Community Composition and Density of Methanogens in the Foregut of the Tammar Wallaby (Macropus eugenii)

The composition of the methanogenic archaeal community in the foregut contents of Tammar wallabies (Macropus eugenii) was studied using 16S rRNA and methyl coenzyme reductase subunit A (mcrA) gene clone libraries. Methanogens belonging to the Methanobacteriales and a well-supported cluster of uncultivated archaeon sequences previously observed in the ovine and bovine rumens were found. Methanogen densities ranged from 7.0 × 10⁵ and 3.9 × 10⁶ cells per gram of wet weight.Kangaroos and wallabies belong to the marsupial family Macropodidae and are native to Australia. Because of their geographical isolation, macropod marsupials have evolved separately from other herbivorous animals, such as ruminants, but like ruminants, macropods have a complex gut microbiome that includes fungi, archaea, bacteria, and protozoa to coordinate plant biomass breakdown (11). The macropod foregut is functionally analogous to the rumen, yet for reasons unknown, macropod species produce relatively low levels of methane compared to ruminants (5, 13, 30).New species of bacteria (21) and protozoa (2-4) have been indentified in the macropod foregut, and the presence of fungi has also been reported (5). Preliminary studies have shown that methanogens are present in the kangaroo foregut (1) but can be absent or at levels below detection limits (22). This study represents the first attempt to describe the diversity of methanogens residing in the macropod foregut by using 16S rRNA and methyl coenzyme reductase A (mcrA) clone libraries in combination with quantitative real-time PCR.     

Process‐relevant concentrations of the leachable bDtBPP impact negatively on CHO cell production characteristics

The biopharmaceutical industry has invested considerably in the implementation of single‐use disposable bioreactors in place of or in addition to their stainless steel‐counterparts. This new wave of construction materials for disposable bioprocess containers encompass a plethora of uncharacterized secondary compounds that, when in contact with the culture media, can leach, contaminating the bioprocess. One such cytotoxic leachable already receiving attention is bis(2,4‐di‐tert‐butylphenyl)‐phosphate (bDtBPP), a breakdown product of the secondary antioxidant Irgafos 168 in polyethylene‐film based bags. This compound has been demonstrated to inhibit cell growth at concentrations ranging from 0.12 to 0.73 mg/L across an array of cell lines. Here we demonstrate that a further two CHO cell lines exhibit sensitivity to bDtBPP exposure at concentrations lower than that previously reported (0.035–0.1 mg/L). Furthermore, these inhibitory concentrations reflect bDtBPP levels found to leach early into the bioprocess, exposing reactor inoculums to serious risk. Quantitative label‐free LC‐MS/MS revealed that irrespective of cell line or concentration of bDtBPP, 8 proteins were found to be commonly differentially expressed in response to exposure to the compound highlighting biological processes related to cellular stress. Although the glycoprofile of the recombinant antibody remains primarily unchanged, we demonstrate that this compound when spiked at meaningful concentrations 72 h into culture considerably reduces the maximum cell density achieved. Studies like this reinforce the requirement for the complete characterization of all potential leachable compounds from disposable materials to assess their risk not only to the patient but also to the production pipeline itself. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1547–1558, 2016     

The biosynthesis of rubber: Incorporation of isopentenyl pyrophosphate into purified rubber particles by a soluble latex serum enzyme

1. The rubber particles in Hevea brasiliensis latex have been partially purified by `washing' with buffer solution, and separated into active fractions of different particle size. 2. The enzyme responsible for incorporating isopentenyl pyrophosphate into rubber is distributed between the surface of the rubber particles and the aqueous serum phase of the latex. The enzyme at the surface can be removed or inactivated if the rubber particles are washed sufficiently with buffer solution. Enzyme in the serum phase can be concentrated by fractional precipitation with ammonium sulphate. 3. To incorporate isopentenyl pyrophosphate into rubber in vitro, active rubber particles are required as well as enzyme and soluble cofactors. The activity of the rubber particles per unit surface area increases with diminishing particle size.     

In vitro inhibition of human influenza A virus replication by chloroquine

Inhibition of the human cytomegalovirus UL97 kinase by maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to maribavir map to UL97, however additional mutations that also confer resistance to the drug were mapped to UL27. These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo.     

Nuclear localization of the Escherichia coli cytolethal distending toxin CdtB subunit

Cytolethal distending toxin (CDT) is a heterotrimeric protein toxin produced by several bacterial pathogens. Cells exposed to CDT die from either activation of the mitotic checkpoint cascade or apoptosis. Introduction of the purified CdtB subunit, a homologue of mammalian type I DNase, into cells mimics the action of the CDT holotoxin. Mutant CdtBs lacking DNase activity are devoid of biological activity. Chromosomal DNA appears to be the CDT target; thus, nuclear translocation of CdtB must precede cytolethal activity. Examination of the CdtB sequence indicates the presence of putative candidate bipartite nuclear localization signals (NLS). Here, we examine the functionality of the two potential NLS sequences found in the Escherichia coli CdtB-II. Nuclear translocation of EcCdtB-II was examined by monitoring the localization of an EcCdtB-II-EGFP fusion in Cos-7 cells. Our results indicated that EGFP-EcCdtB-II localized to the nucleus. The candidate EcCdtB-II-II NLS sequences were modified by site-directed mutagenesis such that tandem arginine residues were changed to threonine and serine respectively. Mutation of both putative NLS sequences had no effect on EcCdtB-II-associated DNase activity; however, cell cycle arrest and nuclear localization were significantly impaired in cells that received CDT reconstituted from the EcCdtB-II-DeltaNLS mutants. When HeLa cells were electroporated with the EcCdtB-II-DeltaNLS1 and the EcCdtB-II-NLS double mutants, toxicity was not observed, whereas the activity of EcCdtB-II-DeltaNLS2 was similar to that of wild-type EcCdtB-II. These data indicate that the putative NLS sequences are important for CDT-mediated action arrest and that they are likely to function in the nuclear translocation of EcCdtB-II.     

Effect of finishing diets on Escherichia coli populations and prevalence of enterohaemorrhagic E. coli virulence genes in cattle faeces

AIM: To determine the effect of different carbohydrate-based finishing diets on fermentation characteristics and the shedding of Escherichia coli and enterohaemorrhagic E. coli (EHEC) virulence genes in cattle faeces. METHODS AND RESULTS: The size of faecal E. coli populations and fermentation characteristics were ascertained in three experiments where cattle were maintained on a range of finishing diets including high grain, roughage, and roughage + molasses (50%) diets. Increased E. coli numbers, decreased pH and enhanced butyrate and lactate fermentation pathways were associated with grain diets, whereas roughage and roughage + molasses diets resulted in decreased concentrations of ehxA, eaeA and stx(1) genes, this trend remaining at lairage. In one experiment, faecal E. coli numbers were significantly lower in animals fed roughage and roughage + molasses, than animals fed grain (4.5, 5.2 and 6.3 mean log10 g(-1) digesta respectively). In a second experiment, faecal E. coli numbers were 2 log lower in the roughage and roughage + molasses diets compared with grain-fed animals prior to lairage (5.6, 5.5 and 7.9 mean log10 g(-1) digesta respectively) this difference increasing to 2.5 log at lairage. CONCLUSIONS: The type of dietary carbohydrate has a significant effect on E. coli numbers and concentration of EHEC virulence genes in faeces of cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a better understanding of the impact finishing diet and commercial lairage management practices may have on the shedding of E. coli and EHEC virulence factors, thus reducing the risk of carcass contamination by EHEC.