Fratercula arctica) chicks (n=65) and adults (n=64) and the stomach contents of their main prey, Atlantic herring  to investigate a previously studied food chain. We compared conventional and molecular-derived chick diet, tested the similarity between the diets of puffin adults and chicks, and determined whether herring prey can be detected in puffin diet samples. There was high variability in the coverage of prey groups between 16S and CO1 markers. We identified more unique prey with our 16S compared to CO1 barcoding markers (51 and 39 taxa respectively) with only 12 taxa identified by both genes. We found no significant difference between the 16S-identified diets of puffin adults (n=17) and chicks (n=41). Our molecular method is more taxonomically resolved and detected chick prey at higher frequencies than conventional field observations. Many likely planktonic prey of herring were detected in feces from puffin adults and chicks, highlighting the impact secondary consumption may have on the interpretation of molecular dietary analysis. This study represents the first simultaneous molecular investigation into the diet of multiple components of a food chain and highlights the utility of a multi-locus approach to diet reconstruction that is broadly applicable to food web analysis.The predator-prey interactions within food chains are used to both characterize and understand ecosystems. Conventional methods of constructing food chains from visual identification of prey in predator diet can suffer from poor taxonomic resolution, misidentification, and bias against small or completely digestible prey. Next-generation sequencing (NGS) technology has become a powerful tool for diet reconstruction through barcoding of DNA in stomach content or fecal samples. Here we use multi-locus (16S and CO1) next-generation sequencing of DNA barcodes on the feces of Atlantic puffin ( Ecosystems are characterized and understood from the fundamental relationships between predator and prey. When linked vertically and horizontally these relationships form food webs, which depict how energy flows through ecosystems and demonstrate how various components of the web interact. The consideration of an ecosystem in its entirety, and not simply an assemblage of independent parts, is the cornerstone of the widely-accepted ecosystem-based management paradigm ,4,5,6,7,a priori knowledge of diet. The use of DNA within feces or stomach contents provides a snapshot of predator diet and has not only been shown to provide a better estimate of diet than conventional methods [de novo diet description.Conventional methods of constructing food chains rely on diet studies which visually identify prey during feeding events or within stomachs, pellets, or feces. These techniques often suffer from misidentification of similar-looking prey, underrepresentation of soft-bodied (within stomach contents) or small (when observed in the field) prey, and low taxonomic resolution, where prey cannot be precisely identified due to distance (during observations) or digestion (within stomach contents) . Biochem methods ,10,11,12The application of DNA barcoding in diet studies has increased considerably with the advent of next-generation sequencing (NGS) technology. It is now possible to identify even the rarest prey from multiple predators to species, genus, or family level in a single sequencing run while maintaining the ability to trace back each prey to the sample from which it came. NGS has been used in barcoding diet studies for fur seals , little Fratercula arctica) of Machias Seal Island and their main prey, Atlantic herring (Clupea harengus) as a model system to investigate how molecular methods can be used to describe food chains as these two species represent a simple food web where two conventional methods of studying diet have been employed. Puffin chick diet is known from hundreds of hours of observing adults provisioning their chicks as part of a long-term seabird research program [Here we use Atlantic puffins  as well as the Canadian Wildlife Service .To assess the effectiveness of conventional field observations as an appropriate method to study puffin diet, we compared diet as estimated from 68 hours of field observations and through DNA within fecal samples of puffin chicks in the 2009 breeding season (May-August) on Machias Seal Island , New Brunswick, Canada. Field observations were conducted in three hour stints with binoculars from observation blinds according to Machias Seal Island’s research protocols ,31 wherec oxidase subunit 1 (CO1). The degenerate primers 16S1F and 16S2R amplify a ~180-270bp region of 16S and have been shown to amplify prey DNA from the feces of penguins . To coms, squid  that sucWe used a pooled massively parallel sequencing (MPS) approach following the protocol of Puritz et al. . To alloStomach and fecal samples can contain significant amounts of DNA from the host species due to sloughing of cells in the digestive tract . Host DNDNA from stomach contents and reference samples was isolated with a CTAB protocol . DNA wasAmplification of fecal DNA with 16S MID-tagged sequencing primers was achieved in 30ul reactions containing 6ul undiluted template, 0.2mM dNTP, 1X bovine serum albumin , 5mM MgSO4, 0.25uM of each primer, and 1.25 of blocking primer, 1X High Fidelity Buffer, and 1.2 units Platinum® Taq DNA Polymerase High Fidelity (Life Technologies). DNA from fish stomach contents was amplified with 16S MID-tagged sequencing primers in 30ul reactions containing 3ul undiluted template, 0.2mM dNTP, 5mM MgSO4, 0.25 uM of each primer, 1.25uM of blocking primer, 1X High Fidelity Buffer, and 1.2 units Platinum® Taq DNA Polymerase High Fidelity. DNA from samples intended for the reference database of potential prey was amplified with 10ng of template, 0.2mM dNTP, 0.5mM MgSO4, 0.25uM of each forward and reverse 16S primer, 0.04 units of Taq DNA polymerase, and 1X ThermoPol Buffer (New England Biolabs). Thermocycling protocol for 16S began at 94°C for 10 minutes followed by 35 cycles of 94°C for 15s, 55°C for 15s, and 68°C for 30s, with a final extension of 68°C for 5 minutes (BioRad C-1000). Amplification with CO1 for both sample types followed similar component and cycling conditions as the 16S with the following modifications: 3ul undiluted template was used, BSA was omitted, there was no blocking primer, and annealing temperature was set at 53°C. All amplicons were visualized under UV light in 2% agarose using SYBR Safe (Life Technologies), and those that amplified were cleaned with Agencourt AMPure XP (Beckman Coulter) at a 0.9 beads to 1 PCR product ratio. DNA concentration was determined with dsDNA BR assays on a Qubit 2.0 Fluorometer (Life Technologies). PCR was repeated for samples with less than 20ng of 16S-amplified DNA then pooled after cleaning with QIAEX II Gel Extraction Kit following the manufacturer’s instructions.All pipetting was done with barrier tips, negative controls were used in every PCR, small aliquots of reagents were used, and lab benches were cleaned between uses. Adult, chick, and herring diet samples were DNA-extracted on different days, several weeks in advance of the PCR component of this project.We made considerable efforts to obtain a sufficient number and an even temporal distribution of samples throughout the sampling period. However, a limiting step in our protocol was the extraction of high quality DNA. Amplification success of herring stomach contents and puffin fecal samples ranged from 47% to 71%. Insufficient homogenization during DNA extraction and PCR inhibitors are two potential explanations for the large number of samples that could not be successfully PCR amplified. There was also considerable variation in sample coverage. Although sequencing success of submitted samples was over 94%, only 61-84% of these samples produced a sufficient number of sequences to be included in calculations of frequency of occurrence (see next section). This variation may be a result of MID tag-induced amplification and sequencing bias , imprecihwww.postgresql.org). Representative sequences were chosen as the first sequence appearing in jMHC data output belonging to a unique MOTU. All CO1 representative MOTUs were queried using Barcode of Life Database (BOLD) online identification tool [Samples were quantified and pooled into a single equimolar library and sequenced by Génome Québec Innovation Centre at McGill University in Montreal, Quebec, Canada. The library was sequenced unidirectionally on half a pico titre plate using the Roche GS-FLX (454) platform. Bases were recalled using Pyrobayes  and sequion tool . All repion tool  was usedWe used PRIMER 6 (PRIMER-E Ltd)  for multWe used the frequency with which a prey was detected across samples, the frequency of occurrence (FOO), to quantify diet because the proportion of prey sequences found within a sample does not necessarily correlate quantitatively with proportions of prey consumed . We limiAfter initial filtering for quality and length, 104,313(16S) and 49,218 (CO1) reads were demultiplexed with over 98% (16S) and 96% (CO1) of sequences successfully matched to herring stomach content samples (n=44) or fecal samples from puffin adults (n=64) and chicks (n=65). We assembled 5759 unique CO1 amplicons >80bp into 132 MOTUs at the 11bp cutoff and 16, 710 unique 16S amplicons >75bp were assembled into 273 MOTUs at the 12bp cutoff. MOTUs were identified to 78 unique prey taxa from kingdom Animalia, and one green alga, one brown alga, three diatoms, two water molds, three bacteria, a protozoan, and several contaminating taxa . All non-contaminant identified taxa are listed in Sebastes fasciatus). An additional 46 samples contained at least one amplicon from each locus. Within these 66 samples, coverage of krill, shrimp, crabs and lobsters, insects, gastropods, and annelids and nemerteans was higher with 16S (Hediste diversicolor) and possibly a snail from the Lacuna genus which has one known Gulf of Maine representative with direct development [We identified more unique prey with 16S than CO1 barcoding markers , were detected in both puffin sample types. Only adult puffin fecal samples contained representatives from the annelid class Clitellata and two unidentified fish taxa  plus an assortment of invertebrate taxa. The assemblage of prey found exclusively in chick diet was less varied than that of adults, comprising three species of fish and a fly that the chicks presumably eat while in their nest burrow (Brachycera). Many of the invertebrate species found in adult and chick puffin fecal samples were also observed in herring . Four spEvadne spinifera 100% and E. nordmanni 43%), crabs , copepods (Temora longicornis 87% and Mertridia lucens 28%), barnacles , insects , the Atlantic jackknife clam (Ensis directus 22%), and Northern krill (Meganyctiphanes norvegica 20%) . Acadian redfish were present in roughly one-third of all adult samples but only 4% of chick samples. Ragworms were detected more often in adult diet  whereas the Slender ragworm was found in only 12% of adult samples but in 34% of chick samples. Shrimp and krill were much more prevalent in adult than chick diet . We also detected taxa  that are unlikely or impossible target prey for adult and chick puffins  . Herringica 20%) . The nexAcartia longiremis, Balanus sp., Calanoida, Calanus finmarchicus, Calanus glacialis, Cancer sp., Evadne nordmanni, Homarus americanus, Great spider crab, Metridia longa, Mytilus sp., Ophiopholis aculeata, Paguridae, Semibalanus balanoides), were removed  and of puffin chicks (n=41) could not be distinguished statistically . The analysis was limited to only 16S-identified animal taxa (n=40) and puffin fecal samples collected within the common sampling period (13 June – 29 July) from which a minimum of 50 sequences were obtained . The simPomatomus saltatrix and Rock gunnel, Pholis gunnellus), that had been identified in chick diet in previous years of field observations on Machias Seal Island but not in 2009, as well as fish species never before identified through field observations (Giant wrymouth (Cryptacanthodes maculatus), Threadfin rockling (Gaidropsarus ensis), Atlantic cod , Scallop snailfish (Liparis inquilinus), Haddock (Melanogrammus aeglefinus), and Acadian redfish (S. fasciatus)  ). Our use of multi-locus pyrosequencing to reconstruct the diets of puffins and one of their primary food sources allowed for a more complete puffin food chain than known to date . The numHemitripterus americanus) and Grubby , and add Thysanoessa sp. to the ‘krill’ category, which was previously thought to comprise only Northern krill. The diet of chicks when assessed by molecular methods is more highly resolved taxonomically than when assessed by conventional field observations . Gulf ofThysanoessa raschii) and 12 copepod species and genera. Through DNA sequencing of stomach contents we identified three cladoceran species, eight fish species/genera, three crab species/genera, one shrimp species, and