Prevotella ihumii sp. nov., a new bacterium isolated from a stool specimen of a healthy woman.

Prevotella ihumii strain Marseille-P3385T (= CSURP3385T; = DSM106428T) is a new species isolated from a fresh stool specimen of a healthy woman.

Noncontiguous finished genome sequences and description of Bacteroides mediterraneensis sp. nov., Bacteroides ihuae sp. nov., Bacteroides togonis sp. nov., Bacteroides ndongoniae sp. nov., Bacteroides ilei sp. nov. and Bacteroides congonensis sp. nov. identified by culturomics.

Culturomics is a concept developing different culture conditions in order to enlarge our knowledge of the human microbiota through the discovery of previously uncultured bacteria. This enabled us to isolate six new species of the Bacteroides genus: Bacteroides mediterraneensis strain Marseille-P2644, Bacteroides ihuae strain Marseille-P2824, Bacteroides togonis strain Marseille-P3166, Bacteroides ndongoniae strain Marseille-P3108, Bacteroides ilei strain Marseille-P3208 and Bacteroides congonensis strain Marseille-P3132. Those bacteria are Gram-negative anaerobic bacilli. We describe here their phenotypic features, together with phylogenetic analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectrum, fatty acid composition, and genome sequencing and annotation.

Noncontiguous finished genome sequences and descriptions of Actinomyces ihuae, Actinomyces bouchesdurhonensis, Actinomyces urinae, Actinomyces marseillensis, Actinomyces mediterranea and Actinomyces oralis sp. nov. identified by culturomics.

The taxonogenomic approach, including the culturomics techniques, is now currently used to isolate and characterize new bacteria. These approaches notably allowed us to discover six new species of the Actinomyces genus: Actinomyces ihuae strain SD1, Actinomyces bouchesdurhonensis strain Marseille-P2825, Actinomyces urinae strain Marseille-P2225, Actinomyces marseillensis strain Marseille-P2818, Actinomyces mediterranea strain Marseille-P3257 and Actinomyces oralis strain Marseille-P3109. Each is the type strain of the corresponding bacterial species. 16S ribosomal RNA gene sequence comparison was used to classify these strains among the Actinomyces genus. These strains are all Gram positive, rod shaped and facultative aerobic. We describe the main characteristics of each bacterium and present their complete genome sequence and annotation.

Genome sequence and description of Paenibacillus ihuae strain GD6 sp. nov., isolated from the stool of a 62-year-old Frenchman.

Paenibacillus ihuae strain GD6 (=CSUR P892 = DSMZ 45751T) is the new type strain collected from the stool of a 69-year-old Frenchman admitted to an intensive care unit and receiving a 10-day course of imipenem at the time of stool collection. This is a Gram-positive, facultative anaerobic, rod-shaped bacterium. We describe here the features of this organism, together with its complete genome sequence and annotation. The genome size is 6 719 043 bp with 49.6% G+C content and contains 6211 protein-coding and 65 sRNA genes, including four 5S rRNA genes, one 16S rRNA gene and one 23S rRNA gene.

Taxonogenomic description of four new Clostridium species isolated from human gut: 'Clostridium amazonitimonense', 'Clostridium merdae', 'Clostridium massilidielmoense' and 'Clostridium nigeriense'.

Culturomics investigates microbial diversity of the human microbiome by combining diversified culture conditions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene identification. The present study allowed identification of four putative new Clostridium sensu stricto species: 'Clostridium amazonitimonense' strain LF2T, 'Clostridium massilidielmoense' strain MT26T, 'Clostridium nigeriense' strain Marseille-P2414T and 'Clostridium merdae' strain Marseille-P2953T, which we describe using the concept of taxonogenomics. We describe the main characteristics of each bacterium and present their complete genome sequence and annotation.

Noncontiguous finished genome sequences and description of Bacillus massiliglaciei, Bacillus mediterraneensis, Bacillus massilinigeriensis, Bacillus phocaeensis and Bacillus tuaregi, five new species identified by culturomics.

