The coordination of pollen tube (PT) growth, guidance and timely growth arrest and rupture mediated by PT-pistil interaction is crucial for the PT to transport sperm cells into ovules for double fertilization. The plasma membrane (PM) represents an important interface for cell–cell interaction, and PM proteins of PTs are pioneers for mediating PT integrity and interaction with pistils. Thus, understanding the mechanisms underlying these events is important for proteomics.Using the efficient aqueous polymer two-phase system and alkali buffer treatment, we prepared high-purity PM from mature and germinated pollen of rice. We used iTRAQ quantitative proteomic methods and identified 1,121 PM-related proteins (PMrPs) (matched to 899 loci); 192 showed differential expression in the two pollen cell types, 119 increased and 73 decreased in abundance during germination. The PMrP and differentially expressed PMrP sets all showed a functional skew toward signal transduction, transporters, wall remodeling/metabolism and membrane trafficking. Their genomic loci had strong chromosome bias. We found 37 receptor-like kinases (RLKs) from 8 kinase subfamilies and 209 transporters involved in flux of diversified ions and metabolites. In combination with the rice pollen transcriptome data, we revealed that in general, the protein expression of these PMrPs disagreed with their mRNA expression, with inconsistent mRNA expression for 74% of differentially expressed PMrPs.This study identified genome-wide pollen PMrPs, and provided insights into the membrane profile of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils. These pollen PMrPs and their mRNAs showed discordant expression. This work provides resource and knowledge to further dissect mechanisms by which pollen or the PT controls PMrP abundance and monitors interactions and ion and metabolite exchanges with female cells in rice.The online version of this article (doi:10.1186/s12870-016-0961-7) contains supplementary material, which is available to authorized users. Pollen, consisting of the large vegetative cell (VC) and immobile male gametes (sperm cells) enclosed in the VC, represents an innovative phenotype of plants during evolution. Pollen is tolerant to dehydration and can transport the male gamete over a long distance with the help of wind and/or animals, important forces driving the distribution of plants in the land. Once landing on the stigma of pistils, pollen hydrates, germinates and gives rise to a tip-growing pollen tube (PT). The tube further journeys within the pistil and finally arrives in the synergids of embryo sacs, where it arrests growth and ruptures to release sperm cells for double fertilization. This process requires maintenance of PT integrity and PT–pistil interaction to guide the PT toward the embryo sac and regulate the timely growth arrest of the PT and rupture for immobile gamete release, thereby precisely guaranteeing the one PT to one ovule relationship .Arabidopsis, ANX1 and ANX2, members of the plant-specific RLKs of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily [anx1anx2 double mutants show precocious rupture. Overexpression of ANXs caused PT growth inhibition [+ transporters, so a novel RLK signaling pathway mediates K+ homeostasis is required for PT growth and integrity [Arabidopsis LRR-RLKs MDIS1, MIK1 and MIK2 form heteromers, and the complex functions as a receptor of LURE1, the defensin-like cysteine-rich peptide from synergids, to regulate PT guidance and perception [Arabidopsis LRR-RLK, PRK6, was identified as a LURE1 receptor and functions in guiding PT tip growth [The plasma membrane (PM) represents the semi-permeable barrier for selective flux of ion and metabolites across the membrane and an important interface for cell–cell interaction. PM proteins, especially receptor-like kinases (RLKs) and transporters/channels of PTs are the pioneers for mediating PT integrity and interaction with pistils . In Arabubfamily , 4. PTs hibition . RUPO, tntegrity . Studiesntegrity . The Ararception . Anotherp growth . Furtherp growth . Chemocyp growth . ZmEA1, p growth . Thus, aArabidopsis, AtACA9, a Ca2+ATPase for Ca2+ efflux is required for PT growth and PT–synergid contact [Arabidopsis cyclic nucleotide gated Ca2+ channel 7 (AtCNGC7) and 8 for Ca2+ influx redundantly regulate the initiation of PT tip growth [in vitro [+ channel in Arabidopsis, strongly reduced K+ influx, which resulted in impaired pollen germination and PT growth [+ influx channel KZM1, which led to excessive influx of K+, thereby causing PT rupture [+ transporter signaling pathway has been revealed in PTs [Ion fluxes across the PM and the ion gradient in PTs are well known and are crucial for PT growth . In Arab contact . Arabidop growth . AtCNGC1in vitro . DisruptT growth . PTs lacT growth . The mai rupture . Moreoved in PTs .Despite these promising findings, our knowledge of RLKs and transporters/channels  that function in PT growth and interaction with pistils is limited, and a detailed understanding of these components at the omic-wide level and proteomic characteristics of pollen and PT PM is lacking. A systematic knowledge of RLKs and transporters in the PM is crucial for an in-depth understanding of the mechanisms underlying PT growth and interaction with pistils.Here, we prepared PMs from mature pollen grains (MPGs) and germinated pollen grains (GPGs) and dissected PM proteins by using iTRAQ quantitative proteomics. We identified 1,121 PM-related proteins (PMrPs) (matched to 899 loci), with 192 differentially expressed during pollen germination, and revealed 37 RLKs and 209 transporters in the proteome. All PMrPs and differentially expressed PMrPs featured signal transduction, transporters, wall remodeling/metabolism and membrane trafficking functions. Further comparison of proteomic and transcriptomic data revealed that PMrPs are in general discordant with their mRNA levels, with inconsistent mRNA profiles for 74% of differentially expressed PMrPs. These results provide insights into the proteomic characteristics of pollen PM and the profile of RLKs and transporters in the membrane.Oryza sativa L. ssp. japonica) was planted under natural conditions in Beijing. Mature pollen grains (MPGs) were collected at anthesis stage by using a modified vacuum cleaner outfitted with nylon meshes. For germination experiments, fresh collected MPGs were transferred into liquid germination medium 2 · 4H2O, 3 mg/L VB1, 10% PEG4000, 250 mM sucrose) immediately and cultured with gentle shaking at room temperature (~30 °C) for about 15 min. Under this condition, more than 90% of MPGs synchronously germinated to generate polar-growing PTs. Germinated pollen grains (GPGs) were collected by centrifugation at 1000 × g at 4 °C for 5 min. All collected MPGs and GPGs were used immediately or stored at −80 °C.Rice cultivar Zhonghua 10  by use of the high-speed bench top homogenizer FastPrep-24 . The homogenate was differentially centrifuged at 1,500 × g for 5 min, 12,000 × g for 20 min and then 31,000 × g for 15 min to remove cell debris, mitochondria and other organelle contaminants, respectively. The resulting supernatant was centrifuged at 100,000 × g for 1 h with use of BECKMAN Optima L-80XP  to collect pellets . MSVs were resuspended in PM isolation buffer  and used to enrich PM vesicles by use of an aqueous polymer two-phase system . All pro+-ATPase (PMA2) from Nicotiana plumbaginifolia (1:5000 dilution) [Arabidopsis thaliana; histone H1 , DEAD-box ATP-dependent RNA helicase , glyceraldehyde-3-phosphate dehydrogenase , and flotillin like protein  from Oryza sativa. Optical density of Western blot bands was quantified by using Image-Pro Plus v6.0 .Proteins were separated by 10% SDS-PAGE. For Western blot analysis, proteins in gels were electrotransferred onto a PVDF membrane  with 25 mM Tris, 192 mM glycine and 20% methanol and incubated with the primary rabbit antibodies for PM Hilution) ; mitochoRapiGest SF surfactant  at a final concentration of 0.2% (w/v) for denaturation and enzymatic digestion enhancing, then reduced with 10 mM TCEP, pH 8.0 at 56 °C for 1 h followed by alkylation with 50 mM iodoacetamide in the dark . Pretreated proteins were digested with trypsin at a ratio of 1:50 (w/w) (Roche) at 37 °C for 16 h, and resulting peptides were labeled with iTRAQ reagents. Experimental repeats were designed as follows: experiment 1 was a mixture of 115 tag-labeled MPG and 117 tag-labeled GPG samples; experiment 2 was a mixture of 117 tag-labeled MPG and 115 tag-labeled GPG samples. Experiment 1 and 2 were lyophilized and subjected to strong cation exchange (SCX) fractionation.Protein digestion and iTRAQ labeling were performed according to the iTRAQ reagents chemistry reference guide  with a few modifications. Briefly, proteins (100 μg) from purified PM vesicles were supplemented with SCX fractionation was performed with an AKTA Purifier 10 HPLC system . The lyophilized samples were resuspended in solvent A  and fractionated with a PolySULFOETHYL A column  at a flow rate of 200 μl/min through a linear gradient  of solvent B  followed by 60-100% solvent B for 10 min, and 100% solvent B for 15 min. Each separated sample was pooled to 16 fractions and lyophilized for LC-MS/MS analysis.Each SCX faction was reconstituted with 100 μL 0.1% formic acid in water. After the removal of salt on a Vydac C18 SPE cartridge by centrifugation, the desalted fraction was supplemented with 50 μL 60% acetonitrile and dried with a speed-vac. Each dried fraction was solved in 20 μL 0.1% formic acid, and 10 μL of the solution was injected for nanoLC-MS/MS analysis by use of AB SCIEX Triple TOF 5600 MS  equipped with a splitless Eksigent nano Ultra 2D Plus nanoLC system and a cHiPLC-Nanoflex microchip system . The cHiPLC-system used changeable microfluidic traps (200 μm × 5 mm) and analytical columns (75 μm × 150 mm) packed with ChromXP C18  for online separation analysis. Sample loading, trapping and desalting involved 100% of mobile phase A  at a flow rate of 2 μL/min for 10 min. Peptide elution was started with 5% mobile phase B , then the gradient increased linearly to 24% in 70 min at a flow rate of 300 nL/min. The total gradient length was 120 min. MS data acquisition was performed in the information dependent acquisition (IDA) mode. Triple TOF 5600 MS was operated with a resolving power of 30,000 (FWHM) for TOF MS scans. IDA survey scans were acquired in 250 ms with mass range of m/z 350–1250. As many as 30 product ion scans were collected for 100 ms with mass range of m/z 100–1500, if exceeding a threshold of 120 cps (counts/s) and with a charge state of +2 to +5. Dynamic exclusion was set for 18 sec. Collision energies were calculated on-the-fly for all precursor ions by using empirical equations based on mass and charge (Rolling CE on), and the Enhance iTRAQ function was turned on to improve the efficiency of the collision-induced dissociation.The raw data files (*.wiff) generated by Triple TOF 5600 were analyzed by using ProteinPilot 4.0 , which involved two different algorithms; Paragon and Pro Group.Oryza sativa nonredundant database ; Sample Type-iTRAQ 4plex labeled; Cys Alkylation-Iodoacetamide; Digestion-Trypsin; Instrument-Triple TOF 5600; Quantitate; Bias Correction; Background Correction; Biological modifications. Precursor mass