a species and genus each of both barnacle and krill.We saw a similar improvement on the taxonomic resolution of herring prey using a DNA-based approach compared to stomach content analyses. In published studies of juvenile herring diet , prey itAurelia aurita), as this prey would be digested completely, leaving nothing for identification. We also detected several taxa that were either visually misidentified as common prey or absent from the diet in 15 years of field observations of puffins , Scallop snailfish, Haddock, and Acadian redfish). As many of the fish caught by puffins are small (~5-15cm), accurate identification can be difficult in the field. Shrimp from the Hippolytidae family, for instance can be easily mistaken for krill, a common prey item. While shrimp were not recorded in chick diet during 2009 field observations, one species  was found as a dropped prey item in the colony that year , offering further evidence that field observations suffer from misidentification error. Our molecular method identified prey not detected or not accurately identified by conventional methods. DNA from insects, echinoderms, amphipods, bivalves, gastropods, polychaetes, and cnidarians was present in herring stomach contents. None of these groups had been observed in previous studies, which may either be due in part to a change in herring diet in the 30 years since the latest published study was conducted, or reflect the increased resolution of a DNA-derived diet. Some taxa, however, would have never been detected in herring diet, such as the Common jellyfish  is important for the effective management of these stocks, as the impact of non-human predators on fish has historically been severely underestimated . The natA. longiremis as well as larval stages of the Crevice brittlestar (Ophiopholis aculeata) and Paguridae crabs are components of plankton that are too small to be target prey for provisioning puffins. The adult forms of these species have never been seen in chick diet, nor do they look sufficiently similar to taxa that have been identified in the field that they could have been be mistaken . Further, these three taxa were also detected in herring samples, implicating secondary consumption as the explanation for these records. While field observations did not accurately identify or quantify chick diet, they did provide a necessary framework from which to interpret our molecular diet data. The fish and shrimp species found in chick diet but not observed in the field were most likely misidentified as one of the more common fish prey or as krill, in the case of shrimp. However, the DNA-identified taxa without visual equivalents that had not been identified through field observations are either a result of secondary consumption, or are newly-identified puffin prey. Adult puffins catch and hold prey in their bills as they propel themselves underwater and upon return to the colony they drop the intact prey in their nest burrow, making very unlikely any transfer of non-targeted prey to their chicks. The copepod Balanus sp., Semibalanus balanoides), copepods , cladocerans , crabs , and a brittlestar (Crevice brittlestar) from our picture of the diet of chicks  detected in herring diet have been shown to be absent from areas where bottom depth was less than 45 metres [Corophium volutator , an oligochaete (Nais elinguis)) and intertidal (an amphipod (Gammarellus angulosus) and Rockweed (Ascophyllum nodosum)) habitats. The presence of these taxa may reflect a coastal foraging habitat of adult puffins, or secondary consumption that was not confirmed due to insufficient overlap between adult puffin and herring diet samples. We recommend future DNA-based diet studies aim to control for confounding secondary consumption through the investigation of prey diet but also by ensuring sufficient number and overlap of predator diet samples across the sampling period so that temporal differences in prey availability are not confused with secondary consumption.Taxa identified in adult puffin diet may also provide evidence for foraging habitat. Several taxa present in adult diet occupy freshwater (a rotifer (Bdelloidea), a diatom (Ammodytes species (dubius or americanus) were present in our diet samples. Additionally, there were many instances where prey could not be resolved to species or genus level because those species had not been previously sequenced at our loci of interest. In several cases the taxon assignment was so general that it was not useful in describing diet at all  and short amplicons (necessary to function effectively on degraded DNA) did pose some problems resolving a few taxa. For both 16S and CO1, the fragment produced from the universal primers was not sufficiently polymorphic to identify which of the two Gulf of Maine a priori knowledge of diet is available, to increase the likelihood of capturing the true assemblage of prey present in predator diet and improve the chance of accurately describing the trophic interactions within a food web.  Although our method identified many more taxa than field observations, an estimated 6 species are present but undetected in our samples . Frequency of occurrence (FOO) is listed for taxa found in samples with at least 50 sequences per marker per sample. Taxa identified in samples that produced less than 50 sequences receive an NF (no FOO). When a taxon was identified (as a MOTU) with both markers, the total number of samples was used for FOO calculation  otherwise sample size of FOO calculation depends on the marker from which a taxon was identified.(DOCX)Click here for additional data file.Table S2Description of prey categories used in field observations for 2009 MSI puffin chick diet and corresponding DNA taxa. Number of prey items identified from field observations of provisioning adults on Machias Seal Island 1995-2010 . Descri(DOCX)Click here for additional data file.Table S3Summary of juvenile herring diet from published stomach content analyses. Most abundant or frequently observed prey types (numbers) or copepods species/genera (letters) are listed in ascending order.(DOCX)Click here for additional data file.Table S4Summary of study sample sizes.(DOCX)Click here for additional data file.


Among scrapie cases, the CH1641 source in particular appears to have certain biochemical properties similar to the BSE strain. In France, several natural scrapie cases were identified as “CH1641-like” natural scrapie isolates in sheep and goats. The Tg(OvPrP4) mouse line expressing the ovine prion protein is a sensitive model for studying and identifying strains of agents responsible for scrapie and BSE. This model is also very useful when studying specific scrapie source CH1641, known to be not transmissible to wild-type mice despite the similarity of some of its biochemical properties to those of the BSE strain. As it is important to be able to fully distinguish CH1641 from BSE, we herein report the histopathological data from CH1641 scrapie transmission experiments compared to specific cases of “CH1641-like” natural scrapie isolates in sheep, murine scrapie strains and BSE. In addition to the conventional vacuolar lesion profile approach and PrP As the disease progresses, it is admitted that PrPc is converted into an abnormal protease-resistant scrapie prion protein , a cell-surface sialoglycoprotein with a concentrated ß-sheet conformation, that then accumulates in the diseased brain d” where d stands for disease-related, is regarded as the most specific element of TSEs, also called prion diseases.Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats and chronic wasting disease in mule deer. The neuropathological changes typical of TSEs are vacuolar damage, neuron loss, astrogliosis and abnormal deposition of the disease-specific form of the normal cellular prion protein   . The lesposition , which iposition , often wposition . Apart flso seen . Some welso seen . These also seen . As alre plaques . The amyres in 87V-infected transgenic mice nor in CH1641-, TR316211-, O104- or O100-infected mice  or mice-adapted scrapie strains derived from wild-type mice. ted mice . HoweverAF84 mAb .d brain mapping data for the natural “CH1641-like” group showed more similarities than divergences, which may be attributed at least in part to the intensity of PrPd accumulation, as suggested by the comparative data between 1st and 2nd passage experiments with TR316211 and O104 isolates. Nonetheless, the vacuolar lesion profiles for CH1641 and TR316211 appeared similar to each other but clearly different from the O104 and O100 group. It is therefore possible to group strains in accordance with the biochemical study grouping In previous publications on CH1641 scrapie source transmission studies, only biochemical data and no histopathological data were provided. In these studies, CH1641 transmission data were analyzed in comparison to the natural “CH1641-like” group  and not the “CH1641-like” isolate (O111), in addition to classical murine scrapie strains and BSE strains  d brain mappings and PrPd deposition type. Ongoing third-passage experiments should provide some explanations for some of the unexpected evolutions in the histopathological data recorded, especially for the 79A and 87V strains. While waiting for the results of this third-passage experimental study, we have considered analyzing the present data further by developing a differential PET-blot analysis. In an attempt to find ways of unifying the biochemical and histopathological data on strain typing, we decided to pursue the histopathological analysis of the brains of TgOvPrP4 mice infected with these scrapie sources. An initial set of experimental data underwent an epitopic PET blot analysis, developed on the basis of the comparative use of SAF84 and P4 mAbs that recognized two different epitopes of the prion protein. Initial findings showed that it is possible to reproduce a classification based on P4 reactivity. The BSE, C506M3, Chandler and 79A groups showed P4-detected PrPres molecules, unlike the 87V, CH1641 and TR316211 group. Some mice from the O100 experiment had PrPres molecules detected using P4, implying individual variations within the experimental group, an implication also suggested by the wide variations in lesion profiles consistent with biochemical analyses res labeling was less intense than that detected using SAF84. All the regions that accumulated PrPres detected using P4 were systematically SAF84–positive too, whereas not all the regions that accumulated PrPres detected using SAF84 were positively labeled using P4 mAb. This may indicate that only specific brain regions or cell types may express the PrPres molecules detected using P4, whereas more brain regions and cell types express PrPres molecules detectable only with SAF84. We cannot exclude the possibility that the P4 antibody may be less sensitive. However, this observation was reproducible using another prion antibody recognizing the same epitopic region recognized by P4 (data not shown). To better characterize the type of cells concerned, a detailed differential immunohistochemical analysis of these specific regions will be necessary. This would actually be a suitable way of apprehending the biological origin of these different forms of PrPd detected in situ. As observed in the sheep species, these different kinds of immunoreactivity probably reveal different levels of naturally-occurring enzymatic cleavage of the PrPd, which may depend on the strain of agent and the type of brain cell considered in situ, in other words the PrPres molecules themselves. It does not investigate the “natural truncation” of PrPd molecules that exists at cellular level. An epitopic PET Blot analysis is an original way of obtaining an approximation in situ of the length of the PrPres molecules accumulated in the brain of mice experimentally infected with different prion strains. The advantage is that this method can be used to characterize a variety of PrPres molecules—similar to detection by WB—directly in situ within the brain depending on the prion strains. Unfortunately, we cannot at this stage identify, of all the PrPres molecules detected in situ using SAF84, which ones may be related to the additional PrPres fragment (named PrPres #2) described recently by our group res product detected using SAF84 mAb specifically in the “CH1641-like” isolates compared to 79A, Chandler and C506M3 strains and transmitted to TgOvPrP4 mice, helps differentiate these prion groups.The data on the BSE and scrapie murine-adapted strains  transmitted in a second passage experiment to TgOvPrP4 mice showed clear differences between strains in terms of survival periods, lesion profiles, PrPMany more experiments related to these biochemically-defined groups, together with the use of several other possible anti-prion antibodies, are already planned. Importantly, this complementary histopathological tool already provides data showing strain properties concordant with the biochemical characterization of this particular group of prion agents.All procedures were carried out in compliance with the guidelines laid down by the Regional (CREEA no. 98) and French (Decree 87–848) Ethical Committees and European Community Directive 86/609/EEC. Animal experiments were performed in the ANSES animal facilities which have the relevant approval to carry out animal work (A 69 387 0801) by licensed people working in the animal experiment unit .The different scrapie sources used are presented in st passage) or 1% (2nd passage) (wt/vol) brain homogenates in 5% glucose in distilled water. This transgenic mouse model (TgOvPrP4) overexpresses in the brain, between 1.5 and 6 times the levels of the PrPARQ ovine prion protein under the control of the neuron-specific enolase promoter Groups of 12 female TgOvPrP4 mice d following procedures published in detail elsewhere d as previously described with pre-treatments designed to enhance PrPd detection using SAF84  or P4 monoclonal antibody  which maps to the regions 167RPVDQY172 and 93WGQGGSH99 of ovine PrP respectively On death, the mouse brains were removed and processed either for biochemical (frozen at −20°C) or histochemical  detection of PrPin situ the resistant form of abnormal PrP (PrPres) after digestion with a high concentration of proteinase K (PK), the PET-blot method was used as previously described 2). Finally, NBT/BCIP  was used to visualize the reaction product (dark blue deposits). PET-blot membranes were assessed using a stereo-microscope  linked to an image analysis workstation .To visualize


