Impact of Tumor-Infiltrating Lymphocytes on Disease Progression in Human Papillomavirus-Related Oropharyngeal Carcinoma.

OBJECTIVE: We aim to explore the prognostic value of tumor-infiltrating lymphocytes (TILs) in the primary tumor and metastatic lymph nodes of patients with HPV(+)OPSCC. We hypothesize that TILS density at both sites is associated with disease-free survival in HPV(+)OPSCC. STUDY DESIGN: Matched case-control study among HPV(+)OPSCC patients who underwent intent-to-cure surgery. Cases developed locoregional or distant recurrence. Controls were matched based on age, sex, pathologic T, N, and overall stage, year of surgery, type of adjuvant treatment received, and the Adult Comorbidity Evaluation-27 (ACE-27) score. SETTING: Single tertiary care center, May 2007 to December 2016. METHODS: Tumoral TILs (tTILs) density was defined as % TILs; stromal TILs (sTILs) density was defined as absent/sparse or moderate/dense crowding. Associations between TILs and time to disease progression were assessed using Cox regression models. RESULTS: Forty-four case-control pairs (N = 88) were included: 42 (48%) AJCC pStage I, 39 (44%) pStage II, and 7 (8%) pStage III. tTILs density ≥10% (hazard ratio [HR] 0.41, 95% confidence interval [CI] 0.17-0.99, p = .048) and a moderate/dense sTILs density (HR 0.21, 95% CI 0.06-0.75, p = .016) in the primary tumor were significantly associated with decreased risk of progression. TILs density in the lymph node was associated with decreased risk of progression but did not reach statistical significance. The tTILs and sTILs density correlated strongly between the primary tumor and lymph node. Concordance between the pathologists' was moderate (60%-70%). CONCLUSIONS: In HPV(+)OPSCC, a higher density of tumoral and stromal TILs in the primary tumor and possibly the lymph node may predict a lower risk of disease progression.

Using fisher-contributed secondary fins to fill critical shark-fisheries data gaps.

Developing-world shark fisheries are typically not assessed or actively managed for sustainability; one fundamental obstacle is the lack of species and size-composition catch data. We tested and implemented a new and potentially widely applicable approach for collecting these data: mandatory submission of low-value secondary fins (anal fins) from landed sharks by fishers and use of the fins to reconstruct catch species and size. Visual and low-cost genetic identification were used to determine species composition, and linear regression was applied to total length and anal fin base length for catch-size reconstruction. We tested the feasibility of this approach in Belize, first in a local proof-of-concept study and then scaling it up to the national level for the 2017-2018 shark-fishing season (1,786 fins analyzed). Sixteen species occurred in this fishery. The most common were the Caribbean reef (Carcharhinus perezi), blacktip (C. limbatus), sharpnose (Atlantic [Rhizoprionodon terraenovae] and Caribbean [R. porosus] considered as a group), and bonnethead (Sphyrna cf. tiburo). Sharpnose and bonnethead sharks were landed primarily above size at maturity, whereas Caribbean reef and blacktip sharks were primarily landed below size at maturity. Our approach proved effective in obtaining critical data for managing the shark fishery, and we suggest the tools developed as part of this program could be exported to other nations in this region and applied almost immediately if there were means to communicate with fishers and incentivize them to provide anal fins. Outside the tropical Western Atlantic, we recommend further investigation of the feasibility of sampling of secondary fins, including considerations of time, effort, and cost of species identification from these fins, what secondary fin type to use, and the means with which to communicate with fishers and incentivize participation. This program could be a model for collecting urgently needed data for developing-world shark fisheries globally. Article impact statement: Shark fins collected from fishers yield data critical to shark fisheries management in developing nations.

