Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle.

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

A Label-free Selected Reaction Monitoring Workflow Identifies a Subset of Pregnancy Specific Glycoproteins as Potential Predictive Markers of Early-onset Pre-eclampsia

Pre-eclampsia (PE) is a serious complication of pregnancy with potentially life threatening consequences for both mother and baby. Presently there is no test with the required performance to predict which healthy first-time mothers will go on to develop PE. The high specificity, sensitivity, and multiplexed nature of selected reaction monitoring holds great potential as a tool for the verification and validation of putative candidate biomarkersfor disease states. Realization of this potential involves establishing a high throughput, cost effective, reproducible sample preparation workflow. We have developed a semi-automated HPLC-based sample preparation workflow before a label-free selected reaction monitoring approach. This workflow has been applied to the search for novel predictive biomarkers for PE.To discover novel candidate biomarkers for PE, we used isobaric tagging to identify several potential biomarker proteins in plasma obtained at 15 weeks gestation from nulliparous women who later developed PE compared with pregnant women who remained healthy. Such a study generates a number of “candidate” biomarkers that require further testing in larger patient cohorts. As proof-of-principle, two of these proteins were taken forward for verification in a 100 women (58 PE, 42 controls) using label-free SRM. We obtained reproducible protein quantitation across the 100 samples and demonstrated significant changes in protein levels, even with as little as 20% change in protein concentration. The SRM data correlated with a commercial ELISA, suggesting that this is a robust workflow suitable for rapid, affordable, label-free verification of which candidate biomarkers should be taken forward for thorough investigation. A subset of pregnancy-specific glycoproteins (PSGs) had value as novel predictive markers for PE.The identification of clinically relevant plasma biomarkers with diagnostic and/or predictive value continues to challenge the proteomics field. Whereas once the biomarker pipeline was described as a two part discovery and validation process, there is increasing consensus that an intermediate step is required in which the proteins identified in the discovery phase are technically verified in 50 to 200 samples. This verification step identifies false positives from the discovery phase and allows prioritization of proteins to be taken into large-scale clinical validation studies (1). Although commercial ELISA kits may be used in this phase, these are unavailable for many proteins, are expensive, and may lack specificity. In addition, sample requirements may be too high to perform ELISA on all candidates, especially if many proteins are identified as potential markers by low powered, high penetration discovery workflows.Selected reaction monitoring (SRM)¹ mass spectrometry has great potential as an alternative verification method (2–6) as it can be multiplexed, customized, and is highly specific. This potential has not been exploited to date, largely because of technical issues developing a low-cost, reproducible workflow encompassing plasma and serum preparation and LC/MS analysis with the capability to measure protein levels reproducible in hundreds of samples. With traditional stable isotope dilution SRM (SID-SRM), the high cost of accurately quantified, purified stable isotope encoded peptides or proteins may be prohibitive for the verification of multiple peptides from many proteins. Label-free relatively quantitative methods are increasingly popular in discovery proteomics but to a much lesser extent in targeted SRM studies (7, 8).For any SRM method, sample preparation workflows must balance the extent of enrichment and fractionation to enable quantification of lower abundance proteins, against increased technical variability (which is influenced by the number of sample handling steps) and reduced multiplexed potential as a consequence of fractionating peptides from the protein of interest into several distinct fractions. It is also essential that the true technical variation in the workflow is quantitatively evaluated from freezer to MS analysis, rather than just the variation within the LC-SRM part of the experiment. As a paradigm for a label-free SRM assay, we developed our workflow and applied it to the verification of candidate biomarkers that indicate the risk of pre-eclampsia (PE).PE affects 2–8% of pregnancies, and is characterized by hypertension and proteinuria, which may progress to severe maternal complications or death (9). Because delivery of the infant is the only effective intervention, a third of babies are born premature and fetal or newborn mortality is increased three- to 10-fold (10). Its complex etiology involves abnormal placentation, an altered immune response and a sensitized maternal vascular endothelium (11). Prediction of the condition in early pregnancy would allow prevention strategies, such as low dose aspirin, to be targeted to high risk women. In first-time pregnant women, a group particularly at risk, biomarkers continue to fall short of a test that would be useful or cost effective in clinical practice (12–14). Better-performing novel biomarkers are required.The aim of this study was to identify candidate predictive biomarkers for PE and then develop a verification assay using mass spectrometry to determine whether these should be taken forward into more extensive and expensive validation studies. Initial discovery experiments were employed using a pooled sample iTRAQ approach using two different MS platforms to increase plasma proteome coverage. Among the set of proteins discovered, we then developed a label-free SRM assay for relative quantification of CXCL7 (Platelet basic protein; PBP) and members of the Pregnancy specific glycoprotein (PSG) family in a 100-sample set from the international SCreeningfOr Pregnancy Endpoints (SCOPE) study (www.scopestudy.net). Our workflow allowed the specificity and linearity of response for each peptide to be determined, along with true technical variability. Although absolute concentration and LOD/LOQ cannot be calculated using this approach, we aimed to test the hypothesis that a label-free SRM approach could provide a rapid, robust, and efficient screen of candidate plasma biomarkers.     