In a new article published in the journal Aging, Borras et al. report that Ras-GRF1 homozigous deletion increases both median and maximum longevity in laboratory mice  includinRas protein, the founding member of the small GTP binding protein superfamily, is found mutated in 30% of all human tumors and, in mammals, consists of three genes and four gene products . Many of these proteins are ubiquitously expressed but regulated by a multitude of specific Guanine nucleotide Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs). In fact, even though this protein superfamily has the endogenous capability to hydrolyze the bound GTP, GEFs and GAPs, respectively catalyze the activating and the inactivating reactions  suggestiOther than the REM and CDC25 domain, capable of exchanging the Ras-bound GDP, full length RasGRFs contain, in fact, a PH domain that can interact with the NGF receptor TrkA  and an IRas-GRF1 was previously implicated in beta cell langherans islet development, glucose homeostasis , learninRas-GRF1 is normally expressed in brain , pancreatic cells and skeletal muscle . MessengRasGrf1−/− mice. Survival curves revealed a marked increase (20%) in the average lifespan of RasGrf1−/− male mice  and mice with RasGrf1−/− showed better motor coordination than controls. At the molecular level they find: a) Increased Expression of the 16S rRNA in RasGrf1−/− mice. 16S rRNA is one of the mitochondrial rRNA and mitochondrial gene expression and function has been demonstrated to positively correlate with longevity in organisms from yeast to human [RasGrf1−/− mice. Sirtuins play a role in a variety of diseases [RasGrf1−/− mice.In their study, Borras et al.  find incto human , 42, 43 diseases , but thediseases . c) MainRasGRF1−/− mice with respect to the wild type of the same age, d) RasGRF1−/− mice displayed reduced IGF1 activity and blood metabolomic analysis showed clear similarities with calorie-restricted animals. IGF1 activity has a consistent effect on the aging process [Reduced glucose uptake is associated with aging . They an process , suggest process . Higher  process . Borras In yeast the Ras, Tor and Sch9 signaling pathways are partly responsible for integrating nutrient inputs into cell growth, division and aging . ImpairmOrthologs of genes that function downstream of Ras in yeast have also been implicated in longevity extension in mice. Deletion of the mouse adenylate cyclase type 5 and the consequently PKA downregulation extends lifespan, stress resistance and mediates upregulation of SODs  and miceIt is therefore tempting to propose that the pro-longevity effect of the homozygous Ras-GRF1 deletion observed by Borras is due to a reduced activity of the Ras or an analogous pathway and to consider that the increased longevity observed may be evidence for the conserved role of the Ras pathway in the aging process in organisms ranging from yeast to mammals . HoweverIn summary, the identification of a pro-aging role of Ras in aging therapy is an important and welcomed discovery especially considering that drug companies have identified a number of compounds capable to inhibit GEFs such as Brefeldin A (large ArfGef), SecinH3  and NSC23766 (RacGef) , 37, 38.


Reactive oxygen species are implicated in the physiopathogenesis of salt-induced hypertension and the C242T polymorphism of the p22-phox gene has been associated with higher superoxide production. This study investigated the impact of this polymorphism on the relationship between urinary sodium excretion (USE) and blood pressure levels in an urban Brazilian population.We cross-sectionally evaluated 1,298 subjects from the city of Vitoria-ES, located in the Southeast region of Brazil, by clinical history, physical examination, anthropometry, analysis of laboratory parameters, USE measurement and p22-phox C242T polymorphism genotyping.No significant differences in studied parameters were detected between the studied genotype groups (CC vs. CT+TT). Systolic blood pressure exhibited significant correlation with USE only in T allele carriers , while diastolic blood pressure and hypertension status correlated with USE in both genotypes albeit more weakly in subjects with CC genotype  than in T carriers . Regression analyses adjusted for confounding factors showed that USE remained independently associated with systolic (p<0.001) and diastolic blood pressure (p<0.001) and hypertension status (p = 0.004) only in T allele carriers. Finally, higher diastolic and systolic blood pressure levels were detected in T allele carriers than in CC genotype individuals in the highest tertile of USE.The p22-phox 242T allele is associated with higher blood pressure levels among subjects with higher USE in an urban Brazilian population. Oxidative stress, exerted by intracellular accumulation of reactive oxygen species, has been implicated in the physiopathogenesis of salt-induced hypertension To date, several polymorphisms of the p22-phox (CYBA) gene have been identified Urinary sodium excretion is usually used as the standard method for evaluation of sodium intake in clinical and epidemiological studies The study was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Institutional Review Board of the Espírito Santo Federal University. All participants read and signed informed consent.A cross-sectional study of risk factors for cardiovascular diseases was performed in the urban population of Vitoria, Brazil, using the WHO-MONICA project guidelines 2). Height was measured in centimeters and weight in kilograms using a calibrated balance.Blood pressure was measured using a standard mercury sphygmomanometer on the left arm after 5 min rest, in the sitting position. Systolic and diastolic blood pressures were calculated from three readings with a minimal interval of 5 min. Hypertension was defined as systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg. Glucose, total cholesterol, lipoprotein fractions, triglycerides, uric acid and creatinine were assayed by standard techniques in 12-h fasting blood sample. Diabetes mellitus was defined as a fasting glucose ≥126 mg/dL and/or use of hypoglycemic drugs Urine was collected from participants during a 12-h period (from 7∶00 PM to 7∶00 AM) during the night before the clinic visit. Sodium and potassium concentrations were measured in the 12-h urine by flame photometry. All measurements were performed in the same laboratory and the same commercial kits were used in all investigations. Previous data have shown that 12-h urine collected at night can be used as a reliable tool to estimate 24-h excretion of sodium and potassium Genomic DNA was extracted from leukocytes in samples of whole blood, following a standard salting-out procedure Data were analyzed using SPSS 15.0™. Descriptive statistical results are given as means ± SEM. All continuous variables presented normal distribution as assessed by the Kolmogorov–Smirnov test. A chi-square test was used to compare categorical variables and to test for Hardy–Weinberg disequilibrium. Unpaired t-test was used to compare continuous variables. Bivariate correlations between variables were examined by using Pearson correlation coefficient for normally distributed data and Spearman rank correlation coefficient for non-normally distributed data. Fisheŕs z-test was used to compare correlation coefficients. Linear and logistic regression analyses were used to evaluate the independent predictors of blood pressure levels and hypertension, respectively. Variables that exhibited significant correlation at bivariate analysis and urinary sodium excretion were included as independent variables in regression analyses. Significance was accepted if p<0.05.2 = 0.78). The clinical, anthropometric and laboratory characteristics of studied subjects according to C242T genotypes are shown in Genotypic frequencies of the C242T polymorphism  were in accordance with the Hardy-Weinberg equilibrium , while diastolic blood pressure correlated with urinary sodium excretion in both genotype groups, albeit more weakly in CC genotype than in T carriers . In addition, significant correlation coefficients between hypertension status and urinary sodium excretion were detected in both genotype groups albeit also more weakly in CC genotype than in T carriers . Further correlation analysis between clinical/laboratory variables and blood pressure measurements/hypertension was performed in order to determine potential confounding factors in the relationship between blood pressure/hypertension status and urinary sodium excretion in both genotype groups .To confirm whether the p22-phox C242T polymorphism influenced the relationship between urinary sodium excretion and blood pressure and hypertension status, linear and logistic regression analysis were performed. Noticeably, urinary sodium excretion remained independently associated with systolic and diastolic blood pressure  and hypeThe next step was to evaluate the frequencies of the p22-phox C242T polymorphism and the clinical and laboratory features of all studied subjects according to the tertiles of urinary sodium excretion . IncreasIn the present report, we found that the p22-phox C242T polymorphism modulated the relationship between urinary sodium excretion and blood pressure levels in Brazilian general population. Remarkably, only in presence of the T allele, urinary sodium excretion showed an independent association with systolic blood pressure, diastolic blood pressure and hypertension status. In addition, higher diastolic and systolic blood pressure levels were detected in T allele carriers than in CC genotype individuals in the highest tertile of urinary sodium excretion. Overall, these findings indicate that the p22-phox 242T allele might be a potential marker of increased blood pressure under higher salt intake.Although the reasons by which the 242T influenced the interaction between sodium intake and blood pressure levels were not apparent in the present study, some explanations can be proposed. High daily salt intake impairs vasodilatation and enhances vasoconstriction, resulting in reduction of regional blood flow and elevation of blood pressure in healthy and hypertensive individuals et alet al  presented a remarkable smaller sample size than that of the present paper , which strengthens the validity of our data. Despite the differences, these data indicate that the p22-phox C242T polymorphism might be a potential marker of susceptibility to blood pressure response to high sodium intake.Our study did not measure salt sensibility, but our main findings are in the same line of those reported by Castejon Some limitations to our study need to be addressed. First, we only performed a single measure of urinary sodium excretion, which might not be fully representative of sodium intake. Nevertheless, a previous study evaluating the urban population of Vitória showed that urinary sodium excretion estimated by a single 12-h urine collection had a good correlation with sodium intake assessed by questionnaires In conclusion, the present study provided novel evidence that the p22-phox 242T allele is associated with higher blood pressure levels among subjects with higher USE in an urban Brazilian population. However, further studies in other populations are warranted to confirm the current evidence.Table S1Bivariate correlation coefficients between blood pressure/hypertension status and clinical/laboratory variables.(DOC)Click here for additional data file.


