Sward type alters enteric methane emissions, nitrogen output and the relative abundance of the rumen microbial ecosystem in sheep.

Observed improvements in animal and sward performance, coupled with a desire for more sustainable pasture-based feeding systems has triggered a surge in the implementation of more botanically diverse pastures. However, thus far, there has been limited research investigating the effects of botanically diverse sward types on enteric methane (CH4) or nitrogen (N) excretion, alongside the ruminal microbiota and fermentation profile, in sheep. Hence, this study investigates the effect of sward type on CH4 production and N excretion, in addition to assessing the rumen microbiome, volatile fatty acid (VFA) proportions, and ammonia nitrogen (NH3-N) concentration in sheep. A 5×5 Latin square design experiment was implemented to investigate five dietary treatments; perennial ryegrass (Lolium perenne L.;PRG) only or PRG plus white clover (Trifolium repens L.;PRG+WC), red clover (Trifolium pratense L.;PRG+RC), chicory (Chicorium intybus L.;PRG+Chic) or plantain (Plantago lanceolata L.;PRG+Plan). Diets were mixed at a ratio of 75% PRG and 25% of the respective companion forage and 100% PRG for the PRG treatment, on a dry matter basis. Twenty castrated male sheep were housed in metabolism crates across five feeding periods. Methane measurements were acquired utilizing portable accumulation chambers. Rumen fluid was harvested using a transoesophageal sampling device. Microbial rumen DNA was extracted and subjected to 16S rRNA amplicon sequencing and fermentation analysis. Data were analysed using PROC MIXED in SAS. Results show that animals consuming PRG+WC ranked lower for CH4 production (g/d) than sheep offered PRG, PRG+Chic or PRG+Plan (P<0.01) while the addition of any companion forage ranked CH4 yield (g/kg DMI) lower (P<0.001) than PRG. There was a moderate positive correlation between DMI and CH4 (g/d; r=0.51). Ruminal NH3-N was lowest in animals consuming the PRG diet (P<0.01). There was a greater abundance of Methanobrevibacter and reduced abundance of Methanosphaera (P<0.001) in sheep offered PRG, compared with any binary sward. On average, herb diets (PRG+Chic or PRG+Plan) reduced urinary nitrogen concentration of sheep by 34% in comparison to legume diets (PRG+WC or PRG+RC) and 13% relative to the PRG diet (P<0.001). Sheep offered PRG+Chic had a greater dietary nitrogen use efficiency than PRG+RC (P<0.05). This study demonstrates the potential for sward type to influence rumen function and the microbial community, along with CH4 and N output from sheep.

Health-associated changes of the fecal microbiota in dairy heifer calves during the pre-weaning period.

Introduction: Neonatal calf diarrhea is a multifactorial condition that occurs in early life when calves are particularly susceptible to enteric infection and dysbiosis of the gut microbiome. Good calf health is dependent on successful passive transfer of immunity from the dam through colostrum. There are limited studies on the developing gut microbiota from birth to weaning in calves. Methodology: Therefore, the objective of this study was to examine the effect of immune status and diarrheal incidence on the development of the fecal microbiota in Jersey (n = 22) and Holstein (n = 29) heifer calves throughout the pre-weaning period. Calves were hand-fed a colostrum volume equivalent to 8.5% of their birthweight, from either the calf's dam (n = 28) or re-heated mixed colostrum (≤2 cows, ≤1d; n = 23) within 2 h of birth. All calves were clinically assessed using a modified Wisconsin-Madison calf health scoring system and rectal temperature at day (d) 0, d7, d21, or disease manifestation (DM) and weaning (d83). Weights were recorded at d0, d21, and d83. Calf blood samples were collected at d7 for the determination of calf serum IgG (sIgG). Fecal samples were obtained at d7, d21/DM [mean d22 (SE 0.70)], and at weaning for 16S rRNA amplicon sequencing of the fecal microbiota. Data were processed in R using DADA2; taxonomy was assigned using the SILVA database and further analyzed using Phyloseq and MaAsLin 2. Results and discussion: Significant amplicon sequence variants (ASVs) and calf performance data underwent a Spearman rank-order correlation test. There was no effect (p > 0.05) of colostrum source or calf breed on serum total protein. An effect of calf breed (p < 0.05) was observed on sIgG concentrations such that Holstein calves had 6.49 (SE 2.99) mg/ml higher sIgG than Jersey calves. Colostrum source and calf breed had no effect (p > 0.05) on health status or the alpha diversity of the fecal microbiota. There was a relationship between health status and time interaction (p < 0.001), whereby alpha diversity increased with time; however, diarrheic calves had reduced microbial diversity at DM. No difference (p > 0.05) in beta diversity of the microbiota was detected at d7 or d83. At the genus level, 33 ASVs were associated (adj.p < 0.05) with health status over the pre-weaning period.

