Talaromyces produce useful biomass-degrading enzymes and secondary metabolites. However, these enzymes and secondary metabolites are still poorly understood and have not been explored in depth because of a lack of comprehensive genetic information. Here, we report a 36.51-megabase genome assembly of Talaromyces pinophilus strain 1–95, with coverage of nine scaffolds of eight chromosomes with telomeric repeats at their ends and circular mitochondrial DNA. In total, 13,472 protein-coding genes were predicted. Of these, 803 were annotated to encode enzymes that act on carbohydrates, including 39 cellulose-degrading and 24 starch-degrading enzymes. In addition, 68 secondary metabolism gene clusters were identified, mainly including T1 polyketide synthase genes and nonribosomal peptide synthase genes. Comparative genomic analyses revealed that T. pinophilus 1–95 harbors more biomass-degrading enzymes and secondary metabolites than other related filamentous fungi. The prediction of the T. pinophilus 1–95 secretome indicated that approximately 50% of the biomass-degrading enzymes are secreted into the extracellular environment. These results expanded our genetic knowledge of the biomass-degrading enzyme system of T. pinophilus and its biosynthesis of secondary metabolites, facilitating the cultivation of T. pinophilus for high production of useful products.Species from the genus  Talaromyces pinophilus, formerly designated Penicillium pinophilum, is a fungus that produces biomass-degrading enzymes such as α-amylase1, cellulase2, endoglucanase3, xylanase2, laccase4 and α-galactosidase2. In one study, a blended enzyme cocktail produced by T. pinophilus and Chrysoporthe cubensis improved the efficiency of hydrolysis of glucan and xylan in sugarcane bagasse for glucose and xylose production, compared with enzymes from a single strain2. A relatively high level of β-glucosidase activity is observed in T. pinophilus under solid state fermentation5. Therefore, T. pinophilus is considered a potential alternative to Trichoderma reesei for cellulase production and efficient biomass hydrolysis. T. pinophilus produces a variety of medically useful metabolites such as 3-O-methylfunicone, which is used to inhibit mesothelioma cell motility6, and talaromycolides 1–3, 5 and 11, which inhibit the growth of the human pathogen methicillin-resistant Staphylococcus aureus7.T. pinophilus 1–95 was isolated from the soil of a dried, ploughed field in Wuzhou, China. This strain produces a highly efficient, calcium-independent α-amylase1. Application of calcium-independent α-amylase in starch conversion avoids problems caused by addition of calcium ions8. Additionally, we found that T. pinophilus 1–95 produces 1.21 ± 0.30 U/mL of filter-paper cellulase, 10.72 ± 0.74 U/mL of carboxymethylcellulose cellulase, 0.71 ± 0.02 U/mL of p-nitrophenyl-β-cellobioside cellulase, 0.27 ± 0.01 U/mL of p-nitrophenyl-β-glucopyranoside cellulase and 41.93 ± 2.84 U/mL of xylanase activities in submerged flask cultivation (data not shown). However, a comprehensive understanding of the biomass-degrading enzyme system in this fungus is still lacking.The fungal strain de novo whole-genome assembly of T. pinophilus strain 1–95, a nearly complete genome sequence of a high biomass-degrading enzyme-producing species in the genus Talaromyces. Carbohydrate-active enzyme (CAZyme) genes and secondary metabolism gene clusters were observed in the sequenced genome. Comparative genomic analysis suggested that T. pinophilus harbors more biomass-degrading enzymes and secondary metabolites than other related filamentous fungi. In addition, the predicted secretory protein patterns of T. pinophilus 1–95 were analyzed.We describe the T. pinophilus 1–95 (CGMCC No. 2645), isolated from soil in a dried, ploughed field in Wuzhou, China, was performed using a combination of single molecule real-time (SMRT) DNA sequencing and next generation sequencing technology. A high-quality genome sequence of T. pinophilus 1–95 was generated on the PacBio RS II platform. Approximately 1.94 Gb of clean subreads, with sequences from a single pass of a polymerase on a single strand of an insert within a SMRTbell template and no adapter sequences with an N50 size of 10,045 bp and average length 8,102 bp were generated. Additionally, a paired-end (PE) library with a 500-bp average insert size was constructed using the Illumina HiSeq 4000 platform, and 3.88 Gb clean, short-sequence PE reads were generated with a length of 125 bp. Reads were used to correct wrong bases in the assembled genome sequence on the PacBio RS II platform. Finally, a 36.51-Mb genome of T. pinophilus 1–95 was generated with 159-fold coverage. This size was in accordance with the estimated genome size of 28–36 Mb for three Talaromyces species11. The genome was covered by nine scaffolds, including eight large scaffolds (accession number CP017344-CP017351 in GenBank) without gaps , GeneMark-ES (http://exon.gatech.edu/GeneMark/), Genewise (http://www.ebi.ac.uk/Tools/psa/genewise/), SNAP14 and an unsupervised learning system program Glean version 1 (https://sourceforge.net/projects/glean-gene/). The number of coding genes was significantly higher than other filamentous fungi that produce biomass-degrading enzymes , were annotated in the Gene Ontology (GO) database (http://geneontology.org/), 12,828 (95.21%) in the UniProt database (http://www.uniprot.org/), 12,946 (96.09%) in the NCBI non-redundant (NR) protein sequences database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/) and 4437 (32.93%) in the Clusters of Orthologous Groups of proteins database (http://www.ncbi.nlm.nih.gov/COG/). A total of 6817 (50.6%) genes belonging to 331 pathways were also annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg.jp/)  search of all-vs.-all findings showed that T. pinophilus 1–95 and T. cellulolyticus Y-94 shared 10,260 orthologous proteins, accounting for 76.16% of the total proteome, with an average amino acid sequence identity of 98.28%. In contrast, low identity was observed to other sequenced Talaromyces species17, Trichoderma sp.18, Penicillium sp.20 and Aspergillus sp.22, ranging from 70% to 88% , 35 families of glycosyl transferases (GTs), 13 families of carbohydrate esterases (CEs), 10 families of auxiliary activities (AAs), 5 families of polysaccharide lyases (PLs) genes and 19 families of carbohydrate-binding modules (CBMs)  Fig. . FurtherMs) Fig. .Figure 3T. cellulolyticus Y-94 and P. oxalicum HP7-1, larger numbers of BGLs were classified into GH families 1 and 3 in the T. pinophilus 1–95 genome , 8 β-1,4-endoglucanases  and 29 β-glucosidases  were included as cellulases. Of these, the known cellulases included Cel7A-2 (TP09412), Cel5A (TP03457), Cel5B (TP07499), Cel7B (TP08514) and Bgl3A (TP09042). Compared with known filamentous fungi used for cellulase production, i.e., T. pinophilus 1–95 genome was rich in hemicellulose-degrading enzymes (97 genes) assigned into 29 predicted CAZyme families; this finding was compared to 77 genes in T. cellulolyticus Y-94 and 80 genes in P. oxalicum HP7–1, which includes Xyn11A (TP00436) and Xyn10A (TP06900), encoding the important β-1,4-endoxylanases. This result supports the high xylanase activity of T. pinophilus 1–95. The predicted hemicellulases in T. pinophilus 1–95 were divided into 19 types by substrate specificities. The large differences among T. pinophilus 1–95, T. cellulolyticus Y-94 and P. oxalicum HP7–1 broadly covered most of the listed hemicellulase types (see Supplementary Table T. pinophilus 1–95 possessed more β-D-xylosidases (EC 3.2.1.37), acetyl xylan esterases (EC 3.1.1.72) and feruloyl esterases (EC 3.1.1.73) than the two others. β-D-xylosidases hydrolyze xylobiose or linear xylooligosaccharides to the monomer xylose. Acetyl xylan esterases liberate acetic acid esterifying position 2 and 3 on mono- and di-O-acetylated β-1,4-linked D-xylopyranosyl residues in xylan chains. Feruloyl esterases liberate trans-ferulic acid from 5-O-feruloylated L-arabinofuranosyl residues. These enzymes facilitate the hydrolysis of hemicellulose26. In contrast, the T. pinophilus 1–95 genome possessed a similar number of pectin-degrading enzymes as T. cellulolyticus Y-94 and P. oxalicum HP7–1 , which mainly hydrolyze α-1,4-glucosidic linkages from the nonreducing ends of starch chains with the release of β-D-glucose28; and a 1,4-α-glucan branching enzyme (EC 2.4.1.18) that cleave α-1,4 glucosidic linkages of glucan chain, and then transfer the cut end to 6-position of glucose residue within the cleaved or another glucan chain, resulting the generation of an α-1,6 glucosidic linkage29. Comparative analysis indicated that T. pinophilus 1–95 had more α-glucosidases and glucoamylases than the other investigated fungi  containing zinc-finger structures, such as the Zn2Cys6 type, the C2H2 type and CCHC type, followed by the winged helix repressor DNA-binding family (97 members) , the cellulase transcription activator CLR-2 (TP10486), the starch degradation regulator AmyR (TP09286) and the xylan degradation regulator XlnR (TP02627) (Table T. pinophilus 1–95.Transcription factors (TFs) are essential for modulating diverse biological processes by regulating gene expression. In total, 943 TFs were found in the predicted proteome of T. pinophilus 1–95 had a wealth of secondary metabolites using the AntiSMASH web service30. A total of 68 secondary metabolism gene clusters harboring 401 putative genes were identified. The predicted products of 52 secondary metabolism gene clusters were classified into 8 different types: 28 T1 polyketide synthase (T1PKS) gene clusters, 9 non-ribosomal peptide synthase (NRPS) gene clusters, 9 terpene gene clusters, 2 Nrps-T1pks gene clusters, 1 phosphonate gene cluster, 1 T1pks-Indole gene cluster, 1 T1pks-Nrps gene cluster and 1 Terpene-T1pks gene cluster; the remaining 16 gene clusters synthesized other unknown secondary metabolites , comprising 831 classical and 372 nonclassical secretory proteins , Cel7B (TP08415), Cel5A (TP13457), Cel5B (TP07499), Cel5C (TP08784), Cel45A (TP06957) and Bgl3A (TP09042). We also identified 62 hemicellulose-degrading enzymes and 6 pectin-degrading enzymes, including eight β-1,4-endoxylanases, nine acetyl xylan esterases, four α-galactosidases, 10 α-L-arabinofuranosidases, five α-L-fucosidases, one endo-1,4-β-mannanase and four feruloyl esterases, as well as two pectin esterases, two tannases, one pectate lyase and one pectin lyase , glucoamylases (5 coding genes) and α-glucosidases (13 coding genes) among species we compared. BGLs are important for releasing inhibition of cellulase activity36. Furthermore, the predicted secretome of T. pinophilus 1–95 showed that approximately 50% of CWDEs and starch-degrading enzymes were secreted into the extracellular region, including major cellulases, hemicellulases and amylases. This result indicated a promising application of T. pinophilus in biorefining. These results also demonstrated that T. pinophilus 1–95 is more excellent cellular machinery for biomass-degrading enzymes than that of P. oxalicum HP7–1 and T. cellulolyticus Y-94 as previously observed35, meriting further study.Comparative genome analysis indicated that the most closely related species to Talaromyces, Trichoderma, Penicillium and Aspergillus, indicated that T. pinophilus 1–95 possessed the most secondary metabolism gene clusters except for A. niger and A. oryzae. T. pinophilus 1–95 had more T1PKs than Trichoderma sp., Penicillium sp. and Aspergillus sp., and fewer NRPS than Aspergillus species. These results suggested that T. pinophilus 1–95 has potential for producing bioactive secondary metabolites. Although thus far, several bioactive secondary metabolites have been extracted and characterized from T. pinophilus7, according to the genomic data, additional secondary metabolites could be generated using this species.Comparative analysis to 10 filamentous fungi from four genera, Talaromyces. The result provided new insights for a comprehensive understanding of the biomass-degrading enzyme system of Talaromyces at the genome level. Detailed comparative genomic analysis revealed a complex biomass-degrading enzyme system in T. pinophilus, indicating its promising application in biomass biorefineries. This study provides a genome-sequence basis for developing strategies that use T. pinophilus as a microbial cell factory for production of high-value enzymes and secondary metabolites.In summary, this study provided a nearly complete genome sequence for the genus T. pinophilus 1–95 was isolated from soil in a dried, ploughed paddy field in Wuzhou, China1 and was deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 2645. Total DNA extraction from mycelia was performed using a phenol-chloroform method with some modifications37. Mycelia were ground in liquid nitrogen and put in 1 mL lysate reagent  per 100 mg mycelia powder. Genomic DNA was collected by centrifugation at 11,300 × g for 10 min.T. pinophilus strain 1–95 genome was sequenced using a PacBio RS II platform and Illumina HiSeq 4000 platform at the Beijing Genomics Institute . Four SMRT cells zero-mode waveguide arrays of sequencing, were used by the PacBio platform to generate the subreads set. PacBio subreads (length < 1 kb) were removed. The program Pbdagcon (https://github.com/PacificBiosciences/pbdagcon) was used for self-correction. Draft genomic unitigs, which are uncontested groups of fragments, were assembled using the Celera Assembler38 against a high-quality corrected circular consensus sequence subreads set. Order, distance and orientation of unitigs and combined scaffolds were generated using software SSPACE39. An upgraded draft genome was obtained after filling or reducing as many captured gaps as possible using software PBJelly40. To improve the accuracy of the genome sequences, GATK (https://www.broadinstitute.org/gatk/) and SOAP tool packages 42 were used to make single-base corrections.The 43 used reasonable PE sequence data from Illumina libraries to perform comprehensive variant detection and improve genome assembly.A DNA library of 500 bp inserts was constructed and PE sequenced. For generated HiSeq reads, Q20, representing the probability of the incorrectness of the corresponding base call, was detected. If Q20 reads accounted for less than 60%, they were discarded. Software PilonT. pinophilus 1–95 genome were predicted independently with the gene prediction programs Augustus , GeneMark-ES (http://exon.gatech.edu/GeneMark/), Genewise (http://www.ebi.ac.uk/Tools/psa/genewise/), SNAP14, and the unsupervised learning system program Glean (https://sourceforge.net/projects/glean-gene/) version 1. Augustus and SNAP, using default parameters, were trained on gene models for A. oryzae, P. oxalicum, T. marneffei and T. stipitatus  and Swiss-Prot and TrEMBL databases (http://www.mrc-lmb.cam.ac.uk/genomes/madanm/pres/swiss2.htm)  and KEGG database (http://www.kegg.jp/) version 76, were used to assign general protein function profiles. We used cut-off E-value ≤ 1e-5, overlap 0.4 and identity 30. InterProScan5 (http://www.ebi.ac.uk/interpro/), stand-alone version 55, and GO (http://geneontology.org/) were also used to annotate the predicted proteome. TFs were predicted based on InterPro IDs in the Fungal Transcription Factor Database (http://ftfd.snu.ac.kr/). The hmmsearch program in the HMMER 3.1b2 package (http://hmmer.org/), was used to search all predicted proteomes with the family-specific hidden Markov model profiles of CAZymes from the dbCAN database26. Primary results were processed with an E-value threshold of 1E-7. Protein kinases and phosphatases were detected using hmmsearch based on the Eukaryotic Kinase and Phosphatase Database (http://ekpd.biocuckoo.org/). Membrane transport proteins were classified and identified by a BLASTp search against the transport classification database44, with E-value threshold 1E-10, overlap 0.4 and identity of 30. AntiSMASH30 was used to annotate secondary metabolism gene clusters.For functional annotation of translated proteins in the 45. We selected 2,082 single-copy genes from 118,099 genes in 11 fungal genomes. MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) version 3.7 with default parameters was used to perform multiple sequence alignment of single-copy genes. A neighbor-joining tree was calculated using TreeBeST46 with bootstrapping set to 100. The phylogenetic tree was visualized using SVGKit (http://svgkit.sourceforge.net/) and PERL scripts.An all-against-all pairwise BLASTp similarity search was performed using proteomes from 11 filamentous fungi  and for glycosylphosphatidyl inositol anchors use web server PredGPI (http://gpcr.biocomp.unibo.it/predgpi/). Software tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) version 1.3 was used for transfer RNA prediction using the T. pinophilus 1–95 genome with option C and other default parameters.The total set of 13,472 proteins of Supplementary Infromation for SREP-16-39329Dataset 1Dataset 2Dataset 3