Globally, it is estimated that nearly 10 million people sustain severe brain injuries leading to hospitalization and/or death every year. Amongst survivors, traumatic brain injury (TBI) results in a wide variety of physical, emotional and cognitive deficits. The most common cognitive deficit associated with TBI is memory loss, involving impairments in spatial reference and working memory. However, the majority of research thus far has characterized the deficits associated with TBI on either reference or working memory systems separately, without investigating how they interact within a single task. Thus, we examined the effects of TBI on short-term working and long-term reference memory using the radial 8-arm maze (RAM) with a sequence of four baited and four unbaited arms. Subjects were given 10 daily trials for 6 days followed by a memory retrieval test 2 weeks after training. Multiple training trials not only provide robust training, but also test the subjects' ability to frequently update short-term memory while learning the reference rules of the task. Our results show that TBI significantly impaired short-term working memory function on previously acquired spatial information but has little effect on long-term reference memory. Additionally, TBI significantly increased working memory errors during acquisition and reference memory errors during retention testing 2 weeks later. With a longer recovery period after TBI, the robust RAM training mitigated the reference memory deficit in retention but not the short-term working memory deficit during acquisition. These results identify the resiliency and vulnerabilities of short-term working and long-term reference memory to TBI in the context of robust training. The data highlight the role of cognitive training and other behavioral remediation strategies implicated in attenuating deficits associated with TBI. Traumatic brain injury (TBI), resulting in temporary or permanent impairment of cognitive abilities and physical functioning, is a major public health problem around the world  is a particularly useful task with which to characterize TBI-induced deficits in both reference and working memory during learning and/or memory retrieval  for three separate experiments  weighing 270–300 g were used for the current experiment. Rats were individually housed in plastic cages (48 × 27 × 16 cm) containing hardwood bedding. Animal quarters were maintained at constant temperature (22 ± 1°C) and relative humidity (40–50%) with a 12 h light/dark cycle (lights on at 8 AM). Food  and water were available Rats were trained on an eight-arm radial arm maze as previously described  and then placed on a stereotaxic frame. A midline incision was made to expose the skull. Animals were subjected to a 6 mm diameter craniotomy centered over the right somatosensory cortex, equidistant from bregma and lambda. Craniotomy was carefully performed to ensure that the dura remained intact. Controlled cortical impact (CCI) injury was induced using a cortical impact device  with a pneumatic steel impactor tip (5 mm diameter) angled at 22° from the sagittal plane and perpendicular to the site of injury . Performance on the radial arm maze during the retention test was analyzed by using unequal variance, one-tailed F = 27.83, p < 0.001, n = 4 per condition] and decreases in both working  and reference  memory errors during the second half of training compared to the first half  = 4.735, p < 0.001, n = 3–4 per condition], in addition to a decrease in reference memory errors during the second half of training . Although there were no differences in percent correct between Sham and TBI groups, overall they performed worse than subjects in experiment 1, which were trained prior to surgery . In addition, both groups made significantly more working memory errors  = 4.191, p < 0.05], while the TBI group overall made significantly more working memory errors compared to Shams throughout the training . The difference in memory acquisition between groups was also reflected in their retention test 2 weeks later: the TBI group had a significantly lower percent correct score compared to Shams  = 19.34, p < 0.001, n = 3–6 per condition] without any differences between TBI and Sham groups. Overall, subjects in experiment 3 did not perform significantly different from subjects in experiment 1, which were trained prior to surgery . Again, both TBI and Sham groups showed significant reductions in working  and reference  memory errors during the second half of training compared to the first half  = 11.09, p < 0.01]. During retention testing 2 weeks after training, there were no differences in retention between conditions  after TBI, the acquisition of new spatial information is impaired. We identify deficits in short-term working memory during training and in long-term reference memory during retention testing 2 weeks post-training. Similarly, with a longer recovery period, we find that deficits in short-term working memory continue to be prevalent during training. However, these subjects show no deficit on the long-term memory retention at 2 weeks. These data suggest that some of the long-term memory deficits associated with TBI can be remediated with longer recovery time and robust training, but lingering deficits in short-term working memory remain. Addressed here, we explore the functional significance of a short-term working memory deficit and raise the question as to other potential remediation factors applied to improving memory deficits associated with TBI.Several studies have shown a reversal of short-term memory deficits in overtrained transgenic mice and an elimination of the spatial memory deficits with subsequent re-training (Daumas et al., In experiments 2 and 3, TBI groups demonstrated no deficits in overall percent correct during the acquisition phase compared to Shams Figures , 2B. ParSince many previous studies on the effects of TBI on memory specifically chose a working memory paradigm to examine, their results are limited to this one particular memory function. In our application of the RAM, we were able to assess both reference and working memory systems. The dissociation between reference and working memories is more difficult to accomplish with other tasks, which can be adapted using protocols for either memory system, but not both simultaneously (Yoshihara and Ichitani, In experiment 2, animals had a limited time to recover before beginning RAM training. Though they showed significant improvement in their overall scores Figure , both grIn experiment 3, where subjects had 6 weeks to recover, we show that memory retrieval remained intact after injury Figures . We findThe CCI model we use is known to affect several different brain regions including parietal and medial frontal cortices involved in memory and spatial localization (Dixon et al., Much effort has been applied to finding the deficits associated with TBI in the hope that better treatment options will be developed. Overall, it appears that the injury itself requires several weeks to reach a stable recovery state (Soblosky et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The genes encoding mgp2–10 are organized on a 40 kb syntenic block in the tsetse genome, have similar exon-intron arrangements, and share regions of amino acid sequence similarity. Expression of mgp2–10 is female-specific and high during milk secretion. While knockdown of a single mgp failed to reduce fecundity, simultaneous knockdown of multiple variants reduced milk protein levels and lowered fecundity. The genomic localization, gene structure similarities, and functional redundancy of MGP2–10 suggest that they constitute a novel highly divergent protein family. Our data indicates that MGP2–10 function both as the primary amino acid resource for the developing larva and in the maintenance of milk homeostasis, similar to the function of the mammalian casein family of milk proteins. This study underscores the dynamic nature of the lactation cycle and identifies a novel family of lactation-specific proteins, unique to Glossina sp., that are essential to larval development. The specificity of MGP2–10 to tsetse and their critical role during lactation suggests that these proteins may be an excellent target for tsetse-specific population control approaches.In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland) and provided in mother's milk during lactation. Interference with at least two milk proteins has been shown to extend larval development and reduce fecundity. The goal of this study was to perform a comprehensive characterization of tsetse milk proteins using lactation-specific transcriptome/milk proteome analyses and to define functional role(s) for the milk proteins during lactation. Differential analysis of RNA-seq data from lactating and dry (non-lactating) females revealed enrichment of transcripts coding for protein synthesis machinery, lipid metabolism and secretory proteins during lactation. Among the genes induced during lactation were those encoding the previously identified milk proteins ( Tsetse flies are the sole vector for African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Transcriptome and proteome analyses were utilized to examine the underlying mechanisms of tsetse lactation that occur during each reproductive cycle. These analyses revealed a dramatic shift to the synthesis of milk proteins during lactation and a novel milk-specific protein family. All members of this family were co-localized, shared sequence similarity and were expressed at 40× basal levels during milk secretion. Suppression of gene from this lactation-associated family impaired progeny development by reducing milk protein content and altering milk homeostasis. These novel genes represent an excellent target for tsetse-specific reproductive-based control mechanisms. In addition, the characterization of tsetse milk production revealed multiple factors that are functionally analogous between tsetse and mammalian lactation. Tsetse reproductive biology is unusual among insects. Female tsetse give birth to a fully mature third instar larva (viviparity) after an extended intrauterine gestation. This reproductive strategy limits the capacity of tsetse mothers to only 8–10 offspring per lifetime The nutritional components of tsetse milk consist mainly of proteins and lipids emulsified in an aqueous base Glossina morsitans morsitansin silico approach, and the microsyntenic genomic organization of nine tsetse-specific milk proteins (MGP2–10) that we propose represent a highly divergent, novel protein family. siRNA-based knockdown analysis was employed to examine the functional roles of the MGP2–10 proteins during tsetse reproduction. Since MGP2–10 are tsetse-specific and have substantial influence over tsetse fecundity, we discuss their potential for exploitation in novel population reduction approaches. Lastly, we discuss our findings in light of milk secretions described from other lactating organisms.In this study, a satellite paper to our report on the whole genome sequence of the tsetse species Wigglesworthia, Sodalis and Wolbachia) specific sequences and cleanup resulted in a 4% reduction in the total number of sequences identified in lactating flies and a 5.1% reduction for dry flies, respectively  were expressed at least 10-fold higher in lactating versus dry flies  was also higher in dry flies, reflecting the yolk protein synthesis that occurs between bouts of lactation (The majority of the contigs (94%) were more abundant in dry (non-lactating) compared to lactating flies . Multiplt damage . Lipid mng flies . Expressactation . Contigsactation . Given tasmase1, mgp1–10 and transferrin). Removing these reads yielded only a 2.6% reduction in the dry fly dataset, but a drastic reduction of 47.2% was observed in the lactating fly dataset , glyoxylate/hydroxypyruvate reductase (an enzyme that converts glycerate to hydroxypyruvate), choline-phosphate cytidylyltransferase  and multiple ribosomal proteins  value and are likely present in milk at low levels or may be products from the larval gut . Most of PGRP-LB , suggestGlossina fuscipes fuscipes  and Glossina pallidipes , using RT-PCR with