Surgery Is an Effective Option after Failure of Chemoradiation in Cancers of the Anal Canal and Anal Margin.

BACKGROUND: Surgery for anal canal cancer (ACC) and anal margin cancer (AMC) is the only curative option after failure of chemoradiotherapy (CRT). This study aimed to determine the efficacy of surgery for ACC or AMC after failed CRT. METHODS: This was a single-centre, retrospective study of 161 patients initially treated with CRT. We compared the survival rates of patients successfully treated by CRT with those of patients whose CRT failed (both surgically salvaged and treated palliatively). RESULTS: Thirty-one patients underwent surgery with curative intent, 20 received palliative treatment after failure of CRT, and 110 had effective CRT. The 5-year overall survival (OS) rate was significantly higher among patients with successful CRT than among patients who underwent surgery with curative intent (86 vs. 66%, p < 0.001). On the other hand, the 5-year OS of patients treated with curative surgery was significantly better than that of patients who underwent palliative treatment (66 vs. 13.5%, p < 0.001). The postoperative morbidity and mortality rates were 32 and 3%, respectively. Considering patients with failed CRT, curative surgery was the only factor prognostic of favourable OS in the multivariate analysis. CONCLUSION: Curative surgery after failure of CRT for ACC or AMC remains an effective treatment to improve survival in two-thirds of cases, resulting in high but manageable morbidity.

Mitochondrial genome evolution in yeasts: an all-encompassing view.

Mitochondria are important organelles that harbor their own genomes encoding a key set of proteins that ensure respiration and provide the eukaryotic cell with energy. Recent advances in high-throughput sequencing technologies present a unique opportunity to explore mitochondrial (mt) genome evolution. The Saccharomycotina yeasts have proven to be the leading organisms for mt comparative and population genomics. In fact, the explosion of complete yeast mt genome sequences has allowed for a broader view of the mt diversity across this incredibly diverse subphylum, both within and between closely related species. Here, we summarize the present state of yeast mitogenomics, including the currently available data and what it reveals concerning the diversity of content, organization, structure and evolution of mt genomes.

Lachancea quebecensis sp. nov., a yeast species consistently isolated from tree bark in the Canadian province of Québec.

A thorough sampling of maple, oak, birch, and apple tree bark in North America yielded a set of isolates that represent a yeast species not yet formally described. The strains obtained were all isolated from the Canadian province of Québec. These four isolates have identical electrophoretic karyotypes, distinct from other species of the genus Lachancea, and are most closely related to the formally recognized species Lachancea thermotolerans according to the D1/D2 domain of the LSU rDNA gene and 5.8S­ITS region. Previous studies revealed the existence of a population of strains closely related to L. thermotolerans, with unique D1/D2 sequences and the ability to grow on melibiose, which is also true for these isolates. The sequences obtained here (for the D1/D2, and 5.8S­ITS region) are identical among the four strains, and in a phylogenetic analysis of the D1/D2 region, the strains form a distinct clade with the previously described population closely related to L. thermotolerans, composed of isolates from Japan, as well as from the provinces of Ontario and Québec in Canada. On the basis of select physiological and phylogenetic characteristics, a novel ascosporogenous yeast species, Lachancea quebecensis sp. nov., is proposed. The type strain LL11_022T ( = CBS 14138T = CLIB 1763T = UCDFST 15-106T) was isolated from maple tree bark in the Station Duchesnay, QC region of Québec, Canada. The MycoBank number is MB811749.

Multilocus sequence typing reveals evidence of homologous recombination linked to antibiotic resistance in the genus Salinispora.

