colitis . By analESM 1(DOCX 17 kb)


Saccharomyces cerevisiae (Baker’s yeast). It has been shown previously that TSS selection in S. cerevisiae is altered in Pol II catalytic mutants defective in a conserved active site feature known as the trigger loop. Pol II trigger loop mutants show growth phenotypes in vivo that correlate with biochemical defects in vitro and exhibit wide-ranging genetic interactions. We assessed how Pol II mutant growth phenotypes and TSS selection in vivo are modified by Pol II genetic interactors to estimate the relationship between altered TSS selection in vivo and organismal fitness of Pol II mutants. We examined whether the magnitude of TSS selection defects could be correlated with Pol II mutant-transcription factor double mutant phenotypes. We observed broad genetic interactions among Pol II trigger loop mutants and General Transcription Factor (GTF) alleles, with reduced-activity Pol II mutants especially sensitive to defects in TFIIB. However, Pol II mutant growth defects could be uncoupled from TSS selection defects in some Pol II allele-GTF allele double mutants, whereas a number of other Pol II genetic interactors did not influence ADH1 start site selection alone or in combination with Pol II mutants. Initiation defects are likely only partially responsible for Pol II allele growth phenotypes, with some Pol II genetic interactors able to exacerbate Pol II mutant growth defects while leaving initiation at a model TSS selection promoter unaffected.Transcription initiation by RNA Polymerase II (Pol II) is an essential step in gene expression and regulation in all organisms. Initiation requires a great number of factors, and defects in this process can be apparent in the form of altered transcription start site (TSS) selection in  Initiation, the first step in transcription, is highly conserved, with regulation requiring a great number of factors , promoter melting, and transcription start site (TSS) selection. The integration of these and other factors determines the efficiency of any particular promoter and the sequences that will be used to initiate transcription. Much remains to be understood about the functions of GTFs and how they integrate with Pol II activity during initiation. The effects of Pol II activity and roles of GTFs in initiation are especially visible in the process of TSS selection in S. cerevisiae, where most promoters use multiple start sites, and this usage is sensitive to a number of factors. Therefore, examination of mutant effects on TSS selection allows a window to the initiation process in vivo.Pol II is essential for expression of all protein-coding genes, and determining how the combined defects of Pol II activity mutants in all steps of the Pol II cycle (S. cerevisiae), is highly conserved throughout evolution but is only found at a subset of promoters. TSS selection at TATA element-dependent promoters in S. cerevisiae involves recognition of transcription start sites positioned 40–120 nucleotides (nt) downstream from the TATA box and the use of multiple start sites at most promoters, whether they are classified as TATA-containing (contains consensus TATA box) or not  or not  (H. Jin revisiae . These rTFIIB (encoded by SUA7 in S. cerevisiae) and TFIIF  contribute to TSS selection. TFIIB bridges the TATA binding protein (TBP)-promoter DNA complex and Pol II, and likely stabilizes open complex formation through interacting with single-stranded DNA sequences in the PIC; TFIIF guides and stabilizes Pol II binding during assembly of PIC and appears to functionally interact with TFIIB, and may directly regulate Pol II activity , a mobile portion of the Pol II active center, have altered elongation rates in vitro. One class of these Pol II mutants confers faster elongation rates [termed gain of function (GOF)] in vitro, another class confers slower elongation rates [termed loss of function (LOF)], and the two classes are generally mutually suppressive when combined. These Pol II catalytic activity mutants conferred various phenotypes both in vitro and in vivo, including altered TSS selection, RNA splicing, presumed termination or processing defects, and chromosome segregation defects . Just as with the severity of their TSS defects, these Pol II mutants have growth defects in vivo that correlate with the extent of Pol II activity alteration. Mutants that have more severely altered activity in vitro (both fast and slow) show greater growth defects, more genetic interactions, and greater alterations to gene expression profiles in vivo. Growth defects can be suppressed when Pol II LOF and GOF mutations are combined within the same enzyme; similarly, there is mutual suppression of Pol II mutant TSS distribution defects at ADH1 in the double mutant, indicating a correlation between TSS defects and general growth defects  . Fragments containing target alleles were cloned into the yeast integrating vector pRS306 . For Pol II trigger loop mutants, MPA sensitivity is predictive of upstream start site defects at ADH1 (Mycophenolic acid (MPA) lowers cellular concentration of GTP by inhibition of IMPDH activity and induces expression of lys2-128∂ allele , a phenotype referred to as Spt− (Suppressor of Ty). The gal10∆56 allele . Mutants that alter readthrough from gal10∆56 or otherwise increase GAL7 expression show galactose resistance on YPRafGal media, a phenotype referred to as GalR.Strains used in this study contain the 32P-labeld oligonucleotide priming downstream of ADH1 start sites in 15 µl total reaction volume. Reverse-transcription reaction was performed by M-MLV reverse-transcriptase (Fermentas) in the presence of RNase Inhibitor (Fermentas) in 45 µl total reaction volume. Products were precipitated, digested with RNase A, and separated in 8% acrylamide gel made with 19:1 acrylamide:bisacrylamide (Bio-Rad), 1XTBE, and 7M urea, followed by visualization by phosphorimaging (Bio-Rad) and quantification with ImageQuant 5.1 software (GE).Primer extension assays were performed as previously described  and withR or Spt− phenotypes are thus shown as red in the heatmaps. Calculated score difference tables were turned into heatmaps using GENE-E (http://www.broadinstitute.org/cancer/software/GENE-E/index.html).Growth on each media were scored using a 0–5 scoring system . To indicate growth difference in different growth conditions, all mutants on YPD, YPD 37°, YPRaf, SC-Leu phenotypes were normalized to WT on each plate by subtraction (mutant score−WT score). Negative numbers (slower growth) are shown as blue, positive numbers (faster growth) are shown in red, and inviable double mutants are in dark gray. Growth differences on SC-Leu+MPA were normalized to growth difference on SC-Leu by subtracting the difference on SC-Leu from the difference on SC-Leu+MPA, thus rendering net growth difference due to MPA sensitivity/resistance. Differences on YPRafGal and SC-Lys (WT growth is zero) were normalized to growth difference on YPD and SC-Leu (“standard growth condition” controls for these phenotyping media) by dividing the difference on YPRafGal or SC-Lys by ratio of WT growth to mutant growth on YPD or SC-Leu to quantify resistance phenotypes. Mutants that have GalADH1 TSS distribution as a proxy for in vivo initiation defects. To quantify changes in TSS distribution at ADH1, signals from ADH1 TSSs were placed into six bins and alterations in the fraction of TSSs present in each bin were determined relative to the WT distribution , sua7-3 (encodes R78C in TFIIB) alleles that have been shown to confer downstream TSS shifts by the Hampsey group  and were recreated in our laboratory based on the published description by ADH1 TSS distribution are shown in tfg2∆146-180 has stronger upstream shifts at ADH1 than tfg2∆261-273, whereas tfg2∆233-248 has little effect, consistent with the work of sua7-1 and sua7-3 show strong downstream shifts as shown previously   .tfg2 alleles were combined, the most severe GOF mutant rpo21/rpb1 G1097D exhibited a strong negative interaction with tfg2∆146-180 or tfg2∆261-273 . LOF Pol II mutants (downstream TSS shifting mutants) and tfg2∆146-180 and tfg2∆261-273 (upstream TSS shifting mutants) did not result in suppression of growth defects of Pol II LOF mutants on standard rich or defined media in contrast to mutual suppression of growth defects when Pol II GOF and LOF (opposite TSS shifting mutants) are combined  have been combined , in contrast to rescue of lethal Pol II LOFs when combined with GOF mutants , we observed apparent lack of additivity in growth defects of strong downstream shifting sua7 alleles combined with Pol II GOFs. The double mutants generally showed growth defects in between those of the single mutants, suggesting directional suppression or epistasis, but not mutual suppression, where double mutants would be expected to grow better than either single mutant . These effects were more pronounced on YPD medium than on YPRaf or defined medium (SC-Leu). We also observed partial suppression of the sensitivity to increased temperature  of sua7-1, sua7-3, and sua7-58A5 alleles by Pol II GOF alleles . The weak downstream shifting allele sua7-70A5 enhanced growth defects with Pol II GOF alleles, distinct from the other stronger sua7 alleles tested.When sua7-Pol II GOF strains, Pol II LOF mutants were exquisitely sensitive to defects in TFIIB, as widespread synthetic lethality or sickness was observed between sua7 alleles and Pol II LOF mutants. sua7-1, sua7-3, and sua7-58A5 showed very strong negative interactions with all LOF Pol II mutants tested, with most double mutants being inviable. A weak downstream shifting allele, sua7-70A5, showed negative but weaker interactions with Pol II LOF alleles. Both classes of mutant, Pol II LOF and sua7 alleles, alter TSS distributions in a similar fashion, suggesting exacerbated TSS defects might underlie observed synthetic genetic interactions (see below). These results indicate that combinations of Pol II and GTF mutants exhibiting the same polarity of TSS defects can lead to exacerbation of growth defects, wherein aggravated initiation defects may be a major contributor of the observed growth defects. The strongest genetic interaction between Pol II mutants and GTF alleles was observed when Pol II LOF mutants and strong sua7 downstream shifting alleles were combined , suggesting that Pol II LOF mutants are much more sensitive to initiation defects than GOF mutants. Combinations of mutants with opposing TSS distribution defects resulted in partial, but not necessarily mutual, suppression of single mutant growth defects on generic media.In contrast to the milder phenotypes of in vivo . We observed sensitivity to mycophenolic acid (MPA) for tfg2 alleles, likely corresponding