Metarhizium anisopliae, chitindegradation is also used to breach the host cuticle during infection. In view ofthe putative role of NAGases as virulence factors, this study explored thetranscriptional profile and evolution of putative GH20 NAGases(MaNAG1 and MaNAG2) and GH3 NAGases(MaNAG3 and MaNAG4) identified inM. anisopliae. While MaNAG2 orthologs are conserved inseveral ascomycetes, MaNAG1 clusters only with Aspergilllus sp.and entomopathogenic fungal species. By contrast, MaNAG3 and MaNAG4 werephylogenetically related with bacterial GH3 NAGases. The transcriptionalprofiles of M. anisopliae NAGase genes were evaluated in sevenculture conditions showing no common regulatory patterns, suggesting that theseenzymes may have specific roles during the Metarhizium lifecycle. Moreover, the expression of MaNAG3 andMaNAG4 regulated by chitinous substrates is the firstevidence of the involvement of putative GH3 NAGases in physiological cellprocesses in entomopathogens, indicating their potential influence on celldifferentiation during the M. anisopliae life cycle.Cell walls are involved in manifold aspects of fungi maintenance. For severalfungi, chitin synthesis, degradation and recycling are essential processesrequired for cell wall biogenesis; notably, the activity ofβ-N-acetylglucosaminidases (NAGases) must be present for chitin utilization. Forentomopathogenic fungi, such as  Chitin is the second most abundant polymer on Earth and its recycling from carapaces,cuticles and fungal cell walls impacts on carbon and nitrogen cycles. The chitinpolymer is composed of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) subunits  and its NAGases are classified into three glycoside hydrolase (GH) families, 3, 20, and 84,on the basis of their amino acid sequence similarities 2-3 substrates. This report furthersupports the existence of GH3 NAGases in other fungal species, especially inascomycetes, considering their expansion of chitinolytic machinery genes . In these species, thechitinolytic system has, probably, two main biological functions: Firstly, as chitinis the major component of fungal cell walls, chitin-degrading enzymes act on thecell wall remodeling, which is necessary for hyphal vegetative growth , are processes that require chitin degradation  (A.nidulans (XP_659106) (T.atroviride (EHK40646 and EHK46127)  was used as a query in the tBLASTn search (R. miehei CAU-432(AGC24356), the only fungal GH3 family member with NAGase activity to date were cultured under seven different growth conditions prior to RNAextraction: i) Cove’s Complete medium (MCc) containing(w/v) 1% glucose, 0.6% NaNO3, 0.15% casein hydrolisate, 0.05% yeastextract, 0.2% peptone, pH 7.0 plus 2% (v/v) salts solution  and 0.04% (v/v) Trace Elements Solution  (ii) 0.25% GlcNAc in minimum mediumcomposed of 0.6% NaNO3 (w/v) plus 0.25% GlcNAc) (w/v) as carbohydratesource, with salts and trace element solutions (iii) 1%Chitin in minimum medium composed of 0.6% NaNO3 (w/v)plus 1% crystalline chitin from crab shells as a carbohydrate source, with saltsand trace element solutions  for 72 h at 28 °C, then washedwith sterile distilled water and filtered through Miracloth andfrozen in liquid nitrogen for total RNA extraction; iv)Autolysis: medium for mycelium autolysis induction (1% glucose(w/v) and 0.6% NaNO3 (w/v), sustained for 9 days) (v) Sporulation: on MCc agar plates for conidia RNAextraction; vi) Blastospores: Inoculation of5104 conidia/mL on ADAMEK medium for blastospore production , shaking for 64 h at28 ºC (vii)Appressoriuminduction medium: 5105 conidia/mL wasinoculated in 0.004% yeast extract solution on 500 glass coverslips for 16 h at28 ºC .at28 ºC ; vii)ApM. anisopliae cells harvested underall seven different growth conditions was performed in triplicate. Samples wereground using a mortar and pestle in liquid nitrogen, prior to standard RNAextraction using Trizol Reagent .Residual DNA was removed with DNase . Thereafter,extracted RNAs were passed through RNeasy Cleanup columns . RNA samples were quantified using a Qubit fluorometer , and stored at -80 °C. One microgram oftotal RNA was used for cDNA synthesis using MMLV-RT enzyme . All procedures were performed according to themanufacturer’s instructions.Total RNA extraction from Polymerase chain reactions were carried out on ABI-7500 Real-Time PCR System. Platinum SYBR Green qPCRSuperMix-UDG  was used to monitordsDNA synthesis. Each biological sample was analyzed in technical triplicates;no-template and no-reverse transcriptase controls were included.TableS1). Five housekeeping genes were evaluated:act (γ-actin), gapdh , tef1-α (translation elongationfactor 1-α), trp1 (tryptophan biosynthesis enzyme), andtub (α-tubulin). The efficiency of each reference geneacross samples was analyzed using geNorm version 3.5 .Primers for qPCR assays were designed using VECTOR NTI software  (-DDCt method (p < 0.05) were performed to determine statisticaldifferences among 2-DDCt values of the seven experimentalconditions.Melting curves from each qPCR reaction were analyzed to confirm specificity ofthe synthesized products and absence of primer dimers. Relative transcriptexpressions were analyzed by Cq (quantification cycle) values, applying the2t method . ResultsM. anisopliae genome, usingNagA from A. nidulans and NAG1 and NAG2 from T.atroviride as queries, resulted in the identification of twoputative GH20 NAGases, named MaNAG1  and MaNAG2 . All otherfungal GH20 NAGase sequences and GH20 conserved domain sequences used as queriesresulted in alignments with the same two previously detected contigs. Therefore,MaNAG1 and MaNAG2 are most probably the only M. anisopliaeputative GH20 NAGases. The GH20 family domain (IPR015883) and the conservedmotif of GH20 proteins (H/N-x-G-A/C/G/M-D-E-A/I/L/V) were found in both MaNAG1and MaNAG2 sequences . Additionally, the putative GH20 NAGasesalso exhibited a chitobiase/beta-hexosaminidase N-terminal domain (IPR029018)(The survey of NAGase genes of the R029018).M. anisopliae genome allowed theidentification of seven positive matches. However, phylogenetic analysis clearlyrevealed that only two sequences, named MaNAG3  and MaNAG4  could be putative GH3 NAGases.Furthermore, these sequences exhibit higher similarity with bacterial GH3NAGases and the RmNag GH3 . MaNAG3 and MaNAG4 share a conserveddomain with GH3 family members (IPR001764) and exhibit the conserved sequencemotif of GH3 proteins (K-H-F/I-P-G-H/L-G-x-x-x-x-D-S/T-H). Furthermore, MaNAG3 and MaNAG4sequences present a conserved GH3 C-terminal domain (IPR002772) , raising the possibility of functionalequivalence.The GH3 domain screening of the R002772) . The othM. anisopliae putative NAGasesare listed in A.nidulans NagA (68 kDa) (T. atrovirideNag1 (73 kDa) (T. harzianum P1 Nag1 (72 kDa) , containingthe highest expected number of introns (4) and theoretical molecular mass (98.71kDa), with N-glycosylation translational modification signals on six sites. Incontrast, MaNAG4 ORF size is 2,057 bp, the theoretical molecular mass is 60.67kDa and the pI of predicted mature protein is 5.6. The predicted molecular massof MaNAG4 is similar to most known bacterial GH3 NAGases, as S.thermoviolaceus NagA (60 kDa) , were recognizedin all of GH20 orthologs .Twenty-six MaNAG1 and MaNAG2 orthologs were identified in 15 filamentous fungigenomes. Most of them are single copy of each putative GH20 NAGase of M. anisopliae and the other fifteen ascomycetes revealed anearly duplication event in GH20 NAGases, resulting in two distinct main clades. This cluster also formed a statistically supported cladewith species from the Aspergillus genus. In contrast, MaNAG2exhibits a more diverse evolutionary history, with orthologs present inTrichoderma sp., Fusariumsp, Neurospora sp., and Magnaporthe sp.Interestingly, the present evolutionary analysis revealed that both NAG1 andNAG2 from the mycoparasite T. atroviride, used in theM. anisopliae genome screening, are evolutionarily morerelated to MaNAG2 . All 15 filamentous fungi have MaNAG3orthologs. However, the M. acridum gene ortholog was notincluded in the phylogenetic analysis, since it was not properly annotated inthe M. acridum genome. In turn, onlyTrichoderma sp., Aspergillus sp., and theentomopathogens C. militaris and B. bassianahave MaNAG4 orthologs.Twenty-three MaNAG3 and MaNAG4 orthologs were identified on the filamentous fungigenomes examined. Conserved sequence motifs of GH3 proteins (K-H-F / I-P-G-H /L-G-x-x-x-x-D-S / T-H) were found in all of them, however, few amino acidresidues substitutions were observed . Additionally, geneduplication of MaNAG3 and MaNAG4 orthologs was not observed.The phylogenetic tree revealed that MaNAG3 and MaNAG4 orthologs formed twodistinct clusters . Both MaS. thermoviolaceus NagA, Clostridiumparaputrificum NagZ, Alteronomonas sp. HexA,V. furnissii NagZ, Thermotoga maritma NagAand T. neapolitana CbsA) grouped into distinct clades fromfungal NAGases. This is probably due to the fact that some bacterial NAGases donot necessarily have GlcNAc hydrolysis specificity over chitooligosaccharides.For example, E. coli NagZ cleaves GlcNAc from muropeptidespresent in the bacterial cell wall  and GH3 family (MaNAG3 andMaNAG4) of M. anisopliae genome wereanalyzed.Virulence determinants are the main focus in the study of entomopathogenic fungi. As chitM. anisopliae by gel-filtration,with a pI of 6.4 and molecular mass of 110-120 kDa. We hypothesize that thisM. anisopliae purified enzyme could be the MaNAG1 presentedhere, based on the predicted pI (6.07) and molecular mass (66.98 kDa) of MaNAG1,likely forming a homodimer. In fact, some fungal GH20 NAGases  clustered together with characterized β-glucosidases, again suggesting thepossibility of similar function  would repressexpression of chitinolytic enzymes by their own activity products in M.anisopliae (catabolic repression)   in T.reesei, the product of which, an MaNAG3 ortholog, holds suggestedNAGase activity. The phylogenetic analyses indicate that MaNAG3 and T.reesei NAG3 are phylogenetically related  exhibits 63%identity with characterized GH84 from Penicillium chrysogenum(XP_002557703.1). The P. chrysogenum GH84 NAGase not only exhibitactivity against GlcNAc substrates, but also hydrolyzes substrates withgalacto-configuration and exhibits transglycosylation activity(Our results are in agreement to previous suggestions of the presence of GH3 NAGasesin fungi (RmNag) ( related . The exiM. anisopliae and entomopathogenic fungi. Thisanalysis will allow the selection of genes for further functional characterizationto elucidate the process and to identify redundancies and specificities. The viewthat chitinase diversity is merely redundant may not correct (M. anispoliae putative GH20 NAGase genesrevealed induced transcript production in the presence of chitin, potentially in theextracellular milieu. The detection of MaNAG3 andMaNAG4 putative genes is the first evidence for the presence ofa possible GH3 family of NAGases in entomopathogenic fungi. MaNAG3and MaNAG4 expression is responsive to chitinous substrates,suggesting their potential influence on cell differentiation during the M.anisopliae life cycle.In conclusion, this study explored relevant evolutionary aspects of putative GH3 andGH20 NAGase genes and the expression analysis highlighted possible functions forthese genes in


Scientific Reports 10.1038/s41598-018-26198-7, published online 18 May 2018Correction to: The Acknowledgements section in this Article is incomplete.“We thank Sara Olenich, Kayley Abell-Hart and Thanh Von for technical assistance. This study was funded by the Osher Foundation, and in part by NCCIH RO1 AT009267.”should read:“We thank Sara Olenich, Kayley Abell-Hart and Thanh Von for technical assistance. This study was supported by the Osher Foundation (H.M.L.), NCCIH RO1 AT009267 (H.M.L.), Susan G. Komen Foundation PDF16376814 (J.B.), National Institutes of Health (NIH) R35 CA210057 (J.J.Z.) and Breast Cancer Research Foundation (J.J.Z.).”


