The four Chineseherbal medicines were intragastricallyadministered to Sprague-Dawley rats,respectively, to prepare drug-containing serumsof corresponding Chinese herbs. BMSCs wereisolated, cultured, and exposed to culturesolution containing 1%, 5%, 10%,and 15% (v/v) Radix Astragali-, Salvia-,Herba Epimedii-, and SaussureaInvolucrata-containing serum, respectively.TGF-β1-induced BMSCs were addressed in thesame manner. Collagen type II protein wasassessed by immunofluorescence methods. Toassess whether the drug-containing serums hadpositive effects on the proliferation of BMSCsand TGF-β1-induced BMSCs, MTT method wasassessed. The proliferation of BMSCs wassignificantly enhanced when exposed to culturesolutions containing 1% and 5% RadixAstragali-, 1% and 5% Salvia-, 5%Herba Epimedii-, and 1%, 5%, and10% Saussurea Involucrata-containing serum.The proliferation of TGF-β1-induced BMSCswas significantly enhanced when exposed to1%, 5%, and 15% Radix Astragali-,10% and 15% Salvia-, 5%, and15% Herba Epimedii-, and 1%, 5%,and 10% Saussurea Involucrata-containingserum.Traditional Chinesemedicine can promote the proliferation of bonemarrow-derived mesenchymal stem cells (BMSCs).We chose four “Kidney-tonifying”Chinese herbal medicines, Radix Astragali, Salvia,Herba Epimedii, and Saussurea Involucrata, toevaluate whether they had positive effects onthe proliferation of BMSCs andTGF- Bone marrow-derived mesenchymal stem cells (BMSCs) are derived from the mesoderm and within bone marrow . BMSCs hβ (TGF-β) family members are potent stimulators of bone formation and have been shown to be positioned in the bone repair site as well as embryonic bone and cartilage formation site [β family members played important roles in promoting the expression of cartilage-specific gene [Osteoarthritis is one of the most common progressive diseases, which affects at least one-third of adults over the age of 55 years . And theion site . Recent fic gene –11. Atractylodis macrocephalae, a tonic traditional Chinese herb, could enhance chondrogenic differentiation in BMSCs and the mechanism was dependent on the Sonic Hedgehog (Shh) pathway [β1 [According to the theories of TCM, the “Kidney” controls the bone metabolism and generates the marrow. Therefore, the use of “Kidney-tonifying” TCM herbs can contribute to bone health. He at al.  reported pathway . A lateshway [β1 .β1-induced chondrogenic differentiation of BMSCs. All the four TCM herbs were often applied to treat osteoarthritis, articular cartilage injury, and osteoporosis with a long history and significant curative effect.Here, we choose four kinds of Chinese herbal medicine, including Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata, to analyze whether they have positive effects on the proliferation of BMSCs as well as TGF-Sprague-Dawley rats  were obtained from the animal center of Nantong University . All rats were housed in the Nantong University animal care facility and were maintained under pathogen-free conditions. Rats were reared in strict accordance with standard operating procedures. Ambient temperature was kept at 22–25°C, humidity of 45% to 55%, light cycle was 12 h light, 12 h darkness. Timely water addition and feeding were managed daily and litter was cleaned once every two days. All experimental procedures were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China. The experimental procedures were approved by the Ethics Committee of the Institute of Wuxi Hospital of Traditional Chinese Medicine.Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata were provided by the traditional Chinese medicine pharmacy of Wuxi Hospital of TCM . Radix Astragali 90 g, Salvia 45 g, and Herba Epimedii 27 g (these amounts were three-day doses for an adult) were immersed in 500 mL distilled water for 40 minutes, boiled, decocted for 1 hour, and condensed to 60 mL, respectively. Saussurea Involucrata 5.5 g (this amount was a three-day dose for an adult) was cut into pieces and immersed in 500 mL 75% ethanol for 30 days. Then the liquid was filtered out, volatilized at 74°C, and condensed to 60 mL. Crude concentrations of Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata in the Chinese herbal solutions were 1.5 g/mL, 0.75 g/mL, 0.45 g/mL, and 0.09 g/mL, respectively. Chinese herbal solutions were placed in a bottle, sealed, sterilized, and stored at 4°C.μm micro porous membrane, and stored at −20°C.The Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata groups (5 rats each group) were intragastrically administered with corresponding Chinese herbal solutions  for 3 consecutive days . The control group was intragastrically administered with normal saline (NS). One hour after the final oral administration on the third day, rats were anesthetized by intraperitoneal injection with 3 mg/100 g sodium pentobarbital . Blood was harvested from the heart and centrifuged at 2500 r/min for 25 minutes. The supernatant was harvested, deactivated in a water bath at 56°C for 30 minutes, filtered through a 0.22 2 environment. The media were changed at 24 hour for the first time and then changed after each 72 hours. The cells were subcultured at a ratio of 1 : 3, and the third generation of cells was prepared for downstream-related experiments. Cell morphology and proliferation were analyzed daily with inverted phase contrast microscopy .4–6-week-old SD rats were sacrificed by cervical dislocation. Both femora and tibias were isolated and rinsed with phosphate-buffered saline . Whole bone marrow cells were collected by repeated washing the bone marrow cavity with low-glucose Dulbecco's modified Eagle's medium . The flushed mixture was centrifuged, washed, and resuspended in low-glucose DMEM containing 15% fetal bovine serum  and 1% penicillin-streptomycin . The resuspended cells were seeded into flasks and incubated in a 37°C, 5% COμg/L transforming growth factor β1 . Medium was replaced twice a week. Cell morphology and proliferation were analyzed daily with inverted phase contrast microscopy .BMSCs of three passages were used for chondrogenic differentiation. Cells were cultured for 2 weeks in chondrogenic medium, consisting of low-glucose DMEM supplemented with 2 mg/L insulin , 3 mg/L transferring , 1 mmol/L sodium pyruvate , 100 nmol/L dexamethasone , and 10 β1-induced BMSCs were fixed with 4% paraformaldehyde  for 30 minutes and pretreated with triton-X100  for 15 minutes. Rabbit polyclonal antibody against collagen type II  and fluorescein isothiocyanate- (FITC-) conjugated anti-rabbit antibodies  were used for fluorescent detection of the specific collagen. Fluorescence images were observed and recorded with a fluorescence microscope . Uninduced BMSCs were used as negative control.Immunocytochemistry was also carried out after 2 weeks of chondrogenic differentiation. For an immunofluorescence stain, TGF-β1-induced BMSCs was treated with the same manner above.The third generation of BMSCs were seeded and exposed to culture solution containing 15% FBS plus 1%, 5%, 10% and 15% (v/v) Radix Astragali-, Salvia-, Herba Epimedii-, and Saussurea Involucrata-containing serum, respectively, serving as experimental groups. 15% FBS plus serum of NS group served as control groups. Freshly prepared DMEM containing 15% FBS was used as blank group. Each experimental group and control group were accompanied by a corresponding blank group. The culture media were replaced every 48 hours. TGF-4/mL cells per well. And then 20 μL MTT 2,5-diphenyltetrazolium bromide; 5 mg/mL; Sigma, USA) was added to each well and the cells were incubated at 37°C for 4 hours. After that, the supernatant was carefully discarded, and 150 μL dimethyl sulfoxide  was added to each well with gentle shaking for 10 minutes. After the blue crystals were dissolved in DMSO, the optic density (OD) values were measured with an enzyme linked immunosorbent assay reader  at wave length of 492 nm. Five parallel wells were set in each group. An average value of the five wells was calculated and used in following formulas: value of Chinese herbal − enhanced cell proliferation = OD of experimental group − OD of blank group; value of control cell proliferation = OD of control group − OD of blank group.On the 7th day after intervention with drug-containing serums of Chinese herbal, the culture fluid was removed. Cells were seeded in 96-well plates at a density of 1 × 10t-test was used to analyze the differences between the values of Chinese herbal-enhanced cell proliferation and control cell proliferation in each group. All data were statistically analyzed using SPSS 16.0 software . A probability value of P < 0.05 was considered statistically significant.All experiments were conducted with a sample size of five and values were reported as mean ± standard deviation. The Student's β1-induced BMSCs exhibited larger cell bodies and multiple cytoplasmic projections and appeared as a polygonal-shape. After 5–8 days the cell bodies increased and the projections extended, exhibiting flattened morphology with the nucleolus split  concentrations of Radix Astragali-, Salvia-, Herba Epimedii-, and Saussurea Involucrata-containing serum, respectively. OD values of experimental, control, and blank groups were detected with MTT assay after 7 days. The results showed that cell proliferation of BMSCs was significantly enhanced when exposed to culture solution containing 1% and 5% Radix Astragali-containing serum, 1% and 5% Salvia-containing serum, 5% Herba Epimedii-containing serum, and 1%, 5%, and 10% Saussurea Involucrata-containing serum .β1-induced BMSCs were exposed to culture medium containing 1%, 5%, 10%, and 15% (v/v) concentrations of Radix Astragali-, Salvia-, Herba Epimedii-, and Saussurea Involucrata-containing serum, respectively. OD values of experimental, control, and blank groups were detected with MTT assay after 7 days. The results showed that cell proliferation of TGF-β1-induced BMSCs was significantly enhanced when exposed to culture solution containing 1%, 5%, and 15% Radix Astragali-containing serum, 10% and 15% Salvia-containing serum, 5% and 15% Herba Epimedii-containing serum, and 1%, 5%, and 10% Saussurea Involucrata-containing serum  of Chinese herbal-containing serum, 1% and 5% Radix Astragali-containing serum, 1% and 5% Salvia-containing serum, 5% Herba Epimedii-containing serum, and 1%, 5%,and 10% Saussurea Involucrata-containing serum could significantly enhance the proliferation of BMSCs. There was no dose-dependent manner between the proliferation of BMSCs and Chinese herbal drug-containing serum concentrations in our study. Moreover, with respect to the higher concentration of 15%, a lower concentration of 1% or 5% of Chinese herbal drug-containing serum could be more effective in increasing BMSCs proliferation, especially a concentration of 5% in our study.Actually, our result was consistent with some previous findings that the influence of Chinese medicine on the proliferation of BMSCs was indeed not dependent on the concentrations. A previous study investigated the proliferative effect of Astragaloside IV (AS-IV) in MSCs. AS-IV was one of the major and active components of the Radix Astragali. They are found in the concentration range of 10 to 200 mM AS-IV; the proliferation rates demonstrated optimal effect at 50 mM, much higher than the 200 mM . AnotherκB induction [β1-induced chondrogenic differentiation of BMSCs. Perhaps it could provide a new research idea for Salvia in the treatment of bone diseases. Actually, a Korean study has reported that tanshinone IIA could enhance the commitment of C2C12 cells into osteoblasts and their differentiation through synergistic cross talk between tanshinone IIA-induced