Nonenzymatic Glycosylation of Lepidopteran-Active Bacillus thuringiensis Protein Crystals

We used high-pH anion-exchange chromatography with pulsed amperometric detection to quantify the monosaccharides covalently attached to Bacillus thuringiensis HD-1 (Dipel) crystals. The crystals contained 0.54% sugars, including, in decreasing order of prevalence, glucose, fucose, arabinose/rhamnose, galactose, galactosamine, glucosamine, xylose, and mannose. Three lines of evidence indicated that these sugars arose from nonenzymatic glycosylation: (i) the sugars could not be removed by N- or O-glycanases; (ii) the sugars attached were influenced both by the medium in which the bacteria had been grown and by the time at which the crystals were harvested; and (iii) the chemical identity and stoichiometry of the sugars detected did not fit any known glycoprotein models. Thus, the sugars detected were the product of fermentation conditions rather than bacterial genetics. The implications of these findings are discussed in terms of crystal chemistry, fermentation technology, and the efficacy of B. thuringiensis as a microbial insecticide.     

Neutron structure factors of in-vivo deuterated amorphous protein C-phycocyanin

Neutron powder diffraction measurements of fully deuterated protein C-phycocyanin have been made at three temperatures, 295, 200, and 77 K, using dry and partially hydrated samples. The average coherent structure factors and the corresponding radial distribution functions d(r) are determined. The changes in d(r) functions observed in hydrated samples depend strongly on the level of hydration and most of these changes are due to water-protein interactions. At 0.365 gram D₂O per gram of protein, the water crystallized into hexagonal ice at 200 K and below, but at 0.175 gram D₂O per gram of protein, no crystallization of water was observed. At the higher hydration a peak appears in the radial distribution function which indicates that the average distance of the water molecule in the first hydration shell from the amino acid residues is 3.5 Å.     

A population model for the alkali fly at Mono Lake: depth distribution and changing habitat availability

The densities of alkali fly larvae and pupae were measured in relation to depth and substrate type at six locations around Mono Lake. Samples representing a mixture of different bottom features were taken to a depth of 10 m (33 ft) using SCUBA. This is at or near the depth limit of fly larvae and pupae. The biomass of larvae and pupae on hard substrate were maximum and approximately equal at depths of 0.5 m and 1 m, substantially lower at intermediate depths of 3 m and 5 m, and over an order of magnitude further reduced at 10 m. Densities of flies on hard or rocky substrates (mainly calcareous tufa deposits), were significantly greater than those found on soft substrates such as mud or sand, at all but the greatest depth surveyed.Bathymetric maps of the areas of hard and soft substrate occurring at different lake depths were used to estimate the fly population size over the whole lake, based on the density distribution of larvae and pupae with depth on different substrates. The mapped areas of soft and hard substrates were also calculated for different lake levels, and applying the same procedure, a population model comparing the abundance of flies at different lake levels was developed. This habitat-based population model predicts that the abundance of the alkali fly is maximized at 6380 ft (1945 m) lake surface elevation. Most of the tufa substrate submerged at this lake level will become exposed and unavailable as habitat as the lake declines to 6370 ft (1942 m). In late 1991, the lake level was just over 6374 ft (1943 + m).     

Fine structure of aphid stylet routes in plant tissues in correlation with EPG signals