tolerance was 0.05 Da and fragment mass tolerance was 0.1 Da. As part of the Paragon analysis method, false discovery rate analysis was performed by searching the decoy database to assess the rate of inaccurately identified proteins.Paragon is a search engine that uses feature probabilities and sequence temperature values to identify peptides from MS/MS spectra . DatabasParagon search results were further processed by the Pro Group algorithm to determine the smallest justifiable set of detected proteins. Each detected protein has an unused protein score, a measurement of all the peptide evidence for a protein that is not better explained by a higher ranking protein, and this score is the true indicator of protein confidence. Unused protein scores 2.0, 1.3, 1.0, and 0.47 correspond to peptide confidence 99, 95, 90, and 66% respectively , as shown in the ProteinPilot Software Beta Help (AB SCIEX).p-value ≤ 0.05 and GPG/MPG ≥ 1.50 or GPG/MPG ≤ 0.67.Protein quantitative analysis was also performed by use of ProteinPilot 4.0. The software calculates protein expression change ratios between different samples based on the relative intensities of iTRAQ-labeled peptides. Only ratios from the spectra that are distinct to each protein or protein form were used, to eliminate any masking of changes in expression due to peptides shared between proteins. For each protein expression change ratio reported, the program calculates a p-value that indicates the probability of randomly detecting a ratio different from 1. If an expression change ratio is extremely well determined, a real change can be detected even when the ratio is not very different from 1. To obtain a more accurate quantified result, we chose bias correction and background correction when searching the database by Paragon. Specifically, the criteria for determining MPGs and GPGs differentially expressed PM proteins are two experiments expressed, http://web.expasy.org/protparam/). The matched loci IDs were obtained from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml). Cellular component, biological process, and molecular function for proteins were annotated by using gene ontology  or WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html). Protein transmembrane domains (TMDs) were predicted by using HMMTOP 2.0 (http://www.enzim.hu/hmmtop/). Modifications related to membrane localization of a protein including glycosylphosphatidyl inositol (GPI) attachment, prenylation, myristoylation, and palmitoylation were predicted by using the big-PI Predictor (http://mendel.imp.ac.at/gpi/gpi_server.html), PrePS-Prenylation Prediction Suite (http://mendel.imp.ac.at/PrePS/), N-Myristoyltransferase , and CSS-Palm 3.0 , respectively. Protein annotations were comprehensively evaluated by using a combination of NCBI (http://www.ncbi.nlm.nih.gov/), RGAP 7 (http://rice.plantbiology.msu.edu/), and ARAMEMNON 7.0 (http://aramemnon.uni-koeln.de/).Protein molecular weight (MW) and isoelectric point (pI) were calculated by using the ProParam tool of Expasy  lysate to carbonate buffer-washed PM (CPM) and one was almost undetectable in EC lysates but was most abundant in CPM  < 1%, unused ≥ 1.3 and two or more unique peptides matched, we identified 1,474 proteins with FDR 0.07% in experiment 1, and 1,284 proteins with FDR 0.08% in experiment 2 , posttranslational modification (PTM), and subcellular location. The analysis of TMDs showed that 1,137/1,979 proteins 57.5%) had at least one TMD  Fig. . PTMs in7.5% had We collected function information for the 1,121 PM-related proteins (PMrPs) from NCBI, RGAP, ARAMEMNON, and GO databases and analyzed by functional categories to gain insight into the biological process occurring in or around pollen PM. These proteins could be organized into 9 categories  identified in experiment 1 were quantified and 1,147/1,284 (89%) in experiment 2 were quantified  between MPGs and GPGs, with 119 abundance-increased and 73 abundance-decreased in GPGs  were reproducibly identified in the two independent experiments. Using the cut-off of fold change in expression (GPG/MPG) ≥ 1.5 or ≤0.67 and http://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml) showed 12% of these genes distributed in chromosome (chr) 1; 10% each in chr 2, 3, and 4; 8% each in chr 5, 6, 7 and 8; 6% each in chr 9 and 10, and 7% each in chr 11 and 12. The genomic loci of pollen PMrPs and the differentially expressed PMrPs showed a significant chromosome bias. They were enriched on chr 1, 2 and 3, but not on the other chromosomes  and decreased (Sar) in levels in GPGs or had no change in level (Band_7) between MPGs and GPGs in iTRAQ data Fig. . The exp2+, phospholipid, auxin, abscisic acid (ABA), gibberellic acid (GA) and phosphorylation cascades , extensin (n = 1), leucine-rich repeat , proline-rich extensin like receptor kinase , receptor-like cytoplasmic kinases , S-domain  and unknown receptor kinase  with 1 not assigned  with about 10,000 representative and putative non-redundant transporters. By blast searching this database, we identified 209 transporters (matched to 161 loci) in the rice pollen PM proteome; these transporters involved 33 families (Table 2+ (n = 12), H+ (n = 38), K+ (n = 22), Cl− (n = 6), Mg2+ (n = 5), sugar (n = 24), phospholipids (n = 3), amino acid/oligopeptide (n = 16), phosphate (n = 7) and sulfate (n = 3) , one K+ transporter (gi|125533127), 3 sugar transporters , one ABC transporter (gi|218188091), 2 phospholipid transporters (gi|40253457 and gi|53793271), 2 oligopeptide transporters (gi|215697740 and gi|90265689) and 3 other transporters , and abundance-decreased transporters were another 7 H+ transporters , 3 K+ transporters , one Mg2+ transporter (gi|115454637), 2 sugar transporters (gi|222622219 and gi|115434360), 3 ABC transporters  and 2 other transporters (gi|90399194 and gi|75253347)  system was used to build a transporter classification database , median (GPGs))/MAX )  with corresponding transcripts; 317 of these were pollen-preferential  [Lycopersicon esculentum) [Arabidopsis thaliana) [Arabidopsis thaliana) [Zea mays; in evolution, these LRR-RLKs appeared to differentiate from the dicot members AtPRP1-2 and 4–5, LePRK1-2 and PiPRK1 before the monocot–dicot split. gi|115486303 and gi|4680345 are in a clade with LePRK3, AtPRK3 and AtPRK6 and gi|125547150, gi|115480655, gi|125531685, gi|125561357 and gi|218201938 are in a clade with AtMIK1 and 2, which indicates their differentiation after the monocot–dicot split. The remaining 4 are in an independent clade without functionally known dicot homologs  . We reveinflate) , pollen–ulentum) , PT elonhaliana)  and PT thaliana) . Four ofArabidopsis – LIP1 and LIP2 – are anchored to the PM of the PT tip via palmitoylation, and they can perceive the female signal – LURE1 – attraction [S-locus glycoproteins (SLGs) that display genotype-specific sequence polymorphisms. S receptor kinases were proposed to be responsible for the allelic specific self-incompatibility response along with SLGs [RLCK is another large subfamily of RLKs that lack the transmembrane domain . Two poltraction . SD-RLKsith SLGs . We iden2+, H+, K+ and Cl− are critical for pollen germination and PT growth [2+, K+, H+, Cl−, water, sugar, amino acids, oligopeptides and phospholipids  that promote H+ exflux in the subapical region [+ influx in the PT apex are unknown, but the nonspecific cation channels are generally considered responsible for apical H+ influx [+ influx. Besides the PMAs, 13 VHAs and 3 H+-PPases were revealed, with the VHA subunit H (gi|218199814) significantly increased and subunit A (gi|115444549) and C (gi|115437984 and gi|115465801) decreased in levels in GPGs; they may also affect PT growth by sustaining cytosolic pH homeostasis [The increased pH from the apical to the subapical region functions as an important node in regulating diverse activities in PT, such as the wall plasticity, vesicle transport and actin dynamics . The pH l region . In rice+ influx . The rict A gi|1144549 and+ is an essential mineral for pollen germination and PT growth [+ channels were required for PT growth [Arabidopsis [+ transporters ; only OsSUT1 and OsSUT3 transcripts were detectable in pollen [Moreover, we revealed 5 Mgydration . Sucroseydration , 61. Then pollen . We idenn pollen . The ricn pollen , but so n pollen . We idenn pollen . ABC tran pollen . ABCG trn pollen . We firsFinally, we identified other transporters in the rice PM (Additional file We revealed 1,121 PMrPs and 192 differentially expressed PMrPs during rice pollen germination. Pollen presynthesizes a set of PM proteins for PT growth and interaction with pistils. The pollen PM protein levels are in general discordant with their mRNA levels, and their genes showed biased distribution in rice chromosomes. The pollen PM have a large set of RLKs mediating cell–cell interaction and transporters for flux of diversified ions and metabolites Fig. . This st


Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids , which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis.The filamentous fungus IMPORTANCETrichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance. Competition in nature drives adaptation of physiological processes to the specific conditions in the microbial habitats. Thus, abilities to economically respond to the availability and nature of substrates to sustain growth, as well as to changing light conditions, are among the most crucial competencies for survival and evolutionary success.Trichoderma reesei is a prolific producer of cellulases and is broadly utilized in research and industry  analysis, with sexual development as a challenging but also promising tool.In the course of sexual development,  mitoses . Interes mitoses  due to aT. reesei QM6a, it was considered possible that its defect might be a result of repetitive laboratory cultivation .The genome sequence of strain CBS999.97 was assembled from 454 and paired-end Illumina reads to generate 1,087 contigs that were ordered into 167 scaffolds, totaling 32.47 Mb, with an N50 of 1.22 Mb. Alignment of the high-throughput-sequencing reads from CBS999.97 to the QM6a reference genome identified 466,083 single nucleotide polymorphisms (SNPs) between the strains, 36,955 of which are predicted to be nonsynonymous (data available at P = 0.0), “stress response” (P = 2.23 × 10−13), “energy” (P = 1.54 × 10−11), “protein synthesis” (P = 2.05 × 10−10), “regulation of C compound and carbohydrate metabolism” (P = 1.25 × 10−7), “fungal-cell-type differentiation” (P = 2.11 × 10−6), “signal transduction mechanisms” (P = 2.11 × 10−6), and “transcription” (P = 8.62 × 10−5) (Data Set S1). In contrast, genes containing more than 10 nonsynonymous SNPs are enriched in “secondary metabolism” (P = 4.91 × 10−7), “DNA repair” (P = 1.26 × 10−3), and “photoperception and -response” (P = 4.45 × 10−3) (Data Set S1).The SNPs were not randomly distributed among genes or gene groups. The 2,390 predicted genes with no nonsynonymous SNPs are strongly enriched in core physiological functions. The most strongly enriched functions include “metabolism” , representing an additional CKI present in T. reesei but not in T. atroviride, T. virens, or Neurospora crassa .We also checked for genomic regions with increased occurrence of nonsynonymous SNPs, which might indicate chromosomal areas