Bradyrhizobium canariense, isolated from root nodules of Lupinus microanthus and Lupinus angustifolius, and 1 strain of Bradyrhizobium japonicum isolated from root nodules from Lupinus angustifolius in Algeria. These sequences add to the known diversity of this agronomically important genus.We report here the whole-genome sequences of 14 strains of Bradyrhizobium canariense , mungbean (Vigna radiata), cowpea (Vigna unguiculata), and siratro (Macroptilium atropurpureum)  is rather recent (Lupinus angustifolius and Lupinus micranthus (Papilionoideae: Genisteae), collected at 2 sites in the National Park El-Kala . For whole-genome sequencing, DNA libraries were generated with a Nextera XT kit . Sequencing was performed on a MiSeq sequencer (Illumina) in three different runs generating 2 × 250-bp paired-end reads (version 2 chemistry) and 2 × 250-bp and 2 × 300-bp paired-end reads (version 3 chemistry). Quality control of the reads was assessed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Genome assemblies were computed with SPAdes genome assembler 3.10  and 8,379,024 bp (UBMAN05), with G+C contents from 62.94%  to 63.06% (UBMA122), and Bradyrhizobium japonicum UBMA197 had a larger genome, at 10,442,239 bp, with 63.3% G+C content. Genome coverage varied from 46-fold (UBMA510) to 255-fold (UBMA060). PlasmidFinder  , 4. Bactrpureum) , 2, 5. Brpureum) , Bradyrhs Poland , 7, Itals Poland , and Mors Poland . While dr recent . The 15 ler 3.10  and analler 3.10 . For BraidFinder  and PlasidFinder  detectedidFinder  identifiAll genome sequences have been deposited at GenBank under the accession numbers reported in


The effect of degradable starch microspheres (DSM) on the intrahepatic distribution of a low molecular weight marker, 99Tcm-labelled methylene diphosphonate (MDP), was studied in rats with hypovascular HSN liver tumours. MDP was injected regionally, via the hepatic artery, alone or co-administered with DSM, with or without subsequent occlusion of either the hepatic artery or the portal vein. Tumour vascularity was measured with 57Co-labelled microspheres. Co-injection with DSM immediately significantly increased hepatic retention of marker in both tumour (T) (median 22.40 (range 16.82-39.58)% injected dose) and normal liver (N) (9.08 (4.85-12.59) %ID) the greater effect seen in T (P < 0.01). After DSM degradation, very little MDP remained in N (0.61 (0.28-1.40) %ID) but there was significant retention in T (10.01 (6.73-20.28) %ID, P < 0.01). Clamping the hepatic artery had minimal effect on the retention of MDP when administered alone. Regional injection of 16.5 microM 57Co microspheres resulted in a N:T ratio of 2.25:1. Concomitant injection of the 40 microM DSM was 57Co microspheres reversed this ratio to 1:2. The results indicate that DSM selectively enhances the retention of MDP to a hypovascular hepatic tumour, not by causing intra-tumour stasis, but by directing a greater arterial flow to hypovascular areas in the liver.


Primary chemotherapy in operable breast invasive carcinoma enables tumour reduction and conservative surgery. In order to search for one or more biological factors capable of predicting tumour behaviour under primary chemotherapy, and subsequent patient survival, an immunohistochemical study was performed with specific antibodies to p53, c-erbB-2 (Her-2/neu), Mib1 (antiKi-67), pS2, GST pi, oestrogen receptors (ERs) and progesterone receptors (PRs). Core biopsies, obtained before primary chemotherapy, were available from a series of 128 breast invasive carcinomas treated between January 1985 and April 1989, with a median follow-up of 93.3 months. Univariate statistical analysis showed that negative ER detection by immunohistochemistry (IHC) was highly correlated with chemosensitivity (P = 0.001). A high percentage of Mib1-positive tumour cells (> 40%), as well as initial tumour size less than 4 cm, were also correlated with tumour responsiveness to chemotherapy (P = 0.009 and P = 0.03). By multivariate analysis IHC-ER, Mib1 and initial tumour size were independent predictors, the last parameter being the most important. Concerning subsequent patient survival, c-erbB-2 overexpression, as detected by IHC, was significant with respect to overall survival (OS) (P = 0.0006), disease-free interval (DFI) (P = 0.03) and metastasis-free interval (MFI) (P = 0.008) by univariate analysis. Furthermore, c-erbB-2 was the major independent prognostic factor for OS and MFI by multivariate analysis.


A sentence was omitted from Section 2.2 of the Methods . The corrected paragraph should read: DMEM, bovine serum and penicillin/streptomycin were purchased from Hyclone . Nilotinib (TasignaR)  was obtained as a gift from Novartis pharmaceuticals . Imatinib  and dasatinib  were purchased from ChemieTeck Inc. . Paclitaxel, vincristine, doxorubicin, colchicine, p-aminophenylmethylsulfonyl fluoride, bovine serum albumin, dimethyl sulfoxide (DMSO) and 1--3,5-diphenylformazan (MTT), the monoclonal mouse antibody against P-gp (P7965), the secondary horseradish peroxidase-labeled anti-goat or anti-mouse IgG were purchased from Sigma-Aldrich Chemical Co. . A polyclonal goat antibody against human MRP7 (D-19) was purchased from Santa Cruz Biotech Inc.<br/>. A polyclonal antibody against human MRP1/ABCC1 was kindly provided by Dr. Akiyama  [21]. A monoclonal antibody BXP-34 against BCRP was acquired from Signet Laboratories Inc. . [3H]-paclitaxel  was purchased from Moravek Biochemicals .


Two tris-chelated water-coordinated units are bridged by a 2,4-dihydroxypyrimidine-5-carboxylate dianion, which is disordered about a center of inversion. The metal center has a monocapped square-antiprismatic coordination.The water-coordinated neodymium(III) atom in the centrosymmetric title compound, [Nd DOI: 10.1107/S1600536808001499/ci2552Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


B. microti infection (7).”In “Transfusion-Associated Babesiosis after Heart Transplant” by Joseph Z. Lux et al., errors occurred in the text. On page 118, left column, lines 20–22, the sentence should read “he became symptomatic during the typical 2- to 8-week incubation period for transfusion-transmitted http://www.cdc.gov/ncidod/eid/vol8no12/02-0149.htm.The corrected text appears online at


The 4,4′-bipyridine ligands bridge the metal ions, forming one-dimensional chains along different directions, which are further connected by formate ligands into a topologically (1010.124.14)(10)3 three-dimensional network.In the title compound, [Cd(HCOO) DOI: 10.1107/S1600536809009969/jh2074Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


From January 1983 to December 1986 seventy-six previously untreated children with non-Hodgkin's lymphoma (NHL) were treated by combination chemotherapy. Burkitt's lymphoma patients were ineligible. The treatment regimens include intermittent chemotherapy and for non-localized patients, prophylactic central nervous system chemotherapy. Intrathoracic non-Hodgkin's lymphoma patients also had cranial prophylactic radiotherapy. Sixty-six patients (86.8%) achieved complete remission. Two year failure-free survival rate was 82.1% for localized (stage I and II) NHL and 53.3% for non-localized (stage III and IV) NHL patients. Failure-free survival did not differ significantly for the two major histologic diagnoses, but two year survival rate was lower in diffuse poorly differentiated lymphoblastic than undifferentiated non-Burkitt's lymphoma (50% versus 66.8% respectively). Failure-free survival rate was 53.7% in mediastinal disease and, 73.2% in abdominal disease at 24 months. Relapse rate was higher in mediastinal cases (46.1%) than primary abdominal cases (24.3%) at 24 months. Eleven (13.5%) died of treatment related sepsis. Although the overall survival rate was 72.4% at 2 years we need novel or more intensive programmes for mediastinal and non-localized disease.


Correction for:10.1371/journal.pmed.0030184Wagstaff SC, Laing GD, Theakston RDG, Papaspyridis C, Harrison RA (2006) Bioinformatics and multiepitope DNA immunization to design rational snake antivenom. PLoS Med 3(6): e184. doi:In the Supporting Information section of this paper, Figure S1 and Figure S2 were incorrectly linked. Figure S1 and Figure S2 labeled and in the order originally intended are provided here.Figure S1(48 KB PDF).Click here for additional data file.Figure S210.1371/journal.pmed.0050209.sg002 (32 KB PDF).Found at doi:Click here for additional data file.


