Aloe pulcherrima Gilbert & Sebsebe is one of the endemic Aloe species traditionally used for the treatment of malaria and wound healing in central, Southern and Northern part of Ethiopia. The aim of the current study was, therefore, to isolate active compounds from roots of A. pulcherrima and evaluate for their antibacterial and antiplasmodial activities using standard test strains. Bioassay-guided sequential extraction and column chrom-atographic separation were employed for the isolation of bioactive pure compounds. The structures of the compounds were determined by 1D and 2D NMR spectro-scopic techniques. Disk diffusion method was employed to evaluate the antibacterial activities of the isolated compounds against four bacterial strains specifically . The malaria SYBR Green I-based in vitro assay technique was used for in vitro antiplasmodial activity evaluation of the compounds against chloroquine resistant (D6) and -sensitive (W2) strains of P. falciparum. Three compounds, chrysophanol, aloesaponarin I and aloesaponarin II were isolated from the acetone extracts of roots of A. pulcherrima. Evaluation of antibacterial activities revealed that aloesaponarin I and aloesaponarin II had significant activities against all the bacterial strains with inhibition zone diameters ranging from 18–27 mm as compared to the reference drug (gentamicin), which displayed inhibition zone diameter ranging between 20 mm (B. subtilis) and 25 mm (P. aeruginosa). The isolated compounds showed moderate in vitro antiplasmodial activity against both chloroquine resistant (W2) -sensitive (D6) strains. Isolation of chrysophanol, aloesaponarin I and aloesaponarin II from roots of A. pulcherrima is the first report of its kind. The finding could be used for further comprehensive evaluation of the isolated compounds for their antibacterial and antimalarial activities besides consideration of the same for potent drug development.Medicinal plants with documented traditional uses remain an important source for the treatment of a wide range of ailments. Evidence shows that majority of the Ethiopian population are still dependent on traditional medicine.  Medicinal plants represent the oldest and remain an indispensable source of novel and effective pharmaceuticals. The advent of phytochemistry and pharmaceutical chemistry has enhanced the ability to utilize active compounds isolated from plants, or their synthetic equivalents in medicine. This is mainly because of the broader degree of chemical diversity and novelty possessed by medicinal plants than any other sources , 2.The development of multi-drug resistance in the microbial world is posing serious challenges in the treatment of infectious diseases caused by several arrays of pathogenic microorganisms especially in the intensive care units –5. In suAloe pulcherrima Gilbert & Sebsebe against selected etiological agents of bacterial and malarial diseases. The genus Aloe (family Asphodelaceae) is the largest genus in the family comprising over 500 species . Analytical TLC was performed on pre-coated silica gel 60 F254 plates, and UV-254/365 nm chamber (UV-Tec) was used for detection of spots on TLC. UV spectra were recorded on a Specord S600 . 1H NMR (600 MHz) and 13C NMR (150 MHz) were recorded on a Bruker Avance 500 spectrometer using the residual solvent peaks as a reference. HSQC and HMBC spectra were obtained using the standard Bruker software. Mueller Hinton agar and nutrient broth were used for antibacterial activity test while Indochina W2 (chloroquine-resistant) and the Sierra Leone D6 (chloroquine-sensitive) Plasmodium falciparum strains were used for antiplasmodial activity test.Analytical grade solvents , was collected from uncultivated land of ‘Saka Chokorsa’ district of Jimma zone , South Western Ethiopia, in April, 2015. This research undertaking is part of an ongoing study being carried out on plants of Asphodelacae family found in Ethiopia. A. pulcherrima is a wild plant collected from none protected area of the study site. Furthermore, this study did not involve endangered or protected species. Accordingly, no specific permission was required for collection and investigation of the aforementioned plants. Identification of the plant was made by professional botanist at Jimma University (Dr. Kitessa Hundera), and a voucher specimen was deposited at the Herbarium of Biology Department, Jimma University under voucher number AP001/2015. Roots of the plant were air-dried and fine powdered to ease the subsequent extraction processes.Root of A. pulcherrima (810 g) were sequentially extracted with n-hexane, chloroform, acetone and methanol (2 x 2 L each) by percolation at room temperature. The extracts were concentrated using rota vapour under reduced pressure to yield a brown crude products weighing 2.0 g (0.25%), 3.2 g (0.39%), 4.9 (0.61%) and 4 g (0.49%), respectively. The extracts were screened for their antibacterial activity against four bacteria strains namely S. aureus (ATCC 25923), E. coli (ATCC 35218), P. aeruginosa (ATCC 27853) and B. subtilis (ATCC 6633), and extract with better activity was subjected to column chromatography. Based on the antibacterial activity of the acetone extract, it was further subjected to column chromatography (column size: 60 cm length and 4 cm diameter) on oxalic acid impregnated silica gel (200 g) eluting with n-hexane containing increasing amounts of ethyl acetate to afford 24 major fractions (ca. 200 mL each). Fractions 8–10 (2% ethyl acetate in n-hexane) that showed yellow fluorescing spots were combined and further purified using column chromatography (column size: 40 cm length and 2 cm diameter), eluted with increasing gradient of ethyl acetate in n-hexane to give chrysophanol . Fractions 20–29 (10–20% ethyl acetate in n-hexane) contained mixtures and showed two major fluorescing compounds when viewed under UV light at 366 nm, which were combined and subsequently subjected to repetitive column chromatography (column size: 60 cm length and 2 cm diameter), eluted with increasing gradient of ethyl acetate in n-hexane to yield aloesaponarin II  and aloesaponarin I  (The air-dried root samples of 16.3 mg) .in vitro antibacterial activity of the crude extracts and isolated compounds were determined against four bacteria strains listed above. The antibacterial activities of the crude extracts and pure compounds against these bacteria strains were carried out following a standard procedure as used earlier by Dagne et al , containing SYBR green I  was added directly to the plates and gently mixed using the Beckman Coulter Biomek 2000 automated laboratory workstation . The plates were then incubated for 5–15 min at ambient temperature in the dark. Parasite growth inhibition was quantified by measuring the per-well relative fluorescence units (RFU) of SYBR green 1 dye using the Tecan Genios Plus  with excitation and emission wave-lengths of 485 nm and 535 nm, respectively, and with the gain set at 60. Differential counts of relative fluorescence units (RFUs) were used in calculating IC50 values for each drug using Prism 4.0 windows software. A minimum of three separate determinations were carried out for each sample. The reference anti-malarial drug, chloroquine, was tested along the test compounds.Two strains of o assays . The culA. pulcherrima, acetone resulted in good extraction product, and the acetone extract also showed superior antibacterial activity (1), aloesaponarin I (2) and aloesaponarin II (3) (Of the four solvents used for extraction of the air dried roots of n II (3) .1 was isolated as a yellow amorphous powder with an Rf value of 0.68 (8% ethyl acetate in n-hexane). In 1H NMR spectrum, two highly downfield shifted signals at δH 12.13 and 12.02 due to chelated hydroxyl protons involved in hydrogen bonding and the downfield shifted carbonyl signals at δC 194.4 and 184.6 in 13C NMR spectrum , 7.80  and 7.37  with ABX spin system were assigned to H-5, H-6 and H-7 of mono-substituted ring C of a 1,8-dihydroxyanthraquinone, respectively. The presence of two meta-coupled aromatic protons resonating at δH 7.21  and 7.55  assigned to H-2 and H-4, respectively, with the biosynthetically expected aromatic methyl (δH 2.46) group being at C-3 of the di-substituted ring A. The 1H NMR data, therefore, suggested that compound 1 is 1,8-dihydroxy-3-methyl anthraquinone. In consistency with the aforementioned spectral data, the 13C NMR spectrum revealed 15 carbon signals; five aromatic methines , seven aromatic quaternary carbons , two carbonyl carbons (δC 194.4 and 184.6) and a methyl (δC 24.7) group. Based on these and comparison with the data in the literature [1 was identified as 1,8-dihydroxy-3-methylanthracene-9,10-dione trivial name chrysophanol (1). Compound 1 is widely distributed in different plant families including family Asphodelaceae [Penicillium islandicum Sopp, as fungal metabolite [Compound spectrum  clearly delaceae , Rhamnactabolite .2 was isolated as yellow amorphous solids with an Rf value of 0.50 (in 20% ethyl acetate in n-hexane). It has UV-VIS (MeOH) λmax at 220, 272, 310 and 420 nm. The 1H NMR (500 MHz) showed highly deshielded signal at δH 12.93 due to chelation of the hydroxyl proton involved in hydrogen bonding at C-1 (δC 163.6), a three-proton singlet at δH 4.06 (δC 53.3) and a shielded carbonyl (δC 170.7), indicating the presence of a methoxyl ester. The downfield shifted methyl signal observed at δH 2.98 (δC 22.0) due to the peri-effect allowed its placement at C-8 (δC 148.1). In addition, the 1H NMR spectrum further showed the presence of three mutually coupled aromatic protons with an ABX pattern centered at δH 7.31 , 7.62  and 7.77  of ring A as in compound 3 (discussed in the next section). In ring C, the singlet aromatic proton at δH 7.80 showed HMBC correlation with C-6, C-7, C-8a and C-10 which is in agreement with its placement at C-5 of tri-substituted  in ring C. The substituents in this ring, a methyl group (δH 2.98 at C-8), a hydroxyl group (at C-6) and carbomethoxy group (at C-7) are evident from the NMR spectra , four aromatic methine carbons , two oxygenated aromatic quaternary carbons (δC 163.6 and 162.6), six non-oxygenated aromatic quaternary carbons , a methoxyl (δC 53.3) and a methyl (δC 22.0) carbons. Based on the above spectral data, compound 2 was identified as 3,8-dihydroxy-1-methyl-2-anthraquinoic acid methyl ester, trivial name aloesaponarin I (2). It was previously reported from the stem of A. saponaria [Furthermore, the ral data  revealedCompound 3 was a yellow crystalline solid characterized by Rf value of 0.37 (in 50% ethyl acetate in n-hexane) and melting point of 190–192 0C. It has UV-VIS (MeOH) λmax at 226, 272, 285 and 427 nm, an indication for the presence of anthraquinone moiety. The presence of 15 carbon signals in 13C NMR spectrum and five aromatic proton signals in 1H NMR , 7.67  and 7.59  and two meta-coupled protons resonating at δH 7.41  and 7.00 . The 13C NMR spectrum showed 15 carbon signals; methines, seven aromatic quaternary carbons, two carbonyl carbons and a methyl group. The only notable difference is that of a methyl group down-field shifted (δH 2.66) in compound 3 indicating its peri-position to carbonyl group. In addition, the presence of only one chelated hydroxyl group (at δH 12.93), allowed the unequivocal placement of the methyl group at C-8. This was further supported by the HMBC 4JC,H correlation observed between methyl protons and carbonyl carbon, C-9 (δC 189.4). Therefore, the structure of compound 3 was characterized as 1,6-dihydroxy-8-methylanthracene-9, 10-dione, trivial name aloesaponarin II (3). This compound has been reported from several other Aloe species, including stem of A. saponaria Haw. [Aloe species [A. pulcherrima, which makes our finding the first report of its kind. Besides Aloe species, aloesaponarin II (3) was reported from the culture broth of marine actinomycetes [3) was first reported from A. saponaria Haw. I. It was later isolated from a recombinant and terrestrial Streptomycete, suggesting the wide distribution of the same compound both in plants and microbes. This ensures its accessibility for sustainable utilization of the same compound for treatment of various ailments.n 1H NMR  