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution. Indeed, in some globular proteins, the full tuneable range can be accessed by the set of site-saturating substitutions at an individual "rheostat" position. However, in proteins with intrinsically disordered regions (IDRs), most functional studies-which would also detect tuneability-used multiple substitutions or small deletions. In disordered transcriptional activation domains (ADs), studies with multiple substitutions led to the "acidic exposure" model, which does not anticipate the existence of rheostat positions. In the few studies that did assess effects of single substitutions on AD function, results were mixed: the ADs of two full-length transcription factors did not show tuneability, whereas a fragment of a third AD was tuneable by single substitutions. In this study, we tested tuneability in the AD of full-length human class II transactivator (CIITA). Sequence analyses and experiments showed that CIITA's AD is an IDR. Functional assays of singly-substituted AD variants showed that CIITA's function was highly tuneable, with outcomes not predicted by the acidic exposure model. Four tested positions showed rheostat behavior for transcriptional activation. Thus, tuneability of different IDRs can vary widely. Future studies are needed to illuminate the biophysical features that govern whether an IDR is tuneable by single substitutions.

Simulated pressure changes in LacI suggest a link between hydration and functional conformational changes.

The functions of many proteins are associated with interconversions among conformational substates. However, these substates can be difficult to measure experimentally, and determining contributions from hydration changes can be especially difficult. Here, we assessed the use of pressure perturbations to sample the substates accessible to the Escherichia coli lactose repressor protein (LacI) in various liganded forms. In the presence of DNA, the regulatory domain of LacI adopts an Open conformation that, in the absence of DNA, changes to a Closed conformation. Increasing the simulation pressure prevented the transition from an Open to a Closed conformation, in a similar manner to the binding of DNA and anti-inducer, ONPF. The results suggest the hydration of specific residues play a significant role in determining the population of different LacI substates and that simulating pressure perturbation could be useful for assessing the role of hydration changes that accompany functionally-relevant amino acid substitutions.

Breed and ruminal fraction effects on bacterial and archaeal community composition in sheep.

While the breed of cattle can impact on the composition and structure of microbial communities in the rumen, breed-specific effects on rumen microbial communities have rarely been examined in sheep. In addition, rumen microbial composition can differ between ruminal fractions, and be associated with ruminant feed efficiency and methane emissions. In this study, 16S rRNA amplicon sequencing was used to investigate the effects of breed and ruminal fraction on bacterial and archaeal communities in sheep. Solid, liquid and epithelial rumen samples were obtained from a total of 36 lambs, across 4 different sheep breeds (Cheviot (n = 10), Connemara (n = 6), Lanark (n = 10) and Perth (n = 10)), undergoing detailed measurements of feed efficiency, who were offered a nut based cereal diet ad-libitum supplemented with grass silage. Our results demonstrate that the feed conversion ratio (FCR) was lowest for the Cheviot (most efficient), and highest for the Connemara breed (least efficient). In the solid fraction, bacterial community richness was lowest in the Cheviot breed, while Sharpea azabuensis was most abundant in the Perth breed. Lanark, Cheviot and Perth breeds exhibited a significantly higher abundance of epithelial associated Succiniclasticum compared to the Connemara breed. When comparing ruminal fractions, Campylobacter, Family XIII, Mogibacterium, and Lachnospiraceae UCG-008 were most abundant in the epithelial fraction. Our findings indicate that breed can impact the abundance of specific bacterial taxa in sheep while having little effect on the overall composition of the microbial community. This finding has implications for genetic selection breeding programs aimed at improving feed conversion efficiency of sheep. Furthermore, the variations in the distribution of bacterial species identified between ruminal fractions, notably between solid and epithelial fractions, reveals a rumen fraction bias, which has implications for sheep rumen sampling techniques.