degenerate primers . The sequence obtained from the flesh fly may represent either a class of proteins distinct from the tsetse MGP family, or could be highly divergent ancestral sequence to MGP2–10 genes found in tsetse.BLASTx searches of the NCBI nucleotide collection failed to recover orthologous sequences to the MGP2–10 from any organism. Partial gene sequences encoding MGPs were identified from two other tsetse species,  primers , S6. Minc region . The phymgp3,7,8 . All mgptructure , despitetructure ; Fig. S5ab initio as no homologous protein structures were available. The ab initio structure predictions from the four programs revealed that MGPs usually form multiple α-helices (6–10 per protein). However, no functional insights were provided by the I-TASSER program, since MGPs lack structural similarity to other characterized proteins  along the coding sequences for these genes. This type of analysis is usually conducted on orthologous genes in different species or on multiple alleles within a species, but utilization of this on paralogous genes could provide insight into regions critical to their function. When dN/dS substantially exceeds 1, evidence for positive selection ( = adaptive evolution) is inferred. In contrast, dN/dS = 1 implies neutral evolution, while dN/dS values closer to zero provide evidence for negative or purifying selection. Sequences were translated and multiple alignments were performed in ClustalX Fixed Effects Likelihood; www.DataMonkey.org; Examination of amino acid alignments identified several regions of moderate conservation across MGP2–10 from G. morsitans, the secretory peptide and conserved region 1 appear to be largely under negative selection  were similar to those observed for mgp2–10 in G. morsitans. Transcript abundance was lower in teneral females and in females with developing intrauterine embryos, becoming progressively greater through larvigenesis  had no effect on the number of pupae produced per female, the length of pregnancy, or the incidence of pupal emergence  reduced the number of pupae deposited per female by 10–15% and extended the duration of pregnancy by 2–3 d, but no difference was observed in the incidence of adult eclosion  compared to siGFP treated controls (0.56±0.04 mg protein/5 µl milk). We hypothesized that these proteins may serve a function in maintaining milk lipid stability. To explore this possibility, we assessed the stability of milk emulsification after MGP knockdown utilizing an emulsion stabilization assay. Knockdown of mgp5,7–9 resulted in an increased rate of separation of the aqueous and lipid fractions of the milk by over two-fold (Bradford assay of the milk protein content indicated that simultaneous knockdown of two-fold . These fGlossina sp.) are vectors of African trypanosomes and reduction of this insect population is a highly effective control strategy. The success of population reduction based control efforts largely is due to the low reproductive capacity of tsetse, which is viviparous yields few progeny per female over their lifespan.Human African Trypanosomiasis  is a fatal disease that affects millions of people in sub-Saharan Africa. A disease caused by closely related parasites in animals, known as African Animal Trypanosomiasis , devastates agricultural and livestock production systems as well as land use in Africa. There are no prophylactic drugs that are affordable and highly efficacious, and simple tools for diagnosis or mammalian vaccines for control of HAT or AAT are lacking. Tsetse . Expression of these genes is tightly regulated through the transition between dry and lactating periods in order to optimize resource allocation for milk production. We also analyzed milk collected from larval guts using a proteomics approach both to characterize its composition and to verify secretion and transfer from mother to offspring of the nine milk proteins in addition to Transferrin, aSMASe1 and MGP1. Among the proteins identified as lactation products, MGP2–10 represent a group of secreted proteins unique to tsetse. Using a knockdown approach we showed that tandem suppression of multiple Glossina austeniMelophagus ovinus, a viviparous sheep ked mgp1–10 likely constitutes over 95% of the protein content in tsetse milk  that develop in actively secreting milk gland cells, and then degenerate during an involution period following parturition asmase1, mgp1–10 and transferrin transcript levels during lactation indicate that the expression of these 12 genes which constitutes less than 0.0005% of the contigs from the de novo library, represents over 45% of the RNA-seq library. In contrast, in dry flies, these same gene transcripts represent less than 2.6% of the library. A heavy investment in specific genes during reproduction is not uncommon The extreme elevation of transferrin, asmase1 and mgp1 expression during lactation were expected since these proteins are recognized as major constituents of tsetse milk asmase1 in lactating females reduces fecundity and severely impacts progeny fitness Increases in bmm, is increased during lactation, while expression of most other lipid metabolism genes are suppressed or expressed at levels equivalent to those seen in dry flies. Such minimal transcript variation is perhaps not surprising in light of recent studies on insect lipolysis, which reveal that most control occurs at the post-translational level, either through insulin signaling or through other factors that interact with the surface of the lipid droplet Lipids, specifically triacylglycerides, constitute the other major nutritional components in tsetse milk midway and bmm, while other such genes associated the lipid metabolism show little variation perilipin1 and perilipin2 transcripts are elevated in dry versus lactating flies and perhaps these proteins, which interact with lipid droplets, are necessary to accommodate the drastic increases in fat body volume that occurs during the involution periods that separate lactation cycles. In general, our results regarding expression of lipid metabolism genes support our previous studies that bmm-mediated lipolysis plays a critical role in regulating lipid homeostasis during pregnancy An argument for post-translational regulation is further supported by our recent study showing that insulin and juvenile hormone signaling both act to coordinate lipid metabolism in tsetse mothers through transcriptional regulation of select lipolytic/lipogenic genes including Drosophila. As a member of transforming growth factor beta (TGF β) superfamily of growth factors, activin may be signaling growth of a specific tissue, possibly the milk gland, during lactation. In addition to the role in Drosophila, activin is key in regulating growth of the mammary gland during lactation in multiple mammals and has a critical role in breast cancer glyoxylate/hydroxypyruvate reductase, grhpr, may provide additional substrates to maintain homeostasis of proline, the main nutrient source in tsetse hemolymph cct, has been linked to changes in lipid droplet size Removal of reads mapping to the 12 abundant milk protein genes during RNA-seq analysis allowed for the identification of three additional genes that were enriched and highly expressed during milk production. Dawdle is an activin signaling molecule that has been linked to synaptic growth at the neuromuscular junction versus lactating flies. This difference is likely due to the fact that transcript levels for most genes are reduced in lactating flies at the expense of generating lactation-specific proteins. In dry flies, transcript elevation for genes associated with digestive processes likely corresponds to the increased bloodmeal size in flies not harboring an intrauterine larva Our transcriptome data revealed that the majority of genes are expressed at higher levels in dry Wigglesworthia and Sodalis, Recent studies focusing on MGP2 and MGP3 failed to verify their transfer to the nursing larvae since antisera were not available mgp2–10 during lactation and reduced expression after parturition indicate that these proteins are under similar transcriptional regulation and that they may also serve as a source of proteins for larval nutrition mgp5,7–9 transcripts were suppressed by 60–70%, suggesting that MGPs, based on total transcript abundance, likely account for 70–75% of the total protein content of tsetse milk. The MGP2–10 proteins also contain all amino acids, supporting the notion that they function as a complete protein resource for the developing larva. Furthermore, multiple phosphorylation sites associated with each protein suggests that MGP2–10 may also serve as a source of phosphate in tsetse milk. The lack of predicted glycosylation sites on MGP2–10 is not surprising since carbohydrate levels are extremely low in tsetse milk We identified seven new milk gland proteins, MGP4–10, that are similar to MGP2–3. MGP2–10 each contains a conserved secretory signal and multiple sites throughout three moderately-to-highly conserved regions with several residues under apparent strong purifying selection. Structural analysis of these MGPs failed to provide functional insights, but did reveal that these proteins are likely globular, consisting of multiple α-helices. Further study is necessary to conclusively determine the structures of these novel proteins. The coordinated high expression levels observed for mgp7–9 results in acceleration of lipid separation from the aqueous phase of milk. This suggests that MGP7–9 (and likely the other MGPs) may represent the previously-documented, unidentified low molecular weight proteins associated with tsetse milk lipids Previous studies have shown that low molecular weight proteins interact with lipids in tsetse milk mgp2–10 may have expanded by multiple gene duplication events from a common ancestor. It is possible that ancestral duplication events yielded two separate groups which may have been subsequently expanded as a result of unequal genetic crossing-over with the mgp2,4,5,6,9,10 being encoded on the antisense strand. Predicted three-dimensional structures between MGP2–10 is similar including multiple α-helices and a globular protein tertiary arrangement. mgp2–10 are under nearly identical transcriptional regulation showing increased expression during tsetse fly lactation and rapid decline during involution. These proteins also exhibit functional redundancy as a source of secreted amino acids in the milk and in sustaining lipid-protein homeostasis within the aqueous milk base. Although MGP2–10 have varying levels of amino acid similarities (18–91%), there are conserved regions they share outside of the secretory peptide. Specifically, 23 sites are under purifying selection (8 in the secretory peptide coding sequence and 15 dispersed throughout the remaining portions of the sequence), and these are likely critical to the functional role of MGP2–10 during tsetse lactation. Collectively, similarities between MGP2–10 indicate that these proteins constitute a novel family in tsetse similar to other highly divergent protein families, including caseins Similarities among MGP2–10 suggest that these proteins represent a highly divergent lactation-specific protein family from tsetse flies. These genes are localized to a single 40 kb chromosomal loci, have similar gene structures and their phylogeny correlates with their chromosomal organization indicating that Our previous work demonstrated that several mechanisms underlying tsetse lactation parallel characteristics of mammalian lactation. First, both systems have highly specialized lactating cells that cycle through periods of high productivity during lactation to low activity following involution in vivo and in vitroMammalian genomes contain no orthologous sequences to the nine novel tsetse MGPs. However, MGPs might function analogously to caseins in mammalian milk. Caseins are the major amino acid and calcium source for the mammalian neonate S1-casein in goats S1-casein, respectively. Offspring receiving milk from α-casein-null mothers experience delayed growth and life-long body size reduction, but only transient effects on physical and behavioral development Few studies have examined the effects of