The three closely related species that currently comprise the genus Salinispora were analyzed using a multilocus sequence typing approach targeting 48 strains derived from four geographic locations. Phylogenetic congruence and a well-supported concatenated tree provide strong support for the delineation of the three species as currently described and the basal relationship of Salinispora arenicola to the more recently diverged sister taxa S. tropica and S. pacifica. The phylogeny of the initial region of the rpoB gene sequenced was atypical, placing the related genera Micromonospora and Verrucosispora within the Salinispora clade. This phylogenetic incongruence was subsequently ascribed to a homologous-recombination event in a portion of the gene associated with resistance to compounds in the rifamycin class, which target RpoB. All S. arenicola strains produced compounds in this class and possessed resistance-conferring amino acid changes in RpoB. The phylogeny of a region of the rpoB gene that is not associated with rifamycin resistance was congruent with the other housekeeping genes. The link between antibiotic resistance and homologous recombination suggests that incongruent phylogenies provide opportunities to identify the molecular targets of secondary metabolites, an observation with potential relevance for drug discovery efforts. Low ratios of interspecies recombination to mutation, even among cooccurring strains, coupled with high levels of within-species recombination suggest that the three species have been described in accordance with natural barriers to recombination.

Marine Actinobacteria from the Gulf of California: diversity, abundance and secondary metabolite biosynthetic potential.

The Gulf of California is a coastal marine ecosystem characterized as having abundant biological resources and a high level of endemism. In this work we report the isolation and characterization of Actinobacteria from different sites in the western Gulf of California. We collected 126 sediment samples and isolated on average 3.1-38.3 Actinobacterial strains from each sample. Phylogenetic analysis of 136 strains identified them as members of the genera Actinomadura, Micromonospora, Nocardiopsis, Nonomuraea, Saccharomonospora, Salinispora, Streptomyces and Verrucosispora. These strains were grouped into 26-56 operational taxonomic units (OTUs) based on 16S rRNA gene sequence identities of 98-100 %. At 98 % sequence identity, three OTUs appear to represent new taxa while nine (35 %) have only been reported from marine environments. Sixty-three strains required seawater for growth. These fell into two OTUs at the 98 % identity level and include one that failed to produce aerial hyphae and was only distantly related (≤95.5 % 16S identity) to any previously cultured Streptomyces sp. Phylogenetic analyses of ketosynthase domains associated with polyketide synthase genes revealed sequences that ranged from 55 to 99 % nucleotide identity to experimentally characterized biosynthetic pathways suggesting that some may be associated with the production of new secondary metabolites. These results indicate that marine sediments from the Gulf of California harbor diverse Actinobacterial taxa with the potential to produce new secondary metabolites.

Salinispora pacifica sp. nov., an actinomycete from marine sediments.

A polyphasic analysis was carried out to clarify the taxonomic status of four marine actinomycete strains that share a phylogenetic relationship and phenotypic characteristics with the genus Salinispora. These strains formed a distinct lineage within the Salinispora 16S rRNA and gyrB trees and were found to possess a range of phenotypic properties and DNA:DNA hybridization values that distinguished them from the type strains of the two validly named species in this genus, Salinispora tropica (CNB-440(T), ATCC BAA-916(T)) and Salinispora arenicola (CNH-643(T), ATCC BAA-917(T)). The combined genotypic and phenotypic data support this conclusion. It is proposed that the strains be designated as Salinispora pacifica sp. nov., the type strain of which is CNR-114(T) (DSMZ YYYYT = KACC 17160(T)).

Microdiversity and evidence for high dispersal rates in the marine actinomycete 'Salinispora pacifica'.

In July of 2006 and January of 2008, a total of 671 marine sediment samples were collected at depths from 5 to 2012 m throughout the Fijian islands and selectively processed for the cultivation of marine actinomycetes belonging to the genus Salinispora. The primary objectives were to assess the diversity, distribution and phylogeny of 'S. pacifica', the least well studied of the three species in the genus. Employing a sequential screening method based on antibiotic sensitivity, RFLP patterns, and 16S rRNA and ITS sequence analyses, 42 of 750 isolates with Salinispora-like features were identified as 'S. pacifica'. These strains represent the first report of 'S. pacifica' from Fiji and include 15 representatives of 4 new 'S. pacifica' 16S rRNA sequence types. Among the 'S. pacifica' strains isolated, little evidence for geographical isolation emerged based on 16S, ITS or secondary metabolite biosynthetic gene fingerprinting. The inclusion of isolates from additional collection sites and other Salinispora spp. revealed a high degree of dispersal among 'S. pacifica' populations and phylogenetic support for the delineation of this lineage as a third species.

