Plasmid DNA fermentation strain and process‐specific effects on vector yield,quality, and transgene expression

Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication. We also investigated the impact of differences in epigenetic dcm methylase‐directed cytosine methylation on plasmid production, transgene expression, and immunogenicity. While Escherichia coli plasmid production yield and quality are unaffected, dcm− versions of CMV and CMV‐HTLV‐I R promoter plasmids had increased transgene expression in human cells. Surprisingly, despite improved expression, dcm− plasmid is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e., production strain) early in product development and that dcm− strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial. Biotechnol. Bioeng. 2011;108: 354–363. © 2010 Wiley Periodicals, Inc.     

Detecting microRNA activity from gene expression data

Background  

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions.     

Causalities of war: The connection between type VI secretion system and microbiota

Microbiota niches have space and/or nutrient restrictions, which has led to the coevolution of cooperation, specialisation, and competition within the population. Different animal and environmental niches contain defined resident microbiota that tend to be stable over time and offer protection against undesired intruders. Yet fluxes can occur, which alter the composition of a bacterial population. In humans, the microbiota are now considered a key contributor to maintenance of health and homeostasis, and its alteration leads to dysbiosis. The bacterial type VI secretion system (T6SS) transports proteins into the environment, directly into host cells or can function as an antibacterial weapon by killing surrounding competitors. Upon contact with neighbouring cells, the T6SS fires, delivering a payload of effector proteins. In the absence of an immunity protein, this results in growth inhibition or death of prey leading to a competitive advantage for the attacker. It is becoming apparent that the T6SS has a role in modulating and shaping the microbiota at multiple levels, which is the focus of this review. Discussed here is the T6SS, its role in competition, key examples of its effect upon the microbiota, and future avenues of research.     

Resource-related variables drive individual variation in flowering phenology and mediate population-level flowering responses to climate in an asynchronously reproducing palm

Many tropical plant species show wide intra-population variation in reproductive timing, resulting in the protracted presence of flowering and fruiting individuals. Various eco-evolutionary drivers have been proposed as ultimate causes for asynchronous phenology, yet little is known about the proximate factors that control reproductive onset among individuals or that influence the proportion of trees producing new inflorescences within a population. We employed a nine-year phenological record from 178 individuals of the hyperdominant, asynchronously flowering canopy palm, Oenocarpus bataua (Arecaceae)¸ to assess whether resource-related variables influence individual- and population-level flowering phenology. Among individuals, access to sunlight increased rates of inflorescence production, while the presence of resource sinks related to current investment in reproduction—developing infructescences—reduced the probability of producing new inflorescences. At the population level, climate anomalies induced by El Niño Southern Oscillation (ENSO) affected the proportion of the population producing inflorescences through time. Moreover, the effects of ENSO anomalies on flowering patterns depended on the prevalence of developing infructescences in the population, with stronger effects in periods of low developing-infructescence frequency. Taken together, these results suggest that resource-related variables can drive phenological differences among individuals and mediate population-level responses to larger-scale variables, such as climate anomalies. Consequently, a greater focus on the role of resource levels as endogenous cues for reproduction might help explain the frequent aseasonal phenological patterns observed among tropical plants, particularly those showing high intra-population asynchrony.     

Carbon uptake in Eurasian boreal forests dominates the high-latitude net ecosystem carbon budget

Arctic-boreal landscapes are experiencing profound warming, along with changes in ecosystem moisture status and disturbance from fire. This region is of global importance in terms of carbon feedbacks to climate, yet the sign (sink or source) and magnitude of the Arctic-boreal carbon budget within recent years remains highly uncertain. Here, we provide new estimates of recent (2003–2015) vegetation gross primary productivity (GPP), ecosystem respiration (R_eco), net ecosystem CO₂ exchange (NEE; R_eco − GPP), and terrestrial methane (CH₄) emissions for the Arctic-boreal zone using a satellite data-driven process-model for northern ecosystems (TCFM-Arctic), calibrated and evaluated using measurements from >60 tower eddy covariance (EC) sites. We used TCFM-Arctic to obtain daily 1-km² flux estimates and annual carbon budgets for the pan-Arctic-boreal region. Across the domain, the model indicated an overall average NEE sink of −850 Tg CO₂-C year⁻¹. Eurasian boreal zones, especially those in Siberia, contributed to a majority of the net sink. In contrast, the tundra biome was relatively carbon neutral (ranging from small sink to source). Regional CH₄ emissions from tundra and boreal wetlands (not accounting for aquatic CH₄) were estimated at 35 Tg CH₄-C year⁻¹. Accounting for additional emissions from open water aquatic bodies and from fire, using available estimates from the literature, reduced the total regional NEE sink by 21% and shifted many far northern tundra landscapes, and some boreal forests, to a net carbon source. This assessment, based on in situ observations and models, improves our understanding of the high-latitude carbon status and also indicates a continued need for integrated site-to-regional assessments to monitor the vulnerability of these ecosystems to climate change.     