to upstream TSS shifts causing inability to induce IMD2 in the presence of MPA, with tfg2∆146-180 the most MPA-sensitive. MPA sensitivity has been shown to correlate well with upstream TSS shifts of a subset of Pol II mutants, including those tested here . In contrast, MPA sensitivities of tfg2 alleles were suppressed when combined with Pol II LOFs; similarly, MPA sensitivities of Pol II GOFs were suppressed when combined with all sua7 alleles, being more apparent in stronger sua7 downstream shifting alleles. Therefore, MPA phenotypes of combinations of GTF alleles and Pol II mutants appeared additive or suppressive depending on the nature of the allele class: when upstream shifting alleles were combined, MPA sensitivity was exacerbated; when an upstream shifting mutant was combined with a downstream shifting mutant, MPA sensitivity was alleviated ; however, this could partially be alleviated when combined with Pol II GOF mutants . Most Pol II GOF mutants do show Spt− phenotype as measured by suppression of lysine auxotrophy (Lys−) in the presence of lys2-128∂ allele , but there is only partial correlation with upstream shifting TSS defects. tfg2∆146-180 enhanced Spt− phenotypes of all moderate Pol II alleles, whereas the other tfg2 alleles did not show apparent interaction with Pol II alleles for the Spt− phenotype. Stronger downstream shifting sua7 alleles suppressed Spt− phenotypes of Pol II GOF alleles  . tfg2∆146-180 and tfg2∆261-273 mutants shifted ADH1 TSSs upstream relative to all single Pol II alleles tested, indicating exacerbation of Pol II GOF alleles that shift ADH1 TSSs upstream on their own and suppression of Pol II LOF alleles that shift TSSs downstream on their own  does not correlate with suppression of Pol II mutant generic growth defects. Taken together, these results suggest that TSS defects may contribute to Pol II mutant growth defects, and Pol II activity alterations can partially compensate for defects in GTFs, but initiation defects are not likely to be the main or only drivers of observed Pol II allele growth phenotypes.We observed that the TSS defects of GTF mutants and Pol II mutants were uniformly additive or suppressive, depending on the direction of TSS shifts of individual mutants, but not epistatic  showed allele-specific genetic interactions with GOF Pol II alleles and suppressed the Spt− phenotypes of Pol II alleles. RTF1, CTR9, PAF1 (genes encoding subunits of the Paf1C complex) showed stronger genetic interactions with Pol II LOF alleles and exhibited mild Spt− phenotypes on their own but suppressed the Spt− phenotypes of Pol II alleles. However, SGF73 (encoding a subunit of the histone-modifying SAGA complex) showed genetic interactions with both classes of alleles, enhanced MPA resistance of Pol II LOF alleles, and suppressed Spt− phenotypes of Pol II alleles .Inspired by the discovery of a Pol II genetic interactor,  alleles . We founheir own , nor didheir own . The genS. cerevisiae and their relationships have been probed here to understand the requirements for normal Pol II initiation. Previous analyses showing that distinct classes of TSS mutant exist in yeast (upstream and downstream shifting), supporting a scanning model for identification of TSSs. When mutants of differing TSS shift class have been combined  has wide-ranging genetic interactions with Pol II mutants in a manner correlating with class of Pol II mutant (GOF or LOF) . Unlike combinations of GTF alleles and a subset of previously examined rpb alleles, TSS defect suppression is partially uncoupled from growth defect suppression for oppositely acting TSS alleles. Negative growth interactions between GTF alleles and Pol II alleles correlated with exacerbated TSS defects but were of much greater strength for downstream shifting GTF TSS alleles and Pol II LOF alleles than for upstream shifting GTF TSS alleles and Pol II GOF alleles. These observations suggest that S. cerevisiae growth is much more sensitive to defects in initiation that result from decreased initiation efficiency. Second, sub1∆ is thus far unique in the strong epistasis observed between Pol II GOF alleles and sub1∆ for TSS selection at ADH1 (Class II). This indicates that sub1∆ defects are distinct from those of TFIIB alleles, and that while sub1∆ appears to be bypassed in Pol II GOF alleles, sua7 defects are not, although there is some observable suppression of specific phenotypes in sua7-Pol II GOF mutant strains. Third, other tested genetic interactors with Pol II do not generally have TSS defects on their own or modulate Pol II TL mutant TSS defects (Class III), suggesting that the strong correlation between TSS defects and a very broadly Pol II interacting mutant, sub1∆, is relatively unique. Genetic interactions we observe between these factors and Pol II mutants may be originated (caused) by other transcriptional defects.We found that, unlike previous combinations of TSS shifting alleles, suppression of TSS defects by Pol II mutant-GTF combinations could be partially separated from their effects on growth. Furthermore, we found that previous observations regarding the relationship of  defects . First, ADH1 transcription mutants appear to alter the probability of usage of YR sequences that are used by WT present in the start region. Although Pol II GTFs might be positioned within the Pol II PIC to interact with sequences and “read” for the start site, the primary determinants for TSS preference are the −1/+1 bases that are located deep in the active site and not bases juxtaposed to hypothetical or predicted GTF locations in the PIC (these would mostly be upstream sequences). Moreover, the types of TSS changes observed for GTF mutants are phenocopied by mutations in the Pol II trigger loop, suggesting they might arise from similar types of defects in transcription. What could these similar defects be?Although our experiments suggest that Pol II activity–dependent growth defects can be uncoupled from observed TSS defects, open questions remain concerning the mechanisms by which Pol II start sites are determined. RNA polymerases prefer to initiate at YR  sites, where the initiating nucleotide of an RNA is a purine, with a pyrimidine just upstream. A crystal structure of a viral RNA polymerase suggested that this sequence preference was likely due to purine stacking between the initiating NTP and a purine at the −1 position on the template strand (meaning a pyrimidine at −1 on the transcribed strand), and a set of very recent bacterial RNAP structures confirm this for multisubunit RNAPs  depending on how much an allele deviates from WT catalytic activity for Pol II alleles or on how strong they appear genetically . In other words, different mutants may not necessarily alter initiation sequence preference, but instead may shift the initiation probability of use in a polar fashion within some set of already usable start sites. In this view, Pol II initiation efficiency may cooperate with a directional scanning process that has its own rate. Pol II with increased catalytic activity enables initiation earlier within the scanning window, increasing catalytic efficiency (initiation probability) of earlier usable start sites usages, and shifting TSS usage distribution upstream.The altered patterns of starts observed in most upstream or downstream shifting start site mutants appear to be stereotypical to each class, meaning the positions of starts that are more likely used at ADH1 TSS distributions in Pol II-GTF mutant strains indicate that individual defects of each allele are present in the double mutant strains. How might we understand these defects? Defects in sua7 are consistent with defects in initiation efficiency and likely represent reduced TFIIB functions. TFIIB function in concert with the Pol II active site might represent communication between TFIIB and the active site as has been proposed . An alternative model in which a smaller region of melted DNA translocates along with the open PIC toward the start region may also be possible, but almost nothing is known of the organization of nucleic acids in such hypothetical complexes or how translocation of the putative bubble would be controlled during initiation. In translocating Pol II elongation complexes, GTFs are not present and interactions between Pol II, both DNA strands, and nascent RNA organize the upstream edge of the transcription bubble.The mechanism driving nd GAL10 , is univTFIIB or TFIIF studied here for ADH1 TSS selection, suggesting that they each function separately as part of a concerted process to promote efficient TSS selection.Genetic analyses have allowed us to examine the relationships between Pol II activity mutants, known initiation factors, and candidates for possible modifiers of Pol II initiation activity. Our experiments indicate that Pol II genetic interactors need not perturb TSS selection, and that initiation defects are likely only a partial driver of Pol II allele growth phenotypes. Altered Pol II activity through TL defects do not bypass or appear epistatic to the alleles of


Aberrant promoter DNA methylation can serve as a predictive biomarker for improved clinical responses to certain chemotherapeutics. One of the major advantages of methylation biomarkers is the ease of detection and clinical application. In order to identify methylation biomarkers predictive of a response to a taxane-platinum based chemotherapy regimen in advanced NSCLC we performed an unbiased methylation analysis of 1,536 CpG dinucleotides in cancer-associated gene loci and correlated results with clinical outcomes.We studied a cohort of 49 patients (median age 62 years) with advanced NSCLC treated at the Atlanta VAMC between 1999 and 2010. Methylation analysis was done on the Illumina GoldenGate Cancer panel 1 methylation microarray platform. Methylation data were correlated with clinical response and adjusted for false discovery rates.Cav1 methylation emerged as a powerful predictor for achieving disease stabilization following platinum taxane based chemotherapy . In Cox regression analysis after multivariate adjustment for age, performance status, gender, histology and the use of bevacizumab, CAV1 methylation was significantly associated with improved overall survival (HR 0.18 (95%CI: 0.03–0.94)). Silencing of CAV1 expression in lung cancer cell linesby shRNA led to alterations in taxane retention.CAV1 methylation is a predictor of disease stabilization and improved overall survival following chemotherapy with a taxane-platinum combination regimen in advanced NSCLC. CAV1 methylation may predict improved outcomes for other chemotherapeutic agents which are subject to cellular clearance mediated by caveolae. With the exception of patients whose tumors