Evolution of secondary metabolite genes in three closely related marine actinomycete species.

The marine actinomycete genus Salinispora is composed of three closely related species. These bacteria are a rich source of secondary metabolites, which are produced in species-specific patterns. This study examines the distribution and phylogenetic relationships of genes involved in the biosynthesis of secondary metabolites in the salinosporamide and staurosporine classes, which have been reported for S. tropica and S. arenicola, respectively. The focus is on "Salinispora pacifica," the most recently discovered and phylogenetically diverse member of the genus. Of 61 S. pacifica strains examined, 15 tested positive for a ketosynthase (KS) domain linked to the biosynthesis of salinosporamide K, a new compound in the salinosporamide series. Compound production was confirmed in two strains, and the domain phylogeny supports vertical inheritance from a common ancestor shared with S. tropica, which produces related compounds in the salinosporamide series. There was no evidence for interspecies recombination among salA KS sequences, providing further support for the geographic isolation of these two salinosporamide-producing lineages. In addition, staurosporine production is reported for the first time for S. pacifica, with 24 of 61 strains testing positive for staD, a key gene involved in the biosynthesis of this compound. High levels of recombination were observed between staD alleles in S. pacifica and the cooccurring yet more distantly related S. arenicola, which produces a similar series of staurosporines. The distributions and phylogenies of the biosynthetic genes examined provide insight into the complex processes driving the evolution of secondary metabolism among closely related bacterial species.

Spatial and temporal dynamics of SAR11 marine bacteria across a nearshore to offshore transect in the tropical Pacific Ocean.

Surveys of microbial communities across transitions coupled with contextual measures of the environment provide a useful approach to dissect the factors determining distributions of microorganisms across ecological niches. Here, monthly time-series samples of surface seawater along a transect spanning the nearshore coastal environment within Kane'ohe Bay on the island of O'ahu, Hawai'i, and the adjacent offshore environment were collected to investigate the diversity and abundance of SAR11 marine bacteria (order Pelagibacterales) over a 2-year time period. Using 16S ribosomal RNA gene amplicon sequencing, the spatiotemporal distributions of major SAR11 subclades and exact amplicon sequence variants (ASVs) were evaluated. Seven of eight SAR11 subclades detected in this study showed distinct subclade distributions across the coastal to offshore environments. The SAR11 community was dominated by seven (of 106 total) SAR11 ASVs that made up an average of 77% of total SAR11. These seven ASVs spanned five different SAR11 subclades (Ia, Ib, IIa, IV, and Va), and were recovered from all samples collected from either the coastal environment, the offshore, or both. SAR11 ASVs were more often restricted spatially to coastal or offshore environments (64 of 106 ASVs) than they were shared among coastal, transition, and offshore environments (39 of 106 ASVs). Overall, offshore SAR11 communities contained a higher diversity of SAR11 ASVs than their nearshore counterparts, with the highest diversity within the little-studied subclade IIa. This study reveals ecological differentiation of SAR11 marine bacteria across a short physiochemical gradient, further increasing our understanding of how SAR11 genetic diversity partitions into distinct ecological units.

Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater.

While marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems. Here, we sought a means to enhance culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. Using a raw seawater sample collected from the tropical Pacific Ocean, a subsample was diluted in seawater growth medium to create 576 2-ml dilution cultures containing 5 cells each and incubated for a high-throughput culturing (HTC) experiment, while another portion was cryopreserved in 10% glycerol. After 10 months, a cryopreserved aliquot was thawed and used to create a second cultivation experiment of 480 2-ml cultures containing 5 cells each and 470 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from the raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (92%) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater environmental sample were isolated by both approaches.IMPORTANCE High-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing them in the presence of cryoprotectants, such as glycerol, has been shown to be an effective method of storing viable cells over long time periods (i.e., years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as an inoculum for high-throughput cultivation experiments. We found that SAR11 and other abundant marine bacteria could be isolated from seawater that was previously cryopreserved for nearly 10 months at a rate of culturability similar to that of the same seawater used fresh, immediately after collection. Our findings (i) expand the potential of high-throughput cultivation experiments to include testing when immediate isolation experiments are impractical, (ii) allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and (iii) enable cultivation experiments across a wide range of other conditions that would benefit from having source inocula available over extended periods of time.

High Complexity and Degree of Genetic Variation in Brettanomyces bruxellensis Population.

Genome-wide characterization of genetic variants of a large population of individuals within the same species is essential to have a deeper insight into its evolutionary history as well as the genotype-phenotype relationship. Population genomic surveys have been performed in multiple yeast species, including the two model organisms, Saccharomyces cerevisiae and Schizosaccharomyces pombe. In this context, we sought to characterize at the population level the Brettanomyces bruxellensis yeast species, which is a major cause of wine spoilage and can contribute to the specific flavor profile of some Belgium beers. We have completely sequenced the genome of 53 B. bruxellensis strains isolated worldwide. The annotation of the reference genome allowed us to define the gene content of this species. As previously suggested, our genomic data clearly highlighted that genetic diversity variation is related to ploidy level, which is variable in the B. bruxellensis species. Genomes are punctuated by multiple loss-of-heterozygosity regions, whereas aneuploidies as well as segmental duplications are uncommon. Interestingly, triploid genomes are more prone to gene copy number variation than diploids. Finally, the pangenome of the species was reconstructed and was found to be small with few accessory genes compared with S. cerevisiae. The pangenome is composed of 5,409 ORFs (open reading frames) among which 5,106 core ORFs and 303 ORFs that are variable within the population. All these results highlight the different trajectories of species evolution and consequently the interest of establishing population genomic surveys in more populations.

Effect of arsenite and growth in biofilm conditions on the evolution of Thiomonas sp. CB2.

Thiomonas bacteria are ubiquitous at acid mine drainage sites and play key roles in the remediation of water at these locations by oxidizing arsenite to arsenate, favouring the sorption of arsenic by iron oxides and their coprecipitation. Understanding the adaptive capacities of these bacteria is crucial to revealing how they persist and remain active in such extreme conditions. Interestingly, it was previously observed that after exposure to arsenite, when grown in a biofilm, some strains of Thiomonas bacteria develop variants that are more resistant to arsenic. Here, we identified the mechanisms involved in the emergence of such variants in biofilms. We found that the percentage of variants generated increased in the presence of high concentrations of arsenite (5.33 mM), especially in the detached cells after growth under biofilm-forming conditions. Analysis of gene expression in the parent strain CB2 revealed that genes involved in DNA repair were upregulated in the conditions where variants were observed. Finally, we assessed the phenotypes and genomes of the subsequent variants generated to evaluate the number of mutations compared to the parent strain. We determined that multiple point mutations accumulated after exposure to arsenite when cells were grown under biofilm conditions. Some of these mutations were found in what is referred to as ICE19, a genomic island (GI) carrying arsenic-resistance genes, also harbouring characteristics of an integrative and conjugative element (ICE). The mutations likely favoured the excision and duplication of this GI. This research aids in understanding how Thiomonas bacteria adapt to highly toxic environments, and, more generally, provides a window to bacterial genome evolution in extreme environments.

Arenamides A-C, cytotoxic NFkappaB inhibitors from the marine actinomycete Salinispora arenicola.

