The discovery of nucleotide diversity captured as single feature polymorphism (SFP) by using the expression array is a high-throughput and effective method in detecting genome-wide polymorphism. The efficacy of such method was tested in rice, and the results presented in the paper indicate high sensitivity in predicting SFP. The sensitivity of polymorphism detection was further demonstrated by the fact that no biasness was observed in detecting SFP with either single or multiple nucleotide polymorphisms. The high density SFP data that can be generated quite effectively by the current method has promise for high resolution genetic mapping studies, as physical location of features are well-defined on rice genome. The publicly available genome sequence information of rice The nucleotide diversity across a genome is the source of most of the phenotypic variation. Such DNA polymorphism is the basis for development of molecular markers, an indispensable tool in genetic mapping studies. In general, the high resolution fine mapping of genes is often limited by lack of sufficient number of polymorphic molecular markers. This problem is compounded with traits controlled by multi-genes because in several such studies QTL locus can't be resolved to a workable resolution that could be feasible for predicting the candidate gene(s) associated with traits of interests. The sequence comparison of Nipponbare and 93-11 genome has shown high degree of polymorphisms ranging from a SNP/∼300 bp to an indel/kbp single feature polymorphism (SFP) There are several ways through which polymorphisms are identified across the genome. The most direct method is to re-sequence the PCR amplicons, the DNA fragments containing alleles from the inbred parents. Similarly EST sequences also provide the direct way to compare the sequence and provide useful information about polymorphism. These methods although straight forward in approach but are quite labor intensive and lack high-throughput. The high density oligonucleotide expression arrays, designed for transcript profiling, have been used successfully as an effective tool for DNA genotyping to measure numerous polymorphic loci in yeast a priori of their sequence information. The rice varieties used in this study were Cypress (CP), LaGrue (LG) and RT0034 (RT); first two belong to japonica The objective of present study was to test the efficacy of such DNA-based gene-chip approach to identify polymorphism in rice. Here we demonstrate that hybridization of probes, generated from labeling of g-DNA, to rice whole genome expression array (Affymetrix) is quite sensitive in predicting SFP The biotin labeled probes generated from labeling of g-DNA was hybridized to Affymetrix rice expression array . Following hybridization the preliminary data quality was assessed from GCOS1.3 software (Affymetrix) generated expression report according to guidelines  set for such experiments. The average background, noise (RawQ) and the call rate was comparable among all the three rice varieties viz. Cypress (CP), LaGrue (LG) and RT0034 (RT) and also among their biological replicates (data not shown). However to get better assessment of data quality the raw intensity data of only perfect match (PM) probes/features of rice varieties viz. CP, LG and RT were log2 transformed and studied by density plots  and pairwww.boiconductor.org) and SAM procedure in R language software essentially as described estimated FDR and the FDR that is determined after sequencing is referred to the observed FDR. The SFPs called at different threshold (delta) in the three datasets viz. CP&LG (a), CP&RT (b) and LG&RT (c) is presented in The background corrected and quantile-normalized log2 intensity values of all PM features of triplicate data of each rice variety were subjected to SFPs call by using the siggene package (The distribution of gene-chip predicted SFPs (at ≥10% estimated FDR) among polymorphic probesets in the three datasets is shown in To test the sensitivity of gene chip predicted SFPs data we verified them by sequence comparison. To the best of our knowledge no sequence information was available for rice varieties Cypress (CP), LaGrue (LG) and RT0034 (RT) and due to this limitation fragments flanking the SFPs were amplified from their respective genome  and their sequences was compared to verify polymorphism, if any. To simplify and maintain uniformity we validated SFPs whose corresponding 25mer probe/feature sequence had unique location, 100% identity and distributed randomly on all 12 chromosomes (supplementary Since a significant proportion (∼25%) of probes having known sequence variation in both the data set escaped from being predicted as SFP at the given analyzed FDR, we explored the possibility of finding such SFPs at less stringent estimated FDR. In CP&LG dataset at 11.9% estimated FDR the number of predicted SFP nearly doubled to 9093 compared to those predicted at 9.5% estimated FDR . From thThe higher the value of d-stat of SFP, the greater is the likelihood of being predicted true In genetic mapping studies theoretically a probeset/gene having either one or multiple SFP will provide the same information if such probeset/gene having their defined position on genome is being exploited as molecular marker. According to result presented in SNPs (single nucleotide polymorphisms) are the most frequent form of polymorphism observed in any organism. Given their wide application in genetic fine mapping studies, it was of interest to analyze how many of probes/features having known SNP was detected by this method. From available sequence information in CP&LG dataset 46 probes were having SNP but only 29 were predicted correctly indicating 63% detection efficiency . On the The objective of present study was to find SFPs in CP&LG and CP&RT datasets as varieties in these two pairs constitute parents of two different mapping populations created to map QTLs/genes associated with milling yields trait