Acoustic sequences in non‐human animals: a tutorial review and prospectus

Animal acoustic communication often takes the form of complex sequences, made up of multiple distinct acoustic units. Apart from the well‐known example of birdsong, other animals such as insects, amphibians, and mammals (including bats, rodents, primates, and cetaceans) also generate complex acoustic sequences. Occasionally, such as with birdsong, the adaptive role of these sequences seems clear (e.g. mate attraction and territorial defence). More often however, researchers have only begun to characterise – let alone understand – the significance and meaning of acoustic sequences. Hypotheses abound, but there is little agreement as to how sequences should be defined and analysed. Our review aims to outline suitable methods for testing these hypotheses, and to describe the major limitations to our current and near‐future knowledge on questions of acoustic sequences. This review and prospectus is the result of a collaborative effort between 43 scientists from the fields of animal behaviour, ecology and evolution, signal processing, machine learning, quantitative linguistics, and information theory, who gathered for a 2013 workshop entitled, ‘Analysing vocal sequences in animals’. Our goal is to present not just a review of the state of the art, but to propose a methodological framework that summarises what we suggest are the best practices for research in this field, across taxa and across disciplines. We also provide a tutorial‐style introduction to some of the most promising algorithmic approaches for analysing sequences. We divide our review into three sections: identifying the distinct units of an acoustic sequence, describing the different ways that information can be contained within a sequence, and analysing the structure of that sequence. Each of these sections is further subdivided to address the key questions and approaches in that area. We propose a uniform, systematic, and comprehensive approach to studying sequences, with the goal of clarifying research terms used in different fields, and facilitating collaboration and comparative studies. Allowing greater interdisciplinary collaboration will facilitate the investigation of many important questions in the evolution of communication and sociality.     

Seasonal shedding of multiple Cryptosporidium genotypes in California ground squirrels (Spermophilus beecheyi)

Twelve percent of 853 California ground squirrels (Spermophilus beecheyi) from six different geographic locations in Kern County, Calif., were found to be shedding on average 44,482 oocysts g of feces(-1). The mean annual environmental loading rate of Cryptosporidium oocysts was 57,882 oocysts squirrel(-1) day(-1), with seasonal patterns of fecal shedding ranging from <10,000 oocysts squirrel(-1) day(-1) in fall, winter, and spring to levels of 2 x 10(5) oocysts squirrel(-1) day(-1) in summer. Juveniles were about twice as likely as adult squirrels to be infected and shed higher concentrations of oocysts than adults did, with particularly high levels of infection and shedding being found among juvenile male squirrels. Based on DNA sequencing of a portion of the 18S small-subunit rRNA gene, there existed three genotypes of Cryptosporidium species in these populations of squirrels (Sbey03a, Sbey03b, and Sbey03c; accession numbers AY462231 to AY462233, respectively). These unique DNA sequences were most closely related (96 to 97% homology) to porcine C. parvum (AF115377) and C. wrairi (AF115378). Inoculating BALB/c neonatal mice with up to 10,000 Sbey03b or Sbey03c fresh oocysts from different infected hosts did not produce detectable levels of infection, suggesting that this common genotype shed by California ground squirrels is not infectious for mice and may constitute a new species of Cryptosporidium.