Three new cyclohexadepsipeptides, arenamides A-C (1-3), were isolated from the fermentation broth of a marine bacterial strain identified as Salinispora arenicola. The planar structures of these compounds were assigned by detailed interpretation of NMR and MS/MS spectroscopic data. The absolute configurations of the amino acids, and those of the chiral centers on the side chain, were established by application of the Marfey and modified Mosher methods. The effect of arenamides A and B on NFkappaB activity was studied with stably transfected 293/NFkappaB-Luc human embryonic kidney cells induced by treatment with tumor necrosis factor (TNF). Arenamides A (1) and B (2) blocked TNF-induced activation in a dose- and time-dependent manner with IC(50) values of 3.7 and 1.7 microM, respectively. In addition, the compounds inhibited nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production with lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Moderate cytotoxicity was observed with the human colon carcinoma cell line HCT-116, but no cytotoxic effect was noted with cultured RAW cells. Taken together, these data suggest that the chemoprevention and anti-inflammatory characteristics of arenamides A and B warrant further investigation.

Correction to: Adaptation in toxic environments: comparative genomics of loci carrying antibiotic resistance genes derived from acid mine drainage waters.

The original version of this article unfortunately contains a mistake.

Adaptation in toxic environments: comparative genomics of loci carrying antibiotic resistance genes derived from acid mine drainage waters.

Several studies have suggested the existence of a close relationship between antibiotic-resistant phenotypes and resistance to other toxic compounds such as heavy metals, which involve co-resistance or cross-resistance mechanisms. A metagenomic library was previously constructed in Escherichia coli with DNA extracted from the bacterial community inhabiting an acid mine drainage (AMD) site highly contaminated with heavy metals. Here, we conducted a search for genes involved in antibiotic resistance using this previously constructed library. In particular, resistance to antibiotics was observed among five clones carrying four different loci originating from CARN5 and CARN2, two genomes reconstructed from the metagenomic data. Among the three CARN2 loci, two carry genes homologous to those previously proposed to be involved in antibiotic resistance. The third CARN2 locus carries a gene encoding a membrane transporter with an unknown function and was found to confer bacterial resistance to rifampicin, gentamycin, and kanamycin. The genome of Thiomonas delicata DSM 16361 and Thiomonas sp. X19 were sequenced in this study. Homologs of genes carried on these three CARN2 loci were found in these genomes, two of these loci were found in genomic islands. Together, these findings confirm that AMD environments contaminated with several toxic metals also constitute habitats for bacteria that function as reservoirs for antibiotic resistance genes.

Whole-Genome Sequencing and Intraspecific Analysis of the Yeast Species Lachancea quebecensis.

The gold standard in yeast population genomics has been the model organism Saccharomyces cerevisiae. However, the exploration of yeast species outside the Saccharomyces genus is essential to broaden the understanding of genome evolution. Here, we report the analyses of whole-genome sequences of nineisolates from the recently described yeast species Lachancea quebecensis. The genome of one isolate was assembled and annotated, and the intraspecific variability within L. quebecensis was surveyed by comparing the sequences from the eight other isolates to this reference sequence. Our study revealed that these strains harbor genomes with an average nucleotide diversity of π = 2 × 10(-3) which is slightly lower, although on the same order of magnitude, as that previously determined for S. cerevisiae (π = 4 × 10(-3)). Our results show that even though these isolates were all obtained from a relatively isolated geographic location, the same ecological source, and represent a smaller sample size than is available for S. cerevisiae, the levels of divergence are similar to those observed in this model species. This divergence is essentially linked to the presence of two distinct clusters delineated according to geographic location. However, even with relatively similar ranges of genome divergence, L. quebecensis has an extremely low global phenotypic variance of 0.062 compared with 0.59 previously determined in S. cerevisiae.

Treatment that follows guidelines closely dramatically improves overall survival of patients with anal canal and margin cancers.