p38 activation and BMP-2-induced Smad activation. They hypothesized that the osteogenic activity of tanshinone IIA might lead to the development of therapeutics that benefit bone regeneration and fracture healing [Salvia was a well-known Chinese herb used in the treatment of diseases including osteoporosis. The precise mechanism of Salvia in treating osteoporosis was unclear. Tanshinone IIA was one of the major active phytochemicals isolated from Salvia. It has been reported that tanshinone IIA could inhibit osteoclast differentiation and suppresses bone resorption through disruption of the actin ring, and the precise inhibitory mechanism of Tanshinone VI on osteoclast differentiating might be through inhibiting RANKL expression and NFnduction . Kwak etnduction  reported healing . Gjb2, Adam17, Adar, Alp1, Bmp10, Bmpr1a, Btrc, Cdh1, Cdkn2a, Fgf21, Fgfr2.Herba Epimedii is a commonly used traditional Chinese herb for “strengthening the kidney” and providing nutrition to bone. The result of our present study confirmed that Herba Epimedii could increase the proliferation of BMSCs and improve the chondrogenic differentiation of BMSCs in an appropriate circumstance. However, the mechanism was not clear. Interestingly, previous studies have demonstrated that Herba Epimedii could improve osteogenic differentiation in BMSCs , 24. Icaβ1-induced BMSCs. Perhaps this finding could expand the application of Saussurea Involucrata in terms of bone regeneration and fracture healing. Of course, this requires more in-depth study of the molecular mechanisms.Saussurea Involucrata is a rare and precious Chinese herb, most of which grow in 4000 meters above sea level in Tianshan mountain in China. As a traditional Chinese herbal medicine, Saussurea Involucrata has been used in the treatment of rheumatoid arthritis, impotence, irregular menses, and so forth . The finβ1-induced BMSCs in vitro. Nevertheless, one limitation existed in our study was that the mechanisms had not been explored. It is the content of our future research. TCM has a long history and a notable curative effect. We suggest that analyzing TCM using modern western research methods can provide a direction for future TCM investigation and contribute to the health of people worldwide.In conclusion, our study revealed that Radix Astragali, Salvia, Herba Epimedii, and Saussurea Involucrata could enhance the proliferation of BMSCs and TGF-


MUC5B rs35705950 variants and determine new plausible candidate variants for IPF among four different European populations. We genotyped 26 IPF candidate loci in 165 IPF patients from four European countries, such as Czech Republic (n = 41), Germany (n = 33), Greece (n = 40), France (n = 51), and performed association study comparing observed variant distribution with that obtained in a genetically similar Czech healthy control population (n = 96) described in our earlier data report. A highly significant association for a promoter variant (rs35705950) of mucin encoding MUC5B gene was observed in all IPF populations, individually and combined . Another non-coding variant, rs7934606 in MUC2 was significant among German patients  and combined European IPF cases . The network analysis for these variants indicated gene–gene and gene–phenotype interactions in IPF and lung biology. With replication of MUC5B rs35705950 previously reported in U.S. populations of European descent and indicating other plausible polymorphic variants relevant for IPF, we provide additional reference information for future extended functional and population studies aimed, ideally with inclusion of clinical parameters, at identification of IPF genetic markers.Idiopathic pulmonary fibrosis (IPF) affects lung parenchyma with progressing fibrosis. In this study, we aimed to replicate  Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive form of fibrosing interstitial pneumonia of unknown cause that predominantly affects lung parenchyma, leading to progressive worsening of dyspnea and lung function . In pathMUC5B and MUC2) or of pro-inflammatory cytokines  and also in cell signaling and innate immunity processes (TLR3 and TOLLIP) involved in inflammatory and profibrotic pathways (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585032/table/T1/), we have described the approach for their simultaneous investigation using a novel mass spectrometry based matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) multiplexed genotyping assay and reported their gene frequencies in healthy Czech (European) population  of 26 IPF candidate loci. Here, we have performed a comparative study for association of these IPF candidate genetic variants among four different European  populations and have ammended it by network prediction for gene–gene/gene–phenotype interactions in IPF and lung biology. We suggest that future extended and replicative studies following hereby described approach could enable better understanding of IPF pathogenesis, and if further supported by patient laboratory and clinical data, it could help to nominate novel disease markers.In this study, 165 IPF patients from four European populations comprising 41 Czechs , 33 Germans , 40 Greeks , and 51 French , representing Central, Southern, and Western Europe, were enrolled (Table CO), and/or restrictive pulmonary deficit, exclusion of other known causes of interstitial lung disease (ILD). For comparisons of genotype, allele frequency, and carriage rate (phenotype frequency) of analyzed genomic variants and case–control association study, we have utilized the data on distribution of these variants in 96 Czech healthy controls  based MALDI-TOF MS assay. The assay consists of an initial locus-specific PCR amplification followed by SBE using mass-modified dideoxynucleotide terminators of an oligonucleotide primer that anneals immediately upstream of the target polymorphic site. The distinct mass of extended primer traces the alternative alleles using MassARRAY Typer 4.0.20. For quality control (QC) step, we determined data missing rate per individuals and missing rate per SNP. Also, for QC of SNP genotyping, positive and negative template control samples were included in each assay plate. Any assay found as positive in negative template control were removed from the study.The details of the panel comprising 26 IPF candidate loci and genotyping procedure in IPF cases have been described previously . In brie2) test or Fisher exact test, as applicable. SNPs within HWE (p > 0.05) and sufficiently common [minor allele frequency (MAF) > 5%] in studied population were included. Carriage rate (phenotype frequency) was calculated as number of individuals carrying one (or two) copies of a particular allele on one or both  chromosomes. Association of SNPs minor alleles with IPF susceptibility were evaluated by Fisher’s exact test providing odds ratio (OR), 95% confidence interval (CI), and level of significance (p). For Bonferroni correction of multiple comparison , a stringent approach with p-value < 0.05/100 (5 × 10−4) was considered as significant.Each SNP was tested for Hardy–Weinberg equilibrium (HWE) by Pearson’s Chi-square (χp < 0.05) and their interaction with IPF and other phenotypes, such as lung disease, lung injury, and lung function was performed using Phenolyzer, a tool for phenotype-based prioritization of candidate genes in human diseases (Prediction of gene–gene network for plausible candidate variants (diseases . The candiseases .PRKCE rs628877 (p = 0.02 in combined IPF cases with F_MISS = 0.008), IL-4 rs2243250 , IL-4 rs2070874 , IL-4Rα rs1801275 , and MAPT rs1981997 . Further, with MAF threshold check, TP53 rs12951053 was found with MAF = 0.04, F_MISS = 0 in Greek IPF cases. Thus, keeping the QC stringent conditions, these six SNPs were removed from further analysis, for which 20 variants remained  using allelic (multiplicative) genetic model revealed a total of nine SNPs for IPF susceptibility. Among these, three SNPs were shared among different IPF populations: first, rs35705950*T within promoter region of Mucin5B (MUC5B) was highly significant among all the IPF populations – Czech ; German ; Greek ; French ; and combined IPF cases  (Table Mucin2 (MUC2) was significant for German , Greek , French , and combined IPF  cases. The third variant, rs1799899*A located in exon region of Transferrin (TF) gene was significant for Germans , French , and in combined IPF  cases. Prediction of loss-/gain-of-function for functional variant TF rs1799899 suggested it as a probably damaging mutation (Polyphen score: 0.869 and SIFT score: 0.07).The primary analysis (Tumor protein 53 (TP53) ; (ii) rs4277405*C in Angiotensin converting enzyme (ACE) ; and (iii) rs4459609*C in ACE  and one each in Greek: Telomerase reverse transcriptase (TERT) rs2736100*G ; French: ATPase, type 11A (ATP11A) rs1278769*A ; and in total IPF cases: Interleukin-1 α (IL-1α) rs1800587*T   rs12602273*G in 2 test and additive test of logistic regression analysis with similar panel and population distribution/stratification of significant risk variants, and an exceptional addition of rs11191865*A (p = 0.046) in Oligonucleotide/oligosaccharide-binding fold containing 1 (OBFC1) exclusively in total IPF set under χ2 test.These findings of allelic model were in concordance with Pearson’s χMUC5B and MUC2 in same gene family , transcriptional interaction among MUC2 and TP53 , and all identified candidate genes belonged to the same biosystem . Further prediction based on their records in several databases , gene–gene interaction and interaction with phenotypes  has , German (1.55 × 10−5), Greek (1.13 × 10−6), French (5.28 × 10−10), and in combined IPF population (1.80 × 10−11); whereas MUC2 rs7934606*A was significant in German (4.03 × 10−4) and in combined IPF (3.73 × 10−5) . Among IPF cases, the allele frequency and carriage rate for rs35705950*T were lowest in Czech  and highest in French ; while rs7934606*A has lowest frequencies in Czech  and highest in Germans .p < 5 × 10−4), we report a high association between MUC5B rs35705950*T and IPF susceptibility in all the four analyzed populations among Czechs: [OR (95% CI) p] 3.77 (7.47–1.9) 1.62 × 10−4, Germans: 4.83 (9.79–2.39) 1.55 × 10−5, Greeks: 5.46 (10.82–2.76) 1.13 × 10−6, and French: 6.77 (12.65–3.62) 5.28 × 10−10 along with all patients from this study as a whole: 5.23 (8.94–3.06) 1.80 × 10−11. Thus, we replicate previous findings and provide an insight for predominant association of gel-forming mucin-encoding gene variants with IPF in Europeans and populations of European descent. The role of mucin variants was also supported with high significance of MUC2 rs7934606 among Germans  and combined European IPF cases . The identified nine variants among MUC5B, MUC2, TF, TP53, ACE, TERT, ATP11A, and IL-1α (significant at p < 0.05) were interactive at gene–gene and gene–phenotype level in network analysis. The present study for inter-population comparison is imperative with respect of comparing the distributions of plausible IPF-associated gene variants and, thus, may provide starting point(s) for further investigations of biological  and translational  aspects.This study is a first report of comparative distribution of genotypes and alleles of 26 candidate gene variants implicated in mucin production, cell-cycle regulation, pro-inflammatory, and profibrotic signaling pathways pertinent to IPF pathobiology among patients from four populations across Europe . Employing stringent statistical approach  on chromosome 11q15.5, MUC5B is the most predominant in the normal distal airway epithelium and is widely associated with sporadic IPF and familial interstitial pneumonia (MUC5B