suggeste species  but not omycetes , aloesapin vitro antibacterial and antiplasmodial activities and the results were promising (2 and 3), compounds with closer structural feature in substituent pattern, displayed strong activity against all the test bacterial strains (inhibition zone diameter ranging between 18–27 mm). The growth inhibitory potential of compound 2 was even greater than the reference drug  against B. subtilis  and chloroquine-sensitive (D6) strains of P. falciparum. The highest activity was exhibited by compound 3 followed by compound 2 , allowing them to involve in multiple biological oxidative processes . Accordiromising . The antsubtilis . This insubtilis  may have species . In the mpound 2 . In gene2) and aloesaponarin II (3) isolated from another Aloe species (A. secundiflora) [3) having strong activity against S. aureus and aloesaponarin I (2) with the most activity against the chloroquine-resistant (W2) strain of P. falciparum. In contrary to the same report [E. coli and P. aeruginosa, an activity almost equal to the activity of commercial antibiotic (gentamicin). The two aloesaponarins  had comparable antibacterial activities with extracts of other traditional medicinal plants reported earlier from the same study region [Aloe vera leaf gel against S. aureus and E. coli reported elsewhere [Earlier study on the antibacterial and antiplasmodial activities of aloesaponarin I (diflora)  also inde report , our resnarin II  isolatedy region , 27. Howlsewhere . Aloesaplsewhere .P. aeruginosa  displayed higher activity against many of the bacterial strains tested. In some cases, however, the crude extracts appear to have even better activity as revealed by the strongest activity (inhibition zone diameter of 24 mm) against ruginosa . The bet targets . In geneA. pulcherrima has resulted in identification of three anthraquinones namely chrysophanol, aloesaponarin I (2) and aloesaponarin II (3). The isolated compounds displayed strong in vitro antibacterial activity against four bacteria strains  with aloesaponarin I (2) being the most active compound against the tested bacteria strains. It even showed better activity than the reference drug (gentamicin) against B. subtilis. Although not as strong as antibacterial activity, the isolated compounds displayed moderate antiplasmodial activity against both chloroquine resistant and -sensitive malaria parasites . The observed antimicrobial activities of the isolated compounds could give insight about the potentials of these anthraquinones as lead compound in development of antibacterial drugs. Further in vivo antibacterial and antiplasmodial tests are recommended to make conclusive decision on their potential candidacy in the development of antibacterial and antiplasmodial drugs.Phytochemical investigation of the roots of S1 Fig(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Oryza sativa L. ‘Dongjin’) under inorganic phosphate (Pi)-deficient conditions. OsMYB5P protein is localized to the nucleus and functions as a transcription activator in plant development. Overexpression of OsMYB5P in rice and Arabidopsis  increases tolerance to phosphate starvation, whereas OsMYB5P knock-out through RNA interference increases sensitivity to Pi depletion in rice. Furthermore, shoots and roots of transgenic rice plants overexpressing OsMYB5P were longer than those of wild plants under both normal and Pi-deficient conditions. These results indicate that OsMYB5P is associated with the regulation of shoot development and root- system architecture. Overexpression of OsMYB5P led to increased Pi accumulation in shoots and roots. Interestingly, OsMYB5P directly bound to MBS (MYB binding site) motifs on the OsPT5 promoter and induced transcription of OsPT5 in rice. In addition, overexpression of OsMYB5P in Arabidopsis triggered increased expression of AtPht1;3, an Arabidopsis Pi transporter, in shoots and roots under normal and Pi-deficient conditions. Together, these results demonstrate that overexpression of OsMYB5P increases tolerance to Pi deficiency in plants by modulating Pi transporters at the transcriptional level in monocots and dicots.Myeloblastosis (MYB) transcription factors play central roles in plant developmental processes and in responses to nutrient deficiency. In this study, OsMYB5P, an R2R3-MYB transcription factor, was isolated and identified from rice ( Wei) lines . When 7-i) lines   lines  Figs. Ini) lines . When thi) lines . Laterali) lines .To understand the function of OsMYB5P in Pi uptake, we measured the Pi concentration of OsMYB5P-OX, OsMYB5P-RNAi, and WT plants under high-Pi or Pi-deficient conditions  S1E, S2 Figs. ThPTs induce transcriptional expression of many Pi starvation-inducible (PSI) genes by Pi starvation [OsPTs gene expression, we performed an in silico analysis of the presumptive promoter region using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Several OsPTs promoters include putative cis-acting regulatory elements of the MYB binding site (MBS), which is typically associated with abiotic stress responses  assay using nucleus protein extracts of WT and OsMYB5P-OX transgenic plants. After immunoprecipitation with an antiserum against OsMYB5P  with biotin-labeled oligonucleotides corresponding to promoter fragments containing MBS motifs (OsPT5-MBS1 and OsPT5-MBS2) and recombinant OsMYB5P-GST or GST proteins . Like the phenotype of OsMYB5P-RNAi plants, growth of the shoot and primary root in the ospt5 mutant was more inhibited than in WT plants in high-Pi and Pi-deficient media OsMYB5P ws plants . These rArabidopsis, the Pht1 family contains nine members, AtPht1;1 to AtPht1;9, which play major roles in the regulation of phosphate uptake in Pi acquisition and translocation [Arabidopsis Pi-starvation response are also known from other families: AtPht2;1, AtPht3;1, AtPht3;2, and AtPht3;3 [OsMYB5P directly regulates phosphate transporters through binding at the promoter in rice  on the promoter [OsPT2 is regulated by OsPHR2 in Pi-starvation responses [OsPT5 may, in part, account for the observed Pi uptake in shoots and roots of OsMYB5P-overexpressing transgenic plants under Pi-deficient conditions, as evidenced by the greater upregulation of this gene in such plants than in the WT . (B) Seven-day-old WT, and three independent OsMYB5P-OX seedlings were grown vertically for 7 d on high Pi (1.25 mM KH2PO4) or Pi deficient (0.0125 mM KH2PO4) media. Scale bar indicates 5 cm. (C and D) Graphical representation of the shoot (C) or primary root (D) length of seedlings depicted in (B). Error bars represent mean ± SD of n = 10 replicates of 3 seedlings for each experiment. (E) Inorganic Pi concentrations were measured in the shoots and roots of plants under both high Pi and Pi deficient conditions. Error bars represent mean ± SD of n = 6 replicates of 10 seedlings for each experiment. Asterisks represent significant differences from the WT .(A) Transcriptional expression of (TIF)Click here for additional data file.S2 FigOsMYB5P in different OsMYB5P-RNAi transgenic plants by northern blot analysis. Total RNA was extracted from WT and three independent OsMYB5P-RNAi seedlings grown under high Pi conditions. rRNA used a loading control. (B) Seven-day-old WT, and three independent OsMYB5P-RNAi seedlings were grown vertically for 7 d on high Pi (1.25 mM KH2PO4) or Pi deficient (0.0125 mM KH2PO4) media. Scale bar indicates 5 cm. (C and D) Graphical representation of the shoot (C) or primary root (D) length of seedlings depicted in (B). Error bars represent mean ± SD of n = 10 replicates of 3 seedlings for each experiment. (E) Inorganic Pi concentrations were measured in the shoots and roots of plants under both high Pi and Pi deficient conditions. Error bars represent mean ± SD of n = 6 replicates of 10 seedlings for each experiment. Asterisks represent significant differences from the WT .(A) Transcriptional expression of (TIF)Click here for additional data file.S3 FigOsACTIN1 was used for normalization. Error bars represent the mean ± SD of three technical replicates. Asterisks represent significant differences from the WT .Total RNA was extracted from shoots and roots of seedlings grown under high Pi or Pi deficient conditions. Expression of (TIF)Click here for additional data file.S4 FigSeven-day-old seedlings were grown for 7 d in high Pi or Pi deficient media, after which shoots (A) and roots (B) were sampled separately. Error bars represent mean ± SD of n = 10 replicates of 3 seedlings for each experiment. Asterisks represent significant differences from the WT .(TIF)Click here for additional data file.S5 Figospt5 mutant. (B) Total RNA was extracted from shoots and roots of ospt5 mutant grown under high Pi conditions. Expression of OsACTIN1 was used for normalization. Error bars represent the mean ± SD of three technical replicates. Asterisks represent significant differences from the WT .(A) Schematic illustration is a representation of the location of T-DNA insertions in (TIF)Click here for additional data file.S6 Figospt5 seedlings were grown vertically for 3 weeks on high Pi (1.25 mM KH2PO4) or Pi deficient (0.0125 mM KH2PO4) media. Scale bar indicates 5 cm. (B and C) Graphical representation of the shoot (B) or primary root (C) length of seedlings depicted in (A). Error bars represent mean ± SD of n = 10 replicates of 3 seedlings for each experiment. (D) Inorganic Pi concentrations were measured in the shoots and roots of plants under both high Pi and Pi deficient conditions. Error bars represent mean ± SD of n = 6 replicates of 10 seedlings for each experiment. Asterisks represent significant differences from the WT .(A) Seven-day-old WT, OsMYB5P-OX, OsMYB5P-RNAi and (TIF)Click here for additional data file.S7 FigEscherichia coli using 0.5 mM IPTG at 30 °C for 3 h. The SDS-PAGE gel with GST and OsMYB5P-GST proteins was stained using Coomassie brilliant blue. M denotes a protein size marker. (B) Immunoprecipitation assay with mouse anti-OsMYB5P antibody. To detect the endogenous OsMYB5P in rice, total proteins were extracted from rice WT, OsMYB5P-OX, and OsMYB5P-RNAi plants, and then immunoprecipitated with mouse anti-OsMYB5P monoclonal antibody. The arrow and arrowhead indicate endogenous OsMYB5P protein  and a non-specific band (upper panel), respectively.(A) Induction of OsMYB5P protein from (TIF)Click here for additional data file.S8 FigOsMYB5P chimeric plasmid structure. The full-length cDNA of OsMYB5P is under the control of a 35S promoter, and linked to the hygromycine resistance gene (hpt) and the green fluorescent protein (Egfp). (B) Expression of OsMYB5P in Arabidopsis OsMYB5P transgenic plants by northern blot analysis. Total RNA was extracted from 11 representative transgenic lines. rRNA is a loading control.(A) Schematic diagram of the (TIF)Click here for additional data file.S9 FigTotal lateral root number (A) or length (B) on primary root of plants shown in (TIF)Click here for additional data file.S10 FigArabidopsis WT and OsMYB5P-OX seedlings grown under high Pi or Pi deficient conditions. Expression levels of AtTUBULIN2 were used for normalization. Bars represent the mean ± standard error of three technical replicates. Asterisks represent significant differences from the WT .Total RNA was extracted from shoots and roots of (TIF)Click here for additional data file.S11 FigThe two types of MYB domain repeats on the N terminus of OsMYB5P are indicated with red (R2) and blue (R3). The alignment of the DNA-binding (R2 and R3) domains with the amino acid sequence of rice R2R3-MYB transcription factors involved in Pi starvation responses was performed using CLUSTAL W. Identical amino acids are shaded black, and similar amino acids are shaded in gray.