Microbial culturomics, which investigates microbial diversity by combining diversified culture conditions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rDNA identification, allowed to identify five new species within the Bacillus genus. Bacillus massiliglaciei strain Marseille-P2600T, Bacillus mediterraneensis strain Marseille-P2384T, Bacillus massilinigeriensis strain Marseille-P2366T, Bacillus tuaregi strain Marseille-P2489T and Bacillus phocaeensis strain SIT16T are each the type strain of the corresponding bacterial species. These strains, the genomes of which are described here, are facultative anaerobic Gram-positive bacilli. Here, we describe the main characteristics of each bacterium and present their complete genome sequence and annotation.

'Actinomyces provencensis' sp. nov., 'Corynebacterium bouchesdurhonense' sp. nov., 'Corynebacterium provencense' sp. nov. and 'Xanthomonas massiliensis' sp. nov., 4 new species isolated from fresh stools of obese French patients.

We report the main characteristics of 'Actinomyces provencensis' strain SN12T sp. nov., 'Corynebacterium bouchesdurhonense' strain SN14T sp. nov., 'Corynebacterium provencense' strain SN15T sp. nov. and 'Xanthomonas massiliensis' strain SN8T sp. nov., which were all isolated from stool samples from obese French patients.

'Pseudoflavonifractor phocaeensis' gen. nov., sp. nov., isolated from human left colon.

We describe here characteristics of 'Pseudoflavonifractor phocaeensis' strain Marseille-P3064T, which was isolated from a human left colon wash sample.

"Negativibacillus massiliensis" gen. nov., sp. nov., isolated from human left colon.

We report here the main characteristics of "Negativibacillus massiliensis" strain Marseille-P3213T, isolated from a human left-colon wash sample.

Noncontiguous finished genome sequence and description of Fusobacterium massiliense sp. nov. isolated from human duodenum.

The strain Marseille-P2749T (= CSUR P2749 = DSM 103085) was isolated as part of culturomics study from a liquid duodenum sample from a French man. Bacterial cells were Gram-negative bacilli, fusiform shaped and non-spore forming, and they grew in microaerophilic and anaerobic atmosphere. Its genome is 1 809 169 bp long and contains 1646 protein-coding genes. The DNA G+C content was 27.33 mol%. This strain exhibited a 95.9% sequence similarity with Fusobacterium periodonticum, the phylogenetically closest species with standing in nomenclature. Strain Marseille-P2749T is suggested to be a novel species belonging to the genus Fusobacterium, for which the name Fusobacterium massiliense sp. nov. is proposed.

Paenibacillus phocaensis sp. nov., isolated from the gut microbiota of a healthy infant.

Paenibacillus phocaensis sp. nov. strain mt24T (= CSUR P2238 = DSM 101777) is a Gram-negative, facultative anaerobic, spore-forming and motile bacilli. This strain was isolated from the stool sample of a healthy infant from Niger. Its genome was estimated to a size of 5 521 415 bp with a 53.54% GC content. It contains 4835 protein-coding genes and 89 RNAs, among which two were 16S rRNA genes. There were also 101 genes (2.09%) identified as ORFans.

Butyricimonas phoceensis sp. nov., a new anaerobic species isolated from the human gut microbiota of a French morbidly obese patient.

Butyricimonas phoceensis strain AT9 (= CSUR 1981 = DSM 100664) was isolated from a stool sample from a morbidly obese French patient living in Marseille using the culturomics approach. The genome of this Gram-negative-staining, anaerobic and non-spore forming rod bacillus is 4 736 949 bp long and contains 3947 protein-coding genes. Genomic analysis identified 173 genes as ORFans (4.5%) and 1650 orthologous proteins (42%) not shared with the closest phylogenetic species, Butyricimonas virosa. Its major fatty acid was the branched acid iso-C15:0 (62.3%).

Noncontiguous finished genome sequence and description of Murdochiella massiliensis strain SIT12 sp. nov.

Murdochiella massiliensis strain SIT12 (= CSUR P1987 = DSM 29078) is the type strain of M. massiliensis sp. nov. This bacterium was isolated from the stool of a healthy 2-year-old Senegalese boy. M. massiliensis is an anaerobic, Gram-positive coccus. The genome size of M. massiliensis strain SIT12 is 1 642 295 bp with 48.9% G+C content and assembled into two scaffolds.

OGX-427 inhibits tumor progression and enhances gemcitabine chemotherapy in pancreatic cancer.