promoter-variant rs35705950 SNP predicted change in transcription factors binding sites (disruption of E2F and creation of HOX9 and PAX2) and the risk T-allele has been strongly associated with increased MUC5B expression in the lung tissue. The relative high frequency of rs35705950*T risk allele  observed in this study are in concordance with several reports, including populations of European descent . To explain the mucin-expressing structures in IPF pathogenesis, Seibold et al. analyzed ciliated, basal, and alveolar type II cells in lung tissue and reported predominant expression of MUC5B in pseudostratified mucociliary epithelium comprised of basal epithelial cells and mucus cells in distal airway. Further, it is suggested that mucociliary dysfunction in the distal airway causing honeycomb cyst may play a role in the development of progressive fibroproliferative lung disease (MUC2 rs79834606 as significantly associated in IPF (p = 3.8 × 10−6) with MAF  0.54 in IPF cases and 0.41 in controls . The SNP rs1799899 marks a Gly/Ser change located in TF gene encoding a glycoprotein involved in iron ion transport and removal of certain organic matter and allergens from serum. As this is the first implication of TF gene in context of IPF, in parallel to the studies of its possible functional role, this association requires replication  rs4277405 and (iii) rs4459609 located in promoter region of ACE-II suggested with critical profibrotic role in IPF (TERT and (v) rs1278769 in 3′-UTR of ATP11A were significant. Several studies have reported the telomerase gene mutation causing short telomerase as risk factor and poor survival in IPF  reported in this study must be replicated in accordance to the guidelines for conductance of genetic association studies  do increase IPF risk among the four European populations, two of which (Czech and Greek) have not been studied before in this regard at all. Additionally, the nominated variants in TF and other variants of TP53, ACE, TERT, ATP11A, and IL-1α may also contribute to IPF susceptibility. Despite our panel of 26 gene variants was designed across pertinent pathobiological pathways, it did not include others from the wide range of plausible IPF-associated SNPs regulating immune and fibrotic functions, such as master regulator TGF-β, TNF-α, full spectrum of TLRs, MHC (HLA) variants, and also SNPs in regulatory microRNAs (miRNAs) (Our findings provide evidence that gene variants involved in mucin production ((miRNAs) , 33, 34.MUC5B promoter region variant (rs35705950) with IPF disease among Europeans. In addition, it suggests further IPF-associated polymorphisms: MUC2 (rs79834606) and TF (rs1799899) variants in general, and the other six  in individual European populations. Their further investigation for disease association among extended patient cohorts is, therefore, warranted. At the same time, findings of our present study represent reference information to be utilized for future extended functional and population studies as well as for translational research of the nominated variants aiming at characterization of biomarkers and/or novel therapeutic targets.The present study confirms and further extends strong association of The study was approved by the ethical committee of the University Hospital, Olomouc; University Medical Center, Freiburg; Medical School University, Athens; and University Hospital, Paris. After approval of the study by ethical committee, the consent of human participants visiting the centres was taken in written for usage of biological sample for research purpose in future.MP, GZ, JM-Q, JP, VK, DB, and DV conceived and designed the work; AK, VZ, LK, JP, EB, HN, VL, GZ, VK, and MP contributed to data acquisition; AK performed data analysis and interpretation; AK and MP drafted the manuscript after its revision for important intellectual context by all authors; MP and AK finalized the article. All authors have read and approved the final manuscript and agreed to be accountable for all aspects of the work.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.


Biological functions are carried out by groups of interacting molecules, cells or tissues, known as communities. Membership in these communities may overlap when biological components are involved in multiple functions. However, traditional clustering methods detect non-overlapping communities. These detected communities may also be unstable and difficult to replicate, because traditional methods are sensitive to noise and parameter settings. These aspects of traditional clustering methods limit our ability to detect biological communities, and therefore our ability to understand biological functions. To address these limitations and detect robust overlapping biological communities, we propose an unorthodox clustering method called SpeakEasy which identifies communities using top-down and bottom-up approaches simultaneously. Specifically, nodes join communities based on their local connections, as well as global information about the network structure. This method can quantify the stability of each community, automatically identify the number of communities, and quickly cluster networks with hundreds of thousands of nodes. SpeakEasy shows top performance on synthetic clustering benchmarks and accurately identifies meaningful biological communities in a range of datasets, including: gene microarrays, protein interactions, sorted cell populations, electrophysiology and fMRI brain imaging. Molecules, cells and tissues carry out biological processes through physical interaction networks124568310111213101516182021a priori community titles. In this context, labels are unique bits of information that are assigned randomly and used to track cluster membership. SpeakEasy differs from previous label propagation algorithms, because nodes update their labels on the basis of their neighbors’ labels, while subtracting the expected frequency of these labels, based on their popularity in the complete network. This process combines a bottom-up approach to clustering (using neighboring information) with a top-down approach (using information from the whole network). This dual approach facilitates accurate community detection in many types of biological networks  clusters in biological networks. SpeakEasy is related to earlier label propagation algorithms2324networks  because not require manual tuning of clustering parameters for good results. Second, it can cluster networks with any type of links  or any type of network structure . SpeakEasy is highly scalable and can quickly cluster networks with hundreds of thousands of nodes. Third, because it is very efficient, the stochastic clustering process can be repeated many times to detect robust clusters that are not generated by data artifacts or noise. The repeated clustering process also allows SpeakEasy to identify multi-community nodes, whose membership tends to oscillate between different clusters. Finally, users can select overlapping or non-overlapping output, as is appropriate for their applications.In addition to accurate cluster detection (see Results section), community detection via SpeakEasy has several practical advantages for biological applications. For instance, since the number of communities in a dataset is rarely known in advance, SpeakEasy automatically predicts the number of communities and does For an intuitive example of how SpeakEasy identifies communities, we illustrate the clustering process on a demonstration network . This neWe use three approaches to determine the accuracy of SpeakEasy community detection. First, we test its performance on a large set of synthetic networks with carefully controlled characteristics, wherein the true clusters are known. Then we apply it to real-world networks, wherein the true clusters are unknown . In thisds)  benchmarks, which are widely used to test overlapping and non-overlapping clustering methodsds) . SpeakEa 25303132. Additio 25303132. Thus, Sm). When multi-community nodes are tied to many communities , community detection becomes more difficult The LFR benchmarks accurately represent certain aspects of social and biological networks, but are limited in other aspects. For example, networks in the LFR benchmarks have low transitivity and null assortativity (propensity for hubs to connect to hubs)ds metric that was shown to be more consistent with other metrics than original Q metric38ds values from SpeakEasy  indicate that it tends to detect more small and highly dense clusters than GANXiSds scores for the two biological networks in this test set (‘dolphins’ and ‘c.elegans’). These modularity values are approach those of methods that directly attempt to maximize modularityWe compare modularity values from SpeakEasy to those from another label propagation algorithm, GANXiS, because that method showed the best overlapping clustering performance in a recent comparison of clustering methodset al.et al.42433233Because a single protein may be part of more than one protein complex (set of bound proteins that work as a unit), Discovery of protein complexes directly benefits from development of methods which detect overlapping communities. We test SpeakEasy community detection of overlapping protein complexes, using two well-studied high-throughput protein interaction networks 46We find that applying SpeakEasy once again, to each of these broad categories of cell types, identifies sub-communities with higher correspondence to the tissue of origin and cell type, considered together . Thus, sSeveral cellular or molecular processes can generate correlated gene expression , including cell-type variation, transcription factors, epigenetic or chromosome configuration21Therefore, we use SpeakEasy to detect overlapping and non-overlapping coexpressed gene sets in two datasets that are commonly used to address many biological questions: The Human Brain Atlas (HBA)We also generate overlapping clusters from both the HBA and CCLE datasets. Overlapping coexpressed gene sets may be useful in biological studies because gene coexpression is driven by overlapping mechanismsExtracellular neuronal recording with single electrodes, tetrodes, or high density multichannel electrode arrays can detect the activity of multiple nearby neurons. However, these combined responses must be separated into responses of specific neurons. This blind source separation process is known as “spike sorting”, because each spike is assigned to a particular theorized neuron. Single neurons often generate relatively unique signatures (i.e. spike waveform shapes and amplitude distributions on multiple adjacent electrodes), and emerge as clusters in the matrix of waveform correlations.To realistically test spike sorting, it is important to match noise levels in real brain recordings. Therefore, we use real depth-electrode recordings generate a simulated time-series of spikes in which the true spike times and unique neuronal sources are known see . CompariFunctional magnetic resonance imaging (fMRI), obtained while a subject is at rest (rs-fMRI), is a valuable tool in understanding of systems-level changes in a variety of domains, including neurodegenerative diseaseCommunities identified in control and PD groups contain biologically similar sets of brain regions  from repeated runs with different initial conditions. The ability to generate many partitions is useful because it can quantify the stability of each cluster . It is a1858How to cite this article: Gaiteri, C. et al. Identifying robust communities and multi-community nodes by combining top-down and bottom-up approaches to clustering. Sci. Rep.5, 16361; doi: 10.1038/srep16361 (2015).