The availability of genome sequence information of Nipponbare All the computationally predicted SFPs  resulting from differential hybridization of probes is a unique high-throughput approach for both genotyping and polymorphism discovery in a single assay a priori of their sequence analyses by deselecting SFPs having d-stat value close to permuted cut-off. Although a caution must be exercised in such approach as one may loose a considerable number of true SFPs also.In the present study we tested the feasibility of such gene-chip based approach for polymorphism discovery in rice by hybridizing probes, generated from labeling of g-DNA, to rice whole genome expression array (Affymetrix). From verification of sequence information of predicted SFP conducted in two independent datasets viz. CP&RT and CP&LG, we found that SFPs can reliably be predicted in rice with ∼75% detection sensitivity . Such a The rice varieties CP and LG are in japonica  Based on predicted SFPs, traditionally one can generate molecular markers once the fragment flanking SFP of interest is amplified and sequenced. Such type of approach can be useful to further narrow down the genetic interval of already identified QTLs. However given their higher sensitivity of detection  togetherMany of the plant traits of economic importance are generally controlled by numerous loci and to fine map genes associated with such traits is not trivial in terms of both time and resources. An alternative approach to speed up the process of gene discovery associated with quantitative traits that complements map-based cloning is association studies where candidate gene diversity is evaluated across natural populations and polymorphisms that correlate with phenotypic variation are identified The three rice varieties used in this study include Cypress, LaGrue and RT0034; first two belong to japonica while later to indica subspecies respectively. These rice varieties are the parents of two mapping population developed by RiceCAP www.affymetrix.com), designed for gene expression analyses, and contains probes to query 49,824 transcripts representing two rice cultivars with 48,564 from japonica and 1,260 from indica cultivar. The array is believed to represent about 46,000 distinct rice genes however the probesets from japonica is 54,168 and 1347 from indica subspecies. The arrays were designed using NCBI UniGene Build #52,  incorporating predicted genes from GenBank® and the TIGR Os1 v2 data set (Affymetrix). Each probeset is represented by 11 perfect matches (PM) and an equal number of mismatch (MM) probes/features and each probe is 25 bp long. The array contains ∼630,000 PM probes/features from rice. The PM and MM probes positioned next to each other constitute a probe pair and each probe pair is distributed randomly but with defined position on the array or gene-chip.The rice genome array (http://www.biotech.missouri.edu/dnacore/). The hybridization and washing was performed according to standard RNA protocol as described in Affymetrix manual.DNA was isolated from leaves by CTAB method www.bioconductor.org). The log2 transformed intensity value of each feature was subsequently used to derive SFP call by publicly available siggene package (www.bioconductor.org) and scripts and codes The data quality was assessed from expression report generated by GCOS1.3 software using the default parameter set for rice genome array. The raw intensity values of all the probes present on the chip was transferred from .cel files into .txt file by using the tools available on GCOS1.3 software. The intensity values of only PM (perfect match) probes/features  were subsequently extracted and subjected to data analyses as described www.affymetrix.com). Primers were designed on the basis of known sequence information of Nipponbare genome (TIGRv3) by using the blast tool of Gramene (www.gramene.org). The thermal cycle program for PCR were 95°C for 2 min, 28 cycles of 95°C for 15 sec, 58°C for 15 sec, 72°C for 30 sec and with final extension at 72°C for 5 min. The PCR conditions were; 1× of 10× Ex Taq buffer (Mg+), 125 µM of each dNTPs, 0.5 units of DNA polymerase, 0.5 µM each primer, 1 µl of genomic DNA to a final volume of 40 µl. In order to get DNA fragments without any error during amplification, PCR was performed with hot-start Ex Taq DNA polymerase  having proof reading activity. PCR products showing single bands were purified  and subjected to sequencing by Applied Biosystems 3730 DNA Analyzer using Prism Big Dye Terminator cycle sequencing chemistry at DNA core facility.DNA fragments of 200–300 bp flanking the SFP regions were amplified from genomic DNA. The sequence information of features/probes was obtained from Affymetrix  on Nipponbare genome (TIGRv3). In the present study any variation in sequence ranging from substitution or indel involving single (SNPs) to multiple bases were considered while determining the nature of polymorphism associated with SFPs. High quality single reads, as obtained from ABI chromatograms, were used for sequence comparison with publicly available vectorNTI tools (Invitrogen).The Affymetrix rice whole genome expression array has been designed mainly on the sequence from japonica sub-species cv. Nipponbare. The sequences of all ∼630,000 25mer PM probes/features sequences present on the array were compared  against Nipponbare genome build 4Table S1Sequence verified SFPs in CP& LG dataset. Changes in sequences are highlighted in blue and black; - indicates deletion; CP = Cypress and LG = LaGrue.(0.03 MB XLS)Click here for additional data file.Table S2Sequence verified SFPs in CP&RT dataset. Changes in sequences are highlighted in blue and black; - indicates deletion; CP = Cypress and RT = RT0034.(0.05 MB XLS)Click here for additional data file.Table S3In-silico predicted SFPs and their distribution. Nip = Nipponbare, −SFP(Nip>93-11), +SFP (93-11>Nip), non SFP (Nip = 93-11)(0.02 MB XLS)Click here for additional data file.