Despite many advances in oncology, almost all patients with pancreatic cancer (PC) die of the disease. Molecularly targeted agents are offering hope for their potential role in helping translate the improved activity of combination chemotherapy into improved survival. Heat shock protein 27 (Hsp27) is a chaperone implicated in several pathological processes such as cancer. Further, Hsp27 expression becomes highly upregulated in cancer cells after chemotherapy. Recently, a modified antisense oligonucleotide that is complementary to Hsp27 (OGX-427) has been developed, which inhibits Hsp27 expression and enhances drug efficacy in cancer xenograft models. Phase II clinical trials using OGX-427 in different cancers like breast, ovarian, bladder, prostate and lung are in progress in the United States and Canada. In this study, we demonstrate using TMA of 181 patients that Hsp27 expression and phosphorylation levels increase in moderately differentiated tumors to become uniformly highly expressed in metastatic samples. Using MiaPaCa-2 cells grown both in vitro and xenografted in mice, we demonstrate that OGX-427 inhibits proliferation, induces apoptosis and also enhances gemcitabine chemosensitivity via a mechanism involving the eukaryotic translation initiation factor 4E. Collectively, these findings suggest that the combination of Hsp27 knockdown with OGX-427 and chemotherapeutic agents such as gemcitabine can be a novel strategy to inhibit the progression of pancreas cancer.

Heat shock protein 27 confers resistance to androgen ablation and chemotherapy in prostate cancer cells through eIF4E.

One strategy to improve therapies in advanced prostate cancer (PC) involves targeting genes that are activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. Heat shock protein 27 (Hsp27) expression becomes highly upregulated in PC cells after androgen withdrawal or chemotherapy, in which it functions as a cytoprotective chaperone to confer broad-spectrum treatment resistance. The purpose of this study is to elucidate anti-apoptotic pathways regulated by Hsp27 that are activated during PC progression. Using two-hybrid experiment, we found that Hsp27 was having a major role in the protein translational initiation process. Furthermore, using complementary DNA (cDNA) microarray analysis, 4E binding protein 1 was identified as being proportionately and highly regulated by Hsp27. These data led us to analyze the protein synthesis initiation pathway, which is a prerequisite for cell growth and proliferation. Using northern and western blot analysis, we found that Hsp27 downregulation decreased eukaryotic translation initiation factor 4E (eIF4E) expression at the protein, but not mRNA, level. The cytoprotection afforded by Hsp27 overexpression was attenuated by eIF4E knockdown using specific eIF4E short interfering RNA (siRNA). Co-immunoprecipitation and co-immunofluorescence confirmed that Hsp27 colocalizes and interacts directly with eIF4E. Hsp27-eIF4E interaction decreases eIF4E ubiquitination and proteasomal degradation. By chaperoning eIF4E, Hsp27 seems to protect the protein synthesis initiation process to enhance cell survival during cell stress induced by castration or chemotherapy. Forced overexpression of eIF4E induces resistance to androgen-withdrawal and paclitaxel treatment in the prostate LNCaP cells in vitro. These findings identify Hsp27 as a modulator of eIF4E and establish a potential mechanism for the eIF4E-regulated apoptosis after androgen ablation and chemotherapy. Targeting Hsp27-eIF4E interaction may serve as a therapeutic target in advanced PC.

Oligonucleotide microarray analysis of estrogen receptor alpha-positive postmenopausal breast carcinomas: identification of HRPAP20 and TIMELESS as outstanding candidate markers to predict the response to tamoxifen.

The estrogen receptor alpha (ER alpha) status of breast tumors is used to identify patients who may respond to endocrine agents such as tamoxifen. However, ER alpha status alone is not perfectly predictive, and there is a pressing need for more reliable markers of endocrine responsiveness. In this aim, we used a two-step strategy. We first screened genes of interest by a pangenomic 44 K oligonucleotide microarray in a series of ten ER alpha-positive tumors from five tamoxifen-treated postmenopausal patients who relapsed (distant metastasis) and five tamoxifen-treated postmenopausal patients who did not relapse, matched with respect to age, Scarff-Bloom-Richardson grade, lymph node status, and macroscopic tumor size. Genes of interest (n=24) were then investigated in an independent well-characterized series of ER alpha-positive unilateral invasive primary breast tumors from postmenopausal women who received tamoxifen alone as adjuvant hormone therapy after primary surgery. We identified four genes (HRPAP20, TIMELESS, PTPLB, and MGC29814) for which high mRNA levels were significantly associated with shorter relapse-free survival (log-rank test). We also showed that hormone-regulated proliferation-associated 20 kDa protein (HRPAP20) and TIMELESS are 17beta-estradiol-regulated in vitro and are ectopically expressed in OH-Tam-resistant cell lines. In conclusion, these findings point to HRPAP20 and TIMELESS as promising markers of tamoxifen resistance in women with ER alpha-positive breast tumors.