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Non-small-cell lung cancer (NSCLC) is one of the leading causes of cancer-related death worldwide. Although epigenetic deregulation is known to be important for tumor progression, the molecular mechanisms in NSCLC remain unclear. Here, we found that G9A (known as EHMT2), a histone methyltransferase responsible for mono- or di-methylation of histone 3 (H3) lysine 9 (K9), is significantly upregulated in NSCLC. Knocking down G9A or pharmacological inhibition of its activity suppressed tumor cell growth, colony formation, invasion and migration. Furthermore, G9A exerts these functions by repressing CASP1 expression. Knocking down CASP1 in G9A-deficient cell restored capacities of tumor cell invasion and migration. Mechanistically, G9A silences the CASP1 promoter activity by increasing H3K9me2 around its promoter. Finally, high expression of G9A or low expression of CASP1 is correlated with poor overall survival in lung adenocarcinoma. Overall, our study uncovers a novel mechanism of G9A promoting tumor cell growth and invasion by silencing CASP1, and implies that G9A may serve as a therapeutic target in treating NSCLC. Non-small-cell lung cancer (NSCLC) is the major type of lung cancer. It is a heterogeneous disease; many different oncogenic mutations have been identified. Epigenetic deregulation is implicated in tumor development.5 It is overexpressed in many types of cancer,10 and its higher expression is associated with poor survival of cancer patients.11 Mechanistically, G9A acts as a transcriptional repressor to silence gene expression.13 For example, G9A interacts with Snail, a transcriptional factor, and is critical for Snail-mediated E-cadherin repression in human breast cancer.14 Moreover, hypoxic stress induced accumulation of G9A leads to increased H3K9me2 and repression of its target genes to promote cell survival.15 However, G9A also functions as a transcriptional activator depending on its interacting cofactors.16 For example, G9A can epigenetically activate the serine–glycine synthesis pathway to sustain cancer cell survival and proliferation.17 However, its role in NSCLC is not well understood. Identification of its key target genes or pathways will help to understand the molecular mechanism of tumorigenesis and metastasis in NSCLC.G9A/EHMT2 is a histone lysine methyltransferase that specifically mono- and dimethylates Lys9 of histone H3 .19 Specifically, CASP1 mediated inflammasome activation regulated immune response and disease pathogenesis.20 In addition, CASP1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria.22 However, the function and regulation of CASP1 in NSCLC is poorly understood.CASP1, also known as caspase 1, belongs to the family of CASP proteins, which are cysteine proteases regulating many cellular processes, such as apoptosis, inflammation and necrosis, etc.In this study, we examined the biological function of G9A in NSCLC cells, and identified one of its key target genes, CASP1. We also uncovered the molecular mechanism of how G9A represses CASP1 to promote tumor cell growth and invasion. Finally, we analyzed whether G9A or CASP1 could serve as prognostic biomarkers in lung adenocarcinoma (LUAD). In addition, our study suggests that G9A may be a therapeutic target for NSCLC.To examine whether G9A expression is dysregulated in NSCLC, we compared its expression between normal and cancer samples using the mRNA-Seq data of LUAD from the TCGA database. We found that G9A is significantly upregulated in tumor samples compared with the normal control in LUAD . In addiWe also examined the expression of G9A in lung cancer using the oncomine database, and found that G9A is upregulated in LUAD regardless of EGFR or KRAS mutation status . OverallTo investigate the function of G9A in NSCLC cells, we knocked down the level of G9A protein significantly in PC9 and A549 cells by selecting cells stably expressing G9A shRNA , and fouConversely, when overexpressing G9A in PC9 and A549 cells stably expressing G9A shRNA , we obseSimilarly, colony formation and sphere formation abilities were reduced upon stable G9A knockdown in both PC9 and A549 cells . Furtherin vivo, we carried out xenograft assays in nude mice, and found that tumors derived from cells stably expressing G9A shRNA showed significantly slower growth rates and smaller tumor sizes, compared with those derived from cells expressing the control shRNA , colon adenocarcinoma (COAD) and lung squamous cell carcinoma (LUSC) from the TCGA database . We founIn conclusion, our results indicate that CASP1 overexpression suppresses G9A-mediated cell invasion and migration. The expression of CASP1 is either positively or negatively associated with distinctive pathways in cancer.24G9A is a histone methyltransferase that increases the level of H3K9me2 around the promoter region of its target genes, and interacts with other co-repressors to silence target gene expression.To investigate whether depletion of G9A reduced the level of H3K9me2 around the CASP1 gene promoter, we carried out chromatin immunoprecipitation assay using anti-G9A and anti-H3K9me2 antibodies, followed by qPCR to examine the relative enrichment of H3K9me2 around the promoter region of CASP1 gene, compared with the input . We founTo study whether G9A represses the CASP1 promoter activity, we cloned the CASP1 gene promoter into pGL3 vector, carried out luciferase reporter assays and found that G9A overexpression significantly suppressed the promoter activity of CASP1 promoter, and this effect was dosage dependent . OverallμM) of BIX-01294, which is a small molecular inhibitor of G9A widely used in other cancer studies.25To study whether pharmacological inhibition of G9A activity can inhibit tumor cell growth, invasion and migration, we treated PC9 and A549 cells with various concentrations ,26 and carried out Kaplan–Meier survival analysis. We found that high expression of G9A is associated with poor overall survival in LUAD .It is possible that G9A regulates other genes and pathways in NSCLC; for example, G9A was found to mediate tumor metastasis by epigenetically repressing the cell adhesion molecule EPCAM in lung cancer cells.31 and induce cell pyroptosis.32 Interestingly, several NALP proteins are also differentially expressed in NSCLC. How various components in the inflammasome affect tumorigenesis in NSCLC may need further investigation.Our study also indicates that CASP1 suppresses tumor cell invasion and migration in NSCLC cells. CASP1 is one of components of the inflammasome complex, which also includes PYCARD, NALP and caspase 5 . They function in the innate immune system, and can activate inflammatory processOverall, our results identify a novel mechanism by which G9A enhances tumor cell proliferation and invasion by silencing CASP1 expression, and suggests that G9A may serve as a therapeutic target in NSCLC.All animal experiments were performed using male BALB/C nude mice (4–5 weeks old). The mice were purchased from the SLAC Laboratory Animal Center  and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal experimental protocols performed in this study were approved by the Institutional Animal Care and Use Committee at Tongji University.2.Human NSCLC cell lines, PC9 and A549, were grown in DMEM medium . Culture media contain 10% FBS  supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml) . The cells were incubated at 37 °C in a humidified atmosphere of 5% COG9A siRNAs were synthesized by Ribobio Inc. . Transfections were performed with Lipofectamine 2000  according to the manufacturer's protocol. Total RNA or cell lysates were prepared 48 h after transfection and were used for real-time RT-PCR or western blotting (WB).Sequences for siRNAs targeting G9A were as follows: #1: 5′-CCAUGCUGUCAACUACCAUdTdT-3′ (sense) and 5′-AUGGUAGUUGACAGCAUGGdTdT-3′ (antisense); and #2: 5′-GAACAUCGAUCGCAACAUCdTdT-3′ (sense) and 5′-GAUGUUGCGAUCGAUGUUCdTdT-3′ (antisense); and #3: 5′-GCUAUGAGGCUACUGAGUAdTdT-3′ (sense) and 5′-UACUCAGUAGCCUCAUAGCdTdT-3′ (antisense).CASP1 siRNAs were synthesized by GenePharma Inc. . The sequences for siRNAs targeting CASP1 were as follows: #1: 5′-GGUGUGGUUUAAAGAUUCATT-3′ (sense) and 5′-UGAAUCUUUAAACCACACCTT-3′ (antisense); #2: 5′-GAAGACUCAUUGAACAUAUTT-3′ (sense) and 5′-AUAUGUUCAAUGAGUCUUCTT-3′ (antisense); and #3: 5′-CUCUCAAGGAGUACUUUCUTT-3′ (sense) and 5′-AGAAAGUACUCCUUGAGAGTT-3′ (antisense).G9A and CASP1 overexpression plasmids and the control plasmid (GV141) were purchased from GeneChem Inc. . Plasmids were transfected into cells using Lipofectamine 2000  according to the manufacturer's protocol.33PC9 and A549 cells were infected with the lentiviral supernatant containing the lentiviral construct for G9A shRNA or the control shRNA . Puromycin was added to the cells for killing uninfected cells. Multiple single colonies were selected and expanded. Total RNA and cell lysates from these colonies were prepared and used for real-time RT-PCR or WB to validate the G9A knockdown effect in these cells. The detailed procedure was described as previously described.The oligonucleotide sequences used to make the shRNA against G9A were as follows: the forward oligo: GATCCGGCACACATTCCTGACCAGAGATGGTACCATCTCTGGTCAGGAATGTGTGTTTTTG and the reverse oligo: AATTCAAAAACACACATTCCTGACCAGAGATGGTACCATCTCTGGTCAGGAATGTGTGCCG. The two oligos were annealed and cloned into the lentiviral vector. The G9A-targeting sequence within these oligos is CACACATTCCTGACCAGAGAT.33Cells were seeded into 96-well plates at a density of 1000 cells per well. For different treatment conditions described in the paper, each condition was replicated six times. At different time points, and cell proliferation assays were performed as previously described.33Cell invasion assays were performed as previously described.33 The assays were performed in triplicate.Cells were cultured in six-well plates until the cell density reached 80% confluence, and cell migration assays were performed as previously described.μg/ml) for 12 days, and colony formation assays were performed as previously described.33 The assays were performed in triplicate.Cells were seeded into 10-cm dishes at a density of 1000 cells per dish and cultured in the presence of puromycin  according to the manufacturer's instructions. Stained cells were analyzed using a BD Accuri C6 Flow Cytometer . Results were plotted using .The proximal promoter region (1.5 kb) of CASP1 gene was cloned into the pGL3 vector using the following PCR primers for genomic DNA amplification: the forward primer, 5′-CTAGGCTAGCACAGCAGCACTCCATTACTCAGTA-3′ and the reverse primer, 5′-GCCCTCGAGTAGCCTGCATCAGGTAGTGTATCC-3′. Luciferase reporter assays were carried out using a dual-luciferase reporter assay system , according to the manufacturer's instructions.33 GAPDH was used as a loading control. The following primary antibodies were used: G9A , H3K9me2 , total histone H3 , caspase 3  and PARP1 , p-ERK , ERK , GAPDH , β-Actin . The following secondary antibodies were used: anti-rabbit IgG  and goat polyclonal anti-mouse IgG .WB was performed as previously described.6 cells per ml were resuspended in 1 × binding buffer, and 100 μl of the cell suspension was mixed with 5 μl FITC and 5 μl PI using the FITC Annexin V apoptosis detection kit , according to the manufacturer's instructions. Stained cells were analyzed using an Accuri C6 flow cytometer .Cells were washed twice in cold 1 × PBS twice. Then, 1 × 1033Total RNA extraction from cells and real-time RT-PCR were performed as previously described.The PCR primers used were as follows: G9A forward, 5′-TACACCACTCATTGGGGATG-3′ G9A reverse, 5′-GGGAAGAGGGGAATGACTTT-3′ GAPDH forward, 5′-GAGTCAACGGATTTGGTCGT-3′ GAPDH reverse, 5′-TTGATTTTGGAGGGATCTCG-3′ CASP1 forward, 5′-GCTTTCTGCTCTTCCACACC-3′ and CASP1 reverse, 5′-TCCTCCACATCACAGGAACA-3′.34 The updated protocol can be found at http://research.hudsonalpha.org/Myers/. Anti-EHMT2/G9A antibody  and anti-Histone H3 dimethyl Lys9 (H3K9me2) antibody  were used. Purified DNA was resuspended in EB buffer for subsequent SYBR green-based real-time PCR. The following primers, which cover the proximal promoter region of CASP1 gene, were used: CASP1 #1 (+140)-Forward, GAACAGTGGTTCACATACTC; CASP1 #1 (+300)-Reverse, GCCTGCATCAGGTAGTGTAT; CASP1 #2 (−100)-Forward, GTGAGCCAAGGTCAAATAAC; CASP1 #2 (+100)-Reverse, GGGTAATGTATGTCCCTGTG; CASP1 #3 (−300)-Forward, CCTAGCAATTTGGGAGACCA; and CASP1 #3 (−100)-Reverse, TGCAGCCTCCACTTCCCAGG; CASP1 #4 (−1000)-Forward, GACACGTCTTACATGGGTGC; CASP1 #4 (−800)-Reverse, ATGAAGGCTGGTGATATGGT.ChIP experiments were performed as previously described.6 cells suspended in 100 μl PBS were injected subcutaneously into the right axillary region of 5-week-old nude mice. Tumor size was measured every 3–5 days using a digital caliper, and tumor volume (v) was calculated based on this formula: v=1/2(length × width2). After 5 weeks, xenograft tumors were isolated, photographed and fixed. The immunohistochemistry experiments were performed using the following primary antibody: Ki67  by Wuhan Google Technology Co. Ltd. . The H&E-stained slides were scanned using a Leica SCN400  and analyzed using SlidePath Gateway LAN software .For the xenograft tumor growth assay, a total of 1 × 10BIX-01294 was purchased from MedChemExpress . PC9 and A549 cells were plated in six-well plates until the cell density reached 30–40% confluence, and the cells were then treated with different concentrations of BIX-01294 for various lengths of time.http://cancergenome.nih.gov) and the firebrowse (http://firebrowse.org). Differentially expressed genes were analyzed as previously described.33 Pearson's correlation was applied to analyze the correlation coefficient between two genes in the normal or cancer samples.The RNA-Seq data of LUAD were obtained from the TCGA project  using gene expression correlation data downloaded from cBioportal .Comparison of pathway analysis was carried out in R .Gene expression and survival information for NSCLC patients were from the released database (2015 version) downloaded from t-test was used. For comparisons among multiple groups, one-way ANOVA was used. For all analyses, a P-value of <0.05 was considered statistically significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.A comparison is made between the outcomes of the control condition and one of the treatment conditions unless stated otherwise. For comparisons between two groups, Student's