under specific evolutionary pressure. Regions with 5 or more genes in close vicinity in the genome sequence containing 10 nonsynonymous SNPs were considered. Indeed, we found 19 regions that fit this definition of being under evolutionary pressure (Data Set S1). In several cases, these regions are located at the ends of scaffolds, indicating that they are close to repetitive regions. Checking overlaps with CAZyme clusters, we only found the region around TR_107835, an as-yet-uncharacterized carbohydrate esterase of family 10, to reside within a region of increased abundance of nonsynonymous SNPs. This result is consistent with the finding that metabolic functions are enriched among genes with a particularly low abundance of nonsynonymous SNPs. Interestingly, http://genome.jgi.doe.gov/Trire2/Trire2.home.html)  .10. Considering the 9,127 genes predicted for the genome, this would mean 9 genes would be expected to differ between FF1a, -b, and -c and QM6a. However, the sequence analysis revealed 106 genes from CBS999.97 after 10 backcrossing steps—a considerably higher number of consistently retained genes in overlapping regions (Table S1). Therefore, we performed an additional backcrossing round and selected 20 female fertile and 20 female sterile strains. We chose 17 SNP-containing genes representing all three overlapping genomic regions and analyzed whether the CBS999.97 or the QM6a version of the respective gene was present in these progeny. This analysis clearly showed a significant correlation of female fertility with the presence of the CBS999.97 version of six genes encoding factors designated FS4, FS5, FS6, FS7, FS8, and FS9 on scaffold 6 and no significant correlation with the genes on the other scaffolds (Table S2). This finding was in line with our hypothesis of one locus being responsible for the female fertility defect in QM6a. Consequently, the genomic region spanning the loci of these genes was found to be responsible for female fertility in the backcrossing strains and to contain the defective gene in QM6a  showed considerable differences between strains with the CBS999.97 background and all strains with the QM6a background . This anPodospora anserina. Therefore, we were interested in whether any mating type-specific differences in substrate utilization would be consistent in the two phenotypically different strain backgrounds of T. reesei QM6a and CBS999.97. The general strain comparison shown in N-acetyl-l-glutamic acid, β-hydroxybutyric acid, and 2-aminoethanol . Slightly better growth was observed for MAT1-2 strains on α-d-lactose, α-d-glucose, d-arabitol, d-mannose, l-pyroglutamic acid, l-glutamic acid, and interestingly, also on d-cellobiose and d-trehalose . As several of the latter substrates are associated with cellulose or hemicellulose degradation, relevance of mating type for efficient plant cell wall degradation cannot be excluded.Mating type can regulate genes associated with metabolism, including sugar transporters , in Podol-proline (158% ± 25%); l-proline can be used as a carbon or a nitrogen source and several intermediates of proline metabolism are either shared or feed directly into arginine and glutamate metabolism (l-Aspartic acid (137% ± 11%), l-alanine (144% ± 8%), l-serine (148% ± 8%), and l-asparagine (166% ± 11%) also increased the growth of QM6a relative to that of CBS999.97. d-Galacturonic acid (270% ± 88%) was especially efficiently metabolized, which is consistent with the enhanced capability to degrade hemicellulose and/or pectin. Better growth on carbon sources that are building blocks of hemicellulose and metabolites of its degradation  was also observed for QM6a .We wanted to evaluate the different strain backgrounds in more detail and so compared CBS999.97 MAT1-1 and CBS999.97 MAT1-2 with QM6a, FF1, and FF2 ; Fig. S3tabolism . l-AsparWe compared QM6a with the sexually fully competent strains of both mating types (FF1 and FF2) in order to evaluate if there was any metabolic competence that was associated with the genes retained from CBS999.97 during backcrossing. Some of the genes reside in a genomic cluster correlating with female fertility of FF1 and FF2, as discussed above.d-galacturonic acid (190% ± 17%), xylitol (117% ± 3%), l-rhamnose (125% ± 6%), d-arabinose (147% ± 4%), and l-arabinose (119% ± 2%) . The latter carbon sources are constituents of or metabolites in the degradative pathway of hemicellulose via the pentose phosphate pathway, suggesting differences in this pathway between QM6a and the backcrossed strains. With respect to amino acids, only growth on l-proline (139% ± 3%) clearly increased in QM6a, while growth on the other amino acids was not significantly different. As hemicellulose metabolism is enhanced in the female fertile FF strains and in strains with the QM6a background relative to that of CBS999.97, we conclude that the differences in hemicellulose metabolism are due to differences in background rather than to differences to female fertility.The female fertile FF1 and FF2 strains grew better than the female sterile QM6a on T. reesei, and hence, we included in our analysis the two strains derived from the wild isolate, CBS999.97 MAT1-1 and CBS999.97 MAT1-2  among the genes of cluster 1. In contrast, the expression of genes representing CBM1, CBM4, and CBM63 increased in QM6a. CAZymes in these families were found to bind xylan, different glucans, and amorphous cellulose.The genes for some specific CAZyme families are found only in one of the clusters, suggesting enhanced relevance of particular CAZyme families to the physiology of CBS999.97 or QM6a. Of the carbohydrate binding module (CBM) families, CBM13 was found in all three clusters, while all genes representing CBM20  and CBM48 (binding to glycogen) were in cluster 1, i.e., their expression was increased in CBS999.97. As both bound substrates may be involved in storage and energy metabolism, these functions are likely to be important for CBS999.97's ability to grow on cellulose. Accordingly, functional category (FunCat) analysis also revealed an enrichment of genes involved in “metabolism of energy reserves” (The expression of both putative pectin acetylesterases of carbohydrate esterase (CE) family 12 is upregulated in CBS999.97. In contrast, the expression of the seven genes in CE family 5 and CE family 7 increased in the QM6a background. These gene families encode acetyl xylan esterases acting on xylan, among other substrates. This finding is in agreement with the enhanced regulation of CBM4 in this background, which is involved in binding xylan, among other substrates. Additionally, better growth was observed on carbon sources representing hemicellulose building blocks in the Biolog assay .In the glycoside hydrolase families, up- and downregulation often occurred within the groups of regulated genes in a given strain background, reflecting fine-tuning of a specific degradative function. The expression of all four members of glycoside hydrolase (GH) family 28, which are involved in galacturonan degradation, increased in CBS999.97. In contrast, the results of the Biolog assay suggest that enhanced degradation of pectin occurred in strains in the QM6a background, indicating that regulation of the necessary enzymes in QM6a might be better tailored to the utilization of pectin as a substrate. Accordingly, several transcription factors for which a function in plant cell wall degradation was shown are differentially regulated between QM6a and CBS999.97 .hpr1 (upregulated in MAT1-1) and hpr2 (upregulated in MAT1-2). Only 3 other genes were differentially transcribed more than 2-fold (P < 0.01). The expression of TR_121284, encoding an as-yet-uncharacterized protein related to a Saccharomyces cerevisiae N-terminal amidase that is involved in the N-end rule protein degradation pathway, is considerably reduced in MAT1-1. The expression of TR_121499, encoding a SANT family protein, and TR_106178, encoding a putative RNA binding protein, was also reduced in MAT1-1 strains. As no CAZyme genes or other characterized metabolism-related genes were significantly differentially regulated between MAT1-1 strains and MAT1-2 strains, no major differences are predicted for substrate degradation by strains with similar genetic backgrounds but different mating types.In the analysis of differential gene expression between MAT1-1 and MAT1-2, we found the expected differential regulation of the pheromone receptor genes P value of <0.01. Applying this criterion, we found 894 downregulated genes and 424 upregulated genes in strains with the CBS999.97 background relative to those with the QM6a background (Data Set S5).We found considerable differences in gene regulation between strains with the QM6a and CBS999.97 backgrounds, and we set a more stringent threshold of 5-fold differential expression with a P = 4.35 × 10−6), particularly “amino acid metabolism” (P = 1.48 × 10−9), “assimilation of ammonia and metabolism of the glutamate group” (P = 9.64 × 10−4), and “metabolism of aspartate and the aspartate family,” as well as “degradation of aspartate” (P < 10−5 for both). The regulation of amino acid metabolism clearly reflects the differences in carbon source utilization preferences of CBS999.97 and QM6a as detected in the Biolog phenotype microarrays (see above). The lower growth rates of CBS999.97 on proline, l-aspartic acid, l-alanine, l-serine, and l-asparagine were all consistent with the downregulation of genes involved in metabolism of these amino acids and also, in part, their downstream pathways in CBS999.97 relative to QM6a (Data Set S5).A considerable proportion of metabolism-related genes showed increased transcript abundance in the QM6a background relative to their levels in the CBS999.97 background, along with enrichment in genes involved in energy-related processes and oxidative stress response , “purine metabolism” (P = 2.65 × 10−3), “C compound and carbohydrate metabolism” (P = 1.20 × 10−3), and, here specifically, “sugar, glucoside, polyol, and carboxylate catabolism” (P = 1.89 × 10−3), as well as “C-2 compound and organic acid metabolism” (P = 2.21 × 10−3). “Fatty acid metabolism” (P = 2.90 × 10−3), functions related to “energy” (P = 9.40 × 10−8), “respiration” (P = 4.09 × 10−4), ribosomal proteins and translation (P < 10−3), “cellular transport” (P = 1.55 × 10−5), and “oxidative stress response” (P = 9.64 × 10−4), and functions in “oxygen and radical detoxification” (P = 4.21 × 10−4) were further enriched categories among genes downregulated in CBS999.97. Genes upregulated in the CBS999.97 background showed enrichment in “extracellular metabolism and protein degradation” (P = 10−3), “translation initiation” (P = 4.09 × 10−3), and “photoperception and -response” (P = 3.38 × 10−3).Moreover, enrichment was observed in genes involved in “metabolism of the pyruvate family and lxr3, lxr4, gal1, gal10, and bga1. Moreover, hxk1, which is essential for growth on fructose by T. reesei . In contrast, the transcript abundances of cel3B/TR_121735 and bgl3i/TR_47268 were strongly increased and the transcript level of the beta galactosidase gene bga1 was 92-fold reduced (Data Set S5). The gene encoding the regulator BglR showed considerably higher transcript levels in CBS999.97.The carbon source utilization data revealed increased growth on hemicellulose and