Animal-to-Animal Variation in Fecal Microbial Diversity among Beef Cattle

The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.The gastrointestinal tracts (GIT) of beef cattle are colonized by microorganisms that profoundly impact animal physiology, nutrition, health, and productivity (5). The GIT microbiota potentially impact food safety via pathogen shedding (13) by interacting with organisms such as Salmonella and competing for resources in the GIT. Cattle intestinal microbiota also play an important role in methane emissions, with U.S. beef cattle alone contributing an estimated 3.87 million metric tons of methane into the environment each year, both from rumen and large-intestine fermentations (7). Although the bovine fecal microbiota have been well characterized using culture-based methods, these techniques are necessarily limited to characterizing bacteria that can be grown in the laboratory. Culture-independent methods can reveal community members that are recalcitrant to culture. Only a handful of deep-sequencing studies have been done using culture-independent 16S rRNA-based methods (1, 11, 12, 14), all with dairy cattle, which have a fundamentally different diet and metabolism from beef cattle. Despite the potential contributions of the beef cattle GIT microbiota to animal health, food safety, and global warming, these communities remain poorly characterized. With the advent of pyrosequencing technology, researchers now have the tools to characterize these important communities. Pyrosequencing will allow rapid characterization of large-sample data sets (1). However, the taxonomic information generated by rapid sequencing is approximate by necessity (9), and full-length 16S-rRNA sequencing remains the “gold standard” method. Accordingly, we have characterized fecal bacteria from six feedlot cattle by full-length capillary sequence analysis of 11,171 16S rRNA gene clones (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Bacterial diversity of six feedlot beef cattle. Gray bars represent the percentages of all 16S sequences that were assigned to each taxonomy. Colored dots represent the percentages of 16S sequences from each library that were assigned to each taxonomic group. Asterisks indicate unclassified members of the named taxon. Panel A shows the data for the first 99% of all the sequences. Panel B shows the data for the remaining 1% of sequences. Note differences in scales for panels A and B.Rectal grab fecal samples (n = 6) were collected according to institutional animal care guidelines. All animals were female cross-bred MARCIII beef heifers, 6 to 8 months of age, 214 to 241 kg, housed in the same feedlot pen for 2 months prior to fecal collection, and fed the same typical feedlot beef production growing rations consisting of 61.6% corn silage (41.3% dry matter), 15.2% alfalfa hay, 20.9% corn, and 2.3% liquid supplement.Total fecal DNA was isolated from homogenized samples using MoBio UltraClean fecal kit (Carlsbad, CA). PCR was performed using 27F and 1392R primers (11). Amplification consisted of 25 cycles, with an annealing temperature of 55°C. Amplicons from three reactions per sample were pooled (8), cloned using the Invitrogen TOPO TA cloning kit (Carlsbad, CA), and sequenced bidirectionally with M13 primers using an ABI 3700 sequencer (17). Low-quality and chimeric sequences (6) were excluded from further analysis. Distance matrices were compiled from ClustalW alignments (18) in PHYLIP (4). Pairwise estimates of shared richness were calculated using EstimateS, version 8.2 (R. K. Colwell; http://purl.oclc.org/estimates). DOTUR (16) was used to identify operational taxonomic units (OTUs) and to generate rarefaction curves (Fig. ​(Fig.2),2), richness and evenness estimates, and Shannon''s and Simpson''s diversity indices (Table ​(Table1).1). A 97% similarity cutoff and an 85% similarity cutoff for estimating OTUs were used to approximate species and class-level designations (15). Taxonomies were assigned to one member of each OTU using the RDP “classifier” tool (19), and the RDP taxonomic information was used for Fig. ​Fig.11 and ​and3.3. Common bovine taxa were identified based on inclusion in all three U.S. culture-independent studies (this study and references 1 and 11).Open in a separate windowFIG. 2.Rarefaction curves for six feedlot beef cattle. OTUs were assigned at the 85% DNA sequence similarity level. For comparison purposes, all six curves were truncated after 1,321 sequences.Open in a separate windowFIG. 3.Phylum-level distribution of bacterial sequences from six beef feedlot cattle. Asterisks indicate unclassified members of the named taxon.

TABLE 1.