Our novel direct synthesis of graphene/TiO2 heterostructure is achieved by C60 deposition on transition Ti metal surface using a molecular beam epitaxy approach and O2 intercalation method, which is compatible with wafer scale growth of heterostructures. As-grown heterostructures exhibit inherent photosensitivity in the visible light spectrum with high photo responsivity. The photo sensitivity is 25 times higher than that of reported graphene photo detectors. The improved responsivity is attributed to optical transitions between O 2p orbitals in the valence band of TiO2 and C 2p orbitals in the conduction band of graphene enabled by Coulomb interactions at the interface. In addition, this heterostructure provides a platform for realization of bottom gated graphene field effect devices with graphene and TiO2 playing the roles of channel and gate dielectric layers, respectively.Heterostructures consisting of two-dimensional materials have shown new physical phenomena, novel electronic and optical properties, and new device concepts not observed in bulk material systems or purely three dimensional heterostructures. These new effects originated mostly from the van der Waals interaction between the different layers. Here we report that a new optical and electronic device platform can be provided by heterostructures of 2D graphene with a metal oxide (TiO In graphene field effect transistors (GFET), graphene provides the active layer where the channel carriers are modulated by gate biasing through the dielectric layer. In graphene optical devices a dielectric layer is needed to induce a built-in potential within the structure so that photo-excited electrons can be extracted from the photosensitive materials into the graphene layer on top. Currently the photosensitive 2D materials such as MoS2 or WS2 are widely investigated to enhance photo-responsibility. Recent studies of graphene/photosensitive 2D heterostructures have revealed new physical phenomena such as Moire patterns582/Gr heterostructure resulted from Van Hove singularities2 has been considered since it is a well-known material that has been widely used for light harvesting2 a very attractive material for the realization of both electronic and photonic devices. The schematic diagram of our growth approach is shown in 2 dielectric layer. We demonstrate this dual functionality by depositing two top metal contacts on the Gr/TiO2 heterostructure to serve as source and drain electrodes. The gating voltage is applied through the underlying TiO2 on n-type SiC in a bottom gated FET configuration. With the same device configuration, the photosensitivity under visible light illumination is measured by the change of photoconductivity with zero gate voltage. We believe that the observed photo response at zero gate voltage is caused by the optical transitions between the valence band maximum, associated O 2p orbitals in TiO2, and the conduction band minima, formed by the C 2p orbitals of the graphene in the Gr/TiO2 heterostructure. The schematic diagrams of device and measurement configurations are shown in 2 heterostructure have been conducted in air.Graphene (Gr) is a very attractive material for diverse optoelectronic and electronic device applications due to its extraordinary transport properties, broad spectral-bandwidth, and fast response time12360 flux dependence were studied. The growth temperature was kept at 1400 °C during these experiments. First, the C60 flux was held at 1.6 × 10−7 Torr while the growth time varied. As growth time increased, the 2D peak position shifted linearly to lower wavenumber. After 30 min. growth, the 2D peak was located at ~2712 ± 5 cm−1. With longer growth time (90min.), it shifted to 2705 ± 1.5 cm−1. The FWHM of the 2D peak showed similar behavior to peak position. The value of the FHWM decreased from 71 ± 9 cm−1 (30 min. growth) to 56.5 ± 2 cm−1 (90 min. growth). The quality of the graphene was estimated from the ratio of the D and G peaks. The growth time dependence of the D/G ratio also exhibited similar behavior to position and FWHM of the 2D peak. Increased growth time resulted in a decrease of the D/G ratio from 0.32 ± 0.07 (30 min. growth) to 0.23 ± 0.01 (90 min. growth). The C60 flux dependence with two different fluxes was also evaluated. The growth was done at 1400 °C for 2 hr. With increasing flux from 0.8 × 10−7 to 1.6 × 10−7 Torr, we observed improvement of graphene quality with a decrease of the D/G ratio and 2D FWHM by 40% and 20%, respectively. The peak position also shifts to lower wavenumber from 2706 cm−1 to 2703 cm−1. Next, the effect of O2 intercalation at 500 °C at various times was studied with Raman analysis. The 2D peak position shifted slightly to lower wavenumber. A 4hr. intercalation led a 2D peak shift of only 5 cm−1, to lower wavenumber. The 2D FWHM showed entirely different behavior. Initially it broadened compared to that of as-grown graphene  and then started to narrow . The graphene quality variation was estimated by comparing the D/G ratio before and after intercalation. We did not observed any variation of the ratio for intercalation up to 60 min. but a significant increase was observed beyond 60 min.To investigate the graphene growth on Ti metal surfaces, the growth time and C2 surfaces. 60 deposition on both TiO2 (red color curve) and Ti (blue color curve). As seen in 3/2 and Ti 2p1/2 peaks for TiO2 are located at 459.1 eV and 464.8 eV respectively. However, our results  shows no O 1s peak corresponding to TiO2. Therefore, we conclude that C60 deposition results in graphene and TiC formation on both Ti and TiO2. The formation of TiC upon C60 deposition could be explained by a carbon thermal reduction reaction2 reacts with carbon to form TiC and releases CO gas above 1300 °C. The high substrate temperature (1400 °C) during C60 deposition could initiate carbon thermal reduction reactions between molecular C60 and the TiO2 surface.Graphene growth was attempted on both Ti and TiO results  show the2 has been obtained from oxidation of TiC in the temperature range from 623 K to 1073 K in air2 was accomplished with an O2 intercalation procedure. Intercalation was conducted under controlled O2 pressure (10−4 Torr) in the XPS chamber (details in Method). 2 intercalation progressed. Besides peaks for as-grown graphene at 284.7eV and TiC at 281.5 eV, three peaks associated with C-O (286.1 eV), C = O (288.1 eV), and the π → π* (291.5 eV) transition were observed in the C 1s spectra (not shown here). Since oxygen-carbon bonding was observed in the as-grown sample as well, the existence of C and O bonding might be due to air exposure during transfer from the growth chamber to the XPS chamber. In as-grown graphene/Ti structure, graphene peak is located at 284.7 eV with ~1.2 eV FWHM. However, the shift of peak position to 284.2 eV and the FHWM narrowing from 1.2 eV to 1 eV were observed as intercalation proceeds. We attribute the shift in binding energy to substrate interactions between the Ti and TiO2. The conversion of TiC into TiO2 during intercalation is shown in 4+ cation doublet located at 458.8 eV and 464.6 eV and the metallic type bonding (titanium carbide) Ti0 peaks located at 454.7 eV and 460.9 eV were observed. These peaks are associated with Ti 2p3/2 and Ti 2p1/2 transitions, respectively. The evolution of chemical composition at different temperatures and intercalation times is also shown in 4+ and Ti3+ along with TiC peaks  and its peak intensity increases with further intercalation. We calculated the activation energy for TiO2 conversion by O2 intercalation and graphene etching by the O2 (detail in Method). The calculated activation energy for TiO2 conversion is about 0.29 eV while activation for graphene etching is 0.41 eV, which indicate that TiO2 conversion is the dominant reaction in our intercalation experiments.Intercalation between epitaxial graphene and its substrate has been widely studied as a method of decoupling the strong interaction from the substrate14151660 deposition on Ti metal and O2 intercalation were also investigated with Raman measurements. 2 exposure. In the as-grown sample, Raman measurements show the typical Raman fingerprint of graphene with the presence of the 2D, G, and D peaks. Importantly, a fit to the 2D peak gave a single Lorentizan at ~2700 cm−1. Typically, C60 deposition on SiC resulted in Bernal stacked few layer graphene−1. This suggests that graphene on Ti metal is not Bernal stacked but is very similar to turbostractic stacking of graphene2 intercalation (2 layer (Eg = 3.2 eV) after O2 intercalation enhances the transmittance of visible incident Raman laser into SiC substrate. It results in Raman activation of SiC substrate and convolution of SiC and graphene Raman spectrum. The formation of TiO2 after intercalation was confirmed by observation of TiO2 vibration modes , an A1g and B1g mode doublet at 516 cm−1, and a B1g mode at 399 cm−12, A1, and E1 modes (black colored annotations), while two distinct TiO2 related Raman peaks (orange color annotations) were observed after O2 intercalation. The peaks at ~143 cm−1 and ~640 cm−1 are attributed to the Eg mode (O-Ti-O bending vibration) of anatase TiO2.The Con modes . TiO2 ha2/n-SiC heterostructures we fabricated bottom gated FET devices. Ti/Au contacts were deposited for source and drain contacts on top of the 1 cm × 1 cm samples.  The channel length was about 6 mm. The schematic diagram of GFET structure is shown in 2. The gate response was measured for a gate voltage range of ±20 V and is shown in g ≈ +3 V, which indicates that the intercalated graphene exhibits hole doping. d ≈ 2.9 mA at Vg = 15 V and Vd = 0.5 V). These results might be due a high capacitance due to the TiO2 dielectric layer.To explore the electrical properties of our Gr/TiO2 has been widely used as photosensitive material. However, its large bandgap (3.2 eV) has restricted its use to ultraviolet applications since the photo response is only activated under ultraviolet radiation. Several approaches have been proposed to extend its photo response to a broader range such as dye sensitization, doping, and coupling with semiconductors23242/n-SiC heterostructures at room temperature (RT). The schematic diagram of measurement is shown in −1 conductance. Upon illumination, the device showed an increased current flow through the source-drain contacts with increased current flow at high bias voltage. The conductance of the graphene improved to 0.06 Ω−1, indicating a visible light photoresponse in our heterostructure. The calculated photo-responsivity of the heterostructure is about 278 mAW−1, which is much higher than that reported for modified graphene photo-detectors such as graphene metal junctions (6.1 mAW−1)−1)−1)g = −20 V, the graphene photoconductivity resulted in a conductance of 0.052 Ω−1 while it increased to 0.06 Ω−1 at 20 V. The difference between negative and positive gate voltage might be related to variations of net current flow through the channel. The response to turning the light on and off was measured under 0.2 V bias between source-drain as shown in −1, which is lower than that of our photo detector. Since the large bandgap (3.2 eV) of TiO2 does not allow for a photo-response to visible light, the photo-response observed here might originate from the Gr/TiO2 heterostructure. A possible mechanism for the enhanced visible light photoresponse seen in our Gr/TiO2 