harbor a targetable driver mutation, response rates following first-line chemotherapy in patients with advanced non-small lung cancer (NSCLC) remain poorWe have recently identified reduced protein expression of the mitotic checkpoint gene CHFR as powerful predictor of taxane sensitivity in NSCLC The detection of aberrant promoter methylation and subsequent epigenetic silencing of genes involved in the cellular response to chemotherapy has been proposed as a qualitative biomarker for chemotherapy response To identify novel predictive methylation markers for improved outcomes after taxane-based chemotherapy in NSCLC, we performed an unbiased methylation analysis of 1,536 CpG dinucleotides on the Illumina GoldenGate methylation array and correlated results with clinical outcome data amongst NSCLC patients who had received platinum/taxane chemotherapy.The study was approved by the Institutional Review Board of Emory University and the Research and Development Committee of the Atlanta VAMC. Waivers for informed consent requirements were granted due to the retrospective and blinded nature of the clinical data to protected health information (PHI). Patients with stage IV NSCLC who received first-line treatment with a platinum-taxane combination between the years 1999–2010 were initially identified from the local cancer registry at the Atlanta VAMC. We had previously correlated CHFR expression with clinical outcomes in this cohort Paraffin blocks were cut in sections of 5 uM thickness. One slide was stained with hematoxylin and eosin (H&E) and analyzed by microscopy for tumor type and tumor cell percentage. Only samples with at least 40% viable tumor cell content were used for subsequent analysis. At least 2 additional unstained sections were obtained for DNA extraction.DNA was extracted from slides 2 and 3 using the E.Z.N.A™ FFPE DNA extraction kit from Omega Biotek . RNA was extracted from slides 4 and 5 using the E.Z.N.A™ FFPE RNA extraction kit from Omega Biotek . Nucleic acid content was quantified using an Eppendorf Biophotometer Plus  with Hellma Tray Cell .mRNA was reversed transcribed using a mix of random hexamer and oligo-dT primers using the Super Script III First strand synthesis kit (LifeTechnologies). qRT-PCR was carried out at a Tm of 55C on a Step One Plus thermocycler (Life Technologies). Primer sequences are available upon request.High-throughput methylation profiling was done using the Illumina GoldenGate Methylation Cancer Panel I microarray platform. DNA quality control by picogreen, bisulfite-conversion by the EZ DNA methylation kit  and array hybridization, according to manufacturer's specifications, were performed by Emory Integrated Genomics Core facility.Data preprocessing and normalization of intensities used the methylumi bioconductor package The multivariate survival analysis was conducted by entering sex, race, ECOG performance status, age, race, histology, and Avastin use into a Cox proportional hazard model.A549, HOP-62 and EKVX NSCLC cell lines were grown in RPMI media, supplemented with 10% fetal calf serum (Invitrogen). All cell lines were a gift from Dr. Paula Vertino who originally obtained the lines from American Type Culture Collection (ATCC), Manassas, VA. Cell lines were authenticated by STR analysis by Biosynthesis Inc. .Small hairpin RNA (shRNA) against Caveolin-1 and non-effective scrambled shRNA in pRFP-C-RS vector were obtained from Origene . 1.5 µg of vector were transfected into A549, Hop-62 and EKVX cell lines using Lipofectamine-2000 (Invitrogen). Stably transfected clones were selected and expanded after incubation in Puromycin containing selection media . Successful transfection of the clones was determined by fluorescent microscopy and visualization of red fluorescence as well as by subsequent immunoblot for caveolin-1.Cells were lysed in 1x cell lysis buffer , containing Complete protease inhibitor and Phostop (Roche) and 1 mM PMSF. Cells were sonicated briefly and lysates clarified by centrifugation. Following SDS-PAGE and semi-dry transfer the following antibodies were used: Caveolin-1 , beta-actin (1∶10000 Sigma), E-cadherin (1∶1000 BD), MDR1 , Focal-adhesion kinase (FAK) , phospho-Y397-FAK .Cell lines indicated were seeded at 1000 cells per well in a 6 well cluster plate. 24 hours post seeding, the cells were treated with docetaxel at 50 nM for 1 hour. The media was exchanged and colonies were allowed to develop for up to three weeks. The cells were fixed with 4% formaldehyde/PBS and stained with crystal violet for imaging and analysis. Image J software was used to quantify the results by measuring the area fraction of each well containing colonies after applying a threshold to the images to eliminate background. Data are reported as fold change over mock treated control.A549 cells lines bearing either shCAV1 or scrambled control were seeded to 75% confluence in 6 well dishes. 16 hours post seeding, the cell monolayers were scratched with a 20–200 ul pipette tip and imaged under 5X power at the indicated time points. The denuded area was measured using Image J software. Technical triplicates were averaged and values reported as area recovered compared to the zero-hour time point.A549 stable cell lines bearing RFP-expressing shCAV1 or scramble controls were enriched to near 100% purity using a BD-FACS Aria cell sorter and subsequently seeded into Cellview 35 mm glass bottom dishes (Greiner Bio-One). The cells were allowed to attach to the plate overnight. The dish was set on a PE Ultraview spinning disc confocal live cell imaging system. Cells were treated with 2 µM Flutax-1(Santa Cruz Biotechnology) and imaged at 20X in both red and green channels at maximum speed. Three fields of each condition were imaged. After 50 cycles, the media containing Flutax was removed and replaced with Flutax-free media. The samples were imaged for an additional 35–50 cycles. The resulting image sets were analyzed using Cell Profiler software. Analysis of Flutax uptake and turnover, termed Flutax flux, was accomplished by selecting all red positive cells as regions of interest and measuring the change of intensity in the green channel in said regions. This intensity change was measured over 50 cycles . Reported values are intensities averaged over three fields. Areas under the curves were calculated by trapezoid rule www.kmplot.com) We utilized datasets of 1,715 tumors which had previously been profiled by Affymetrix microarray analysis  for death: 0.18 (95%CI: 0.03–0.94), p = 0.04) , highligPromoter methylation generally results in epigenetic silencing of gene transcription In order to determine the biologic relevance of a loss of Cav1 expression, we stably transfected A549, HOP-62 and EKVX NSCLC cell lines with a shRNA against CAV1 or non-silencing scrambled controls. In A549 cells, loss of CAV1 was associated with a distinct morphologic change towards spindle cell shape, consistent with EMT . At the EMT has long been considered to be an important mediator of chemo-resistance in cancer In order to determine if loss of CAV1 expression alters intracellular kinetics of taxane in- and efflux, we performed life-cell imaging after addition of the fluorescently labeled taxol-derivative Flutax-1 (Santa Cruz Biotechnologies). We detected a statistically higher area under the curve (AUC) for Flutax1 in CAV1 deficient cells, suggesting that either increased taxane influx or decreased efflux are likely responsible for the observed increased cytotoxicity in the colony formation assays . MultidrIn order to test the hypothesis that CAV1 specifically plays a role in mediating chemoresistance rather than being associated with poor prognosis independently from treatment, we analyzed existing genomically and clinically characterized datasets. After adjusting for stage high CAV1 expression correlated with inferior overall survival in patients who received chemotherapy , but did not predict survival in patients who did not receive chemotherapy  . These fThis is the first report to show a correlation between promoter methylation of CAV1 with favorable outcomes following combination chemotherapy with paclitaxel and carboplatin in NSCLC. These findings are important because they may help establish CAV1 methylation as clinically relevant biomarker, which could aid in the selection of treatments that lead to a higher likelihood of survival. Our results suggest that CAV1 methylation is a better discriminator for clinical outcomes than mRNA expression. Similar findings have been previously observed for MGMT methylation vs. expression as predictor for response to alkylating agents in glioblastoma Before CAV1 methylation can be firmly established as predictive marker for taxane sensitivity in lung cancer, three important questions need to be discussed. Is it mechanistically plausible that CAV1 mediates taxane resistance? Is CAV1 silencing specifically associated only with taxane sensitivity or do intracellular pharmacokinetics of other chemotherapeutics converge on the same mechanism? Finally, could CAV1 methylation or expression be a prognostic marker that is associated with favorable prognosis independent of treatment.CAV1 serves as integral part of caveolae, special lipid rafts which play major functions in cell signaling and endocytosis Even though our data show that CAV1 silencing increases taxane- but not platinum sensitivity the possibility exists that intracellular pharmacokinetics of other drugs used in lung cancer therapy such as gemcitabine or etoposide may be dependent on CAV1 mediated mechanisms as well. This is due to the fact that no cellular in-or efflux mechanisms have so far been described that are exclusively specific for taxanes which could at least in part explain the observations that lung cancer patients with high CAV1 expression levels had inferior survival compared to those with low expression when gemcitabine based chemotherapy was given While we have established CAV1 methylation is a predictive biomarker for taxane based chemotherapy response in lung cancer, a possible prognostic value independent from treatment needs to be considered as well. In NSCLC, CAV1 overexpression has been associated with higher disease stage and inferior survival in patients with adenocarcinoma, but a robust correction of the survival data for stage and treatment was not done in these studies In summary, we have shown that CAV1 methylation is associated with high rates of stable disease and improved overall survival in patients with advanced NSCLC following chemotherapy with platinum-taxane based regimens. CAV1 methylation could serve as biomarker for taxane sensitivity and could help identify subsets of patients who are more likely to benefit from this cytotoxic chemotherapy.