casein knockdown/knockout in mammals. In mice, knockout lines have been developed for α-, β- and κ-casein This study provides the first complete examination of the mechanisms underlying tsetse fly lactation. In general, our results show that the majority of genes have lower expression during lactation with the exception of those directly involved in milk production. The combination of transcriptomic and proteomic analyses reveals there are 12 major milk gland proteins, which comprise ∼47% of the transcriptome of lactating flies, along with multiple minor protein constituents of tsetse milk. We have provided an overview of the combined results of this study . FurtherG. morsitans morsitans were reared at Yale University and the Institute of Zoology at the Slovak Academy of Sciences (SAS). The other two species  were reared at SAS. Flies were maintained on blood meals provided through an artificial feeding system at 48 h intervals S. crassipalpis, acquired from Ohio State University were reared according to standard procedures Colonies of Total RNA was extracted from individual flies or dissected tissues using Trizol reagent , following the recommended protocol. RNA was treated twice with the TURBO DNA Free kit  to remove DNA, alcohol precipitated to remove residual salt, and further cleaned using the RNeasy kit . Total RNA (2–3 µg) was pooled from 10 flies extracted individually for each treatment. Sample quality and concentration was determined using a Bioanalyzer 2100 . Library construction was performed using standard protocols for Illumina mRNA-Seq sequencing by the W. M. Keck Foundation Microarray Resource at the Yale School of Medicine. Each single-end library was sequenced on one lane of the Genome Analyzer II platform .WigglesworthiaWolbachia (unpublished) and SodalisG. morsitans. Following symbiont specific sequence removal, remaining sequences were trimmed in CLC Genomics (CLC Bio) to remove ambiguous nucleotides. Contig libraries were constructed using Abyss D. melanogaster) and Vectorbase An. gambiae). Each read from each library was compared by BLASTn to the assembled coding sequences (CDS) using a word size of 25, m8 output and low complexity filter turned off. CDS coverage and CDS number of read “hits” from each library were computed from the BLAST output file. A hit was only considered significant if it had 97% or better identity to its target and no more than one gap. The same read could be mapped up to three different CDS to the extent that their BLAST scores were identical. Expression levels were determined using CLC Genomics Workbench . Reads were mapped to our de novo assembly with an algorithm allowing only two mismatches and a maximum of 10 hits per read. RPKM was used as a measure of gene expression To determine Illumina read quality, FastQC analysis was performed on the transcriptomes generated from dry and lactating flies. Due to the prevalence of tsetse symbiont sequences in the reads, a specific quality control step was included to reduce bacterial sequence reads using the known whole genome sequence data from 2O to a final volume of 100 µl. Samples were stored at −80°C until analysis. Peptides were separated with a Waters nanoAcquity UPLC system (75 µm×150 mm BEH C18 eluted at 500 nl/min at 35°C) with Buffer A  and Buffer B . A linear gradient was established with 5% Buffer B, increasing to 50% Buffer B at 50 minutes and finally to 85% Buffer B at 51 minutes. MS/MS was acquired with an AB Sciex 5600 Triple Time-of-Flight mass spectrometer using 1 microscan followed by four MS/MS acquisitions. Neutral loss scans were obtained for 98.0, 49.0 and 32.7 amu. Seven separate 1 µl injections at an estimated 0.351 µg/µl concentration for a total of 2.457 µg on the column were used for analysis.Pulled glass capillary tubes were used to collect milk samples by negative pressure from the guts of feeding third instar larvae, which were microscopically dissected from the uterus of pregnant females. Samples were stored in 1× protease inhibitor cocktail (Sigma-Aldrich). Proteins were precipitated with 10% trichloroacetic acid (Fisher Scientific) at 4°C overnight, collected by centrifugation  and washed two times with ice-cold acetone. Protein pellets were briefly dried and dissolved in 10 µl of protein pellet buffer . Trypsin digestion was performed at 37°C for 12–16 h following dilution with distilled H10P), where P is the absolute probability that a match is random. Matches were considered significant when the probability of a random match fell below 5% . Therefore, Mascot scores greater than 68 were above the significance threshold when searching the newly assembled library. Proteins were considered to be successfully identified when two or more peptides matched the same predicted protein and the Mascot score exceeded the significance threshold. The exponentially modified protein abundance index (empai) was employed to estimate levels of protein species based on the number of species detected compared to the number of possible peptides for specific protein Mascot algorithm was used to analyze uninterrupted MS/MS spectra mgp2–10 were obtained by mapping Illumina high-throughput reads against G. m. morsitans genomic scaffolds in the CLC Genomics software package. Nucleotide and predicted protein sequences were aligned using PROMALS3D Sarcophaga crassipalpis, sequences were obtained from a previous EST project mgp2–10 from other tsetse species  were obtained from female cDNA by RT-PCR followed by cloning into T-vector plasmid (Invitrogen) and sequenced at the DNA Analysis Facility at Yale University . Pairwise phylogenetic tree construction and bootstrap analysis (10000 replicates) were performed using the MEGA4/5 sequence analysis suite Chromosomal organization of genes and full length mRNA sequences for For sex- and tissue-specific RT-PCR expression analyses, total RNA isolated from males and females and from dissected tissues was used as template for the Superscript III reverse transcriptase kit following the manufacturer's protocols (Invitrogen). Fat body and milk gland were analyzed as a combined samples since complete separation is nearly impossible due to the intricate association of these organs. PCR was performed with gene-specific primer pairs  using thtubulin) relative to the average value for the control treatment or newly emerged flies, yielding the delta Ct value. All experiments were analyzed in triplicate and subject to ANOVA followed by Bonferroni correction and Dunnett's test.For pregnancy-specific transcript abundance determination, qPCR analyses were performed using a CFX PCR detection system . Data were analyzed with CFX manager software version 3.1 (Bio-Rad). Primer sequences used were the same as used in RT-PCR analyses . Comparamgp gene, was also compared to the reference RNA library/G. morsitans genome tubulin transcripts. For combined knockdown studies, siMGP constructs were mixed to yield a sample concentration of at least 600 ng/µl for each siRNA targeting a specific MGP transcript. Fecundity following MGP knockdown was assessed as previously described Short interfering RNAs (siRNA) consisting of two Duplex sequences  were dest/ABS0) is plotted versus time based on the exponential model (ABSt = ABS0 e−kt). For this model, ABSt denotes absorbance at any time t, ABS0 is the initial absorbance, and k is the rate of absorbance decline in %/h.Emulsification assays were based on milk turbidity measurements. For this assay, milk was acquired from the guts of actively feeding larvae as before and diluted 10× prior to the assay. Samples were vortexed for 1 min at 10,000 rpm, and absorbance of the diluted emulsion was measured at 500 nm. Changes in absorbance were measured hourly for 10 h. Results were analyzed based on the slope of a regression, where ln Click here for additional data file.Figure S2Distribution of reads per contig in RNA-seq libraries with at least 2 mapped reads and 25 total reads between the two sample sets. Lactating (A) and dry (B) contigs are displayed in descending amount of number of reads per contig.(TIF)Click here for additional data file.Figure S328S expression during the first two tsetse gonotrophic cycles. Transcript levels were determined by qPCR with a CFX PCR detection system  and data were analyzed with CFX manager software version 3.1 (Bio-Rad). Data represent the mean ± SE of three replicates and was normalized to tubulin.Time course of (TIF)Click here for additional data file.Figure S4superoxide dismutase, SOD). (B) Heat shock protein genes. (C) Lipid metabolism genes . (D) Yolk protein genes. *, indicates significantly different between lactating and dry flies based on Kal's test followed by Bonferroni correction.Summary of specific genes that are differentially expressed in lactating compared to dry flies. (A) Antioxidant enzyme genes ((TIF)Click here for additional data file.Figure S5Glossina morsitans milk gland protein 2–10 (MGP2–10) and Sarcophaga crassipalpis milk gland protein-like protein (Sc-MGP-like protein). Multiple alignment was performed with ClustalX and optimized in BioEdit. Blue indicates at least 75% similarity between sequences and gray indicates 75% similarity between the classes of amino acids. Blue line above alignment indicates region of high sequence similarity.Amino acid analysis of MGP2–10. Multiple alignment of full length amino acid sequences of (TIF)Click here for additional data file.Figure S6Glossina morsitans morsitans, G. pallidipes and G. fuscipes milk gland proteins and Sarcophaga crassipalpis milk gland protein-like protein amino acid sequences. (A) Initial sequence alignment was completed using PROMALS3D server  Amino acid phylogeny of partial overlapping region of (TIF)Click here for additional data file.Figure S7Glossina morsitans morsitans. Structures predicted by Quark (126), I-TASSER Predicted protein structure of (TIF)Click here for additional data file.Figure S8G. fuscipes, G. pallidipes and the flesh fly, Sarcophaga crassipalpis in relation to total RNA content. (A) Transcript levels in tsetse mother with the indicated progeny developing in the ovary or uterus (B) Transcript levels in the flesh fly. Transcript levels were determined by qPCR. Data represent the mean ± SE of three replicates and was normalized to nadh subunit 2 (G. fuscipes), 28S  and Rp49 . *, denotes significant difference from control following ANOVA with Tukey's test at P<0.01.Transcript levels of milk gland protein and milk gland protein-like genes in (TIF)Click here for additional data file.Table S1Quantitative PCR primers and siRNA used for injection.(XLSX)Click here for additional data file.Table S2Glossina morsitans.Complete results for RNA-seq analysis comparing lactating and dry tsetse flies, (XLSX)Click here for additional data file.Table S3Results of RNA-seq analysis with statistical differences between lactating and dry determined by a Kal's test with Bonferroni correction, P<0.05.(XLSX)Click here for additional data file.Table S4Trypsin genes decreased in dry flies in comparison to lactating flies.(XLSX)Click here for additional data file.Table S5Results for RNA-seq data comparing lactating and dry flies following removal of reads for milk-specific contigs.(XLSX)Click here for additional data file.Table S6Results of RNA-seq data with statistical differences between lactating and dry flies determined by a Kal's test with Bonferroni correction, P<0.05 following removal of reads for milk-specific contigs.(XLSX)Click here for additional data file.Table S7Complete results for proteomic analysis of tsetse milk secretion.(XLSX)Click here for additional data file.Table S8Amino acid composition of milk gland proteins 2–10 as percent amino acids per protein. Total represents the average percent across MGP2–10. Highlighted are the essential amino acids.(XLSX)Click here for additional data file.Text S12 ratios from RNA-seq and qPCR values for seventeen genes. The Pearson's correlation coefficient (0.934) and goodness of fit (R2 = 0.872) were high, indicating a high degree of correlation between RNA-seq and qPCR fold changes between dry and lactating flies.Validation of RNA-seq data with qPCR. Correlation of log(DOCX)Click here for additional data file.