These EBV mutants established latent infection in secondary lymphoid organs of infected huNSG mice for at least 3 months, but did not cause tumor formation. Low level viral persistence in the absence of EBNA3A or EBNA3C seemed to be supported primarily by proliferation with the expression of early latent EBV gene products transitioning into absent viral protein expression without elevated lytic replication. In vitro, EBNA3A and EBNA3C deficient EBV infected B cells could be rescued from apoptosis through CD40 stimulation, mimicking T cell help in secondary lymphoid tissues. Thus, even in the absence of the oncogenes EBNA3A and 3C, EBV can access a latent gene expression pattern that is reminiscent of EBV persistence in healthy virus carriers without prior expression of its whole growth transforming program.The oncogenic Epstein Barr virus (EBV) infects the majority of the human population and usually persists within its host for life without symptoms. The EBV oncoproteins nuclear antigen 3A (EBNA3A) and 3C (EBNA3C) are required for B cell transformation in vitro and are expressed in EBV associated immunoblastic lymphomas in vivo. In order to address the necessity of EBNA3A and EBNA3C for persistent EBV infection in vivo, we infected NOD- Epstein Barr virus (EBV) was discovered 54 years ago as the first human candidate tumor virus. In vitro, EBV can readily transform human B cells into indefinitely growing cell lines. In this transformation process, multiple EBV proteins play an important role, such as the two oncogenes EBNA3A and especially EBNA3C. To address the necessity of these oncogenes in vivo, we investigated EBNA3A and 3C deficient EBV for their persistence in mice with reconstituted human immune system components (huNSG mice), an in vivo model for EBV infection, associated tumorigenesis and immune control. Here, we show that EBV devoid of EBNA3A or EBNA3C was able to establish latent infection in vivo. The persistence was characterized through proliferation with the expression of early latent EBV gene products transitioning into absent viral protein expression, but without tumor formation. Furthermore, we were able to rescue in vitro infected B cells with EBNA3A or EBNA3C deficient EBV from apoptosis by mimicking T cell help. This might allow the virus to access the gene expression pattern that is thought to also mediate persistence in healthy EBV carriers and does not seem to require prior expression of all growth transforming viral proteins. Epstein Barr virus (EBV) was discovered as the first human candidate tumor virus in Burkitt lymphoma 54 years ago , 2. EBV INK4a up-regulation is blocked primarily by EBNA3C with the help of EBNA3A [Accordingly, EBV latency III establishment during primary B cell infection in vitro and the resulting outgrowth of LCLs were taken as surrogates for EBV persistence. Upon B cell infection by EBV, the transient expression of the two viral Bcl-2 homologues BHRF1 and BALF1 initially ensures the survival of EBV harboring cells . During f EBNA3A –13. Therf EBNA3A , 15. Twof EBNA3A , 17. In f EBNA3A –21. ThesTherefore, we investigated EBNA3A and EBNA3C deficient EBV viruses for their persistence in mice with reconstituted human immune system components (huNSG mice), an in vivo model for persistent EBV infection, EBV associated lymphomagenesis and cell-mediated immune control of EBV . Surprisscid γcnull mice with reconstituted human immune system compartments (huNSG mice) are suitable models to study persistent EBV infection and immune control in vivo [5 Raji-infectious units (RIU) of 3AKO, 3CKO or wildtype EBV (wt). EBV DNA was readily detected in the spleen and lymph nodes of the majority of both 3AKO and 3CKO infected mice 5 weeks p.i.  and EBNA3C knock-out EBV (3CKO) were previously shown to be unable to establish indefinitely growing LCLs in vitro , and onl in vivo , 24. In eks p.i. . The vireks p.i. . In orded groups . Howeverd groups . Hence, d groups . Active d groups . The numted mice . EBER+ cnfection  and likenfection . EBV infl groups . Despite+ T cells and result in splenomegaly [+ T cells was significantly increased in 105 RIU of 3CKO infected animals 5 weeks p.i. compared to uninfected animals. In low dose (105 RIU) 3AKO infected mice a similar trend for an increase of splenic CD8+ T cells was observed (+ T cells compared to wt infected mice (6 RIU) infection with 3AKO and 3CKO resulted in a significantly higher number of splenic CD8+ T cells in both groups compared to the uninfected group  in the presence of CD40L (as inferred from the difference in culture conditions with or without CD40L) was significantly higher in 3CKO infected B cells 2 and 3 weeks p.i. as well as in 3AKO infected B cells 3 weeks p.i. compared to wt infected B cells (+ 7AAD- early apoptotic cells in the absence of CD40L (as inferred from the difference in culture conditions with or without CD40L) were significantly increased after 3, but not 2 weeks in 3CKO infected cultures compared to wt infected cultures  was produced from EBNA3A knock-out (3AKO), EBNA3C knock-out (3CKO) or wildtype (wt) BACs maintained in human embryonic kidney HEK293 cells  as described elsewhere . The GFPscid γcnull (NSG) mice and HLA-A2 transgenic NSG were obtained from the Jackson Laboratories and maintained under specific pathogen-free conditions. Newborn mice (1–5 days old) were irradiated with 1 Gy using a Cs or x-ray source and after 5 to 7 hours reconstituted intrahepatically with 1–3 x 105 CD34+ human hematopoietic progenitor cells (HPCs) purified from human fetal liver (HFL) tissue obtained from Advanced Bioscience Resources. Preparation of HFL tissue and isolation of human CD34+ cells were performed as described previously [5 RIU of EBV wt, 3AKO or 3CKO or with 1 x 106 RIU of EBV 3AKO or 3CKO and followed for 5 to 6 or 12 weeks respectively.NOD-eviously , 24. Humeviously . Mice we+ cells were depleted with 100 μg of monoclonal antibody against human CD4  via intraperitoneal inoculation for 3 consecutive days just prior to EBV infection. To deplete CD4+ cells for the duration of 5 weeks, the same injection procedure was repeated every 2 weeks.CD4Splenic tissue was processed using a QIAamp DNA Tissue Kit (QIAGEN) and total DNA from whole blood was extracted using the NucliSENS EasyMAG System (bioMérieux), both according to the manufacturer’s recommendations. TaqMan (Applied Biosystems) real-time PCR was used to quantify EBV DNA as previously described , with mo4Cl. Spleens were mechanically disrupted and filtered through a 70 μm cell strainer before separation of mononuclear cells on Ficoll-Paque gradients. Cell suspensions were stained with antibodies for 30 min on ice, washed, and analyzed on FACSCanto or LSR Fortessa cytometers (BD Biosciences). Analysis of flow cytometric data was performed with FlowJo (Tree Star). The absolute numbers of leukocytes in each category were computed from the white blood cell count measured with a Beckman Coulter AcT diff Analyzer. Early apoptotic cells were assessed by incubating cells with AF647 annexin V (BioLegend) and 7AAD (BioLegend) diluted in annexin binding buffer for 15 min at room temperature in the dark, 400 μl binding buffer was added and the staining was directly analyzed on FACSCanto.All fluorescently labeled antibodies were purchased from BD Biosciences, BioLegend and Invitrogen (Thermo Fisher Scientific) . Lysis oINK4a , rabbit anti-CD3 , mouse anti-CD20  and rabbit anti-CD20 . For immunofluorescence, the AF488 donkey anti-rabbit IgG (Jackson ImmunoResearch), Cy3 donkey anti-mouse IgG (Jackson ImmunoResearch), DyLight549 horse anti-mouse IgG (Vectrolabs) and DAPI (Sigma) were used. In situ hybridization (ISH) for EBV-encoded RNAs (EBER-ISH) was done as described previously, employing diaminobenzidine (DAB) chromogen  as substrate [2 using the image analysis software HISTO (Biomas). EBNA2, LMP1 and CD138 stainings, EBER ISH as well as EBNA2 plus CD3, EBNA2 plus CD20, EBNA2 plus Ki67 and EBNA2 plus p16INK4a co-stainings were analyzed with a Vectra3 automated quantitative pathology imaging system (PerkinElmer) using Vectra and InForm software analysis (PerkinElmer). Snapshots were taken using a scanning protocol created with InForm tissue segmentation to recognize the tissue. Briefly, the stained slides were loaded onto the Vectra slide scanner, 1 to 10 images with 20 × objective lens (0.5 micron/pixel) were taken with a CCD using the scanning protocol. Images taken were used to set up an algorithm in InForm to recognize and count EBNA2, LMP1 or CD138 positive cells, respectively. Signal cross-talk was eliminated using one chromogen only  control stains. If the algorithm was >90% confident that the signal was positive, the cells were counted and the number of positive cells per 1mm2 was determined. InForm was also used to set up an algorithm to calculate the percentage of EBNA2 plus Ki67, EBNA2 plus CD20 and EBNA2 plus CD3 double positive cells of all EBNA2 positive cells. The percentage of EBNA2 and p16INK4a double positive cells of all EBNA2 positive cells was quantified by an independent observer.Tissue was fixed in 10% saline buffered formalin and paraffin embedded. For immunohistochemistry and immunofluorescence, 3 μm sections were processed on a Leica BOND-MAX or Bond-III automated immunohistochemistry system. Stainings were performed with monoclonal mouse anti-EBNA2 , mouse anti-LMP1 , mouse anti-BZLF1 , mouse anti-CD138 , rat anti-EBNA1 , rabbit anti-Ki67 , rabbit anti-p16ubstrate . Quantifubstrate . The numHuman B cells were isolated from spleen suspensions of infected mice or from human leucocyte concentrates (blood bank Zurich) using CD19 microbeads (Miltenyi Biotec) according to the manufacturer’s recommendations.6 magnetically sorted human B cells from human leukocyte concentrates were infected with wt, 3AKO or 3CKO EBV with the addition of irradiated (140Gy) 3T3 fibroblast expressing CD40L or not [10L or not . Irradia+ fraction of infected mice or infected buffy coats. To remove contaminating genomic DNA, a 15min on-column DNAse treatment was included during the RNA isolation . Purified RNA was reverse transcribed using GoScript Reverse Transcriptase (Promega) for 1h at 42°C according to the recommendations of the manufacturer using a primer mix combining previously described gene-specific RT primers [GAPDH expression found in mouse samples of the used experiments.RNA was isolated using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s recommendations either of the magnetically enriched CD19 primers  at conce primers , equal R primers . Cq valuStatistical analysis and graph preparation was performed with Prism software (GraphPad). Two-tailed Mann-Whitney U, unpaired two-tailed t-test with Welch's correction, two-way ANOVA tests with Bonferroni correction, paired t-test or Fisher’s exact test for actual numbers were used. A p value of < 0.05 was considered statistically significant.All animal protocols were conducted according to the Swiss Animal Welfare Act, Tierschutzgesetz (TSchG) and approved by the veterinary office of the canton of Zurich, Switzerland . All studies involving human samples were reviewed and approved by the cantonal ethics committee of Zurich, Switzerland (protocol KEK-StV-Nr.19/08). The used human samples were anonymized.S1 Fig EBNA2 and CD20 or  EBNA2 and CD3 of splenic sections of huNSG mice infected with either (A) 105 RIU of wt, 3AKO or 3CKO 5 weeks p.i. or (B) 106 RIU of 3AKO or 3CKO 6 weeks p.i.. (C) Frequency of EBNA2+ cells with or without CD20 or CD3 expression in mice infected with either 105 RIU of wt, 3AKO or 3CKO 5 weeks p.i. or 106 RIU of 3AKO or 3CKO. (D) EBER ISH and CD20 staining of splenic section of huNSG mice infected with either 105 RIU of 3AKO or 3CKO 12 weeks p.i..Staining for (TIF)Click here for additional data file.S2 Fig(A) blood CD8+ T cells / ml and (B) blood CD4+ T cells / ml of huNSG mice infected with either 105 RIU of wt, 3AKO or 3CKO 5 weeks p.i. (n = 14-17/group) or 106 RIU of 3AKO or 3CKO 6 weeks p.i. (n = 7-13/group) or non-infected control (mock) huNSG mice as determined by flow cytometry and white blood cell counting with a hemocytometer  Pooled data from 4 low and 2 high infectious dose experiments with the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by Mann-Whitney U test.The change between the beginning and the end of the experiment (Δend-start) in the number of (TIF)Click here for additional data file.S3 Fig+ CD8+ blood and splenic  T cells or HLA-DR+ CD4+ blood and splenic  T cells of huNSG mice infected with either 105 RIU of wt, 3AKO or 3CKO 5 weeks p.i. (n = 14-21/group) or 106 RIU of 3AKO or 3CKO 6 weeks p.i. (n = 7-13/group) or non-infected control (mock) huNSG mice was determined by flow cytometry. (A-H) Pooled data from 4 low and 2 high infectious dose experiments with mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U test.The frequency of HLA-DR(TIF)Click here for additional data file.S4 Fig(A) Representative immunohistochemistry staining for CD138  and the (B) quantification of CD138+ cells/mm2 in splenic sections from huNSG mice infected with 106 RIU of 3AKO or 3CKO at 6 weeks p.i. and from non-infected control (mock) huNSG mice. Pooled data from 2 experiments represented with the mean ± SEM, Mann-Whitney U test. (C) Representative immunohistochemistry staining for BZLF1  in splenic sections of huNSG mice infected with 106 RIU of 3AKO or 3CKO 6 weeks p.i. and non-infected control (mock) huNSG mice.(TIF)Click here for additional data file.S5 FigBZLF1 normalized to GAPDH as determined by RT-qPCR in purified human CD19+ B cells infected with wt, 3AKO or 3CKO with or without irradiated fibroblasts (FB) either expressing CD40L or not 3 weeks p.i. (n = 6–13). Pooled data from 3 experiments represented with the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney U test.Relative mRNA expression of (TIF)Click here for additional data file.S6 Fig(A) Splenic endpoint viral DNA load and (B) viral DNA load per gram lymph node tissue determined by qPCR of huNSG mice inoculated with a CD4 specific antibody and infected with 105 RIU of 3AKO or 3CKO for 5 weeks (n = 4-6/group). Values for mice in which no viral DNA was detected are plotted on the X-axis. (C) Blood DNA viral load over time determined by qPCR of huNSG mice infected with 105 RIU of 3AKO or 3CKO 5 weeks p.i. (n = 4-6/group). Horizontal dashed line indicates the viral load of 3 times the lower limit of quantification (LLOQ). Horizontal dotted line indicates the LLOQ. (A-B) Data from 1 experiment is displayed with geometric mean for splenic and lymph node viral load and SEM for blood viral load, *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni correction for blood viral load and Mann-Whitney U test for splenic and lymph node viral load.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Streptococcus pneumoniae strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for β-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive S. mitis strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain23F-1 clone, which is widespread among international penicillin-resistant S. pneumoniae clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain S. mitis 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documenting the existence of distinct mutational pathways for the development of penicillin resistance.Penicillin-resistant  Streptococcus pneumoniae strains (referred to here as penicillin-resistant S. pneumoniae [PRSP] strains) worldwide, which have been recognized since the early 1980s, represent a paradigm for the evolution of a pathogen due to the selective pressure of antibiotic use. In 1997, the Pneumococcal Molecular Epidemiology Network (PMEN) was established for the classification of 16 global resistant clones based on multilocus sequence typing (MLST) (http://web1.sph.emory.edu/PMEN/). Spain23F-1 is the most successful PRSP clone, and representatives of this clone were first identified in Spain in 1984  are the main cause of penicillin resistance. PBPs, the target enzymes of β-lactam antibiotics, form a rather stable covalent complex via their active-site serine, and these acylated PBPs are enzymatically inactive. Mutations in the PBPs of penicillin-resistant laboratory mutants and clinical strains decrease the affinity to the antibiotic inhibitor molecule, and thus, the strains can still function in the presence of increased drug concentrations. The identification of the amino acid changes involved in resistance has been the focus of multiple investigations, since, in combination with structural information, knowledge of these changes will help to provide an understanding of the enzymatic function of PBPs and might facilitate the design of new inhibitors.S. mitis is one of the main donors of such mosaic blocks  and extends through the 3′ end. This sequence block is highly related to pbp2x of the sensitive S. mitis M3 strain first wrongly described as S. oralis, interspersed with two characteristic diverse sequence regions  was almost identical, differing by 2 nucleotides (nt) and a cluster of 6 nt, resulting in 3 amino acid (aa) changes. These alterations were also present in pbp2x of strain RO76 (pbp2xRO76), which was almost identical to pbp2x of the Spain14-5 clone (11 nt and 3 aa changes). pbp2x of strain IR164 (pbp2xIR164) was similar to that of the Taiwan19F-14 clone (4 nt and 2 aa changes). Finally, the pbp2x genes of strains IR148 and IR174 (pbp2xIR148 and pbp2xIR174), which were identical to each other, differed from the pbp2x gene of the Spain6B-2 clone by 18 nt and 4 aa, but only 2 nt and 2 aa changes were located within the common mosaic block.The S. mitis 658 .Noteworthy was the presence of sequences related to the sequence of itis 658 , green, pbp2x of low-level-resistant S. mitis strain 578 which belongs to the family of mosaic genes related to the pbp2x gene of sensitive reference strain S. mitis 658 (pbp2x variant from strain RO67 (pbp2xRO67), which was almost identical to pbp2x of the high-level-resistant S. mitis B6 strain after codon 329 (4 nt and 3 aa differences). The single pbp2x variant from an Iranian strain (strain IR158) contained sequence blocks related only to the sequences of reference S. mitis strains M3 and 658 (pbp2x gene from Hungarian strain Hu7 of a different ST (not shown).The second group consisted of seven variants derived from 10 Romanian strains . They alitis 658 . Moreove and 658 ; it was S. mitis B6, were distinct from PBP2x of the sensitive S. pneumoniae R6 strain , followed by a linker region and a C-terminal extension (residues 635 to 750) which is folded into two PASTA domains . Mutatio6 strain . However23F-16 and Tennessee23F-4 clones, where S389 was not altered, contained the signature mutations of this family: T338A, L364F, I371T, S389L, N417K, N444S, L510T, T513N, and N605T  contained completely distinct mutations. First, T338 was changed to P, as in PBP2x of all group 2 Romanian strains, and it possessed the three alterations M289T, A369V, and V456A.PBP2x of strain RO116 .S. mitis B6: T338A, I366M, A369V, I371T, E378G, A507T, S531Q, and a cluster of 5 aa changes at sites 595 and 597 to 600. There were three differences between these two PBP2x variants: L328F was present in RO67 and RO56 but not in S. mitis B6, site 595 was changed to L in S. mitis B6 but was Y in RO67, and only PBP2x of S. mitis B6 carried S494Y. PBP2x of IR158 was the only one without a change at site 338, but it contained the mutation H394L, which is close to the second conserved active-site motif S395SN, plus the mutations A369V and S389L.PBP2x of strain RO67 shared all mutations with PBP2x of S. pneumoniae strains isolated from two geographic areas, Romania and Iran. Both countries reported extremely high rates of occurrence of PRSP between 1990 and early in this century, but only limited data on the underlying changes in PBPs are available  extended into the region encoding the N-terminal domain of the protein farther than in any other variants, a phenomenon observed so far only in PBP2x of penicillin-resistant S. mitis and S. oralis strains  occur in particular pbp2x variants of the 23F family (The presence of sequence blocks of variable length highly related to the sequence of the sensitive reference strain pbp2x variants were not found in other PRSP strains when the NCBI databases (containing nucleotide and draft genome sequences) were searched by BLAST analysis. Since the isolates were mainly of serogroup 19, it is tempting to assume that they are genetically related.The second group consisted only of PBP2x variants from 10 Romanian isolates that expressed unusually high levels of penicillin resistance and for which penicillin MICs were 4 to 8 μg/ml, a phenotype prevalent in Romania . RelatedS. mitis reference strains were reflected by their different positions within the protein structure (1138–23F-16 and Tennessee23F-4 clones), suggesting that it is not important for the resistance phenotype. On the other hand, structural data revealed that the L389 mutation produces a destabilizing effect that generates an open active site  in combination with the results of biochemical and microbiological tests confirmed that T371, A338, and T605 are critical for resistance  and in all group 2 PBP2x variants and is associated with high-level cefotaxime resistance in S. pneumoniae  contribute to the high resistance level of the Romanian strains. The mutation A338, which was frequent in the 23F family, reduces the acylation efficiency for cefotaxime  by a factor of 2, whereas a 5-fold reduction was observed for strains with the P338 mutation . Susceptibilities to antibiotics were determined by the agar dilution method recommended by CLSI (Nine strains isolated in 2011 (8 strains) and 2008 (1 strain) in Tehran hospitals and 15 strains from Romania were included in the present study . Serotyp by CLSI  in the c by CLSI , and by Taq polymerase (Eurogentec) or DreamTaq polymerase (Fischer) according to the manufacturers' instructions. The PBP2x gene fragments of strains from Iran were amplified with the primers pn2xup and pn2xdown  (S. mitis 578 (KY292539), and S. mitis 658 (KY292535), from the genomes of clones Spain23F-1 (FM211187), Spain6B-2 (NC_014498), Spain14-5 (FWTC01000077), Finland 6B-12 (FWWA01000144), South Africa19A-13 (FWSX01000072), Taiwan19F-14 (NZ_FWSZ00000000), Taiwan23F-15 (FWSS01000062), and Poland23F-16 (FWSU01000014), and from S. mitis B6 (NC_013853) and S. mitis M3 (LROV01000013).Chromosomal DNAs from streptococci were isolated as described previously . PCR propn2xdown  and, in pn2xdown  and in SM779356) , S. mitiPBP2x sequences were aligned by use of the ClustalX2 program  and furtpbp2x genes from the strains analyzed in this study are MF506856 to MF506879 ( The GenBank accession numbers for the MF506879 .