Ca) produced action potential duration (APD) alternans seen clinically at slower pacing rates. Using single-cell clamps of voltage, fluxes, and state variables, we determined that alternans onset was Ca2+-driven rather than voltage-driven and occurred as a result of decreased RyR inactivation which led to increased steepness of the sarcoplasmic reticulum (SR) Ca2+ release slope. Iterated map analysis revealed that because SR Ca2+ uptake efficiency was much higher in control atrial cells than in cAF cells, drastic reductions in kiCa were required to produce alternans at comparable pacing rates in control atrial cells. These findings suggest that RyR kinetics may play a critical role in altered Ca2+ homeostasis which drives proarrhythmic APD alternans in patients with AF.Atrial fibrillation (AF) is the most common cardiac arrhythmia, but our knowledge of the arrhythmogenic substrate is incomplete. Alternans, the beat-to-beat alternation in the shape of cardiac electrical signals, typically occurs at fast heart rates and leads to arrhythmia. However, atrial alternans have been observed at slower pacing rates in AF patients than in controls, suggesting that increased vulnerability to arrhythmia in AF patients may be due to the proarrythmic influence of alternans at these slower rates. As such, alternans may present a useful therapeutic target for the treatment and prevention of AF, but the mechanism underlying alternans occurrence in AF patients at heart rates near rest is unknown. The goal of this study was to determine how cellular changes that occur in human AF affect the appearance of alternans at heart rates near rest. To achieve this, we developed a computational model of human atrial tissue incorporating electrophysiological remodeling associated with chronic AF (cAF) and performed parameter sensitivity analysis of ionic model parameters to determine which cellular changes led to alternans. Of the 20 parameters tested, only decreasing the ryanodine receptor (RyR) inactivation rate constant (ki Atrial fibrillation is an irregular heart rhythm affecting millions of people worldwide. Effective treatment of this cardiac disorder relies upon our detailed knowledge and understanding of the mechanisms that lead to arrhythmia. Recent clinical observations have suggested that alternans, a phenomenon where the shape of the electrical signal in the heart alternates from beat to beat, may play an important role in this process, but the underlying mechanisms remain unknown. In this study, we use computational models to conduct a detailed examination of the causes and contributors to alternans associated with human atrial fibrillation. We find that in atria remodeled by atrial fibrillation, alternans appears near resting heart rates because several aspects of calcium cycling are disrupted in the atrial cells. In particular, the release and uptake of calcium from the cellular storage compartment, the sarcoplasmic reticulum, becomes imbalanced, leading to alternation in calcium signals from beat to beat. These findings provide important insights into the mechanisms of proarrhythmic alternans in human atrial fibrillation which may be used to develop novel therapeutic targets and treatment strategies in the future. Atrial fibrillation (AF) is currently the most common cardiac rhythm disorder, posing a significant medical and economic challenge for the US health care system et al. reported differences in the rate dependence of action potential duration (APD) alternans in patients, with APD alternans occurring at pacing rates near rest in AF patients but only at fast pacing rates in controls et al. also found that APD alternans always preceded AF initiation, indicating that alternans may play an important role in establishing the arrhythmogenic substrate and creating vulnerability to AF. Thus, a better understanding of AF arrhythmogenesis will likely depend upon identification of the mechanism driving atrial alternans at heart rates near rest.Beat-to-beat alternation in the shape of cardiac electrical signals, a phenomenon called alternans, has been observed in the atria of AF patients, but the mechanism underlying these alternans is not known 2+ handling in atrial myocytes is known to play a crucial role in the generation of AF triggers and in AF maintenance 2+ cycling instabilities have been shown to underlie ventricular alternans in heart failure 2+ handling abnormalities, or other electrophysiological changes that occur in AF, lead to APD alternans. We identified a critical change in the kinetics of the ryanodine receptor (RyR) that was responsible for APD alternans onset at slower pacing rates, and subsequently aimed to elucidate the mechanistic relationship between this disruption in RyR kinetics and alternans onset. To this end, we employed single-cell clamping of ionic model parameters and iterated map analysis in order to dissect the mechanisms which drive alternans in atrial tissue, as well as to provide important insights into the pathophysiological changes that contribute to the development of alternans in AF patients.Interestingly, in AF patients the slope of the APD restitution curve was <1 during APD alternans onset at slow pacing rates. This suggests that a cellular mechanism other than voltage-driven instability underlies APD alternans at heart rates near rest et al. to induce APD alternans in AF patients In order to investigate ionic mechanisms in human AF that contribute to the generation of atrial APD alternans at the tissue level, we created a computer model of human atrial tissue incorporating ionic remodeling associated with chronic AF (cAF), as described in Ca) had the greatest effect on alternans onset CL in the human cAF-remodeled tissue  version of the cAF model Ca was decreased by 50% in the cAF model , APD alternans onset data from the human AF tissue model agreed well with data from persistent AF patients. Significant APD alternans began at 400-ms CL  at the onset CL  vs. the even (short) beat (2+ load before release (0.288 vs. 0.273 mM), higher peak RyR open probability (RyRo) (9.0e-4 vs. 4.7e-4), a larger intracellular Ca2+ transient (CaT) amplitude (Δ[Ca2+]i = 0.13 vs. 0.067 µM), similar L-type Ca2+ (LCC) current (integrated over one beat: 144 vs. 140 mC/F), and increased Na+/Ca2+ exchanger (NCX) current . The positive coupling between transmembrane potential (Vm) and Ca2+, with INCX as the primary electrogenic current, is consistent with experimental findings alt model provided the best agreement with clinical APD alternans data  was clamped to either the even or odd beat waveforms, alternans in both APD and CaT were eliminated (<−99%), demonstrating that the alternans were driven by SR Ca2+ instability (column 4 of i), RyR open probability (RyRo), junctional Ca2+ ([Ca2+]j), and SR Ca2+ release flux (JSRCarel)  . All fiv2+ ([Ca2+]sl) and sub-sarcolemmal Na+/Ca2+ exchanger current (INCXsl). Clamping [Ca2+]sl to the even beat eliminated all alternans; clamping to the odd beat greatly reduced APD and CaT alternans , although large alternation in SR load persisted , while clamping to the odd beat waveform resulted in elimination of all alternans  voltage clamp conditions so that changes in CaT alternans would be due solely to changes in Ca2+ homeostasis rather than bidirectional coupling between Vm and Ca2+. After clamping each ionic model at a CL of 400 ms until steady state was reached, we perturbed [Ca2+]SR and tracked SR load and SR Ca2+ release on the subsequent clamped beats  ionic models are depicted in alt model  at the beginning of a clamped beat and compared the unperturbed  and perturbed (dotted line) traces for each model (2+ regulation of the RyR (causing more opening), as well as to the increased concentration gradient between the SR and junctional compartments. In both the cAF and cAFalt models, these changes led to increased peak [Ca2+]j  and RyR opening  as a result of more Ca2+-induced Ca2+ release . However, the amount of positive feedback differed between the cAF and cAFalt ionic models. Positive feedback amplifies changes in release inputs, such as SR load; therefore, in the cAF model, where [Ca2+]j is higher and positive feedback is stronger, the increase in [Ca2+]SR produced a slightly greater change in release  during the rising phase of [Ca2+]j (t<48 ms) than in the cAFalt model  occurs. In the cAFalt model, negative feedback is decreased both directly, via reduction of kiCa, and indirectly, via reduction in [Ca2+]j that occurs as a result of decreased SR load. This causes prolongation of the Ca2+ release event and a larger peak [Ca2+]j  depends upon RyR inactivation by junctional Ca2+. However, recent work suggests that termination of release does not rely on direct Ca2+-dependent inactivation of the RyR but rather on local SR Ca2+ depletion 2+] decreases causing changes in RyR opening and closing rates.The results in column 1 of 34  is decreased from 100% to 50% , steady-state Ca2+ concentrations change modestly as compared to the original RyR formulation , peak [Ca2+]j is reduced by 10.5%  and delayed, and total release increases by 3.6% . When [Ca2+]NSR is perturbed in the Sato-Bers models by +20 µM, Ca2+ release increases more in the cAFalt model than in the cAF model  has the opposite effect as in Qu et al.2+ alternans.For each version of the human atrial cell model (cAF and control), we calculated the SR Caes , see , the thrCa are explored for the cAF model in Ca value (100%), as CL is decreased from 700 ms to 200 ms (−10 ms increments), 2+ cycling, coinciding with the iterated map parameter values residing very close to the theoretically predicted boundary given by Ca is set at 50% of the default cAF value , a similar trend is observed. However, the 