Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.     

Complete genome sequence of Ignisphaera aggregans type strain (AQ1.S1)

Ignisphaera aggregans Niederberger et al. 2006 is the type and sole species of genus Ignisphaera. This archaeal species is characterized by a coccoid-shape and is strictly anaerobic, moderately acidophilic, heterotrophic hyperthermophilic and fermentative. The type strain AQ1.S1(T) was isolated from a near neutral, boiling spring in Kuirau Park, Rotorua, New Zealand. This is the first completed genome sequence of the genus Ignisphaera and the fifth genome (fourth type strain) sequence in the family Desulfurococcaceae. The 1,875,953 bp long genome with its 2,009 protein-coding and 52 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Predation of <Emphasis Type="Italic">Halyomorpha halys</Emphasis> (Hemiptera: Pentatomidae) from Web-Building Spiders Associated with Anthropogenic Dwellings

The brown marmorated stink bug, or Halyomorpha halys, is an invasive pest in North America and Europe that causes severe agricultural damage and nuisance problems for homeowners; and it is originally from China, Taiwan, and the Republic of Korea. While the natural enemy community of H. halys has been evaluated in several agroecosystems, it has not been examined where H. halys overwinters in anthropogenic structures. The aims of the current study were to evaluate 1) whether spider webs commonly found in the home and yard can successfully ensnare H. halys, 2) whether entanglement resulted in consumption by spiders inhabiting the webs, and 3) how frequently H. halys becomes entangled in webs under ambient conditions. To accomplish this, adult H. halys were introduced into webs in and near anthropogenic structures in West Virginia and Maryland, United States, and the behavior of spiders was observed for 5-min intervals at 0, 1, 2, and 24 h after introduction. In addition, a survey of webs was performed to determine the frequency with which spiders naturally capture H. halys inside buildings and in the landscape. Overall, the study found seven spider families in anthropogenic structures. Adult H. halys that were introduced into the webs of Theridiidae, Pholcidae, or Agelenidae had a greater than 50% chance of being ensnared and consumed. Adult H. halys were found naturally most often in webs of Theridiidae. Webs with a funnel or cob web architecture had the greatest probability of ensnaring H. halys, while those with orb structures resulted in the fewest caught. In the wild, 13–20% of spider webs contained dead H. halys. Our results suggest that spiders may be an important contributing factor for mortality of H. halys at overwintering sites, and spiders in or outside homes may help reduce nuisance problems caused by H. halys.     

Biochanin A improves fibre fermentation by cellulolytic bacteria

Aims

The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity.

Methods and Results

When bovine microbial rumen cell suspensions (n = 3) were incubated (24 h, 39°C) with ground hay, cellulolytic bacteria proliferated, short‐chain fatty acids were produced and pH declined. BCA (30 μg ml^?1) had no effect on the number of cellulolytic bacteria or pH, but increased acetate, propionate and total SCFA production. Addition of BCA improved total digestibility when cell suspensions (n = 3) were incubated (48 h, 39°C) with ground hay, Avicel, or filter paper. Fibrobacter succinogenes S85, Ruminococcus flavefaciens 8 and Ruminococcus albus 8 were directly inhibited by BCA. Synergistic antimicrobial activity was observed with BCA and heat killed cultures of cellulolytic bacteria, but the effects were species dependent.

Conclusions

These results indicate that BCA improves fibre degradation by influencing cellulolytic bacteria competition and guild composition.

Significance and Impact of the Study

BCA could serve as a feed additive to improve cellulosis when cattle are consuming high‐fibre diets. Future research is needed to evaluate the effect of BCA on fibre degradation and utilization in vivo.     

Microbial synthesis of the sweet-tasting plant protein thaumatin

Thaumatin, the intensely sweet-tasting protein from the African plant, Thaumatococcus daniellii, is a potential low-caloric substitute for sugar and an attractive compound for studying the structural aspects of sweetness perception. Since thaumatin has other properties, such as flavour enhancement, it could also be used in products other than low-calorie foods. Recombinant DNA techniques could be used to synthesize this protein in quantities sufficient for such applications.     