Fmr1 knockout (KO) mice results, among other changes, in abnormal dendritic spine formation and altered synaptic plasticity in the neocortex and hippocampus. Clinical features of FXS include cognitive impairment, anxiety, abnormal social interaction, mental retardation, motor coordination and speech articulation deficits. Mouse pups generate ultrasonic vocalizations (USVs) when isolated from their mothers. Whether those social ultrasonic vocalizations are deficient in mouse models of FXS is unknown. Here we compared isolation-induced USVs generated by pups of Fmr1-KO mice with those of their wild type (WT) littermates. Though the total number of calls was not significantly different between genotypes, a detailed analysis of 10 different categories of calls revealed that loss of Fmr1 expression in mice causes limited and call-type specific deficits in ultrasonic vocalization: the carrier frequency of flat calls was higher, the percentage of downward calls was lower and that the frequency range of complex calls was wider in Fmr1-KO mice compared to their WT littermates.Fragile X syndrome (FXS) is a well-recognized form of inherited mental retardation, caused by a mutation in the fragile X mental retardation 1 (Fmr1) gene. The gene is located on the long arm of the X chromosome and encodes fragile X mental retardation protein (FMRP). Absence of FMRP in fragile X patients as well as in  Fragile X syndrome affects approximately 1/4000 males. Due to the mosaicism of the x-linked mutation, symptoms are typically milder in females but about 1/8000 females have significant features of the syndrome Fmr1 expression causes deficits in ultrasonic vocalizations in mouse pups. USV emission is an important parameter for social communication in mouse pups Mice begin vocalizing shortly after birth, with a peak in vocalization rates occurring around postnatal day 8 (P8). They continue vocalizing, albeit at reduced rates, throughout adulthood Fmr1-KO and WT mice. Our findings revealed specific qualitative and quantitative deficits in the production of isolation induced USVs in pups of a Fmr1-KO mice compared to their wild type littermates.Mouse pups produce different shapes/categories of USVs when they are separated from their mother and siblings Fmr1-KO littermates were used for testing to avoid variability in behavioral performance because of mosaicism of the mutant allele due to X-inactivation in females. All experiments were performed with 8-day-old pups . All mice were weighed before behavioral testing began and the measurements were used for statistical comparison of body weights between Fmr1-KO and WT mice. Behavioral testing was performed by investigators blinded to genotype. Recordings were performed during afternoon sessions (1–5 pm) held constant across mice. After completion of behavioral tests, small pieces of tails were collected from the animals and processed for genotyping .Breeding pairs of B6.129P2-Fmr1tm1Cgr/J mice were purchased from Jackson Laboratory (stock # 003025) and bred by pairing wild type males with heterozygous females in a conventional mouse vivarium at the University of Tennessee Health Science Center using harem breading trios. Pups were kept with the dam until weaning at Postnatal Day 21 (P21). Juveniles were housed by gender in standard plastic cages not exceeding four per cage. All mice used in this study were raised and all experiments were performed in accordance with procedural guidelines approved by the University of Tennessee Health Science Center Animal Care and Use Committee. Principles of laboratory animal care  were followed. Only male WT and For USV recording of social calls Waveform patterns recorded in ten pups from each genotype were examined in depth. We classified a total of 1399 calls from WT and 1543 calls from KO animals. Each call was classified as one of 10 distinct categories, based on internal pitch changes, lengths and shapes, using previously published categorizations Fmr1-KO and wild-type mice.Besides the counts of call types, other parameters analyzed for each test day included 1) the total number of calls 2) the percentage of calls in the different categories, 3) the duration of calls, 4) the average carrier frequency of calls and 5) the frequency modulation depth of calls. We used power spectral density analysis to determine the frequency at peak power of each call. Average values of all parameters were calculated for each mouse and used for the statistical comparison of A mixed-model Analysis of Variance (ANOVA) with repeated measures was performed to analyze genotype dependent effects on the ultrasonic vocalizations, with the genotype as factor and call categories as the repeated measures Fmr1 KO mice (N = 10) were recorded and classified. Based on this classification we identified three deficits in USV calls in pups of Fmr1 KO mice.We classified all USV calls recorded during a 3 minute observation period into 10 types  according to the classification scheme proposed by Scattoni et al. Fmr1-KO compared to their WT littermates  = 3.177, p = 0.001; Newman-Keuls at % downward calls : p<0.001 for WT versus KO; The total number of downward call emissions was reduced in pups of termates , but thaTo compare the average carrier frequencies at which calls were emitted, we compared flat calls, as this category of calls maintains a single carrier frequency throughout the call. The average career frequency measurements were obtained from 10 mice of each genotype, based on averages from 10 flat calls per mouse. The average carrier frequency of flat calls emitted by KO mice  was higher than in WT animals   .Fmr1-KO (N = 10) compared to WT (N = 10) pups [genotype x call interaction: F  = 5.211, p = 0.004; Newman-Keuls at complex call p = 0.002 for WT versus KO; In order to analyze the frequency modulation of calls, i.e. the width of the frequency band covered by frequency modulation within calls, we measured the difference between the lowest and the highest frequency within calls Duration of calls was measured for complex, upward, downward, chevron, shorts, composite and flat calls . The aveFmr1-KO pups to be lighter than their wild type littermates. Body weight was not correlated with the number of USV emitted .The body weights of mouse pups tested for isolation induced USV calls were not significantly different between genotypes (t (20) = −1.864; P = 0.076; Fmr1 expression in a mouse model of fragile X syndrome does not cause a change in the number of isolation induced USVs generated by 8 day pups. Instead, we found that pups of Fmr1 KO mice showed more subtle, call type specific deficits: the carrier frequency of flat calls was higher, the frequency range of complex calls was wider and the percentage of downward calls was lower in Fmr1-KO mice compared to their WT littermates.Here we report that loss of Fmr1-KO mice emitted a lower percentage of downward calls compared to their WT littermates, while the percentages of all other call types were similar in both genotypes. Scattoni et al Pups of Fmr1-KO and WT pups. There are reports of longer and shorter duration of calls produced by different mouse models of autism Fmr1-KO mice are not expected to be at a disadvantage in eliciting the mother's response through USVs.There was no genotype difference in the duration of calls between Fmr1-KO mice are functionally related or caused by related changes in neuronal control mechanisms remains to be shown.The average carrier frequency of flat calls emitted by KO mice were higher compared to their WT littermates. It is unclear if this change has an effect on the response of the mother. Mouse pup calls incorporate some properties that suggest they could serve some of the same functions as the crying of human babies, especially their abilities to elicit parental retrieval behaviors Fmr1-KO mice were frequency modulated over wider frequency ranges than calls emitted by WT pups. Brudzynski et al −/− mutant mice to be less frequency modulated than the calls of their WT littermates. This is in contrast to our findings that pups of Fmr1-KO mice have increased frequency modulation compared to their WT littermates, a difference that remains to be explained. However, the findings indicate that autism related changes in the genetic makeup of the nervous system might affect the control mechanisms for USVs. Linking the specific USV deficits reported here and in other studies to specific neuronal or genetic mechanisms will require more in-depth studies. Currently, the analysis of USVs in mouse models of autism spectrum disorders is still in its infancy.Calls emitted by Fmr1 KO and WT mice.Body weight has been speculated to affect USVs, e.g. through differences in lung capacity Ube3a deficient mouse model of Angelman syndrome Fmr1 KO mice Fmr1-KO mice The neuronal mechanisms controlling human speech articulation and mouse oromotor/vocalization behavior are poorly understood. Several lines of evidence suggest that the cerebellum may play an important role in oromotor and vocalization/articulation in both species. In mice, the cerebellum has been shown to be critically involved in generation of ultrasonic vocalizations


Since the composition of the human microbiome is highly variable both within and between individuals, researchers are increasingly reliant on high-throughput molecular approaches to identify linkages between the composition of these communities and human health. While new sequencing technologies have made it increasingly feasible to analyze large numbers of human-associated samples, the extraction of DNA from samples often remains a bottleneck in the process. Here we tested a direct PCR approach using the Extract-N-Amp Plant PCR Kit to accelerate the 16S rRNA gene-based analyses of human-associated bacterial communities, directly comparing this method to a more commonly-used approach whereby DNA is first extracted and purified from samples using a series of steps prior to PCR amplification. We used both approaches on replicate samples collected from each of five body habitats  from four individuals. With the exception of the tongue samples, there were few significant differences in the estimates of taxon richness or phylogenetic diversity obtained using the two approaches. Perhaps more importantly, there were no significant differences between the methods in their ability resolve body habitat differences or inter-individual differences in bacterial community composition and the estimates of the relative abundances of individual taxa were nearly identical with the two methods. Overall, the two methods gave very similar results and the direct PCR approach is clearly advantageous for many studies exploring the diversity and composition of human-associated bacterial communities given that large numbers of samples can be processed far more quickly and efficiently. There is growing recognition that the human body harbors diverse communities of microbes and that the composition of these microbial  communities can have important effects on human health The most commonly-used approach to determine the phylogenetic and taxonomic structure of any microbial community, including those associated with humans, is to extract and purify DNA from samples , PCR amplify the 16S rRNA gene (or a region of that gene), sequence the resulting amplicons, and then analyze the resulting sequence data. Large numbers of samples can now be analyzed in this manner given that PCR setup can readily be automated using liquid-handling robots and the increasing availability of next-generation DNA sequencing technologies To assess if we could more efficiently and quickly go from sample to amplified DNA suitable for high-throughput 16S rRNA gene sequencing of human-associated bacterial communities, we tested a commercially available direct PCR kit that was originally designed for extraction and amplification of plant DNA. We analyzed samples collected from human mouths (tongue), feces, and three skin locations  using this direct PCR approach and compared the sequence data obtained to replicate samples extracted using a more traditional protocol in order to determine if the two approaches yielded comparable information on bacterial community structure. If so, the direct PCR approach could be useful in situations where high-throughput analyses of human-associated bacterial communities are currently hindered by the time and effort required to conduct standard DNA extractions.Volunteers were made aware of the nature of the experiment and gave written informed consent in accordance with the sampling protocol approved by the University of Colorado Human Research Committee (protocol 0409.13).