Topological constraints and modular structure in the folding and functional motions of GlpG, an intramembrane protease.

We investigate the folding of GlpG, an intramembrane protease, using perfectly funneled structure-based models that implicitly account for the absence or presence of the membrane. These two models are used to describe, respectively, folding in detergent micelles and folding within a bilayer, which effectively constrains GlpG's topology in unfolded and partially folded states. Structural free-energy landscape analysis shows that although the presence of multiple folding pathways is an intrinsic property of GlpG's modular functional architecture, the large entropic cost of organizing helical bundles in the absence of the constraining bilayer leads to pathways that backtrack (i.e., local unfolding of previously folded substructures is required when moving from the unfolded to the folded state along the minimum free-energy pathway). This backtracking explains the experimental observation of thermodynamically destabilizing mutations that accelerate GlpG's folding in detergent micelles. In contrast, backtracking is absent from the model when folding is constrained within a bilayer, the environment in which GlpG has evolved to fold. We also characterize a near-native state with a highly mobile transmembrane helix 5 (TM5) that is significantly populated under folding conditions when GlpG is embedded in a bilayer. Unbinding of TM5 from the rest of the structure exposes GlpG's active site, consistent with studies of the catalytic mechanism of GlpG that suggest that TM5 serves as a substrate gate to the active site.

Rapid protein disappearance rates along the small intestine advantage poultry performance and influence the post-enteral availability of amino acids.

A foundation diet, an intermediate blend and a summit diet were formulated with different levels of soyabean meal, casein and crystalline amino acids to compare 'slow' and 'rapid' protein diets. The diets were offered to male Ross 308 chicks from 7 to 28 d post-hatch and assessed parameters included growth performance, nutrient utilisation, apparent digestibility coefficients and disappearance rates of starch and protein (N) in four small intestinal segments. Digestibility coefficients and disappearance rates of sixteen amino acids in three small intestinal segments and amino acid concentrations in plasma from portal and systemic circulations from the foundation and summit diets were determined. The dietary transition significantly accelerated protein (N) disappearance rates in the distal jejunum and ileum. The transition from foundation to summit diets significantly increased starch digestibility coefficients in the ileum and disappearance rates in all four small intestinal segments. These starch responses were associated with significant enhancements in nutrient utilisation. The dietary transition linearly increased digestibility coefficients and disappearance rates of amino acids in the majority of cases. The summit diet increased plasma concentrations of five amino acids but decreased those of four amino acids relative to the foundation diet to significant extents. Plasma concentrations of free amino acids were higher in the portal than systemic circulations. Rapid protein disappearance rates advantaged poultry performance and influenced post-enteral availability of amino acids. If the underlying mechanisms are to be identified, further research into the impact of protein digestive dynamics on broiler performance is required but appears justified.

Evaluation of ground grain versus pre- and post-pellet whole grain additions to poultry diets via a response surface design.

1. The objective of this study was to compare the effects of pre- and post-pellet whole grain wheat additions to diets on growth performance, gizzard and pancreas development, nutrient utilisation and starch and protein (N) digestibility coefficients in broiler chickens via an equilateral triangle response surface design. 2. The three apical treatments of the equilateral triangle comprised (1A) a standard diet containing 600 g/kg ground wheat, (2B) the same diet containing 600 g/kg pre-pellet whole wheat and (3C) the same diet containing 300 g/kg ground wheat and 300 g/kg post-pellet whole wheat. Seven blends of the three apical diets were located within the triangle to complete the design and a total of 360 male Ross 308 chicks were offered the ten experimental diets from 7 to 28 d post-hatch. Model prediction and response surface plots were generated with R 3.0.3 software. 3. The most efficient FCR of 1.466 was observed in birds offered an almost equal mixture of the pre- and post-pellet whole grain apical dietary treatments, which corresponded to 172 g/kg ground grain, 256 g/kg pre-pellet whole grain, 172 g/kg post-pellet whole grain in a diet containing 600 g/kg wheat. 4. The most efficient energy utilisation (ME:GE ratio of 0.766) was observed in birds offered a blend of the ground grain and pre-pellet whole grain apical dietary treatments which corresponded to a mixture of 384 g/kg pre-pellet whole grain and 216 g/kg ground grain. 5. Pre-pellet whole grain feeding generated the most pronounced responses in increased relative gizzard contents, reduced gizzard pH and increased relative pancreas weights. Consideration is given to the likely differences between pre- and post-pellet whole grain feeding.