50% kiCa cAF model reaches threshold at a lower pacing rate  fails to reach threshold and remains in the stable, no alternans region. This suggests that alternans in control patients, which occur at CL<250 ms 2+. As in the cAF model, the alternans threshold CL in the control model can be adjusted by modulating the value of kiCa (Ca must be decreased much more than in the cAF model in order to reach Ca reduced to 16% vs. 50%). The need for dramatic and possibly unrealistic reductions in kiCa to produce alternans at slow rates in control is consistent with the absence of alternans observed in control patients at CL≥250 ms We next performed the same iterated map analysis for the control atrial cell model with varying CL and kia values . When ki2+ cycling properties of the cAF and control models, we examined the effects of cAF cellular remodeling on iterated map parameters. Stochastic ionic model parameter variation and regression analysis CaL, gKur, koCa, IbarNCX, gto, gK1, and gNa, see 2+ handling . The effects of changing these three parameters from control to cAF values is depicted sequentially in CaL is decreased and then IbarNCX and koCa are increased to cAF values, resulting in an overall decrease in Ca is decreased to the cAFalt value (50%), the large increase in Ca value and pacing rate, SR uptake efficiency  could produce APD alternans at relatively slow pacing rates in a tissue model of persistent/chronic AF. In particular, decreasing kiCa by 50%  produced a good match to clinical data. We next aimed to provide mechanistic insight into why disruption of RyR kinetics, together with other electrophysiological changes occurring in AF, leads to alternans onset at pacing rates near rest. We established that alternans in the cAFalt model at the onset CL were Ca2+-driven rather than voltage-driven, and that they depended upon SR Ca2+ release. Furthermore, CaT alternans occurred in the cAFalt model at relatively long CLs because of steep SR Ca2+ release slope and decreased SR Ca2+ uptake efficiency. Lastly, we demonstrated that the ability to generate alternans at slower pacing rates by modulating kiCa depended upon the negative feedback properties of SR Ca2+ release.The first goal of this study was to identify the electrophysiological changes in human atrial cells that are responsible for the occurrence of APD alternans at heart rates near rest, as observed in AF patients. Using parameter sensitivity analysis, we found that of the 20 electrophysiological model variables tested, only changes in the RyR inactivation rate constant  enhanced alternans when clamped  is not consistent with ERP at the tissue level 2+ release, which was later confirmed through iterated map analysis.Our ionic model variable clamping protocol, which involved separately clamping the even or odd beat waveforms, was used to test for model variables which could robustly suppress alternans when clamped to either of two very different waveforms. An alternative approach would be to clamp model variables to the single unstable, non-alternating waveform obtained using a control algorithm 2+ depletion, rather than Ca2+-dependent RyR inactivation, as the main mechanism of SR release termination alt model relied on Ca2+-dependent RyR inactivation, other termination mechanisms which rely on SR Ca2+ (used in the Sato-Bers RyR model) may have similar effects on SR release slope and alternans susceptibility . APD alternans normalized magnitude (ANM), obtained by dividing the alternans magnitude by the mean APD over the last 10 beats, was used to compare alternans between cells of varying APD. Alternans onset CL was defined as the longest CL for which ANM was greater than 5% Voltage traces from the recording electrode  were ana2+ current (ICaL), rapidly activating potassium current (IKr), SR uptake, or SR release . Model variables were considered critical for alternans if clamping them to either the even or odd beat reduced both APD and CaT alternans magnitudes by >99% of baseline To determine which human atrial ionic model variables drive the occurrence of alternans, we clamped individual ion currents and state variables in a single-cell model paced at a CL exhibiting alternans 2+ cycling properties of the human atrial cell model under different pacing rates and parameter values, the following equation was used to clamp Vm to a generic atrial AP-like waveform so that comparisons between different conditions would not be influenced by variations in Vm:2+ cycling properties in ventricular myocyte models max = 10 mV, Vrest = −75 mV, and APD = 200 ms. CL ranged from 200 to 700 ms.To evaluate the Ca2+ cycling stability in the human atrial cell model via an iterated map analysis et al.2+ content of the cell are tracked from beat to beat. In our analysis, Ca2+ cycling stability depended upon three iterated map parameters: SR Ca2+ release slope . Lastly, To compute the iterated map parameters, a single atrial cell was repeatedly clamped to the AP waveform until model variables reached steady state. Following this, i (column 3), or [Ca2+]SR (column 4). Alternans persisted when Vm or [Ca2+]i was clamped, but clamping [Ca2+]SR eliminated alternans.(TIF)Click here for additional data file.S5 Figure2+ release parameter even beat clamps for the single-cell cAFalt model.SR Ca Traces of transmembrane potential , intracellular Ca2+ , and SR Ca2+  from two consecutive beats are superimposed to show alternans between even (red) and odd (blue) beats. Traces from the even beat at 400-ms CL pacing were used to clamp the relevant variable and are shown in row 4. Clamping RyR inactivated probability , RyR open probability , junctional Ca2+ , or SR Ca2+ release flux  eliminated alternans in Vm and Ca2+.(TIF)Click here for additional data file.S6 Figure2+ release parameter odd beat clamps for the single-cell cAFalt model.SR Ca Traces of transmembrane potential , intracellular Ca2+ , and SR Ca2+  from two consecutive beats are superimposed to show alternans between even (red) and odd (blue) beats. Traces from the odd beat at 400-ms CL pacing were used to clamp the relevant variable and are shown in row 4. Clamping RyR inactivated probability , RyR open probability , junctional Ca2+ , or SR Ca2+ release flux  eliminated alternans in Vm and Ca2+.(TIF)Click here for additional data file.S7 Figurealt model.Sub-sarcolemmal parameter clamps for the single-cell cAF Traces of transmembrane potential , intracellular Ca2+ , and SR Ca2+  from two consecutive beats are superimposed to show alternans between even (red) and odd (blue) beats. Traces from the even or odd beat at 400-ms CL pacing were used to clamp the relevant variable and are shown in row 4. Clamping sub-sarcolemmal Ca2+ ([Ca2+]sl) to the even beat (column 1) eliminated alternans in Vm and Ca2+, but clamping [Ca2+]sl to the odd beat (column 2) produced small alternans in Vm and [Ca2+]i and large alternans in [Ca2+]SR. Clamping sub-sarcolemmal Na+/Ca2+ exchanger current (INCXsl) to the even beat (column 3) eliminated alternans in APD but produced large alternans in [Ca2+]i and [Ca2+]SR. Clamping INCXsl to the odd beat (column 4) eliminated alternans in Vm and Ca2+.(TIF)Click here for additional data file.S8 FigureMultivariable regression between ionic model parameters and alternans threshold CL. (A) Bar graph of regression coefficient magnitudes. Twenty ionic model parameters were varied stochastically over 500 simulations to assess their effects on alternans cycle length (CL). Of the 500 simulations, 83 were excluded from the analysis because alternans threshold CL was below 100 ms or above 750 ms. Linear regression coefficients for each of the parameters are plotted in order of decreasing magnitude, with positive values plotted in red and negative values plotted in blue. Asterisks indicate p<0.05 for the t-statistic. (B) Bar graph of the predicted contribution of parameters to alternans threshold CL in the cAF-remodeled cell. Ten of the twenty parameters used in the regression analysis were altered from control values to represent cAF remodeling . Parameters whose changes were predicted to increase (decrease) the alternans CL are plotted in red (blue). Some unaltered parameters had nonzero predicted contributions to alternans threshold CL due to nonzero sample means from the regression analysis. The alternans threshold CL predicted by regression analysis (245 ms) was very close to the actual alternans threshold CL determined by simulation (244 ms).(TIF)Click here for additional data file.S9 FigureSingle-cell APD restitution in control model. With default model parameter values, APD alternans occurred at 200 ms CL (black). When the RyR inactivation rate constant (kiCa) was reduced to 95%, alternans occurred at slightly longer CLs (red). These results were comparable to alternans onset data from control patients (TIF)Click here for additional data file.S10 FigureAPD and CaT oscillations in single-cell and tissue models with Sato-Bers RyR formulation. Control (black), cAF (red), and cAFalt (dotted red line) versions of the model using the Sato-Bers RyR alt model, the calsequestrin-bound RyR closing rate (k34) was decreased by 50%. APD (A and C) and CaT (B and D) restitution data are plotted showing the mean±SD range . Oscillations in APD and CaT included but were not limited to alternans. Oscillations exhibited the reverse of the rate dependence observed in models using the original RyR formulation, with larger oscillations at longer CL. APD oscillations in these models were diminished as compared to the original models Click here for additional data file.S1 TextSupplemental methods.(PDF)Click here for additional data file.S2 TextSupplemental equations.(PDF)Click here for additional data file.