Studies on dsDNA bacteriophages have revealed that a DNA packaging complex assembles at a special vertex called the ‘portal vertex’ and consists of a portal, a DNA packaging ATPase and other components. AdV protein IVa2 is presumed to function as a DNA packaging ATPase. However, a protein that functions as a portal is not yet identified in AdVs. To identify the AdV portal, we performed secondary structure analysis on a set of AdV proteins and compared them with the clip region of the portal proteins of bacteriophages phi29, SPP1 and T4. Our analysis revealed that the E4 34K protein of HAdV-C5 contains a region of strong similarity with the clip region of the known portal proteins. E4 34K was found to be present in empty as well as mature AdV particles. In addition, E4 34K co-immunoprecipitates and colocalizes with AdV packaging proteins. Immunogold electron microscopy demonstrated that E4 34K is located at a single site on the virus surface. Finally, tertiary structure prediction of E4 34K and its comparison with that of single subunits of Phi29, SPP1 and T4 portal proteins revealed remarkable similarity. In conclusion, our results suggest that E4 34K is the putative AdV portal protein. Briefly, the empty capsids are first assembled from the major capsomers, a step that possibly requires scaffolding function of an AdV protein. However, an AdV scaffolding protein is not yet identified but AdV L4 100K, pVII and pVIII seem to be important for this step8. Due to the presence of a packaging signal located close to the left end of the viral genome between the left inverted terminal repeat and the E1A transcription start site, the viral genome is specifically recognized by AdV proteins that constitute the viral packaging machinery. The AdV packaging proteins include IVa2, L4 33K, L4 22K, L1 52/55K, and possibly, DNA binding protein (DBP)15. IVa2, L4 33K and L4 22K mediate specific recognition of the viral genome25. IVa2 is equivalent to the packaging ATPase proteins of dsDNA containing phages and is essential for genome packaging26. The L4 33K, and possibly L4 22K, are required for efficient genome packaging. The L1 52/55K is likely involved in organization and stabilization of the packaged genome28.Adenovirus (AdV) morphogenesis appears to follow a pathway similar to that observed in dsDNA containing bacteriophages and herpesviruses30. The genome is packaged into precursor capsids by the action of a molecular motor that assembles at a unique vertex, the portal vertex. All known portals share a remarkable structural similarity and are invariable dodecamers of the portal protein assembled into a turbine-like structure with a central channel for the passage of genome during packaging31. The portal is also essential for capsid assembly as it serves as the nucleation point for co-polymerization of the scaffolding protein and the capsomers32. Once the capsids are assembled, a complex of viral packaging ATPase associated with the viral genome and other viral proteins assembles as a ring on the portal and threads the genome into the portal channel. The genome is pushed into the capsids by the ATPase function of the ATPase protein. The ATPase activity is further potentiated by another packaging protein which binds to the ATPase.The process of genome packaging is well characterized for dsDNA phages such as phi29, T4, P22 and SPP110. As mentioned earlier, IVa2 possibly serves as an ATPase providing energy during genome packaging. Precise roles of L4-33K and DBP during genome packaging are not clear, but L4-33K seems to potentiate ATPase activity of IVa29. A structure equivalent to the portal has not been identified in AdV. Despite limited sequence similarity, all the known portal proteins share a remarkable structural similarity33, and as mentioned above, the portals are essential for capsid assembly31. We compared the predicted secondary structures of a number of AdV proteins with the predicted secondary structures of known portal proteins of phages phi29, SPP1 and T4. Secondary structure analysis of E4 34K revealed a region containing a number of α -helices and β-sheets similar to the conserved fold (helix α-3 to α-6) found in the core of portal proteins and referred to as the ‘clip region’35  ratio of the empty and mature particles was determined to be approximately 107 to 101, respectively, suggesting that the fraction with empty particles was not significantly contaminated with mature particles. Uninfected (mock) or HAdV-C5-infected 293 cells were harvested at 36 h post-infection to serve as negative and positive controls, respectively. The extracts of the mock-infected and AdV-infected cells, as well as purified preparations of empty and mature virus particles were separated by SDS–PAGE for immunoblot analysis with anti-E4 34K antibody. In HAdV-C5-infected cells, E4 34K was detected primarily as a monomer; however, a faint band was also seen running above the 50 kDa MW marker  and the small terminase. IVa2 seems to act as the packaging ATPase, whereas the L4 33K (will be referred to as 33K for simplicity) possibly functions as small terminase equivalent and potentiates ATPase activity of IVa2IgG Fig.  or with IgG Fig. .To further validate the above results, we performed confocal microscopy to visualize co-localization of E4 34K with IVa2, 33K and DBP. 293 cells were transfected with various combinations of plasmids pEGFP/IVa2, pEBFP/33K, pmCherry/DBP, pEGFP/E4-34K and pmCherry/E4-34K. Specifically, the fusion proteins were expressed in the following combinations: E4 34K-EGFP and DBP-mCherry, E4 34K-EGFP and 33K-EBFP or IVa2-EGFP and E4 34K-mCherry. The transfected cells were examined by confocal microscopy at 36 h post-transfection. Superimposition of the images from green (E4 34K-EGFP) & red (DBP-mCherry), green (E4 34K-EGFP) & blue (33K-EBFP), and green (IVa2-EGFP) & red (E4 34K-mCherry) channels resulted in yellow, cyan and yellow fluorescence, respectively Fig. . These r25. Since the portal is expected to be presented in both the empty and mature particles, it was not necessary to separate them for the detection of E4-34K by immunogold EM. Purified HAdV-C5 particles were layered onto Formvar-carbon-coated nickel EM grids and treated with anti-E4 34K antibody or normal rabbit IgG. Specific binding of the primary antibody was detected by anti-rabbit antibody conjugated to 6 nm gold particles. Grids were negatively stained with phosphotungstic acid (PTA) and examined by transmission EM (TEM). The anti-E4 34K antibody-treated virions demonstrated the presence of gold particles at a single location  and tertiary structures , a derivative of HEK 293 cells that constitutively expresses Cre recombinase42, was obtained from Dr. Frank Graham, McMaster University, Hamilton, Ontario, Canada and Dr. Andy Bett, Merck, Kenilworth, NJ, United States. These cell lines were maintained in minimum essential medium (MEM) with 10% FetalClone III serum . Viruses used in this study were HAdV-C5 and ADLC8cluc, an HAdV-C5 vector in which the packaging signal is flanked by loxP sites42. HAdV-C5 was obtained from ATCC. ADLC8cluc was obtained from McMaster University, Hamilton, Ontario, Canada and Merck, Kenilworth, NJ, United States. The HAdV-C5 and ADLC8cluc were grown in 293 or 293cre, respectively to purify the mature or empty virion particles using a continuous cesium chloride density gradient and ultra-centrifugation as described earlier43. Virus titers were determined by plaque assay on BHH2C (bovine-human hybrid clone 2 C) cells44. Purification of empty and mature virus particles was performed as described earlier45. Plasmids pEGFP/IVa2, pEBFP/33K and pmCherry/DBP have been described earlier10. Plasmid pEGFP/E4-34K is pcDNA3.1+ expressing E4 34K-EGFP fusion protein . Plasmid pmCherry/E4-34K is pcDNA3.1+ expressing E4 34K-mCherry fusion protein .HEK 293 (p31), a human embryonic kidney cell line transformed with E1 region of HAdV-C510. Anti-DBP antibody clone B646 was kindly provided by Dr. Arnold Levine . Cells extracts or purified virus particles were processed for immunoblotting as described earlier44. The HAdV-C5 hexon mouse monoclonal antibody (clone: 65H6) was purchased from ThermoFisher Scientific and it recognizes a linear epitope.Anti-E4 34K antibody was kindly provided by Dr. Philip Branton . Anti-IVa2 and anti-33K antibodies have been described earlier10. The immunoprecipitated complexes were analyzed by SDS-PAGE followed by immunoblotting with anti-E4 34K antibody.Preparation of nuclear extracts from HAdV-C5-infected cells and co-immunoprecipitation with anti-IVa2, anti-33K and anti-DBP antibodies from nuclear extracts have been described earlierl-lysine. At about 60% confluency, cells were transfected with plasmids pEGFP-IVa2, pEBFP-33K, pmCherry-DBP, pEGFP/E4-34K and pmCherry/E4-34K in various combinations. The transfections were done using Lipofectamine 2000 following manufacturer’s instructions. At 36 h post-transfection, the cells were washed once with PBS followed by fixation with 4% PFA in PBS for 15 min at room temperature. The coverglasses were mounted on glass slides using Fluoro-gel mounting medium . Fluorescence imaging was performed using a Nikon C1+ confocal microscope  with 60× objective. Image analyses were performed using NIS Elements software (Nikon).293 cells were grown on coverglasses  pre-coated with poly-10, with anti-E4 34K antibody as primary antibody and anti-rabbit antibody labeled with 6 nm gold particles as secondary antibody.Purified preparations of HAdV-C5 were used for immunogold electron microscopy, as described elsewhere47. Consensus secondary structures were extracted from both prediction results. For tertiary structure prediction, protein sequences were analyzed using RaptorX Structure Prediction48 and I-TASSER40 guiding model (Phi29 was used as a template) was employed using their online programs with default settings. The final structure images were generated with MacPyMOL (v1.8.4.0).E4 34K homologous proteins in 65 different human and animal AdVs were identified using NCBI BLASTp. Sequences of these proteins were aligned with E4 34K of HAdV-C5 using COBALT (Constraint Based Multiple Alignment Tool) at the NCBI website. Protein sequences of E4 34K and other portal proteins were retrieved from the NCBI website: E4 34K (AP_000228.1), phage phi29 (P04332.1), phage portal protein, SPP1 (GAP04951.1), and bacteriophage T4 Portal Protein (3JA7_L). Sequences were submitted to PSIPRED and I-TASSER through their online websites for secondary and tertiary structure predictions, respectivelyFigure S1


D-amino acid mixtures were found to enhance two non-oxidizing biocides [tetrakis hydroxymethyl phosphonium sulfate (THPS) and NALCO 7330 (isothiazoline derivatives)] and one oxidizing biocide [bleach (NaClO)] against a biofilm consortium from a water cooling tower in lab tests. Fifty ppm (w/w) of an equimass mixture of D-methionine, D-leucine, D-tyrosine, D-tryptophan, D-serine, D-threonine, D-phenylalanine, and D-valine (D8) enhanced 15 ppm THPS and 15 ppm NALCO 7330 with similar efficacies achieved by the 30 ppm THPS alone treatment and the 30 ppm NALCO 7330 alone treatment, respectively in the single-batch 3-h biofilm removal test. A sequential treatment method was used to enhance bleach because D-amino acids react with bleach. After a 4-h biofilm removal test, the sequential treatment of 5 ppm bleach followed by 50 ppm D8 achieved extra 1-log reduction in sessile cell counts of acid producing bacteria, sulfate reducing bacteria, and general heterotrophic bacteria compared with the 5 ppm bleach alone treatment. The 10 ppm bleach alone treatment showed a similar efficacy with the sequential treatment of 5 ppm bleach followed by 50 ppm D8. The efficacy of D8 was found better than that of D4  in the enhancement of the three individual biocides against the biofilm consortium.Different species of microbes form mixed-culture biofilms in cooling water systems. They cause microbiologically influenced corrosion (MIC) and biofouling, leading to increased operational and maintenance costs. In this work, two  Large-scale cooling water systems are widely used to remove heat from industrial equipment using a heat exchanger in chemical manufacturing facilities, power plants and petroleum refineries . NaturalMicrobiologically influenced corrosion is a major problem in various industrial sectors, such as water utilities, oil and gas, and power generation . MIC in Biocide dosing is a common approach to combat microorganisms in cooling water systems . ChlorinD-amino acids are naturally occurring chemicals. D-methionine (D-met), D-leucine (D-leu), D-tyrosine (D-tyr), and D-tryptophan (D-trp) were found to disperse Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus biofilms (D-amino acids can enhance the efficacy of some existing biocides against corrosive biofilms. Lab