Drosophila, ∼50 classes of olfactory receptor neurons (ORNs) send axons to 50 corresponding glomeruli in the antennal lobe. Uniglomerular projection neurons (PNs) relay olfactory information to the mushroom body (MB) and lateral horn (LH). Here, we combine single-cell labeling and image registration to create high-resolution, quantitative maps of the MB and LH for 35 input PN channels and several groups of LH neurons. We find (1) PN inputs to the MB are stereotyped as previously shown for the LH; (2) PN partners of ORNs from different sensillar groups are clustered in the LH; (3) fruit odors are represented mostly in the posterior-dorsal LH, whereas candidate pheromone-responsive PNs project to the anterior-ventral LH; (4) dendrites of single LH neurons each overlap with specific subsets of PN axons. Our results suggest that the LH is organized according to biological values of olfactory input.In  Drosophila, most ORN classes express one specific odorant receptor and send axons to one of ∼50 glomerular targets  expressing a specific odorant receptor (and therefore having precisely defined olfactory tuning properties) send axon projections to discrete and reproducibly positioned glomeruli in the vertebrate olfactory bulb or insect antennal lobe . In Dros targets .Persistent spatial organization deep within the brain is a motif in many sensory systems. For example, adjacent regions of the somatosensory cortex respond to stimuli from neighboring body parts . Does thC. elegans hermaphrodites  that joins the antennal lobe to higher olfactory centers. After registration we found that axons segregate within the iACT (10–15 μm in diameter) according to their birth time and lineage: embryonic-born PNs  are cleaIn summary, these examples give an upper bound on the registration error of a few microns.1–2D1). In contrast, the same labeled PNs had synaptotagmin-HA localized through most of their LH axonal arbours (2–2D2). These findings applied to all PN classes examined  and the maxillary palp. In the antenna, ORNs of each class reside in one of three broad types of sensillum: basiconic, trichoid or coeloconic. Each sensillum type has a distinct morphology, spatial distribution and function  the (E)-2-hexenal activation pattern is rather similar to low concentrations of banana and especially apple extracts but not pineapple extract , a master regulator of male sexual behavior, have identified olfactory channels likely to process sex pheromones during male courtship  Interestingly, fruit odors analyzed in the previous section do not activate this LH region. In fact, projections of these candidate pheromone PNs appear complementary to almost all other PN projections A. SpecifInterestingly, VA1lm and DA1 are the only two glomeruli dually represented by conventional iPNs and by vPNs that project directly to the LH via the mACT. While most PNs are cholinergic and excitatory e.g., , a recenHaving shown that PNs originating from sexually dimorphic glomeruli target discrete LH regions, we next asked whether there are regional volume differences in the LH between the sexes and which PN axons target these regions. Nonrigid registration generates a deformation field that maps arbitrary points between each sample and the reference brain; this can be used to compare volumes of brain regions between sexes.After normalising each brain to remove absolute differences in size, we found significant difference in the relative size of the LH F and MB To understand the anatomical basis of integration of PN input by candidate output neurons of the LH (LHNs), we examined their dendritic arborizations. We generated single-cell clones from three Gal4 lines labeling specific LHNs, and then registered the sample brains to our reference .3 and 7A4). These neurons send one projection to the ventromedial LH, and another to the ventrolateral protocerebrum (VLPR). To find the direction of information flow, we examined the localization of the presynaptic marker synaptotagmin-GFP (2). Although both processes were labeled by synaptotagmin-GFP, much stronger labeling was observed in the VLPR . The LH density maps of NP5194 and acj6 are indeed similar although NP5194 LHNs seem to have slightly more lateral arborization (compare 5-6 and 7B5-6).Six Gal4 lines have been reported that may label LHNs . MARCM a1 and 7C3-4). Synaptotagmin-GFP is relatively enriched in the SLPR, suggesting that this branch is the axonal process (arrow in 2). Density maps indicate that NP6099 neurons target a dorsolateral region of the LH, distinct from acj6 and NP5194 LHNs (5-6).Finally, NP6099-Gal4 is expressed in 2-3 LHNs with cell bodies anterior-ventral to the LH. They send projections to the dorsolateral LH and superior lateral protocerebrum  is evident in the individual tracings in Drosophila brain. We discuss our methodology , the spatial organization of the olfactory system, and the biological implications of these results.We have combined single neuron labeling and image registration methods to provide a comprehensive description of the organization of higher olfactory centers in the C. elegans hermaphrodite , followed by a nonrigid registration. A parameter controlling the spatial smoothness of the warping was selected at a pilot stage to allow sufficient deformation for accurate registration while preventing unrealistic deformations.Single neurons were manually traced from confocal stacks, then transformed to the reference coordinate system. In the LH, tracings for each neuronal class were convolved with a 3D Gaussian kernel with 2.5 μm standard deviation to generate a smoothed density estimate of the number of terminals in each region of space. For the MB, positions of individual boutons for each PN class were convolved with a kernel with σ = 3.5 μm.We first calculated a distance matrix between different classes of neuron in the LH or MB based on the similarity (Pearson correlation coefficient) of their density maps, then used this matrix as the input for clustering by Ward's algorithm. The MB and LH distance matrices were compared using a Mantel test to look for organizational similarities.The relative volume change at each voxel (Jacobian determinant, see We used the potential synapse approach of