lactose degradation-associated substrates. Accordingly, genes involved in lactose degradation and that channel metabolites into the pentose phosphate pathway were differentially regulated between strains with QM6a and CBS999.97 backgrounds , particularly . reesei , was strcat8, cat9, and cat10), as well as two superoxide dismutases (sod1 and sod3), were decreased in CBS999.97, suggesting an altered response to oxidative stress conditions in this strain. Also, 14 genes involved in lipid metabolism, 4 genes involved in nitrogen metabolism, and 11 genes associated with sexual development were downregulated.Additionally, the transcript levels of three genes encoding catalases/peroxidases (P < 0.01). Thirty-five genes were downregulated and 55 genes upregulated in fertile strains compared to their expression in the female sterile QM6a strain (Data Set S6). Among the genes downregulated in female fertile strains, we found the catalase-encoding gene cat7, four genes encoding glycoside hydrolases of families 2, 30, 76, and 95 , the sulfite oxidase gene encoding TR_76601, and two transporter genes (TR_71010 and TR_121441), as well as a putative aquaporin gene . Genes upregulated in female fertile strains compared to their expression in female sterile QM6a include the GH family 5-encoding TR_53731, phospholipase C-encoding TR_111102, and the two transporter genes TR_106556 and TR_70934. Even though sexual development does not occur during growth in liquid medium with cellulose as the carbon source, the transcript abundance of the pheromone precursor gene hpp1 was still considerably (∼14-fold) upregulated in QM6a compared to its expression in female fertile strains.We evaluated differential gene expression between female fertile CBS999.97 strains carrying both mating types, the two fertile lines FF1 and FF2, and the female sterile strain QM6a. We found 90 genes to be differentially regulated by more than 2-fold (P = 7.30 × 10−04), specifically, “metabolism of acetic acid derivatives, thioredoxin, glutaredoxin, or glutathione,” and moreover, in “substrate transport” (P = 1.58 × 10−04), as well as “defense and resistance.”With more than 40-fold upregulation, TR_123084, a putative secreted short-chain dehydrogenase, was the most strongly differentially regulated gene. We investigated whether this gene is coregulated with genes for specific functions, which might hint at its potential physiological relevance. Therefore, we evaluated gene expression patterns in the wild type and mutants with mutations impacting photoreceptors  and compThe finding that genes containing a high number of nonsynonymous SNPs were enriched in functions of secondary metabolism (Data Set S1) indicates that QM6a and CBS999.97 likely differ in secondary metabolite production. This conclusion is supported by the transcriptome analysis, which identified differences in the regulation of genes involved in secondary metabolism in the CBS999.97 background compared to their regulation in the QM6a background, hence suggesting altered capabilities for the production of secondary metabolites. In order to discern differences in the regulation of secondary metabolism, we analyzed the metabolites secreted from cultures grown under different conditions with high-performance thin-layer chromatography (HPTLC). In accordance with the transcriptome data, CBS999.97 showed clear differences from QM6a, especially upon growth in buffered liquid Mandels-Andreotti minimal medium with glucose as the carbon source in standing culture  and B. BQuantitative mass spectrometry (liquid chromatography–tandem mass spectrometry [LC-MS/MS]) multitarget analysis of known fungal toxins and metabolites confirmed clear differences between QM6a and CBS999.97. Trichodimerol  and paraTrichoderma is one of the most widely used groups of fungi for both industrial applications in enzyme production and agricultural applications  and strains CBS999.97 MAT1-1, CBS999.97 MAT1-2  , which uses analysis of variation (ANOVA) to identify statistically significant differential regulation of genes. ANOVA enables reliable comparison of more than two samples by avoiding type I errors . For the analysis in this study, the individual data sets were combined according to the respective research question and treated as replicates . For gene expression pattern analyses and hierarchical clustering, the open source software HCE3.5  was usedncatDB/)  The P va in QM6a .18 column  equipped with a C18 SecurityGuard cartridge (4 mm long and 3-mm i.d.) . Calibration was performed with a serial dilution of a multianalyte stock solution. Using this approach, routine detection and quantification of 710 metabolites was performed .For analysis of secondary metabolite patterns, QM6a and CBS999.97 were grown on agar plates or in standing cultures in petri dishes with Mandels-Andreotti minimal medium with 1% (wt/vol) glucose as the carbon source, either buffered at pH 5.0 with 0.1 M phosphate-citrate buffer or unbuffered. Agar pieces or liquid medium from at least three plates were pooled, and two independent biological replicates were evaluated. High-performance thin-layer chromatography (HPTLC) was performed and the results were visualized as described previously , except PRJNA348760 and GenBank accession number MLIW00000000. Transcriptome data are available at Gene Expression Omnibus under accession number GSE89103.Data for the CBS999.97 genome are available under NCBI BioProject accession number


Chlamydia trachomatis (Ct) can induce scarring disease of the ocular mucosa, known as trachoma, the most common infectious cause of blindness worldwide. We hypothesized that epithelial-mesenchymal transition (EMT) contributes to the fibrotic process in trachomatous scarring. Infection of human conjunctival epithelial cells (HCjE) with Ct activated signaling pathways involved in EMT induction, which was correlated with decreased expression of E-cadherin, guardian of the epithelial phenotype. In addition, Ct infection was associated with increased expression of two mesenchymal cell markers: fibronectin and α-SMA. The DNA methylation statuses of selected regions of E-cadherin, fibronectin, and α-SMA genes revealed that Ct infection was accompanied with changes in DNA methylation of the E-cadherin promoter, while the expression of the two mesenchymal markers was not related with this epigenetic event. Our data suggest that Ct infection of conjunctival epithelial cells induces EMT-like changes that go along with modification of the methylation profile of the E-cadherin promoter and could, as one of the earliest events, contribute to processes triggering conjunctival scarring. Chlamydia trachomatis (Ct) via direct smear infection or mechanical transmission , and posttranslational (through numerous stability- and activity-affecting protein modifications) levels. Recent findings have suggested that epigenetic events are master regulators of expression of all EMT-related genes  cells in vitro by analyzing the expression of selected relevant EMT inducers and marker genes , and to investigate whether alterations in gene expression after Ct infection correlate with changes in their DNA methylation profiles and subsequent gene expression. The involvement of epigenetic mechanisms in EMT process in trachoma would open up possibilities for novel therapeutic approaches in trachoma treatment.The aim of this study was to test the ability of Ct and cell lines) or hazardous chemicals were carried out using standard protective measures and following all local and national regulations.All experiments involving biohazards  were grown in McCoy cells (ATCC® CRL-1696™) according to standard procedures . The cells were disrupted with glass beads and centrifuged at 900 × g for 15 min to remove host cell debris. Inoculation stocks and McCoy cells were tested to be negative for mycoplasma.Stocks of 2, and 1000 U/mL penicillin and 1000 μg/mL streptomycin  at 37°C, under 5% CO2 and 95% humidity. The medium was changed every second day, and the cells were passaged at 70% confluence. Cells were harvested by trypsinization  and seeded at a density of 400,000 cells/flask in 75-cm2 flasks. Confluent cultures (an estimated 3.5 × 106 HCjE cells) were infected with 107 infectious units (IFU) Ct in inoculation medium  per flask and the MOI was calculated to be 3. To promote adhesion, the flasks were centrifuged at 900 × g for 1 h at 37°C. After incubation for 1 h at 37°C, the inoculation medium was changed to keratinocyte serum-free medium without antibiotics, and the cells were incubated for another 72 h. Host cell viability was monitored by lactate dehydrogenase assay and immunoblot analysis for PARP-1, procaspase 3, and active form of caspase 3; no differences were observed between control and Ct-infected cells.hTert-immortalized HCjE cells, kindly provided by Prof. Ilene Gipson , were maintained in keratinocyte serum-free medium  supplemented with bovine pituitary extract, 0.2 ng/mL recombinant human epidermal growth factor, 0.4 mM CaClg. For isolation of DNA, RNA, and proteins, an AllPrep DNA/RNA/Protein Mini Kit  was used according to the manufacturer's instructions. Samples were stored at −80°C until analysis.Cells were harvested by scraping and pelleted at 900 × SNAI1 and ZEB2) or 60°C/60 s (SNAI1 and ZEB2). The relative expression levels of target genes were calculated using the comparative 2−ΔΔCt method after normalization using GAPDH as an endogenous control, as no difference in GAPDH mRNA expression between control and Ct-infected HCjE cells was observed.Reverse transcription was carried out with the RevertAid First Strand cDNA Synthesis Kit  on 1 μg DNAse I-treated RNA, using oligo(dT) primers. qRT-PCR was carried out using Maxima SYBR Green/ROX qPCR Master Mix (Fermentas) on a QuantStudio 3 Real-Time PCR system . The thermal cycles used were as follows: initial denaturation at 95°C/10 min and 40 cycles of two-step PCR at 95°C/15 s and 57°C/60 s  using sequences stored in GenBank under the following accession numbers: TGFβ1 (transforming growth factor beta 1) NM_000660; TGFβ2 (transforming growth factor beta 2) NM_003238; CDH1 (cadherin 1) NM_004360; FN1 (fibronectin 1) NM_212482; ACTA2  NM_001141945; SNAI1  NM_005985; ZEB2 (zinc finger E-box binding homeobox 2) NM_014795; GAPDH  NM_002046. The primer sequences are listed in Table Primers were designed with Primer-BLAST  in PBS for 15 min at 37°C. The cells were permeabilized with PBS containing 0.3% Triton X-100 for 10 min and blocked with 3% bovine serum albumin for 1 h. The coverslips were incubated overnight at 4°C with primary antibodies listed in Table Cells were grown on sterile glass coverslips in 24-well tissue culture plates and infected with 10Ct-infected cells was bisulfite-converted using the EZ-DNA methylation kit  according to manufacturer's instructions. CpG islands (short stretches of DNA in which the frequency of CG dinucleotides is higher than in other regions) were predicted using CpG Island Searcher (http://cpgislands.usc.edu/) , fibronectin , and α-SMA  genes. All primer pairs, methylated and unmethylated, for DNA methylation analysis were designed in MethPrimer (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) and are listed in Table Genomic DNA isolated from control and CDH1, and one CpG in forward and two CpGs in reverse primers for