Drosophila wing growth by fueling a wave front of Fat-Dachsous signaling that recruits new cells into the wing primordium.The secreted morphogen Wingless promotes  Drosophila wing primordium undergoes a dramatic increase in cell number and mass under the control of the long-range morphogens Wingless  and Decapentaplegic . This process depends in part on the capacity of wing cells to recruit neighboring, non-wing cells into the wing primordium. Wing cells are defined by activity of the selector gene vestigial (vg) and recruitment entails the production of a vg-dependent “feed-forward signal” that acts together with morphogen to induce vg expression in neighboring non-wing cells. Here, we identify the protocadherins Fat (Ft) and Dachsous (Ds), the Warts-Hippo tumor suppressor pathway, and the transcriptional co-activator Yorkie  as components of the feed-forward signaling mechanism, and we show how this mechanism promotes wing growth in response to Wg. We find that vg generates the feed-forward signal by creating a steep differential in Ft-Ds signaling between wing and non-wing cells. This differential down-regulates Warts-Hippo pathway activity in non-wing cells, leading to a burst of Yki activity and the induction of vg in response to Wg. We posit that Wg propels wing growth at least in part by fueling a wave front of Ft-Ds signaling that propagates vg expression from one cell to the next.During development, the  Drosophila, wings develop at the larval stage from wing primordia. Recently, we discovered that the morphogen Wingless promotes growth of the Drosophila wing by inducing the recruitment of neighboring cells into the wing primordium. Wing cells are defined by the expression of the “selector” gene vestigial. Recruitment depends on the capacity of wing cells to send a short-range, feed-forward signal that allows Wingless to activate vestigial in adjacent non-wing cells. Here, we identify the molecular components and circuitry of the recruitment process. We define the protocadherins Fat and Dachsous as a bidirectional ligand-receptor system that is controlled by vestigial to generate the feed-forward signal. Further, we show that the signal is transduced by the conserved Warts-Hippo tumor suppressor pathway via activation of its transcriptional effector Yorkie. Finally, we propose that Wingless propels wing growth by fueling a wave front of Fat-Dachsous signaling and Yorkie activity that propagates vestigial expression from one cell to the next.Under normal conditions, animals and their various body parts grow until they achieve a genetically predetermined size and shape—a process governed by secreted organizer proteins called morphogens. How morphogens control growth remains unknown. In  Growth is a fundamental property of animal development. Under normal conditions, animals of a given species, as well as their various body parts, achieve a characteristic size, shape, and pattern under tight genetic control. However, the basis of this control is poorly understood.Drosophila wing, the morphogens Wingless  and Decapentaplegic  drive a rapid ∼200-fold increase in cell number and mass that occurs during larval life Morphogens, such as secreted factors of the Wingless/Int (Wnt), Bone Morphogenetic Protein (BMP), and Hedgehog (Hh) families, control growth. For example, in the classic paradigm of the Another system involved in growth is the evolutionarily conserved Warts-Hippo tumor suppressor pathway Drosophila, two protocadherins, Dachsous (Ds) and Fat (Ft), have been implicated as a ligand-receptor pair that acts, via the atypical myosin Dachs (D), to regulate Wts kinase activity In Ft and Ds are also important for planar cell polarity (PCP), in which cells within epithelial sheets adopt a common orientation, e.g. as manifest by their secreting hairs that point in the same direction Drosophila wing growth by morphogen vestigial (vg), the selector gene that defines the wing state vg expressing cells to send a feed-forward (FF) signal that induces neighboring cells to activate vg in response to Wg vg expression from cell to cell in response to Wg spreading from the border cells.Recently, we defined a new mechanism for the control of vg expression and initiate a new cycle of FF signaling. Based on these findings, we posit that Wg (and likely Dpp) promote wing growth by fueling the propagation of a wave front of Ft-Ds signaling that transiently suppresses the Wts-Hpo pathway and elevates Yki activity to recruit new cells into the wing primordium.In our initial analysis of the recruitment process, we speculated that Ft and Ds might be involved in the FF mechanism vg Boundary Enhancer (BE) to generate a stripe of vg expressing “border cells” vg expression in surrounding cells via the vg Quadrant Enhancer (QE)   [4],[5],apterous (ap) in D, but not V, cells ap null discs (henceforth oap discs), the D-V segregation fails, vg and wg expressing border cells are not specified, and the nascent wing primordium is subsequently lost  and ds, itself, are expressed at peak levels in complementary domains that abut at the wing periphery, fj in the vgON domain . Such clones express moderate levels of exogenous Vg, a few fold lower than the peak endogenous level observed in wild type discs, and rescue wing development cell-autonomously fj-lacZ and repress ds-lacZ . Hence, prospective wing cells in these discs behave as if they have constitutively activated the FF signal transduction pathway but can mount only a weak QE response owing to the low levels of cryptic Wg available As described above, imordium  and 2B [o apoft discs is Wg dependent. First, the QE response is abolished in clones of o Dfz2ofz cells, which are unable to transduce Wg (UAS.Nrt-wg clones) drive peak levels of Vg and 5XQE.DsRed expression in o apoft discs, both within the clones and in abutting cells . These results confirm that ods clones serve as ectopic sources of FF signal, capable of inducing QE-dependent vg expression in neighboring cells, provided that the responding cells also receive Wg.In the presence of Nrt-Wg, ame disc . In the F signal : they inoft clones elicited a strictly cell autonomous response, both in Nrt-Wg expressing oap discs  and hence should be potent sources of FF signal; nevertheless they behave as if devoid of the capacity to signal. Note that this failure cannot be attributed to a generic inability of oft cells to send intercellular signals. First, oft clones repolarize their neighbors, whereas o ftods clones do not, indicating that they have the capacity to send the Ds PCP signal oft clones in the wing primordium can also send DSL-Notch, Wg, and Dpp signals  and generate ectopic FF signal in oap discs, as monitored by the induction of 5XQE.DsRed expression in adjacent wild type cells  that wing cells require Ft to generate FF signal and (ii) that non-wing cells require both Ft and Ds to receive the signal.Although wing cells require Ft, but not Ds, to send the FF signal, cells undergoing recruitment are also in position to receive an opposing Ds signal coming from non-wing cells on the other side, raising the possibility that this Ds input may also contribute to activating the QE and recruiting cells into the wing primordium.oap discs and asked if the resulting disparity in Ds signaling across the clone border is sufficient to induce the QE response in surrounding cells.To assess this, we generated Ds over-expressing clones in UAS.ds cells in oap discs generate levels of Ds that are several fold higher than endogenous Ds . In the absence of exogenous Wg, such UAS.ds clones had little effect on surrounding cells, only occasionally inducing 5XQE.DsRed expression just outside the clone (unpublished data). However, when supplemented with exogenous Wg (using co-expression of a UAS.wg transgene), most UAS.ds clones induced 5XQE.DsRed expression both within the clone and in surrounding cells . Hence, if the FF signal is transduced by the Wts-Hpo pathway, manipulations that promote Yki action , as expected if D is not available to block Wts activity in the absence of Ds and/or Ft.First, we examined the consequences of generating ods and od clones in oUAS.wg ap discs. Under these conditions, the ods clones both expressed Vg and induced Vg expression in neighboring wild type cells but failed to induce detectable expression in abutting cells belonging to the od clone, resulting in their exclusion from the rescued wing pouch . In this context, Ds and Ft are thought to function as a ligand-receptor pair, with tissue-wide gradients of Ds signal serving to activate Ft to appropriate levels within each cell To date, Ft-Ds signaling has been studied in two contexts: the control of Yki target genes in tissue growth and the orientation of cell structures in PCP. Most work on tissue growth has focused on Yki target genes that control basic cell parameters, such as survival, mass increase, and proliferation  and non-wing (vgOFF) cells. Further, we posit that these differentials are transduced in cells undergoing recruitment  that work in conjunction with FF propagation see also . And thiDrosophila wing, Wts-Hpo activity and YAP appear to function in these contexts in a manner that is distinct from their generic roles in the regulation of cell survival, growth, and proliferation, namely as part of an intercellular signaling mechanism that specifies cell type. We suggest that this novel employment of the pathway constitutes a new, and potentially general, mechanism for regulating tissue and organ size.Our identification of Ft-Ds signaling, the Wts-Hpo pathway, and Yki as key components of the FF recruitment process provides a striking parallel with the recently discovered involvement of the Wts-Hpo pathway and Yki/YAP in regulating primordial