heterostructures may be provided by Geng, Liu and Yao2/graphene composites they calculated the electronic properties of atomic scale TiO2 clusters on graphene films and predicted anchoring of the clusters by charge rearrangement at the interface due to an interaction between the Ti 3d orbitals of the TiO2 and the p orbital of the graphene sheet. This results in extra states in the bandgap of TiO2 which enable absorption of visible light by the composite and the transfer of photoexcited electrons from the TiO2 valence band to the graphene conduction band. The subsequent separation of electrons and holes in graphene and TiO2 leads to long carrier lifetimes. In addition, since the interface between TiO2 and graphene plays a critical role, they also suggested that increasing the interface contact area could improve the photo response. We speculate that a similar process may be taking place in our comparatively large area heterostructures. The absorption of light takes place primarily in the TiO2 closest to the interface where their density of states is modified by the presence of the graphene layer. Electrons then transfer to the graphene layer and increase the electron density. The separation of electrons and holes reduces recombination and the holes in the TiO2 can screen the gating effect under positive bias while inducing more holes in the graphene layer under negative bias, leading to a reduced photoconductivity at negative bias. This model explains our experimental results shown in the gate response characteristics  with a metal carbide interface layer (TiC). After O2 intercalation the byproduct TiC layer was completely converted into TiO2, which resulted in a graphene/oxide heterostructure. We showed that this heterostructure exhibits dual functional properties, showing both electronic field effect phenomena and an enhanced visible light photosensitity. The field effect phenomena and enhanced photosensitivity were demonstrated in bottom gated GFET devices.We demonstrated that C60 molecules were deposited on the Ti layers in a high temperature molecular beam epitaxy system. The growth temperature and carbon flux were 1400 °C and 1.6 × 10−7 Torr. The growth time was 30 min. For the study of graphene growth on TiO2 surfaces, the as-deposited Ti/n-SiC sample was annealed in an O2 environment at 800 °C for 1 hr. The oxidation of the metallic Ti layer was confirmed by XPS analysis. The graphene growth was performed with the same growth conditions as those for Ti/n-SiC. After graphene growth, the sample was exposed to air while being transferred to the XPS chamber for O2 intercalation experiments.Metallic titanium layers (20 nm) were grown on an n-type SiC wafers by e-beam evaporation. CFor the field effect transistor device tests, Au/Ti metal contacts (source and drain) were deposited on the graphene surface. The photo response experiment was performed by illumination with a white LED at room temperature.2 was estimated by high resolution XPS spectra. The rate constant (k) for TiO2 formation due to intercalation was obtained by analyzing the temporal change in the ratio of TiC to TiO2 atomic concentrations at each intercalation temperature. By fitting an Arrhenius plot (Log (k) vs. 1/T), the activation energy (Eg) for oxidation was calculated. The graphene etching effect during O2 intercalation was monitored by the reduction of the graphene C 1s peak (284.7 eV) intensity. The etching activation energy was also calculated by estimating rate constant (k) and fitting the Arrhenius plot (Log (k) vs. 1/T). The temporal variation of C 1s ratio between as-grown and intercalated samples was used to calculate the rate constant (k) at each intercalation temperature.The temporal change in atomic concentration of TiC and TiO−8 Torr the sample was transferred to the analysis chamber. After initial XPS scans were acquired the sample was transferred back into the load-lock chamber for O2 intercalation. For the intercalation experiments research grade O2 was introduced into the chamber with a leak valve. The background pressure in the chamber was maintained at 1.0 × 10−4 Torr. Once the desired pressure was reached the sample was heated from the backside of the sample holder with a 10.6 μm CO2 IR laser. The laser output power was adjusted until the target temperature was reached. The temperature of the sample was measured with a pyrometer. After the intercalation the chamber was pumped down until a background pressure 3.0 × 10−8 Torr was reached. The sample was then transferred back into the analysis chamber for XPS data acquisiton.After graphene growth, the sample was transferred to the load-lock of the XPS system. When the background pressure of the load lock chamber reached 3.0 × 107/2 (84.0 eV) peak with an overall resolution of ~1 eV. Each spectrum was fit with a Shirley background followed by fitting convoluted Gaussian and Lorentzian peak shapes for each chemical state in the region of interest. For the C 1s graphene and Ti 2p metal peaks an additional asymetry parameter was added to the Gaussian-Lorentzian convolution. The fitting procedure consisted of a Levenberg-Marquardt routine that minimizes χ2.The XPS data was acquired with an Scienta Omicron standard Mg Kα (1253.6 eV) X-ray source and Argus hemispherical analyzer. High resolution scans of the C 1s, O 1s, and Ti 2p regions were acquired at 20 eV pass energy with 0.1 eV steps. The binding energy scale was calibrated against the Au 4fHow to cite this article: Park, J. et al. Approach to multifunctional device platform with epitaxial graphene on transition metal oxide. Sci. Rep.5, 14374; doi: 10.1038/srep14374 (2015).


This study focused on a collection of strains representing Ophiostoma piliferum, Ophiostoma pluriannulatum and related species that cause blue-stain; these fungi colonize the sapwood of trees and impart a dark stain. This reduces the value of the lumber. The goal was to examine the mtDNA rns intron landscape for these important blue stain fungi in order to facilitate future annotation of mitochondrial genomes (mtDNA) and to potentially identify mtDNA introns that can encode homing endonucleases which may have applications in biotechnology.Based on previous studies, it was suspected that the mitochondrial Comparative sequence analysis identified five intron insertion sites among the ophiostomatoid fungi examined. Positions mS379 and mS952 harbor group II introns, the mS379 intron encodes a reverse transcriptase, and the mS952 intron encodes a potential homing endonuclease. Positions mS569, mS1224, and mS1247 have group I introns inserted and these encode intact or eroded homing endonuclease open reading frames (ORF). Phylogenetic analysis of the intron ORFs showed that they can be found in the same insertion site in closely and distantly related species.rns introns), strains representing O. pilifera, O. pluriannulatum and Ophiostoma novae-zelandiae could not be resolved. Phylogenetic studies suggest that introns are gained and lost and that horizontal transfer could explain the presence of related intron in distantly related fungi. With regard to the mS379 group II intron, this study shows that mitochondrial group II introns and their reverse transcriptases may also follow the life cycle previously proposed for group I introns and their homing endonucleases. This consists of intron invasion, decay of intron ORF, loss of intron, and possible reinvasion.Based on the molecular markers examined  contains supplementary material, which is available to authorized users. Syd.] and some are plant pathogens , there is evidence that an rns GII intron could be associated with inducing hypovirulence . An Erlenmeyer flask containing fifty ml of PYG was inoculated with agar blocks (~1 × 1 mm) derived from an agar plate culture and the liquid cultures were incubated in the dark at 20 °C for 4–6 days. The protocol used for DNA extraction was previously described by Hausner et al. (2EDTA·2H2O (pH 8.0), 1.5 M NaCl, 2 % (w/v) cetyltrimethylammonium bromide (CTAB)] was added along with 3 g of glass beads and 660 μl of 20 % (w/v) sodium dodecyl sulfate (SDS). This mixture was vortexed for about 2 min. Thereafter, the lysate was incubated at 55 °C for 2 h. Cell debris and contaminants were removed by chloroform extraction and centrifuging for 20 min at 2000 rpm. The top aqueous layer was recovered and 2.5 volume of ice cold 95 % ethanol was added to precipitate the nucleic acids. Finally, the nucleic acids were recovered by centrifuging for 30 min at 3000 rpm. The DNA pellets were washed with 1 ml of 70 % ethanol and resuspended in 300 µl TE buffer [10 mM Tris–HCl (pH 7.6) and 1 mM Na2EDTA·2H2O (pH 8.0)].Fungal strains examined in this study are listed in Table r et al. . Brieflyrns sequences were previously described by Hausner and Wang . Automated fluorescent DNA sequence analysis was performed by the Manitoba Institute of Cell Biology DNA Sequencing Facility .Primers and conditions for the polymerase chain reaction (PCR) for obtaining the internal transcribed spacer (ITS) regions and mtDNA and Wang  and Hafeand Wang , respectand Wang . Primers Hausner  and Hafe Hausner ; additio Hausner . All PCRhttp://doua.prabi.fr/software/cap3). The resulting contigs were used as templates for designing new primers. Once completed, the rns sequences were applied to the basic local alignment search tool  to identify similar sequences. Sequences were compiled and aligned by the online multiple sequence alignment program, MAFFT . The literature was also consulted with regard to comparing introns identified in this study with introns and their putative folds, characterized in other studies  were also consulted. Intron sequences  were applied to RNAweasel  alignments were analyzed with three tree building methods  and the bootstrap option was implemented (2000 replicates) in order to assess the level of support for the tree topologies generated by the respective methods. With regard to NJ analysis, the maximum composite likelihood method and its defaults were selected along with the complete deletion of gaps option. For the PARS method, the complete deletion option was also selected. In the ML method, the best model was first determined with the “best model” function as implemented in MEGA. Therefore, for ML analysis, the Whelan and Goldman plus Freq. model was selected and the complete deletion option was selected to remove segments of the alignments that contained gaps.rns gene among the strains of Ophiostoma examined in this study. Based on previous reports (Hafez and Hausner rns genes that do not have insertions and rns derived amplicons greater than 1.2 kb have insertions (introns). Among the 23 strains surveyed, only the following strains, based on the size of rns-derived PCR amplicons, appear to have insertions within the rns gene: Ophiostoma californicum WIN(M)505 , Ophiostoma pluriannulatum WIN(M)539 [(Hedgc.) Syd. & P. Syd.], Ophiostoma carpenteri WIN(M)855 (J. Reid & Georg Hausner), Ophiostoma perfectum WIN(M)823 [(R.W. Davidson) de Hoog], and O. piliferum WIN(M)972. The sizes of the amplicons were as follows: O. perfectum WIN(M)823 yielded a 7 kb PCR amplicon while O. pluriannulatum WIN(M)539, O. piliferum WIN(M)972, O. californicum WIN(M)505, and O. carpenteri WIN(M)855 yielded rns-derived PCR amplicons of about 5 kb 823 contains three introns at the following positions 972 and O. californicum WIN(M)505 have