FN1 and ACTA2. The reaction mixture for MSP contained Maxima SYBR Green/ROX qPCR Master Mix (Fermentas), 4 μL of bisulfite-converted DNA , and 0.5 μM of M primers in a final volume of 10 μL. MSP was conducted on the QuantStudio 3 Real-Time PCR system. PCR cycling conditions were as follows: initial denaturation step at 95°C/10 min followed by 40 cycles of denaturation at 95°C/15 s and annealing and elongation at 58°C/60 s for CDH1 and FN1 or 55°C/60 s for ACTA2. Reference primers, which do not contain CpGs and exclusively amplify bisulfite-converted DNA, were used as an endogenous control for normalization of the MSP results and as a control for efficient bisulfite conversion. After normalization, the amount of the methylated product was estimated in comparison to in vitro methylated DNA, bisulfite-converted and amplified under the same conditions, which was assumed to be 100% methylated. In vitro methylation was performed using CpG Methylase M.SssI (E2011) , which completely methylates all cytosine residues.MSP analysis was conducted using methylated (M) primers containing two CpGs in each primer (forward and reverse) for Human methylated and non-methylated DNA standards (D5014) (Zymo Research) were bisulfite-converted according to manufacturer's instructions. To generate a range of methylated:non-methylated DNA standards, methylated and non-methylated templates were mixed at 0:100, 25:75, 50:50, and 100:0 ratios. PCR amplification and MS-HRM analysis were carried out sequentially on a QuantStudio 3 Real-Time PCR system. Multiplex PCR with a mix of all four primers  was used to cover all possible variants in methylation status. PCR was carried out in a 10-μL reaction mixture consisting of 5 μL 2 × MeltDoctor HRM Master Mix (Applied Biosystems), 0.15 μM of each primer, and 1 μL bisulfite-converted template . The amplification consisted of initial denaturation at 95°C/10 min, followed by 45 cycles of three-step PCR and a final elongation step at 72°C/7 min. The three-step PCR program consisted of 5 cycles of denaturation at 95°C/30 s, annealing at 58°C/30 s and elongation at 72°C/60 s; 5 cycles of denaturation at 95°C/30 s, annealing at 53°C/30 s and elongation at 72°C/60 s; and 35 cycles of denaturation at 95°C/30 s, annealing at 47°C/30 s and elongation at 72°C/60 s. The HRM program consisted of temperature ramping from 60–95°C by 0.025°C/s with florescence acquisition at each temperature increment. HRM Software v3.1 (Applied Biosystems) was employed for end-product analysis. Peak heights of standards, obtained from difference curves aligned against an unmethylated control (0%), were plotted against the percentage of methylation to obtain a standard curve and linear regression equation. After normalization, the degree of methylation of each sample was quantitatively calculated from the linear regression equation.Bisulfite-modified DNA was amplified using M primers in a 30-μL reaction mixture that included 15 μL 2 × AmpliTaq Gold 360 Master Mix (Applied Biosystems), 0.5 μM of each primer, and 4 μL of bisulfite-converted template . Amplification was carried out on an Eppendorf Mastercycler Pro , using the following thermal cycles: initial denaturation at 95°C/10 min, 45 cycles of three-step PCR, and final elongation at 72°C/7 min. The three-step PCR program consisted of 5 cycles of denaturation at 95°C/30 s, annealing at 62°C/30 s and elongation at 72°C/60 s; 5 cycles of denaturation at 95°C/30 s, annealing at 57°C/30 s and elongation at 72°C/60 s; and 35 cycles of denaturation at 95°C/30 s, annealing at 51.5°C/30 s and elongation at 72°C/60 s. Purification of PCR products and Sanger sequencing of bisulfite-converted DNA were carried out by Eurofins Genomics AT . The sequencing results were analyzed using the BiQ Analyzer software .t-test. For data with non-normal distribution, the Mann–Whitney U test was applied. The null hypothesis was rejected at p < 0.05. “*” Indicates statistically significant difference compared to control cells .Experiments were performed in triplicate and all results are presented as the mean ± SD. IBM SPSS Statistics for Windows, Version 20.0  was used for statistical analysis. The Shapiro–Wilk test was used to determine whether samples followed normal distribution. For normally distributed data, two groups were compared using Student's Ct induces changes in expression profiles of proteins that could trigger a transition of the conjunctival epithelium toward a mesenchymal state, thus contributing to trachomatous scarring. To this end, we analyzed the expression of two TGFβ genes, known as main EMT inducers, using qRT-PCR  CpG island that covers the region from −406 to +1,329 with regard to the position of the TSS. The region selected for DNA methylation analysis encompasses the promoter sequence and TSS, and its genomic position, as well as those of the primers used in this study, is shown in Figure Ct infection initiated a 4% increase in DNA methylation, from 11.5% in control to 15.5% in Ct-infected cells  after Ct infection  located at –635 to +360 from the TSS. We selected a region within the gene body (first exon) for DNA methylation analysis. Positions of the selected region and primers are presented in Figure Ct, these CpG positions were almost completely unmethylated (0.5% of methylation) . Therefore, the MSP results reflect a precise quantification of changes in DNA methylation of the selected region. Based on the MSP and HRM data, it can be concluded that slight demethylation of 3–4% of the selected DNA region in FN1 accompanies Ct infection.The ) Figure . HRM ana Figures . HoweverACTA2 gene contains a CpG island located at –289 to +883 from the TSS. The region that was selected for DNA methylation analysis of ACTA2 is located in the gene body (first exon), as shown in Figure Ct results in a decrease in ACTA2 methylation, although to a lower extent than in MSP analysis (from 15% in control to 11% in Ct-infected cells) , indicating that MSP results give an adequate estimation of the ACTA2 DNA methylation level in the selected region.The  Figures . BisulfiCt infection strongly induces mRNA expression of TGFβ, particularly TGFβ2, in HCjE cells, in line with the finding that lung epithelial cells show enhanced secretion of TGFβ after infection with Ct  (Lee et al., Ct produces NUE methyltransferase, which is able to methylate mammalian histones (Pennini et al., Ct infection and a spectrum of epigenetic changes in the host cell.Our results indicate that EMT could be an additional underlying mechanism of fibrotic processes in trachoma. Using an Ct infection might induce EMT in host cells through DNA methylation changes could play a particularly important role in the development of new therapeutic approaches to trachoma treatment, based on the reversibility of both processes of EMT and DNA methylation. Targeting Ct-induced EMT by demethylating agents, such as the DNA methyltransferase inhibitor azacitidine, might have significant therapeutic potential. However, the main obstacle of epigenetic drugs is their complex network of functions and concomitant variety of side effects associated with systemic application. This could be avoided using gene editing by targeted DNA demethylation and activation of the E-cadherin gene that is essential for maintaining epithelial cell identity. This approach seems as a promising methodology for use in trachoma treatment, as it would potentially enable reversing or at least stopping scar progression in ocular Ct infection.If confirmed, our finding that Initiated the project: TB. Conceptualization: TB, AI, MV, ES, and NG. Conceived and designed the experiments: NG, MV, AI, ES, and TB. Performed the experiments: JR, NG, ES, AI, SD, NS, AU, EG, and MM. Analyzed the data: JR and NG. Contributed reagents/materials/analysis tools: TB and MV. Writing – original draft: JR, NG, MV, AI, ES, and TB. Writing – review and editing: JR, AI, NG, ES, SD, NS, AU, EG, MM, MV, and TB.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.


Chronic HCV Patients taking PEG-IFN-α/R from different ethnic groups have different probabilities of reaching a sustained viral response (SVR). There are many influence factors, such as HCV genotype, IL-28B single-nucleotide polymorphisms (SNP), Fibrosis 4 index (FIB-4), and aspartate aminotransferase-to-platelet ratio index (APRI) score. But the baseline factors in relation to treatment outcome was still not much clear.We evaluated data from 231 chronic HCV patients with or without liver fibrosis and their antiviral efficacy after treatment with pegylated interferon plus ribavirin (PEG-IFN-α/R) for 24–48 weeks. IL-28B SNP and HCV genotypes were analyzed with genome sequencing using pyrosequencing.Sustained viral response (SVR) rates of patients with HCV 1b and 2a genotypes were 52.25% (58/111) and 75.28% (67/89) (P < 0.01). SVR rates of patients with IL-28B rs8099917 TT, rs12979860 CC and rs12980275 AA were 92.41% (25/27), 92.86% (26/28) and 88.89% (24/27) separately. We found that SVR rates in HCV 1b and 2a patients were only 31.0 and 39.4% if their FIB-4 > 3.25. In addition, when their APRI > 2, only 30.3% of HCV 1b patients and 50.2% of HCV 2a patients could obtain SVR.There were high proportion of HCV genotype 1b and 2a in Northwest China. In both HCV 1b and 2a genotypes, patients with protective-genotype of IL-28B were more likely to obtain SVR. However, those with significant fibrosis or cirrhosis were less likely, no matter their genotype. Combined factors of HCV genotype, IL-28B genotype, FIB-4 and ARPI may indicate high prediction and clinical value regarding treatment with PEG-IFN-α/R and prognostic evaluation of chronic hepatitis C patients. Hepatitis C is an infectious disease that is widely spread geographically. It has been reported that about 180 million people are infected with the hepatitis C virus (HCV) worldwide, accounting for about 3% of the current population . The appPatients taking PEG-IFN-α/R from different ethnic groups have different probabilities of reaching a sustained viral response (SVR). In the United States, the rate of obtaining SVR in the black population is almost 50% less than of white patients, and the probability of obtaining SVR in white patients with HCV genotype 1 treated by PEG-IFN-α/R is approximately 42–53% . In ChinDetermination of HCV genotype is important to predict response to antiviral therapy and time of treatment . Early cLiver fibrosis had been suggested being closely associated with the risk of HCC development in chronic hepatitis C patients . The eraTo determine the favorable patients for treatment with PEG-IFN-α/R, HCV genotype, IL-28B SNP, Fibrosis 4 (FIB-4) index and aminotransferase-to-platelet ratio index (APRI) score we retrospectively analyzed with SVR in this study.All patients with CHC were enrolled for this research with signed informed consents following the protocols approved by the Institutional Review Board of the Fourth Military Medical University Table . InclusiWe collected data on 230 patients with chronic HCV infection who were seen at Xijing Hospital from October 2013 to February 2016. The average age was 46.71 years , with 112 male and 118 female patients. Patients had been treated with standard of care for 24–48 weeks. PEG IFN-α/RBV: The recommended dose of PEG IFN-α-2a (Pegasys Roche Shanghai) for chronic