Glucocorticoids are potent regulators of the innate immune response, and alteration in this inhibitory feedback has detrimental consequences for the neural tissue. This study profiled and investigated functionally candidate genes mediating this switch between cell survival and death during an acute inflammatory reaction subsequent to the absence of glucocorticoid signaling. Oligonucleotide microarray analysis revealed that following lipopolysaccharide (LPS) intracerebral administration at striatum level, more modulated genes presented transcription impairment than exacerbation upon glucocorticoid receptor blockage. Among impaired genes we identified ceruloplasmin (Cp), which plays a key role in iron metabolism and is implicated in a neurodegenative disease. Microglial and endothelial induction of Cp is a natural neuroprotective mechanism during inflammation, because Cp-deficient mice exhibited increased iron accumulation and demyelination when exposed to LPS and neurovascular reactivity to pneumococcal meningitis. This study has identified genes that can play a critical role in programming the innate immune response, helping to clarify the mechanisms leading to protection or damage during inflammatory conditions in the CNS. The presence of infection is recognized by receptors for specific elements called the pathogen-associated molecular patterns (PAMPs) that are produced by microorganisms. Recognition of these PAMPs by antigen-presenting cells is the first step of a complex inflammatory reaction that characterizes the innate immune response Glucocorticoids (GCs) are potent endogenous anti-inflammatory molecules. Activated GC receptors (GRs) are able to interfere with the transactivation potential of the p65 NF-κB subunit as well as AP-1. Other mechanisms explaining the effects of GCs on gene transcription have been proposed, including the competition between GRs and NF-κB for nuclear coactivators, such as CREB-binding protein and p300 (CBP/p300) Intracerebral LPS administration causes a strong and time-dependent transcriptional activation of inflammatory genes in microglial cells ipsilateral to the site of injection and this acute reaction is not associated with neuronal damage or demyelination in vivo model to identify the key players involved in the delicate balance between cell survival and death during a natural innate immune reaction in the CNS. While the use of neurotoxins is limited to access the contribution of the innate immunity to brain damage due to either direct or indirect microglia activation by their ability to cause neuronal death, the model used in the present study is unique and appropriated in this regard. The comparison between LPS alone and LPS combined to RU486 offers an adequate paradigm because the deregulated innate immune reaction is directly responsible for the cell fate. Functional analysis of the differentially regulated genes between the two conditions led to the selection of ceruloplasmin (Cp) as a candidate protective gene and to the investigation of its role in neuroinflammatory contexts, providing a model to illustrate the beneficial mechanisms engaged by the CNS innate immune response.Here we aimed to determine the genes that are differentially regulated by LPS in the presence or absence of the GR signaling. We took advantage of this unique Our design allowed the detection of 345 probe sets representing 286 different genes (gene IDs) significantly modulated 12 h after intrastriatal LPS injection in the ipsilateral side of the injection . These probes are listed in the Regarding gene expression comparing LPS effects in presence or absence of RU486, 235 probe sets (representing 200 different gene IDs) were selected according to our strategy  , genes with GR-mediated repression on LPS induction  and other transcripts modulated by GR signaling. In this particular case, RU486 essentially prevented gene upregulation  or downregulation (C1qa and Hexb) by LPS. Several genes were selected for validation by The expression pattern of C1qa and Hexb is highly suggestive of ubiquitous constitutive expression, which was selectively repressed by the endotoxin in the ipsilateral side of injection . BecauseAccording to our double labeling procedure, Cd44, Tnfsf9 and Cd83 are expressed mainly by microglial cells, Lcn2 by endothelial cells and Saa3 transcript by microglia and few astrocytes. In contrast, Stat1 is mainly produced by neurons .Cp gene encodes a protein with ferroxidase activity and loss of function mutations in this gene leads to accumulation of iron and human neurodegenerative disease  and Cp-/-(n = 7) group, but not in the group of Cp+/-mice (n = 3). Since the highest variance was associated with Cp+/-genotype, we removed this group from the statistical analysis to avoid misrepresentation and substantial reduction of power. In this regard, Selp mRNA levels were significantly different between Cp+/+ and Cp-/-according to student's t-test (p<0.05), but not when ANOVA is used to compare the three genotypes. Nevertheless, a significant linear trend between Selp mRNA levels and Cp deficiency was reached (p<0.05) when assigning 0, 1 and 2 values to Cp-/-, +/-and+/+genotypes, respectively. These statistical tests did not reveal any significant changes in Nfkbia mRNA levels among two or three genotypes.Since blood vessels are strategically positioned to produce Cp protein during systemic infection, we evaluated whether this APR modulates gene expression in vascular-associated elements during meningitis. culation . Neverth+/+ mice . We then+/+ mice . In cont+/+ mice  D, E, F The capacity of GR to repress NF-κB/AP-1 activity and gene transcription is believed to be a key intracellular process mediating the powerful anti-inflammatory properties of synthetic and endogenous GCs. Such a mechanism was elegantly explored by gene profiling using cell-culture system and the GR agonist Dexamethasone (Dex) in vivo or in vitro models. Some of our results may be in sharp contrast to those previously reported because the paradigm used here to access the role of endogenous glucocorticoids does not block the inflammatory process.Therefore our results do not rule out the possibility that GR antagonism increases the inflammatory reaction in the CNS, especially at later time-points. These data do suggest that genetic profile is no longer respected in the absence of GR signaling on the cerebral immune system. Although the effects of GRs on the regulation of genes encoding APRs were expected Mycobacterium tuberculosisOur gene profiling data indicated that endogenous GCs are required to