introns inserted at positions mS952 and mS1224. O. carpenteri WIN(M)855 has one intron at position mS952. O. pluriannulatum WIN(M)539 also has an intron at mS952 in addition to an intron inserted at mS569.As summarized in Fig. Ophiostoma species . Sequences for strains representing Leptographium and Grosmannia were included as outgroups. All Ophiostoma species were clearly separated from the outgroup species. The taxonomy of O. piliferum is a complex issue 823 revealed to be the most complex with regard to intron arrangements  Georg Hausner, J. Reid & Klassen] with strong bootstrap support and both of these sequences are related to the mS379 ORFs from the Ophiostoma minus [(Hedgc.) Syd. & P. Syd.] rns genes. The phylogenetic tree also shows that mS379 RT ORFs are related to an RT ORF that exists in the cox1 gene of Podospora anserina. Although, at lower node support values, basal to the above grouping, are additional RT ORFs mostly encoded within cox1 introns with the exception of the Nitella hyalina rns intron-encoded RT ORF. A node that received high bootstrap support (94 %) suggests monophyly for RT ORFs encoded within bacterial genomes such as Pseudomonas syringae, Pseudomonas putida, Pseudomonas aeruginosa, Ktedonobacter racemifer, and the cox1 and cox3 intron-encoded RT ORFs from various fungi, brown algae  and liverwort (Marchantia polymorpha) along with the fungal mS379 ORFs.The ORF Finder program identified an intact GIIA RT ORF 510 aa) in the 0 aa in tO. perfectum WIN(M)823 and O. pluriannulatum WIN(M)539, showed that the ORF is degraded in O. perfectum due to some frame shift mutations but otherwise nearly identical to the intact sequence found in O. pluriannulatum. A data set was assembled by extracting related sequences from GenBank depending on the O. perfectum WIN(M)823 sequence data as a query. The alignment was analyzed with three programs contained within MEGA6 . Peltigera malacea was used as an outgroup  and O. pluriannulatum WIN(M)539 (286 aa) and for O. californicum WIN(M)505 (323 aa) but for O. pluriannulatum WIN(972) (173 aa) the ORF only has one LAGLIDADG domain. The latter single domain ORF may be an indication that this ORF has eroded due to mutations that generated premature stop codons. The phylogenetic tree shows that the O. carpenteri GII intron-encoded LHE ORF (representing the other mS952 ORFs uncovered during this study) is found in a variety of fungi including several members of the Ophiostomatales: Ophiostoma stenoceras [(Robak) Melin & Nannf.], Ophiostoma brevicolle [(R.W. Davidson) de Hoog & R.J. Scheff.], O. torulosum, Leptographium species and O. minus. A LHE sequence from the nad5 i2 ORF from Rhizoctonia solani was used as the outgroup 505 (319aa) and O. piliferum WIN(M)972 (441aa) suggests that these ORFs are closely related the ms1224 intron ORFs that exists in Ophiocordyceps sobolifera [AB027350], Agrocybe aegerita [AAB50391] and Leptosphaeria maculans [FP929115]. The clade is supported with high node support values. The rns i4 intron (=mS1210) ORF from C. parasitica joins the above clade at a node that received only moderate support (72 %). The remaining nodes representing deeper branching patterns within this tree received only poor support values and thus, distant relationships cannot be extracted from this analysis.The mS952 GII intron LHE ORF from rpenteri 22 aa andO. perfectum (rns i3 ORF) as a query in blastp. The Amoebidium parasiticum rnl-i2 ORF was used as the outgroup in this analysis  due to frameshift mutations but 418 aa could be assembled. The O. perfectum mS1247 ORF grouped with mS1247 ORFs from O. sobolifera [AB027350], L. maculans [FP929115] and Chaetomium thermophilum [JX139037]. However, the node support values are moderate for the clade which included all four mS1247 ORF sequences; but the node linking the O. sobolifera with O. perfectum received strong support (99 %). With regard to deeper nodes, the phylogenetic tree received poor node support values so no further conclusions could be reached as to possible relationships of the mS1247 with other LAGLIDADG type ORFs.Another data set was constructed by using the mS1247 intron ORF sequence from Ophiostoma species sampled in this study, many lacked introns within the rns gene, but a few members did contain introns . This is suggestive of either rapid gain or rapid loss of introns. Gain of introns could be achieved by horizontal transfer or by vertical transmission of these introns, random loss of introns could be achieved by the original intron containing allele being replaced by a cDNA version of the gene  due to the presence/absence of introns and associated ORFs. Also, introns appear to be inserted at the same positions among related and distantly related fungi, which is a reflection of the homing mechanism promoted by the intron-encoded homing endonucleases. Homing endonucleases recognize long target sites and therefore, require conserved regions for their long term survival and for moving laterally from one species to another 832 rns gene is 7 kb in length and has three introns inserted in highly conserved regions within the rns gene. The mS379 intron is a GII intron, type AI, that was previously reported in three O. minus strains by Hafez and Hausner (O. perfectum WIN(M)823 intron at mS379 encodes a complete RT ORF that is inserted into domain II of the GII intron. The latter is unusual as organellar GII intron ORFs are typically located in domain IV , a life cycle of invasion and degeneration for HEGs and their hosting GI introns. Our study suggests that RT ORFs in GII introns can follow a similar life cycle; i.e. invasion into an empty spot, followed by slow degeneration initially of the ORF, and eventually complete loss of the ORF, and presumably, complete loss of the intron, regenerating a potential site available for reinvasion.The analysis of sequence data has shown that the  Hausner . In thesand Burt  predicteOphiostoma mS379 RT ORF within a grouping that includes sequences from brown algae, bacteria, plants and fungi. Thus, it appears this intron ORF has a complex evolutionary history that could include horizontal transfers. It has been shown that GII intron ORFs probably co-evolve with their host introns but these composite elements can evolve independently from the host genomes that encode them due to lateral transfers 823 is the mS569 GI intron, type C2, with a LHE ORF. This intron is also found in O. pluriannulatum WIN(M)539. Hafez et al. 855, O. pluriannulatum WIN(M)927, O. subannulatum WIN(M)539, and O. californicum WIN(M)505. This GII intron was previously found in other Ophiostoma species by Mullineux et al. 972 and O. californicum WIN(M)505 have a GI intron, type C2, at position S1224 and a LHE as an IEP. This intron and ORF are also found in rather distantly related fungi, L. maculans, O. sobolifera, A. aegerita and C. parasitica 823 is located at position mS1247. The mS1247 intron is a GI, type C2, encoding a LHE ORF. So far within Ophiostoma, the mS1247 intron has only been observed in O. perfectum. Hafez et al.  and GII introns . Among intron-encoded ORFs, the LAGLIDADG family appears to have invaded four of these introns, with the mS379 GII intron maintaining an RT ORF. Intron landscapes provide information for those that annotate mtDNAs or for those searching for sources of polymorphisms. In addition, ribozymes and intron-encoded ORFs (HEases and RTs) have applications in biotechnology, genome editing, or as functional genomics tools (Stoddard With regard to the Stoddard . Intron Stoddard .Phylogenetic investigations suggest that the intron ORFs evolve rapidly and thus, most trees had poorly supported topologies. In general, among members of the Ophiostomatales, introns appear to be gained and lost frequently. The intron ORFs tree topologies do not follow the expected host species/genome trees, which hints at the possibilities of horizontal transfers of these elements among distantly related lines. Movement of introns along with their IEPs is facilitated by their encoded proteins that target specific sites in cognate intronless alleles (Colleaux et al.


Fungal secretome consists of various functional groups of proteins, many of which participate in nutrient acquisition, self-protection, or manipulation of the environment and neighboring organisms. The least characterized component of the secretome is small secreted proteins (SSPs). Some SSPs have been reported to function as effectors, but most remain to be characterized. The composition of major secretome components, such as carbohydrate-active enzymes, proteases, lipases, and oxidoreductases, appear to reflect the lifestyle and ecological niche of individual species. We hypothesize that many SSPs participate in manipulating plants as effectors. Obligate biotrophs likely encode more and diverse effector-like SSPs to suppress host defense compared to necrotrophs, which generally use cell wall degrading enzymes and phytotoxins to kill hosts. Because different secretome prediction workflows have been used in different studies, available secretome data are difficult to integrate for comprehensive comparative studies to test this hypothesis. In this study, SSPs encoded by 136 fungal species were identified from data archived in Fungal Secretome Database (FSD) via a refined secretome workflow. Subsequently, compositions of SSPs and other secretome components were compared in light of taxa and lifestyles. Those species that are intimately associated with host cells, such as biotrophs and symbionts, usually have higher proportion of species-specific SSPs (SSSPs) than hemibiotrophs and necrotrophs, but the latter groups displayed higher proportions of secreted enzymes. Results from our study established a foundation for functional studies on SSPs and will also help understand genomic changes potentially underpinning different fungal lifestyles. R)-gene mediated defense as avirulence factors  rich in cysteine residues; and (e) presence of a conserved motif within effector candidates like in oomycete and fungal effectors  with individual refined secretomes as queries against BLAST databases of these enzyme sets. Fungal oxidoreductases were downloaded from Fungal Peroxidase Database  and conserved SSPs (CSSPs) were annotated using pre-computed InterPro terms, which were retrieved from Comparative Fungal Genomics Platform 2.0  clustering. Resulting data were clustered via MCL with the inflation option 1.4 for high granularity  to 1940  and the highest being 11.0% . Both P. jirovecii and M. oryzae belong to the phylum Ascomycota, illustrating high degrees of variance within individual phyla  and one Ascomycota species (P. jirovecii) displayed large proportions  and a saprotroph (Kluyveromyces lactis), did not have any SSSPs. The remaining SSPs were encoded by at least two species and were termed conserved SSPs (CSSPs), which may correspond to general fungal proteins or elicitors.Effector-like SSPs were divided into two classes. Proteins of 300 aa or shorter that appeared to be encoded only by one species were considered species-specific SSPs (SSSPs); a total of 8275 SSSPs were found from 133 species. The fasta formatted SSSP sequences available in Supplementary Data Sheet InterPro domain analysis was performed to predict potential functions of both SSSPs and CSSPs. Most SSPs, 7960 out of 8275 (96.2%), displayed no defined InterPro terms with only 315 SSPs being