The next generation of protein super-fibres: robust recombinant production and recovery of hagfish intermediate filament proteins with fibre spinning and mechanical–structural characterizations

Native hagfish intermediate filament proteins have impressive mechanical properties. However, using these native fibres for any application is impractical, necessitating their recombinant production. In the only literature report on the proteins (denoted α and ɣ), heterologous expression levels, using E. coli, were low and no attempts were made to optimize expression, explore wet-spinning, or spin the two proteins individually into fibres. Reported here is the high production (~8 g l⁻¹ of dry protein) of the hagfish intermediate filament proteins, with yields orders of magnitude higher (325–1000×) than previous reports. The proteins were spun into fibres individually and in their native-like 1:1 ratio. For all fibres, the hallmark α-helix to β-sheet conversion occurred after draw-processing. The native-like 1:1 ratio fibres achieved the highest average tensile strength in this study at nearly 200 MPa with an elastic modulus of 5.7 GPa, representing the highest tensile strength reported for these proteins without chemical cross-linking. Interestingly, the recombinant α protein achieved nearly the same mechanical properties when spun as a homopolymeric fibre. These results suggest that varying the two protein ratios beyond the natural 1:1 ratio will allow a high degree of tunability. With robust heterologous expression and purification established, optimizing fibre spinning will be accelerated compared to difficult to produce proteins such as spider silks.     

Effects of predation environment and food availability on somatic growth in the Livebearing Fish Brachyrhaphis rhabdophora (Pisces: Poeciliidae)

Variation in somatic growth rates is of great interest to biologists because of the relationship between growth and other fitness‐determining traits, and it results from both genetic and environmentally induced variation (i.e. plasticity). Theoretical predictions suggest that mean somatic growth rates and the shape of the reaction norm for growth can be influenced by variation in predator‐induced mortality rates. Few studies have focused on variation in reaction norms for growth in response to resource availability between high‐predation and low‐predation environments. We used juvenile Brachyrhaphis rhabdophora from high‐predation and low‐predation environments to test for variation in mean growth rates and for variation in reaction norms for growth at two levels of food availability in a common‐environment experiment. To test for variation in growth rates in the field, we compared somatic growth rates in juveniles in high‐predation and low‐predation environments. In the common‐environment experiment, mean growth rates did not differ between fish from differing predation environments, but the interaction between predation environment and food level took the form of a crossing reaction norm for both growth in length and mass. Fish from low‐predation environments exhibited no significant difference in growth rate between high and low food treatments. In contrast, fish from high‐predation environments exhibited variation in growth rates between high and low food treatments, with higher food availability resulting in higher growth rates. In the field, individuals in the high‐predation environment grow at a faster rate than those in low‐predation environments at the smallest sizes (comparable to sizes in the common‐environment experiment). These data provide no evidence for evolved differences in mean growth rates between predation environments. However, fish from high‐predation environments exhibited greater plasticity in growth rates in response to resource availability suggesting that predation environments may exhibit increased variation in food availability for prey fish and consequent selection for plasticity.     

Inferential biases linked to unobservable states in complex occupancy models

Modeling of species distributions has undergone a shift from relying on equilibrium assumptions to recognizing transient system dynamics explicitly. This shift has necessitated more complex modeling techniques, but the performance of these dynamic models has not yet been assessed for systems where unobservable states exist. Our work is motivated by the impacts of the emerging infectious disease chytridiomycosis, a disease of amphibians that is associated with declines of many species worldwide. Using this host‐pathogen system as a general example, we first illustrate how misleading inferences can result from failing to incorporate pathogen dynamics into the modeling process, especially when the pathogen is difficult or impossible to survey in the absence of a host species. We found that traditional modeling techniques can underestimate the effect of a pathogen on host species occurrence and dynamics when the pathogen can only be detected in the host, and pathogen information is treated as a covariate. We propose a dynamic multistate modeling approach that is flexible enough to account for the detection structures that may be present in complex multistate systems, especially when the sampling design is limited by a species’ natural history or sampling technology. When multistate occupancy models are used and an unobservable state is present, parameter estimation can be influenced by model complexity, data sparseness, and the underlying dynamics of the system. We show that, even with large sample sizes, many models incorporating seasonal variation in vital rates may not generate reasonable estimates, indicating parameter redundancy. We found that certain types of missing data can greatly hinder inference, and we make study design recommendations to avoid these issues. Additionally, we advocate the use of time‐varying covariates to explain temporal trends in the data, and the development of sampling techniques that match the biology of the system to eliminate unobservable states when possible.     

Synthesis,characterization, and biological activity of poly(arginine)‐derived cancer‐targeting peptides in HepG2 liver cancer cells

The solid‐phase synthesis, structural characterization, and biological evaluation of a small library of cancer‐targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose‐regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3–12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14–46%) and crude purities >95% as analyzed by liquid chromatography–mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure–activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose‐regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose‐response (0–1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer‐targeting delivery agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.