Oligonucleotides: Current Trends and Innovative Applications in the Synthesis,Characterization, and Purification

Oligonucleotides (ONs) are gaining increasing importance as a promising novel class of biopharmaceuticals. Thanks to their fundamental role in gene regulation, they can be used to develop custom‐made drugs (also called N‐to‐1) able to act on the gene expression at pre‐translational level. With recent approvals of ON‐based therapeutics by the Food and Drug Administration (FDA), a growing demand for high‐quality chemically modified ONs is emerging and their market is expected to impressively prosper in the near future. To satisfy this growing market demand, a scalable and economically sustainable ON production is needed. In this paper, the state of the art of the whole ON production process is illustrated with the aim of highlighting the most promising routes toward the auspicated market‐size production. In particular, the most recent advancements in both the upstream stage, mainly based on solid‐phase synthesis and recombinant technology, and the downstream one, focusing on chromatographic techniques, are reviewed. Since ON production is projected to expand to the large scale, automatized multicolumn countercurrent technologies will reasonably be required soon to replace the current ones based on batch single‐column operations. This consideration is supported by a recent cutting‐edge application of continuous chromatography for the ON purification.     

Metabolic and water loss rates of two cryptic species in the African velvet worm genus Opisthopatus (Onychophora)

Velvet worms (Onychophora) are characterised by a dearth of mechanisms to retain water, yet recently identified cryptic species are located in areas with seemingly different climates. Using flow-through respirometry, this study determined the metabolic, water loss and cuticular water loss rates of two cryptic species of Opisthopatus cinctipes s.l. from locations that differ in their current climate. When controlling for trial temperature and body mass, velvet worms from the drier and warmer site had significantly lower water loss rates than the wetter and cooler site. Mass-corrected metabolic rate and cuticular water loss did not differ significantly between the two sites. The scaling exponent for the relationship between log metabolic rate and log body mass for O. cinctipes s.l. declined with an increase in temperature from 5 to 15 °C. Females in the two cryptic Opisthopatus species had higher metabolic, water loss and cuticular water loss rates than males, which may represent the increased energetic demands of embryonic growth and development in these viviparous taxa.     

Uncovering symbiont‐driven genetic diversity across North American pea aphids

Heritable genetic variation is required for evolution, and while typically encoded within nuclear and organellar genomes, several groups of invertebrates harbour heritable microbes serving as additional sources of genetic variation. Hailing from the symbiont‐rich insect order Hemiptera, pea aphids (Acyrthosiphon pisum) possess several heritable symbionts with roles in host plant utilization, thermotolerance and protection against natural enemies. As pea aphids vary in the numbers and types of harboured symbionts, these bacteria provide heritable and functionally important variation within field populations. In this study, we quantified the cytoplasmically inherited genetic variation contributed by symbionts within North American pea aphids. Through the use of Denaturing Gradient Gel Electrophoresis (DGGE) and 454 amplicon pyrosequencing of 16S rRNA genes, we explored the diversity of bacteria harboured by pea aphids from five populations, spanning three locations and three host plants. We also characterized strain variation by analysing 16S rRNA, housekeeping and symbiont‐associated bacteriophage genes. Our results identified eight species of facultative symbionts, which often varied in frequency between locations and host plants. We detected 28 cytoplasmic genotypes across 318 surveyed aphids, considering only the various combinations of secondary symbiont species infecting single hosts. Yet the detection of multiple Regiella insecticola, Hamiltonella defensa and Rickettsia strains, and diverse bacteriophage genotypes from H. defensa, suggest even greater diversity. Combined, these findings reveal that heritable bacteria contribute substantially to genetic variation in A. pisum. Given the costs and benefits of these symbionts, it is likely that fluctuating selective forces play a role in the maintenance of this diversity.     

The ecology of Bactrocera tryoni (Diptera: Tephritidae): what do we know to assist pest management?

The distribution, systematics and ecology of Bactrocera tryoni, the Queensland fruit fly, are reviewed. Bactrocera tryoni is a member of the B. tryoni complex of species, which currently includes four named species, viz. B. tryoni ssp., B. neohumeralis, B. melas and B. aquilonis. The species status of B. melas and B. aquilonis is unclear (they may be junior synonyms of B. tryoni) and their validity, or otherwise, needs to be confirmed as a matter of urgency. While Queensland fruit fly is regarded as a tropical species, it cannot be assumed that its distribution will spread further south under climate change scenarios. Increasing aridity and hot dry summers, as well as more complex, indirect interactions resulting from elevated CO₂, make predicting the future distribution and abundance of B. tryoni difficult. The ecology of B. tryoni is reviewed with respect to current control approaches (with the exception of sterile insect technique (SIT) which is covered in a companion paper). We conclude that there are major gaps in the knowledge required to implement most noninsecticide‐based management approaches. Priority areas for future research include host–plant interactions, protein and cue‐lure foraging and use, spatial dynamics, development of new monitoring tools, investigating the use of natural enemies and better integration of fruit flies into general horticultural IPM systems.     