Serratia marcescens is a ubiquitous Gram-negative opportunistic pathogen. This announcement describes the isolation and genome annotation of S. marcescens T5-like siphophage Slocum. Terminal repeats, 170 protein-coding genes, and 23 tRNAs were predicted in the 112,436-bp Slocum genome. Serratia marcescens is a ubiquitous Gram-negative opportunistic pathogen. This announcement describes the isolation and genome annotation of S. marcescens T5-like siphophage Slocum. Terminal repeats, 170 protein-coding genes, and 23 tRNAs were predicted in the 112,436-bp Slocum genome. Serratia marcescens is a Gram-negative bacillus that is ubiquitous throughout nature and is a member of the family Enterobacteriaceae , was cultured aerobically at 30°C and 37°C in LB (BD), and phages were propagated via the soft-agar overlay method (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). After trimming was performed using the FastX Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit), the raw Slocum contig was assembled using SPAdes v3.5.0, with default parameters, to 409.3-fold coverage  (Bacteriophage Slocum originated from filtered (0.2-μm filter) municipal wastewater in Bryan, Texas. The host, y method . After py method . Phage gy method , and theoverage 810. Rho-ioverage 8. Functiooverage 8–15. Addiverage 8–. Structuverage 8–. The genverage 8–. All lisaxy-pub) , 21. UnlEscherichia phage T5 (GenBank accession no. NC_005859) and T5-like Escherichia phage slur09 (GenBank accession no. LN887948), with 27 to 29% nucleotide identity to slur09 and several Salmonella phages, fragmented across the genome.The phage Slocum genome consists of 112,436 bp of double-stranded DNA, with a G+C content of 44.8%. With 170 predicted protein-coding genes and 23 predicted tRNA genes, the genome protein coding density is 88%. Additionally, 12,521-bp predicted direct terminal repeats were used as the boundary for genome reopening. At the amino acid level, phage Slocum shares 81 similar proteins with MN095770, BioProject accession no. PRJNA222858, SRA accession no. SRR8892204, and BioSample accession no. SAMN11411459.The genome sequence and associated data for phage Slocum were deposited under GenBank accession no.


Gemcitabine/nab-paclitaxel and FOLFIRINOX demonstrated significantly increased survival compared with gemcitabine in metastatic pancreatic ductal adenocarcinoma (PDAC): objective response rate (ORR) 23 and 31.6%, progression-free survival (PFS) 5.5 and 6.4 months, overall survival (OS) 8.7 and 11.1 months. Present phase II study evaluated recommended first-line triplet FIr/FOx schedule.010%, p130%, power 80%, α5%, β20%. Projected ORR: I step, 1/10; II 5/29. Schedule: 12h-timed-flat-infusion/5-fluorouracil 750-800-900 mg/m2 d1-2,8-9,15-16,22-23; irinotecan 120-140-160 mg/m2 d1,15; oxaliplatin 70-80 mg/m2 d8,22; every 4 weeks, according to clinical parameters , liver function). Activity and efficacy were evaluated, and compared using log-rank; limiting toxicity syndromes (LTS), using chi-square.Simon two-step design: pP 0.022).Twenty-nine consecutive patients were enrolled, according to primary/intermediate/secondary Cumulative Illness Rating Scale (CIRS). Median age 62; elderly 13 (44.7%); PS2 3 (10.4%), secondary CIRS 5 (17.2%). Primary endpoint was met: ORR 53% (7/13 patients) as-treated, 50% intent-to-treat. Cumulative G3-4 toxicities: diarrhea 17%, asthenia 14%, hypertransaminasemy 7%, mucositis 7%, vomiting 3%, anemia 3%, thrombocytopenia 3%. LTS were 27.5% overall, 38.4% in elderly. At 3 months median follow-up, PFS 4 months, OS 11 months. PS2 patients showed significantly worse OS (Intensive first-line triplet FIr/FOx is tolerable at modulated doses, and confirms high activity/efficacy in metastatic PDAC. Patients’ careful selection, and exclusion of PS2, can maintain safety profile and efficient dose intensity. Fixed-dose rate gemcitabine (10 mg/m2/minute) maximized intracellular concentrations of the phosphorylated active forms of gemcitabine, and may substitute standard infusion over 30 minutes [Metastatic pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with approximately 6 months median overall survival (OS) –3. In th minutes , 5.BRCA 2 or PALB mutations).Even if phase III studies proposing gemcitabine-based associations failed to improve OS –16, exceThe association of nab-paclitaxel to gemcitabine reported highest clinical benefit in patients with PDAC, and significantly raised up OS 8.7 months, PFS 5.5 months, and ORR 23%, compared with gemcitabine alone , 23. IntThe most relevant issue limiting the feasibility of addition of more drugs in a chemotherapy combination is the proper design of the schedule assuring the balance between tolerability for individual patients and effective received dose intensity (DI) of each drug in order to obtain the expected efficacy of the combination. Intensive regimens based on triplet chemotherapy in MCRC, and FOLFIRINOX regimen in metastatic PDAC, frequently required proper clinical management of toxicity and treatment modulations due to moderate/severe toxicities , 25.PM to 10AM) timed-flat-infusion (TFI) 5-FU (TFI/5-FU), without leucovorin, associated to irinotecan (CPT-11) and OXP, according to a weekly alternating schedule, also added to bevacizumab [Over the last 10 years, we developed triplet chemotherapy regimen according to FIr/FOx schedule, characterized by 12-hour , lung 2 (6.8%), lymph nodes 22 (75.8%), local recurrence 2 (6.8%), cutaneous/subcutaneous tissue 1 (3.4%), peritoneal carcinomatosis 12 (41.3%), bone 2 (6.8%). Metastatic site was single in 2 patients (6.8%), multiple in 27 (93.1%). Single metastatic sites: lymph nodes 2 patients (6.8%). Liver metastases were single in 1 patient (3.4%), multiple in 17 (58.6%).Five patients underwent surgery of primary pancreatic tumor. One patient received adjuvant gemcitabine, 1 patient underwent radiotherapy associated with capecitabine.Baseline CA19.9 measurement was normal in 3 patients, elevated in 26.In the first step, according to two-steps Simon’s design , assuminThe phase II study was performed among the projected 29 patients. In the intent-to-treat analysis, 14 patients were evaluable, 13 patients did not received at least 3 cycles of treatments, and 2 patients were on-treatment at data cut-off. OR were 7 out of 14 patients, ORR 50%    ; PFS was not significantly different (P = 0.078) , even if OS was trendy worse in patients with other than head pancreatic tumor location , even if OS was trendy worse in patients with liver metastases , median OS 11 months (0-33); among 12 patients with other than head pancreatic tumor locations, median PFS was 2 months (0-21), median OS 3 months (0-25). PFS and OS were not significantly different (Ten patients (34.4%) received, at least, a second line treatment: FIr/FOx re-challenge in 1 patient (3.4%); gemcitabine/nab-paclitaxel association in 8 patients (27.5%); intra-arterial chemotherapy in 1 patient (3.4%). Two patients (6.8%) received a third line treatment: gemcitabine in 1 patient (3.4%); capecitabine in 1 patient (3.4%).All evaluable patients with an increased Ca19.9 baseline value had a >20% decrease: 58% ≥50% decrease, 42% ≥70% and 90% decrease. CA19-9 levels were not significantly correlated with PFS and OS.Median number of administered cycles was 2 (range 1-11).2/w, 70.4% of projected-DI (pDI); CPT-11 56 (30-80) mg/m2/w, 70%; OXP 29 (11-40) mg/m2/w, 72.5%  mg/m2/w, 79%; OXP 33.5 (0-40) mg/m2/w, 83.75%. In yE patients, median rDIs per cycle were: 5-FU 1500 (375-1800) mg/m2/w, 83.3% of pDI; CPT-11 64 (30-80) mg/m2/w, 80%; OXP 34 (11-40) mg/m2/w, 85%.Median received dose intensities (rDI) per cycle were: 5-FU 1268.5 375-1800) mg/m75-1800 mTable Overall, LTS were observed in 8 patients 27.5%) , and OS 11 months , in the preliminary analysis of efficacy. Activity and clinical outcome of FIr/FOx exceeded that reported with gemcitabine: median OS 5.7 months, and 1-year OS rate 20%, median PFS 2.08 months, ORR 5.4% .P < 0.001) [Recently, phase III trials evaluating more intensive first-line chemotherapy regimen, such as gemcitabine plus nab-paclitaxel and triplet FOLFIRINOX, demonstrated to be much more, and equivalently effective, with significantly increased survival benefit over standard gemcitabine alone in metastatic PDAC patients : ORR 23%< 0.001) . OS rate< 0.001) , 27.Our present real life study on consecutive, unselected patients showed that 13 patients (44.8%) were not evaluable in the ITT analysis because they did not received at least 3 cycles of treatment, thus confiming that, even today, the primary challenge of clinical management of metastatic PDAC patients is to start and safely perform at least 3 cycles of intensive chemotherapy treatment, to evaluate activity contributing to increase clinical outcome.P = 0.022), thus confirming overall benefit in clinical outcome achieved by metastatic PDAC patients with PS 0-1, treated with gemcitabine-based combinations [Among patients treated with intensive triplet FIr/FOx regimen, PFS and OS were not significantly different among patients treated with modulated and standard drug dosage, due to clinical parameters requiring treatment modulations. PS 2 significantly affected worse OS , the safety profile was less favourable than that reported in the gemcitabine arm [Median rDIs were >80% of the projected dose for each drug, also in elderly patients. Cumulative G3-5 toxicities were prevalently represented by: diarrhea (17%), stomatitis/mucositis (6%), asthenia (14%), nausea (3%), vomiting (3%), hypoalbuminemia (3%), hypokalaemia (7%), hypertransaminasemia (7%), hyperbilirubinemia (3%), hypercreatininemia (3%), anemia (3%), neutropenia (17%), thrombocytopenia (3%). In the previously reported phase II trial proposing intensive first-line triplet chemotherapy plus bevacizumab according to FIr-B/FOx regimen, as first-line treatment in MCRC patients, cumulative G3-4 toxicities were similar: diarrhea (28%), stomatitis/mucositis (6%), asthenia (6%), hypertension (2%), hypertransaminasemy (4%), neutropenia (10%). Triplet FIr/FOx, even associated to bevacizumab, determined only 10% G3-4 neutropenia, while FOLFOXIRI schedule, added or not to bevacizumab , 27, prebine arm . More ad2 plus nab-paclitaxel 125 mg/m2 association, 3 out of 4 weeks [In the phase III trial, gemcitabine 1000 mg/m 4 weeks , prevale 4 weeks .In the present study, LTS were observed in 27.5% of individual patients and in 38.4% of elderly patients treated with FIr/FOx regimen. The innovative clinical evaluation of LTS, consisting of at least the LT associated or not to other G2 or LT, introduced to better evaluate, in the individual patient, the presence of LT alone, LTS-ss, or the association of major toxicities in different sites, LTS-ms, showed that: overall, they were 3.4% and 