hepatitis C was 180 ug per time, once a week, subcutaneous injection of the abdomen or thigh. The dose of RBV was determined by the genotype of virus: the dose for genotype 2 or 3 was 800 mg a day for 24 weeks, and the dose of genotype 1 was 1000–1200 mg daily according to body weight, for 48 weeks. We mainly investigated outcomes after 24 weeks.There are many serological markers for HCV or evidence of liver disease including HCV RNA was used to understand the activity of virus replication, ALT, AST, Total bilirubin, direct bilirubin, indirect bilirubin, albumin, globulin, choline esterase, alkaline phosphatase, phosphatase and abdominal ultrasound were used for evaluation of liver damage, CT or MRI was used to be clear of the extent of liver damage. Liver biopsy is the gold standard for evaluation of liver inflammation and fibrosis staging in patients. In addition, there were also patient compliance issues. After careful consideration we chose these common and easy to get markers as ALT and AST for liver damage, FIB-4 and APRI to evaluate liver fibrosis. Use of viral content to evaluate curative effect was according to the 2014 European Association for the Study of the Liver Recommendations on Treatment of Hepatitis C and the Guideline of Prevention and Treatment of Hepatitis C , 17. We For DNA extraction for IL-28B gene detection, we used a blood genomic DNA extraction kit . For HCV RNA extraction, we used the MinElute column QIAamp method and the virus genome DNA/RNA extraction kit (Tiangen). All extracted DNA/RNA were then immediately used for gene detection or stored at −80 °C.Primer sequences were designed by using DNAMan 6.0.3.99  for IL-28B gene amplification, including IL-28B rs8099917, IL-28B rs12979860 and IL-28B rs12980275 genes . Sequencing primers were designed by the Q24 PyroMark. HCV polymorphism sequencing primers were also provided by Qiagen.9/L), where ULN is the upper limit of normal value. Fibrosis-4 index was based on values of ALT, AST, platelet count and patient age. This index can be used to diagnose liver fibrosis (similar to least significant fibrosis using METAVIR F scoring system ≥2) [9/L] × AST [IU/L]) 1/2.The degree of liver fibrosis was evaluated with APRI score, which can be used for the assessment of liver cirrhosis . APRI scstem ≥2) . A signiP < 0.05 was considered to be statistically significant.We used Pearson chi-squared and Kruskal-Wallis tests to analyze the qualitative data with SPSS 19.0 . A logistic regression model was used to analyze the correlation between SNPs  and SVR of patients. Odds ratio (OR) was used to describe the correlating degree of disease and exposed factors. OR tests were two-sided tests, in which P > 0.05).HCV genotypes of 230 patients with chronic HCV infection were sequenced, with results shown in Table P > 0.05).Fifty-one patients with HCV were included in the IL-28B gene polymorphism loci analyses Table . Of the P < 0.01). In patients with HCV genotype 1b, 70.67% (53/75) did not reach SVR  and 75.28% (67/89), with differences being statistically significant , with OR  of11.02 (3.98–23.16) for rs8099917, 10.20 (5.23–22.14) for rs12979860 and 10.08 (2.98–18.59) for rs12980275. Figure We used SNP Stats Software (tart.htm ) for corWe combined the results of IL-28B SNP and HCV genotype and analyzed the correlation between these factors. We found that 12/51 patients with HCV genotype 1b infection obtained SVR, in which 8/12 cases were rs12979860 CC . Thirty-nine patients had APRI score > 2, including 11 patients with genotype 1b and 8 patients with genotype 2a infection. In this patient group, 30.3% with HCV genotype 1b (11/19 × 58/111) and 50.2% with genotype 2a (8/12 × 67/89) reached SVR (P < 0.05). Although we could not prove that genotype affected the ability to reach SVR in patients with cirrhosis (APRI > 2), we did observe that APRI score > 2 significantly affected SVR. Details are shown in Tables Because there were only 190 out of 231 patients did PLT detection, and APRI and FIB-4 were calculated according to results of patients’ PLT. So APRI score and FIB-4 index were calculated in 190 of 231 patients with chronic HCV infection. Sixty patients had FIB-4 index > 3.25, including 32 patients with genotype 1b infection and 21 patients with genotype2a infection. In this patient group, 31.0% of patients with HCV genotype 1b (19/32 × 58/111) and 39.4% of patients with genotype 2a (11/21 × 67/89) reached SVR . That means more than sixty percent of patients are suitable for PEG-IFN-α/R treatment. Well, the major limitation in this part of study is that the sample amount was relatively too small to determine the role of IL-28B gene polymorphism in antiviral efficacy. However, our findings were in accordance with previous data [Furthermore, our SNP analysis results of 51 patients with chronic HCV infection  and sequencing results of 230 patients with chronic HCV infection showed that more patients had IL-28B rs8099917 TT versus rs8099917 GG, more had IL-28B rs12979860 CC versus TT, and more had IL-28B rs12980275 AA versus GG in Northwest China, similar to some previous reports , 25. Preous data –29.Interestingly, we found that differences in cirrhosis progression between 1b and 2a were statistically significant, confirming that HCV genotype can influence the progression of liver cirrhosis. FIB-4 index > 3.25 or APRI score > 2 indicated that patients had significant liver fibrosis or even cirrhosis . In thisThere were high proportion of HCV genotype 1b and 2a in Northwest China. In both HCV 1b and 2a genotypes, patients with protective-genotype of IL-28B were more likely to obtain SVR. However, those with significant fibrosis or cirrhosis were less likely, no matter their genotype. Combined factors of HCV genotype, IL-28B genotype, FIB-4 and ARPI potentially have a very high prediction and clinical value regarding treatment with PEG-IFN-α/R and prognostic evaluation of chronic hepatitis C patients.


Pseudomonas aeruginosa is a major cause of bacterial keratitis (BK) worldwide. Inappropriate or non-optimal antibiotic chemotherapy can lead to corneal perforation and rapid sight loss. In this study, we tested the hypothesis that P. aeruginosa strain PAO1 invades primary human corneal fibroblasts (hCFs) in vitro and persists intracellularly, despite chemotherapy with antibiotics used commonly to treat BK. In rank order, ciprofloxacin, levofloxacin and polymyxin B showed the highest activity against planktonic PAO1 growth , followed by gentamicin and ofloxacin . These bactericidal antibiotics (50–200 μg/mL concentrations) all killed PAO1 in the extracellular environment of infected hCF monolayers. By contrast, the bactericidal antibiotic cefuroxime and the bacteriostatic antibiotic chloramphenicol failed to sterilize both PAO1 broth cultures, even at a concentration of ≥200 μg/mL) and infected hCF monolayers. Statistically, all antibiotics were able to prevent LDH release from PAO1-infected hCF monolayers at both concentrations tested. Intracellular Pseudomonas were significantly reduced  following treatment with ciprofloxacin, levofloxacin and ofloxacin, whereas gentamicin, polymyxin B and cefuroxime failed to clear intracellular bacteria over 24 h. Intracellular Pseudomonas infection was resistant to chloramphenicol, with hCF death observed by 9 h. Eventual growth of remaining intracellular Pseudomonas was observed in hCF after removal of all antibiotics, resulting in re-infection cycles and cell death by 48 h. All of the antibiotics reduced significantly (P < 0.05) IL-1β secretion by hCF infected with a Multiplicity Of Infection (MOI) = 1 of PAO1. With higher MOI, no pro-inflammatory effects were observed with antibiotic treatment, expect with polymyxin B and ofloxacin, which induced significant increased IL-1β secretion (P < 0.001). The findings from our study demonstrated that bactericidal and bacteriostatic antibiotics, routinely used to treat BK, failed to eradicate Pseudomonas infection of hCFs in vitro and that their bactericidal efficacies were influenced by the cellular location of the organism. Pseudomonas aeruginosa is the leading cause of bacterial keratitis (BK) associated with contact lens wear  in vitro and subsequently invade. We also reported that hCFs were a source of IL-1β secretion in response to challenge with P. aeruginosa in vitro  in vitro could persist intracellularly, despite chemotherapy with antibiotics used commonly to treat BK, e.g., fluoroquinolone , aminoglycoside (gentamicin), polypeptide (polymyxin B), semisynthetic cephalosporin (cefuroxime) and synthetic bacteriostatic (chloramphenicol) antibiotics. We have screened and compared the pharmacodynamics, antimicrobial and anti-inflammatory activities of these different antibiotic therapies currently available for clinical use in the United Kingdom to treat BK  was obtained from the National Collection of Industrial, Food and Marine Bacteria (NCIMB), United Kingdom. PAO1 was grown in Luria-Bertani  broth and on nutrient agar (Oxoid) at 37°C.2 at 37°C.Corneal epithelium was scraped from corneo-scleral rims of healthy donors and the stromal layer was dissected and digested with collagenase type-1  for 3 h at 37°C. Digested stroma was cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 5% (v/v) decomplemented Fetal Calf Serum , 100 Units/mL of penicillin and 100 Units/mL of streptomycin (Life Technologies) and 0.5 μg/mL of amphotericin B (Lonza) and fibroblasts were characterized as described previously . The numIn experiments to examine the efficacy of different antibiotics against extracellular and intracellular PAO1, hCFs were cultured in antibiotic-free DMEM supplemented with 5% (v/v) dFCS for at least one passage prior to bacterial challenge, in order to ensure the absence of possible traces of antibiotics.Patients provided written informed consent in accordance with the Declaration of Helsinki to use surplus corneal tissue specimens for research via the NHS Blood Transplant Eye Retrieval Service. Protocols were approved by the NRES Committee South Central - Berkshire 06/Q1602/56.in vitro study were based on those commonly used in United Kingdom clinical practice, namely the bactericidal antibiotics ciprofloxacin , cefuroxime 0.5% (w/v) , polymyxin B , gentamicin , ofloxacin (0.3% w/v) , levofloxacin (0.5% w/v)  and the bacteriostatic antibiotic chloramphenicol 0.5% (w/v) . A broth dilution assay was done to determine PAO1 susceptibility to each antibiotic. Various concentrations of antibiotic  were added to equal volumes of LB broth medium with various concentrations of bacteria /mL). Absorbance  λ600 nm) as a measure of bacterial growth was assessed hourly up to 9 h and then at 24 h of incubation at 37°C with shaking (200 rpm). Comparison of the 24 h OD readings for bacterial growth with and without antibiotics was used to calculate the lowest concentration of antibiotic that completely reduced OD growth by 100% and the concentration of antibiotic that reduced OD growth by 50%.The antibiotics chosen for this 5 hCF/well) with different Multiplicities Of Infection  of PAO1. At given time-points, cell monolayers were washed gently four times with phosphate buffered saline (PBS), pH7.4 and associated PAO1 bacteria were quantified by viable counting on nutrient agar after hCF lysis with a buffer of PBS containing 0.1% (w/v) saponin and 0.1% (v/v) dFCS, as described previously , then treated with antibiotics and IL-1β cytokine was quantified from 9 h antibiotic-treated-hCF supernatants using the Meso Scale Discovery (MSD) electro-chemiluminescence assay , following the manufacturer’s instructions.The cytotoxicity of CIP, CXM, PMB, GEN, OFX, LVX and CHL (50 and 200 μg/mL) alone to hCFs and of PAO1 infection  with and without antibiotics, was examined after 0, 1, 3, 6, 9, and 24 h of incubation. Release of lactate dehydrogenase (LDH) was measured using the Pierce LDH Cytotoxicity Assay Kit (Thermo-Scientific) and the percentage cytotoxicity was calculated following the manufacturers’ instructions.t-test. P-values < 0.05 denoted significance.Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test or by unpaired in vitro model of primary hCF cells derived from the stroma, to evaluate current antibiotic treatments used in the United Kingdom to treat eye infections. Initially, we used a broth dilution method to calculate the lowest concentration of GEN, CXM, OFX, PMB, CIP, CHL and LVX antibiotics that completely reduced OD growth of P. aeruginosa strain PAO1 (tested at various concentrations of ∼105 – ∼107 CFU/mL) by values of 50 and 100%. In rank order, the fluoroquinolones CIP and LVX and polymyxin PMB showed the highest antimicrobial activities with 100% reduction in OD with a concentration ≤10 μg/mL and 50% reduction in OD with a concentration ≤1 μg/mL (Table 1). This was followed by GEN and OFX (100% reduction in OD with concentrations ≤50 μg/mL and 50% reduction in OD with concentrations ≤10 μg/mL) and lastly by CXM and CHL treatments, which showed the lowest activities (≥200 μg/mL) and failed to sterilize the PAO1 broth cultures .In the current study, we used an Table 1). For some of the antibiotics, e.g., CIP, LVX and PMB, these concentrations were significantly higher than the concentrations that could completely inhibit bacterial growth as judged by the broth dilution assay, but could be seen as potentially representing accumulated antibiotic concentrations in clinical use with repeated dosing of infected eye tissue. hCF cultures were infected with PAO1 , antibiotics added at time = 0 h and extracellular growth of the organism quantified (CFU) over time . Concentrations of 50 and 200 μg/mL of CIP, LVX, PMB, GEN and OFX killed all PAO1  in the extracellular environment by 1 h . Treatment with CXM at 50 μg/mL resulted in a >99% reduction of extracellular bacterial CFU by 6 h (P < 0.05), but this was not maintained and bacterial CFU increased thereafter to levels similar to bacterial growth observed in PAO1-infected monolayers not treated with antibiotics . CXM treatment only killed extracellular PAO1 when used at a concentration of 200 μg/mL (P < 0.05 up to 6 h and P < 0.001 at 24 h), with sterility occurring between 6–9 h . By contrast, CHL was ineffective at killing PAO1 at both concentrations  (P > 0.05), with extracellular CFU of PAO1 increasing over time to levels  similarly observed in PAO1-infected monolayers not treated with antibiotics .Next, we examined the bactericidal ability of the antibiotic panel to control PAO1 extracellular infection of cultured hCF cells. The antibiotic concentrations to be tested were chosen as 50 and 200 μg/mL based on their ability to reduce planktonic growth of PAO1 in broth . We next examined whether the antibiotic treatments protected hCF monolayers from PAO1-induced cell death. In general, antibiotics alone were not cytotoxic to hCFs in vitro, except for treatment with PMB, which increased LDH release by 30–40% by 24 h, and with OFX to a lesser extent (∼10% increased LDH release) . By visual inspection, PAO1-infected hCF monolayers treated and maintained with the antibiotics CIP, LVX, GEN and PMB were intact after 24 h. OFX-treated hCF cells showed signs of apoptosis by morphology, i.e., cell rounding and shrinkage, although the monolayers were still intact. By contrast, CXM or CHL treatments failed to protect hCF monolayers from PAO1 infection, with cell death and monolayer detachment occurring between 9 and 24 h.PAO1 infection is cytotoxic to hCFs Figure 2). Monolayers were infected with PAO1 , antibiotics were added at 3 h and LDH release measured at 9 and 24 h. At a MOI = 1, there was no significant difference at 9 h in the cytotoxicity levels between CIP and GEN concentrations and no antibiotic (P > 0.05), whereas cytotoxicity levels were increased marginally following LVX, OFX, CXM, CHL (P < 0.05) and significantly with PMB (P < 0.0001) treatments. By 24 h, significant reductions in cytotoxicity were observed with CIP, LVX, GEN and OFX (P < 0.0001), PMB and CXM (P < 0.05) and CHL (P < 0.01).The protective effect of antibiotics was confirmed by measuring LDH release (P < 0.001) and OFX (P < 0.05) and with 200 μg/mL concentration only of CXM (P < 0.05). With PMB treatment, there was no significant difference in cytotoxicity levels with the 50 μg/mL concentration (P > 0.05), but the 200 μg/mL concentration increased cytotoxicity significantly (P < 0.001). By 24 h, highly significant reductions in cytotoxicity were observed with 50 and 200 μg/mL of CIP, LVX, PMB, GEN, OFX and CXM (P < 0.0001). CHL used at a concentration of 50 μg/mL had no significant effect on LDH release at either 9 or 24 h (P > 0.05), but percentage cytotoxicity was reduced with the higher concentration tested (P < 0.05) .At a MOI = 10, there was a significant reduction by 9 h in percentage cytotoxicity with 50 and 200 μg/mL concentrations of CIP, LVX and GEN (P < 0.0001) and GEN and OFX (P < 0.01) and with CHL . By contrast, PMB used at 200 μg/mL significantly increased cytotoxicity (P < 0.05). By 24 h, a 50 μg/mL concentration of CIP, LVX, OFX, PMB, GEN and OFX significantly reduced cytotoxicity (P < 0.0001), but both CXM and CHL had no significant effect (P > 0.05). With the higher concentration of 200 μg/mL, significant reductions were observed for all antibiotic treatments .At a MOI = 100, there was a significant reduction by 9 h in percentage cytotoxicity with concentrations of 50 and 200 μg/mL of CIP and LVX . The numbers of invasive PAO1 were quantified as ∼5 × 102 CFU/monolayer with an initial MOI = 1, ∼103 CFU/monolayer for an initial MOI = 10 and ∼5 × 104 CFU/monolayer for an initial MOI = 100 .Recently, we have shown with adherence and gentamicin exclusion assays that y invade . In the Figure 3), after which antibiotics (50 or 200 μg/mL) were added to the cultures. The numbers of intracellular PAO1 were then quantified after 1.5, 4.5, 7.5, and 24 h of antibiotic treatment. A clear rank order for antibiotic efficacy was observed against intracellular PAO1 in vitro. The fluoroquinolones CIP, LVX and OFX demonstrated the highest antimicrobial activity . Treatment with CIP and LVX (50 and 200 μg/mL) killed >99.99% of all intracellular bacteria by 3 h, regardless of MOI used to infect the monolayers . Treatment with OFX (50 or 200 μg/mL) eradicated >99.99% of intracellular bacteria by 3 h from hCF monolayers infected initially with an MOI = 1 . With increasing MOI = 10–100, between ∼102 – ∼103 intracellular bacteria were recovered by 24 h after OFX (50 or 200 μg/mL) treatment, which still represented high antimicrobial activity of >99.9% reduction in CFU .We next tested the hypothesis that the ability of PAO1 to invade hCFs enabled the organism to evade antibiotic chemotherapy, thereby creating a potential reservoir for reactivation of bacterial infection. In order to examine the effects of antibiotics on intracellular bacteria, hCF monolayers were infected with PAO1  for 3 h to allow bacterial invasion to occur (3 – ∼105 intracellular bacteria were recovered after treatment for 3 h with GEN or PMB (50 and 200 μg/mL), and these intracellular PAO1 numbers essentially remained unchanged by 24 h . CXM treatment at 50 μg/mL could not clear the infection (intracellular or extracellular), and exponential increases in bacterial CFU were observed , and the numbers of intracellular PAO1 enumerated at 3 h (∼103 – ∼105 CFU) in the presence of 200 μg/mL of CXM remained essentially unchanged by 24 h . These experiments confirmed also that PAO1 was resistant to CHL, with exponentially increasing numbers of bacteria recovered over time intracellular and extracellularly . With OFX, intracellular PAO1 were recovered from infected hCF-monolayers at 24 h  and after antibiotic removal, intracellular CFU numbers showed similar exponential increases up to 24 h . With these three treatments, extracellular bacteria were recovered by 9 h onward after antibiotic removal and CFU numbers increased exponentially . For both PMB and GEN, which failed to clear intracellular bacteria after 24 h , removal of the antibiotics resulted in increased CFU numbers intracellularly and early recovery of extracellular bacteria . Regardless, after removal at 24 h of all the different antibiotics, by 24–48 h later, all the monolayers were destroyed as a consequence of uncontrolled bacterial growth. All the bacteria recovered at 48 and 96 h now represented growth in host cell-free cultures.Clinically, a high incidence of recurrent BK has been observed in patients after cessation of antibiotic treatment . We nextP. aeruginosa PAO1 induces significant extracellular release of the cytokine IL-1β . Baseline levels of IL-1β in uninfected monolayers without antibiotic treatments were ≤0.25 pg/mL . Addition of some of the antibiotics, i.e., PMB, OFX, CXM and CHL, did statistically increase IL-1β release from uninfected monolayers, but the levels never exceeded 6 pg/mL  IL-1β levels (≥40 pg/mL) in the presence of bacteria . With MOI = 100, ∼50–60 pg/mL IL-1β was induced  and none of the antibiotics used at a concentration of 50 μg/mL statistically reduced IL-1β release (Supplementary Table P > 0.05) measured, whereas addition of 200 μg/mL of OFX was pro-inflammatory, significantly (P < 0.001) increasing IL-1β release by ∼2-fold (>100 pg/mL) compared to no antibiotic, infected hCF cells  are administrated hourly to the eye. However, these agents are diluted by the tears with the majority draining through the nasolacrimal duct   , but has (MDR-P) . CXM treistrated . In our losporin .in vitro infection model, we observed eventual intracellular growth of P. aeruginosa after cessation of antibiotic treatment, followed by pathogen release and cell death. It has been reported that high levels of recurrent BK follow the cessation of antibiotic treatment  production , which iin vitro Pseudomonas infection of a monoculture model of primary hCFs. Both antibiotics eliminated extracellular and intracellular bacteria and reduced a cardinal inflammatory cytokine signal, which is observed in BK. The study also suggested that antibiotic concentration regimens must be sufficiently prolonged to eliminate P. aeruginosa intracellular infection, in order to minimize the risk of emergent bacteria that could initiate new cycles of infection.In summary, our study identified the fluoroquinolone CIP, followed by LVX, as the most effective antibiotics for treating MdMC, PH, and MC designed the experimental research. MdMC performed the experiments. MdMC, PH, and MC interpreted and analyzed the data. MC, MdMC, and PH wrote the manuscript. MC and PH contributed equally as PIs to this study.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.