Uso de Aletas Secundarias Proporcionadas por Pescadores para Llenar Vacíos Importantes de Información sobre las Pesquerías de Tiburones Resumen Con frecuencia no se evalúan las pesquerías de tiburones del mundo en desarrollo ni cuentan con un manejo activo de sustentabilidad. Uno de los principales obstáculos para esto es la falta de información sobre las especies y la composición de los tamaños en las capturas. Probamos e implementamos una estrategia nueva y potencialmente aplicable en todas partes para la recolección de estos datos: la entrega obligatoria de las aletas secundarias de bajo valor económico (aletas anales) obtenidas de los tiburones desembarcados por parte de los pescadores y el uso de estas aletas para reconstruir las especies y tamaños en la captura. Usamos identificaciones genéticas de bajo costo e identificaciones visuales para determinar la composición de las especies y aplicamos una regresión lineal a la longitud total y a la de la base de la aleta anal para la reconstrucción del tamaño en captura. Probamos la viabilidad de esta estrategia en Belice, primero en un estudio de prueba de concepto y después subiendo al nivel nacional para la temporada de pesca de tiburón 2017-2018 (1,786 aletas analizadas). Se registraron 16 especies en esta pesquería. Las más comunes fueron Carcharhinus perezi, C. limbatus, Rhizoprionodon terraenovae y R. porosus (consideradas como un grupo) y Sphyrna cf. tiburo. Las últimas tres especies fueron desembarcadas principalmente por encima del tamaño maduro, mientras que con las dos primeras especies lo hacían por debajo del tamaño maduro. Nuestra estrategia demostró ser efectiva en la obtención de información crítica para el manejo de la pesquería de tiburones y sugerimos que las herramientas desarrolladas como parte de este programa puedan ser exportadas a otras naciones en esta región y aplicadas casi de manera inmediata si existen los medios para comunicarse con los pescadores e incentivarlos a proporcionar las aletas anales. Fuera del Atlántico Occidental tropical, recomendamos una mayor investigación de la viabilidad del muestreo de aletas secundarias, incluyendo la consideración del tiempo, esfuerzo y costo de la identificación de especies a partir de estas aletas; cuál tipo de aleta secundaria utilizar; y los medios mediante los cuales comunicarse con los pescadores e incentivarlos a participar. Este programa podría ser un modelo para la recolección de información de necesidad urgente para las pesquerías del mundo en desarrollo.

Caldicellulosiruptor saccharolyticus transcriptomes reveal consequences of chemical pretreatment and genetic modification of lignocellulose.

Recalcitrance of plant biomass is a major barrier for commercially feasible cellulosic biofuel production. Chemical and enzymatic assays have been developed to measure recalcitrance and carbohydrate composition; however, none of these assays can directly report which polysaccharides a candidate microbe will sense during growth on these substrates. Here, we propose using the transcriptomic response of the plant biomass-deconstructing microbe, Caldicellulosiruptor saccharolyticus, as a direct measure of how suitable a sample of plant biomass may be for fermentation based on the bioavailability of polysaccharides. Key genes were identified using the global gene response of the microbe to model plant polysaccharides and various types of unpretreated, chemically pretreated and genetically modified plant biomass. While the majority of C. saccharolyticus genes responding were similar between plant biomasses; subtle differences were discernable, most importantly between chemically pretreated or genetically modified biomass that both exhibit similar levels of solubilization by the microbe. Furthermore, the results here present a new paradigm for assessing plant-microbe interactions that can be deployed as a biological assay to report on the complexity and recalcitrance of plant biomass.

Survival and activity of individual bioaugmentation strains.

Successful application of bioaugmentation for enhanced degradation of environmental pollutants is often limited by the lack of methods to monitor the survival and activity of individual bioaugmentation strains. However, recent advancements in sequencing technologies and molecular techniques now allow us to address these limitations. Here a complementing set of general applicable molecular methods are presented that provides detailed information on the performance of individual bioaugmentation strains under in situ conditions. The approach involves genome sequencing to establish highly specific qPCR and RT-qPCR tools for cell enumerations and expression of involved genes, stable isotope probing to follow growth on the target compounds and GFP-tagging to visualize the bioaugmentation strains directly in samples, all in combination with removal studies of the target compounds. The concept of the approach is demonstrated through a case study involving degradation of aromatic hydrocarbons in activated sludge augmented with the bioaugmentation strain Pseudomonas monteilii SB3078.

A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus, lacking flagella, has unusual growth physiology.

A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.