24.1%, respectively; among elderly patients, they were all LTS-ms 38.4%. LTS were not significantly represented by LTS-ms compared to LTS-ss, even if the small number of enrolled patients requires further analyses. LTS-ms were mostly represented by diarrhea, mucositis, asthenia, neurotoxicity, thrombocytopenia and/or anemia, associated to nausea, vomiting, anorexia and/or neutropenia, hypokaliemia, hypoalbuminemia, hypertransaminasemia. In the individual patient, limiting and moderate toxicities often characterized LTS, previously observed in 44% MCRC patients treated with FIr-B/FOx, and equally involving single or multiple sites .DPYD, UGT1A1, ABCB1, CYP3A4, specifically influencing fluoropyrimidines and CPT-11-related adverse events, justifying inter-patients variability in safety profile, may help selection of patients fit for triplet chemotherapy, and may predict the occurrence of individual LTS, prevalently gastrointestinal [Pharmacogenomic analysis evaluating 5-FU degradation rate (FUDR) and/or detection of a panel of DNA Single Nucleotide Polimorphisms (SNPs) involving different genes, such as testinal , 41, 42.Reported data confirmed that intensive regimens, such as FIr/FOx, frequently required proper clinical management of toxicity and treatment modulations due to moderate/severe toxicities. Careful selection of eligible patients, based on age, PS, comorbidity index, and monitoring of individual safety, also according to LTS in individual patients, are major parameters to optimize clinical management of metastatic PDAC. More, close monitoring of patients, expertise with a particular regimen, and toxicity management, remained the physician-related factors, that can guide personalized selection of first-line regimens in individual PDAC patients.Patients were eligible if they had clinical and/or histological/cytological confirmed diagnosis of measurable metastatic PDAC; age ≥18 years, specifically <65 years (non-elderly), ≥65 <75 years (yE), and ≥75 years (oE); WHO PS ≤ 2; adequate hematological, renal and hepatic functions; life expectancy more than 3 months.Ineligibility criteria: pregnancy and breast-feeding; uncontrolled severe diseases; cardiovascular disease ; thromboembolic disease, coagulopathy, pre-existing bleeding diatheses; sensory and/or motor polineuropathy; surgery within the previous 28 days; previous adjuvant chemotherapy or radiotherapy completed less than 6 months before. CIRS was used to evaluate the comorbidity status . Primaryin label for metastatic PDAC treatment in Italian public hospitals, and published in Gazzetta Ufficiale Repubblica Italiana . The study was approved by the Regional Review Board , and conducted in accordance with the Declaration of Helsinki. All patients provided written, informed consent.Treatment was approved by Agenzia Italiana del Farmaco (AIFA) for administration This was a single-arm phase II study evaluating safety and activity of weekly alternating 5-FU, CPT-11, and OXP (FIr/FOx) as first-line treatment of metastatic PDAC.2/die, over 12-hour (from 10:00 p.m to 10:00 a.m.), days 1-2, 8-9, 15-16 and 22-23; CPT-11 , 120-140-160 mg/m2, administered over 90 minutes as an intravenous infusion in 250 ml of NaCl 0.9%, days 1 and 15; OXP  over 2-hours as an intravenous infusion in 250 ml of dextrose 5%, at the dose of 70-80 mg/m2, days 8 and 22. Cycles were repeated every 4 weeks. 5-FU was administered by a portable pump  using a venous access device. According to patients’ fitness, and particularly in patients with PS 2, and/or ≥75 years, secondary CIRS stage, and/or abnormal liver functional laboratory tests, such as ≥ G2 hypertransaminasemia at baseline, drugs’ doses were modulated in individual patients as reported, providing a received DI >75% for each drug, reported as active and efficacious in previous studies of triplet chemotherapy regimens [FIr/FOx association consisted of 5-FU associated to alternating CPT-11 or OXP according to the following weekly schedule: TFI/5-FU , 750-800-900 mg/mregimens , 26–31.Physical examination and routine laboratory studies were performed at baseline and every week on-treatment, including complete blood cell count, electrolytes, liver and renal function tests, urine examination and coagulation function; tumor markers every cycle; electrocardiogram every cycle, and echocardiogram at baseline, and every 3 cycles.Primary end-point was ORR; secondary end-points were toxicity, PFS, OS. ORR was evaluated according to RECIST criteria ; PFS andLTS, consisting of at least a LT associated or not to other limiting or G2 toxicities, were evaluated as previously reported , 32. TheCorrelations between maximum decrease from baseline in CA19.9 level and PFS and OS were analyzed, to assess possible relationships between CA19.9 and clinical outcomes.0 was considered as the estimated activity reported with gemcitabine alone (median ORR 10%), and confirmed with the association of gemcitabine plus erlotinib (ORR 8.6%) [1 as the projected ORR using the present intensive triplet combination, according to FIr/FOx schedule, increasing the activity ≥ 20% in metastatic PDAC patients, as reported with FOLFIRINOX (ORR 31.6%), and with the association of gemcitabine and nab-paclitaxel in the phase I/II trial  [This phase II study was planned according to two-steps Simon’s design : assuminRR 8.6%) , 16; p1 I trial) –24.FIr/FOx intensive triplet chemotherapy in metastatic PDAC patients preliminary showed high activity. Present schedule was feasible in non-elderly and elderly patients, with PS 0-1, with manageable toxicities, at proper CPT-11, OXP, and 5-FU doses. LTS were prevalently characterized by diarrhea, mucositis, asthenia, anemia, neurotoxicity and/or thrombocytopenia, associated to nausea, vomiting, anorexia and/or neutropenia. Elderly patients preliminary showed trendy, but not significantly worse OS. Adequate selection of suitable patients, based on clinical parameters, aimed to maintain safety profile at efficacious DIs, will verify if more intensive approaches, such as triplet chemotherapy regimen could increase OS, compared to historical gemcitabine control, in the clinical practice management of metastatic PDAC patients, and if it could be a treatment option in locally advanced/borderline resectable pancreatic cancer patients.


Also, the data include the bond lengths and angles of [Ln2(IMBA)6(dmp)2] .In this data article, we present the FT-IR and PXRD data of the lanthanide complexes constructed by 4-iodo-3-methylbenzoic acid (IMBA) and 4,7-dimethyl-1,10-phenanthroline (dmp). Detailed structure analysis, luminescence and sensing properties were discussed in our previous study, “Highly Luminescent Lanthanide Complexes as Bifunctional Sensor for Et Specifications tableValue of the data•This structure information would be valuable for FT-IR analysis of lanthanide complexes.•This data would be worthy for further investigation of the PXRD properties.•This data provide a new process to synthesize two ligands coordinated lanthanide complexes.1[Ln2(IMBA)6(dmp)2] ,  are isostructural. They crystallize in triclinic space group P-1 (no. 2). These complexes are dinuclear cluster structures which contain two lanthanide ions , six deprotonated IMBA and two dmp, forming an electroneutral unit (1a that incubation in aqueous solution for as long as the sensing time (1 h) was in line with the as-synthesized sample and calculated data, confirming that the sensor 1a is a highly stable 6(dmp)2]·(1a). Yield: 37% based on Eu3+. Anal. Calcd (%): C, 39.92; H, 2.645. Found (%): C, 40.13; H, 2.633. FT-IR (−1): 3450 (m), 2947 (w), 1620 (s), 1584 (w), 1438 (s), 1403 (m), 1305 (m), 1193 (w), 1033 (m), 934 (w), 872 (m), 789 (s), 747 (w), 550 (w), 482 (w).[Eu3. FT-IR  (KBr pel2(IMBA)6(dmp)2] (1b). Yield: 34% based on Gd3+. Anal. Calcd (%): C, 39.74; H, 2.633. Found (%): C, 39.61; H, 2.618. FT-IR (−1): 3435 (m), 2995 (w), 1648 (w), 1592 (s), 1542 (m), 1495 (w), 1438 (s), 1305 (m), 1152 (w), 1033 (s), 943 (m), 872 (s), 789 (s), 739 (m), 600 (w), 550 (w), 488 (m).[Gd8. FT-IR  (KBr pel2(IMBA)6(dmp)2] (1c). Yield: 35% based on Tb3+. Anal. Calcd (%): C, 39.68; H, 2.629. Found (%): C, 39.79; H, 2.637. FT-IR (−1): 3443 (m), 2980 (w), 1627 (w), 1592 (s), 1542 (m), 1494 (w), 1430 (s), 1312 (w), 1152 (w), 1033 (s), 934 (w), 872 (m), 824 (m), 789 (s), 739 (w), 550 (w), 488(w).[Tb7. FT-IR  3·6H2O , dmp and IMBA at a molar ratio of 1:1:1.5 at 333 K for 120 h. The colorless block single crystals of 1a–1c were collected by filtration, and mounted on a glass fiber Lanthanide complexes Single crystal X-ray diffraction data were obtained on an instrument of Bruker SMART 1000 CCD, at wavelength of 0.71073 Å (Mo-Ka radiation) at 25 °C. The structures were refined by full-matrix least-squares methods with SHELXL-97 module. Phase purity of bulk samples were tested by PXRD, on a DMAX2200VPC diffractometer


Oribacterium sp. strain C9, isolated from the rumen of a steer grazing on Rhodes grass in Rockhampton, Queensland, Australia.We report the 3.7-Mb genome sequence of  Oribacterium sp. strain C9, isolated from the rumen of a steer grazing on Rhodes grass in Rockhampton, Queensland, Australia. This draft genome consists of 3,720,024 bp with a 42.8% G+C content, 3,130 predicted coding DNA sequences (CDSs), and 67 RNAs.We report the 3.7-Mb genome sequence of  Oribacterium was first proposed by Jean-Philippe Carlier  in Australia , using an anaerobic in vitro culture with 0.8% yeast extract. DNA sequencing was performed at Macrogen (South Korea) on a HiSeq 2500 instrument  (2 × 100-bp paired-end sequencing) to produce 58,649,712 total reads with 15× coverage. Raw reads were trimmed with Trimmomatic version 0.32   version 4 (https://www.ncbi.nlm.nih.gov/genome/annotation_prok) . The average nucleotide identity (ANI) was calculated using the algorithm described by Yoon et al. (Oribacterium sp. strains P6A1 (GenBank accession number JNKO00000000), WCC10 (FOPH00000000), NK2B42 (AUJX00000000), FC2011 (JNJM00000000), and KHPX15 (FNRG00000000) (from the Hungate1000 project [O. parvum strain ACB1T (AFZC00000000), and O. sinus F0268 (ACKX00000000), respectively.The 16S rRNA gene sequence was found to be 95% identical to that of in ACB1T  and Oribn et al.  with theOribacterium sp. strains P6A1, WCC10, NK2B42, FC2011, and KHPX15, O. sinus F0268, and O. parvum strain ACB1T are 28.4%, 24.9%, 24.4%, 24%, 24.1%, 21.1%, and 26.1%, respectively. Having both ANI values lower than 95% and dDDH values lower than 70% indicated that isolated strain C9 is a species distinct from the other isolates.The digital DNA-DNA hybridization (dDDH) was determined using the Genome-to-Genome Distance Calculator (GGDC) version 2.1 with default settings . The esthttp://www.cazy.org/) (http://csbl.bmb.uga.edu/dbCAN/)  (zy.org/)  and dbCA/dbCAN/) , we idenOribacterium sp. strain C9 has been deposited in DDBJ/EMBL/GenBank under the accession number MUHW00000000, BioProject number PRJNA362832, and BioSample number SAMN06249574. The genomic raw sequencing reads are available in the Sequence Read Archive (SRA) database under accession number SRR8449972.The draft genome sequence for


Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA  and CFTR protein (Western blotting) level. MicroRNAs  are from 19 to 25 nucleotide-long noncoding RNAs that regulate gene expression by targeting mRNAs, leading to a translational repression or mRNA degradation ,4. SinceEpigenetic regulation of expression of cystic fibrosis transmembrane conductance regulator (CFTR) gene by miRNAs has been recently explored by different groups in cystic fibrosis (CF) primary bronchial epithelial cells in vitro or from bronchial brushings ex vivo. Expression of miR-145 and miR-494 was found anti-regulated with that of CFTR . The effPeptide nucleic acids (PNAs) are DNA analogues of outstanding properties ,13,14, sThe objective of this study was to design a PNA targeting miR-145, determine its activity in inhibiting miR-145, and verify whether it induces an increase of CFTR production. The PNA was conjugated to a poly-arginine tail, since this type of constructs was previously used by our group for the delivery of PNA into cell lines ,25,26. AIn Homo sapiens (H. sapiens), Pan troglodytes (Chimp), Macaca Mulatta (Rhesus), Saimiri sciureus (Squirrel), Mus musculus (Mouse), Rattus norvegicus (Rat), Oryctolagus cuniculus (Rabbit), Sus scrofa (Pig), Bos taurus (Cow), Felis catus (Cat), Canis lupus familiaris (Dog), Myotis lucifugus (Brown bat) and Loxodonta africana (Elephant). In red the differences with respect to the Homo sapiens sequence are underlined. The CFTR sequence complementary to miR-145-5p seed region is boxed. The region showing the highest level of homology (reaching 100% homology in some positions) belongs to the seed-complementary stretch, as also shown in 8) peptide was conjugated at N-terminus of the PNA chain since it induces an efficiency in the delivery which approaches 100% , as elsewhere published 4+, 1255.97 (1256.07) [MH5]5+, 1046.81 (1046.9) [MH6]6+, 897.41 (897.48) [MH7]7+, 785.36 (785.42) [MH8]8+, 698.21 (698.26) [MH9]9+.R8-PNA-a145-MUT: sequence H-R8-AGAGATGCCTTGGAGAAC-Gly-NH2; Rt = 10.23 min, HRMS: calculated MW: 6272.4698 g/mol; m/z found : 1569.70 (1569.84) [MH4]4+, 1255.97 (1256.07) [MH5]5+, 1046.81 (1046.9) [MH6]6+, 897.41 (897.48) [MH7]7+, 785.36 (785.42) [MH8]8+, 698.21 (698.26) [MH9]9+.R8-PNA-a509: sequence H-R8-CTACCCACAGACGTACCA-Gly-NH2; Rt = 11.17 min, HRMS: calculated MW: 6097.4456 g/mol; m/z found : 1525.95 (1526.07) [MH4]4+, 1220.96 (1221.06) [MH5]5+, 1017.64 (1017.71) [MH6]6+, 872.40 (872.47) [MH7]7+, 763.48 (763.54) [MH8]8+, 678.76 (678.81) [MH9]9+, 610.98 (611.03) [MH10]10+R8-PNA-a494: sequence H-R8-TTTCCCGTGTATGTTTCA-Gly-NH2; Rt = 12.30 min, HRMS: calculated MW: 6146.4044 g/mol; m/z found : 1538.19 (1538.32) [MH4]4+, 1230.75 (1230.86) [MH5]5+, 1025.80 (1025.88) [MH6]6+, 879.40 (879.47) [MH7]7+, 769.60 (769.66) [MH8]8+, 684.20 (684.25) [MH9]9+, 615.88 (615.93)[MH10]10+R8-PNA-a433: sequence H-R8-CGGGGAACCCTTCAAGAT-Gly-NH2; Rt = 10.47 min, HRMS: calculated MW: 6208.4525 g/mol; m/z found : 1036.14 (1036.22) [MH6]6+, 888.26 (888.33) [MH7]7+, 777.35 (777.41) [MH8]8+, 691.09 (691.15) [MH9]9+.2/air in DMEM/F12 medium  supplemented with 10% fetal bovine serum , 100 units/mL penicillin and 100 μg/mL streptomycin  and 1% NEEA (100×) . To determine the effect on proliferation, cell growth was monitored by determining the cell number/mL using a Z2 Coulter Counter .Calu-3 ,30 and NCultured cells were trypsinized and collected by centrifugation at 1500 rpm for 10 min at 4 °C, washed with PBS, lysed with Tri-Reagent , according to manufacturer’s instructions. The isolated RNA was washed once with cold 75% ethanol, dried and dissolved in nuclease free pure water before use. RNA from human tissue sections was extracted and purified with miRNeasy FFPE Kit  according to the manufacture's procedures. For miRNA quantification using real-time RT-qPCR reagents, the primers and probes  were obtained from Applied Biosystems . Reverse transcriptase (RT) reactions were performed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems); real-time PCR was performed according to the manufacturer’s protocols . The quaGene expression analysis was performed by RT-qPCR. 300 ng of the total RNA was reverse transcribed by using random hexamers. Quantitative real-time PCR (qPCR) assays were carried out using gene-specific double fluorescently labeled probes. Primers and probes used to assay CFTR (Assay ID: Hs00357011_m1) gene expression were purchased from were from Applied Biosystems, (Applied Biosystems). The relative expression was calculated using the comparative cycle threshold method and, as reference genes, the human RPL13A (Assay ID: Hs03043885_g1) ,53. g for 10 min at 4 °C. Protein concentration was determined by the method of Lowry after precipitation with 5% Trichloroacetic acid (TCA), utilizing bovine serum albumin as a standard. For CFTR analysis 20 μg of total protein was heated in Laemmli buffer (Bio-Rad) at 37 °C for 10 min and loaded onto a 3 to 8% Tris-acetate gel (Bio-Rad). The gel proteins were transferred to PVDF membrane (Bio-Rad) by using Trans Blot Turbo (Bio-Rad) and processed for Western blotting by using mouse monoclonal antibody, clone 596, against NBD2 domain of CFTR  at a dilution of 1:2500 by an overnight incubation at 4 °C. After washes, membranes were incubated with horseradish peroxidase-coupled anti-mouse immunoglobulin  at room temperature for 1 h and after washes the signal was developed by enhanced chemiluminescence . After membranes stripping, β-Actin monoclonal antibody (Sigma-Aldrich) was used to confirm the equal loading of samples [Cell pellets were lysed in 1% Nonidet P40 (IGEPAL), 0.5% sodium deoxycholate, 200 mM NaCl, 10 mM Trizma base, pH 7.8, 1 mM EDTA plus protease inhibitor mixture and 1 mM PMSF for 30 min in ice. Lysates were cleared by centrifugation at 10,000×  samples . Annexin V and Dead Cell assay on Calu-3 cells, untreated and treated for 72 h with 2 μM PNA-a145, 5 μM Stattic together with 10% DMSO, were performed with “Muse”  method, according to the instructions supplied by the manufacturer. This procedure utilizes Annexin V to detect PS (PhosphatidylSerine) on the external membrane of apoptotic cells. A dead cell marker is also used as an indicator of cell membrane structural integrity. It is excluded from live, healthy cells, as well as early apoptotic cells. Four populations of cells can be distinguished in this assay. Cells were washed with sterile PBS 1X, trypsinized, suspended and diluted (1:2) with the one step addition of the Muse Annexin V & Dead Cell reagent. After incubation of 10 min at room temperature in the dark, samples were put on ice, vortexed and analyzed. Data from prepared samples are acquired and recorded utilizing the Annexin V and Dead Cell Software Module (Millipore) .t test. Statistical significance was defined with p < 0.05  and p < 0.01 .Results are expressed as mean ± standard deviation (S.D.) Comparisons between groups were made by using paired Student’s


Brassica campestris L. ssp. chinensis var. rosularis Tsen). However, there is little information on the global gene expression of BRs under LT stress in wucai. In this study, the molecular roles of 24-epibrassinolide (EBR) after exogenously application, were explored by RNA sequencing under LT conditions.Brassinosteroids (BRs) have a positive effect on many processes during plant growth and development, and in response to various abiotic stressors. Low-temperature (LT) stress constricts the geographic distribution, growth, and development of wucai (According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, photosynthesis was significantly enriched after spraying EBR under LT. The transcripts encoding the photosystem II (PSII) oxygen-evolving enhancer protein, photosystem I (PSI) subunit, light-harvesting chlorophyll protein complexes I and II, and ferredoxin were up-regulated after the application of EBR. Transcripts encoding several key enzymes involved in chlorophyll biosynthesis were also up-regulated, accompanied by significant differences in the contents of 5-aminolevulinic acid (ALA), porphobilinogen (PBG), protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-proto IX), protochlorophyllide (Pchl), and photosynthetic pigments. Notably, transcriptional and physiological analyses revealed that under LT stress, plant responses to EBR involved a major reorientation of photosynthesis, as well as porphyrin and chlorophyll metabolism.This study explored the role of EBR as an LT stress tolerance mechanism in wucai. At the transcription level, LT tolerance manifests as an enhancement of photosynthesis, and the amelioration of porphyrin and chlorophyll metabolism. Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variant of non-heading Chinese cabbage (Brassica campestris L.), a crucial species in the Brassicaceae family  [ψo / (1– ψo)]. Maximum quantum yield of primary photochemistry was calculated as: φPo = Fv / Fm = (Fm - Fo) / Fm [In vivo chlorophyll formulae . The flumeasured . The folFo) / Fm . Other pp < 0.05. Analyses were conducted using SPSS v19.0 for Windows . Figures were plotted using GraphPad Prism v7.0 and Origin Pro v9.1 software .All of the data from the 4 treatments were subjected to an analysis of variance (ANOVA). The mean separation was performed using the Fisher’s protected least significant difference (LSD) test with a significance level of Additional file 1: Figure S1. The quality control of six samples. A-F represent LT-1, LT-2, LT-3, LT + EBR-1, LT + EBR-2, and LT + EBR-3, respectively.Additional file 2: Figure S2. The reads mapping of six samples. A-F represent LT-1, LT-2, LT-3, LT + EBR-1, LT + EBR-2, and LT + EBR-3, respectively.Additional file 3: Figure S3. The 20 DEGs we randomly selected for qRT-PCR assay in EBR-mediated LT stress.Additional file 4: Figure S4. The results of identifying EBR concentration.Additional file 5: Table S1. Summary of sequence assembly after illumine sequencing.Additional file 6: Table S2. Number of reads sequenced and mapped to the Brassica rapa genome.Additional file 7: Table S3. The expression patterns of the cold acclimation and cold-induced genes.Additional file 8: Table S4. The expression patterns of the BR-responsive genes.Additional file 9: Table S5. Primers of verifying genes.Additional file 10: Table S6. Primers of photosynthesis genes.Additional file 11: Table S7. DEGs of photosynthesis–antenna proteins and photosynthesis.Additional file 12: Table S8. Parameters derived from the OJIP transient for use in the current study.