coordinate the ability of LPS for inducing expression of genes involved in the viral and interferon response. These include components of viral dsRNA sensing pathway Ifih1 (MDA-5), Ikbke (iKK/IKKε) and Irf7 Although GR signaling is necessary to support innate immune response gene expression, our results should not be interpreted as a newly described pro-inflammatory role of GCs. Many recent studies pointed that GC exposure may increase CNS inflammation and Sorrells and Sapolsky suggested a conciliatory manner to interpret both anti- and pro-inflammatory effects of GCs based on early, delayed, acute or chronic regimen It is interesting to note that many regulated genes that exhibited a particular profile  are related to brain development and not necessarily to myeloid functions. These data suggest alternative mechanisms underlining the actions of GC and TLR4 signaling on gene repression in the CNS. Our factorial design also allowed us to report a list of genes that follows a GR-blockage main effect, revealing genes that respond to RU486 and are not changed by the inflammatory reaction.in situ hybridization could not clearly corroborate this result.As depicted by the The endotoxin was able to downregulate a number of genes in a GR-dependent manner. These include C1qa and Hexb genes, but the meaning of their localized suppression during neuroinflammation has yet to be determined. Inhibition of the classical complement pathway could be a natural mechanism to avoid a precipitated antibody mediated cell lysis, whereas Hexb may have uncharacterized immune properties modulating neuronal functions. Indeed, Hexb gene disruption in mice leads to neurological deficits, which can be partially prevented by the transplantation of bone marrow stem cells taken from wild type mice The gene profiling results clearly pointed out that APR gene induction was a prominent feature of the ability of GRs to modulate the inflammatory transcriptional programs. The role of APRs during infection is not well understood, but a subset of genes is implicated in iron homeostasis. For instance, Lcn2 plays an important role in iron acquisition during infection Cp-/-mice and not their control littermates are quite interesting, because they underline the critical neuroprotective role of Cp in an inflammatory context. We propose that Cp produced by microglial cells is released to the extracellular space for controlling iron homeostasis in this critical period, when white matter elements are more vulnerable. Hence, these findings support an important new role for Cp in mediating neuroprotection in the course of an acute innate immune process. Such neuroprotective properties of Cp are not associated with changes in microglial reactivity to LPS.The physiological relevance of this process has yet to be determined, but it could be related to limit the iron uptake by bacteria or to avoid an exacerbated accumulation of reactive iron (II) levels. The data that LPS caused CNS injury in S. Pneumoniae is a leading cause of meningitis worldwide. It is proposed that high levels of bacteria in the blood targets the endothelium in order to reach brain compartments Cp-/-exhibited a very different vascular reactivity to the circulating bacteria. In fact, Cp deficiency was associated with a robust expression of Selp transcript in large blood vessels and arteries. Selp controls key events related to leukocyte cell recruitment and this adhesion molecule is a determinant modulator of vascular permeability to bacterial elements We also observed that Cp is highly expressed in cells lining the blood vessels. In consequence, it was attractive to test the hypothesis that Cp could be a mediator of barrier function, protecting the brain against circulating pathogens or potentially damaging molecules derived from their infection. Cp-deficient mice generated exciting data regarding the ability of acute-phase proteins to make the difference between cell survival and death during a natural and timely sensitive innate immune reaction in the CNS.Endogenous GCs play a critical role for modulating the innate immune response in the CNS. Ablation of GR signaling exacerbated the expression of a small subset of genes, which are implicated in cell recruitment and prolonged immune activity. Surprisingly, expression of many genes related to host defense were critically impaired upon RU486 treatment, a mechanism that could explain unusual bacterial infections in individuals taking this compound as abortion pill and the beneficial effects of an acute therapy with GCs. This gene profiling experiment led us to investigate the role of Cp in the course of an acute LPS challenge. Experiments using Cp-/-) in C57Bl/6 background Cp+/-) were used to generate Cp+/+, +/- and -/-mice. These mice were genotyped by PCR of DNA isolated from ear punching. Animal breeding and experiments were conducted according to Canadian Council on Animal Care guidelines, as administered by the Laval University Animal Care Committee. Efforts were made for minimizing animal number utilization; 3 to 6 animals per group were used in each experimental group, except for infection protocol (19 to 20 mice per group).Adult male C57Bl/6  were acclimated to standard laboratory condition. Ceruloplasmin-deficient mice  were killed 2 and 5 days after intrastriatal (IS) infusion with saline or LPS.In protocols for a comparative study of the effects of endogenous GCs, C57Bl/6 mice received an i.p. injection of vehicle (DMSO-50 µl) or RU486 (50 mg/kg b.w.) and were submitted to surgery 4 h later. Just after the surgery the animals received a second i.p. injection of vehicle or RU486 to guarantee high brain levels of the drug. In a second protocol mice received different doses of RU486  in similar conditions in order to test dose-dependent effects. The mice receiving intraparenchymal injections were anesthetized with a mixture of ketamine and xylazine and placed in a stereotaxic apparatus (David Kopf Instruments). The right caudate putamen was reached, using a small cannula  at the coordinates 0.0 mm anteroposterior, −2.0 mm lateral, and −3.0 mm dorsoventral according to a mouse brain atlas For gene array and qPCR assay, mice were anesthetized under isofluorane and blood was drawn via cardiac puncture before head decapitation. Brains were removed rapidly from the skulls and placed in cold phosphate buffered saline (PBS) solution. A brain region defined