Retroactive Maintains Cuticle Integrity by Promoting the Trafficking of Knickkopf into the Procuticle of Tribolium castaneum

Molting, or the replacement of the old exoskeleton with a new cuticle, is a complex developmental process that all insects must undergo to allow unhindered growth and development. Prior to each molt, the developing new cuticle must resist the actions of potent chitinolytic enzymes that degrade the overlying old cuticle. We recently disproved the classical dogma that a physical barrier prevents chitinases from accessing the new cuticle and showed that the chitin-binding protein Knickkopf (Knk) protects the new cuticle from degradation. Here we demonstrate that, in Tribolium castaneum, the protein Retroactive (TcRtv) is an essential mediator of this protective effect of Knk. TcRtv localizes within epidermal cells and specifically confers protection to the new cuticle against chitinases by facilitating the trafficking of TcKnk into the procuticle. Down-regulation of TcRtv resulted in entrapment of TcKnk within the epidermal cells and caused molting defects and lethality in all stages of insect growth, consistent with the loss of TcKnk function. Given the ubiquity of Rtv and Knk orthologs in arthropods, we propose that this mechanism of new cuticle protection is conserved throughout the phylum.     

Effect of disorder and polarization sequences on two-dimensional spectra of light-harvesting complexes

Photosynthesis Research - Two-dimensional electronic spectra (2DES) provide unique ways to track the energy transfer dynamics in light-harvesting complexes. The interpretation of the peaks and...     

Effects of Wild Pig Disturbance on Forest Vegetation and Soils

In North America, wild pigs (Sus scrofa; feral pigs, feral swine, wild boars) are a widespread exotic species capable of creating large-scale biotic and abiotic landscape perturbations. Quantification of wild pig environmental effects has been particularly problematic in northern climates, where they occur only recently as localized populations at low densities. Between 2016 and 2017, we assessed short-term (within ~2 yrs of disturbance) effects of a low-density wild pig population on forest features in the central Lower Peninsula of Michigan, USA. We identified 16 8-ha sites using global positioning system locations from 7 radio-collared wild pigs for sampling. Within each site, we conducted fine-scale assessments at 81 plots and quantified potential disturbance by wild pigs. We defined disturbance as exposure of overturned soil, often resulting from rooting behavior by wild pigs. We quantified ground cover of plants within paired 1-m² frames at each plot, determined effects to tree regeneration using point-centered quarter sampling, and collected soil cores from each plot. We observed less percent ground cover of native herbaceous plants and lower species diversity, particularly for plants with a coefficient of conservatism ≥5, in plots disturbed by wild pigs. We did not observe an increase in colonization of exotic plants following disturbance, though the observed prevalence of exotic plants was low. Wild pigs did not select for tree species when rooting, and we did not detect any differences in regeneration of light- and heavy-seeded tree species between disturbed or undisturbed plots. Magnesium and ammonium content in soils were lower in disturbed plots, suggesting soil disturbance accelerated leaching of macronutrients, potentially altering nitrogen transformation. Our study suggested that disturbances by wild pigs, even at low densities, alters short-term native herbaceous plant diversity and soil chemistry. Thus, small-scale exclusion of wild pigs from vulnerable and rare plant communities may be warranted. © 2020 The Wildlife Society.     

Honokiol: A non-adipogenic PPARγ agonist from nature

Background

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators.

Methods

We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists.

Results

The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain.

Conclusion

We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo.

General significance

This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.     

Holding behavior of Chinook salmon (Oncorhynchus tshawytscha) and steelhead (O. mykiss) smolts, as influenced by habitat features of levee banks, in the highly modified lower Sacramento River, California

Using acoustic telemetry methods on large numbers of tagged fish, we studied how the holding behavior of Chinook salmon and steelhead smolts could be related to habitat features and spatial and temporal variables on a highly altered section of the Sacramento River. We viewed downstream migration as a process in which fish transition between moving and holding states, and used a binomial and negative binomial Generalized Linear Model to analyze two aspects of holding: 1) probability of holding, and 2) holding time. For Chinook salmon, the probability of holding increased as wood size and fine substrates increased; holding time increased as overhead shade increased. For steelhead, holding behavior was only weakly related to habitat variables, in contrast to the strong relationships with spatial and temporal variables. For both species, the probability of holding increased when distance from the release location decreased and instream flows decreased. We found support for three main findings: 1) spatial and temporal factors have considerably greater influence on Chinook salmon and steelhead smolt holding behavior than nearshore habitat features; 2) holding behaviors of Chinook salmon smolts are influenced more strongly by habitat features than steelhead smolts; and 3) incorporation of habitat features such as large woody material and overhead shade should be considered when conducting nearshore bank rehabilitation projects to increase cover from predators and provide velocity refuge, improving holding habitat during downstream migration.     