An update of xanthones encountered in lichens is proposed as more than 20 new xanthones have been described since the publication of the compendium of lichen metabolites by Huneck and Yoshimura in 1996. The last decades witnessed major advances regarding the elucidation of biosynthetic schemes leading to these fascinating compounds, accounting for the unique substitution patterns of a very vast majority of lichen xanthones. Besides a comprehensive analysis of the structures of xanthones described in lichens, their bioactivities and the emerging analytical strategies used to pinpoint them within lichens are presented here together with physico-chemical properties (including NMR data) as reported since 1996. H-xanthen-9-one scaffold [Xanthones are ubiquitous polyphenolic compounds displaying a common 9scaffold . Bioactiscaffold  likely tscaffold . Thus, mscaffold . Even thscaffold , variousAs with most lichen metabolites , the bioet al. [Most lichen xanthones arise through the folding of a polyketide intermediate as described by Cacho et al. , resultiPyrenochaeta terrestris [ortho carboxybenzophenone that might follow several metabolic fates. A first possibility is the 1,4-addition of a B-ring phenol to the A-ring dienone followed by dehydration and decarboxylation to access ravenelin-like xanthones after a final oxidation [Aspergillus variecolor, a skeleton thus far unknown from lichens.In contrast, a limited number of structures arise via a distinct biosynthetic pathway that leads to the ravenelin skeleton, with the methyl group in position 3. This biosynthetic scheme begins with the widespread anthraquinone emodin as a precursor  Figure . To begirrestris ,10. The rrestris . Deeper rrestris , who proxidation . This reHypericum androsaemum) or 1,3,5-trihydroxyxanthone (Centaurium erythraea) [It is noteworthy that the xanthone nucleus of plants is of mixed biosynthetic origin with the A-ring being acetate-derived whereas shikimic acid pathway–derived 3-hydroxybenzoic acid gives rise to the C-ring . Aromatiythraea) .Anthocleista djalonensis [Croton cuneatus [, Cupania cinerea [, Feroniella lucida [, Minquartia guianensis [Zanthoxylum microcarpum [Z. valens [Hypericum ascyron [Penicillium [Penicillium patulum [Ulocladium [Penicillium patulum [Given these different biosynthetic pathways, only few lichen xanthones are known from non-lichenized organisms. However, some can be produced by higher plants, such as lichexanthone , vineto ascyron ). Likewiicillium , norlich patulum  and the ocladium ; 1,3,6-t patulum . Secalon patulum .Vismia parviflora [Weddelina squamulosa [Penicillium strain [Cassia obtusifolia [Astrocystis sp. BCC 22166 [Cassia occidentalis [Phomopsis sp. [Talaromyces bacillosporus [Engyodontium album [O-methyl-2-deprenylrheediaxanthone B ). It can therefore be stated that xanthones are highly unique to each realm, legitimating joint efforts on higher plants, non-lichenized fungi and lichens to widen the chemical diversity of these privileged structures.Likewise, very few xanthones are common to higher plants and fungi, with some such examples being 1,7-dihydroxyxanthone  1,8-dihusifolia  and fungCC 22166 ), pinseldentalis  and sevepsis sp. , Talaromlosporus  and the um album ), 6-O-meillardii  and fungpsis sp. ), as welca. 2000 occurrences of xanthones sensu lato . These considerable efforts might be explained by the discovery of promising leads such as the anti-angiogenic molecule Vadimezan (AS 404) which is currently undergoing phase III clinical trials as a tumor vascular-disrupting agent [Recent data regarding the number of naturally occurring xanthones are scarce, with the last numbered record of 278 xanthones listed by Vieira and Kijjoa more than 10 years ago . By Janung agent ,36, as wng agent ,38. ObviStructural diversity of lichen xanthones mostly stems from variations in the orientation and degree of chlorination of the norlichexanthone or ravenelin core, as well as from the position and extent of methylation of the phenolic groups. Elix and Crook tremendously advanced the understanding of chlorination and methylation processes affecting the xanthone core. Through the example of 40 different species of lichens, theoretical biosynthetic schemes mirroring the sequence of biogenetic events leading to the joint occurrence of xanthones among lichens were constructed . This reet al. in 2002 [Aspergillus nidulans, through the identification of prenyltransferase genes belonging to the fungal indole prenyltransferases, so far known for their involvement in the prenylation of amino acids [Further structural modifications might be taken into account to extend the chemodiversity of lichen xanthones. Important contributors to the chemical diversity of xanthones are prenyl groups which are most often incorporated as dimethylallyl moieties . General in 2002 . Underlyno acids . Glycosyno acids  and hirtno acids . Blennoria sp. [ortho and para C positions [The diversity of lichen xanthones is also extended through some dimeric xanthones. Although the identification of key dimerization processes still warrants further investigation, xanthone dimers are most likely obtained after the biaryl linkage of monomers. This hypothesis is preferred to that of a tandem biosynthetic pathway which would involve a side-by-side cyclization of a double-length polyketide, especially since the discovery of the long-sought-after monomeric units of secalonic acids  within the fungus oria sp. . Enzymatositions .Reductive dearomatizations are sometimes observed on xanthones to yield dihydro-, tetrahydro- or hexahydroxanthones. To date, in lichens, such reduced species were only observed from dimeric xanthones: secalonic acids, hirtusneanoside and eumitrin A1 (bis tetrahydroxanthones) and eumitrin A2, B and T . Overall, a vast majority of xanthones reported from lichens have a monomeric and fully aromatized structure.Lecanora dispersa [Lecanora rupicola. This lichen is known to produce metabolites of various polyketide classes: lecanoric acid (depsides), hematommic and orsellinic acids (monocyclic phenols), eugenitol and sordidone (chromones) and arthothelin (xanthone). The whole chemosyndrome could be produced from axenically grown mycobionts, with the notable exception of arthothelin [Physconia distorta but not by its isolated mycobiont [Pyrenula japonica and P. pseudobufonia revealed the biosynthesis of xanthones that cannot be evidenced from the lichen as a whole (see further), suggesting their significance in the pre-lichenized condition [Even though xanthones from free-living fungi are well known, a plant-fungus collaboration has been suggested for several lichen xanthones. As an example, the typical lichen xanthone 2,7-dichlorolichexanthone could be isolated from the lichen dispersa . Howeverhothelin , suggestycobiont . Adverseondition ,52.Dehalococcoides mccartyi that has gathered attention because of its ability to dechlorinate a large array of anthropogenic pollutants [p-dioxins (dioxins), such bacterial strains might be of paramount interest for bioremediation purposes [A total of 72 xanthones containing one to four chlorine atoms are currently known from lichens. Even though recent studies have shed light on chlorinated xanthone biosynthesis, no study has focused on their degradation. Dechlorination of organochlorines can be achieved by some bacterial strains, referred to as organohalide respirers. Such bacteria comprise the famous llutants ,54,55. Lllutants ,57,58. Tllutants , which illutants ,61,62. Hpurposes . Acting purposes ,63.Lecidella asema, without the need for matrix assistance [in situ [Lecidella asema shows an exhaustive chemical profile  to perform separation and therefore to collect retention time and UV/Vis data as obtained with more traditional dereplication protocols [However, regarding the specific example of lichen xanthones, the lack of mutually supportive data such as chromatographic retention times and/or UV/Vis spectra precludes the differentiation of isomers and is a severe limitation of such methods. Possible ways to overcome such limitations while keeping an rotocols . Even throtocols . Such inrotocols ,83. Thisrotocols .® was dedicated to the identification of lichen metabolites from an original database of 550 compounds, including HPLC retention indices, TLC-Rf values, UV/visible colors of TLC spots and thalline reactions to guide their identification [Lichenized and non-lichenized Ascomycetes) [Together with analytical achievements, informatics tools were also developed to alleviate the dereplication holdup. A computer software named Wintabolitesfication . A most mycetes) , a databmycetes) .Lecanora schofieldii [O-methylnorlichexanthone , 2,4-dicalderae ), 2,5-diia fusca  among otulescens  and Phyldatinica ), 4,5,7-leprosum ) and 2,4rpallida ). Chlorirpallida . One migBesides these fully rationalized structures, a limited number of metabolites exhibit structural variations that are more or less difficult to explain. As an example, demethylchodatin displays a methoxy group in position 4 that might be introduced by a xanthone oxidase on this trichlorinated norlichexanthone derivative. Another unusual modification of the lichexanthone series is the occurrence of two acetyl groups in erythrommone. The chemical structures of xanthones with trivial names discussed in this manuscript are enlisted in Rinodina thiomela.However, 22 lichen xanthones display unusual structural features that make their biosynthetic intermediates tricky to unravel  [Plasmodiumfalciparum and Trypanosoma brucei [Aedes aegypti [Very few lichen xanthones have been investigated to date for their bioactivities. Lichexanthone exerts a weak activity against rculosis  and M. aM. aurum . DespiteM. aurum . Lichexa: 21 μM) . No antia brucei . Lichexaa brucei  and indua brucei . Lichexaa brucei . At last aegypti .lck tyrosine kinase at 200 μg/mL [50 values ranging from 0.3 to 12 μM [Norlichexanthone was shown to display promising cytotoxic activities . Indeed,00 μg/mL  and inhito 12 μM . Norlichto 12 μM . Dayan ato 12 μM . Alongsito 12 μM .Haematomma fluorescens as a response to a 365 nm UV light exposure, emphasizing its protective role as a light filter [As most structural classes of lichen metabolites, xanthones display strong UV-absorbing properties, predominantly in the wavelength range of UVA . Indeed,t filter . Depositt filter ,110. Suct filter . A Time-t filter .The past 20 years witnessed the isolation of 23 further lichen xanthones. It is noteworthy that those new xanthones displayed original substitution patterns and revealed original moieties compared to the previous state of the art.Pyrenula japonica and P. pseudobufonia afforded a series of five new ravenelin-type lichen xanthones, the first reports of such xanthones from aposymbiotically grown lichen mycobionts. A first report elucidated the structures of 1,5,8-trihydroxy-3-methylxanthone, 1,8-dihydroxy-5-methoxy-3-methylxanthone and 1,7-dihydroxy-3-methylxanthone [Cassia occidentalis [Cultures of the spore-derived mycobionts of xanthone  while a xanthone . Notablydentalis ) and alsdentalis . Surprisdentalis . Besidesdentalis .Umbilicaria proboscidea [d-glucopyranose unit anchored at position O-7, whereas umbilicaxanthoside B stands for a C2,C8-diprenylxanthone with a disaccharide β-d-glucopyranose-(1→4)-β-d-glucopyranose moiety at O-7. These compounds were the first prenylated xanthones to be isolated from a lichen source and glycosides are not frequently encountered in lichens [O-glycosides corresponding to linolenoyl, lineoyl, palmitoleoyl, oleoyl, palmitoyl, eicosenoyl and stearoyl esters of both umbilicaxanthosides A and B [Two highly unusual glycosylated prenylxanthones were subsequently identified from the Ural lichen boscidea . Umbilic lichens . Besides A and B . FragmenCladonia incrassata [Sporormiella minimoides [Staphylococcus aureus even though the paucity of the compound precluded the determination of a minimal inhibitory concentration [The last monomeric xanthone isolated so far from a lichen source is cladoxanthone A , obtained from crassata . The occcrassata . It is nnimoides   as well as antifungal (Microbotryum violaceum) and antialgal agent (Chlorella fusca) [Gliocladium sp. T 31 streamlined the isolation of secalonic acid D as the cytotoxic metabolite against four cell lines in a highly variable range of 0.03–15 μM, suggesting selective pharmacodynamic properties [50 of