cell populations in vertebrates, notably the segregation of trophectoderm and inner cell mass in early mammalian embryos (i) Flp/FRT mediated mitotic recombination Hsp70.flp transgene . Wing discs from mature third instar larvae were dissected, fixed, and processed for immuno-fluorescence by standard methods, using anti-Vg, anti-Wg, anti-HA, and anti-βGal antisera  by heat shock induced expression of an vg QE activity was monitored by expression of 1XQE.lacZ and 5XQE.DSRed reporter transgenes as well as by the expression of Vg protein in the absence of DSL-Notch signaling fj-lacZ enhancer trap allele P1fjvg expression. All four assays gave similar results, with the 5XQE.DSRed and fj-lacZ reporters showing the greatest sensitivity.http://flybase.bio.indiana.edu/) The following amorphic mutant alleles and transgenes were employed Exel6006, UA071ds, 2D60bds, E1ex, P1fj, 15ft, P21fz, C1Dfz2 , 83b27Rvg, and X1wts.Mutant alleles: apintraUAS.N, UAS.Nrt-wg, UAS.wg, Tubα1>GFP,y+>vg, C765.Gal4, nub.Gal4, Tubα1>Gal80>Gal4, GSUAS.ds, UAS.ft, UAS.d, UAS.yki, Hsp70.GFP.Transgenes: Exact genotypes, by Figure panel:56f fjP1/+.y w 5XQE.DsRed/y w Hsp70.flp; FRT39 ap(2A) 56f fjP1/FRT39 ap56f.y w 5XQE.DsRed/y w Hsp70.flp; FRT39 ap(2B) 2D60b FRT39 ap56f vg83b27R/+.y w 5XQE.DsRed/y w Hsp70.flp; ds(2C) 2D60b FRT39 ap56f vg83b27R/FRT39 ap56f.y w 5XQE.DsRed/y w Hsp70.flp; ds(2D) 56f vg83b27R 5XQE.DsRed/FRT39 ap56f vg83b27R fjP1; Tubα1>flu-GFP,y+>vg/+.y w Hsp70.flp/y w Hsp70.flp; ap(2E) 56f vg83b27R 5XQE.DsRed/ds2D60b FRT39 ap56f vg83b27R; Tubα1>flu-GFP,y+>vg/+.y w Hsp70.flp/y w Hsp70.flp; ap(2F) 56f/Hsp70.flu-GFP FRT39 ap56f fjP1; Tubα1>flu-GFP,y+>vg UAS.Nrt-flu-wg/C765.Gal4.y w 5XQE.DsRed/y w Hsp70.flp; FRT39 ap(2G) 56f/ds2D60b FRT39 ap56f vg83b27R; Tubα1>flu-GFP,y+>vg UAS.Nrt-flu-wg/C765.Gal4.y w 5XQE.DsRed/y w Hsp70.flp; FRT39 ap(2H) UA071 FRT39 ap56f/dsUA071 FRT39 ap56f; UAS.wg/+.y w 5XQE.DsRed/y w Hsp70.flp; ds(3A) 15 FRT39 ap56f/dsUA071 ft15 FRT39 ap56f fjP1.y w 5XQE.DsRed/y w 5XQE.DsRed; ft(3B) UA071 ft15 FRT39 ap56f/dsUA071 ft15 FRT39 ap56f fjP1; Tubα1>CD2,y+>Gal4/+.y w 5XQE.DsRed/y w 5XQE.DsRed; ds(3C) 15 FRT39 ap56f/ft15 FRT39 ap56f; fzP21 Dfz2C1 FRT2A/Hsp70.CD2 Hsp70.flu-GFP FRT2A.y w 5XQE.DsRed/y w Hsp70.flp; ft(3D) 15 FRT39 ap56f/ft15 FRT39 ap56f; UAS>CD2,y+>Nrt-flu-wg C765.Gal4/+.y w 5XQE.DsRed/y w Hsp70.flp; ft(3E) UA071 FRT39 ap56f/Hsp70.flu-GFP Tubα1.Gal80 FRT39 ap56f fjP1.y w 5XQE.DsRed/y w Hsp70.flp Tuba1.Gal4 UAS.GFPnls; ds(4A) 15 FRT39 ap56f/Hsp70.flu-GFP Tubα1.Gal80 FRT39 ap56f fjP1; UAS.wg/+.y w 5XQE.DsRed/y w Hsp70.flp; ft(4B) UA071 ft15 FRT39 ap56f fjP1/Hsp70.flu-GFP Tubα1.Gal80 FRT39 ap56f; C765.Gal4/+.y w 5XQE.DsRed/y w Hsp70.flp; ds(4C) UA071 FRT39 ap56f/Hsp70.flu-GFP FRT39 ap56f; UAS.Nrt-flu-wg/C765.Gal4.y w 5XQE.DsRed/y w Hsp70.flp; ds(4D) 15 FRT39 ap56f/Hsp70.flu-GFP FRT39 ap56f; UAS.Nrt-flu-wg/C765.Gal4.y w 5XQE.DsRed/y w Hsp70.flp; ft(4E) UA071 FRT39 ap56f/Hsp70.flu-GFP FRT39 ap56f; UAS>CD2,y+>Nrt-flu-wg C765.Gal4/1XQE.lacZ.y w Hsp70.flp/y w Hsp70.flp; ds(4F) UA071 Tubα1.Gal80 FRT39 ap56f vg83b27R/Hsp70.flu-GFP FRT39 ap56f fjP1; UAS.ft/Tuba1.Gal4.y w 5XQE.DsRed/y w Hsp70.flp; ds(5A) 15 Tubα1.Gal80 FRT39 ap56f/Hsp70.flu-GFP FRT39 ap56f fjP1; UAS.ft/Tubα1.Gal4.y w 5XQE.DsRed/y w Hsp70.flp; ft(5B) 56f fjP1/FRT39 ap56f UAS.flu-wg; UAS.ft/Tubα1>Gal80,y+>Gal4.y w 5XQE.DsRed/y w Hsp70.flp UAS.GFPnls; FRT39 ap(5C) 56f 1XQE.lacZ/dsUA071 FRT39 ap56f; UAS.ds/Tubα1>Gal80,y+>Gal4 UAS.wg.y w 5XQE.DsRed/y w Hsp70.flp UAS.GFPnls; ap(5D) 56f/Hsp70.flu-GFP Tubα1.Gal80 FRT39 ap56f fjP1; UAS.d/UAS.wg.y w 5XQE.DsRed/y w Hsp70.flp Tuba1.Gal4 UAS.GFPnls; FRT39 ap(6A) 56f UAS.flu-wg/FRT39 ap56f fjP1; Tubα1>Gal80,y+>Gal4 UAS.yki/+.y w 5XQE.DsRed/y w Hsp70.flp UAS.GFPnls; FRT39 ap(6B) 56f/ap56f UAS.flu-Nrt-wg; FRT82 wtsx1/FRT82 Hsp70.flu-GFP.y w Hsp70.flp/y w Hsp70.flp; nub-Gal4 FRT39 ap(6C) e1 FRT39 ap56f/Hsp70.flu-GFP FRT39 ap56f fjP1; UAS.wg/C765.Gal4.y w 5XQE.DsRed/y w Hsp70.flp; ex(6D) UA071 Hsp70.flu-GFP FRT39 ap56f/dGC13 FRT39 ap56f fjP1; UAS.wg/C765.Gal4.y w 5XQE.DsRed/y w Hsp70.flp; ds(7A) GC13 FRT39 ap56f fjP1/dGC13 FRT39 ap56f; Tubα1>flu-GFP,y+>vg UAS.Nrt-flu-wg/C765.Gal4.y w 5XQE.DsRed/y w Hsp70.flp; d(7B) UA071 dGC13 FRT39 ap56f/Hsp70.flu-GFP Tuba1.Gal80 FRT39 ap56f fjP1; UAS.wg/+.y w 5XQE.DsRed/y w Hsp70.flp Tuba1.Gal4 UAS.GFPnls; ds(7C) UA071 FRT39 ap56f/dsUA071 FRT39 ap56f; UAS.Nrt-flu-wg/Tubα1>Gal80,y+>Gal4.y w 5XQE-DsRed/y w Hsp70.flp; ds(S1A) 56f UAS>CD2,y+>Nrt-flu-wg/nub-Gal4 FRT39 ap56f; FRT82 wtsx1/FRT82 Hsp70.flu-GFP.y w Hsp70.flp/y w Hsp70.flp; ap(S1B) 56f 1XQE.lacZ/FRT39 ap56f; UAS>CD2,y+>Nrt-flu-wg C765.Gal4/UAS.yki.y w Hsp70.flp/y w Hsp70.flp; ap(S1C) (S2A) as (4E).15 FRT39 ap56f/Df(2L)Exel6006 Hsp70.flu-GFP FRT39 ap56f; UAS.Nrt-flu-wg/C765.Gal4.y w 5XQE-DsRed/y w Hsp70.flp; ft(S2B) 15 Tuba1.Gal80 FRT39 ap56f/Hsp70.flu-GFP FRT39 ap56f; UAS>CD2,y+>Nrt-flu-wg C765.Gal4/1XQE.lacZ.y w Hsp70.flp/y w Hsp70.flp; ft(S2C) cx4 FRT39 ap56f/Hsp70.flu-GFP Tubα1.Gal80 FRT39 ap56f; UAS.Nintra/1XQE.lacZ.y w Hsp70.flp Tubα1.Gal4 UAS-GFPnls/y w Hsp70.flp; wg(S3A) 15 wgcx4 FRT39 ap56f/Hsp70.flu-GFP Tubα1.Gal80 FRT39 ap56f; lqf1227 Hsp70-CD2 FRT2A UAS.Nintra/+.y w 5XQE-DsRed/y w Hsp70.flp Tubα1.Gal4 UAS-GFPnls; ft(S3B) 15 FRT39 ap56f/Tubα1.Gal80 FRT39; UAS.wg/+.y w Hsp70.flp Tubα1.Gal4 UAS-GFPnls/y w Hsp70.flp; ft(S3C) 15 FRT39 ap56f/Tubα1.Gal80 FRT39; UAS.dpp/+.y w omb-lacZ/y w Hsp70.flp Tubα1.Gal4 UAS-GFPnls; ft(S3D) Figure S1Quadrant enhancer activity is Wingless dependent in oap discs that lack either Dachsous or Warts or that over-express Yorkie. (A) A UAS.Nrt-wg clone in a o apods disc. Both Vg and 5XQE.DsRed are expressed at peak levels in the clone and in surrounding cells that abut the clone, as observed for UAS.Nrt-wg clones in o apoft discs , Vg is strongly up-regulated in the UAS.Nrt-wg clones and abutting cells. (C) UAS.Nrt-wg clones generated in an oUAS.yki ap disc; same outcome as in (A), except a 1XQE.lacZ transgene was used instead of the 5XQE.DsRed transgene.po discs . (B) UAS(3.99 MB TIF)Click here for additional data file.Figure S2Exceptional cases of local non-autonomous Quadrant enhancer activity associated with oft clones can be attributed to induction by their sibling 2×+ft clones. (A) A oft clone (marked by the absence of GFP) associated with local, non-autonomous activity of the 5XQE.DsRed transgene (appears yellow in A') in an o UAS.Nrt-wgap disc. Note that this non-autonomous expression is associated with a sibling 2×+ft clone . In this experiment, 26/43 oft clones were associated with strictly cell-autonomous QE activity , and in the remaining 10/17 cases, 9/10 had no detectable twin, and 1/10 had a twin clone located elsewhere. Thus, the majority of oft clones analyzed in this experiment showed a strictly cell-autonomous response, and in 7/8 cases in which local, non-autonomous 5XQE.DsRed expression was observed and a 2×+ft twin survived, the twin spot was associated with the 5XQE.DsRed expression. Based on these results, we attribute the exceptional cases of non-autonomous 5XQE.DsRed expression associated with oft clones to signaling by their 2×+ft sibling clones, a conclusion further supported by experiments in panels (B) and (C). (B) A oft clone generated and marked as in (A), except under conditions in which its sibling 2×+ft clone died, owing to homozygosity for Df(2L)Exel6006. Note the strictly cell-autonomous expression of the 5XQE.DsRed transgene. 39/45 clones generated in this experiment behaved in this way; 6/45 showed local non-autonomy. We have not determined how quickly the sibling 2×+ Df(2L)Exel6006ft clones die after being generated in this experiment; it is possible that rare 2×+ Df(2L)Exel6006ft clones survive long enough to induce self-sustaining vg and 5XQE.DsRed expression in neighboring cells prior to their loss. (C) A UAS.Nrt-wg 2×+ft clone  and its oft sibling clone (marked by the absence of GFP) in an oap disc. Note the association of the 2×+ft clone with ectopic Vg expression as well as the local induction of Vg expression by Nrt-Wg in neighboring cells . This result corroborates the evidence shown in (A) and (B), that 2×+ft clones generated in 1×+ apoft discs have the capacity to induce 5XQE-DsRed and vg expression.ty as in : of thesft+ twin , and the(2.19 MB TIF)Click here for additional data file.Figure S3oft clones can send Delta/Serrate/Lag2 (DSL), Wingless, and Decapentaplegic signals. (A) A intra wgoUAS.N clone generated in an oap disc. intraN encodes a constitutively active form of Notch; clones of intra wgoUAS.N cells in oap discs up-regulate the expression of the Notch ligands Delta and Serrate and activate Notch in adjacent cells, as visualized by the induction of a ring of ectopic, Wg-expressing D-V border cells encircling the clone . These ectopic border cells suffice to initiate the long-range propagation of QE-dependent vg expression in surrounding cells, as indicated by the broad halo of 1XQE-lacZ expression. (B) A intra wgo ftoUAS.N clone generated an oap disc. Essentially the same experiment shown in (A), except that the clones are also oft. The result is the same (except that a 5XQE-DsRed reporter was used in place of the 1XQE-lacZ reporter), indicating that cells in the clone can send DSL signals to the surround, even though they are devoid of Ft. (C) A oUAS.wg ft clone generated in a wild type disc. QE-dependent vg expression depends on the level of Wg input. As a consequence UAS.wg clones up-regulate Vg expression in surrounding cells within the wing pouch, as seen in this example, even though the clone is also oft. (D) A oUAS.dpp ft clone generated in a wild type disc. Ectopic Dpp expressed by the clone has induced ectopic omb-lacZ expression in the surround, even though the clone is oft.(3.03 MB TIF)Click here for additional data file.