annotated with 97 different terms , one biotroph (Mixia osmundae), and one saprotroph  had higher AT-contents at 52, 72, 44, and 60%, respectively  and B. graminis (Ascomycota) have the highest numbers. In contrast, two species in Ustilagomycotina (Ustilago maydis and Sporisorium reilianum) and three Ascomycota species  encode small numbers of SSSPs, similar to those in saprotrophs. In addition, wood-decaying necrotrophs and hemibiotrophs have similar numbers of SSSPs.The number of SSSPs within species ranged from 0 to 466. Although not all plant-associated fungi have higher numbers of SSSPs than saprotrophs and animal pathogens Figure , in geneU. maydis and S. reilianum) contained greater numbers of SSSPs compared to other members. In Ascomycota, the range was widest in Pezizomycotina with many outliers being present at both sides of the mean. Three hemibiotrophs, including M. oryzae, Leptosphaeria maculans, and Mycosphaerella graminicola, one nectrotroph Stagonospora nodorum, one biotroph B. graminis, and one saprotroph Pyronema confluens encode much larger SSSPs than the mean. Saccharomycotina and Taphrinomycotina encode noticeably smaller SSSPs than Pezizomycotina.The range of SSSPs numbers was also analyzed with regard to taxonomic positions Figure . The meaT. deformans, the only biotroph in Taphrinomycotina, also shows a high number of SSSPs. However, A. gossypii, the only biotroph in Saccharomycotina, has a lower number and proportion of SSSPs compared to other members  were conserved in 91 species, all belonging to Pezizomycotina or Agricomycotina (Figure Botrytis cinerea and Fusarium spp. have undergone duplication events. The wide distribution of the genes encoding cerato-platanin domain proteins in species with pathogenic lifestyles suggested that their products play an important role in pathogenesis. However, numbers of this group were also found in 4 symbionts, 2 nematophagous fungi, and 19 saprotrophs, suggesting other roles associated with them. The gene family identified using the B. graminis effector candidate BEC1040 has gone through 16 duplications and 12 losses, and its numbers were present in 50 species in Ascomycota and Punctularia strigosozonata in Basidiomycota (Supplementary Figure We predicted 13 families of CSSPs containing PHI-base effectors Table . Among ta Figure . In totaRapid progresses in sequencing fungal genomes, in combination with various “omics” tools, have facilitated large-scale comparative genomic analyses to uncover the genetic and evolutionary basis of various traits or functions of fundamental and practical significance. In this study, we developed a pipeline for mining SSPs as effector candidates from fungi with different lifestyles and taxonomic positions in order to conduct their kingdom-wide comparative analysis. It has been commonly hypothesized that biotrophs and symbionts secrete more effectors than necrotrophs, as biotrophic associations require the modulation of the host defense system to keep host cells alive for nutrient acquisition while preventing the launch of strong defense responses. Since necrotrophs utilize CAZymes and toxins to kill host cells to obtain nutrient, such manipulations of host defense likely play less critical roles. Because only a small number of fungal proteins in selected plant pathogens have been identified as effectors, we tested this hypothesis by comparing SSSPs as effector candidates.F. graminearum (Brown et al., S. reilianum and U. maydis are similar to those reported by Schirawski et al. (F. graminearum was compared to the data by Brown et al. (We refined secretomes to identify SSSPs as previously reported in analyzing the secretome of i et al. . The corn et al. , their 5n et al.  for kingM. oryzae is a zinc metalloprotease that acts as an avirulence factor in its host rice (Zhang and Xu, F. graminearum suppresses callose formation in wheat and is required for host infection (Blümke et al., The roles of fungal effector proteins regarding lifestyles are previously discussed by Lowe and Howlett  and Lo PL. bicolor (Martin et al., Tuber melanosporum (Martin et al., Neurospora crassa, a saprotrophic fungus that has been reported to be associated with pine trees in harsh conditions and even pathogenic (Kuo et al., In general, phytopathogenic fungi tend to have larger secretomes than non-pathogens. Although, Lowe and Howlett  suggesteC. fulvum and P. confluens. However, only the proteins secreted with canonical pathways were considered in this analysis, which means the minimum size of protein is bound to the length of signal peptide that is 15 to 40 aa (Choo et al., Another concern is that the number of small proteins could be affected by the minimum protein size used to annotate each genome. However, we strictly used the annotated data associated with published genomes, thus we can assume that the numbers of annotated proteins were comparable to each other. In addition, the majority of genome data were also from JGI and Broad Institute, which followed the conventional JGI annotation process and Broad Gene Finding Methods, respectively. If the minimum cutoff length for gene prediction was stated, only short peptides without EST support were eliminated. Since, the majority of fungal genome studies did not show the cutoff size for protein-coding gene prediction, we analyzed and compared the length distribution of the annotated proteomes for validation (the last column of Supplementary Table C. fulvum effector Ecp2 (Stergiopoulos et al., B. graminis, e.g., BEC1040, BEC1019, and BEC1005, were found in fungi having different lifestyles other than biotroph. These observations were further supported by the facts that these proteins are involved in fungal development, and resemble metalloprotease and glucanase, respectively (Pliego et al., Many genes coding candidate effectors in plant pathogens and virulence-associated genes of animal pathogens have been reported to reside in gene-sparse, AT-rich, and telomere-proximal genomic regions (Schmidt and Panstruga, In conclusion, different secretome components reflect lifestyle-associated genomic adaptations in fungi. Results from this comparative study provide new insights into the genetic basis and molecular evolution of fungal lifestyles and also establish a solid foundation for future discovery and functional validation of effectors.KK and YL designed this project. KK, JJ, HS and GC performed computational analyses. JC and KC provided the secretome and the genome data for analyses. KK, JJ, SK, and YL wrote the manuscript. SK and YL supervised the research. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Together, our data suggest that CDH23-C is a CAMSAP3/Marshalin-binding protein that can modify MT networks indirectly through its interaction with CAMSAP3/Marshalin.Cadherin-related 23 (CDH23) is an adhesive protein important for hearing and vision, while CAMSAP3/Marshalin is a microtubule (MT) minus-end binding protein that regulates MT networks. Although both CDH23 and CAMSAP3/Marshalin are expressed in the organ of Corti, and carry several protein-protein interaction domains, no functional connection between these two proteins has been proposed. In this report, we demonstrate that the C isoform of CDH23 (CDH23-C) directly binds to CAMSAP3/Marshalin and modifies its function by inhibiting CAMSAP3/Marshalin-induced bundle formation, a process that requires a tubulin-binding domain called CKK. We further identified a conserved N-terminal region of CDH23-C that binds to the CKK domain. This CKK binding motif (CBM) is adjacent to the domain that interacts with harmonin, a binding partner of CDH23 implicated in deafness. Because the human Usher Syndrome 1D-associated mutation, CDH23 R3175H, maps to the CBM, we created a matched mutation in mouse CDH23-C at R55H. Both  CDH23 is widely expressed in various tissues including brain, heart, lung, kidney, nose, eye, and ear1CDH23 is known to cause various degrees of hearing impairment and blindness, including Usher Syndrome 1D6CDH23, an atypical cadherin protein, belongs to the cadherin superfamily that plays a major role in cell adhesion. CDH23 isoforms  and minus end tracking proteins (−TIPs), each of which targets the corresponding end of the MT and assists in maintaining the dynamic nature of the cytoskeletal network. In contrast to +TIPs, for which many have been identified, only a few −TIPs have been reported. Previously, we identified Marshalin (KIAA1543), a MT minus-end binding protein, as a potential CDH23-associated protein through a membrane-based yeast two hybridization screeningin vitro pull-down assays and in vivo CAMSAP3/MT bundle formation methodologies. We demonstrate that the CKK domain of CAMSAP3/Marshalin binds to the N-terminus of CDH23-C, which we named the CKK binding motif (CBM). Interestingly, a missense mutation has been found within the CBM of CDH23 (R3175H) in Usher Syndrome 1D patientsIn this study we investigated the relationship between CAMSAP3/Marshalin and CDH23-C proteins, as the two proteins are co-expressed in many cells including cochlear hair cells. Since both CDH23 and CAMSAP3/Marshalin carry several protein-protein interaction domains, we systemically characterized the authenticity of interactions between CAMSAP3/Marshalin and CDH23-C using both Previously, we showed that overexpression of CAMSAP3/Marshalin-Ld  resulted in dramatic reorganization of MT networks into bundles composed of both CAMSAP3/Marshalin and tubulinp = 0.0013) and CDH23-C2-GFP (p = 0.0037): by 21.3% for CDH23-C1 and by 20% for CDH23-C2, respectively. Thus, type I cells dominated when OK cells co-expressed CAMSAP3/Marshalin-Ld and CDH23-C isoforms. In contrast, a majority of cells co-expressing CAMSAP3/Marshalin-Ld and Ceacam16 showed type III MT patterns similar to the expression patterns of cells expressing CAMSAP3/Marshalin-Ld and GFP . Thus, CDH23-C1-GFP co-expression interferes with CKK’s ability to induce MT bundles, similar to that seen for full-length CAMSAP3/Marshalin-Ld.To understand the molecular mechanisms underlying the inhibitory role of CDH23-C isoforms on CAMSAP3/Marshalin-induced MT bundle formation, we first determined the functional domains that are responsible for the CDH23-C and CAMSAP3/Marshalin interaction. CAMSAP3/Marshalin-Ld was identified as a potential CDH23-associated protein using the membrane-based yeast two-hybridization (Y2H) approachin vitro pull-down assays. GST-tagged CDH23-C1 and CDH23-C2 fusion proteins were expressed in bacteria and immobilized on glutathione-agarose. The glutathione resins with GST-alone or GST-CDH23-C isoforms were then incubated with bacterial lysates containing the CKK domain, and bound proteins were eluted and detected on SDS-PAGE. The presence of GST, and GST-CDH23-C isoforms were detected by Coomassie Blue staining ), exon 68 (Cdh23 (Δ108)), and NBM (Cdh23 (Δ143) (see Methods for details), did not abolish binding between CKK and CDH23 (Cytoplasmic regions of CDH23 have been reported to interact with other proteins including harmonin through the PDZ domain-binding motif (PBM), exon 68, or the N-terminus domain binding motif (NBM) as shown in Fig. 1C1718nd CDH23 . This rend CDH23  were mixnd CDH23 , top, whnd CDH23 , bottom.Our data demonstrated that CDH23-C binds to CAMSAP3/Marshalin’s CKK domain but the minimum binding region did not include one of the harmonin binding sites (NBM). To learn more about the