Structural and Dynamic Properties of Allergen and Non-Allergen Forms of Tropomyosin

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Novel scabies mite serpins inhibit the three pathways of the human complement system

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.     

A rapid one-step purification of NADPH-cytochrome c (P-450) reductase from rat liver microsomes

NADPH-cytochrome c (P-450) reductase from liver microsomes of phenobarbital-treated rats has been purified in a single step by affinity chromatography on agarose-hexane-adenosine 2',5'-diphosphate. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay, and radioimmunoassay the protein obtained by this single step procedure is as pure as that isolated by multicolumn procedures.     

Technoeconomic analysis of five microalgae-to-biofuels processes of varying complexity

The economics surrounding five algae-to-fuels process scenarios were examined. The different processes modeled were as follows: an open pond producing either triacylglycerides (TAG) or free fatty acid methyl ester (FAME), a solar-lit photobioreactor producing either FAME or free fatty acids (FFA), and a light emitting diode irradiated (LED-lighted) photobioreactor producing TAG. These processes were chosen to represent both classical and esoteric approaches presented in the open literature. Viable (or suggested) processing techniques to liberate and purify (and convert) the microalgal triacylglycerides were then modeled to accompany each growth option. The investment and cost per kg of fuel or fuel precursor for each process was determined. The open pond produced TAG at ～$7.50/kg, while the process using the LED-lit photobioreactor produced TAG at ～$33/kg. The scenario containing the solar-lit photobioreactor produced FAME at ～$25/kg, while the open pond produced FAME at ～$4/kg. The scenario containing the solar-lit photobioreactor produced FFA at ～$29/kg. The open pond scenarios appear to be closest to the $1/kg pricepoint at this time, and thus are the most viable economic options. Future technological advancements that reduce the cost of bioreactor vessels, LED lighting, and solvent recovery, may reduce the oil production costs of these scenarios to a more attractive level.     

Genomic mapping of chromosomal region 2p15-p21 (D2S378-D2S391): integration of Genemap'98 within a framework of yeast and bacterial artificial chromosomes

The region of chromosome 2 encompassed by the polymorphic markers D2S378 (centromeric) and D2S391 (telomeric) spans an approximately 10-cM distance in cytogenetic bands 2p15-p21. This area is frequently involved in cytogenetic alterations in human cancers. It also harbors the genes for several genetic disorders, including Type I hereditary nonpolyposis colorectal cancer (HNPCC), familial male precocious puberty (FMPP), Carney complex (CNC), Doyne's honeycomb retinal dystrophy (DHRD), and one form of familial dyslexia (DYX-3). Only a handful of known genes have been mapped to 2p16. These include MSH2, which is responsible for HNPCC, FSHR, the gene responsible for FMPP, EFEMP-1, the gene mutated in DHRD, GTBP, a DNA repair gene, and SPTBN1, nonerythryocytic beta-spectrin. The genes for CNC and DYX-3 remain unknown, due to lack of a contig of this region and its underrepresentation in the existing maps. This report presents a yeast- and bacterial-artificial chromosome (YAC and BAC, respectively) resource for the construction of a sequence-ready map of 2p15-p21 between the markers D2S378 and D2S391 at the centromeric and telomeric ends, respectively. The recently published Genemap'98 lists 146 expressed sequence tags (ESTs) in this region; we have used our YAC-BAC map to place each of these ESTs within a framework of 40 known and 3 newly cloned polymorphic markers and 37 new sequence-tagged sites. This map provides an integration of genetic, radiation hybrid, and physical mapping information for the region corresponding to cytogenetic bands 2p15-p21 and is expected to facilitate the identification of disease genes from the area.