tests showed that 1 ppm (w/w) D-tyr and 100 ppm D-met individually enhanced the efficacy of low concentrations of THPS and ADBAC  biocides, respectively in the mitigation of the Desulfovibrio vulgaris (a sulfate reducing bacterium) biofilm on carbon steel, achieving better efficacies than higher concentrations of THPS and ADBAC (D-tyr at low concentrations (2–5 ppm) were found to enhance ciprofloxacin in the mitigation of anaerobic P. aeruginosa biofilms by achieving better efficacies than higher concentrations of ciprofloxacin  and equimass D-tyr, D-met, D-trp, D-leu, D-serine (D-ser), D-threonine (D-thr), D-phenylalanine (D-phe), and D-valine  (labeled as D8) were evaluated as biocide enhancers for bleach (active component: NaClO), THPS, and NALCO 7330 (active components: 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one) against a field biofilm consortium on C1018 carbon steel coupons retrieved from a water cooling tower.biofilms . D-aminond ADBAC . D-tyr afloxacin . It was  effects . In thisFigure 1A). The coupons were shipped overnight in capped vials to minimize deterioration of the biofilms. The biofilm morphology of the consortium on a coupon surface before the removal tests was observed using an SEM (scanning electron microscopy) . The detailed procedure of the coupon preparation for the observation under SEM was reported before . Other chemicals used in this study were purchased either from Fisher Scientific  or Sigma–Aldrich . The biofilm treatment lab tests were conducted aerobically. Before each lab test, the PBS  buffer solution, tweezers, test tubes, and pipette tips were autoclaved at 121°C for 20 min. D-amino acid solutions were sterilized with a 0.22 μm Stericup filter . All experiments were conducted at least three times for accuracy.C1018 carbon steel coupons covered with biofilms were retrieved from a water cooling tower in a US chemical manufacturing facility after field exposure of 3 weeks . Biofilms were treated with non-oxidizing biocides (THPS and NALCO 7330 separately) with and without a D-amino acid mixture. The test matrix is shown in Table 1.Before the 3-h biofilm removal test, planktonic cells and the field fluid on the coupons were rinsed off using a pH 7.4 PBS buffer solution. Then, coupons were placed in weighing dishes with 100 ml of the PBS buffer with added treatment chemicals for 3 h in a biosafety cabinet at 25°C  method. MPN test kits were purchased from Biotechnology Solutions . Three liquid culture media, namely the modified Postgate’s B (MPB) medium for SRB (sulfate reducing bacteria), the standard bacterial nutrient broth for general heterotrophic bacteria (GHB), and the phenol red dextrose (PRD) medium for acid producing bacteria (APB), were used for MPN. After the biofilm removal test, coupons were rinsed with the PBS buffer solution to remove any loosely attached planktonic cells and treatment chemicals. The biofilm was scrapped off a coupon into a test tube with 10 ml PBS buffer using a small sterile brush. The scraped-off biofilm, the brush and the 10 ml PBS buffer were placed in the test tube. A vortex mixer was used for 30 s to suspend all the sessile cells evenly in the liquid before the liquid was used for MPN serial dilutions and incubation at 37°C. Each MPN enumeration was repeated twice for reproducibility. The commonly used Live and dead sessile cells in biofilms on coupons were examined under confocal laser scanning microscopy (CLSM) . The information on the staining procedure was described in a previous work . The ImaFigure 1A) were treated with bleach and D-amino acid mixtures. The operation was conducted in the biosafety cabinet with a cardboard box cover to prevent UV degradation due to exposure to light . Since the bleach can react with D-amino acids, an abiotic chemical compatibility test was conducted first. Five ppm bleach  was mixed with 10 ppm D-amino acid mixture in deionized water without inoculation for 3 h at 25°C. After that, the free chlorine concentration was measured using the “SenSafe Free Chlorine Water Check” test strips .The field coupons covered with biofilms . The company used bacterial tag-encoded semi-conductor sequencing with 515F-GTGCCAGCMGCCGCGGTAA and 806R-GGACTACHVGGGTWTCTAAT as primers for the analysis. Samples were amplified for semi-conductor sequencing employing a forward primer and a reverse fusion primer. The amplification products were analyzed using eGels . Sequencing was performed with the Ion Torrent PGM following manufacturer protocols . The metagenomics data reveal that there were many species in the biofilm consortium. Achromobacter marplatensis, Lacibacter cauensis, and Sphingobacterium sp. are APB  and D8 alone (p = 0.036) treatments both achieved 1-log GHB sessile cell reduction compared with the no treatment control. Compared with the no treatment control, no APB and SRB sessile cell reductions were observed with the 50 ppm D4 alone or 50 ppm D8 alone treatment. The 15 ppm THPS alone treatment only achieved 1.5-log GHB sessile cell reduction compared with the no treatment control (p = 0.032). The cocktail of 15 ppm THPS + 50 ppm D4 led to extra 1-log reduction of APB sessile cell count compared with the 15 ppm THPS alone treatment (p = 0.011). The treatment of 15 ppm THPS + 50 ppm D8 achieved extra 1.5-log reductions of APB sessile cell count (p = 0.008) and extra 1-log reduction of SRB (p = 0.005) and GHB (p = 0.004) in sessile cell counts, respectively compared with the 15 ppm THPS alone treatment. The outcome of the 15 ppm THPS + 50 ppm D8 treatment was similar to that of the 30 ppm THPS alone treatment. This means 50% reduction in THPS dosage was achieved. Results here indicate that D8 at the same concentration was more powerful than D4 in the enhancement of THPS against the biofilm consortium.Figure 4 shows the CLSM biofilm images after different treatments using THPS. There were many live cells (green dots) on the no treatment control coupon . Biofilm images after the 50 ppm D4 alone treatment, and the 50 ppm D8 alone treatment are shown in Figures 4B,C. The 15 ppm THPS alone treatment led to many dead cells (red dots) among live cells . After the 15 ppm THPS + 50 ppm D4 treatment , there were fewer live cells compared with the 15 ppm THPS alone treatment. After the 15 ppm THPS + 50 ppm D8 treatment , there were much fewer live cells. Dead cells were abundant after the 30 ppm THPS alone treatment . Figure 4H shows the numbers of live and dead sessile cells calculated from CLSM images in Figure 4 using the ImageJ software. The results indicate that the mixture of D-amino acids alone had a very minor ability for the biofilm removal (p = 0.032). The cocktail of 15 ppm THPS + 50 ppm D8 achieved a better efficacy than the cocktail of 15 ppm THPS + 50 ppm D4 (p = 0.007). The efficacy of the cocktail of 15 ppm THPS + 50 ppm D8 was similar to that of the 30 ppm THPS alone treatment (p = 0.108). The CLSM data in Figure 4 supported the MPN data in Figure 3.Figure 5), the 15 ppm NALCO 7330 alone treatment had a similar efficacy as the 15 ppm THPS alone treatment. The cocktail of 15 ppm NALCO 7330 + 50 ppm D4 led to extra 1-log reduction in GHB sessile cell count in comparison with the 15 ppm NALCO 7330 alone treatment (p = 0.006). The 15 ppm NALCO 7330 + 50 ppm D8 resulted in extra 1.5-log reductions of APB (p = 0.009) and GHB (p = 0.003) sessile cell counts and extra 1-log reduction of SRB (p = 0.008) sessile cell count in comparison with the 15 ppm NALCO 7330 alone treatment. The cocktail of 15 ppm NALCO 7330 + 50 ppm D8 achieved a similar efficacy to that achieved by the 30 ppm NALCO 7330 alone treatment. This means that 50 ppm D8 could reduce the biocide concentration by 50%. Results here demonstrate that the D-amino acid mixtures enhanced these non-oxidizing biocides at lower concentrations by achieving a similar efficacy as higher concentrations of non-oxidizing biocides. The CLSM images in Figure 6 corroborated the sessile cell count results in Figure 5. With treatments of 15 ppm NALCO 7330 + 50 ppm D8 and 30 ppm NALCO 7330 alone, much fewer live cells were observed compared with those in the 15 ppm NALCO 7330 alone treatment. Figure 6E shows the numbers of live and dead sessile cells calculated from CLSM images in Figure 6 using the ImageJ software. The cocktail of 15 ppm NALCO 7330 + 50 ppm D8 achieved a better efficacy than the cocktail of 15 ppm NALCO 7330 + 50 ppm D4 (p = 0.006). The efficacy for the cocktail of 15 ppm NALCO 7330 + 50 ppm D8 was similar to that for the 30 ppm NALCO 7330 alone treatment (p = 0.309). The enhanced non-oxidizing biocides with D-amino acid mixtures confirmed that a biocidal stress is necessary for D-amino acids to disperse the biofilm consortium  were mixed with 5 ppm bleach  in a dark environment for 3 h at 25°C. The free chlorine test strips can detect levels of ClO- from 0 to 6 ppm, with a color range from white to dark blue. A white color means 0 ppm ClO-. Figure 7 suggests that the D-amino acids depleted the ClO- after 3 h. Thus, bleach and D-amino acids should not be dosed together.Chlorine is an oxidizing agent that can react with amino acids . In the p = 0.035), 50 ppm D8 alone (p = 0.035), and 5 ppm bleach alone (p = 0.029) treatments  all achieved 1 log GHB sessile cell reduction in comparison with the no treatment control . When 5 ppm bleach and 50 ppm D4 or D8 were mixed to treat for 4 h zero-log reductions in APB, SRB, and GHB sessile cell counts were achieved in comparison with the treatment using 5 ppm bleach alone (lasting 2 h). The results confirmed that the chlorine reaction with D-amino acids rendered D-amino acids ineffective. Thus, a sequential treatment method was applied.At the end of the 4-h biofilm removal test, 50 ppm D4 alone  and GHB (p = 0.002) sessile cell counts in comparison with the 5 ppm bleach alone treatment. The sequential treatment of 5 ppm bleach followed by 50 ppm D8 achieved extra 1-log reductions of APB (p = 0.001), SRB (p = 0.021), and GHB (p = 0.001) sessile cell counts in comparison with the 5 ppm bleach alone treatment. The 10 ppm bleach alone treatment showed a similar efficacy with the sequential treatment of 5 ppm bleach followed by 50 ppm D8. This means that 50 ppm D8 could reduce the bleach dose by 50%.In the sequential treatment, the coupon was first immersed in 100 ml of the PBS buffer containing 5 ppm bleach. After 2 h, it was retrieved and placed into 100 ml fresh PBS buffer containing a 50 ppm mixture of Figure 9 supported the sessile cell count results in Figure 8. There were numerous live cells on the coupons from the no treatment control, the 50 ppm D4 alone treatment, and the 50 ppm D8 alone treatment as seen in Figures C. Many live sessile cells also appeared in the combination treatments using bleach + a D-amino acid mixture . Fewer live cells appeared in sequential treatments  in comparison with the bleach alone treatment . The CLSM images also showed the efficacy of the sequential treatment of 5 ppm bleach followed by 50 ppm D8  was close to that of the 10 ppm bleach alone treatment . Figure 9J shows the numbers of live and dead sessile cells calculated from CLSM images in Figure 9. The sequential treatments of 5 ppm bleach followed by 50 ppm D4 (p = 0.030) and 5 ppm bleach followed by 50 ppm D8 (p = 0.012) achieved a better efficacy than the 5 ppm bleach alone treatment. The efficacy for the sequential treatment of 5 ppm bleach followed by 50 ppm D8 was similar to that for the 10 ppm bleach alone treatment (p = 0.092).The CLSM analysis in Figure 9 shows that 50 ppm D8 showed a better efficacy than 50 ppm D4. This was likely because different microbial species in the biofilm consortium responded to different D-amino acids. Thus, a mixture of more D-amino acids was better  at 50 ppm enhanced two non-oxidizing biocides (THPS and NALCO 7330) against a field biofilm consortium from a water cooling tower. D8 was found to be more effective than D4 at the same concentration to enhance the biocides. Fifty ppm D8 enhanced 15 ppm THPS and 15 ppm NALCO 7330 by achieving similar efficacies as biocides at 30 ppm. A sequential treatment was tested for bleach due to its reactivity with D-amino acids. The sequential treatment of 5 ppm bleach followed by 50 ppm D8 achieved extra 1-log reductions in APB, SRB, and GHB sessile cell counts in comparison with the 5 ppm bleach alone treatment. The 10 ppm bleach alone treatment showed a similar efficacy with the sequential treatment of 5 ppm bleach followed by 50 ppm D8. This work demonstrated that 50 ppm D8 reduced the biocide dosages by 50% while achieving the same efficacies.In this work, two different Conceived and designed the experiments: HA-M and TG. Performed the experiments: RJ and YL. Analyzed the data: RJ and YL. Wrote and polished the paper: RJ, YL, HA-M, and TG.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.