Differences in the Composition of the Rumen Microbiota of Finishing Beef Cattle Divergently Ranked for Residual Methane Emissions.

With the advent of high throughput technology, it is now feasible to study the complex relationship of the rumen microbiota with methanogenesis in large populations of ruminant livestock divergently ranked for enteric emissions. Recently, the residual methane emissions (RME) concept has been identified as the optimal phenotype for assessing the methanogenic potential of ruminant livestock due to the trait's independence from animal productivity but strong correlation with daily methane emissions. However, there is currently a dearth of data available on the bacterial and archaeal microbial communities residing in the rumens of animals divergently ranked for RME. Therefore, the objective of this study was to investigate the relationship between the rumen microbiota and RME in a population of finishing beef cattle. Methane emissions were estimated from individual animals using the GreenFeed Emissions Monitoring system for 21 days over a mean feed intake measurement period of 91 days. Residual methane emissions were calculated for 282 crossbred finishing beef cattle, following which a ∼30% difference in all expressions of methane emissions was observed between high and low RME ranked animals. Rumen fluid samples were successfully obtained from 268 animals during the final week of the methane measurement period using a trans-oesophageal sampling device. Rumen microbial DNA was extracted and subjected to 16S rRNA amplicon sequencing. Animals ranked as low RME had the highest relative abundances (P < 0.05) of lactic-acid-producing bacteria (Intestinibaculum, Sharpea, and Olsenella) and Selenomonas, and the lowest (P < 0.05) proportions of Pseudobutyrivibrio, Butyrivibrio, and Mogibacterium. Within the rumen methanogen community, an increased abundance (P < 0.05) of the genus Methanosphaera and Methanobrevibacter RO clade was observed in low RME animals. The relative abundances of both Intestinibaculum and Olsenella were negatively correlated (P < 0.05) with RME and positively correlated with ruminal propionate. A similar relationship was observed for the abundance of Methanosphaera and the Methanobrevibacter RO clade. Findings from this study highlight the ruminal abundance of bacterial genera associated with the synthesis of propionate via the acrylate pathway, as well as the methanogens Methanosphaera and members of the Methanobrevibacter RO clade as potential microbial biomarkers of the methanogenic potential of beef cattle.

Enteric methane research and mitigation strategies for pastoral-based beef cattle production systems.