by dashed lines in in situ hybridization histochemistry, histology and immunolabeling, animals were deeply anesthetized via an intraperitoneal injection of a mixture of ketamine hydrochloride and xylazine and then rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in PBS, pH 7.6, at 4°C. Brains were removed rapidly from the skulls, postfixed for 2–4 d, and then placed in a solution containing 20% sucrose overnight at 4°C. The frozen brains were mounted on a microtome (Reichert-Jung) and cut into 25-μm coronal sections. The slices were collected in cold cryoprotectant solution and stored at −20°C.For S. Pneumoniae strain capsular type 6A DBL6A was used in an experimental model of pneumococcal infection passive of meningitis. The mice  were anesthetized and received an i.p. injection of the working suspension (50–100 C.F.U./ml) in a 100 µl volume . Expression values from the CEL files generated from scanning were obtained using RMA algorithm Real-time RT-PCR was performed as described in 35S-labeled riboprobes were synthesized as described before , anti-glial fibrillary acidic protein (GFAP) and anti-neuron specific nuclear protein (NEUN) labeled cells of myeloid (macrophages and microglia), astroglial and neuronal lineages, respectively. Dual labeling was performed as described in Immunohistochemistry was combined with the Iron (III) staining was performed on free-floating sections as a described improved method for Perls' stain Cp deficient mice. Double immunofluorescence staining was performed in sequential manner; primary antibody against Cp detection with Alexa488-conjugated goat anti-rabbit was followed by incubation of primary antibodies against CD31, GFAP, IBA1, or MAC-2 that were detected with immunoglobulin antibodies conjugated to Cy3.Immunolabeling was performed on free-floating sections following standard procedures detailed in text S1. Cp immunohistochemistry staining specificity was verified using brain sections of Photomicrographs were taken with the same exposure time using a digital camera (QIMAGING) mounted directly on a microscope (Nikon Eclipse 80i). Confocal laser scanning microscopy was performed with a BX-61 microscope equipped with the Fluoview SV500 imaging software 4.3 (Olympus America Inc). Confocal images were acquired by sequential scanning using a two-frame Kalman filter and a z-separation of 1 µm.S. pneumoniae infection protocol were analyzed at the level of hindbrain. Quantification of area without Plp1 transcript signal (demyelinated regions and areas associated with lesions) was performed as previously described Semi-quantitative analyses of hybridization signals were performed as described previously Stereological analysis was performed as previously described Photomicrographs were processed to enhance contrast and image quality using Adobe Photoshop 8 (Adobe Systems) and were assembled using Adobe Illustrator (Adobe Systems). The image edition was kept to a minimum to avoid artifacts. Pictures representing different groups received equivalent image treatment.Comparison of group means was performed using an one-way ANOVA followed by a Tukey's HSD procedure as post hoc multiple comparisons test. Two or three factor ANOVA was performed using Bonferroni's correction when applicable and also according to presence or absence of interaction between factors. Student's t-test was applied for comparison between two groups according to homogeneity of variances. All the analyses were carried out with SPSS software version 11.0, except for Logrank test and Logrank test of trend to compare survival curves, which were performed using Prism 4 software.Dataset S1Excel Spreadsheet; LPS modulated genes(0.06 MB XLS)Click here for additional data file.Dataset S2Excel Spreadsheet; LPS Vs LPS/RU486 differential gene expression(0.06 MB XLS)Click here for additional data file.Figure S1Gene expression exploration data(2.86 MB PDF)Click here for additional data file.Figure S2C1qa and Serping1 expression profile(0.19 MB PDF)Click here for additional data file.Figure S3Cellular site of transcript synthesis(0.42 MB PDF)Click here for additional data file.Figure S4Cp expression and IBA1-Vs MAC2-positive cells(0.44 MB PDF)Click here for additional data file.Figure S5Time-dependent extracellular Cp expression(0.31 MB PDF)Click here for additional data file.Text S1Supplementary Materials and Methods(0.18 MB PDF)Click here for additional data file.


Here, a group of Oryza sativa L. genes encoding calmodulin (CaM) and CaM-like (CML) proteins that do not possess functional domains other than the Ca2+-binding EF-hand motifs was analyzed.A wide range of stimuli evoke rapid and transient increases in [CaCam genes and thirty two additional CML genes were identified. Extensive analyses of the gene structures, the chromosome locations, the EF-hand motif organization, expression characteristics including analysis by RT-PCR and a comparative analysis of Cam and CML genes in rice and Arabidopsis are presented.By functional analyses and BLAST searches of the TIGR rice database, a maximum number of 243 proteins that possibly have EF-hand motifs were identified in the rice genome. Using a neighbor-joining tree based on amino acid sequence similarity, five loci were defined as 2+-signals in regulating cellular responses to stimuli and this family of proteins likely plays a critical role as their transducers.Although many proteins have unknown functions, the complexity of this gene family indicates the importance of Ca Due to the activities of Ca2+-ATPases and Ca2+-channels on the cellular membrane, rapid and transient changes of its cytosolic concentrations are possible. In plant cells, a wide range of stimuli trigger cytosolic [Ca2+] increases of different magnitude and specialized character  and CBL3 [GenBank: NP_643248] as query sequences against the rice genome were conducted. Nucleotide and amino acid sequences as well as information regarding each gene of interest were obtained. Gene annotations at the Rice Annotation Project Database (RAP-DB) [Searches of the rice genome at The Institute of Genomic Research (TIGR)  for Inte(blastp)  using th(RAP-DB)  were alsAAA32763]; HvCaM [GenBank: AAA32938]; T-CaM1 [GenBank: AAC49578]; ZmCaM [GenBank: CAA52602]; SCaM1 [GenBank: AAA34013]; PCM5 [GenBank: AAA85155]; MmCaM [GenBank: NP_033920]; CMD1p [GenBank: AAA34504].If necessary, predictions of coding regions were verified using available EST and cDNA sequences. Deduced sequences of proteins identified by InterProScan as containing an EF hand were subjected to phylogenetic analysis. Alignments were performed by the multiple sequence alignment program ClustalX  using deDeduced amino acid sequences CaM and CaM-like proteins were aligned with one another by Align  and the Cam and CML genes were identified by performing BLAST searches of the Oryza sativa EST database and by searching UniGene entries corresponding to all genes at GenBank [ESTs corresponding to  GenBank . ExpressOryza sativa L. tissues were ground in liquid nitrogen using chilled mortars and pestles. Total RNA was isolated according to [15 primers and PCR was carried out using forward and reverse oligonucleotide primers  as given in Table OsCam1-1; OsCam1-2; OsCam2; and OsCam3 and a program of 94°C for 2 minutes, 58°C for 1 minute, and 72°C for 2 minute for OsCam1-3. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining and UV fluorescencing. All enzymes and chemicals for RT-PCR were purchased from Promega .rding to  and usedBB and TB participated in database searches and extensive analyses of the gene and protein sequences. BB carried out the laboratory work and prepared figures and tables. TB performed data analysis and interpretation, and drafted the manuscript. Both authors read and approved the final manuscript.A list of all identified EF-hand-containing proteins as an Excel spreadsheet.Click here for fileThe alignment for the phylogenetic tree in Figure Click here for fileThe alignment for the phylogenetic tree in Figure Click here for fileThe alignment for the phylogenetic tree in Figure Click here for file


Secondary involvement of the thyroid gland is rare. Often the origin of the tumor is difficult to identify from the material obtained by fine-needle aspiration cytology. Renal cell carcinoma of the clear-cell type is one of the more common carcinomas to metastasize to the thyroid gland. Somatic mutations of the von Hippel-Lindau tumor suppressor gene are associated with the sporadic form of this tumor. We aimed to illustrate the potential utility of DNA based technologies to search for specific molecular markers in order to establish the anatomic site of origin.A 54-yr-old Caucasian male complaining of a rapidly increasing neck tumor was diagnosed as having a clear-cell tumor by fine-needle aspiration cytology. A positive staining for cytokeratin as well as for vimentin and CD10 in the absence of staining for thyroglobulin, calcitonin and TTF1 suggested a renal origin confirmed by computed tomography.VHL gene.Using frozen RNA, obtained from cells left inside the needle used for fine needle aspiration cytology, it was possible to identify a somatic mutation (680 delA) in the VHL gene in material derived from thyroid aspirates might provide additional information to immunocytochemical studies and therefore plays a contributory role to establish the final diagnosis. Moreover, in a near future, this piece of information might be useful to define a targeted therapy.In the presence of a clear-cell tumor of the thyroid gland, screening for somatic mutations in the  Metastasis to the thyroid is an uncommon occurrence. In clinical series, the frequency of secondary involvement of the thyroid is considerably lower than the frequency of 25% found at autopsy of patients with disseminated malignancies ,2.The increasing number of reported cases of secondary tumors to the thyroid, as suggested by two studies from the Mayo Clinic referring to distinct periods ,3, is prVHL gene in DNA derived from the leftover cells in the needle used for FNAC might be helpful to establish the origin of the primitive tumor.Secondary involvement of the thyroid gland by renal cell carcinoma (RCC) clear-cell type (CCRCC) is rare; however CCRCC is one of the more common neoplasms to metastasize to the thyroid gland . Since mA 54-yr-old Caucasian male was referred for evaluation of a rapidly increasing neck tumor noticed for the first time four months earlier. He complained of sporadic dysphagia without weight loss. He was a heavy smoker until recently but had no previous medical history. He worked as a house painter, for seven years, in his thirties. The physical examination revealed an asymmetric multinodular goiter with a left dominant nodule with firm consistency and no palpable regional nodes. Serum TSH was 1.3 μUI/ml , T4 7.3 μg/dl  and T3 126 ng/dl . Antimicrosomal and antithyroglobulin antibodies were negative. A thyroid ultrasound demonstrated a multinodular gland with the right and left lobes measuring 6 × 4 × 3 cm and 9 × 7 × 6 cm respectively. A computed tomography (CT) scan of the neck showed a slight tracheal deviation to the right without compression. A Tc-99 m scintigraphy disclosed irregular uptake in both lobes of the thyroid and a large cold nodule in the left lobe. FNAC from both lobes revealed a clear-cell carcinoma with an immunocytochemical profile suggestive of a secondary tumor from the kidney (refer to section pathology).FNAC results prompted a clinical and radiographic investigation. An abdominal CT scan revealed a tumor of the left kidney measuring in greatest diameter 10 cm. Bone scan, chest computed tomography scan, liver ultrasound and laboratory data were normal and there was no evidence of other distant metastases.Initial treatment included a left radical nephrectomy and a total thyroidectomy. One year later, there was evidence for cervical nodal metastases. The patient was then submitted to right radical neck dissection with internal jugular vein ligation and section of spinal accessory nerve and left modified radical neck dissection type III. At histological examination, only right nodes were metastatic.