The expression of retinoblastoma (Rb), c-Myc and Bcl-2 proteins was studied by immunohistochemical methods in 104 cases of renal adenocarcinoma. One tumour was completely negative for Rb protein and altered expression pattern was detected in 36% of cases. A low fraction of Rb-positive nuclei was related to high grade (P = 0.016) and high mitotic index (P = 0.012). Twenty-eight per cent of the tumours expressed c-Myc in cancer cell nuclei and 87% showed cytoplasmic positivity. Cytoplasmic expression of c-Myc was related to high grade (P = 0.002), while nuclear expression of c-Myc was related to small tumour diameter (P = 0.034), low T category (P = 0.04), low mitotic index (P = 0.019) and expression of c-ErbB-2 (P = 0.0007). Overexpression of c-myc predicted favourable outcome in M0 tumours (P = 0.0157). Bcl-2 was expressed in 20% of tumours and it was related to small tumour size (P < 0.0001), low T category (P < 0.0001), lack of venous invasion (P = 0.008), node negativity (P = 0.015) and absence of metastasis (P = 0.017). In multivariate analysis the expression of Rb, Bcl-2 and c-Myc had no independent prognostic value over T category (P < 0.001), mitotic index (P = 0.008) and combined nuclear grade (P = 0.056).


P= 0.08). A negative association was found between ARs and cell proliferative activity: MIB-1 scores were higher (25.4%) in AR-negative than in AR-positive cases . A strong positive association (P= 0.0001) was found between ARs and oestrogen receptors (ERs). In univariate analysis, ARs (as well as ERs and PGRs) were not correlated with overall survival; tumour histological grade (P= 0.02), size (P= 0.01), p53 expression (P= 0.0008) and MIB-1 scores (P= 0.0003) had strong prognostic value. In multivariate survival analysis, only p53 expression (P= 0.002) and histological grade (P= 0.02) retained independent prognostic significance. In conclusion, the lack of association between AR and most clinicopathological features and survival, together with the absence of prognostic value for ER/PGR status, suggest that MBCs are biologically different from female breast carcinomas and make it questionable to use antihormonal therapy for patients with MBC. © 1999 Cancer Research CampaignAndrogen receptor (AR) expression was retrospectively analysed in 47 primary male breast carcinomas (MBCs) using a monoclonal antibody on formalin-fixed, paraffin-embedded tissues. AR immunopositivity was detected in 16 out of 47 (34%) cases. No association was found with patient age, tumour stage, progesterone receptor (PGR) or p53 protein expression. Well-differentiated MBCs tended to be AR positive more often than poorly differentiated ones (


P for trend = 0.022, adjusted odds ratio 1.20, 95% confidence interval 1.07–1.33 respectively. No association was found between HHV-6 and ALL, CML or RAEB. © 1999 Cancer Research CampaignThe relationships between acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL), chronic myeloid leukaemia (CML) and refractory anaemia with excess of blasts (RAEB) and human herpes virus (HHV)-6 antibody level were investigated in a multicentre case-control study. An association between increased HHV-6 seropositivity and geometric mean titre ratio with AML was shown:


We have studied the cytotoxicity of bleomycin (4--10 u/kg/day for 6 days) given by continuous i.p. infusion (using an osmotic minipump) compared to daily i.p. bolus administration, against P388 leukaemic spleen colony-forming-units(LCFU-S). Continuous i.p. bleomycin at 8 u/kg/day caused a 0.5 log greater reduction of LCFU-S than did an identical dose given by intermittent bolus administration. The infusion minipump provided constant bleomycin plasma levels of 0.62 +/- 0.03 mu/ml and a total plasma AUC (area under the plasma decay curve) of 89.0 mu.h/ml for 6 days at 8 u/kg/day. Intermittent bolus bleomycin at 8 u.kg/day had a terminal-phase plasma t1/2 of 15 min and a total 6-day plasma AUC of 90.8mu.h/ml. These pharmacokinetic data validate the osmotic minipump as a constant drug-delivery system, and suggest that the two administration schedules resulted in equal total bleomycin dosages. Although high peak bleomycin plasma levels (i.e. 32 mu/ml) were achieved with the intermittent bolus administration, continuous-infusion bleomycin's greater inhibition of LCFU-S was probably related to the drug's schedule-dependent cell-killing characteristics. The results of this study provide further rationale for the continuing use of infusion bleomycin schedules in cancer patients.


P < 0.0001), 100 nM NPY (P < 0.0001), 100 nM VIP (P < 0.001), 100 nM CGRP (P < 0.001), 100 nM SP (P < 0.01), and 0.1 mM histamine (P < 0.01), whereas 0.1 mM 5-HT, mM acetylcholine, and 1 μM isoproterenol             (β-adrenergic agonist) had no effect. Proliferation(incorporation of tritiated thymidine) was increased by CGRP (P = 0.004) and histamine (P < 0.02), but decreased by isoproterenol (P = 0.002), 5-HT (P = 0.003), and acetylcholine (P < 0.05). The percentage of multinucleate cells was decreased after isoproterenol (2.5%), and increased after 5-HT (21.3%), GABA (15%), and histamine (15.1%). Compared to controls, thymulin in the supernatant was decreased after challenge with acetylcholine (52%), isoproterenol (71%), 5-HT (73%), and histamine (84%). This study demonstrates direct effectsof neuropeptides and neurotransmitters on functional aspects of cultured thymic epithelial cells.To determine if major thymic neuropeptides and neurotransmitters can directly influence the functional activity of cultured rat thymic epithelium, neuropeptides and neurotransmitters were applied, and intercellular communication, proliferation, and thymulin secretion assessed. After injections of a mixture of lucifer yellow dextran (too large to pass gap junctions) and cascade blue (which does) into single cells, some neuropeptides decrease dye coupling: 0.1 mM GABA (


The atomic arrangement consists of an infinite long-chain polyphosphate organization. Chains, with a period of four PO4 tetrahedra, run along the a-axis direction. Two other polymorphs of this phosphate are known, in space groups P21/n and C2/c.The title compound, potassium yttrium polyphosphate, KY(PO DOI: 10.1107/S1600536808013925/br2073Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The 4,6-dihydroxybenzene-1,3-disulfonate anion, acting as a counter-ion, is also located on the mirror plane. The crystal packing is stabilized by O—H⋯O hydrogen bonds, forming a three-dimensional supramolecular network.In the title compound, [Zn(CH DOI: 10.1107/S1600536810006525/hy2283Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Since 1997, the largest epidemic of highly pathogenic avian influenza (H5N1) ever recorded has caused 172 human and several billion bird deaths. Recently administered questionnaires determined that live poultry exposures have declined by ≈63% in Hong Kong since 2004 and that, in Vietnam, domestic backyard exposures to poultry are likely more important than retail exposures. MappingIn Hong Kong, random household telephone interviewing of 1 adult >17 years of age selected by Kisch grid (which randomizes selection of persons within households) was conducted from December 2005 through 2006 from a list of 5,000 numbers. Simultaneously in Vietnam, stratified cluster sampling was carried out throughout 2 districts in each of 5 northern provinces. Within 3 of these provinces, 1 district with and 1 without an HPAI epidemic history were selected. Within each district, 1 urban and 1 rural commune each provided 100 households randomly selected from electoral rolls. Kisch grid selected 1 adult from each household for face-to-face interviews.Respondents estimated their live poultry purchase frequency and touching at purchase ; 64% of respondents were women and 36% were men; their median age, 44 years . VietnamIn Hong Kong in 2006, 18,586 standardized purchases averaged 10.56 chickens/household/year . This is a territory-wide gender-adjusted rate of 11.05 chickens/household/year, which indicated that 22,673,000 live chickens were purchased during the preceding year, 41% fewer than in 2004. Households buying poultry bought an average of 15.6 chickens/household/year. Among respondents personally buying, 7.5% touched the poultry during purchasing (compared with 11% in 2004), giving ≈1,700,500 exposures/year. Adjustment for touching frequency and gender differences in reported touching  gave ≈1,110,900 contacts  for Hong Kong in 2006, or 0.76 exposures/buying household/year . Applied retrospectively to Hong Kong 2004 data, this gave an adjusted exposure rate of 8.6% (95% CL 6.8–10.3), ≈3,311,300 contacts, and exposure frequency of 2.07 exposures/buying household/year (0.62/person/year) in 2004. These adjusted estimates indicate an absolute exposure decline of 3.7% (95% CL 2.25%–4.91%), a relative decline of 43% between 2004 and 2006. Less purchasing and touching reduced annualized buying exposures by 63% overall.2 = 45.57, df = 4, p<0.001), after adjustment for gender proportion and reported touching, was 63% (62%–64%). Estimated exposures in the surveyed provinces from buying were ≈13,097,000 × 0.63 = ≈8,251,000 exposures/year. When these rates were used, national per capita exposure estimates (assuming 4.49 persons/household) from touching when buying are ≈62,479,000 exposures/year, 2.24 exposures/person/year in buying households, 0.76 exposures/person/year overall.In Vietnam, respondents reported 10,659 standardized purchases, averaging 5.36 chickens/household/year, giving a gender-adjusted  rate of 5.43 chickens/household/year. Estimated number of live birds purchased in the sampled provinces  was 13,097,000 chickens per year. Buying households  buy on average 15.97 chickens per year, comparable to the Hong Kong 2006 purchase rate. Touching frequency during purchasing  that raised poultry, 92 (5%) ceased keeping poultry from February 2005 through February 2006 . HousehoWhile 34% (32%–36%) of households buy live chickens, 53% (52%–54%)  raise live poultry at home, and 12% (10%–13%) do both. Assume a 53% national average and, conservatively, that all persons within households rearing backyard poultry have at least weekly physical contact with their birds, bird eggs, or feces. Household size in the surveyed districts averages 3.38 persons . Thus, 224,685,500 exposures/year would occur from backyard poultry in surveyed districts, an average exposure within backyard poultry raising households of ≈175 exposures/person/year. Households buying live poultry have 8,251,000 /820,080 = 10.1 exposures/household/year (2.99 exposures/person/year) from these purchases. Total purchase-related plus backyard exposure events then equal  + 224,685,500 = 232,968,300 exposures/year. Average household exposure is therefore ≈96 exposures/household/year (28 exposures/person/year) in sampled districts. If daily backyard exposure occurs, then there are ≈1,581,081,300 total exposures, ≈655 exposures/household/year (194 exposures/person/year). Nationally, average household size is 4.49 persons. Hence, between ≈2,322,546,000 (weekly contact) and ≈15,882,953,000 (daily contact) exposures/year, ≈127–869 exposures/household/year occur nationally. If multiple contacts occur daily, these figures would be much higher.Epidemic and nonepidemic district-buying frequency CL overlapped (exposure 3.4 [1.9–4.8] chickens/household/year vs. nonexposure 5.8 [4.5–7.0] chickens/household/year). Dual adjusted touching frequencies were 69% (62%–76%) in epidemic and 60% (57%–63%) in nonepidemic districts, respectively. Backyard poultry were more common in epidemic districts (71% [67%–75%] vs. 45% [42%–48%]), where keeping poultry declined 17% (14%–20%) compared with 8% (6%–10%) in nonepidemic districts. Epidemic and nonepidemic districts had comparable average incomes .In Hong Kong, government import restrictions have reduced poultry availability by 41% from 2004 to 2006. Purchase and touching declines prompted by health education messages have together reduced exposure by ≈60%.www.worldbank.org/data/quickreference/quickref.html), live chickens costs $16.6–$18.0 and $21.8–$31.0  each in Hong Kong and in Vietnam, respectively. Hence, temptation to use sick, dying, or dead poultry is high, increasing the risk for human influenza (H5N1) infection (Fewer Vietnamese households bought live chickens, but those that did so bought at comparable frequencies to Hong Kong 2006 households. Chickens are relatively more expensive in Vietnam. Adjusted for purchasing power parity (Limitations include generalizing from 5 northern Vietnamese provinces to the country as a whole and using arbitrary estimates for backyard exposure frequency. Nonetheless, valuable data are presented on differential exposure patterns.


Objective: Octreotide, a somatostatin analogue, hasbeen shown to prevent angiogenesis in diverse invitro models. We evaluated its effect on retinal neovascularizationin vivo, using a neonatal rat retinopathymodel.                    Methods: We used, on alternating days, hypoxia(10% O2) and hyperoxia (50% O2) during the first 14days of neonatal rats, to induce retinal neovascularization.Half of the rats were injected subcutaneouslywith octreotide 0.7 μg/g BW twice daily. Atday 18 the eyes were evaluated for the presence ofepiretinal and vitreal hemorrhage, neovascularizationand epiretinal proliferation. Octreotide pharmacokineticsand its effect on serum growth hormone(GH) and insulin-like growth factor I (IGF-I) wereexamined in 28 rats.                    Results: Serum octreotide levels were 667 μg/1 twohours after injection, 26.4 μg/1 after nine hours and3.2 μg/1 after 14 hours. GH levels were decreasedby 40% (p = 0.002) two hours after injection butthereafter returned to baseline. IGF-I levels were unchangedtwo hours after injection and were elevatedby 26% 14 hours after injection (p = 0.02). Epiretinalmembranes were highly associated with epiretinalhemorrhages (p < 0.001), while retinal neovascularizationwas notably associated with vitreal hemorrhages (p <  0.001).                    Conclusions: Twice-daily injections of octreotidefailed to produce sustained decrease in serum GH,but produced rebound elevation of serum IGF-I.Accordingly, no statistically significant effect of injectionson retinal pathology was noted. This finding,however, does not contradict our assumptionthat GH suppression may decrease the severity ofretinopathy.