It has been established that patients with chronic rhinosinusitis with nasal polyps (CRSwNP) often have co-existing asthma.We aimed to test two hypotheses: (i) upper and lower airway inflammation in CRSwNP is uniform in agreement with the united airways concept; and (ii) bronchial inflammation exists in all CRSwNP patients irrespective of clinical asthma status.We collected biopsies from nasal polyps, inferior turbinates and bronchi of 27 CRSwNP patients and 6 controls. All participants were evaluated for lower airway disease according to international guidelines. Inflammatory cytokines were investigated using a Th1/Th2 assay including 14 chemokines and cytokines; tissue concentrations were normalized according to tissue weight and total protein concentration. Individual cytokines and multivariate inflammatory profiles were compared between biopsy sites and between patients and controls.We found significantly higher concentrations of Th2 cytokines in nasal polyps compared to inferior turbinate and bronchial biopsies. In addition, we showed that the inflammatory profile of nasal polyps and bronchial biopsies correlated significantly (p<0.01). From the Th2 cytokines measured, IL-13 was significantly increased in bronchial biopsies from CRSwNP patients with, but not without asthma.Our findings support the united airways concept; however, we did not find evidence for subclinical bronchial inflammation in CRSwNP patients without asthma. Finally, this study indicates for the first time that nasal polyps potentially play an important role in the airway inflammation rather than being a secondary phenomenon. It has been reported that 20–60% of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) have asthma. –3 HoweveOutside China, CRSwNP is an eosinophilic disease characterized by Th2 cytokines such as interleukin (IL)-5, eotaxin and IL-13. ,5 Nasal We here hypothesized that (i) upper and lower airway inflammation in CRSwNP show similar profiles indicating a common pathogenic mechanism. We further hypothesized that (ii) bronchial inflammation is present in all CRSwNP patients irrespective of their clinical asthma status.3OS) criteria for CRSwNP [3OS guideline with nasal steroids and saline irrigations for more than three months without recovery. Topical nasal steroids, but not inhaled steroids, were discontinued four weeks before surgery. A control group comprised non-asthmatic patients undergoing nasal septal surgery and/or turbinate reduction. Lower airway disease was classified according to the Global Initiative for Asthma (GINA) and Global initiative for chronic Obstructive Lung Disease (GOLD) guidelines. [Patients who met the European Position Paper on Rhinosinusitis and Nasal Polyps  < 0.5 liter; trombocytopenia or international normalized ratio (INR) > 1.5.Exclusion criteria were: age below 18 or above 80 years; systemic steroids within 3 months before inclusion; severe psychiatric disorders; need for interpreter; pregnancy or nursing; immunodeficiencies; chronic obstructive pulmonary disease (COPD), sarcoidosis, cystic fibrosis, primary ciliary dyskinesia; systemic vasculitis; unstable cardiovascular disease; dysregulated diabetes, forced expiratory volume in the 1The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Copenhagen County (H-B-2008-106); a written informed consent was obtained from all participants.We collected biopsies from nasal polyps, ITs and bronchii of 27 CRSwNP patients and 6 controls. IT biopsies were unavailable from seven of the CRSwNP patients. All patients and controls were of European or Western Asian descent. For patient characteristics, see During FESS, nasal biopsies (polyp and ITs) were taken endoscopically. Punch biopsies from the IT were taken >2 cm posteriorly to its anterior edge. Subsequently, bronchoscopy was performed and 6–8 biopsies were taken from the subcarinae of the right inferior lobe according to guidelines. BiopsiesBiopsies were homogenized using a Tissuelyser LT . Samples were precooled in liquid nitrogen for at least 30 minutes and then disrupted and homogenized without lysis buffer. One ml of 0.9% NaCl + Protease Inhibitor per 0.1 g tissue  was added after disruption and homogenization. Samples were centrifuged at 3000 rpm for 10 min at 4°C and aliquots were kept at -80°C until analysis. From tissue homogenates, the protein concentration was determined using a standard BCA assay and the measured values were normalized to a concentration of 100 μg total protein per sample (in 25 μl pr well), based on parallel analysis of protein concentrations. Homogenates were analyzed using an ultra-sensitive human chemokine 7-plex assay (cat# K15031C-2) and an ultra-sensitive human Th1/Th2 cytokine assay , according to the protocol from the manufacturer. Included biomarkers were: eotaxin, interleukin (IL)-8, interferon-inducable protein (IP)-10, monocyte chemotactic protein (MCP)-1, MCP-4, macrophage inflammatory protein (MIP)-1β, thymus- and activation-regulated chemokine (TARC), interferon (INF)-γ, IL-10, IL-12p70, IL-13, IL-2, IL-4, IL-5 . All plates were analyzed using a Sector Imager from Mesoscale Diagnostics, LCC.In CRSwNP patients outside China, IL-5 has been shown to be a defining cytokine in CRSwNP; therefore Spearman’s rho was used to identify IL-5-correlated cytokines (Spearman’s rho >0.6 and p<0.001) within nasal polyps. These cytokines were used in the subsequent between-group analysis using the Kruskal-Wallis test. A paired Wilcoxon Signed Rank test was applied for comparison of cytokine concentrations at different airway levels. Furthermore, to increase comparability with previous studies, Spearman’s rho was used for a second analysis of the cytokine correlation between biopsy sites .To prepare data for multivariate analysis, biomarker concentrations were log transformed producing roughly normally distributed data. An exploratory principal component analysis (PCA) on the data from both locations revealed location as the main source of variation. Therefore in order to at least partially remove this source of variation, the log-transformed data were standardized (mean subtracted and divided by standard deviation) for each location separately. The exploratory PCA and bi-plot analysis also showed a high degree of co-variation within the IL-5 correlated cytokines (Th2) and the remaining cytokines (mainly Th1) further supporting that we could treat these as two groups (profiles) in the multivariate analysis (Th1 and Th2).A low Euclidian distance between the patient’s transformed cytokine levels in upper  and lower airway biopsies indicates that the cytokine signal co-varied between the upper and lower airways. We devised a permutation test where the average patient Euclidian distance was compared to the average distance calculated by randomly pairing measurements from the two locations in different patients.  The nullR was used for the PCA and permutation test  and SPSSDemographic data are reported in After normalization for tissue weight and total protein concentration, we found significantly higher concentrations of Th2 cytokines in nasal polyp biopsies in comparison with bronchial biopsies ; however, lower Th2 cytokine concentrations were found at the level of the IT . Most ofWhen applying a univariate correlation analysis, only IL-4 correlated significantly between nasal polyp and bronchial biopsies; however, this was due to three outliers .Finally, using the permutation test we rejected the null hypothesis of no patient specific association between nasal polyp and bronchial inflammation . The correlation between IT and bronchial inflammation was less consistent in patients  and controls .This is the first study that simultaneously investigates inflammatory cytokines in biopsies from the upper and lower airways in the same CRSwNP patients; in addition, this is the first study to compare bronchial inflammation in CRSwNP patients with controls. In CRSwNP patients, we found higher concentrations of Th2 cytokines in nasal polyps compared with bronchial biopsies and ITs and a strong correlation between the upper and lower airway inflammatory profiles was seen. None of the measured cytokines were significantly increased in the bronchi of CRSwNP patients except for IL-13 in CRSwNP patients with asthma. Taken together, these findings indicate that a similar inflammation exists throughout the airways in CRSwNP patients and that nasal polyps might play an important role in this airway inflammation. Our findings could not confirm the presence of subclinical bronchial inflammation in CRSwNP patients without asthma.It has been hypothesized that asthma simply develops in allergic airway disease when the inflammation of the upper airway is expressive enough—a hypothesis that is also feasible in the context of CRSwNP and asthma.  Studies Our study thus indicates that nasal polyps might be an epicenter of airway inflammation in patients with CRSwNP and asthma. However, the decreased inflammatory response of the bronchii and ITs could also have resulted from a hypothetical protective mechanism outside the nasal polyps—a mechanism that might also explain why polyps normally are absent at the level of the ITs and bronchii. Furthermore, our findings might be ascribed to selection bias: as patients are included from the department of otorhinolaryngology, patients with severe upper airway inflammation are selected over those with primarily lower airway inflammation. Finally, we found a trend towards higher bronchial cytokine concentrations with increasing asthma severity indicating that had we included cases with more severe asthma, stronger bronchial inflammation would have been reported .We compared biopsies from three different levels of the airway: nasal polyps, ITs and bronchii. We assumed that the inflammation was comparable across biopsy sites since the entire airway is lined with respiratory pseudostratified epithelium with underlying lamina propria; however, organ specific structures are situated just below these layers such as glands, vascular structures and smooth muscle. We took this heterogeneity between biopsy sites into consideration during biopsy sampling by preserving primarily the superficial layers before snap freezing; thereby we removed most of the organ specific structures while preserving the inflammatory cells that were situated superficially.  In additIt has previously been assumed that a similar inflammatory profile exist throughout the airway in CRSwNP patients with co-existing asthma. However, before the present study, evidence for this assumption was sparse and primarily derived from independent studies of the upper airway in CRS patients and the lower airways in asthma patients. Only Ragab et al. had compared upper and lower airway inflammation in the same patients—and failed to show a correlation.  When we Subclinical upper and lower airway inflammation has been shown in allergic airway disease.  In contiWe found lower concentrations of Th2 cytokines in bronchial biopsies when compared to nasal polyps—with and without standardization for total protein. This could in part be explained by the continuation of inhaled steroids in 60% of the asthmatic CRSwNP patients, whereas nasal steroids were discontinued four weeks before the operation –31; alteFinally, an important limitation was the poor match of atopy between groups and the inclusion of three patients with Samter’s triad. However, when analysing non-atopics only or when excluding the three patients with Samter’s triad, the conclusions from our finding remained unchanged.In conclusion, we found higher Th2 cytokine concentrations in nasal polyps compared with the remaining airway and a strong correlation between the upper and lower airway inflammatory profiles was seen; however, we could not confirm the presence of subclinical bronchial inflammation in CRSwNP patients without asthma. Our findings indicate that a similar inflammation exists throughout the airways in CRSwNP patients and potentially nasal polyps play an important role in this airway inflammation rather than being a secondary phenomenon.S1 TableIn CRSwNP patients who do not take inhalation steroids. P-values represent comparison by a paired Wilcoxon signed rank test.(DOCX)Click here for additional data file.S2 TableAstmatic CRSwNP patients only. P-values represent comparison by a paired Wilcoxon signed rank test.(DOCX)Click here for additional data file.S1 DatasetConcentrations in pg/ml.(SAV)Click here for additional data file.