Complete Genome Sequences of Pseudomonas monteilii SB3078 and SB3101, Two Benzene-, Toluene-, and Ethylbenzene-Degrading Bacteria Used for Bioaugmentation.

Pseudomonas monteilii SB3078 and SB3101 are benzene-, toluene-, and ethylbenzene-degrading strains used for bioaugmentation in relation to treatment of wastewater contaminated with petrochemical hydrocarbons. Complete genome sequencing of the bioaugmentation strains confirms that they are very closely related (100.0% average nucleotide identity). Both strains contain extensive integration of phage elements, with the main difference being insertion of additional phage elements in the SB3078 genome.

Complete Genome of Rhodococcus pyridinivorans SB3094, a Methyl-Ethyl-Ketone-Degrading Bacterium Used for Bioaugmentation.

Here, we present the complete genome of Rhodococcus pyridinivorans SB3094, a methyl-ethyl-ketone (MEK)-degrading strain used for bioaugmentation relating to the treatment of wastewater contamination with petrochemical hydrocarbons. The genome highlights important features for bioaugmentation, including the genes involved in the degradation of MEK.

Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725.

Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of ∼ 80 °C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments.

Phylogenetic, microbiological, and glycoside hydrolase diversities within the extremely thermophilic, plant biomass-degrading genus Caldicellulosiruptor.

Phylogenetic, microbiological, and comparative genomic analyses were used to examine the diversity among members of the genus Caldicellulosiruptor, with an eye toward the capacity of these extremely thermophilic bacteria to degrade the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii, C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA gene phylogeny and cross-species DNA-DNA hybridization to a whole-genome C. saccharolyticus oligonucleotide microarray, revealing that C. saccharolyticus was the most divergent within this group. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid-pretreated switchgrass, only C. saccharolyticus, C. bescii, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman no. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that the cellulolytic species also had diverse secretome fingerprints. The C. saccharolyticus secretome contained a prominent S-layer protein that appears in the cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interactions. Growth physiology also correlated with glycoside hydrolase (GH) and carbohydrate-binding module (CBM) inventories for the seven bacteria, as deduced from draft genome sequence information. These inventories indicated that the absence of a single GH and CBM family was responsible for diminished cellulolytic capacity. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and this argues for continued efforts to isolate new members from high-temperature terrestrial biotopes.

Part I: characterization of the extracellular proteome of the extreme thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS2.

The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. In the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS(2) analysis and prediction servers suggested probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments, all including the C(18) bead technique, demonstrated that two levels of sample purification were necessary to effectively desalt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described, as enzymatic and putative uncharacterized proteins, anticipate consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion, and 11 contained transmembrane domain stretches suggested by Phobius and transmembrane hidden Markov model. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study, involved acetone precipitation followed by the C(18) bead method in which 2.4% (63 proteins) of the predicted proteome was identified, including proteins suggested to have secretion and transmembrane moieties.

Part II: defining and quantifying individual and co-cultured intracellular proteomes of two thermophilic microorganisms by GeLC-MS2 and spectral counting.

Probing the intracellular proteome of Thermotoga maritima and Caldicellulosiruptor saccharolyticus in pure and co-culture affords a global investigation into the machinery and mechanisms enduring inside the bacterial thermophilic cell at the time of harvest. The second of a two part study, employing GeLC-MS(2) a variety of proteins were confidently identified with <1% false discovery rate, and spectral counts for label-free relative quantification afforded indication of the dynamic proteome as a function of environmental stimuli. Almost 25% of the T. maritima proteome and 10% of the C. saccharolyticus proteome were identified. Through comparison of growth temperatures for T. maritima, a protein associated with chemotaxis was uniquely present in the sample cultivated at the non-optimal growth temperature. It is suspected that movement was induced due to the non-optimal condition as the organism may need to migrate in the culture to locate more nutrients. The inventory of C. saccharolyticus proteins identified in these studies and attributed to spectral counting, demonstrated that two CRISPR-associated proteins had increased expression in the pure culture versus the co-culture. Further focusing on this relationship, a C. saccharolyticus phage-shock protein was identified in the co-culture expanding a scenario that the co-culture had decreased antiviral resistance and accordingly an infection-related protein was present. Alterations in growth conditions of these bacterial thermophilic microorganisms offer a glimpse into the intricacy of microbial behavior and interaction.