Predictive energy landscapes for folding membrane protein assemblies.

We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na(+)-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

AI-Based Discovery and CryoEM Structural Elucidation of a KATP Channel Pharmacochaperone.

Pancreatic KATP channel trafficking defects underlie congenital hyperinsulinism (CHI) cases unresponsive to the KATP channel opener diazoxide, the mainstay medical therapy for CHI. Current clinically used KATP channel inhibitors have been shown to act as pharmacochaperones and restore surface expression of trafficking mutants; however, their therapeutic utility for KATP trafficking impaired CHI is hindered by high-affinity binding, which limits functional recovery of rescued channels. Recent structural studies of KATP channels employing cryo-electron microscopy (cryoEM) have revealed a promiscuous pocket where several known KATP pharmacochaperones bind. The structural knowledge provides a framework for discovering KATP channel pharmacochaperones with desired reversible inhibitory effects to permit functional recovery of rescued channels. Using an AI-based virtual screening technology AtomNet® followed by functional validation, we identified a novel compound, termed Aekatperone, which exhibits chaperoning effects on KATP channel trafficking mutations. Aekatperone reversibly inhibits KATP channel activity with a half-maximal inhibitory concentration (IC50) ~ 9 µM. Mutant channels rescued to the cell surface by Aekatperone showed functional recovery upon washout of the compound. CryoEM structure of KATP bound to Aekatperone revealed distinct binding features compared to known high affinity inhibitor pharmacochaperones. Our findings unveil a KATP pharmacochaperone enabling functional recovery of rescued channels as a promising therapeutic for CHI caused by KATP trafficking defects.

AI Based Discovery of a New AKR1C3 Inhibitor for Anticancer Applications.

AKR1C3 is an upregulated enzyme in prostate and other cancers; in addition to regulating hormone synthesis, this enzyme is thought to play a role in the aggressiveness of tumors and in the defense against drugs. We here used an unbiased method to discover new potent AKR1C3 inhibitors: through an AI-based virtual drug screen, compound 4 was identified as a potent and selective enzymatic inhibitor able to translate this activity into a pronounced antiproliferative effect in the 22RV1 prostate cancer cell model. As other known AKR1C3 inhibitors, compound 4 determined a significantly increased activity of abiraterone, a drug approved for advanced prostate cancer. Compound 4 also showed a synergic effect with doxorubicin in osteosarcoma cell lines; specifically, the effect is correlated with AKR1C3 expression. In this research work, therefore, the use of AI allowed the identification of a new structure as an AKR1C3 inhibitor and its potential to enhance the effect of chemotherapeutics.

Funneling and frustration in the energy landscapes of some designed and simplified proteins.

We explore the similarities and differences between the energy landscapes of proteins that have been selected by nature and those of some proteins designed by humans. Natural proteins have evolved to function as well as fold, and this is a source of energetic frustration. The sequence of Top7, on the other hand, was designed with architecture alone in mind using only native state stability as the optimization criterion. Its topology had not previously been observed in nature. Experimental studies show that the folding kinetics of Top7 is more complex than the kinetics of folding of otherwise comparable naturally occurring proteins. In this paper, we use structure prediction tools, frustration analysis, and free energy profiles to illustrate the folding landscapes of Top7 and two other proteins designed by Takada. We use both perfectly funneled (structure-based) and predictive (transferable) models to gain insight into the role of topological versus energetic frustration in these systems and show how they differ from those found for natural proteins. We also study how robust the folding of these designs would be to the simplification of the sequences using fewer amino acid types. Simplification using a five amino acid type code results in comparable quality of structure prediction to the full sequence in some cases, while the two-letter simplification scheme dramatically reduces the quality of structure prediction.

Intensification of Penaeid Shrimp Culture: An Applied Review of Advances in Production Systems, Nutrition and Breeding.