In contrast, neither systemic injection (0.25–0.75 mg/kg) or intrastriatal infusion (30 μM–1 mM) of AZD1446 had a significant effect on DA efflux in WT or DYT1 KI mice. In vitro, we found that AZD1446 had no effect on the membrane properties of striatal spiny projection neurons (SPNs) and did not alter the spontaneous firing of ChI interneurons in either WT or DYT1 KI mice. We did observe that the firing frequency of dopaminergic neurons was significantly increased by AZD1446 (10 μM), an effect blocked by dihydro-beta-erythroidine (DHβE 3 μM), but the effect was similar in WT and DYT1 KI mice. Our results support the view that DYT1 models are associated with abnormal striatal cholinergic transmission, and that the DYT1 KI animals have enhanced sensitivity to nicotine. We found little effect of AZD1446 in this model, suggesting that other approaches to nicotinic modulation should be explored.DYT1 dystonia is an early-onset, hyperkinetic movement disorder caused by a deletion in the gene TOR1A, which encodes the protein torsinA. Several lines of evidence show that in animal models of DTY1 dystonia, there is impaired basal dopamine (DA) release and enhanced acetylcholine tone. Clinically, anticholinergic drugs are the most effective pharmacological treatment for DYT1 dystonia, but the currently used agents are non-selective muscarinic antagonists and associated with side effects. We used a DYT1 ∆GAG knock-in mouse model (DYT1 KI) to investigate whether nicotine and/or a non-desensitizing nicotinic agonist, AZD1446, would increase DA output in DYT1 dystonia. Using  DYT1 dystonia is a form of early onset isolated dystonia  are made up of pentameric assemblies of receptor subunits . They are broadly distributed across the brain. In the striatum, the most commonly expressed of the subunits are α4β2, which make up approximately 70% of striatal nAChR , as appropriate. All members of the staff involved in the experiments have undergone a specific training for handling and working with rodents. All efforts were made to reduce the number of animals used and minimize their suffering.∆E/+; . PCR products were run using a 2% agarose gel. Adult (3–6 months of age) male and female mutant (Tor1A∆E/+) and littermate controls (TorA+/+) were used for the following experiments.Heterozygous DYT1 mutant knock-in mice (Tor1A+/+ and Tor1A∆E/+ littermates were anesthetized with isoflurane (1%–3%) and placed in a stereotaxic apparatus. Unilateral microdialysis cannulae  were implanted vertically above the striatum  at a constant rate of 2 μL/min for the duration of the experiment. Following habituation, samples were collected every 20 min. To determine the effect of nicotine on striatal DA release, following habituation, mice were striatally infused with 10 mM nicotine dissolved in aCSF for 1 h. To examine the effects of systemic AZD1446 on striatal DA, WT mice underwent a dose-response where AZD1446 was either systemically injected  or striatally infused  for 1 h per dose. The genotype-specific effect of AZD1446 on striatal DA release was determined using reverse microdialysis (300 μM AZD1446) for 1 h in WT and DYT1 KI mice. Mice were killed following each procedure. To ensure correct placement of microdialysis probes, brains were post-fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose after 48 h, and sectioned using a freezing microtome. Correct placement was visually determined to be 100% of all mice used.Male Tor1As et al. . Two anc−6 M to 10−9 M.For microdialysate collected during the nicotine infusion experiment, reverse-phase HPLC-ED was used to determine concentration of DA in each sample, according to the protocol of Kilpatrick et al. , a metho−6 M to 10−9 M, and expressed as [DA] in nM.For the AZD infusion and systemic injection microdialysis experiments, DA was measured in microdialysate using HPLC-ED  with an enzyme reactor  and an electrode . The mobile phase consisted of a 50 mM dibasic potassium phosphate buffer (pH 8.5). Microdialysate sample chromatographs were analyzed based on established concentration curves for DA from 102 and 5% CO2, containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 glucose, 25 NaHCO3. Sagittal and coronal slices  were used for medium spiny projecting neurons (SPNs) and ChIs recordings, while horizontal slices (300 μm thickness) were used in order to better preserve substantia nigra. After 30–60 min recovery at room temperature, slices were transferred individually in the recording chamber (0.5–1 mL volume) and continuously superfused with oxygenated Krebs’ medium (2.5–3 mL/min at 32–33°C).Preparation and maintenance of corticostriatal and midbrain slices were carried out as previously described  was used to visualize neurons on a PC monitor. Recordings were performed on individual cells by means of IR-DIC videomicroscopy with an Olympus BX51WI equipped with a 40× water immersion objective . Intracellular recordings were performed blindly using sharp microelectrodes filled with 2 M KCl (40–60 MΩ). This electrophysiological approach was used to retain the fidelity of post-receptor signaling pathways. Glutamatergic excitatory postsynaptic potentials (EPSPs) were evoked with a bipolar electrode in the cortex (V–VI layer) in picrotoxin (50 μM) to block GABA receptors. Synaptic stimuli were delivered at 0.1 Hz, and six events were averaged. Paired-pulse facilitation was assessed by presenting two stimuli, which evoked synaptic responses at ~50% of maximal amplitude, with an interstimulus interval of 50 ms and measuring the ratio of the peak amplitude of the second EPSP (EPSP2).+ internal solution containing (in mM): 125 potassium gluconate, 10 NaCl, 1.0 CaCl2, 2.0 MgCl2, 0.1 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetra-acetic acid (BAPTA), 10 HEPES, 0.3 GTP, 2.0 MgATP; pH adjusted to 7.3 with KOH. Under voltage-clamp conditions, cells were clamped at −60 mV and series resistance (8–15 MΩ) was monitored during the course of the experiment by the peak amplitude of the capacitive transient induced by a −5 mV pulse. Signal acquisition and offline analysis were performed with an Axoclamp 2B (Axon Instruments) and pClamp9 software .Patch clamp recordings, either in cell-attached or whole-cell configuration, were made using borosilicate glass pipettes  pulled on a P-97 Puller . Pipettes resistances ranged from 3 MΩ to 5 MΩ. Membrane currents were continuously monitored and access resistance, measured in voltage-clamp, was in the range of 5–30 MΩ. For cell-attached or whole-cell recordings on ChIs, pipettes were filled with KExtracellular single-unit signals were recorded at 34 ± 0.5°C from dopaminergic neurons of the substantia nigra pars compacta (SNpc) located in a submerged chamber with aCSF flowing at 2.5–3 mL/min, on the stage of an upright microscope , equipped for infrared video microscopy . Glass microelectrodes had a tip resistance of 10–20 MΩ and were filled with aCSF. The recordings were performed with an Axoclamp 900A amplifier , using Clampex 10 software and digitized with a Digidata 1440A. Dopaminergic neurons, selected on the basis of their localization and morphology, were identified by their well-known properties, such as the presence of a regular spontaneous firing (0.5–4 Hz), firing inhibition in response to DA  and Prism 6.0 e (GraphPad). Numerical data are presented as means ± SEM; n values represent independent observations. The evaluation of statistical difference was performed using Student’s (-)Nicotine hydrogen tartrate salt, DA hydrochloride and CNQX were obtained from Sigma-Aldrich , whereas picrotoxin and MK-801 were obtained from Tocris (UK). AZD1446 was obtained from AstraZeneca (Sweden). For electrophysiological experiments, drugs were prepared in stock solutions and bath-applied at known concentrations via a three-way tap system. A complete exchange of the solution in the recording chamber occurred in about 1 min. For microdialysis, nicotine was dissolved in aCSF and pH adjusted to 7.4. AZD1446 was prepared in stock solution in aCSF for striatal infusion and in water for systemic injections.t-test; ANOVA p < 0.05).Previous physiological studies have indicated that nicotinic receptor activation by tonic acetylcholine release is needed to ensure that striatal DA terminals are sensitive to stimulation , DYT1 KI (n = 4); ANOVA, p > 0.05). Since there was no difference seen systemically, we explored local intrastriatal administration of AZD1446 by reverse microdialysis. In WT mice, AZD1446 did not affect extracellular striatal DA across a range of doses from 30 μM to 1000 μM , DYT1 KI (n = 5); ANOVA, p > 0.05). Based on these data and in order to allow for a bidirectional change in DA output in DYT1 KI mice, a 300 μM concentration of AZD1446 was employed to determine if the DYT1 mutation would alter the response of striatal DA efflux to this drug. However, AZD1446 failed to elicit a significant increase in DA output in either WT or DYT1 KI mice .Since DYT1 KI mice showed an increase in striatal DA in response to nicotine, we explored whether AZD1446 could enhance striatal DA efflux when delivered either systemically or intrastriatally in a DYT1 knock-in mouse model. When AZD1446 was given systemically in WT mice, no difference in extracellular DA was observed in doses ranging from 0.25 mg/kg to 0.75 mg/kg  1.308 ± 0.22%; DYT1 KI (n = 23) 1.05 ± 0.5%; ANOVA p > 0.05).In order to characterize the mechanism of action of AZD1446, we performed intracellular recordings on SPNs before and after acute bath-application of known drug concentrations. Low and moderate concentrations of AZD1446 (0.01–5 μM) failed to modify intrinsic properties of SPNs recorded from WT and DYT1 KI mice; in contrast, high concentrations (>5 μM) of AZD1446 produced a significant depolarization linked with an increased membrane resistance Figures . Then, wn = 3) 91.2 ± 3.02%; DYT1 KI (n = 6) 89.2 ± 9.66%; 10 μM: WT (n = 4) 92.2 ± 2.95%; DYT1 KI (n = 4) 91.2 ± 7.30%; ANOVA, p > 0.05). To rule out a possible desensitizing effect, AZD1446 was delivered by means of a single puff, allowing a focal, fast drug perfusion. AZD1446  did not produce a significant effect on firing frequency or membrane potential of ChIs, both in WT and DYT1 KI mice  107.3 ± 2.11%; DYT1 KI (n = 6), 108.1 ± 9.38%; p > 0.05).ChIs are known to play a key role in regulating DA release : 1.321 ± 0.1139, paired t-test p = 0.048; DYT1 KI (n = 4): 1.336 ± 0.07; paired t-test p = 0.0224). No difference was found by comparing the two strains . In accord with the pharmacological profile of AZD1446, pretreatment with the selective α4β2 receptor antagonist DHβE  prevented the excitatory effect of AZD1446 on nigral neurons in both animal groups .Perfusion with AZD1446  caused a modest but significant increase of spontaneous firing activity of recorded cells both in DYT1 KI and WT slices (Figure in vivo. However, the α4β2-specific, non-desensitizing nAChR agonist AZD1446 did not have significant effects on DA efflux systemically or intrastriatally in either WT or DYT1 KI mice. In vitro, AZD1446 did not alter membrane properties or firing of ChIs in DYT1 KI mice. While there was no effect on ChIs, AZD1446 had a small effect on dopaminergic neuron firing which could be blocked with nicotinic antagonist DHβE.The goal of this study was to determine the effect of AZD1446 on striatal function in an animal model of DYT1 dystonia. In this study, we found that DYT1 KI mice are more sensitive than WT animals to nicotine’s DA stimulating effects when the drug is applied directly to the striatum in vivo microdialysis, chronic nicotine has been shown to significantly increase DA efflux in the striatum of WT mice (Salas and De Biasi, Nicotinic receptors have long been known to regulate DA release. Each subunit of the pentameric structure has different properties and contributes to DA efflux differently. α7 nAChR subunits are usually found in a homomeric state and mostly found on dopaminergic neuron terminals (Quick and Lester, Basic physiological studies indicate that tonic acetylcholine release acts on nicotinic receptors on striatal dopaminergic terminals, rendering them sensitive to action potentials (Rice and Cragg, in vitro there was no effect on ChI firing activity in WT or DYT1 KI mice (Figures AZD1446 is a non-desensitizing nicotinic agonist selectively acting on α4β2-containing nAChRs. It is of particular interest because it has been used in human subjects, where it has been found to be generally safe and well tolerated (Mazurov et al.,  Figures , 4. We d Figures .Collectively, our data indicate that AZD1446 is not a particularly promising candidate for development as a treatment for DYT1 dystonia. Nicotinic agents as a class, however, do appear to have substantial promise, as illustrated by the differences in DA release in response to nicotine in DYT1 and WT animals. As such, nicotinic agents likely have a place in the field of DYT1 dystonia and their effects should continue to be studied to help understand more about the disease and possible therapeutics.DGS, AP and CNZ designed the research; MM, FRR, MS and CNZ performed the research; MM, FRR and CNZ analyzed the data; CNZ, KLEJ, DGS and AP wrote the manuscript; DGS and AP obtained funding.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.