Ruminant livestock play a key role in global society through the conversion of lignocellulolytic plant matter into high-quality sources of protein for human consumption. However, as a consequence of the digestive physiology of ruminant species, methane (CH4), which originates as a byproduct of enteric fermentation, is accountable for 40% of global agriculture's carbon footprint and ~6% of global greenhouse gas (GHG) emissions. Therefore, meeting the increasing demand for animal protein associated with a growing global population while reducing the GHG intensity of ruminant production will be a challenge for both the livestock industry and the research community. In recent decades, numerous strategies have been identified as having the potential to reduce the methanogenic output of livestock. Dietary supplementation with antimethanogenic compounds, targeting members of the rumen methanogen community and/or suppressing the availability of methanogenesis substrates (mainly H2 and CO2), may have the potential to reduce the methanogenic output of housed livestock. However, reducing the environmental impact of pasture-based beef cattle may be a challenge, but it can be achieved by enhancing the nutritional quality of grazed forage in an effort to improve animal growth rates and ultimately reduce lifetime emissions. In addition, the genetic selection of low-CH4-emitting and/or faster-growing animals will likely benefit all beef cattle production systems by reducing the methanogenic potential of future generations of livestock. Similarly, the development of other mitigation technologies requiring minimal intervention and labor for their application, such as anti-methanogen vaccines, would likely appeal to livestock producers, with high uptake among farmers if proven effective. Therefore, the objective of this review is to give a detailed overview of the CH4 mitigation solutions, both currently available and under development, for temperate pasture-based beef cattle production systems. A description of ruminal methanogenesis and the technologies used to estimate enteric emissions at pastures are also presented.

Effect of divergence in residual methane emissions on feed intake and efficiency, growth and carcass performance, and indices of rumen fermentation and methane emissions in finishing beef cattle.

Residual expressions of enteric emissions favor a more equitable identification of an animal's methanogenic potential compared with traditional measures of enteric emissions. The objective of this study was to investigate the effect of divergently ranking beef cattle for residual methane emissions (RME) on animal productivity, enteric emissions, and rumen fermentation. Dry matter intake (DMI), growth, feed efficiency, carcass output, and enteric emissions (GreenFeed emissions monitoring system) were recorded on 294 crossbred beef cattle (steers = 135 and heifers = 159; mean age 441 d (SD = 49); initial body weight (BW) of 476 kg (SD = 67)) at the Irish national beef cattle performance test center. Animals were offered a total mixed ration (77% concentrate and 23% forage; 12.6 MJ ME/kg of DM and 12% CP) ad libitum with emissions estimated for 21 d over a mean feed intake measurement period of 91 d. Animals had a mean daily methane emissions (DME) of 229.18 g/d (SD = 45.96), methane yield (MY) of 22.07 g/kg of DMI (SD = 4.06), methane intensity (MI) 0.70 g/kg of carcass weight (SD = 0.15), and RME 0.00 g/d (SD = 0.34). RME was computed as the residuals from a multiple regression model regressing DME on DMI and BW (R2 = 0.45). Animals were ranked into three groups namely high RME (>0.5 SD above the mean), medium RME (±0.5 SD above/below the mean), and low RME (>0.5 SD below the mean). Low RME animals produced 17.6% and 30.4% less (P < 0.05) DME compared with medium and high RME animals, respectively. A ~30% reduction in MY and MI was detected in low versus high RME animals. Positive correlations were apparent among all methane traits with RME most highly associated with (r = 0.86) DME. MY and MI were correlated (P < 0.05) with DMI, growth, feed efficiency, and carcass output. High RME had lower (P < 0.05) ruminal propionate compared with low RME animals and increased (P < 0.05) butyrate compared with medium and low RME animals. Propionate was negatively associated (P < 0.05) with all methane traits. Greater acetate:propionate ratio was associated with higher RME (r = 0.18; P < 0.05). Under the ad libitum feeding regime deployed here, RME was the best predictor of DME and only methane trait independent of animal productivity. Ranking animals on RME presents the opportunity to exploit interanimal variation in enteric emissions as well as providing a more equitable index of the methanogenic potential of an animal on which to investigate the underlying biological regulatory mechanisms.

Kirkwood-Buff-Derived Force Field for Peptides and Proteins: Philosophy and Development of KBFF20.