Intensification of the shrimp sector, also referred to as vertical expansion, has been predominately driven by consecutive incidences of global disease outbreaks, which have caused enormous economic loss for the main producer countries. A growing segment of the shrimp farming industry has opted to use production systems with higher density, biosecurity, and operating control to mitigate the risks posed by disease. However, successful super-intensive shrimp production is reliant on an advanced understanding of many important biological and economic parameters in the farming system, coupled with effective monitoring, to maintain optimal production. Compared to traditional extensive or semi-intensive systems, super-intensive systems require higher inputs of feed, energy, labor, and supplements. These systems are highly sensitive to the interactions between these different inputs and require that the biological and economical parameters of farming are carefully balanced to ensure success. Advancing nutritional knowledge and tools to support consistent and efficient production of shrimp in these high-cost super-intensive systems is also necessary. Breeding programs developing breeding-lines selected for these challenging super-intensive environments are critical. Understanding synergies between the key areas of production systems, nutrition, and breeding are crucial for super-intensive farming as all three areas coalesce to influence the health of shrimp and commercial farming success. This article reviews current strategies and innovations being used for Litopenaeus vannamei in production systems, nutrition, and breeding, and discusses the synergies across these areas that can support the production of healthy and high-quality shrimp in super-intensive systems. Finally, we briefly discuss some key issues of social license pertinent to the super-intensive shrimp farming industry.

Determining the Importance of Macro and Trace Dietary Minerals on Growth and Nutrient Retention in Juvenile Penaeus monodon.

Twelve minerals were screened to identify key dietary minerals important for Penaeus monodon growth. The minerals selected included boron, calcium plus phosphorus (assessed in tandem at a 1:1 ratio), cobalt, copper, magnesium, manganese, potassium, selenium, sodium, strontium and zinc. Twelve purified casein/gelatin-based diets were formulated and contained minerals at two levels: below expected requirements, as attributed by the basal formulation (-) and above expected requirements by adding inorganic minerals (+). The two levels were allocated to dietary treatments in juvenile prawns in accordance with the PB design. A two-level screening analysis was employed where effect of each mineral at level - or + across twelve diets were compared to identify the minerals of importance for culture performance of juvenile prawns. Calcium plus phosphorus (at a 1:1 ratio), magnesium, boron, manganese, selenium and zinc produced the greatest positive effects on weight gain, feed conversion efficiency, biomass gain and nutrient/energy retention. Particularly, boron and manganese significantly increased retention of key macronutrients and energy including gross energy, crude protein and crude lipid. Our study demonstrates the importance of several macro and trace minerals in prawn diets and the pressing need to refine their requirements for P. monodon.

Outlook: Sorghum as a feed grain for Australian chicken-meat production.

This review is an outlook for sorghum as a feed grain for broiler chickens based on a survey of relevant stake-holders and recent research outcomes. Australian grain sorghum production will probably continue to generate a harvest in the order of 2.5 × 106 t of which some 7.9 × 105 t will be used as a feed grain for poultry and pigs. Feed grains are included primarily to provide energy from starch, but energy utilisation by broiler chickens offered sorghum-based diets is relatively inferior, because of incomplete starch digestion. Kafirin, the dominant protein fraction, 'non-tannin' phenolic compounds and phytate are 3 'starch extrinsic' factors in sorghum that compromise starch digestibility and energy utilisation in broiler chickens offered sorghum-based diets. Kafirin concentrations in 6 sorghum varieties were negatively correlated with metabolizable energy to gross energy (ME:GE) ratios (r = -0.891; P < 0.02) or the efficiency of energy utilisation in broiler chickens. Importantly, kafirin proportions of sorghum protein may be increasing with time in Australia. If so, this represents a fundamental challenge to sorghum breeders which presumably could be met by the development of sorghum varieties with different characteristics, especially in relation to the γ- and ß-kafirin fractions. White sorghum varieties contain lower polyphenol concentrations which should be advantageous as concentrations of total phenolic compounds were negatively correlated to ME:GE ratios (r = -0.838; P < 0.04) in 6 sorghum varieties. It would be desirable if more white varieties were to become available. It is suggested that responses to exogenous phytase in birds offered sorghum-based diets would be more robust if sorghum were to contain lower concentrations of kafirin and phenolic compounds. Paradoxically, while better sorghum varieties almost certainly could be developed, it may not necessarily follow that they will command a price premium from poultry and pig producers.