BACKGROUND: To assess relevance of ESMO-ESSO-ESTRO treatment guidelines in a retrospective analysis of patients with anal canal or anal margin cancers. MATERIAL AND METHODS: 155 patients were separated into standard treatment group (STG), treated according to or closely the guidelines, and an altered treatment group (ATG). RESULTS: The median follow-up time was 50.7 months. In the STG, the 5- and 10-year LR-DFS rates were 75.2% and 72.7%; in the ATG, they were 66.8% and 61.2%, respectively. In the STG, the 5- and 10-year OS rates were 81.8% and 68%; in the ATG, they were 63.3% and 49.5%, respectively (p=0.037). In the multivariate analysis, favorable prognostic factors for OS included the standard treatment, age <60, tumor 50.4Gy. CONCLUSION: This study identifies the superiority of treatment according to standard guidelines compared to altered treatment. Our results corroborate the guidelines.

Draft Genome Sequence of Lachancea lanzarotensis CBS 12615T, an Ascomycetous Yeast Isolated from Grapes.

We report the genome sequencing of the yeast Lachancea lanzarotensis CBS 12615(T). The assembly comprises 24 scaffolds, for a total size of 11.46 Mbp. The annotation revealed 5,058 putative protein-coding genes. Detection of seven centromeres supports a chromosome fusion, which occurred after divergence from Lachancea thermotolerans and Lachancea kluyveri.

Adaptation in Toxic Environments: Arsenic Genomic Islands in the Bacterial Genus Thiomonas.

Acid mine drainage (AMD) is a highly toxic environment for most living organisms due to the presence of many lethal elements including arsenic (As). Thiomonas (Tm.) bacteria are found ubiquitously in AMD and can withstand these extreme conditions, in part because they are able to oxidize arsenite. In order to further improve our knowledge concerning the adaptive capacities of these bacteria, we sequenced and assembled the genome of six isolates derived from the Carnoulès AMD, and compared them to the genomes of Tm. arsenitoxydans 3As (isolated from the same site) and Tm. intermedia K12 (isolated from a sewage pipe). A detailed analysis of the Tm. sp. CB2 genome revealed various rearrangements had occurred in comparison to what was observed in 3As and K12 and over 20 genomic islands (GEIs) were found in each of these three genomes. We performed a detailed comparison of the two arsenic-related islands found in CB2, carrying the genes required for arsenite oxidation and As resistance, with those found in K12, 3As, and five other Thiomonas strains also isolated from Carnoulès (CB1, CB3, CB6, ACO3 and ACO7). Our results suggest that these arsenic-related islands have evolved differentially in these closely related Thiomonas strains, leading to divergent capacities to survive in As rich environments.

Population genomic analysis reveals highly conserved mitochondrial genomes in the yeast species Lachancea thermotolerans.

The increasing availability of mitochondrial (mt) sequence data from various yeasts provides a tool to study genomic evolution within and between different species. While the genomes from a range of lineages are available, there is a lack of information concerning intraspecific mtDNA diversity. Here, we analyzed the mt genomes of 50 strains from Lachancea thermotolerans, a protoploid yeast species that has been isolated from several locations (Europe, Asia, Australia, South Africa, and North / South America) and ecological sources (fruit, tree exudate, plant material, and grape and agave fermentations). Protein-coding genes from the mtDNA were used to construct a phylogeny, which reflected a similar, yet less resolved topology than the phylogenetic tree of 50 nuclear genes. In comparison to its sister species Lachancea kluyveri, L. thermotolerans has a smaller mt genome. This is due to shorter intergenic regions and fewer introns, of which the latter are only found in COX1. We revealed that L. kluyveri and L. thermotolerans share similar levels of intraspecific divergence concerning the nuclear genomes. However, L. thermotolerans has a more highly conserved mt genome with the coding regions characterized by low rates of nonsynonymous substitution. Thus, in the mt genomes of L. thermotolerans, stronger purifying selection and lower mutation rates potentially shape genome diversity in contract to what was found for L. kluyveri, demonstrating that the factors driving mt genome evolution are different even between closely related species.