relationship among CAMSAP3/Marshalin, harmonin, and CDH23-C isoforms, we tested whether CDH23-C isoforms could still bind to CAMSAP3/Marshalin in the presence of harmonin. GST-tagged CDH23-C1 and CDH23-C2 proteins were first immobilized on glutathione-agarose along with GST as a negative control. GST-agarose and CDH23-C-GST-agarose were then simultaneously incubated with lysates from bacteria expressing CKK and lysates from bacteria expressing harmonin. In paired comparisons, glutathione-agarose with GST or GST-CDH23-C isoforms were incubated with CKK lysates and uninduced harmonin bacterial lysates, which were derived from the same bacterial transformants without IPTG treatment. The latter serves as the negative control as it contains similar proteins except for harmonin. As shown in CDH23 cause Usher Syndrome type 1D. One disease-associated missense mutation occurs within the intracellular region that changes arginine to histidine at the amino acid position 3175 of CDH23-A1 (R3175H)Cdh23-C1, which is also conserved among all species. We suspected that mutation of this highly conserved, positively charged amino acid might affect CDH23-C and CAMSAP3/Marshalin-Ld binding. Therefore, we introduced the same mutation within our GST-CDH23-C1 construct at position 55 (CDH23-C1_R55H), which corresponds to the R3175H mutation found in Usher Syndrome type 1D patients. This point mutation altered migration of the CDH23-C1 protein compared to the wildtype in SDS-PAGE (p < 0.05) as compared to GFP controls, albeit less effectively than wildtype CDH23-C1. In other words, more cells showed the type III phenotype in OK cells co-expressing CAMSAP3/Marshalin-Ld and CDH23-C1_R55H-GFP than OK cells co-expressing CAMSAP3/Marshalin-Ld and wildtype CDH23-C1-GFP (p < 0.05). Since the R55H mutation is within the CBM of CDH23-C isoform, we also introduced R55H into the CDH23-C1_1–67 construct, and performed the GST pull-down assay. The resulting GST-CDH23-C1_1–67_R55H fragment also exhibited a slight increase in the apparent molecular weight on the gel compared to the WT (p = 0.0141). These data suggest that the mutation within the CBM of CDH23-C isoforms alters the interaction between CDH23-C and CAMSAP3/Marshalin-Ld.Mutations in SDS-PAGE . The incSDS-PAGE , while tSDS-PAGE . The pero the WT , top. Coin vivo MT bundle formation assays within mammalian cells and in vitro GST pull-down assays. We have identified the binding sites between CDH23-C and CAMSAP3/Marshalin-Ld in the N-terminus of CDH23-C (CBM) and the CKK domain of CAMSAP3/Marshalin-Ld, respectively. We further showed that mutation in the CMB of CDH23, which was discovered in an Usher Syndrome 1D patient (R3175H), resulted in a marked decrease in CDH23-C’s ability to interact with CAMSAP3/Marshalin both in vivo and in vitro. These data suggest that CDH23-C plays an important role in regulating MT networks via its interaction with CAMSAP3/Marshalin, and that alteration of this interaction can lead to pathological conditions such as Usher Syndrome.CDH23 is an essential protein for auditory and visual signal transduction and is important for cell adhesion. Function of the cytoplasmic isoforms, however, is not fully understood even though CDH23-C is detected in various tissues and implicated in cochlear hair cell developmentAs an adhesive protein, CDH23 is generally thought to localize to the cell surface. Therefore, it was puzzling when CDH23 was also found in MT-rich areas such as centrosomes and basal bodies that are internal structures1013CDH23 is known to bind to the PDZ domain of harmonin through multiple domains including PBM, exon 68, and NBM18Our data suggest that CDH23-C can regulate MT networks indirectly through its interaction with CAMSAP3/Marshalin. As a new modifier of MT networks, additional studies are required to further define CDH23-C’s roles in various physiological contexts that may ultimately impact our understanding of the fundamental mechanisms associated with hearing and vision.CAMSAP3/Marshalin-Ld or the CKK domain  was inserted into a pcDNA6/V5His vector to introduce a V5-His tag to the C-terminusGFP-Cdh23-C1 and GFP-Cdh23-C2 constructs were kindly provided by Dr. FriedmanGFP-Cdh23-C1 using a QuickChange Site-Directed Mutagenesis Kit (Stratagene) to generate GFP-Cdh23-C1_R55H. For bacterial expression, CKK domains of mouse CAMSAP3/Marshalin-Ld  were inserted into the NheI/SalI site of the pET24(a) vector. Both constructs contained a His-tag at the C-terminus. We also made a construct encoding CKK1 cDNA without the His-tag. Glutathione S-Transferase (GST)-tagged mouse CDH23-A cytoplasmic tail constructs, CDH23, CDH23 (+68), CDH23 , CDH23 (Δ108), and CDH23 (Δ143), were generous gifts from Dr. BartlesCdh23-C1 and Cdh23-C2 fragments were inserted into the EcoRI/XhoI site of the pGEX-4T-2 vector with an N-terminal GST tag to generate the following constructs: Cdh23-C1, Cdh23-C2, Cdh23-C_1–67 (first 67 aa of CDH23-C), CDH23-C_1–67_R55H (first 67 aa of CDH23-C with the R55H mutation).For mammalian expression, the cDNA of mouse 2 Fragment , 1:1,000 for IF.Anti-MarshalinCdh23-C1 and Cdh23-C2 fragments, His-tagged CKK1/2 and harmonin were induced by 1 mM Isopropyl β-D-1-thiogalactopyranoside  in E. coli BL21 (DE3) cells (Invitrogen). Cells were then lysed in PBS containing 1X protease inhibitor (Sigma), 1 mM phenylmethanesulfonylfluoride , 1 mg/ml lysozyme (Sigma), and 370 unit/ml Benzonase (Sigma) by sonication. The bacterial lysates were incubated at 4 °C for 30 minutes, then centrifuged at 14000 rpm for 30 minutes at 4 °C to separate insoluble fragments from supernatant. GST-tagged Cdh23 tail proteins and various Cdh23-C1 and Cdh23-C2 fragments were then immobilized on glutathione-agarose (Thermo) at 4 °C on a rotator for 1.5 hours. Unbound proteins were washed away with chilled PBS. For Cdh23- CAMSAP3/Marshalin pull-downs, Cdh23 bound glutathione-agarose (Cdh23-GST-agarose) was incubated with CKK lysate at 4 °C on a rotator for 1.5 hours or overnight. In some experiments, CKK proteins were purified first using a MagneHis Protein Purification System (Promega) before adding 30–60 ng/ml of CKK protein to the Cdh23-GST-agarose. For pull-downs including harmonin, uninduced harmonin bacterial lysate (no harmonin expression) and induced harmonin bacterial lysate were combined with CKK lysate in a 1:1 ratio, and added the Cdh23-GST-agarose. Following incubation, the resins were washed with chilled PBS, and bound proteins were eluted with 100 mM L-Glutathione (Sigma), and then loaded onto 12% (w/v) polyacrylamide-SDS gels. In some experiments, CKK proteins and harmonin were purified first using a MagneHis Protein Purification System. The imidazole in elution buffer was removed and replaced with PBS using PD MiniTrap G25 . Protein concentrations were measured used a Pierce 600 nm Protein Assay Reagent (Thermo Scientific). Appropriate molar concentrations of CKK1 and harmonin were then added to the Cdh23-C1-GST-agarose. To visualize enriched GST-tagged proteins/fragments, Coomassie Blue (Thermo) was used to stain the gels, or 5% Ponceau S (Sigma) to stain the nitrocellulose membranes. To probe for CKK domains, membranes were incubated with anti-Marshalin followed by HRP-conjugated secondary antibodies and a SuperSignal West Pico Chemiluminescent Substrate (Thermo). Images were taken with a Kodak Imaging System and the intensities of the expected CDH23 and CKK bands measured using Molecular Imaging software from Kodak . For statistical analysis, the signal intensities of CKK were normalized to the intensities of Cdh23-GST bait, and means from 4 independent experiments were plotted. Significance was determined using one-way ANOVA with the Tukey’s multiple comparisons test in Prism 6 (GraphPad).Expression of GST-tagged CDH23 cytoplasmic tail proteins or various 2 in air at 37 °C. 24–45 hours post transfection, cells were treated with colchicine at a concentration of 250 ng/ml for 24 hrs or 1 μg/ml for 3 hrs. We also utilized LLC–PK1–CL4 cells (CL4 cells) cloned from LLC–PK1 cells provided by Dr. Bartles (Northwestern University). Detailed culture conditions for these cells were described in a previous publicationPlasmids encoding V5-His tagged CAMSAP3/Marsalin-Ld or simply its CKK domain were transiently co-transfected with GFP, CDH23-C1-GFP, and CDH23-C2-GFP into opossum kidney (OK) cells as previously describedGFP-Cdh23-C1 or GFP-Cdh23-C1_R55H were harvested 2 days after transfection and lysed in cold lysis buffer  supplemented with 1X protease inhibitor cocktail and 1 mM PMSF (Sigma). Insoluble material was removed by centrifugation at 15,000 g for 15 minutes at 4 °C. Proteins were resolved using 8% SDS-PAGE, followed by immunoblotting using anti-GFP (Rockland). Signals were detected using a SuperSignal West Pico Chemiluminescent Substrate (Thermo). A Kodak Imaging System was used to capture the images.OK cells transiently transfected with How to cite this article: Takahashi, S. et al. Cadherin 23-C Regulates Microtubule Networks by Modifying CAMSAP3’s Function. Sci. Rep.6, 28706; doi: 10.1038/srep28706 (2016).


Tumor PD-L1 expression is associated with increased tumor-infiltrating lymphocytes (TILs) in diverse solid tumors, including lung cancer. In addition, PD-L1 upregulation in tumor and/or stromal immune cells has been associated with increased clinical benefit to PD-1/PD-L1 axis blockers. The significance of PD-L1 expression in different immune cell subpopulations at the tumor microenvironment remains poorly understood. Here, we measured PD-L1 protein specifically in tumor-associated macrophages using objective methods and analyzed its clinical significance in human lung cancer.Using multiplexed quantitative immunofluorescence (QIF) we measured the levels of PD-L1 protein  in 554 stages I-IV FFPE lung carcinomas represented in two tissue-microarrays, one from Yale University  and one from Greece . PD-L1 was measured using multispectral QIF in CD68-positive cells  or in tumor cells  based on fluorescence co-localization. The levels of CD3 , CD8  and CD20  were also determined in the tumor samples. Associations between the macrophage and tumor PD-L1 levels, clinico-pathological variables and survival were analyzed.2]=0.311 in the Yale cohort and R2=0.43 in the Greek collection). Using the median score as cutpoint, elevated macrophage PD-L1 content was significantly associated with high CD8+ and CD20+ cells in both cohorts. In addition, increased macrophage PD-L1 signal was marginally associated with longer overall survival in the Yale set , but not in the Greek group .In lung cancer samples, PD-L1 was detected in both macrophages and tumor cells with a predominant membranous staining pattern. Overall, the levels of PD-L1 in the CD68+ population were lower than in tumor cells in both cohorts . In both collections, the macrophage and tumor PD-L1 signal showed a positive non-linear association (Linear regression coefficient [RPD-L1 is expressed in tumor-associated macrophages of human lung carcinomas. The macrophage PD-L1 signal is lower than in tumor epithelial cells and they show a positive association. Elevated macrophage PD-L1 is prominently associated with increased cytotoxic T lymphocytes and B cell infiltrates in lung tumors, but has limited prognostic value. Further studies will be required to evaluate the possible predictive role of PD-L1 expression in macrophages and other immune cell types.