Abstract. Plant penetration by Aphis fabae (Scopoli) was recorded by the electrical penetration graph (EPG) technique and followed by stylectomy during wave-forms that were suspected of indicating sieve element punctures. The severed stylets in the plant tissue were subsequently processed for transmission electron microscopy (TEM) and sectioned either transverse or longitudinal to the stylets. Two completely serially sectioned probes from the epidermis to the phloem were reconstructed.
In one probe the stylet pathway went to a sieve element and showed many empty branches of salivary sheath material. Breaks in cell walls filled with sheath material demonstrated that the majority of cells bordering the track had been punctured, which supports earlier evidence from EPGs. All types of cells showed punctures and the highest number was found inside the vascular bundle. Very few cells died, which would appear to be important for virus transmission, and in others cellular reactions remained limited to some callose formation. The route of the stylets was intercellular and passed through the secondary wall material. The role of pectinase in intercellular penetration, and previous evidence for intracellular tracks are discussed. Most sieve elements had been punctured but only one was eventually accepted. Thus, reaching a sieve element in a host plant does not automatically imply its acceptance though the reason remains unclear. Gelation of phloem proteins was shown in the stylet canal.
In a second probe, plant cytological and morphological correlations with the EPG were emphasized. Probes by other aphid-plant combinations showed great similarity.     

Understanding the links between snoring,OSA and aortic root pathologies in Marfan syndrome

Sleep and Biological Rhythms -     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Hatching plasticity in a Southeast Asian tree frog mitigates submergence-induced mortality

Environmentally cued hatching has been well-documented in amphibians in response to a wide range of abiotic and biotic factors. The hatching of terrestrial amphibian eggs in response to flooding may be basal within the group, but amphibian lineages in tropical Asia and sub-Saharan Africa have not received as much attention as their Neotropical counterparts. We investigated submergence-induced hatching in Feihyla hansenae, a Rhacophorid tree frog with terrestrial eggs. We quantified natural rates of clutch submergence at our study site in Thailand. Using submergence experiments, we found that embryos are capable of hatching early to escape flooding, and that failure to hatch results in mortality. Among the embryos that were able to hatch early, only the earliest, youngest hatchlings experienced a trade-off in body size that persisted for 6 days, while later, older hatchlings were not significantly smaller than spontaneous hatchlings under control conditions. By incorporating our natural and experimental data into Monte Carlo methods to simulate and compare survival probabilities with and without hatching plasticity, we found an overall 3.1% increase in submergence survival due to hatching plasticity. Our findings support the idea that flooding-induced hatching is widespread across amphibians with terrestrial eggs and highlight the importance of researching understudied tropical regions. As climate change is projected to affect rainfall patterns, the ability of embryos to escape abiotic egg-stage threats may be an indicator of species' ability to flexibly navigate a changing environment.     

Ecomorphological constraints imposed by the kidney component measurements in honeyeater birds inhabiting different environments

The histological renal anatomy of 10 species of honeyeaters was examined quantitatively, using stereology. The kidneys of five species of predominantly wet zone inhabiting birds: the western spinebill Acanthorhynchus superciliosus , White-checked honeyeater Phylidonyris nigra . New Holland honeyeater Phylidonyris novaehollandiae , little wattlebird anthochaera chrysoptera and red wattlebird Anthochaera carunculata , were compared to five predominantly arid zone inhabiting birds: the grey-fronted honeyeater Meliphaga plumula , white-plumed honeyeater Meliphaga penicillata , white-fronted honeyeater Phylidonyris albifrons , spiny-checked honeyeater Acanthogenys rufogularis and yellow-throated miner Manorina flarigula. The kidneys were asymmetrical, with the left kidney being larger than the right kidney. Kidney mass was directly proportional to body mass (coefficient of correlation, r =+0·95), as was kidney volume to kidney mass (r =+1·0). Wet zone honeyeaters generally and a higher percentage and absolute volume of renal cortex, whilst arid zone honeyeaters generally had a significantly higher percentage and absolute volume of renal medulla. There were few differences between species, in either the percentage or absolute volume or luminal surface area of nephron components within the cortex. Within the medulla, wet zone honeyeaters generally had a higher percentage and absolute luminal surface area of collecting ducts, whilst arid zone honeyeaters had a higher percentage and absolute surface area of capillaries. This may be due to factors such as variations in diet and climate between habitats resulting in differences in honeyeater renal morphology which enable arid zone birds to conserve water and wet zone birds to conserve ions more efficiently.