Behavioural Responses of European Roe Deer to Temporal Variation in Predation Risk

In natural environments, predation risk varies over time. The risk allocation hypothesis predicts that prey is expected to adjust key anti‐predator behaviours such as vigilance to temporal variation in risk. We tested the predictions of the risk allocation hypothesis in a natural environment where both a species‐rich natural predator community and human hunters are abundant and where the differences in seasonal and circadian activity between natural and anthropogenic predators provided a unique opportunity to quantify the contributions of different predator classes to anti‐predator behaviour. Whereas natural predators were expected to show similar levels of activity throughout the seasons, hunter activity was high during the daytime during a clearly defined hunting season. According to the risk allocation hypothesis, vigilance should then be higher during the hunting season and during daytime hours than during the non‐hunting season and night‐time hours. Roe deer (Capreolus capreolus) on the edge of Bia?owie?a Primeval Forest in Eastern Poland displayed vigilance behaviour consistent with these predictions. The behavioural response of roe deer to temporarily varying predation risks emphasises the behavioural plasticity of this species and suggests that future studies of anti‐predator behaviour need to incorporate circadian variation in predation pressure as well as risk gradients of both natural and anthropogenic predators.     

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n‐octyl‐oligo‐oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N‐dimethyldodecylamine N‐oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size‐dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.     

Assessment of DNA damage and repair in adults consuming allyl isothiocyanate or Brassica vegetables

Allyl isothiocyanate (AITC) is a dietary component with possible anticancer effects, though much information about AITC and cancer has been obtained from cell studies. To investigate the effect of AITC on DNA integrity in vivo, a crossover study was conducted. Adults (n= 46) consumed AITC, AITC-rich vegetables [mustard and cabbage (M/C)] or a control treatment with a controlled diet for 10 days each. On day 11, volunteers provided blood and urine before and after consuming treatments. Volunteers were characterized for genotype for GSTM1 and GSTT1 (glutathione S-transferases) and XPD (DNA repair). DNA integrity in peripheral blood mononuclear cells was assessed by single-cell gel electrophoresis. Urine was analyzed for 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and creatinine. Ten-day intake of neither AITC nor M/C resulted in statistically significant differences in DNA strand breaks [least squares mean (LSmean) % DNA in tail±S.E.M.: 4.8±0.6 for control, 5.7±0.7 for AITC, 5.3±0.6 for M/C] or urinary 8-oxodG (LSmean μg 8-oxodG/g creatinine±S.E.M.: 2.95±0.09 for control, 2.88±0.09 for AITC, 3.06±0.09 for M/C). Both AITC and M/C increased DNA strand breaks 3 h postconsumption (LSmean % DNA in tail±S.E.M.: 3.2±0.7 for control, 8.3±1.7 for AITC, 8.0±1.7 for M/C), and this difference disappeared at 6 h (4.2±0.9 for control, 5.7±1.2 for AITC, 5.5±1.2 for M/C). Genotypes for GSTM1, GSTT1 and XPD were not associated with treatment effects. In summary, DNA damage appeared to be induced in the short term by AITC and AITC-rich products, but that damage disappeared quickly, and neither AITC nor AITC-rich products affected DNA base excision repair.     

Comparability of Mixed IC50 Data – A Statistical Analysis

The biochemical half maximal inhibitory concentration (IC₅₀) is the most commonly used metric for on-target activity in lead optimization. It is used to guide lead optimization, build large-scale chemogenomics analysis, off-target activity and toxicity models based on public data. However, the use of public biochemical IC₅₀ data is problematic, because they are assay specific and comparable only under certain conditions. For large scale analysis it is not feasible to check each data entry manually and it is very tempting to mix all available IC₅₀ values from public database even if assay information is not reported. As previously reported for K_i database analysis, we first analyzed the types of errors, the redundancy and the variability that can be found in ChEMBL IC₅₀ database. For assessing the variability of IC₅₀ data independently measured in two different labs at least ten IC₅₀ data for identical protein-ligand systems against the same target were searched in ChEMBL. As a not sufficient number of cases of this type are available, the variability of IC₅₀ data was assessed by comparing all pairs of independent IC₅₀ measurements on identical protein-ligand systems. The standard deviation of IC₅₀ data is only 25% larger than the standard deviation of K_i data, suggesting that mixing IC₅₀ data from different assays, even not knowing assay conditions details, only adds a moderate amount of noise to the overall data. The standard deviation of public ChEMBL IC₅₀ data, as expected, resulted greater than the standard deviation of in-house intra-laboratory/inter-day IC₅₀ data. Augmenting mixed public IC₅₀ data by public K_i data does not deteriorate the quality of the mixed IC₅₀ data, if the K_i is corrected by an offset. For a broad dataset such as ChEMBL database a K_i- IC₅₀ conversion factor of 2 was found to be the most reasonable.     

A Low Cost Metal-Free Vascular Access Mini-Port for Artifact Free Imaging and Repeated Injections in Mice

Purpose

Small injection ports for mice are increasingly used for drug testing or when administering contrast agents. Commercially available mini-ports are expensive single-use items that cause imaging-artifacts. We developed and tested an artifact-free, low-cost, vascular access mini-port (VAMP) for mice.

Procedures

Leakage testing of the VAMP was conducted with high speed bolus injections of different contrast agents. VAMP-induced artifacts were assessed using a micro-CT and a small animal MRI (9.4T) scanner ex vivo. Repeated contrast administration was performed in vivo.

Results

With the VAMP there was no evidence of leakage with repeated punctures, high speed bolus contrast injections, and drawing of blood samples. In contrast to the tested commercially available ports, the VAMP did not cause artifacts with MRI or CT imaging.

Conclusions

The VAMP is an alternative to commercially available mini-ports and has useful applications in animal research involving imaging procedures and contrast agent testing.