Plants shape distinct, species-specific microbiomes in their rhizospheres. A main premise for evaluating microbial communities associated with root-soil compartments is their successful separation into the rhizosphere (soil-root interface), the rhizoplane (root surface), and the endosphere (inside roots). We evaluated different approaches  regarding their efficiency to separate microbial cells associated with different root compartments of soil-grown rice using fluorescence microscopy and community fingerprinting of 16S rRNA genes. Vigorous washing detached 45% of the rhizoplane population compared to untreated roots. Additional sonication reduced rhizoplane-attached microorganisms by up to 78% but caused various degrees of root tissue destruction at all sonication intensities tested. Treatment with sodium hypochlorite almost completely (98%) removed rhizoplane-associated microbial cells. Community fingerprinting revealed that microbial communities obtained from untreated, washed, and sonicated roots were not statistically distinguishable. Hypochlorite-treated roots harbored communities significantly different from all other samples, likely representing true endospheric populations. Applying these procedures to other root samples (bean and clover) revealed that treatment efficiencies were strongly affected by root morphological parameters such as root hair density and rigidity of epidermis. Our findings suggest that a careful evaluation of separation strategies prior to molecular community analysis is indispensable, especially when endophytes are the subject of interest. Soils host an important interface where organisms of several taxonomic kingdoms live and interact with each other: the rhizosphere. Plants influence the soil surrounding their roots by nutrient and water uptake as well as the release of rhizodeposits, gasses, and protons, consequently applying selective pressure to microorganisms . In turn2O2 – poplar trees , it now is well established that these three habitats host distinct communities in thaliana , Oryza sa sativa , or Popueltoides . These seltoides , and in eltoides . Even geeltoides , soy beaeltoides , maize  to separate root-associated microbial communities followed by an interpretation of the results based on high-throughput sequencing techniques e.g., . HoweverO. sativa L. (cultivar IR 36) were surface sterilized in 5% NaOCl for 2 min, followed by three washes in autoclaved ultrapure water. The seeds were pre-germinated in the soil used for the experiment for 2 weeks. Individual seedlings were transplanted and cultivated on a Chinese paddy soil  in three replicate pots under controlled climatic conditions (Seeds of nditions . Eight wTable 1) or were washed in 5 ml tubes containing 3 ml of 1 × PBS amended with 0.2% Silwet  on an orbital shaker . Washed roots were then transferred to 15 ml tubes (five roots per treatment) containing 2 ml 0.2% PBS-S and subjected to sonication using a sonication probe  or a sonication bath with adaptive cavitation technique  with two levels of intensity each (Table 1). Tubes containing roots were cooled (+4°C) during sonication steps to prevent heat damage of root tissue and microbial cells. Alternatively, washed roots were treated by submerging in 2 ml 5% NaOCl for 2 min followed by three washes in autoclaved ultrapure water (T7). Washed, sonicated, and bleached roots were divided into two batches and (i) stored at -20°C for DNA extraction and (ii) stored in PBS-EtOH  at -20°C for fluorescence microscopy. The PBS-S solutions remaining in the tubes after washing and sonication treatments  were also divided into two batches and (i) stored at -20°C for DNA extraction and (ii) fixed in 4% formaldehyde-PBS , washed in PBS, and stored in PBS-EtOH  at -20°C.Root segments remained either untreated  and incubated at room temperature for 15 min in the dark. Root segments were destained with sterile ultrapure water for 5 min and mounted in antifading medium . Cover slips were fixed with carbon pads which served as spacer ensuring a parallel placement of the cover slips and prevented destruction of root tissue. Microscopy was performed with a fluorescence microscope  and a 63× objective .z-axis) was selected per image to encompass root surface topology and single layers were recorded with blue- and green excitation every 0.5 μm distance along the z-axis. Subsequently, greyscale images were replaced with false colors and merged to single image projections per microscopic field of view. For cell counting, eight spots were selected along the root longitudinal axis with a distance of approximately 2.5 mm. Consequently, eight image stacks were recorded and analyzed for each of the three biological replicate roots per treatment. Cells were counted by marking every stained cell in the merged z-stack images in selected regions of interest, which were defined using image analysis software . Respective cell numbers were referred to the area of the corresponding region of interest and presented as colonization densities (cells per mm2).Microbial cells colonizing the root segments were first observed with a double excitation filter set  in order to prove positive staining of microorganisms on top of plant tissue-derived autofluorescent background. Greyscale images were then taken with an AxioCam MRm digital camera  and AxioVision 4.8 software . A total range of approximately 20 μm in depth . Aliquots of 750 μl were filtered through polycarbonate filters . Filter sections were embedded in mounting medium containing the general cell stain DAPI  and analyzed by epifluorescence microscopy  using a DAPI filter set  and a 40× objective . Images were taken on 20 spots for each filter section following a line from the center to the outer edge of the polycarbonate filter to account for filtration inhomogeneity. Total cells were counted in these images by marking stained cells using AnalySIS image analysis software . All counting data were extrapolated to cells per root segment in the respective treatment suspension. In parallel, cells associated with fragments of root tissue or soil particles were enumerated. Moreover, the quantity and size of fragments of root tissue and soil particles was assessed.The DNA of all samples (roots and suspensions containing detached cells) was extracted using the NucleoSpin Soil Kit  with buffer SL1 and enhancer SX. Root segments were first ground in liquid nitrogen in micro reaction cups and manually comminuted with spatula. Afterward, buffer and beads were added to the bead beating tube and the extraction was finalized according to manufacturer’s instructions. DNA from detached cells was extracted after filtering the treatment suspension through polycarbonate membranes . The membranes were placed into the aforementioned bead beating tubes and DNA extraction was performed according to the manual. For sample lysis  a swing mill  was used with 25 Hz for 30 s. The DNA of all samples was eluted in 30 μl elution buffer  and stored at -20°C.-1 of each primer and 50 μmol l-1 of each nucleotide.The composition of microbial communities associated with roots and detached from roots was analyzed by terminal restriction fragment length polymorphism (T-RFLP). For the amplification of 16S rRNA genes the bacterial primer set 27F  and 907MspI , and the FAM-labeled fragments were purified using SigmaSpin Post-Reaction Clean-Up columns . Three microliter of digested product were added to a mix of X-Rhodamine MapMarker 1000  and Hi-Di formamide . After incubation at 95°C for 5 min, the samples were analyzed with a 3130 Genetic Analyzer . Raw data was analyzed with the TREX online suite1. Peaks representing sequence lengths shorter than 50 base pairs or longer than 900 base pairs were discarded from the dataset. The denoising process implemented in TREX was used to identify true peaks . 120 ng of PCR product was digested with ue peaks , and theP-values were adjusted for multiple testing according to the default setting of glht. Statistical significance was assumed when adjusted p-values were below α = 0.05. For community profile data, OTU richness per sample was assessed by the calculation of Hill numbers D of order q = 0 and q = 2 based on peak height data, representing unweighted  and abundance-weighted  richness estimates . In our assessment, an untreated rice root segment in 4–6 cm distance to the root tip hosted an average of 2.48 × 104 cells per mm2 , which also included tightly attached rhizosphere soil-associated microorganisms. Washing as a single treatment (T2) resulted in an average of 1.35 × 104 cells per mm2, which were still attached to rice root surfaces, and accounted for a loss of 55% compared to the initial average cell count . By washing plus subsequent probe sonication (T3 and T4), the microbial cell colonization decreased by approximately 75%. The highest cell decrease was found after treatment with the sonication bath at highest energy (T6), reducing the initial colonization density by 78%. No significant differences were found in cell counts between the four sonication treatments tested, although results from probe sonication (T3 and T4) were more homogeneous between replicates than the sonication bath (T5 and T6). In total, the sonication procedures were approximately 1.7 times more effective in cleaning root surfaces from colonizing cells compared to washing alone. In contrast, the treatment with NaOCl (T7) resulted in nearly complete removal of the rhizoplane community , leaving only 2.4% of the initial colonization attached to the rhizoplane. Increasing intensity, probe and especially bath sonication (T5 and T6) led to a significantly heterogeneous distribution of remaining cells within and between replicates . Bath sonication lead to significantly higher numbers of detectable microbial cells in the respective suspensions compared to probe sonications (p < 0.05), with no effect of sonication intensity. Numbers of detached cells ranged from 3.96 × 104 cells per root segment in the suspension of T3 to 1.85 × 105 cells of T5 , in addition to an average of 5.23 × 104 cells already detached during the washing step. Hence, the sonication bath treatments removed 272 and 355% more stainable cells from the rhizoplane with low and high energy, respectively, in comparison to the washing step alone. Similar to the observation on rhizoplanes, no detectable signal was observed in filtered suspensions