Richness and diversity indices for 6 beef feedlot cattle

Relationship of cranial robusticity to cranial form,geography and climate in Homo sapiens

Variation in cranial robusticity among modern human populations is widely acknowledged but not well‐understood. While the use of “robust” cranial traits in hominin systematics and phylogeny suggests that these characters are strongly heritable, this hypothesis has not been tested. Alternatively, cranial robusticity may be a response to differences in diet/mastication or it may be an adaptation to cold, harsh environments. This study quantifies the distribution of cranial robusticity in 14 geographically widespread human populations, and correlates this variation with climatic variables, neutral genetic distances, cranial size, and cranial shape. With the exception of the occipital torus region, all traits were positively correlated with each other, suggesting that they should not be treated as individual characters. While males are more robust than females within each of the populations, among the independent variables (cranial shape, size, climate, and neutral genetic distances), only shape is significantly correlated with inter‐population differences in robusticity. Two‐block partial least‐squares analysis was used to explore the relationship between cranial shape (captured by three‐dimensional landmark data) and robusticity across individuals. Weak support was found for the hypothesis that robusticity was related to mastication as the shape associated with greater robusticity was similar to that described for groups that ate harder‐to‐process diets. Specifically, crania with more prognathic faces, expanded glabellar and occipital regions, and (slightly) longer skulls were more robust than those with rounder vaults and more orthognathic faces. However, groups with more mechanically demanding diets (hunter‐gatherers) were not always more robust than groups practicing some form of agriculture. Am J Phys Anthropol, 2010. © 2009 Wiley‐Liss, Inc.     

The glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension

Glucocorticoids are used as a treatment for a variety of conditions and hypertension is a well-recognized side effect of their use. The mechanism of glucocorticoid-induced hypertension is incompletely understood and has traditionally been attributed to promiscuous activation of the mineralocorticoid receptor by cortisol. Multiple lines of evidence, however, point to the glucocorticoid receptor as an important mediator as well. We have developed a mouse model of glucocorticoid-induced hypertension, which is dependent on the glucocorticoid receptor. To determine the site(s) of glucocorticoid receptor action relevant to the development of hypertension, we studied glucocorticoid-induced hypertension in a mouse with a tissue-specific knockout of the glucocorticoid receptor in the distal nephron. Although knockout mice had similar body weight, nephron number and renal histology compared to littermate controls, their baseline blood pressure was mildly elevated. Nevertheless, distal nephron glucocorticoid receptor knockout mice and controls had a similar hypertensive response to dexamethasone. Urinary excretion of electrolytes, both before and after administration of glucocorticoid was also indistinguishable between the two groups. We conclude that the glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension in our model.     

The neutral lipid composition present in the digestive vacuole of Plasmodium falciparum concentrates heme and mediates β-hematin formation with an unusually low activation energy

In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodium falciparum, a parasite responsible for causing malaria in humans, neutral lipids may have other functions during the intraerythrocytic stage of the parasite life cycle. Specifically, experimental data suggest that neutral lipid structures behave as a catalyst for the crystallization of hemozoin, a detoxification byproduct of several blood-feeding organisms, including malaria parasites. Synthetic neutral lipid droplets (SNLDs) were produced by depositing a lipid blend solution comprised of mono- and diglycerides onto an aqueous surface. These lipid droplets are able to mediate the production of brown pigments that are morphologically and chemically identical to hemozoin. The partitioning of heme into these SNLDs was examined by employing Nile Red, a lipid specific dye. Soluble ferriprotoporphyrin IX was observed to spontaneously localize to the lipid droplets, partitioning in a pH-dependent manner with an estimated log P of 2.6. Interestingly, the pH profile of heme partitioning closely resembles that of β-hematin formation. Differential scanning calorimetry and kinetic studies demonstrated that the SNLDs provide a unique environment that promotes hemozoin formation. SNLD-mediated formation of the malaria pigment displayed an activation energy barrier lower than those of individual lipid components. In particular, lipid droplets composed of diglycerides displayed activation barriers lower than those composed of monoglycerides. This difference was attributed to the greater fluidity of these lipids. In conjunction with the known pattern of lipid body proliferation, it is suggested that neutral lipid structures within the digestive vacuole not only are the location of in vivo hemozoin formation but are also essential for the survival of the parasite by functioning as a kinetically competent and site specific mediator for heme detoxification.     