The influence of the selection of macronutrients coupled with dietary energy density on the performance of broiler chickens.

A total of 360 male Ross 308 broiler chickens were used in a feeding study to assess the influence of macronutrients and energy density on feed intakes from 10 to 31 days post-hatch. The study comprised ten dietary treatments from five dietary combinations and two feeding approaches: sequential and choice feeding. The study included eight experimental diets and each dietary combination was made from three experimental diets. Choice fed birds selected between three diets in separate feed trays at the same time; whereas the three diets were offered to sequentially fed birds on an alternate basis during the experimental period. There were no differences between starch and protein intakes between choice and sequentially fed birds (P > 0.05) when broiler chickens selected between diets with different starch, protein and lipid concentrations. When broiler chickens selected between diets with different starch and protein but similar lipid concentrations, both sequentially and choice fed birds selected similar ratios of starch and protein intake (P > 0.05). However, when broiler chickens selected from diets with different protein and lipid but similar starch concentrations, choice fed birds had higher lipid intake (129 versus 118 g/bird, P = 0.027) and selected diets with lower protein concentrations (258 versus 281 g/kg, P = 0.042) than birds offered sequential diet options. Choice fed birds had greater intakes of the high energy diet (1471 g/bird, P < 0.0001) than low energy (197 g/bird) or medium energy diets (663 g/bird) whilst broiler chickens were offered diets with different energy densities but high crude protein (300 g/kg) or digestible lysine (17.5 g/kg) concentrations. Choice fed birds had lower FCR (1.217 versus 1.327 g/g, P < 0.0001) and higher carcass yield (88.1 versus 87.3%, P = 0.012) than sequentially fed birds. This suggests that the dietary balance between protein and energy is essential for optimal feed conversion efficiency. The intake path of macronutrients from 10-31 days in choice and sequential feeding groups were plotted and compared with the null path if broiler chickens selected equal amounts of the three diets in the combination. Regardless of feeding regimen, the intake paths of starch and protein are very close to the null path; however, lipid and protein intake paths in choice fed birds are father from the null path than sequentially fed birds.

The potential of rapid visco-analysis starch pasting profiles to gauge the quality of sorghum as a feed grain for chicken-meat production.

Thirteen extensively characterised grain sorghum varieties were evaluated in a series of 7 broiler bioassays. The efficiency of energy utilisation of broiler chickens offered sorghum-based diets is problematic and the bulk of dietary energy is derived from sorghum starch. For this reason, rapid visco-analysis (RVA) starch pasting profiles were determined as they may have the potential to assess the quality of sorghum as a feed grain for chicken-meat production. In review, it was found that concentrations of kafirin and total phenolic compounds were negatively correlated with peak and holding RVA viscosities to significant extents across 13 sorghums. In a meta-analysis of 5 broiler bioassays it was found that peak, holding, breakdown and final RVA viscosities were positively correlated with ME:GE ratios and peak and breakdown RVA viscosities with apparent metabolizable energy corrected for nitrogen (AMEn) to significant extents. In a sixth study involving 10 sorghum-based diets peak, holding and breakdown RVA viscosities were positively correlated with ME:GE ratios and AMEn. Therefore, it emerged that RVA starch pasting profiles do hold promise as a relatively rapid means to assess sorghum quality as a feed grain for chicken-meat production. This potential appears to be linked to quantities of kafirin and total phenolic compounds present in sorghum and it would seem that both factors depress RVA starch viscosities in vitro and, in turn, also depress energy utilisation in birds offered sorghum-based diets. Given that other feed grains do not contain kafirin and possess considerably lower concentrations of phenolic compounds, their RVA starch pasting profiles may not be equally indicative.

Reducing agent and exogenous protease additions, individually and in combination, to wheat- and sorghum-based diets interactively influence parameters of nutrient utilisation and digestive dynamics in broiler chickens.