secalonic acid D on the carcinoma KB cells and an inhibition of human topoisomerase 1 with a promising IC50 of 0.16 μg/mL [++-dependent enzymes through competitive inhibition [Regarding dimeric xanthones, known metabolites of the secalonic acid series were first isolated from lichen sources in 2009, anescens . The joianescens . Secalonanescens . Secalonc acid D ,119, wara oryzae . Likewisa fusca) . Severala fusca) . Bioassaoperties . Further16 μg/mL . More re16 μg/mL . As a la16 μg/mL . Anotherhibition . Subsequhibition . As a cohibition ,128.Usnea hirta corresponding to a rhamnoside of an unsymmetrical dimeric tetrahydroxanthone [Staphylococcus aureus and Bacillus subtilis [l-rhamnose and the aglycone named hirtusneanin, the physico-chemical data of which were also reported by Rezanka and Sigler [Hirtusneanoside is a new xanthone dimer isolated from xanthone . If the subtilis . The enzd Sigler .Available physico-chemical properties regarding the newly described lichen compounds are listed below.Cladoxanthone A14H7O4Cl3 (343.94099)CYellow powdermax (MeOH)(log ε): 381 (3.24), 323 (3.50), 259 (4.11) nmUV λmax (CHCl3): 3353, 3056, 1642, 1597, 1450, 800 cm−1IR νCladonia incrassata [Sources: crassata .1,8-Dihydroxy-3-hydroxymethyl-5-methoxyxanthone15H12O6 (288.06339)CYellow needles, mp 221–222 °Cmax (MeOH)(log ε): 387.5 (3.27), 341 (3.84), 271 (4.16), 263 (4.24), 255 (4.29), 236 (4.17)UV λmax (KBr): 3533, 1659, 1634, 1609, 1589, 1493 cm−1IR νPyrenula japonica [Sources: Mycobiont of japonica .1,2,8-Trihydroxy-5-methoxy-3-methylxanthone15H12O6 (288.06339)CYellow needles, mp 214–215 °Cmax (MeOH) (log ε): 421 (3.42), 352 (3.58), 280 (4.27), 263 (4.18), 243 (4.17), 206 (4.23)UV λmax (KBr): 3487, 1663, 1636, 1607, 1576, 1489 cm−1IR νPyrenula japonica [Sources: Mycobiont of japonica .1,7-Dihydroxy-3-methylxanthone14H10O4 (242.05791)CYellow needles, mp 259–260 °Cmax (MeOH) (log ε): 384 (3.84), 290 (3.98), 261 (4.58), 235 (4.45)UV λmax (KBr): 3285, 1653, 1607, 1585, 1483 cm−1IR νPyrenula japonica [Sources: Mycobiont of japonica .1,5,8-Trihydroxy-3-methylxanthone14H10O4 (258.05282)CYellow needles, mp 277–278 °Cmax (MeOH) (log ε): 403 (3.54), 341 (4.01), 272 (4.58), 263 (4.40), 255 (4.47), 236 (4.32)UV λmax (KBr): 3461, 1661, 1633, 1606, 1591, 1497 cm−1IR νPyrenula japonica and Pyrenula pseudobufonia [Sources: Mycobiont of obufonia .1,8-Dihydroxy-5-methoxy-3-methylxanthone15H12O5 (272.06847)CYellow needles, mp 214–215 °Cmax (MeOH) (log ε): 393 (3.54), 340 (4.00), 272 (4.30), 254 (4.49), 235 (4.36)UV λmax (KBr): 3445, 1661, 1631, 1609, 1585, 1489 cm−1IR νPyrenula japonica and Pyrenula pseudobufonia [Sources: Mycobiont of obufonia .Hirtusneanoside40H46O17 (798.27350)CFaint yellow crystals, mp 231–232 °Cc = 0.02 in MeOH)max (MeOH)(log ε): 340 (3.24), 275 (4.01), 230 (4.52) nmUV λmax (CHCl3): 3290, 1735, 1620, 1590, 870 cm−1IR νUsnea hirta [Sources: ea hirta .Hirtusneanine34H36O13 (652.21559)CPale yellow crystals, mp 251–253 °Cc = 0.01 in MeOH)max (MeOH)(log ε): 338 (3.97), 275 (4.04), 231 (4.43) nmUV λmax (CHCl3): 3290, 1734, 1608, 1590, 870 cm−1IR νUsnea hirta [Sources: ea hirta .Umbilicaxanthoside A25H28O11 (504.16316)CYellow needles, mp 114 °Cmax (MeOH) (log ε): 345 (3.90), 310 (4.15), 270 (4.05), 245 (4.50).UV λmax (KBr): 3320, 2945, 2905, 1643 cm−1IR νUmbilicaria proboscidea [Sources: boscidea .Umbilicaxanthone A19H18O6 (342.11034)CUmbilicaria proboscidea [Sources: boscidea .Umbilicaxanthoside B36H46O16 (734.27859)CPale yellow needles, mp 133 °Cmax (MeOH) (log ε): 355 (4.06), 319 (4.01), 271 (3.96), 244 (4.08)UV λmax (KBr): 3350, 2950, 2900, 1640 cm−1IR νUmbilicaria proboscidea [Sources: boscidea .Umbilicaxanthone B24H26O6 (410.17294)CUmbilicaria proboscidea [Sources: boscidea .Linolenoylumbilicaxanthoside B54H74O17 (994.49260)CPale yellow needles, mp 133 °Cmax (MeOH) (log ε): 355 (4.06), 319 (4.01), 271 (3.96), 244 (4.08)UV λmax (KBr): 3350, 2950, 2900, 1640 cm−1IR νUmbilicaria proboscidea [Sources: boscidea .Secalonic acid A32H30O14 (638.16356)CFaint yellow crystals, mp 231–232 °Cc = 0.1 in CHCl3)max (MeOH)(log ε): 338 (4.52), 260 (4.12), 228 (4.32) nmUV λmax (CHCl3): 3464, 1726, 1610, 1585, 1566, 1436, 1233, 1067 cm−1IR νDiploicia canescens [Sources: anescens .Spectral data: .c = 0.14 in CHCl3)Enantiomeric secalonic acid D: similar physicochemical data except Secalonic acid B32H30O14 (638.16356)CFaint yellow crystals, mp 231–232 °Cc = 0.38 in CHCl3)max (MeOH)(log Ɛ): 338 (4.52), 260 (4.12), 228 (4.32) nmUV λmax (CHCl3): 3582, 3014, 1747, 1611, 1589, 1214, 1058, 796, 726 cm−1IR νDiploicia canescens [Sources: anescens .Spectral data: Even though lichens offer the widest diversity of compounds in the fungal realm, the bioactivities of their secondary metabolites remain under-investigated in regards to any other fungi , especia


Cancer still is one of the leading causes of death and its death toll is predicted to rise further. We identified earlier the potential tumour suppressor zygote arrest 1 (ZAR1) to play a role in lung carcinogenesis through its epigenetic inactivation.ZAR1 is epigenetically inactivated not only in lung cancer but also across cancer types, and ZAR1 methylation occurs across its complete CpG island. ZAR1 hypermethylation significantly correlates with its expression reduction in cancers. We are also the first to report that ZAR1 methylation and expression reduction are of clinical importance as a prognostic marker for lung cancer and kidney cancer. We further established that the carboxy (C)-terminally present zinc-finger of ZAR1 is relevant for its tumour suppression function and its protein partner binding associated with the mRNA/ribosomal network. Global gene expression profiling supported ZAR1's role in cell cycle arrest and p53 signalling pathway, and we could show that ZAR1 growth suppression was in part p53 dependent. Using the CRISPR-dCas9 tools, we were able to prove that epigenetic editing and reactivation of ZAR1 is possible in cancer cell lines.We are the first to report that ZAR1 tumour suppressor in cancer.ZAR1 is a novel cancer biomarker for lung and kidney, which is epigenetically silenced in various cancers by DNA hypermethylation. ZAR1 exerts its tumour suppressive function in part through p53 and through its zinc-finger domain. Epigenetic therapy can reactivate the  ZAR1 was reported to be expressed in porcine and bovine brain and testis -dCas9 (100087), DNMT3A-dCas9 (100090). Epigenetic editing of endogenous ZAR1 was performed in the ZAR1 partially methylated Hela, if not mentioned otherwise. ZAR1 RNA guides are #1 ACTTTCGCTCACTTAGCCAG, #2 TGGTTCCCTTACGGATCAGC, #3 GTAGGGAGAAGGACGAAGAG, #4 GTCGCCTATTTAGGGTGCGG, #5 CGCGGCCACCAAGGGCAAGG, and #6 CCGCGGTACAGTGCTCGCTG and are positioned relative to TSS at −402 #1, −230 #2, −133 #3, −3 #4, +120 #5, and +386 #6.CRISPR-Cas9 vector px549 was obtained from Lienhard Schmitz  and adapted for epigenetic editing by inactivation of Cas9 (dCas9 site-directed mutation). 2. I brief, beads were resuspended in two volumes urea buffer  and incubated shaking for 30 min at room temperature. Cysteins were alcylated at 55 mM final concentration of iodoacetamide, shaking at room temperature and in the dark for another 30 min. Peptidolysis was then initiated with 0.5 μg Lys-C  for 3 h shaking at room temperature, followed by dilution to 2 M urea/thiourea, addition of 0.5 μg trypsin (Serva) and an overnight shaking incubation at room temperature. Peptide-containing supernatants were brought to 1% NH3 and loaded onto three-layer SAX tips equilibrated previously with 30 μl of 0.1% NH3. After sequential washes with 30 μl 0.1% NH3 and 30 μl NH3 in 2-propanol, respectively, columns were syringe dried, peptides eluted using 30 μl 80% acetonitrile, 0.1% formic acid and vacuum dried. In-solution chemical labelling was performed as described [2 analysis used an in-house packed 70 μm ID, 15 cm reverse phase column emitter  with a buffer system comprising solvent A  and solvent B . Relevant instrumentation parameters are extracted using MARMoSET [ZAR1-EYFP, ZAR1delZF-EYFP vs. EYFP-empty were overexpressed in HEK293T cells (24 h), and pulldown was performed according to manufacturer's protocol by GFP Trap (ChromoTek). Triplicate sample pairs were processed by off-bead digest, strong anion exchange (SAX) extraction, and dimethyl-labelling, followed by LC-MSescribed , 72. PepMARMoSET  and inclMARMoSET , 75  APS = 16.2 (407) Avg = 12.8, Source: GEO ID: gse36133 Dataset Date: 2012-03-20. Additional file ZAR1 expression in cancer cell lines vs. normal tissues, 1555775_a_at, log2, data Roth vs. Broad, Anova one way. ZAR1 methylation in normal to tumour tissues and cancer cell lines, cg22773661/cg1753764, data Lokk vs. Heyn vs. Esteller, Anova one way. ZAR1 methylation in normal to tumour tissues and cancer cell lines relative to CpG island/shores and for all ZAR1 (cg) reporters from array. T-SNE analysis on Broad and Roth, Transform: zscore, no gene filter, no sample filter, perplexity = 50, Colour mode: Colour by Gene (ZAR1), Transform log 2. Overview on R2 used datasets : Normal Various - Roth - 504 – MAS5.0 - u133p2, Source: GEO ID: gse7307 Dataset Date: 2007-04-09; Cellline CCLE Cancer Cell Line Encyclopedia - Broad - 917 - MAS5.0 - u133p2, Source: GEO ID: gse36133 Dataset Date: 2012-03-20; Normal Tissues - Lokk - 70 - custom - ilmnhm450, Source: GEO ID: gse50192 Dataset Date: 2014-02-26; Tumor Types (landscape) - Heyn - 493 - custom - ilmnhm450, Source: GEO ID: gse76269 Dataset Date: 2017-06-07; Cellline Cancer Pharmacogenomic - Esteller - 1028 - custom - ilmnhm450, Source: GEO ID: gse68379 Dataset Date: 2016-07-05. Figure Wanderer [ZAR1, dataset project: TCGA, data type: 450k Methylation Array, for LUAD lung adenocarcinoma and KIRC Kidney renal clear cell carcinoma. Pan-cancer mRNA RNA-seq using KM Plotter [MethSurv [PhyloP; UCSC genome browser [BioEdit [NCBI RefSeq [Swiss-Model [PhosphoSitePlus [cBioPortal [String v11 [Gene expression, promoter methylation correlation, and Kaplan–Meier calculations were performed using Platform , WandereWanderer , KM Plot Plotter –81, and MethSurv . Gene serv [GSEA . The fol37, data . ZAR1 exZAR1 15555_a_at AP Plotter , Tumor tMethSurv  on TCGA  browser  and BioE[BioEdit  matrix: I RefSeq . Swiss-Mss-Model  predictiioPortal , 90. FigZAR1 promoter (186 bp) were upper primer GGAGAAGGAYGAAGAGGGGTTTTT and lower primer TCCCCCAAAACCRCCATAAAC, and pyrosequencing primer was TGGTAGGAAGGGYGTGGAGG. Primers for RT-PCR were ZAR1 AGCTGGGCAAGGAGCGGCTG and GGTGGGGCCGTTTAGGGTCCA (264 bp), GAPDH TGGAGAAGGCTGGGGCTCAT and GACCTTGGCCAGGGGTGCTA (176 bp), ACTB CCTTCCTTCCTGGGCATGGAGTC and CGGAGTACTTGCGCTCAGGAGGA (226 bp), p21 CCTTGTGCCTCGGTCAGGGGAG and GGCCCTCGCGCTTCCAGGAC (183 bp), p27 GTGCGAGAGAGGCGGTCGTG and TCCACCGGGCCGAAGAGGTT (146 bp), WEE1 CACACGCCCAAGAGTTTGC and CACTTGAGGAGTCTGTCGCA (135 bp) and WEE1 3′UTR primers are: pair 1 CTCCCCCTGAACACTGTGAC and ACTGACACCAATCGAGAAAGT (87 bp), pair 2 CACCAGCCTTTCCAGGGTTA and GGTCACTACAGGGAAAGACACC (92 bp), pair 3 AGCCTTCAATGTACCTGTGTGT and TGCCTACAAAGTGCTCCCAG (93 bp), pair 4 CTGGGAGCACTTTGTAGGCA and AGCAGCAAATTCACAAGGCA (77 bp), pair 5 AGTTTTGTCTTTGCTGTAAACTTGT and CATCAAAAGCAGCTATACATTTCAC (100 bp), pair 6 TGCACCCTTTCCCTCCTTTG and GTCCGGGAAGGACATTACCA (89 bp), pair 7 TGTTTTGCCCGGTTTTTCTCT and GTCAGAAGTCATTCTGGCATTTCA (95 bp), pair 8 TTTGCACTTGTCTTTGACTTGTGT and AGGTAAGCTCAGAGTGACTTTT (70 bp), pair 9 GCCATTTGACTAATAATACTGGCT and ACACAAGTCAAAGACAAGTGC (106 bp).Primers for CoBRA analysis of the Additional file 1: Figure S1. Overview ZAR1 genomic structure, expression pattern and GO-term correlation, Figure S2.ZAR1 promoter is hypermethylated across cancer cell lines. Figure S3.ZAR1 methylation in ovarian carcinoma. Figure S4. Epigenetic inactivation of ZAR1 across human cancers. Figure S5. ZAR1 is conserved across human, mouse, and xenopus with high C-terminal homology and structure prediction. Figure S6. Confirming human ZAR1 RNA binding ability. Figure S7. ZAR1 is cytosolic. Figure S8. Reexpression of ZAR1 alters the transcriptome and reveals association with mRNA 3′end processing. Figure S9. ZAR1 function depends on its zinc-finger. Figure S10. ZAR1 binding partner GO-term association overview. Figure S11. ZAR1 binding partner GO-term association overview. Figure S12. Targeting the ZAR1 genomic region with CRISPR ZAR1 guide RNA oligos. Figure S13. Effective epigenetic editing of ZAR1 with distinct RNA guide combinations upstream the TSS.