The crystal structure contains six sites in the asymmetric unit: two sites are statistically occupied by rare-earth atoms with Gd:Sc ratios of 0.967 (4):0.033 (4) and 0.031 (3):0.969 (3), one site (.m. symmetry) is occupied by Sc atoms, and three distinct sites (two of which with .m. symmetry) are occupied by Ge atoms. The rare-earth atoms form two-dimensional slabs with Ge atoms occupying the trigonal-prismatic voids.Gd DOI: 10.1107/S1600536809007211/mg2062Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


We looked for associations between spontaneous apoptosis, p53 gene mutation, p53 protein accumulation, growth fraction, bcl-2 expression and histological parameters in 64 ovarian, four tubal and three peritoneal carcinomas. Apoptotic cells were detected with the TUNEL method. p53 gene variants were detected by the single-strand conformation polymorphism and were sequenced directly. P53, Ki-67 and bcl-2 protein expressions were detected immunohistochemically. A weighed multiple logistic regression model was applied. Apoptotic index (Al) ranged 0.02–0.18 (mean 0.11); proliferation index (PI) ranged 3–90% (mean 54%). p53 gene mutations were present in 51, p53 protein accumulation in 46, and diffuse bcl-2 expression in 29 of 71 tumours. The AI was positively associated with the presence of p53 gene mutation (P = 0.011). However, the PI included into the analysis did positively influence the AI (P = 0.02) and diminished the association with p53 gene mutation (P = 0.082). The AI was negatively associated with good histological differentiation (P = 0.0006), the serous tumour type (P = 0.002), and diffuse bcl-2 expression (P = 0.025). Strong bcl-2 expression was associated with endometrioid tumour type (P = 0.002). FIGO stage and p53 protein accumulation were the only parameters that influenced overall survival time. Thus, our results suggest that histological tumour type and grade are major determinants of spontaneous apoptosis in ovarian carcinomas;p53 alterations do not adversely but rather positively affect spontaneous apoptosis by increasing growth fraction. This, in turn, suggests p53-independency of spontaneous apoptosis in ovarian carcinomas. © 2000 Cancer Research CampaignChanges in cell survival contribute to tumour development, influence tumour biology and its response to chemotherapy.


Lactobacillus plantarum LQ80 is a strain isolated from liquid feed for pigs. We determined the complete genome sequence of this strain using the PacBio RS II platform. LQ80 contained a single circular chromosome of 3,230,192 bp, with 44.66% G+C content and seven plasmids. Lactobacillus plantarum is found in various environmental niches, e.g., vegetable pickles  (https://www.qiagen.com/us/resources/resourcedetail?id=8b1b2b4f-3dca-4447-8d21-f6fd64c3a729), and a 5-kb library was constructed with shearing. One SMRT cell was used for sequencing on the PacBio RS II platform with a 360-min movie time. The result of 362,945 reads with a mean length of 1,519 bp was assembled with HGAP3, and five circular contigs were obtained, representing five plasmids . From this analysis, another four smaller plasmids were constructed.Strain LQ80 was cultured with de Man-Rogosa-Sharpe medium at 37°C. For the genome sequencing of LQ80, DNA was isolated at the early log phase. DNA was purified using a PowerClean DNA cleanup kit , followed by 20-kb library construction for P6-C4 chemistry with shearing. Five single-molecule real-time (SMRT) cells were used for sequencing on the PacBio RS II platform  with a 180-min movie time. We obtained 320,152 reads with a mean length of 5,268 bp.  (HGAP3) . Four ciThe LQ80 chromosome was 3,230,192 bp in length, with a G+C content of 44.66%. In addition, LQ80 had seven plasmids: pLQ801 , pLQ802 , pLQ803 , pLQ804 , pLQ805 10,218 bp), pLQ806 , and pLQ807 . The complete genome was annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP)  and Micr bp, pLQ8L. plantarum LQ80 have been deposited in GenBank under accession numbers CP028977 (chromosome), CP028978 (pLQ801), CP028979 (pLQ802), CP028980 (pLQ803), CP028981 (pLQ804), CP028982 (pLQ805), CP028983 (pLQ806), and CP028984 (pLQ807).The complete genome sequences of the chromosome and seven plasmids of


Salmonella typhimurium (S. typhimurium) A1-R, a facultative anaerobe that is an auxotroph of leucine and arginine. The tumor-targeting efficacy of S. typhimurium A1-R was demonstrated in vivo and vitro using several malignant cell lines including melanoma, sarcoma, glioma, breast, pancreatic, colon, cervical, prostate, and ovarian cancers. Our laboratory also developed a patient-derived orthotopic xenograft (PDOX) model by implanting patient-derived malignant tumor fragments into orthotopic sites in mice. We reviewed studies of S. typhimurium A1-R against recalcitrant cancers. S. typhimurium A1-R was effective against all PDOX tumor models tested and showed stronger efficacies than chemotherapy or molecular-targeting therapy against some tumors. Furthermore, the synergistic efficacy of S. typhimurium A1-R when combined with chemotherapeutic agents, molecular-targeting agents, or recombinant methioninase was also demonstrated. We suggest potential clinical uses of this S. typhimurium A1-R treatment.We developed tumor-targeting  Streptococcus pyogenes (S. pyogenes). He then developed Coley’s toxin, a mixture of killed S. pyogenes and Serratia marcescens, achieving clinical responses for many malignant tumors [Dr. William B. Coley began bacterial therapy of cancer using t tumors .Live bacteria can actively penetrate tumors to reach lesions distant from blood vessels, where chemotherapeutic drugs cannot be delivered, and damage malignant cells by several cytotoxic mechanisms . ObligatClostridium. butyricum (C. butyricum) M-55 resulted in oncolysis and accumulation in treated tumors [C. novyi-NT induced intratumor infection and necrosis [Salmonella typhimurium (S. typhimurium) VNP20009 attenuated by msbB and purI mutations, showed bacterial colonization of treated melanomas [S. typhimurium showed bacterial colonization in treated tumors after local or systemic administration [In clinical studies, limited antitumor efficacy has been shown thus far. d tumors ,7. Intraelanomas ,9. ObjecS. typhimurium is a facultative anaerobe. Green fluorescence protein (GFP)-labeled S. typhimurium A1-R developed by our laboratory has high tumor-targeting efficacy, due to the leucine–arginine auxotroph, resulting in broad antitumor efficacy and limited adverse effects [S. typhimurium A1-R was quickly eliminated from normal organs including the liver and spleen seven days after intravenous administration [S. typhimurium A1-R remained at high density in CT26 tumors. In addition, tumors treated with S. typhimurium A1-R were significantly smaller than those treated with S. typhimurium VNP20009. The efficacy of S. typhimurium A1-R was demonstrated in orthotopic nude mouse models of prostate [S. typhimurium A1-R was also effective in metastatic cancer models [ effects . In a CTstration . In contprostate , breast prostate ,14, pancprostate ,16, and prostate , as wellprostate  and glioprostate ,20. S. tr models ,22.S. typhimurium A1-R against malignancies in patient-derived orthotopic xenograft (PDOX) nude mouse models, in which human tumors are orthotopically implanted in mice, to examine future clinical applicability.We review in the present report the therapeutic efficacy of We established PDOX tumors as follows: when the original tumors derived from primary sites, the PDOX tumors were implanted into the same primary sites in mice; when the original tumors derived from recurrent or metastatic sites, the PDOX tumors were implanted into original, recurrent, or metastatic sites in mice.S. typhimurium A1-R in PDOX models were identified. All of the 17 studies of human cancer of different histological types were evaluated and the efficacy of S. typhimurium A1-R was compared with the efficacy of chemotherapy  [S. typhimurium A1-R ranged from 5 × 105 colony-forming units (CFUs) to 1.5 × 108 CFUs. Intravenous injection twice weekly with a dose of 5 × 107 CFUs was used in most experiments. S. typhimurium A1-R treatment was performed either as a monotherapy or a polytherapy in combination with chemotherapeutic or molecular-targeting agents, or recombinant methioninase (rMETase), which reduces the plasma methionine on which cancer cells are addicted [s (i.v.) ,37,38,39s (i.v.) ,25, intrs (i.v.) ,28, or is (i.v.) . A singladdicted . PolytheS. typhimurium A1-R was evaluated via culture of resected specimens. A fluorescent microscope was used to detect GFP-expressing S. typhimurium A1-R in tumors grown in PDOX models treated with S. typhimurium A1-R i.t., i.p., i.v., or i.a. injection [S. typhimurium A1-R grown from a Ewing’s sarcoma PDOX tumor [S. typhimurium A1-R were present in STS PDOX tumors, including undifferentiated STS [S. typhimurium A1-R were not detectable in adjacent muscles, suggesting selective tumor-targeting efficacy [The tumor-targeting efficacy of njection ,34,37,38OX tumor . Abundanated STS , pleomorated STS , follicuated STS , melanomated STS ,29,30,34ated STS , a GIST ated STS , and a Cefficacy ,37. The treatment efficacy of S. typhimurium A1-R was confirmed in all PDOX models . SignifiS. typhimurium A1-R showed stronger antitumor efficacy than gemcitabine, cisplatinum, or fluorouracil treatment in the pancreatic PDOX model [S. typhimurium A1-R was more effective than cisplatinum [S. typhimurium A1-R resulted in greater tumor growth inhibition compared to doxorubicin treatment [S. typhimurium A1-R showed stronger efficacy than imatinib in the GIST PDOX model [OX model . In the platinum ,32. In treatment ,35,39. MOX model .S. typhimurium A1-R with chemotherapy [S. typhimurium A1-R had additional efficacy when combined with gemcitabine or gemcitabine plus bevacizumab [S. typhimurium A1-R with temozolomide or vemurafenib significantly reduced tumor growth compared to monotherapy with these agents [S. typhimurium A1-R, rMETase, and cisplatinum was more effective than double therapy using S. typhimurium A1-R with rMETase, or monotherapy of these agents [Synergistic treatment efficacy was observed with combination of otherapy ,36, moleotherapy ,26,29,30otherapy ,35. In pacizumab ,36. Addie agents .S. typhimurium A1-R-treated tumors showed extended necrosis compared to untreated tumors. As an example, S. typhimurium A1-R caused central tumor necrosis to a large extent in the Ewing’s sarcoma PDOX model, while the untreated tumors grew without necrosis (S. typhimurium A1-R treatment resulted in changes in sarcoma cell shape but not necrosis [S. typhimurium A1-R treatment in combination with cisplatinum and rMETase resulted in tumor necrosis. Moreover, S. typhimurium A1-R showed more extensive necrosis when combined with chemotherapy or molecular-targeting agents than S. typhimurium A1-R monotherapy on undifferentiated STS and melanoma PDOX models [S. typhimurium A1-R induced a higher degree of necrosis in several PDOX models including pancreatic cancer, STS, osteosarcoma, and GIST [S. typhimurium A1-R led to more extensive necrosis than intravenous administration in the osteosarcoma PDOX model [Established tumors in the PDOX model had a similar morphologic appearance to the original patient tumor A,B. S. tnecrosis C–G. In anecrosis . S. typhX models ,29,30. Wand GIST ,32,33,37OX model . These rS. typhimurium A1-R compared to the untreated control.None of the PDOX experiments showed adverse effects, in terms of significant weight loss, in mice treated with S. typhimurium A1-R against recalcitrant-caner PDOX models, indicating advantages that S. typhimurium A1-R may have over chemotherapy. Therefore, for rare malignancies or cancers of unknown primary origin, for which effective treatments have not been established, S. typhimurium A1-R treatment can be a good candidate. Moreover, for highly aggressive malignancies such as pancreatic cancer or melanomas, S. typhimurium A1-R was highly effective when combined with chemotherapy or molecular-targeting therapy. In addition, adverse effects were shown to be limited. Therefore, S. typhimurium A1-R treatment has clinical potential. PDOX models are theoretically better at mimicking the human disease than heterotopic tumors, increasing the robustness of drug discovery studies. The present review demonstrates the strong antitumor efficacy of S. typhimurium penetrated the cancer cells in vitro by being attracted to small molecules such as ribose and serine [S. typhimurium A1-R targeted tumors in several PDOX mouse models. The antitumor efficacy of S. typhimurium A1-R against many kinds of cancer cell lines was demonstrated and suggested that S. typhimurium A1-R kills cancer cells directly [S. typhimurium A1-R expanded and burst, resulting in loss of viability [S. typhimurium A1-R, a facultative anaerobe, can grow under anaerobic condition [S. typhimurium A1-R induces central tumor necrosis (Salmonella plays the role of inducing antitumor immune responses in an immunocompetent model [Salmonella enhances both innate and adaptive immunity. Salmonella induces cytokine production, including interferon-γ, via Toll-like receptor 4 signaling [S. typhimurium resulted in recruitment of CD8+ lymphocytes, CD4+ lymphocytes and B lymphocytes as well as macrophages and granulocytes in the tumor [S. typhimurium A1-R was correlated with CD8+ lymphocyte infiltration into treated tumors in a pancreatic cancer syngeneic immunocompetent mouse model [S. typhimurium A1-R acts as a decoy. It induces the cancer cells to leave the chemo-sensitive state of the cell cycle, making the cancer cells highly sensitive to chemotherapy [S. typhimurium A1-R. d serine ,42. The directly ,43,44. Uiability . Importaondition ,44. As anecrosis . In addint model . Salmoneignaling . Upregulignaling ,47. Avogse model . Moreoveotherapy . These fS. typhimurium A1-R against recalcitrant cancer PDOX models. Pre-clinical efficacy studies of S. typhimurium A1-R were completed and only a toxicity test needs to be performed to enable S. typhimurium A1-R to begin phase I clinical studies.The present review discusses the antitumor efficacy of