A new classical nonpolarizable force field, KBFF20, for the simulation of peptides and proteins is presented. The force field relies heavily on the use of Kirkwood-Buff theory to provide a comparison of simulated and experimental Kirkwood-Buff integrals for solutes containing the functional groups common in proteins, thus ensuring intermolecular interactions that provide a good balance between the peptide-peptide, peptide-solvent, and solvent-solvent distributions observed in solution mixtures. In this way, it differs significantly from other biomolecular force fields. Further development and testing of the intermolecular potentials are presented here. Subsequently, rotational potentials for the ϕ/ψ and χ dihedral degrees of freedom are obtained by analysis of the Protein Data Bank, followed by small modifications to provide a reasonable balance between simulated and observed α and ß percentages for small peptides. This, the first of two articles, describes in detail the philosophy and development behind KBFF20.

Kirkwood-Buff-Derived Force Field for Peptides and Proteins: Applications of KBFF20.

Here, we perform structural, thermodynamic, and kinetics tests of the Kirkwood-Buff-derived force field, KBFF20, for peptides and proteins developed in the previous article. The physical/structural tests measure the ability of KBFF20 to capture the experimental J-couplings for small peptides, to keep globular monomeric and oligomeric proteins folded, and to produce the experimentally relevant expanded conformational ensembles of intrinsically disordered proteins. The thermodynamic-based tests probe KBFF20's ability to quantify the preferential interactions of sodium chloride around native ß-lactoglobulin and urea around native lysozyme, to reproduce the melting curves for small helix- and sheet-based peptides, and to fold the small proteins Trp-cage and Villin. The kinetics-based tests quantify how well KBFF20 can match the experimental contact formation rates of small, repeat-sequence peptides of variable lengths and the rotational diffusion coefficients of globular proteins. The results suggest that KBFF20 is naturally able to reproduce properties of both folded and disordered proteins, which we attribute to the use of the Kirkwood-Buff theory as the foundation of the force field's development. However, we show that KBFF20 tends to lose some well-defined secondary structural elements and increases the percentage of coil regions, indicating that the perfect balance of all interactions remains elusive. Nevertheless, we argue that KBFF20 is an improvement over recently modified force fields that require ad hoc interventions to prevent the collapse of intrinsically disordered proteins.

Synthetic Sequencing Standards: A Guide to Database Choice for Rumen Microbiota Amplicon Sequencing Analysis.

Our understanding of complex microbial communities, such as those residing in the rumen, has drastically advanced through the use of high throughput sequencing (HTS) technologies. Indeed, with the use of barcoded amplicon sequencing, it is now cost effective and computationally feasible to identify individual rumen microbial genera associated with ruminant livestock nutrition, genetics, performance and greenhouse gas production. However, across all disciplines of microbial ecology, there is currently little reporting of the use of internal controls for validating HTS results. Furthermore, there is little consensus of the most appropriate reference database for analyzing rumen microbiota amplicon sequencing data. Therefore, in this study, a synthetic rumen-specific sequencing standard was used to assess the effects of database choice on results obtained from rumen microbial amplicon sequencing. Four DADA2 reference training sets (RDP, SILVA, GTDB, and RefSeq + RDP) were compared to assess their ability to correctly classify sequences included in the rumen-specific sequencing standard. In addition, two thresholds of phylogenetic bootstrapping, 50 and 80, were applied to investigate the effect of increasing stringency. Sequence classification differences were apparent amongst the databases. For example the classification of Clostridium differed between all databases, thus highlighting the need for a consistent approach to nomenclature amongst different reference databases. It is hoped the effect of database on taxonomic classification observed in this study, will encourage research groups across various microbial disciplines to develop and routinely use their own microbiome-specific reference standard to validate analysis pipelines and database choice.

Rumen Microbiome Composition Is Altered in Sheep Divergent in Feed Efficiency.