High-resolution oligonucleotide array-CGH applied to the detection and characterization of large rearrangements in the hereditary breast cancer gene BRCA1.

We have developed a new method for detecting and characterizing large rearrangements in the BRCA1 gene based on high-resolution oligonucleotide array-CGH technology. We designed a specific CGH array for the BRCA1 gene and its flanking regions. We then used this approach to analyze nine DNA samples known to contain large deletions and large duplications. When possible, the deleted or duplicated region was sequenced to identify the break point. All the large rearrangements were detected by the new method, and their size was estimated to be within 1--2 kb. This enabled us to develop a simple polymerase chain reaction screening test for other family members. A refined choice of oligonucleotides should improve the precision of the breakpoint determination. Finally, the high resolution of oligonucleotide array-CGH should help to detect new large rearrangements missed by other current methods.

Identification of novel genes that co-cluster with estrogen receptor alpha in breast tumor biopsy specimens, using a large-scale real-time reverse transcription-PCR approach.

The estrogen receptor alpha (ERalpha) plays a critical role in the pathogenesis and clinical behavior of breast cancer. To obtain further insights into the molecular basis of estrogen-dependent forms of this malignancy, we used real-time quantitative reverse transcription (RT)-PCR to compare the mRNA expression of 560 selected genes in ERalpha-positive and ERalpha-negative breast tumors. Fifty-one (9.1%) of the 560 genes were significantly upregulated in ERalpha-positive breast tumors compared with ERalpha-negative breast tumors. In addition to well-known ERalpha-induced genes (PGR, TFF1/PS2, BCL2, ERBB4, CCND1, etc.) and genes recently identified by cDNA microarray-based approaches (GATA3, TFF3, MYB, STC2, HPN/HEPSIN, FOXA1, XBP1, SLC39A6/LIV-1, etc.), an appreciable number of novel genes were identified, many of, which were weakly expressed. This validates the use of large-scale real-time RT-PCR as a method complementary to cDNA microarrays for molecular tumor profiling. Most of the new genes identified here encoded secreted proteins (SEMA3B and CLU), growth factors (BDNF, FGF2 and EGF), growth factor receptors (IL6ST, PTPRT, RET, VEGFR1 and FGFR2) or metabolic enzymes (CYP2B6, CA12, ACADSB, NAT1, LRBA, SLC7A2 and SULT2B1). Importantly, we also identified a large number of genes encoding proteins with either pro-apoptotic (PUMA, NOXA and TATP73) or anti-apoptotic properties (BCL2, DNTP73 and TRAILR3). Surprisingly, only a small proportion of the 51 genes identified in breast tumor biopsy specimens were confirmed to be ERalpha-regulated and/or E2-regulated in vitro (cultured cell lines). Therefore, this study identified a limited number of genes and signaling pathways, which better delineate the role of ERalpha in breast cancer. Some of the genes identified here could be useful for diagnosis or for predicting endocrine responsiveness, and could form the basis for novel therapeutic strategies.

Delivery of antisense oligonucleotide to the cornea by iontophoresis.

We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the design of specific antiangiogenic strategy in diseases of the cornea.

The CGA gene as new predictor of the response to endocrine therapy in ER alpha-positive postmenopausal breast cancer patients.

We recently identified CGA (coding for the alpha subunit of glycoprotein hormones) as a new estrogen receptor alpha (ER alpha)-responsive gene in human breast tumors. Here, we assessed the relationship between CGA status (as determined by real-time quantitative RT-PCR) and the response to tamoxifen therapy in a well-defined cohort of 125 ER alpha-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. CGA overexpression, observed in 37.6% of patients, was associated with good relapse-free survival (P=0.037; univariate analysis). CGA status, combined with ERBB2 status (a marker of poor outcome), was an independent predictor of the response to tamoxifen (P=0.020; multivariate analysis). CGA status, especially when combined with ERBB2 status, may thus provide useful predictive information on tamoxifen responsiveness in breast cancer.