obtained from NaOCl-treated roots indicating the dissociation of double-stranded DNA  and subsequent sonication treatments T3–T6. Respective treatment suspensions were filtered and during enumeration, cells were differentiated between root debris-associated, soil particle-associated, and free-floating microbial cells (nded DNA . In geneTable 2). Suspensions from roots subjected to the washing procedure (T2) featured more cells attached to soil  than to root debris , indicating a well-preserved structure of soil particles removed from the rhizoplane via washing. This effect was reversed when samples were sonicated subsequently to washing. Thus, the proportion of soil associated cells decreased by more than 60% in the suspensions of the sonication probe treatments (T3 and T4) and 90% in the respective samples of the sonication bath treatment (T5 and T6) compared to washing (T2). By contrast, a higher number of cells in the suspensions of the sonication probe treatments were still attached to root fragments accounting for 29 and 39% of all detached cells in T3 and T4, respectively. Following treatments T5 and T6, only very few cells could be observed being associated to either fraction of root tissue (around 10%) or soil particles  although the total number of detached cells had increased in this treatment .While evaluating suspensions of the individual treatments, we recognized microbial cells associated with either root tissue fragments or soil particles. Thus, we further compared the abundance of these cells still attached to soil or root particles after the respective separation treatments, which were in general lower than free-floating microbial cells . All sonication methods were able to compromise rhizoplane integrity . However, a clear difference was observed among the two sonication techniques as clear cuts and losses of larger fragments were obvious for probe sonication (T3 and T4). Bath sonication (T5 and T6) resulted in disruption of tissue within root cell walls and smaller fragments in the respective suspensions. We assessed these microscopic observations quantitatively by determining the abundance and size of root fragments in treatment suspensions (Table 2). Respective counts were higher in the suspensions of sonication bath methods, especially when compared to the washing treatment itself (T2) which was almost devoid of root debris. High intensity probe and bath sonication procedures caused comparable rhizoplane damage, while low intensity sonication bath procedures showed the strongest effect, with high variance within replicates. However, both sonication bath methods tended to produce smaller root tissue fragments compared to the other methods.Root tissue disruption was commonly observed after sonication, as evident from rhizodermis imaging and root tissue debris in treatment suspensions . The highest proportion of soil particles was removed from the root surface after washing (T2). Sonication resulted in the detachment of additional 47–59% soil particles compared to washing alone, while no significant differences between sonication protocols were observed. Aggregates of soil particles detached via the washing treatment  and probe sonication  were observed to be larger compared to soil particles detached via bath sonication. Soil particles still adhering to the rhizoplane were frequently found after sonication with both low and high intensity  is required. In our experiments, all treatment suspensions containing detached microbial cells featured soil particles of different sizes showing the removal of rhizosphere soil from the rhizoplane (0D (14 ± 6 SE), whereas communities detached from roots showed the highest average richness (67 ± 20–105 ± 11). Untreated roots (T1) featured high OTU richness (86 ± 12). However, as evident from the high standard errors of the mean, the OTU richness differed highly between replicates, suggesting that observed β-diversity between root communities is not always the result of the treatment effect. Thus, the inversed Simpson concentration index (2D) was calculated for each community, which puts more emphasis on changes in the abundances of the dominant OTUs. For root samples, a steady decrease in 2D values was found between untreated roots and NaOCl-treated roots with washed and sonicated root samples ranking in between, except T6 . For the root samples, 2D values were higher for the sonication bath treatments than for the probe sonicated roots. For microbial assemblies detached from roots, much higher values for 2D were observed in general, and no clear trend was visible due to high within-sample heterogeneity .In order to assess biological richness and diversity in root samples and treatment suspensions containing detached microorganisms, T-RFLP analyses of 16S rRNA genes were performed. A total of 255 individual peaks in 36 samples  were observed after removal of singletons and the putative plastid peak (TRF 493 bp). Root samples treated with NaOCl had the lowest average OTU richness Figure 4). The highest distance was observed between communities in the suspensions with detached cells obtained from the washing step (samples T2-D) and root-associated communities after NaOCl-treatment most likely representing the endophytic community (samples T7-R). The NMDS analysis was complemented with anosim under 999 permutations, which showed a significant (p < 0.001) difference of the community structures between treatments. Within the ordination space, root samples (including untreated roots) were undistinguishable from root samples obtained after washing and sonication (T1-R–T6-R). Only root-associated microbiota from samples incubated in NaOCl clustered on their own.Non-metric multi-dimensional scaling based on Bray–Curtis-distances revealed a clear separation of populations sampled from remaining roots and treatment suspensions along the first axis .It is often assumed that rhizosphere soil can be removed from the root surface by washing alone . In thisFigure 4), which further indicated that no bacterial groups were preferentially detached from the rhizoplane. Although fingerprinting methods such as T-RFLP may not reach the analytical resolution of high-throughput sequencing, they are well suited to detect changes in β-diversity  from the rhizoplane but failed to completely remove microbial cells thereof. These findings obtained via quantification of single microbial cells were supported by NMDS ordination of T-RFLP data. Bacterial communities in untreated, washed and sonicated roots remained indistinguishable . The problem of root disruption for downstream analysis is twofold: firstly, root endophytes may be incorporated into the rhizoplane sample and bias its community composition. However, based on T-RFLP analysis, root tissue disruption by sonication was not detected as the composition of communities of detached cells (T3-R–T6-R) was different from that of other treatments . Secondly, sonication triggers the loss of cells colonizing outer root layers, and thus, leads to potential changes in the endophytic community . The latter may apply to a number of studies, where sonication was performed in addition to washing and where scanning electron microscopy revealed the immense level of root tissue destruction . The application of a standardized sonication protocol should presumably lead to a sufficient separation into rhizoplane and endorhizosphere communities as demonstrated for a range of plant species . Consequently, it remains difficult to interpret whether observed differences in microbial communities of previous studies are blurred by technical limitations of the separation protocol, especially when appropriate controls are not comparatively evaluated . Such studies would then rather describe the detachable fraction of rhizoplane-colonizing microorganisms and an endosphere compartment that is contaminated by cells resisting sonication. This methodological bias has to be considered, especially when sonication is applied to address research questions investigating mechanisms involved in the selection of endophytes by roots.Regardless of the technique used, sonication seriously compromised the integrity of the outer root layer  in a water bath for 15 min did not result in a decrease of rhizoplane-colonizing bacteria as assessed by quantification of 23S rRNA gene copy numbers  while retaining the integrity of the outer cell layer. Despite the high variability of diversity within treatments [also reported by We observed a high variability of microbial cell numbers among biological replicate root segments. This was already pronounced in untreated samples, and thus, not related to individual separation treatments, which indicates that different roots of individual plants have varying success in recruiting microbial populations from soil. Especially on the surface of relatively young roots (our study: 10 weeks), highly irregular colonization patterns can be expected . The appVicia faba and Trifolium pratense led to different surface cleaning efficacies . Applying the tested protocols to roots of es e.g.,  should bin situ. Studies focusing on endophytes should carefully apply chemical agents such as NaOCl to remove rhizoplane-associated cells from the root surface. We recommend appropriate replication and rigorous testing of selected separation strategies in respect to reproducibility among replicates and species. The presented approach using SYBR-Green-based fluorescence microscopy represents a prime choice for quick and easy control of separation efficiency.The selection of a separation strategy with respect to plant microbiome analysis remains a critical issue and has to be adapted to the respective research questions. Probe and bath sonication may not always represent a strategy that reliably removes microorganisms and soil particles from roots and has the potential to severely compromise root tissue integrity. Future studies that apply sonication should clearly state the necessary details of the sonication device to ensure comparability among studies. Washing of roots may represent the best approach when researchers aim to include the analysis of rhizoplane-colonizing cells TR-H, HS, and TE conducted and interpreted the experiments and wrote the manuscript. SK provided molecular analyses. MF was involved in the conception of the experiments and in writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.