Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

Background

The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A_2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures.

Results

Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A_2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A_2AR, and therefore more suitable for further functional and structural studies.

Conclusion

Large-scale expression of the A_2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.     

A mass mortality of fishes caused by receding water levels in the vegetated littoral zone of the West Kleinemonde Estuary,South Africa

On 15 November 2017 the mouth of the West Kleinemonde Estuary breached following heavy catchment rains and increased river flow. The water level in the estuary following mouth opening decreased by 1.65 m within 24 h, resulting in an almost complete draining of the littoral zone where large beds of the aquatic macrophyte Ruppia cirrhosa and mats of the associated filamentous algae were present. As the water depth within the plant beds decreased, the macrophytes, together with the algal filaments, created an increasingly dense mat, trapping fish that were resident, foraging or passing through the littoral zone. By 16 November 2017 large numbers of fishes belonging to at least 20 species were trapped in pools and depressions within the littoral, as well as within the R. cirrhosa beds and filamentous algal mats in the lower reaches of this system. Other affected taxa included crustaceans, especially isopods, and large numbers of small bivalves attached to macrophyte vegetation. Beneficiaries of the fish kill, in terms of unexpected food availability, included a variety of piscivorous bird species and the Cape clawless otter Aonyx capensis. This is the first documented account of a diverse species fish kill associated with estuary mouth breaching.     

Norian-Rhaetian reefs in Argolis Peninsula,Greece

Summary Upper Triassic to Lower Jurassic shallow-water carbonate sequences of the ‘Pantokrator limestones’ are widely distributed in the Argolis Peninsula, southern Greece. Within this sequence are some reef or reefal structures. In the Mavrovouni Mountains, near Sarmeika, 6 km SE of the ancient theatre of Epidavros (Argolis Peninsula), a Norian-Rhaetian reef complex has been identified. This is the first well-documented Norian-Rhaetian reef in Greece. The main reef builders are coralline sponges (‘sphinctozoans,’ ‘inozoans’, and sclerosponges), followed by dendroid, cerioid, and solitary corals, and algae. The reef type corresponds to a ‘sponge-coral reef’.     

Reproduction in the male honey possum (Tarsipes rostratus: Marsupialia): the epididymis

The epididymis of the adult honey possum, Tarsipes rostratus, is enclosed by a heavily pigmented tunica vaginalis and lies with the testis in a prominent prepenile scrotum. It is connected to the testis by a single ductus efferentis and is lined by approximately equal numbers of cuboidal ciliated and principal cells. It is unusual for marsupials in having no well-defined compartments or fibrous septae and in having extensive convolutions of the duct only at the caudal flexure. Three principal functional zones (initial, middle, and terminal segments) were identified in the epididymis, based on epithelial type and ultrastructural evidence of sperm maturation. Luminal diameter increases progressively throughout the tract, and epithelial height variations (from about 2 to 20 microns) are greatest in the terminal segment. The epithelium itself is remarkably low (maximum of 21.6 microns) compared with that seen in the epididymis of other mammals. The thickness of the peritubular smooth muscle coat increases close to the junction of the epididymis and ductus deferens. Sperm concentrations were estimated from counts of sperm nuclei and thus can be no more than approximations. The figures are consistent, however, with a rapid increase in concentration in the initial segment, indicating extensive fluid resorption. Sperm concentrations appear to peak in the distal zone of the terminal segment, although sampling problems and wide variations in count make such a conclusion only tentative. Principal and basal cells are the predominant cell types in the epididymal epithelium. Basal cells are most abundant in the initial and distal middle segment. Principal cells show structural evidence of active exchange with the luminal contents and have abundant apical stereocilia, the structure of which depends on the epididymal zone. Other cell types occur less commonly in the epithelium. Lipid-rich and phagocytic principal cells are restricted to the middle and distal zones of the middle segment, respectively. Clear cells, restricted to the terminal segment, and halo cells were found in very low numbers. As in some other marsupials, principal cells (possibly specialized for this function) selectively remove cytoplasmic droplets and probably other cellular debris from the luminal contents. In Tarsipes, however, this process is not very efficient, and many discarded droplets pass through to the terminal segment where they form large masses of debris associated with aggregates of degenerating spermatozoa.     

Temporal scaling of molecular evolution in primates and other mammals

Molecular clocks are routinely tested for linearity using a relative rate test and routinely calibrated against the geological time scale using a single or average paleontologically determined time of divergence between living taxa. The relative rate test is a test of parallel rate equality, not a test of rate constancy. Temporal scaling provides a test of rates, where scaling coefficients of 1.0 (isochrony) represent stochastic rate constancy. The fossil record of primates and other mammals is now known in sufficient detail to provide several independent divergence times for major taxonomic groups. Molecular difference should scale negatively or isochronically (scaling coefficients less than 1.0) with divergence time: where two or more divergence times are available, molecular difference appears to scale positively (scaling coefficient greater than 1.0). A minimum of four divergence times are required for adequate statistical power in testing the linear model: scaling is significantly nonlinear and positive in six of 11 published investigations meeting this criterion. All groups studied show some slowdown in rates of molecular change over Cenozoic time. The break from constant or increasing rates during the Mesozoic to decreasing rates during the Cenozoic appears to coincide with extraordinary diversification of placental mammals at the beginning of this era. High rates of selectively neutral molecular change may be concentrated in such discrete events of evolutionary diversification.      

Wasserlinsen als Nutzpflanzen

Duckweeds as crop plants Members of the plant family Lemnaceae (duckweeds) are not only interesting because they represent the smallest flowering plants; they possess also the fastest rates of producing biomass. As aquatic plants, duckweed production is not in competition with other agricultural crops that require fertile land while the cultivation of duckweeds does not contribute to further eutrophication of surface water. Instead, they can be cultivated on municipal or agricultural waste water and remove the nutrients during their propagation and growth. Duckweeds can thus be used for cleaning of waste water and the resulting biomass can be valuable starting material for animal feeds and the production of biofuels. Research focusing on these goals has begun to transfer from research laboratories to pilot plants in different parts of the world, e.g. in New Jersey and North Carolina, USA; Chengdu, P. R. China; and Armidale, Australia.     

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.