To determine whether 3D acquisitions provide equivalent image quality, lesion delineation quality and PI-RADS v2 performance compared to 2D acquisitions in T2-weighted imaging of the prostate at 3 T.This IRB-approved, prospective study included 150 consecutive patients . Two uroradiologists  independently rated image quality and lesion delineation quality using a five-point ordinal scale and assigned a PI-RADS score for 2D and 3D T2-weighted image data sets. Data were compared using visual grading characteristics (VGC) and receiver operating characteristics (ROC)/area under the curve (AUC) analysis.p = 0.269. Lesion delineation was rated good to excellent for 2D  and 3D T2w  without significant differences (p = 0.785). ROC analysis showed an equivalent performance for 2D (AUC 0.580–0.623) and 3D (AUC 0.576–0.629) T2w .Image quality was similarly good to excellent for 2D T2w  and 3D T2w , Three-dimensional acquisitions demonstrated equivalent image and lesion delineation quality, and PI-RADS v2 performance, compared to 2D in T2-weighted imaging of the prostate. Three-dimensional T2-weighted imaging could be used to considerably shorten prostate MRI protocols in clinical practice.• 3D shows equivalent image quality and lesion delineation compared to 2D T2w.• 3D T2w and 2D T2w image acquisition demonstrated comparable diagnostic performance.• Using a single 3D T2w acquisition may shorten the protocol by 40%.• Combined with short DCE, multiparametric protocols of 10 min are feasible. Multiparametric MRI (MP-MRI) of the prostate is increasingly recognised as an accurate tool for the diagnosis of prostate cancer (PCa) . It enabCurrently, combinations of high-resolution, T2-weighted imaging, diffusion-weighted imaging (DWI) and dynamic contrast-enhanced (DCE) imaging are key components for accurate interpretation and reporting of prostate findings , 8. WhilConsequently, the purpose of our study was to determine whether 3D T2-weighted image acquisitions achieve similar image quality, lesion delineation quality and PI-RADS v2 performance compared to standard 2D acquisitions in prostate MRI at 3 T, without an endorectal coil.The local ethics committee approved this prospective study. Between November 2015 and February 2017, 150 consecutive patients fulfilled the inclusion criteria: multiparametric MRI of the prostate due to elevated PSA level and/or a suspicious digital rectal examination and/or local staging of the cancer in case of known prostate cancer. There were no specific exclusion criteria.All patients were examined using a 3-T MRI system . The vendor-supplied combined spine array and body array receive-only coils were used for signal acquisition. After emptying the bladder, the patients were positioned in the feet-first supine position. An antiperistaltic agent, 10 mg of hyoscine butyl-bromide , was injected intramuscularly. In addition, the rectum was filled with ultrasound gel to improve image quality by minimising artefacts.The imaging protocol was performed in accordance with the PI-RADS v2 guidelines, including T2w images, diffusion-weighted imaging (DWI) and dynamic contrast enhancement (DCE) . DetaileThe image acquisition time for the three-parametric protocol was approximately 19 min comprising 11:14 min for 2D T2w, 2:29 min for DWI and 5:02 min for DCE.Qualitative visual image analysis was performed by two different radiologists with at least 5 years of experience in MRI . The image analysis was performed on a dedicated workstation. The images were anonymised and reviewed randomly. To avoid any bias, the two readers independently reviewed each acquired data set and assigned ordinal numeric scores to each examination.Lesions that were noted in the report were independently assessed by each reviewer. Lesion delineation was rated from 1 (poor) to 5 (excellent) and a diagnostic PI-RADS category was assigned for the most suspicious lesion.1 = images non-diagnostic2 = images poor but still interpretable3 = image quality acceptable4 = image quality good5 = image quality excellentA score ranging from 1 to 5 was used to describe the overall image quality:An example of image quality grading is provided in Fig. 1 = non-diagnostic2 = poor but still interpretable3= acceptable4 = good5 = excellentThe readers evaluated the delineation of the lesions and assigned a confidence score ranging from 1 to 5. In patients with more than one lesion, we assessed an index lesion. The index lesion was determined by the largest and best-seen lesion.An example of lesion delineation and characterisation is provided in Fig. For lesion characterisation, the assessed lesions were rated according to the PI-RADS version 2 guidelines . Both reFor lesion diagnosis, only histologically verified cases were considered. Histopathology was obtained as follows: in case a lesion was assigned a PI-RADS 4/5 category, MRI-guided, in-bore 18G core biopsy was performed according to reported standards . If lesip < 0.05 were considered statistically significant.All the statistical analyses were performed with commercially available software. For data collection and management, an Excel 2011  spreadsheet was used. The statistical analysis was performed with Medcalc 15.8 . Values of p < 0.05 were considered statistically significant.Ordinal visual analysis ratings were analysed by visual grading characteristics (VGC) analysis, a non-parametric, rank-invariant statistical method derived from receiver operating characteristics (ROC) analysis. VGC was used to directly determine the difference in image quality between the two T2w image acquisitions . The areBetween November 2015 and February 2017, 150 consecutive patients  met the inclusion criteria. No patients had to be excluded. In all consecutive patients, the examination was conducted without interruptions or side effects.p = 0.894; R2, 0.531, p = 0.351). There was no significant difference between both readers, p = 0.269  judged the image quality for 2D T2w  and 3D T2w  as between good and excellent. Overall imaging quality ratings did not differ between 2D and 3D T2w image acquisition .In our patient cohort, 67 lesions were identified. The lesion delineation was rated as good/excellent by both readers for 2D T2w  and 3D T2w  Fig. . The areFor lesion characterisation, 53 histopathologically verified patients were further evaluated. Histopathology revealed a prostate carcinoma in 40 patients, and the lesion was benign in 13 patients.p = 0.099) between the readers  between the readers  and 0.623 (R2). There was no significant difference  and p = 0.971 (R2).When comparing the diagnostic accuracy rating for the 2D and 3D T2w images according to the PI-RADS criteria, there was no statistically significant difference between the image acquisition methods: p > 0.05. These results suggest the possibility of using a shorter imaging protocol in patients with suspected prostate cancer, with no decrease in diagnostic accuracy.Our study demonstrated that a faster T2w image acquisition by 3D T2w achieved results as good as standard 2D T2w for overall image quality, lesion delineation and lesion characterisation. There was no statistically significant difference between the two sequences, Prostate MRI protocols routinely include a 2D T2-weighted sequence in three orthogonal planes while 3D T2w images are still only considered an adjunct to 2D image acquisitions . These sBefore being applied in the clinical setting, the equivalence of both 2D and 3D acquisitions, in terms of image quality and diagnostic information, needed to be investigated by a head-to-head comparison of both techniques. In our study, we found no difference between the techniques in terms of image quality, lesion delineation and PI-RADS v2 T2w scoring. Our results are supported by a study by Rosenkrantz et al., who showed a substantial agreement between the 2D and 3D sequences for the detection and staging of PCa . The autThe 3D technique may also prove beneficial for further evaluation of the neurovascular bundle and for the assessment of extracapsular extension. Panebianco et al. assessed the value of 2D and 3D T2w to assess neurovascular bundle changes after nerve-sparing radical retropubic prostatectomy with regard to erectile function . They coIn our study, the 3D acquisition required 3 min 45 s, whereas the standard acquisition of three perpendicular 2D T2w planes requires 11 min 14 s. Therefore, the overall protocol in our study could have been shortened by 40% in our scenario  approach has been reduced to a three-parametric standard in the PI-RADS version 2. Several studies had proven that 3D proton MR spectroscopy was of little additional diagnostic value to a three-parameter  core protocol –18. By iOne limitation of our study was the limited number of histologically verified lesions in our prospective and consecutive patient cohort. However, the aim of our study was to compare two T2w sequences with regard to overall image quality, quality of lesion delineation and characterisation. As this was a head-to-head comparison of two different T2w acquisition techniques, these were not embedded in different, full MP-MRI readings. Thus, we did not evaluate 2D T2w + DCE/DWI versus 3D T2w + DCE/DWI separately, as the equivalence of both techniques was sufficiently proven by our methodological approach. On the basis of our results, we may thus assume no differences in a three-parametric reading considering either 3D or 2D T2w acquisitions. Of note, the AUCs for both readers were low for lesion characterisation on both T2w sequences. This was expected as histopathology was obtained solely in MRI-detected PI-RADS 4 and 5 lesions causing a patient selection towards suspicious cases and low accuracies. Still, these results further stress the importance of a multiparametric approach to prostate MRI. The protocol time reductions reported here need to be interpreted with care as they may vary to a substantial degree depending on the choice of acquisition parameters. Despite visual grading characteristics being an established and appropriate method for image comparison studies , 23, 24,In conclusion, a single isotropic 3D acquisition of T2-weighted images in multiparametric prostate MRI is equivalent with regard to image quality, lesion delineation and characterisation compared to the longer 2D acquisition in multiple planes. On the basis of these results, multiparametric prostate MRI protocols shorter than 12 min can be designed that allow the examination of up to two patients more per hour in clinical practice.