Inline ultrafiltration

Inline ultrafiltration (UF) can significantly increase the recoverable mass of biopharmaceutical products when pool tank volumes are limiting. Using relatively small commercially available ultrafiltration cassettes, a proof‐of‐concept study demonstrates that inline UF can significantly increase recoverable mass in an antibody purification process. With ever‐increasing cell culture titers pushing product masses to higher levels, inline UF offers a relatively easy‐to‐implement and less disruptive alternative to installing larger pool tanks and enables more cost‐effective production utilizing facilities designed for smaller bulk sizes. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Verrucomicrobia Are Candidates for Polysaccharide-Degrading Bacterioplankton in an Arctic Fjord of Svalbard

In Arctic marine bacterial communities, members of the phylum Verrucomicrobia are consistently detected, although not typically abundant, in 16S rRNA gene clone libraries and pyrotag surveys of the marine water column and in sediments. In an Arctic fjord (Smeerenburgfjord) of Svalbard, members of the Verrucomicrobia, together with Flavobacteria and smaller proportions of Alpha- and Gammaproteobacteria, constituted the most frequently detected bacterioplankton community members in 16S rRNA gene-based clone library analyses of the water column. Parallel measurements in the water column of the activities of six endo-acting polysaccharide hydrolases showed that chondroitin sulfate, laminarin, and xylan hydrolysis accounted for most of the activity. Several Verrucomicrobia water column phylotypes were affiliated with previously sequenced, glycoside hydrolase-rich genomes of individual Verrucomicrobia cells that bound fluorescently labeled laminarin and xylan and therefore constituted candidates for laminarin and xylan hydrolysis. In sediments, the bacterial community was dominated by different lineages of Verrucomicrobia, Bacteroidetes, and Proteobacteria but also included members of multiple phylum-level lineages not observed in the water column. This community hydrolyzed laminarin, xylan, chondroitin sulfate, and three additional polysaccharide substrates at high rates. Comparisons with data from the same fjord in the previous summer showed that the bacterial community in Smeerenburgfjord changed in composition, most conspicuously in the changing detection frequency of Verrucomicrobia in the water column. Nonetheless, in both years the community hydrolyzed the same polysaccharide substrates.     

A molecular and physiological survey of a diverse collection of hydrothermal vent Thermococcus and Pyrococcus isolates

Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collection assembled by Holger Jannasch at the Woods Hole Oceanographic Institution between 1984 and 1998 were identified and partially characterized by Denaturing Gradient Gel Electrophoresis, 16S rRNA gene sequencing, and by growth tests at different temperatures and on different organic carbon and nitrogen sources. All strains were members of the genera Thermococcus and Pyrococcus. The greatest phylogenetic diversity was found in strains from a single Guaymas Basin core isolated by serial dilution from four different depth horizons of heated sediment incubated at the corresponding in situ temperatures. In contrast, geographically distinct vent locations and sample materials yielded a lower diversity of isolates when enriched under uniform temperature regimes and without prior dilution of the source material.     

S100A8/A9 is not involved in host defense against murine urinary tract infection

Background

Inflammation is commonly followed by the release of endogenous proteins called danger associated molecular patterns (DAMPs) that are able to warn the host for eminent danger. S100A8/A9 subunits are DAMPs that belong to the S100 family of calcium binding proteins. S100A8/A9 complexes induce an inflammatory response and their expression correlates with disease severity in several inflammatory disorders. S100A8/A9 promote endotoxin- and Escherichia (E.) coli-induced sepsis showing its contribution in systemic infection. The role of S100A8/A9 during a local infection of the urinary tract system caused by E. coli remains unknown.

Methodology/Principal Findings

We investigated the contribution of S100A8/A9 in acute urinary tract infection (UTI) by instilling 2 different doses of uropathogenic E. coli transurethrally in wild type (WT) and S100A9 knockout (KO) mice. Subsequently, we determined bacterial outgrowth, neutrophilic infiltrate and inflammatory mediators in bladder and kidney 24 and 48 hours later. UTI resulted in a substantial increase of S100A8/A9 protein in bladder and kidney tissue of WT mice. S100A9 KO mice displayed similar bacterial load in bladder or kidney homogenate compared to WT mice using 2 different doses at 2 different time points. S100A9 deficiency had little effect on the inflammatory responses to E. Coli-induced UTI infection, as assessed by myeloperoxidase activity in bladder and kidneys, histopathologic analysis, and renal and bladder cytokine concentrations.

Conclusions

We show that despite high S100A8/A9 expression in bladder and kidney tissue upon UTI, S100A8/A9 does not contribute to an effective host response against E. Coli in the urinary tract system.     