Hydrogenomics of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus.

Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO(2), and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium.

Polysaccharide degradation and synthesis by extremely thermophilic anaerobes.

Extremely thermophilic fermentative anaerobes (growth T(opt) > or = 70 degrees C) have the capacity to use a variety of carbohydrates as carbon and energy sources. As such, a wide variety of glycoside hydrolases and transferases have been identified in these microorganisms. The genomes of three model extreme thermophiles-an archaeon Pyrococcus furiosus (T(opt) = 98 degrees C), and two bacteria, Thermotoga maritima (T(opt) = 80 degrees C) and Caldicellulosiruptor saccharolyticus (T(opt) = 70 degrees C)-encode numerous carbohydrate-active enzymes, many of which have been characterized biochemically in their native or recombinant forms. In addition to their voracious appetite for polysaccharide degradation, polysaccharide production has also been noted for extremely thermophilic fermentative anaerobes; T. maritima generates exopolysaccharides that aid in biofilm formation, a process that appears to be driven by intraspecies and interspecies interactions.

Impact of substrate glycoside linkage and elemental sulfur on bioenergetics of and hydrogen production by the hyperthermophilic archaeon Pyrococcus furiosus.

Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h(-1) at 90 degrees C. In the absence of S(0), cellobiose-grown cells generated twice as much protein and had 50%-higher specific H(2) generation rates than maltose-grown cultures. Addition of S(0) to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H(2) to H(2)S. In contrast, the presence of S(0) in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H(2) to H(2)S production, with 2.5 times more H(2) than H(2)S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in alpha- or beta-glucan uptake and processing were up-regulated in an S(0)-independent manner. Most differentially transcribed open reading frames (ORFs) responding to S(0) in cellobiose-grown cells also responded to S(0) in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated) were differentially transcribed when S(0) was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars. These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S(0) metabolism and hydrogen production in P. furiosus. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel by fermentative anaerobes.

Colocation of genes encoding a tRNA-mRNA hybrid and a putative signaling peptide on complementary strands in the genome of the hyperthermophilic bacterium Thermotoga maritima.

In the genome of the hyperthermophilic bacterium Thermotoga maritima, TM0504 encodes a putative signaling peptide implicated in population density-dependent exopolysaccharide formation. Although not noted in the original genome annotation, TM0504 was found to colocate, on the opposite strand, with the gene encoding ssrA, a hybrid of tRNA and mRNA (tmRNA), which is involved in a trans-translation process related to ribosome rescue and is ubiquitous in bacteria. Specific DNA probes were designed and used in real-time PCR assays to follow the separate transcriptional responses of the colocated open reading frames (ORFs) during transition from exponential to stationary phase, chloramphenicol challenge, and syntrophic coculture with Methanococcus jannaschii. TM0504 transcription did not vary under normal growth conditions. Transcription of the tmRNA gene, however, was significantly up-regulated during chloramphenicol challenge and in T. maritima bound in exopolysaccharide aggregates during methanogenic coculture. The significance of the colocation of ORFs encoding a putative signaling peptide and tmRNA in T. maritima is intriguing, since this overlapping arrangement (tmRNA associated with putative small ORFs) was found to be conserved in at least 181 bacterial genomes sequenced to date. Whether peptides related to TM0504 in other bacteria play a role in quorum sensing is not yet known, but their ubiquitous colocalization with respect to tmRNA merits further examination.

Sulfur ylides via decarboxylation of carboxymethylsulfonium betaines: a novel and mild protocol for the preparation of oxiranes.

A novel protocol for the generation of sulfur ylides is described. The overall process involves thermal decarboxylation of a carboxymethylsulfonium betaine to give a sulfur ylide that, in the presence of an aldehyde, affords the corresponding terminal oxirane. Yields were found to correlate with the electron deficiency of the aryl aldehyde. In situ generation of betaine in the presence of an aldehyde successfully afforded the desired oxirane in moderate yield, thus demonstrating the feasibility of a catalytic process. [reaction: see text]