ROADTRIPS: Case-Control Association Testing with Partially or Completely Unknown Population and Pedigree Structure

Genome-wide association studies are routinely conducted to identify genetic variants that influence complex disorders. It is well known that failure to properly account for population or pedigree structure can lead to spurious association as well as reduced power. We propose a method, ROADTRIPS, for case-control association testing in samples with partially or completely unknown population and pedigree structure. ROADTRIPS uses a covariance matrix estimated from genome-screen data to correct for unknown population and pedigree structure while maintaining high power by taking advantage of known pedigree information when it is available. ROADTRIPS can incorporate data on arbitrary combinations of related and unrelated individuals and is computationally feasible for the analysis of genetic studies with millions of markers. In simulations with related individuals and population structure, including admixture, we demonstrate that ROADTRIPS provides a substantial improvement over existing methods in terms of power and type 1 error. The ROADTRIPS method can be used across a variety of study designs, ranging from studies that have a combination of unrelated individuals and small pedigrees to studies of isolated founder populations with partially known or completely unknown pedigrees. We apply the method to analyze two data sets: a study of rheumatoid arthritis in small UK pedigrees, from Genetic Analysis Workshop 15, and data from the Collaborative Study of the Genetics of Alcoholism on alcohol dependence in a sample of moderate-size pedigrees of European descent, from Genetic Analysis Workshop 14. We detect genome-wide significant association, after Bonferroni correction, in both studies.     

Polarizable Atomic Multipole X-Ray Refinement: Hydration Geometry and Application to Macromolecules

We recently developed a polarizable atomic multipole refinement method assisted by the AMOEBA force field for macromolecular crystallography. Compared to standard refinement procedures, the method uses a more rigorous treatment of x-ray scattering and electrostatics that can significantly improve the resultant information contained in an atomic model. We applied this method to high-resolution lysozyme and trypsin data sets, and validated its utility for precisely describing biomolecular electron density, as indicated by a 0.4-0.6% decrease in the R- and R_free-values, and a corresponding decrease in the relative energy of 0.4-0.8 Kcal/mol/residue. The re-refinements illustrate the ability of force-field electrostatics to orient water networks and catalytically relevant hydrogens, which can be used to make predictions regarding active site function, activity, and protein-ligand interaction energies. Re-refinement of a DNA crystal structure generates the zigzag spine pattern of hydrogen bonding in the minor groove without manual intervention. The polarizable atomic multipole electrostatics model implemented in the AMOEBA force field is applicable and informative for crystal structures solved at any resolution.     

Fluorescence lifetime imaging reveals that the environment of the ATP binding site of myosin in muscle senses force

Fluorescence lifetime imaging microscopy is used to demonstrate that different loads applied to a muscle fiber change the microenvironment of the nucleotide binding pocket of myosin. Permeabilized skeletal muscle fibers in rigor were labeled with a fluorescent ATP analog, 3′-DEAC-propylenediamine (pda)-ATP (3′-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP), which was hydrolyzed to the diphosphate. Cycles of small-amplitude stretches and releases (<1% of muscle segment length) were synchronized with fluorescence lifetime imaging and force measurements to correlate the effect of force on the lifetime of the ATP analog bound to the actomyosin complex. Analysis of the fluorescence decay resolved two lifetimes, corresponding to the free nucleotide DEAC-pda-ATP (τ₁ = 0.47 ± 0.03 ns; mean ± SD) and nucleotide bound to the actomyosin complex (τ₂ = 2.21 ± 0.06 ns at low strain). Whereas τ₁ did not change with force, τ₂ showed a linear dependence with the force applied to the muscle of 0.43 ± 0.05 ps/kPa. Hence, the molecular environment of the nucleotide binding pocket of myosin is directly affected by a change of length applied at the ends of the fiber segments. These changes may help explain how force modulates the actomyosin ATPase cycle and thus the physiology and energetics of contraction.     

Shaping a Morphogen Gradient for Positional Precision

Morphogen gradients, which provide positional information to cells in a developing tissue, could in principle adopt any nonuniform profile. To our knowledge, how the profile of a morphogen gradient affects positional precision has not been well studied experimentally. Here, we compare the positional precision provided by the Drosophila morphogenetic protein Bicoid (Bcd) in wild-type (wt) embryos with embryos lacking an interacting cofactor. The Bcd gradient in the latter case exhibits decreased positional precision around mid-embryo compared with its wt counterpart. The domain boundary of Hunchback (Hb), a target activated by Bcd, becomes more variable in mutant embryos. By considering embryo-to-embryo, internal, and measurement fluctuations, we dissect mathematically the relevant sources of fluctuations that contribute to the error in positional information. Using this approach, we show that the defect in Hb boundary positioning in mutant embryos is directly reflective of an altered Bcd gradient profile with increasing flatness toward mid-embryo. Furthermore, we find that noise in the Bcd input signal is dominated by internal fluctuations but, due to time and spatial averaging, the spatial precision of the Hb boundary is primarily affected by embryo-to-embryo variations. Our results demonstrate that the positional information provided by the wt Bcd gradient profile is highly precise and necessary for patterning precision.