The objective of the study was to investigate the possibility that tandem inclusions of a reducing agent and a protease may advantage chicken-meat production and to ascertain if the established benefits of including sodium metabisulphite in sorghum-based diets extend to wheat-based diets. The study comprised a 2 × 2 × 2 factorial array of treatments in which either nutritionally iso-nitrogenous and iso-energetic wheat- or sorghum-based diets, without and with sodium metabisulphite (2.75 g/kg), without and with protease (1,000 units/kg) were offered to broiler chickens from 7 to 28 days post-hatch. The effects of dietary treatments on growth performance, nutrient utilisation, protein (N) and starch digestibility coefficients and digestive dynamics were determined. A preliminary investigation into the effects of two treatments on concentrations of free amino acids and glucose in the portal circulation was conducted. There was significant feed grain by sodium metabisulphite interactions (P = 0.03 to 0.005) for parameters of nutrient utilisation (AME, ME:GE ratios, N retention, AMEn). For example, sodium metabisulphite inclusions in sorghum-based diets enhanced AME by 0.18 MJ (12.47 versus 12.29 MJ/kg) but depressed AME by 0.43 MJ (11.88 versus 12.31 MJ/kg) in wheat-based diets. There was a linear relationship between starch:protein disappearance rate ratios in the distal ileum with weight gain (r = -0.484; P = 0.0012) indicating that condensed ratios (or absorption of more protein relative to starch) advantaged growth performance. Concentrations of free amino acids in the portal circulation or the post-enteral availability of certain amino acids, including the branched-chain amino acids, methionine, phenylalanine and threonine, were significantly correlated to FCR. For example, threonine concentrations were negatively correlated to FCR (r = -0.773; P = 0.005). Finally, tandem inclusions of sodium metabisulphite and protease in sorghum-based diets may hold merit but it appears that the established 'energy sparing' effects of sodium metabisulphite inclusions in sorghum-based diets are not duplicated in wheat-based diets.

Performance of broiler chickens offered nutritionally-equivalent diets based on two red grain sorghums with quantified kafirin concentrations as intact pellets or re-ground mash following steam-pelleting at 65 or 97°C conditioning temperatures.

The Liverpool Plains is a fertile agricultural region in New South Wales, Australia. Two sorghums from the 2009 Liverpool Plains harvest, sorghums #3 and #5, were extensively characterised which included concentrations of kafirin and phenolic compounds plus rapid visco-analysis (RVA) starch pasting profiles. Diets based on these two sorghums were formulated to be iso-nitrogenous and iso-energetic and were offered to male Ross 308 broiler chicks from 7 to 28 days post--hatch as either intact pellets or reground mash following steam-pelleting at conditioning temperatures of either 65 or 97°C. Thus the feeding study consisted of a 2 × 2 × 2 factorial array of dietary treatments: two sorghum varieties, two feed forms and two conditioning temperatures. Each of the eight treatments was replicated six times with six birds per replicate cage. Assessed parameters included growth performance, nutrient utilisation, apparent starch and protein (N) digestibility coefficients and disappearance rates from the distal jejunum and distal ileum. Intact pellets supported higher (P < 0.001) feed intakes and weight gains by 9.83 and 9.08%, respectively, than reground mash diets. Feed conversion ratios of broilers offered diets steam-conditioned at 97°C were 2.46% inferior (P < 0.001) in comparison to 65°C diets and both apparent metabolizable energy (AME) and N-corrected AME (AMEn) were compromised. Broilers offered sorghum #3-based diets significantly (P < 0.001) outperformed their sorghum #5 counterparts in terms of weight gain by 3.75% (1,334 versus 1,223 g/bird), FCR by 4.81% (1.524 versus 1.601), AME by 1.06 MJ (13.61 versus 12.55 MJ/kg), ME:GE ratio (ME:GE) by 4.81% (0.806 versus 0.769) and AMEn by 1.03 MJ (12.38 versus 11.35 MJ/kg). The inferiority of sorghum #5 appeared to be associated with higher concentrations of kafirin (61.5 versus 50.7 g/kg) and conjugated phenolic acids, including ferulic acid (31.1 versus 25.6 µg/g). There were no significant differences in jejunal and ileal starch and protein (N) digestibility coefficients between the two sorghums. However, starch to protein (N) disappearance rate ratios from the distal jejunum were significantly (P < 0.001) correlated with ME:GE and AME. The multiple linear regression equations indicated that energy utilisation was enhanced by coupling rapidly digestible protein with slowly digestible starch, which suggests that bilateral bioavailability of starch and protein is pivotal to efficient energy utilisation.