Rumen microbiome composition and functionality is linked to animal feed efficiency, particularly for bovine ruminants. To investigate this in sheep, we compared rumen bacterial and archaeal populations (and predicted metabolic processes) of sheep divergent for the feed efficiency trait feed conversion ratio (FCR). In our study 50 Texel cross Scottish Blackface (TXSB) ram lambs were selected from an original cohort of 200 lambs. From these, 26 were further selected for experimentation based on their extreme FCR (High Feed Efficiency, HFE = 13; Low Feed Efficiency, LFE = 13). Animals were fed a 95% concentrate diet ad libitum over 36 days. 16S rRNA amplicon sequencing was used to investigate the rumen bacterial and archaeal communities in the liquid and solid rumen fractions of sheep divergent for FCR. Weighted UniFrac distances separated HFE and LFE archaea communities from the liquid rumen fraction (Permanova, P < 0.05), with greater variation observed for the LFE cohort (Permdisp, P < 0.05). LFE animals exhibited greater Shannon and Simpson diversity indices, which was significant for the liquid rumen fraction (P < 0.05). Methanobrevibacter olleyae (in liquid and solid fractions) and Methanobrevibacter millerae (liquid fraction) were differentially abundant, and increased in the LFE cohort (P.adj < 0.05), while Methanobrevibacter wolinii (liquid fraction) was increased in the HFE cohort (P.adj < 0.05). This suggests that methanogenic archaea may be responsible for a potential loss of energy for the LFE cohort. Bacterial community composition (Permanova, P > 0.1) and diversity (P > 0.1) was not affected by the FCR phenotype. Only the genus Prevotella 1 was differentially abundant between HFE and LFE cohorts. Although no major compositional shifts of bacterial populations were identified amongst the feed efficient cohorts (FDR > 0.05), correlation analysis identified putative drivers of feed efficiency with Ruminococcaceae UCG-014 (liquid, rho = -0.53; solid, rho = -0.56) and Olsenella (solid, rho = -0.40) exhibiting significant negative association with FCR (P < 0.05). Bifidobacterium and Megasphaera showed significant positive correlations with ADG. Major cellulolytic bacteria Fibrobacter (liquid, rho = 0.43) and Ruminococcus 1 (liquid, rho = 0.41; solid, rho = 41) correlated positively with FCR (P < 0.05). Our study provides evidence that feed efficiency in sheep is likely influenced by compositional changes to the archaeal community, and abundance changes of specific bacteria, rather than major overall shifts within the rumen microbiome.

Replacing Barley and Soybean Meal With By-products, in a Pasture Based Diet, Alters Daily Methane Output and the Rumen Microbial Community in vitro Using the Rumen Simulation Technique (RUSITEC).

Plant based by-products (BP) produced from food and bioethanol industries are human inedible, but can be recycled into the global food chain by ruminant livestock. However, limited data is available on the methanogenesis potential associated with supplementing a solely BP formulated concentrate to a pastoral based diet. Therefore the objective of this in vitro study was to investigate the effects of BP inclusion rate (in a formulated concentrate) to a pasture based diet on dietary digestibility, rumen fermentation patterns, methane production and the prokaryotic microbial community composition. Diets consisted of perennial ryegrass and one of two supplementary concentrates, formulated to be isonitrogenous (16% CP) and isoenergetic (12.0 MJ/ME/kg), containing either 35% BP, barley and soybean meal (BP35) or 95% BP (BP95) offered on a 50:50 basis, however, starch, NDF and fat content varied. The BPs, included in equal proportions on a DM basis, were soyhulls, palm kernel expeller and maize dried distillers grains. The BP35 diet had greater (P < 0.05) digestibility of the chemical constituents DM, OM, CP, NDF, ADF. Greater total VFA production was seen in the BP35 diet (P < 0.05). Daily methane production (mmol/day; +22.7%) and methane output per unit of total organic matter digested (MPOMD; +20.8%) were greatest in the BP35 diet (P < 0.01). Dietary treatment influenced microbial composition (PERMANOVA; P = 0.023) with a greater relative abundance of Firmicutes (adj P < 0.01) observed in the BP35. The Firmicutes:Bacteroidetes ratio was significantly reduced in the BP95 diet (P < 0.01). The relative proportions of Proteobacteria (adj P < 0.01), Succinivibrionaceae (adj P < 0.03) and Succinivibrio (adj P = 0.053) increased in the BP95 diet. The abundance of Proteobacteria was found to be negatively associated with daily methane production (rs, -0.71; P < 0.01) and MPOMD (rs, -0.65; P < 0.01). Within Proteobacteria, the relationship of methane production was maintained with the mean abundance of Succinivibrio (rs, -0.69; P < 0.01). The abundance of the Firmicutes phyla was found to be positively correlated with both daily methane production (rs, 0.79; P < 0.001) and MPOMD (rs, 0.75; P < 0.01). Based on in vitro rumen simulation data, supplementation of an exclusively BP formulated concentrate was shown to reduce daily methane output by promoting a favorable alteration to the rumen prokaryotic community.