Divergent interactions involving the oxidosqualene cyclase and the steroid-3-ketoreductase in the sterol biosynthetic pathway of mammals and yeasts

In mammals and yeasts, oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol, the first cyclic intermediate in sterol biosynthesis. We used a murine myeloma cell line (NS0), deficient in the 17β-hydroxysteroid dehydrogenase type 7 (HSD17B7), as a model to study the potential interaction of the HSD17B7 with the OSC in mammals. HSD17B7 is the orthologue of the yeast steroid-3-ketoreductase (ERG27), an enzyme of ergosterol biosynthesis that plays a protective role towards OSC. Tracer experiments with NS0 cells showed that OSC is fully active in these mammalian cells, suggesting that in mammals the ketosteroid reductase is not required for OSC activity. Mouse and human HSD17B7 were overexpressed in ERG27-deletant yeast cells, and recombinant strains were tested for (i) the ability to grow on different media, (ii) steroid-3-ketoreductase activity, and (iii) OSC activity. Recombinant strains grew more slowly than the control yeast ERG27-overexpressing strain on sterol-deficient media, whereas the growth rate was normal on media supplemented with a 3-ketoreductase substrate. The full enzymatic functionality of mammalian steroid-3-ketoreductase expressed in yeast along with the lack of (yeast) OSC activity point to an inability of the mammalian reductase to assist yeast OSC. Results demonstrate that in mammals, unlike in yeast, OSC and steroid-3-ketoreductase are non-interacting proteins.     

Heat production and body temperature during cooling and rewarming in overweight and lean men

Objective: To compare overweight and lean subjects with respect to thermogenesis and physiological insulation in response to mild cold and rewarming. Research Methods and Procedures: Ten overweight men (mean BMI, 29.2 ± 2.8 kg/m²) and 10 lean men (mean BMI, 21.1 ± 2.0 kg/m²) were exposed to cold air for 1 hour, followed by 1 hour of rewarming. Body composition was determined by hydrodensitometry and deuterium dilution. Heat production and body temperatures were measured continuously by indirect calorimetry and thermistors, respectively. Muscle activity was recorded using electromyography. Results: In both groups, heat production increased significantly during cooling (lean, p = 0.004; overweight, p = 0.006). The increase was larger in the lean group compared with the overweight group (p = 0.04). During rewarming, heat production returned to baseline in the overweight group and stayed higher compared with baseline in the lean group (p = 0.003). The difference in heat production between rewarming and baseline was larger in the lean (p = 0.01) than in the overweight subjects. Weighted body temperature of both groups decreased during cold exposure (lean, p = 0.002; overweight, p < 0.001) and did not return to baseline during rewarming. Discussion: Overweight subjects showed a blunted mild cold‐induced thermogenesis. The insulative cold response was not different among the groups. The energy‐efficient response of the overweight subjects can have consequences for energy balance in the long term. The results support the concept of a dynamic heat regulation model instead of temperature regulation around a fixed set point.     

Phylogenetic relationships of a large marine Beggiatoa

Based upon 16S rRNA sequence and phenotypic similarities, a large, uncultured Beggiatoa sp. from the Bay of Concepción (Chile), is very closely related to the Chilean Thioploca species Thioploca araucae., whose filaments grow as sheathed bundles. The formation of sheathed filament bundles, the key character to distinguish the genus Thioploca from Beggiatoa, places closely related filamentous sulfur-oxidizing bacteria into two different genera, incongruent with 16S rRNA-defined clades.     

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly ¹³C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.     

Marine subsurface eukaryotes: the fungal majority

Studies on the microbial communities of deep subsurface sediments have indicated the presence of Bacteria and Archaea throughout the sediment column. Microbial eukaryotes could also be present in deep-sea subsurface sediments; either bacterivorous protists or eukaryotes capable of assimilating buried organic carbon. DNA- and RNA-based clone library analyses are used here to examine the microbial eukaryotic diversity and identify the potentially active members in deep-sea sediment cores of the Peru Margin and the Peru Trench. We compared surface communities with those much deeper in the same cores, and compared cores from different sites. Fungal sequences were most often recovered from both DNA- and RNA-based clone libraries, with variable overall abundances of different sequence types and different dominant clone types in the RNA-based and the DNA-based libraries. Surficial sediment communities were different from each other and from the deep subsurface samples. Some fungal sequences represented potentially novel organisms as well as ones with a cosmopolitan distribution in terrestrial, fresh and salt water environments. Our results indicate that fungi are the most consistently detected eukaryotes in the marine sedimentary subsurface; further, some species may be specifically adapted to the deep subsurface and may play important roles in the utilization and recycling of nutrients.