Siphoviridae bacteriophage, characterized by an unusual prolate capsid, containing a 72,658-base-pair double-stranded DNA genome with 132 predicted protein-coding genes. Conserved among cluster O bacteriophages, the Ryadel genome contains 31 copies of a unique 17-bp sequence with dyad symmetry.Mycobacteriophage Ryadel is a newly isolated cluster O  Siphoviridae bacteriophage, characterized by an unusual prolate capsid, containing a 72,658-base-pair double-stranded DNA genome with 132 predicted protein-coding genes. Conserved among cluster O bacteriophages, the Ryadel genome contains 31 copies of a unique 17-bp sequence with dyad symmetry.Mycobacteriophage Ryadel is a newly isolated cluster O  Mycobacterium smegmatis mc2155 at 37°C for 48 hours and resulted in small-sized lytic plaques. Ryadel was purified by collecting virus from well-isolated plaques from the direct isolation and two successive rounds of serial dilutions. Negative-staining transmission electron microscopy . Soil samples were placed in 7H9 liquid medium, and the supernatant was passed through a 0.22-µm filter for direct isolation of bacteriophages. Filtered supernatant was incubated with croscopy  of isolahttps://blast.ncbi.nlm.nih.gov/) whole-genome alignment (MG099943) and Catdawg (GenBank accession number KF017002)  (http://phagesdb.org/DNAMaster/), and PECAAN (https://pecaan.kbrinsgd.org/). Mycobacteriophage Ryadel was predicted to contain 132 protein-coding genes, and no tRNA genes were identified by ARAGORN v1.2.38  encode DNA polymerase III sliding clamp beta, Ku-like double-stranded DNA (dsDNA) break-binding protein, and ParB-like dsDNA partitioning protein.Glimmer v3.02 , 7 and Gor.org/) , DNA Mas v1.2.38  or tRNAs v1.2.38 . Putativ v1.2.38  and NCBI v1.2.38 . SimilarMH590592. Raw reads are available in the SRA under accession number SRX4721442.The mycobacteriophage Ryadel genome is available at GenBank under accession number


Few studies have compared the effectiveness of brief training courses on point-of-care ultrasound (POCUS) skill acquisition of novice attending physicians vs. trainees. The purpose of this study was to evaluate the change in POCUS image interpretation skills and confidence of novice attending physicians vs. trainees after a 1-day POCUS training course.A 1-day POCUS training course was held in March 2017 in Japan. A standardized training curriculum was developed that included online education, live lectures, and hands-on training. The pre-course assessment tools included a written examination to evaluate baseline knowledge and image interpretation skills, and a physician survey to assess confidence in performing specific ultrasound applications. The same assessment tools were administered post-course, along with a course evaluation. All learners were novices and were categorized as trainees or attending physicians. Data were analyzed using two-way analysis of variance.p < 0.001). The post-course physician confidence scores in using ultrasound significantly increased in all skill categories for both groups. Both trainees and attending physicians demonstrated similar improvement in their post-course test scores and confidence with no statistically significant differences between the groups. The course evaluation scores for overall satisfaction and satisfaction with faculty members’ teaching skills were 4.5 and 4.6 on a 5-point scale, respectively.In total, 60 learners attended the course, and 51 learners (85%) completed all tests and surveys. The 51 novice learners included 29 trainees  and 22 attending physicians . The mean pre- and post-course test scores of novice trainees improved from 65.5 to 83.9% while novice attending physicians improved from 66.7 to 81.5%  contains supplementary material, which is available to authorized users. Point-of-care ultrasound (POCUS) is defined as ultrasonography at the patient’s bedside that is performed in real-time by a physician caring for the patient . In contMultiple studies have demonstrated the effectiveness of POCUS training courses on knowledge and skills acquisition by different learner groups. These courses have varied in duration, content, delivery, and target audience , 8, 12. Even though POCUS ultrasound training courses have been proven to be effective, , 8, 12 tAs a preparatory step, a 2-day train-the-trainer course was conducted in November 2016 to standardize the educational curriculum for a future POCUS training course in March 2017. The train-the-trainer course curriculum was based on the principles shown to be effective for POCUS skills training –15. The The same twelve POCUS faculty that participated in the train-the-trainer course in November 2016 served as the faculty for a 1-day POCUS training course in March 2017 at the 14th Japanese Society of Hospital General Medicine Semi-Annual Meeting. The 1-day POCUS course was a separate session from the main conference and the enrollment cap was 60 participants. The 1-day POCUS course was geared toward novices learners and included pre-course internet-based modules, live lectures 3.5 h), and hands-on skills training with live models (1.5 h)  and included three focused cardiac ultrasound (FOCUS) sessions, and one session each for deep vein thrombosis, lung/diaphragm, and abdominal ultrasound. The faculty-to-learner ratio was 1:3 during these hands-on sessions. Learning objectives were displayed, and faculty used standardized printed materials as teaching aids at each station . We analyzed differences in examination and survey scores between the trainees and attending physicians. Data analyses were performed using R statistical software (R version 3.1.3.)In total, 60 learners attended the course, and 51 learners (85%) completed all tests and surveys. The 51 novice learners included 29 trainees  and 22 attending physicians  (Table p < 0.001). However, there was no statistically significant difference between the pre-course examination scores (p = 0.80) nor the post-course examination scores (p = 0.43) between trainees vs. attending physicians. Descriptive statistics and an ANOVA table of the examination scores for each application are shown in Additional file The mean pre- and post-course written examination scores for all learners were 66.0% (standard deviation [SD] 12.9) and 82.8% (SD 9.0), respectively. The mean pre-course examination scores of trainees vs. attending physicians were 65.5% (SD 13.0) and 66.7 (SD 13.0), respectively. The mean post-course examinations scores of trainees vs. attending physicians were 83.9% (SD 9.0) and 81.5% (SD 9.0), respectively. Post-course examination scores were significantly improved compared with pre-course examination scores in both groups (es p = 0.3 betweenp < 0.001). However, there were no significant differences in improvement of post-course confidence scores between the trainees vs. attending physicians in all categories . The pre- and post-course self-evaluation survey results are shown in Additional files The mean confidence scores for all learners in the pre-course vs. post-course self-evaluation surveys were: general ultrasound skills, 2.37 (SD 0.90) vs. 3.32 (SD 0.71); focused cardiac ultrasound, 2.56 (SD 0.84) vs. 3.60 (SD 0.71); vascular diagnostics, 1.94 (SD 0.90) vs. 3.55 (SD 0.70); lung/diaphragm ultrasound, 1.77 (SD 0.76) vs. 3.30 (SD 0.70); and abdominal ultrasound, 2.95 (SD 0.97) vs 3.81 (SD 0.75). Post-course confidence scores were significantly higher compared with pre-course scores in all categories for both groups Additional file 2:Pre- and post-course physicians survey. (DOCX 17 kb)Additional file 3:Post-course satisfaction surveys. (DOCX 15 kb)Additional file 4:Descriptive statistics and an ANOVA table of the test scores for pre- and post-course written examinations. (DOCX 19 kb)Additional file 5:Descriptive statistics for the pre- and post-course self-evaluation survey. (DOCX 19 kb)Additional file 6:Analysis of variance table for pre- and post-course self-evaluation survey. (DOCX 22 kb)


Using the Meikirch model as a theoretical underpinning, the present study aimed to examine population health disparities by discerning age variations within and across rural urban areas. Secondary data analysis was conducted using the CDC’s 2017 Behavioral Risk Factor Surveillance System. The study’s outcome variables included physical health and mental health burden . A total sample of 96,568 adults were included with a mean age of 66.05 years (SD = 9.91). Individuals were classified in the following age groups: 43% middle-aged (45-64 years), 33% young-old (65-74 years), and 25% old-old adults (75+ years). The sample was largely female (61%), Non-Hispanic White (86%), and urban (67%). A series of chi-square tests of independence – post hoc tests when applicable – were completed. Overall, rural residents reported a higher prevalence of severe physical and mental health symptom burden. Regarding physical health burden, a significant difference was found within urban settings (X2(4) = 50.74, p < .001), where, unexpectedly, young-old adults reported the best physical health. Regarding mental health burden, a significant difference was found for both urban (X2(4) = 1661.72, p < .001)) and rural settings (X2(4) = 820.65, p < .001), with middle-aged adults reporting greater mental illness and the old-old adults reporting greater mental health resiliency. Findings suggest that a multidimensional framework of health usefully informs public health and clinical service interventions, identifying populations and locations in need .


Bacillus sp. strain YSP-3 was isolated from a salt lake.The halophilic, alkaliphilic bacterium  Bacillus sp. strain YSP-3 was isolated from a salt lake. It grows optimally at 8% (wt/vol) NaCl (pH 9.0). The draft genome is composed of 4,006 predicted genes. Genomic analysis showed that various genes are potentially involved in the adaptation mechanisms for osmotic stress and pH homeostasis.The halophilic, alkaliphilic bacterium  Bacillus sp. strain YSP-3 was isolated from a salt lake in Jilin, China. In sodium bicarbonate-buffered medium (Bacillus aurantiacus K1-5T (97.9  %) and Bacillus populi FJAT-45347T (97.01%). A phylogenetic tree was reconstructed by a neighbor-joining (B. aurantiacus K1-5T and B. populi FJAT-45347T (data not shown), which revealed that YSP-3 belonged to the genus Bacillus. To understand the adaptive strategies for survival under hypersaline conditions, draft genome sequencing of Bacillus sp. YSP-3 was performed using an Illumina platform.The halophilic and alkaliphilic bacterium d medium , growth d medium . The resde novo assembled using MicrobeTrakr plus v. 0.9.1 (http://microbetracker.org/). Genome annotation was processed using NCBI PGAP (www.ncbi.nlm.nih.gov/genome/annotation_prok). The total length of the draft genome sequence was 4,047,843 bp and yielded 12 contigs, with a G+C content of 48.26%. Among the predicted 4,006 genes, 3,910 putative protein-coding genes (CDSs) were identified. Furthermore, 87 complete RNAs, including 10 rRNAs , 72 tRNAs, and 5 noncoding RNAs (ncRNAs) were found.Total genomic DNA (2 μg) was isolated using a DN12 microbial DNA isolation kit  according to the manufacturer’s instructions. A library for genome sequencing was constructed using the NEBNext Ultra DNA library prep kit for Illumina . The genBacillus sp. YSP-3 contains 1 gene cluster  for ectoine biosynthesis from aspartate semialdehyde, 1 glnA gene for l-glutamine biosynthesis from l-glutamate, 9 genes of glycine/betaine ABC transporter, 3 genes of Na+/solute symporter, 3 genes of Na+/alanine symporter, and 2 genes of Na+/proline symporter, indicating that Bacillus sp. YSP-3 may resist osmotic stress when salinity increases by taking up compatible solute  into the cell from the extracellular environment (+ uptake system also implies that strain YSP-3 possibly gains isosmotic cytoplasm though K+ as an osmolyte via a "salt-in strategy" when coping with a rapid osmotic shock (Bacillus sp. YSP-3 contains nine Na+/H+ antiporter genes (+ antiporter gene (+/H+ antiporter gene (Bacillus sp. YSP-3 survival in alkaline environments (+ homeostasis under salinity stress.Analysis of the genome sequence revealed that ironment , 7. The ic shock . Alkalipter gene , and a Kter gene , which mronments . These pBacillus sp. YSP-3 has been deposited at DDBJ/ENA/GenBank under the accession number PDOF00000000.The draft genome assembly of