Sward type alters the relative abundance of members of the rumen microbial ecosystem in dairy cows.

The performance of ruminant livestock has been shown to benefit from the enhanced nutritive value and herbage yield associated with clover incorporation in the grazing sward. However, little research to date has been conducted investigating the effects of mixed swards containing white clover on the composition of the rumen microbiome. In this study, the rumen microbial composition of late lactation dairy cows grazing perennial ryegrass only (PRG; n = 20) or perennial ryegrass and white clover (WCPRG; n = 19) swards, was characterised using 16S rRNA amplicon sequencing. PERMANOVA analysis indicated diet significantly altered the composition of the rumen microbiome (P = 0.024). Subtle shifts in the relative abundance of 14 bacterial genera were apparent between diets, including an increased relative abundance of Lachnospira (0.04 vs. 0.23%) and Pseudobutyrivibrio (1.38 vs. 0.81%) in the WCPRG and PRG groups, respectively. The composition of the archaeal community was altered between dietary groups, with a minor increase in the relative abundance of Methanosphaera in the WCPRG observed. Results from this study highlight the potential for sward type to influence the composition of the rumen microbial community.

Gas or Liquid? The Supercritical Behavior of Pure Fluids.

By definition, the distinction between a gas and a liquid ceases to exist beyond the critical point for pure fluids. Nevertheless, there remains a strong desire to attribute gas-like or liquid-like behavior to fluids corresponding to different parts of the supercritical region, especially as this becomes important for understanding and designing the properties of supercritical fluids. Here, we use a combination of fluctuation solution theory and accurate equation of state data to elucidate an easily accessible dividing line and corresponding transition regime between liquid-like and gas-like behavior in the supercritical region of all pure fluids. Liquid-like behavior in the supercritical region is characterized by a negative skewness in the particle number distribution for an equivalent open system, indicating that particle deletion is favored for liquids, whereas gas-like behavior is characterized by a positive skewness, indicating that particle insertion is favored for gases. Identical behavior is observed either side of the liquid-vapor line. The possible consequences for the behavior of fluids at the critical point are also discussed.

Classical harmonic model for the behavior of pure fluids at the critical point.

We present a simple model to explain the limiting behavior of many thermodynamic properties of pure classical fluids as one approaches the critical point. The model consists of four main aspects: (i) a focus on the thermodynamic properties as described by Fluctuation Solution Theory (FST); (ii) the removal of all intramolecular energy contributions from the FST expressions; (iii) particle fluctuations (and not the energy fluctuations) dominate the thermodynamics close to the critical point; and (iv) the equipartition theorem applies to the resulting low frequency collective modes of the fluid at the critical point. Using this approach, we predict and compare the thermodynamic results for 121 pure fluids as provided by accurate equations of state. The model explains why some ratios of diverging thermodynamic properties remain finite at the critical point, and reliably predicts the value of these ratios for equations of state that mimic both mean field and Ising system types, suggesting universal behavior.

Experimental Investigation of Triplet Correlation Approximations for Fluid Water.