SLPI and elafin: multifunctional antiproteases of the WFDC family

SLPI (secretory leucoprotease inhibitor) and elafin represent the archetypal members of the WFDC [WAP (whey acidic protein) four disulfide core] family of proteins, and were originally characterized as protease inhibitors but have since been shown to possess a wider repertoire of activities. These functions include antimicrobial and immunomodulatory properties, suggesting that these proteins may play key roles in the innate immune response, and indicate the potential to develop some of these proteins as novel therapeutics. Susceptibility to host and bacterial protease cleavage may, however, limit the efficacy of recombinant protein therapies in diseases with a high protease burden such as CF (cystic fibrosis) lung disease. To overcome this problem, further refinement of the native proteins will be required to provide effective treatment strategies.     

Time-course for attainment and reversal of acclimation to constant temperature in two Ceratitis species

Acclimation in the thermal tolerance range of insects occurs when they are exposed to novel temperatures in the laboratory. In contrast to the large number of studies that have tested for the ability of insects to acclimate, relatively few have sought to determine the time-course for attainment and reversal of thermal acclimation. In this study the time required for the Mediterranean fruit fly, Ceratitis capitata Wiedemann, and the Natal fruit fly, Ceratitis rosa Karsch, to acclimate to a range of constant temperatures was tested by determining the chill-coma recovery time and heat knock-down time of flies that had been exposed to novel benign temperatures for different durations. The time required for reversal of acclimation for both Ceratitis species was also determined after flies had been returned to the control temperature. Acclimation to 31 °C for only one day significantly improved the heat knock-down time of C. capitata, but also led to slower recovery from chill-coma. Heat knock-down time indicated that acclimation was achieved after only one day in C. rosa, but it took three days for C. rosa to exhibit a significant acclimation response to a novel temperature of 33 °C when measured using chill-coma recovery time. Reversal of acclimation after return to initial temperature conditions was achieved after only one day in both C. capitata and C. rosa. Adult C. capitata held at 31.5 °C initially exhibited improved heat knock-down times but after 9 days the heat knock-down time of these flies had declined to levels not significantly different from that of control flies held at the baseline temperature of 24 °C. In both Ceratitis species, heat knock-down time declined with age whereas chill-coma recovery time increased with age, indicating an increased susceptibility to high and low temperatures, respectively.     

Characterization of recombinant human acetyl-CoA carboxylase-2 steady-state kinetics

Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, a key metabolite in the fatty acid synthetic and oxidation pathways. The present study describes the steady-state kinetic analysis of a purified recombinant human form of the enzyme, namely ACC2, using a novel LC/MS/MS assay to directly measure malonyl-CoA formation. Four dimensional matrices, in which bicarbonate (HCO₃^?), ATP, acetyl-CoA, and citrate were varied, and global data fitting to appropriate steady-state equations were used to generate kinetic constants. Product inhibition studies support the notion that the enzyme proceeds through a hybrid (two-site) random Ter Ter mechanism, one that likely involves a two-step reaction at the biotin carboxylase domain. Citrate, a known activator of animal forms of ACC, activates both by increasing k_cat and k_cat/K_M for ATP and acetyl-CoA.     

Interspecific responses of wild African carnivores to odour of 3-mercapto-3-methylbutanol,a component of wildcat and leopard urine

The scent of 3-mercapto-3-methylbutanol (3-M-3-MB), a volatile component of leopard (Panthera pardus) and domestic cat (Felis silvestris catus) urine, released at about 10 ng/s from slow-release dispensers, elicited scent-marking from African civet (Civettictis civetta), small-spotted genet (Genetta genetta) and slender mongoose (Galerella sanguinea), as well as African wildcat (F. s. cafra). A female leopard was apparently repelled by the scent. The scent-marking and scent-rubbing by species other than African wildcats and leopards were unexpected and have important implications for the design of studies to investigate chemical communication between wild mammals and the use of camera traps to estimate animal numbers. Videos showing the behaviours referred to in this article are available at; http://www.momo-p.com/showdetail-e.php?movieid=momo161223fs01a; http://www.momo-p.com/showdetail-e.php?movieid=momo161223gs01a; http://www.momo-p.com/showdetail-e.php?movieid=momo161223gg01a.