Verticillium nonalfalfae, a soilborne vascular fungus, shows promise for biocontrol of highly invasive Ailanthus altissima strains. This announcement provides draft genome sequences of the aggressive isolate G1/5 (wild-type strain), the highly aggressive isolate Vert56 (improved strain), and the mildly aggressive isolate I3/2, all obtained from symptomatic A. altissima trees in Austria. Verticillium nonalfalfae, a soilborne vascular fungus, shows promise for biocontrol of highly invasive Ailanthus altissima strains. This announcement provides draft genome sequences of the aggressive isolate G1/5 (wild-type strain), the highly aggressive isolate Vert56 (improved strain), and the mildly aggressive isolate I3/2, all obtained from symptomatic A. altissima trees in Austria. Verticillium nonalfalfae is an important vascular wilt pathogen on several dicotyledonous crops as well as on tree of heaven   –3. This issima) ––7. For telection  and resuKT223526 and WGLJ00000000) (WGLK00000000), and another less pathogenic isolate, I3/2 (GenBank accession numbers KT223527 and SPUV00000000)  with Milli-Q water for elution. Whole-genome sequencing was performed by Vienna BioCenter Core Facilities GmbH  using an Illumina HiSeq 2500 system with a single-end 100-bp setup. Sequencing libraries were prepared according to the Westburg next-generation sequencing (NGS) DNA library prep kit protocol v3.1 . A total of 60.09 million (Vert56), 54.24 million (I3/2), and 57.87 million (G1/5) reads were received, representing in total 6,008.9 million, 5,423.5 million, and 5,786.7 million bases, respectively. Default parameters were used for all software, unless otherwise noted. A read quality check was performed using FastQC v0.11.4) (.4 (11) we strain , and theV. nonalfalfae strains Vert56, I3/2, and G1/5, respectively.Sequencing completeness was estimated using BUSCO (v3.0.2)  based onWGLJ00000000 , SPUV00000000 , and WGLK00000000 . The versions described in this paper are the first versions, WGLJ01000000, SPUV0100000000, and WGLK01000000, respectively. Sequence reads were deposited under SRA project accession numbers SRR8271219, SRR8271218, and SRR8271217, respectively, and BioProject accession number PRJNA507541.These whole-genome shotgun projects have been deposited at DDBJ/EMBL/GenBank under the accession numbers


Dictyostelium discoideum forms a fruiting body consisting of spores and a multicellular stalk. Originally, the chlorinated alkylphenone differentiation-inducing factors (DIFs) -1 and -3 were isolated as stalk cell inducers in D. discoideum. Later, DIFs and their derivatives were shown to possess several biologic activities including antitumor and anti-Trypanosoma properties. In this study, we examined the antibacterial activities of approximately 30 DIF derivatives by using several bacterial species. Several of the DIF derivatives strongly suppressed the growth of the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis and Enterococcus faecium, at minimum inhibitory concentrations (MICs) in the sub-micromolar to low-micromolar range. In contrast, none of the DIF derivatives evaluated had any noteworthy effect on the growth of the Gram-negative bacterium Escherichia coli . Most importantly, several of the DIF derivatives strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant E. faecalis and E. faecium. Transmission electron microscopy revealed that treatment with DIF derivatives led to the formation of distinct multilayered structures consisting of cell wall or plasma membrane in S. aureus. The present results suggest that DIF derivatives are good lead compounds for developing novel antimicrobials.At the end of its life cycle, the cellular slime mold  Dictyostelium discoideum has long been studied as an excellent model system in the fields of cell and developmental biology; at the end of its developmental process, this organism forms fruiting bodies, each consisting of spores and a multicellular stalk [D. discoideum [D. discoideum [The cellular slime mold ar stalk ,2,3. Difar stalk ,5 and moscoideum . DIF-3  [Trypanosoma drugs [In addition to these developmental roles, DIF-1, DIF-3, and several of their derivatives B,C exertomiasis) . Importaomiasis) ,26,27,28ma drugs . MoreoveStaphylococcus aureus, Bacillus subtilis, Enterococcus faecalis and, Enterococcus faecium. In addition, several of the DIF derivatives evaluated strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant enterococci . Our results support the investigation of such DIF derivatives as candidate lead compounds for developing novel antimicrobials.In this study, we investigated the antibacterial activities of DIF derivatives in vitro and show that several of them exert strong antibacterial effects against Gram-positive bacteria, including S. aureus , methicillin-resistant S. aureus , vancomycin-susceptible E. faecalis , vancomycin-resistant E. faecalis , vancomycin-resistant E. faecium , and B. subtilis (ATCC6633) and the Gram-negative bacteria Escherichia coli (NIHJ) and Mycobacterium bovis  were used in this study. DIF derivatives (The Gram-positive bacteria methicillin-susceptible ivatives  were synS. aureus suspension (109 CFU/mL) in Mueller–Hinton broth (MHB) (0.2% (w/v) beef extract, 1.75% (w/v) casein hydrolysate, 0.15% (w/v) starch) into 10 mL of MHB agar (MHB containing 1.7% (w/v) agar; final bacterial density, 107 CFU/mL) and spread this suspension in a plastic culture dish . Paper discs  impregnated with 2 µL of EtOH, DMSO, a 10 mM solution of the DIF or DIF derivative of interest in EtOH or DMSO (approx. 5.5–8 µg DIF/disc), or the purchased penicillin–streptomycin solution (10 units penicillin and 10 µg streptomycin/disc) were placed on the bacterial agar plate. In addition, paper discs  containing minomycin (30 µg/disc), imipenem (10 µg/disc), gentamicin (10 µg/disc), levofloxacin (5 µg/disc), or clindamycin (2 µg/disc) were placed on a MHB bacterial agar plate as controls. The plates were incubated for 20 h at 37 °C, after which the bacterial growth-inhibition zones around each disc was noted.We mixed 0.1 mL of S. aureus in MHB (107 CFU/mL) was spread on an MHB agar plate , after which 2 µL of each 10 mM DIF solution was dropped directly on the agar surface. The plates were incubated for 20 h at 37 °C, after which the bacterial growth-inhibition zones around the discs were noted.In addition, a diffusion assay without paper discs was performed for comparison. Briefly, 10 mL of 5 CFU/mL; 0.1 mL/well) were incubated for 20 to 24 h at 37 °C in 96-well plates  in the presence of vehicle, various concentrations of serially diluted DIF derivatives, or known antibiotics; MIC was defined as the lowest concentration of the additives that inhibited visible bacterial growth.Gram-positive and Gram-negative bacteria in MHB (5 × 10M. bovis was grown in Hayflick modified PPLO (pleuropneumonia-like organisms) broth (0.5% (w/v) glucose, 5% (w/v) beef heart infusion, 2.5% (w/v) yeast extract, 1% (w/v) peptone, 0.5% (w/v) NaCl, 15% (v/v) heat-inactivated horse serum, 50 µg/mL of ampicillin) and used at concentrations of 104 to 107 CFU/mL for the minimum inhibitory concentration (MIC) assay as described; tiamulin  and enrofloxacin (Sigma-Aldrich) were used as positive-control antibiotics.S. aureus (strain ATCC29213) in MHB  was incubated for 1.5 h at 37 °C in 6-well plates (Corning) in the presence of 0.2% DMSO (vehicle) or 4 µM DIF derivative. After incubation, the bacteria were transferred to centrifuge tubes, collected by centrifugation , fixed overnight at 4 °C in 0.5 mL of 2.5% (v/v) glutaraldehyde in 100 mM phosphate buffer (pH 7.4) , and postfixed in 1% (w/v) OsO4 in 100 mM phosphate buffer. Fixed specimens were dehydrated through a graded series of ethanol and embedded in Epon812 . Ultrathin sections were cut by using an ultramicrotome  and stained with uranyl acetate and lead citrate. These sections were examined under a transmission electron microscope .Specimens for transmission electron microscopy were prepared according to standard procedures . BrieflyS. aureus (MSSA: strain 209P) (S. aureus (MRSA: strain MS29202)  from those of the known antibiotics that we used.We first examined the effects of DIF-1, DIF-3 A, and thin 209P) A and metMS29202) B by usinMS29202) A, a largMS29202) B, peniciMS29202) C. These B. subtilis (ATCC6633) and on the Gram-negative bacterium E. coli (NIHJ), and MIC values of DIF derivatives were determined   and enrofloxacin (0.7 µM).We next examined the effects of DIF derivatives on the growth of the Gram-positive bacteria MSSA (strain 209P), MRSA (MS29202), and termined . Several>100 µM) ; M. boviNote that the MIC values of DIF-3 against MSSA and MRSA were comparable to those of DIF-1 , even thvanB and vanA, respectively . Transmission electron microscopy revealed distinct multilayered structures consisting of cell wall and/or plasma membrane in the DIF-treated MSSA cells . This fiD. purpureum [S. aureus (strain IID671), MRSA (IID1677), E. faecalis (ATCC29212), and B. subtilis (ATCC6633) are 1.56 µg/mL (3.4 µM), 3.13 µg/mL (6.8 µM), 50 µg/mL (109 µM), and 0.78 µg/mL (1.7 µM), respectively [Polysphondylium filamentosum [The antibacterial substance AB0022A, a chlorinated dibenzofuran A, was isurpureum  and is sectively , and thumentosum , but theWhen we assessed the MIC values of these dichlorinated dibenzofurans in MRSA (strain MS29202), neither AB0022A nor Pf-2 had any noteworthy inhibitory effect  on the growth of this strain B. In conPenicillium notatum have been isolated from fungi and Actinobacteria [Most of the many antibiotics  identified since the discovery of penicillin in the fungus bacteria ,34,35,36bacteria ,35,36,37D. discoideum has long been used as a model organism in the study of cell and developmental biology. However, slime molds have recently been identified as excellent sources of potential lead compounds for drug discovery and the development of novel medicines [D. discoideum-derived DIFs and their derivative compounds in various types of eukaryotic cells [The cellular slime mold edicines ,38. In tic cells ,16,29.D. discoideum DIF derivatives possess antibacterial activity against Gram-positive bacteria, including S. aureus, E. faecalis, E. faecium, and B. subtilis. In contrast, these DIF derivatives did not inhibit the growth of Mycoplasma or the Gram-negative bacterium E. coli  and E. faecium (containing vanA) were resistant to both vancomycin and ampicillin (E. faecalis and E. faecium cause the great majority of enterococcal infections, and isolates that carry drug-resistance genes such as vanA or vanB can cause serious infections [In the present study, we showed that several  E. coli . Most im E. coli . Note th E. coli B but als E. coli C, wherea E. coli . In addipicillin . Althougfections ,40. OverAlthough the antibacterial activities of the DIF derivatives varied among the Gram-positive strains of bacteria examined in this study , the strS. aureus, Ph-DIF-1, Ph-DIF-3, and Bu-DIF-3 induce the formation of distinct multilayered structures composed of cell wall or membrane (Regarding the mechanism through which DIF derivatives exert their antibacterial effects, DIF derivatives have mitochondrial uncoupling activity in mammalian cells ,18. Givemembrane . AlthougD. discoideum possess strong antimicrobial activities against Gram-positive bacteria including MRSAs and VREs. Our results suggest that the DIF derivatives are good lead compounds for developing novel antimicrobials.In this study, we showed that several derivatives of DIF-1 and DIF-3, chlorinated polyketides found in The following authors hold a patent related to this article:Kubohara, Y.; Kikuchi, H.; Oshima, Y. Antibacterial drugs. Japanese Patent No. 6478378, 15 February 2019.