Triplet correlations play a central role in our understanding of fluids and their properties. Of particular interest is the relationship between the pair and triplet correlations. Here we use a combination of Fluctuation Solution Theory and experimental pair radial distribution functions to investigate the accuracy of the Kirkwood Superposition Approximation (KSA), as given by integrals over the relevant pair and triplet correlation functions, at a series of state points for pure water using only experimental quantities. The KSA performs poorly, in agreement with a variety of other studies. Several additional approximate relationships between the pair and triplet correlations in fluids are also investigated and generally provide good agreement for the fluid thermodynamics for regions of the phase diagram where the compressibility is small. A simple power law relationship between the pair and triplet fluctuations is particularly successful for state points displaying low to moderately high compressibilities.

A Kirkwood-Buff derived force field for alkaline earth halide salts.

The activity and function of many macromolecules in cellular environments are coupled with the binding of divalent ions such as calcium or magnesium. In principle, computer simulations can be used to understand the molecular level aspects of how many important macromolecules interact with ions. However, most of the force fields currently available often fail to accurately reproduce the properties of divalent ions in aqueous environments. Here we develop classical non-polarizable force fields for the aqueous alkaline earth metal halides (MX2), where M = Mg2+, Ca2+, Sr2+, Ba2+ and X = Cl-, Br-, I-, which can be used in bimolecular simulations and which are compatible with the Simple Point Charge/Extended (SPC/E) water model. The force field parameters are specifically developed to reproduce the experimental Kirkwood-Buff integrals for aqueous solutions and thereby the experimental activity derivatives, partial molar volumes, and excess coordination numbers. This ensures that a reasonable balance between ion-ion, ion-water, and water-water distributions is obtained. However, this requires a scaling of the cation to water oxygen interaction strength in order to accurately reproduce the integrals. The scaling factors developed for chloride salts are successfully transferable to the bromide and iodide salts. Use of these new models leads to reasonable diffusion constants and dielectric decrements. However, the performance of the models decreases with increasing salt concentration (>4m), and simulations of the pure crystals exhibited unstable behavior.

SLTCAP: A Simple Method for Calculating the Number of Ions Needed for MD Simulation.

An accurate depiction of electrostatic interactions in molecular dynamics requires the correct number of ions in the simulation box to capture screening effects. However, the number of ions that should be added to the box is seldom given by the bulk salt concentration because a charged biomolecule solute will perturb the local solvent environment. We present a simple method for calculating the number of ions that requires only the total solute charge, solvent volume, and bulk salt concentration as inputs. We show that the most commonly used method for adding salt to a simulation results in an effective salt concentration that is too high. These findings are confirmed using simulations of lysozyme. We have established a web server where these calculations can be readily performed to aid simulation setup.

Peptide nanosponges designed for rapid uptake by leukocytes and neural stem cells.

The structure of novel binary nanosponges consisting of (cholesterol-(K/D) n DEVDGC)3-trimaleimide units possessing a trigonal maleimide linker, to which either lysine (K)20 or aspartic acid (D)20 are tethered, has been elucidated by means of TEM. A high degree of agreement between these findings and structure predictions through explicit solvent and then coarse-grained molecular dynamics (MD) simulations has been found. Based on the nanosponges' structure and dynamics, caspase-6 mediated release of the model drug 5(6)-carboxyfluorescein has been demonstrated. Furthermore, the binary (DK20) nanosponges have been found to be virtually non-toxic in cultures of neural progenitor cells. It is of a special importance for the future development of cell-based therapies that DK20 nanosponges were taken up efficiently by leucocytes (WBC) in peripheral blood within 3 h of exposure. The percentage of live cells among the WBC was not significantly decreased by the DK20 nanosponges. In contrast to stem cell or leucocyte cell cultures, which have to be matched to the patient, autologous cells are optimal for cell-mediated therapy. Therefore, the nanosponges hold great promise for effective cell-based tumor targeting.