°C immediately after collection.Samples were collected from five body habitats  on three men and one woman at a single point in time. These body habitats were selected as they likely represent a broad range of bacterial community types http://www.earthmicrobiome.org/emp-standard-protocols/). This extraction approach involves mechanical lysis, chemical lysis, and DNA purification in a series of 32 steps following the manufacturers' instructions. All 80 samples and 16 negative controls consisting of both reagent blanks and sterile swabs were extracted in a single 96-well plate which requires approximately 6–8 h to process a full plate. For the skin and tongue samples, DNA was extracted directly from individual swab tips placed into the respective wells. For the fecal samples, the four replicate swab tips per individual were placed into a sterile, DNA-free 50 mL conical tube with 2 mL of PCR-grade water . The conical tube was vortexed for 30 s and the resulting fecal slurry was used to load four replicate wells in the 96-well plate with 25 µL of fecal slurry per well. The resulting DNA was then PCR amplified using a primer set (515f/806r) °C, 30 s; 50°C, 1 min; 72°C, 1 min) after an initial denaturation of 3 min. at 95°C. Amplicons from the triplicate reactions were pooled together, visualized on an agarose gel, and quantified using the PicoGreen dsDNA assay . Amplicons from all samples were pooled in equimolar concentrations into a single composite sample that was then cleaned using a single-tube MoBio UltraClean PCR Clean-up Kit , and sequenced at Engencore (University of South Carolina) on a Roche GS-FLX 454 automated pyrosequencer running the Titanium chemistry.DNA was extracted from 80 of the 160 swabs using the approach described in detail in °C for 10 minutes, followed by centrifugation for 5 min at 2500 xg. Extract-N-Amp Plant Dilution solution  was added to the wells at a 1∶1 ratio to the extraction solution and mixed gently by pipetting. The plate was resealed with the mat and stored at 4°C overnight. PCR was conducted in 20 µL triplicate reactions per sample using 10 µL of Extract-N-Amp Ready Mix , 1 µL of the forward and reverse primers (the same primers described above), 5 µL of PCR-grade water, and 4 µL of the Extract-N-Amp sample solutions from the 96-well plate. PCR cycling conditions were identical to those described above, as were the amplicon processing, pooling, cleaning, and sequencing methods. A flow chart comparing the two extraction protocols is shown in The other 80 samples and negative controls (again consisting of reagent blanks and sterile swabs), were prepared for sequencing using a direct PCR approach. Tongue and skin swab tips were placed directly into wells in a 2 mL 96-well Deep Well plate  along with the appropriate negative control samples. The Axygen plate was chosen for its separate well walls, which offered more uniform heating of samples as opposed to plates with shared well walls. The four replicate fecal samples from each individual were processed as described above with 25 µL of fecal slurry placed into each of four replicate wells in the 96-well plate. The plate was processed using the Extract-N-Amp Plant PCR kit  following the manufacturers' instructions except for adjusting the volume of reagents as detailed below. Wells containing fecal slurry received 100 µL of Extract-N-Amp Plant Extraction solution  and wells containing the swab tips  received 250 µL of this solution. The plate was then sealed securely with a 96 round well Impermamat Silicon Sealing Mat , heated in a water bath at 90–95de novo into operational taxonomic units (OTUs) at 97% sequence identity using UCLUST All sequences were processed and sorted using the default parameters in QIIME Gammaproteobacteria. After removal of these negative control OTUs, quality filtering of sequences, and rarefaction to 400 sequences per sample, 142 of the 160 test samples remained (Most (13/16) of the negative control samples associated with the direct PCR approach produced sufficient amplicon yields for sequencing. To correct for this, OTUs constituting greater than 1% of the total negative control sequences were removed from all samples prior to rarefaction and all downstream analyses. In total, 16 OTUs  were removed with 75% of the sequences  belonging to the remained . With thTo evaluate the suitability of the direct PCR protocol and to compare the direct PCR results to the results obtained using the extraction/purification approach, a variety of alpha and beta diversity metrics (both phylogenetic and taxonomic metrics) were calculated from the resulting sequence data. Briefly, alpha diversity is defined here as the number of taxa (OTU richness) or lineages , the two approaches do yield different estimates of some taxon abundances, differences that could be due to the direct PCR approach failing to release DNA from certain cell types. If the goal is to assess the intra-individual variability in bacterial communities within a given body habitat (e.g. time series studies), either method would suffice, but it is important not to mix the methods as the two methods do not yield identical estimates of taxon abundances, particularly in the fecal and tongue samples.When we restrict our analyses to the beta diversity patterns across replicate samples collected from specific body habitats of individuals, we were able to detect significant differences between the methods for nearly all of the fecal samples, some of the tongue samples, and a few of the skin samples  and 3. OOne important aspect of high-throughput sequencing is the variability in results obtained from replicate samples. Samples that are identical and are processed in an identical manner should yield very similar sequence data, but previous work has demonstrated that this is often not the case The direct approach is far faster – going from sample to amplicons suitable for sequencing was at least 6 to 8 times faster with the direct PCR approach than the DNA extraction/purification approach . AlthougOf course, the direct PCR approach does have its limitations that render it unsuitable (or at least less suitable) for certain applications and study designs. The direct PCR approach is likely not appropriate for shotgun metagenomic analyses and, if a given sample is to be used for multiple PCR-based assays, it would probably be useful to extract DNA using a standard approach. However, we have successfully conducted multiple PCR analyses off of Extract-N-Amp dilution solutions stored at 4°C for up to 20 weeks (data not shown), but this is not likely a long term storage option. If DNA needs to be stored for extended periods of time for downstream analyses, the more standard extraction approach would likely be preferable. Also, we do not know how the direct PCR approach would work for other types of PCR-based analyses . Nevertheless, as studies are increasingly moving to very large sample numbers and amplicon sequencing becomes ever faster and cheaper, the direct PCR approach is clearly a valuable option for high-throughput studies examining the structure and diversity of human-associated bacterial communities.Figure S1Average number of OTUs observed for each body habitat of each individual using the direct PCR (dark grey) and standard extraction/purification (light grey) protocols. Bars with asterisks denote comparisons that were statistically significant within an individual . Side brackets with asterisks denote comparisons that were statistically significant across all individuals . Error bars are ± one standard deviation.(TIF)Click here for additional data file.Figure S2PCoA plots illustrating differences in community composition across body habitats (a and c) and no differences based on which protocol was used across individuals (b and d). Plots a and b are based on unweighted UniFrac distances while c and d are based on Bray-Curtis dissimilarity values. Results of ANOSIM tests are presented in the bottom right of each plot.(TIF)Click here for additional data file.Figure S3PCoA plots derived from Bray-Curtis dissimilarities comparing the communities observed using the direct PCR (black circles) and standard extraction/purification (grey circles) protocols. Letters A-D denote individual participants. Results of the ANOSIM testing for statistical differences between methods across individuals are shown for each body habitat. Note that individual C was not included in the underarm analysis as sequences were not obtained for the direct PCR samples.(TIF)Click here for additional data file.Figure S4Variation of Bray-Curtis dissimilarities of replicate samples using the direct PCR (dark grey) and standard extraction/purification (light grey) protocols. Bars with asterisks denote comparisons that were statistically significant within an individual . Only communities from the tongue showed differences between methods across individuals . Error bars are ± one standard deviation.(TIF)Click here for additional data file.Table S1Samples used to evaluate the suitability of the direct PCR protocol for use in high-throughput 16S rRNA gene surveys of the human microbiome. Note that the before subtraction column refers to the number of sequences before removal of negative control OTUs present as 1% or greater of total negative control sequences. For all downstream analyses, samples were rarefied to 400 sequences per sample using sequences after subtraction.(DOC)Click here for additional data file.