Escherichia coli 4s is a Gram-negative bacterium found in the equine intestinal ecosystem alongside diverse other coliform bacteria and bacteriophages. This announcement describes the complete genome of the T7-like E. coli 4s podophage Penshu1. From its 39,263-bp genome, 54 protein-encoding genes and a 179-bp terminal repeat were predicted. Escherichia coli 4s is a Gram-negative bacterium found in the equine intestinal ecosystem alongside diverse other coliform bacteria and bacteriophages. This announcement describes the complete genome of the T7-like E. coli 4s podophage Penshu1. From its 39,263-bp genome, 54 protein-encoding genes and a 179-bp terminal repeat were predicted. Escherichia coli is a Gram-negative bacterium found living among the intestinal microbiome of all mammals. Due to multiple protective abilities, including extreme acid resistance, E. coli colonizes the intestine as a commensal . After trimming using FastX-Toolkit v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/), they were assembled into a single contig at 376.4-fold coverage using SPAdes v3.5.0   wastewater treatment sample from Bryan, TX, using the host . Followithe host . The phathe host . The DNAs v3.5.0 . Contig xy-pub/) , 10. Proxy-pub/) , 12. No xy-pub/) . Protein-pub/) 1417. Rho-i-pub/) 14. Genome--pub/) 14.Escherichia phage ST31 (GenBank accession number KY962008) and 80.1% nucleotide sequence identity with Escherichia phage YZ1 (GenBank accession number MG845865). As for phage T7, Penshu1 has a slippery sequence in the major capsid protein (NCBI accession number QEG09806) that can lead to translation by frameshift of the minor capsid protein (NCBI accession number QEG09807).Penshu1 has a 39,263-bp genome, with a 93.4% coding density and 50.6% G+C content. Analysis predicted 54 protein-coding genes, with 30 being assigned a putative function. The Penshu1 genome was reopened at T7-like direct terminal repeats of 179 bp predicted by PhageTerm . Penshu1MK903281, BioProject accession number PRJNA222858, SRA accession number SRR8893626, and BioSample accession number SAMN11414580.The genome sequence and associated data for phage Penshu1 were deposited under GenBank accession number


HERC1 and HERC2, are present in most metazoan taxa. They encode very large proteins  that contain more than one RCC1-like domain as a structural characteristic. Accumulating evidences show that these unusually large proteins play key roles in a wide range of cellular functions which include neurodevelopment, DNA damage repair, and cell proliferation. To better understand the origin, evolution, and function of the Large HERC family, this minireview provides with an integrated overview of their structure and function and details their physiological implications. This study also highlights and discusses how dysregulation of these proteins is associated with severe human diseases such as neurological disorders and cancer.Homologous to the E6AP carboxyl terminus (HECT) and regulator of chromosome condensation 1 (RCC1)-like domain-containing proteins (HERCs) belong to the superfamily of ubiquitin ligases. HERC proteins are divided into two subfamilies, Large and Small HERCs. Despite their similarities in terms of both structure and domains, these subfamilies are evolutionarily very distant and result from a convergence phenomenon rather than from a common origin. Large HERC genes,  Proteins containing a HECT domain are ubiquitin ligases (E3). These enzymes participate in the ubiquitylation process accepting ubiquitin from a ubiquitin-conjugating enzymes (E2) and catalysing its transfer to the protein to be ubiquitylated . In animAlthough traditionally classified together with the Small HERC proteins, Large and Small HERCs form two distant protein families . Large HMonosiga brevicollis and Salpingoeca rosetta, the emergence of HERC1 occurred in Metazoa. Both proteins are already present in the placozoan Trichoplax adhaerens and in most metazoan phyla, with the absence of HERC1 in certain insect clades . HERC2 induces BRCA1 degradation in breast cancer. This is inhibited either by binding of TUSC4 to HERC2  or by BAHERC1 deletions affect MSH2 protein levels  and certain HERC2 SNPs can interfere OCA2’s expression thus affecting eye, skin, and hair pigmentation , for HERC1: T. adhaerens (XP_002116356.1), Anoplophora glabripennis (Asian long-horned beetle) (XP_018567016.1), Callorhinchus milii (Elephant shark) (XP_007906088.1), Latimeria chalumnae (Coelacanth) (XP_005987767.1), Danio rerio (Zebrafish) (XP_021333511.1), Oreochromis niloticus (Tilapia) (XP_013121789.1), Xenopus tropicalis (Frog) (XP_004916026.1), Gekko japonicus (Gecko) (XP_015275623.1), Gallus gallus (Chicken) (XP_004943789.1), Ornithorhynchus anatinus (Platypus) (XP_028921613), Monodelphis domestica (Opossum) (XP_016284147.1), Sarcophilus harrisii (Tasmanian devil) (XP_023353554.1), Equus caballus (Horse) (XP_023471806.1), Rattus norvegicus (Rat) (XP_017451567.1), Mus musculus (Mouse) (NP_663592.3), Homo sapiens (Human) (NP_003913.3), Pan troglodytes (Chimpanzee) (XP_001174017.1), Canis lupus familiaris (Dog) (XP_544717.3), Felis catus (Cat) (XP_023110993.1), and Lepidosiren paradoxa (Lungfish) . For HERC2: M. brevicollis (XP_001743304.1), S. rosetta (XP_004996155.1), T. adhaerens (XP_002112421), Asian long-horned beetle (XP_018562376.1), Drosophila melanogaster (Fruit fly) (NP_608388.2), Apis mellifera (Honey bee) (XP_01677273), Coelacanth (XP_014343905.1), Zebrafish (XP_021332870.1), Tilapia (XP_013130527.1), Frog (XP_012813156.1), Gecko (XP_015266462.1), Chicken (XP_015133197.1), Platypus (XP_016083014.1), Opossum (XP_007501467.1), Tasmanian devil (XP_023356163.1), Horse (XP_023507879.1), Mouse (NP_001347009.1), Rat (XP_006229362.1), Human (NP_004658.3), Chimpanzee (XP_024204823.1), Dog (XP_022272508.1), Cat (XP_023110801.1), Elephant shark (XP_007889299.1), and Lungfish .Amino acid sequences were aligned using the Mafft FFT-NS-i algorithm . The phyHuman RCC1 (NCBI accession number P18754) and HECT domain from E6AP  three-dimensional structures were modelled using Swiss-Model online software . RLDs anJG-C and JR conceived and designed the manuscript. JG-C, AM-M, and JR analysed the data and performed figures and tables. JG-C, AM-M, JS-G, LP, and JR wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococcus aureus clones and genes encoding antimicrobial resistance and toxins were examined among 120 S. aureus strains from nosocomial infections in tehran, Iran.In the current research, the prevalence of mec, agr, spa, and MLST, the isolates were typed. Antimicrobial susceptibility was examined, based on disk diffusion and PCR method to identify resistance and toxin-encoding genes. Based on the polymorphisms in SCCS. aureus isolates, 85 (70.8%) were methicilin resistant S.aureus (MRSA), and 35 (29.2%) were methicilin sensetive S. aureus (MSSA). The tested isolates contained resistance genes, including ant(4΄)-Ia (90%), aac(6΄)-Ie/aph(2˝) (80%), aph(3΄)-IIIa (30%), erm(A) (26.7%), erm(B) (10.8%), erm(C) (11.7%), msr(A) (40.8%), msr(B) (14.2%), tet(M) (45.8%), and mupA (8.3%). The MRSA strains were clustered into six different clones. The most common genotypes included ST239-SCCmec III/t037 (23.3%), ST239-SCCmec III/t388 (22.5%), ST22-SCCmec IV/t790 (8.3%), ST15-SCCmec IV/t084 (7.5%), ST585-SCCmec III/t713 (5%), and ST239-SCCmec III/t924 (4.2%), respectively. ST182/t196 (8.3%) and ST123/t171 (5%) belonged exclusively to MSSA strains. Overall, 10 (66.7%) and 5 (33.3%) out of 15 isolates with pvl genes were attributed to clones ST22-SCCmec IV/t790 and ST15-SCCmec IV/t084, respectively. ST22-SCCmec IV/t790, ST239-SCCmec III/t037, and ST15-SCCmec IV/t084, were related to high-level mupirocin-resistant phenotypes. Among 120 S. aureus was confirmed in our hospitals, and ST239-SCCmec III/t037 showed a relatively high prevalence in our study. It seems that assessment of resistance and virulence genes in different S. aureus molecular types is necessary for proper antibiotic consumption.The genetic diversity of  Staphylococcus aureus, which is described as a common nosocomial pathogen, is responsible for various diseases, such as food poisoning, osteomyelitis, wound infections, and even fatal conditions, such as endocarditis (S. aureus (MRSA) occurred in the UK  is recognized as a staphylococcal cassette chromosome mec (SCCmec). Generally, SCCmec is categorized into 11 types with respect to mec genes and ccr gene complexes  infections. HA- and CA-MRSA strains can be distinguished with respect to some genotypic, phenotypic, and epidemiological characteristics, as well as virulence factors  genes have significantly limited the availability of antibiotics over the past decades. In addition, the growing emergence of MDR-MRSA strains poses a major global health concern . Wide remupA and mupB genes are responsible for resistance to mupirocin which is used to treat various types of skin diseases caused by S. aureus  including aminoglycoside nucleotidyltransferases, aminoglycoside phosphotransferases and aminoglycoside acetyltransferases . mupA an. aureus . Resistaing site . Thus, i spa, SCCmec and agr techniques in clinical samples taken from patients in Tehran, Iran.This study was conducted to identify antibiotic resistance patterns and the carriage of resistance and virulence genes as well as major MRSA clones by MLST,Sampling, MRSA isolation and antibacterial susceptibility testingThis cross-sectional study included 368 clinical samples from wound, blood, and urine specimens during April-December 2016. Ethics Committee of Shahid Beheshti University of Medical Sciences approved the implementation of this study (IR.SBMU.SM.REC.1395.157). All patients signed written informed consent forms.S. aureus identification was performed, based on the conventional biochemical tests. S. aureus identification was confirmed based on the PCR assay for nucA gene , clindamycin (CD 2 µg), ciprofloxacin (CIP 5 µg), trimethoprim- sulfamethoxazole (TS 2.5 µg), kanamycin (K 30 µg), ceftriaxon (CRO 30 µg), quinupristin-dalfopristin (SYN 15 µg), erythromycin (E 15 µg), amikacin (AK 30 µg), gentamicin (GM 10 µg), tobramycin (TN 10 µg), teicoplanin (TEC 30 µg), penicillin (PG 10 µg), and linezolid (LZD 30 µg)  were determined, based on the CLSI criteria  were considered as the reference strains for the quality control purposes.Using E-test strips (bioMe´rieux), the minimum inhibitory concentrations (MICs) were measured for mupirocin and vancomycin. Resistance to three antibiotic groups or more, besides beta-lactams, was defined as MDR. High mupirocin resistance was defined as antibiotic use ≥ 256 mg/l. Growth in a well containing clindamycin and erythromycin  indicated inducible macrolide-lincosamide-streptogramin B and/or clindamycin resistance phenotypes; otherwise, constitutive MLSB and/or clindamycin resistance phenotype was confirmed . ATCC292Extraction of genomic DNAS. aureus cultures were used on 5% sheep blood agar , based on the protocols of InstaGene Matrix kit . For extracting genomic DNA, pure overnight Resistance and toxin genes profilingetb, tst, pvl, eta) and resistance (tet(M), aac (6΄)-Ie/aph (2˝), mupA, erm(A), msr(A), msr(B), erm(B), erm(C), ant (4΄)-Ia, aph (3΄)-IIIa) genes, PCR assay was carried out. The details of the degenerated primers in this study are described in To identify toxin  and reverse (agr1 to agr4) primers for the agr groups as previously recommended by Gilot et al . Chromas 1.45  was used to edit the sequences. To assign the sequences to specific spa types, the Ridom SpaServer database was searched.On the other hand, lleagues . After tMLST techniqueS. aureus isolates. The internal fragments of housekeeping genes were used to identify the allelic profiles; these genes included gmk, arcC, aroE, glpF, pta,yqiL, and tpi. The isolate was assigned a sequence type (ST) after comparing the sequences with the S. aureus MLST database.Via amplification and sequencing, MLST was carried out on Sampling and antibiotic susceptibility S. aureus. These isolates originated from wound (60%), blood (20.8%) and urinary tract infections (19.2%). Of the 120 S. aureus clinical isolates obtained from the hospitalized patients, 85 (70.8%) were MRSA and 35 (29.2%) were methicillin susceptible S. aureus (MSSA). In this study, out of 368 samples obtained from various clinical specimens, 120 isolates (83 (69.2%) obtained from men and 37 (30.8%) from women) were identified as  in vitro antimicrobial susceptibility tests. All isolates were susceptible to vancomycin, among which 55 (45.8%), 48 (40%) and 17 (14.2%) isolates had a MIC of 0.5, 1 and 2 µg/ml, respectively. None of the isolates were susceptible to all of the antimicrobial agents tested regardingP= 0.145 and 0.128, respectively). The frequency of resistance for MRSA and MSSA isolates to different antibacterial agents are presented in Among the 35 MSSA isolates, no mupirocin resistance was detected, whereas 30 MRSA isolates (35.3%) were mupirocin resistant. Of these mupirocin resistant isolates, 14 (46.7%) and 16 (53.3%) had high and low resistance levels, respectively. All the high-level mupirocin-resistant (HLMUPR) isolates were collected from wound samples. Patients diagnosed with mupirocin-susceptible and -resistant MRSA infections were not significantly different in terms of age and gender  were defined as MDR. The predominant multiple drug resistance profile among the MDR isolates were resistance to 9 and 7 antibiotics found in 75 (62.5%) and 12 (10%) isolates, respectively. Distribution of resistance profile and different clinical sample in S. aureus isolated from nosocomial infections are presented in Of the 120 The distribution of resistance genesant(4΄)-Ia (90%), aac(6΄)-Ie/aph(2˝) (80%), aph(3΄)-IIIa (30%), erm(A) (26.7%), erm(B) (10.8%), erm(C) (11.7%), msr(A) (40.8%), msr(B) (14.2%), tet(M) (45.8%), and mupA (8.3%) (ant(4΄)-Ia, aac(6΄)-Ie/aph(2˝), and mecA genes. Other detected antibiotic resistance genes included tet(M) (64.7%), msr(A) (57.6%), aph(3΄)-IIIa (42.3%), erm(A) (37.6%), msr(B) (20%), erm(C) (16.5%), erm(B) (15.3%), and mupA (11.8%), while 31.4% and 56.7% of MSSA strains were found to respectively carry aac(6΄)-Ie/aph(2˝) and ant(4΄)-Ia. Particularly, the MRSA strains contained more resistance genes, compared to the MSSA isolates.In addition, the frequency of antibiotic resistance genes was measured. The genes included A (8.3%) . The MRSS. aureus isolates were identified as HLMUPR-MRSA, while mupA gene was confirmed in 10 isolates (71.4%). In addition, ant(4΄)-Ia , followed by aac(6΄)-Ie/aph(2˝) , was recognized as the most common aminoglycoside resistance gene. A total of 75 (62.5%) isolates contained ant(4΄)-Ia, as well as aac(6΄)-Ie/aph(2˝). On the other hand, 15 (12.5%) isolates harbored aph(3΄)-IIIa, besides ant(4΄)-Ia. Also, eleven (9.2%) isolates harbored ant(4΄)-Ia, aph(3΄)-IIIa, and aac(6΄)-Ie/aph(2˝) genes, while 10 (8.3%) contained aac(6΄)-Ie/aph(2˝), as well as aph(3΄)-IIIa; however, ant(4΄)-Ia alone was confirmed in 7 (5.8%) strains. Resistance to tetracycline was observed among 84 (70%) S. aureus isolates, 55 (45.8%) of which harbored tet(M) gene. Antibiotic resistance genes showed the highest prevalence among MRSA strains from wound infections.Fourteen (11.7%) out of 30 mupirocin-resistance Virulence gene profilingtst  and etb  genes, respectively . S. aureus isolates harbouring tst gene were isolated from wound , blood , and urinary tract infections  while pvl positive isolates were detected in wound  and blood  samples. Eight isolates carrying pvl and tst genes, simultaneously, had mupA gene.Among toxin-encoding genes, the highest and lowest frequencies were attributed to ectively . In presDistribution of SCCmec typesAccording to SCCmec typing, 66 (77.6%) and 19 (22.4%) MRSA isolates contained SCCmec types III and IV, respectively. No isolate harbored SCCmec type V, II, or I. Based on the multiplex PCR, isolates positive for PVL were attributed to SCCmec type IV, while tst gene was found in MRSA isolates from SCCmec III and IV. The HA-MRSA origin was emphasized by the presence of SCCmec type III.Frequency of agr typesagr typing indicated that type I was the predominant agr type present in 91 isolates (75.8%), followed by type III which was present in 20 isolates (16.7%). agr type II was detected in 9 isolates (7.5%). Among 35 MSSA isolates, 20 and 15 harboured agr types I and III respectively .spa typingS. aureus isolates. spa typing discriminated eight different types: t037 (23.3%), t388 (22.5%), t713 (16.8%), t924 (8.3%), t790 (8.3%), t196 (8.3%) t084 (7.5%) and t171 (5%)  . All theMLSTmec III/t037 was found to be the most prominent MRSA clone identified in this study. Distribution of MRSA clones isolated from nosocomial infections are presented in Apparently, 120 isolates belonged to six different STs including ST239 (65 strains), ST22 (10 strains), ST182 (10 strains), ST15 (9 strains), ST585 (20 strains) and ST123 (6 strains). ST239, ST585, ST182 and ST123 were found in MSSA strains. It should be noted that of these STs, ST182 and ST123 belonged exclusively to MSSA strains. In conclusion, MRSA strains are clustered into six different groups. ST239-SCCmec IV/t790  and ST15-SCCmec IV/t084  clones. Among the isolates under study, 58 (48.3%) isolates harboring tst-1 were distributed in ST239-SCCmec III/t037 , ST239-SCCmec III/t388 , ST15-SCCmec IV/t084 , ST585-SCCmec III/t713 , ST239-SCCmec III/t924 , and ST22-SCCmec IV/t790  clones. Among examined isolates, nine isolates (7.53%) were found to carry the eta gene. The eta positive isolates were distributed in ST15-SCCmec IV/t084 , ST22-SCCmec IV/t790  and ST239-SCCmec III/t037  clones. The etb gene was detected in ST585-SCCmec III/t713  and ST22-SCCmec IV/t790  clones. MRSA clones resistance profile varied. Resistance to mupirocin was detected in all the MRSA clones with the exception of ST585-SCCmec III/t713 clone. Interestingly, mupirocin resistant MSSA isolates belonged to ST182/spa type t196. HLMUPR-MRSA strains were detected in ST22-SCCmec IV/t790 , ST15-SCCmec IV/t084  and ST239-SCCmec III/t037  clones. Fifteen PVL-carrying strains in our study belonged to ST22-SCCB phenotype was detected in ST239-SCCmec III/t037 , ST239-SCCmec III/t388 , ST585-SCCmec III/t713 , ST15-SCCmec IV/t084 , ST239-SCCmec III/t924  and ST22-SCCmec IV/t790  while iMLSB phenotype distributed among 3 major clones ST239-SCCmec III/t388 , ST22-SCCmec IV/t790  and ST15-SCCmec IV/t084 . Of 12 MSSA isolates with cMLSB phenotype, 5 isolates belonged to ST182/t196, 4 isolates belonged to ST239/t924, 2 isolates belonged to ST585/t713 and 1 isolate belonged to ST123/t171. All the iMLSB phenotype among MSSA strains belonged to ST585/t713. Characteristics of MRSA clones are presented in cMLSet al. /aph(2′′) gene (40.3% ) in comparison with other AME genes, in the present study, ant(4΄)-Ia (90%) was the most frequent AME gene in S. aureus isolates. However, in this study, aph(3΄)-IIIa gene frequency rate was higher than the study reported by Ida et al. (8.9%)  and Margtudy (aac′/aph(2′′S. aureus strains (S. aureus isolates were found to be lower. We detected the resistance gene mupA in 10 isolates (8.3%) which is lower than 12.6%  gene that may cause tetracycline resistance was detected in 55 (45.8%) isolates. In accordance with the results of our previous study  a high tet al. (7%) reported low frequency of iMLSB phenotype among S. aureus isolates as well  presented cMLSB phenotype while the frequency of iMLSB phenotype was 10% (12/120). In consistent with our findings, Schreckenberger  as well . Accordiospitals . They fo. in USA  reportedn survey , the fre ) or transposons (erm(A) and erm(B)) and msr genes expressing active efflux pumps mainly msr(A)) (msr(A), erm(A), msr(B),erm(C), and erm(B) genes to be 40.8%, 26.7%, 14.2%, 11.7%, and 10.8% respectively. In contrast to Rashidi Nezhad’s study (erm(A) gene as the predominant gene among the isolates with inducible phenotype and erm(C) among the isolates with the constitutive phenotype, our finding revealed that the msr(A) gene was the most common gene among strains with the constitutive phenotype , followed by erm(A) , erm(C) , erm(B) , msr(B)  while erm(A) , erm(B) , erm(C) , msr(A)  and msr(B)  were much more common among the isolates with inducible phenotype. It is worth mentioning that the frequency of erm and msr genes depends on the bacterial species as well as the geographic region in which the study is carried on. As previously mentioned, resistance to macrolides is encoded by genes often carried on plasmids ( msr(A)) . Our res’s study  who repomec (77.6%), according to the results of SCCmec typing, associated with an MDR pattern among MRSA isolates. This is in consistence with the results reported by Japoni and colleagues , type III (16.7%), and type II (7.5%), respectively. Goudarzi et al. . This spa type was reported from Saudi Arabia, China, Iran as well as among HA-MRSA isolates found in Europe, America and other regions of Asia . The review of the literature reveals that the multiresistant ST239 clone is responsible for at least 90% of HA-MRSA infections in Europe, United States, and some Asian countries, including Kuwait and Malaysia  was the second most commonly detected MRSA clone. A similar result was reported by Ohadian Moghadam and ST30 .mec IV/t790 (8.3%). This clone was associated with high resistance to mupirocin, carrying resistance genes, including mecA, msr(A), ant(4΄)-Ia, msr(B), tet(M), and mupA. In our study, all ST22-SCCmec IV/t790 strains contained pvl genes. There are reports of S. aureus ST22 harboring pvl gene from Iran  was the fourth most commonly detected clone. The low frequency of this clone has been previously reported in 16 European countries . The presence of eight different spa types, i.e., t037, t388, t713, t924, t790, t196, t084, and t171, was also confirmed. For the first time in Iran, STs 182 and 123, as well as spa types t196 and t171, were detected, which might be indicative of the emergence of new clones. Further studies on other neighboring regions, focusing on the emergence of new circulating clones, are necessary to reach an overall understanding of dynamic MRSA clones in Iran and the Middle East.To sum up, the present findings showed that MRSA isolates have various genetic backgrounds in our hospitals and involve six major clones. Certain molecular types were associated with some resistance and virulence genes (e.g.,