® Kit . Cell block (CB) sections were stained with hematoxilin-eosin. Immunocytochemistry was performed on CB sections, using an avidin-biotin method with diaminobenzidine as the chromogen for the following antibodies: thyroglobulin, calcitonin, vimentin, CD10, TTF1  and cytokeratin AE1/AE3 .Both thyroid lobes were sampled. Smears were air dried and acetone fixed and stained with May-Grünwald-Giemsa (MGG) and Papanicolaou (PAP) stains, respectively. An additional sample was fixed in formalin and processed as a cell-block using the Shandon CytoblockSmears consisted of a clear, large cell neoplasia with large nuclei, prominent nucleoli and finely vacuolated cytoplasm, with indistinct borders. The tumor cells were arranged in aggregates of variable size and shape, many of them centered by thin walled capillaries. This intimate relationship of neoplastic cells and vessels was better appreciated on cell-block sections. These cells were immunoreactive for pancytokeratin AE1/AE3), vimentin and CD10 and were negative for thyroglobulin, thyroid transcription factor1 (TTF1) and calcitonin , according to the manufacturer's instructions. Half of the RNA was reversed transcribed with Superscript  in 20 μl reaction volume with random primers and cDNA kept frozen. To screen for VHL mutations in thyroid aspirates, 2,5 μl of first-strand cDNA was used as a template for PCR using primers designed by us .Somatic DNA was extracted from samples corresponding to renal carcinoma and thyroid metastases obtained during surgeries and immediately frozen with liquid nitrogen until nucleic acid extraction using TRIzol Reagent . Genomic DNA was also obtained from peripheral venous blood and isolated by a manual method adapted from Bowtell .VHL mutations was performed by single-strand conformational polymorphism analysis (SSCP). To further characterize the abnormal pattern observed in the SSCP, PCR purified products were either sequenced directly using the ABI PRISM® BigDye™ Terminator Cycle Sequencing Ready Reaction Kit  and the ABI PRISM 310 Genetic Analyser or subcloned into pGEM®-T Easy Vector , and subsequently sequenced using the ABI PRISM® BigDye™ Terminator Cycle Sequencing Ready Reaction Kit  and the ABI PRISM 310 Genetic Analyser. Restriction analysis, using the restriction endonuclease BstZ17 I  was also performed.DNA samples were amplified by PCR using primers previously described . The scrVHL gene was identified in thyroid aspirates, renal carcinoma, thyroid metastases and lymph node metastases. It causes a frame-shift and creates a premature stop predicting a truncated pVHL  of the VHL Fig. . ConstitMetastatic disease to the thyroid gland from distant sites is rare. Breast and lung carcinoma are the most frequently identified sources of secondary thyroid carcinoma found at autopsy while renal cell carcinoma comprises over 50% of secondary thyroid malignancies discovered clinically . Clear-cMetastases of a clear-cell tumor present similar morphologic features regardless the origin of the primitive tumor. In a patient with a past history of malignancy elsewhere, the finding of a clear-cell tumor on thyroid aspirates will prompt the hypothesis of a secondary tumor. The sudden enlargement of the thyroid in an otherwise healthy individual, as it was the case in our patient, makes the diagnosis of metastatic disease challenging for the cytopathologist and reinforces the need of immunohistochemical studies ,7. A posRCC is a highly treatment-resistant tumor type and surgery remains the standard approach. Pending the extent of the metastatic disease and the general condition of the patient, the surgical management of metastases is justified as long term survivals have been reported -19. ConsVHL mutations account for the majority of sporadic CCRCCs [VHL gene in different samples of tumor DNA including frozen cDNA obtained from the cells left inside the needle used for fine-needle aspiration cytology. cDNA had been obtained and kept frozen in the setting of an independent ongoing study to seek for molecular markers in thyroid aspirates.Since somatic c CCRCCs ,12 and mVHL mutations has been described in patients with CCRCCs, mutations cluster within exon 2 [VHL mutations may originate from exposure to exogenous carcinogens [VHL might be involved. Hypermethylation studies were not conducted thus we can not exclude inactivation of the wild type allele and on the other hand, contamination with normal tissue can not be ruled out. Despite the previous comment, the frequency of biallelic inactivation of VHL in sporadic renal cell carcinomas was found to be unexpectedly low [The same heterozygous mutation, a 680 delA, (codon 156/exon 3), was observed in all tumor samples and absent in constitutional DNA. The tumor was therefore defined as sporadic. Although a broad spectrum of somatic n exon 2 ,12,20,21cinogens . Accordiedly low .VHL mutations in thyroid aspirates may help in distinguishing metastatic renal cell carcinoma from other primary clear-cell tumors. VHL mutations have not been identified in the thyroid except in MEN 2 associated medullary thyroid carcinoma [VHL gene has been identified in follicular carcinomas of the thyroid [The possibility to screen for arcinoma  although thyroid . Herein,VHL gene regulates the oxygen-dependent expression of genes involved in the cellular response to oxygen deprivation. VHL interacting with the transcription factors hypoxia inducible factors HIF 1α and HIF 2α mediates transcriptional activation of genes involved in angiogenesis, erythropoesis and anaerobic metabolism. Given that multiple pathways contribute to tumor growth, it is conceivable that anti-tumor activity may be increased by agents targeting multiple pathways [The pathways -28.VHL gene may be helpful to establish a diagnosis as well as to make a decision concerning emerging targeted therapies [Thus, identification of a somatic mutation in the herapies .CCRCC – clear cell renal cell carcinoma; FNAC – fine needle aspiration cytology; LOH – loss of heterozygosity; MEN 2 – multiple endocrine neoplasia type 2; RCC – renal cell carcinoma; VHL – von Hippel LindauThe author(s) declare that they have no competing interests.MJB – Provided clinical care for the patient, coordinated studies and draft the manuscript; EM – cytopathologist who carried out the immunocytochemical studies and interpreted results; PC – carried out the molecular genetic studies; JRS – conducted total thyroidectomy; ES – conducted the nephrectomy; ALC – performed the histopathological analysis; LS – critical revision.All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


FOXP2 is a transcription factor implicated in the development and neural control of orofacial coordination, particularly with respect to vocalisation. Observations that orthologues show almost no variation across vertebrates yet differ by two amino acids between humans and chimpanzees have led to speculation that recent evolutionary changes might relate to the emergence of language. Echolocating bats face especially challenging sensorimotor demands, using vocal signals for orientation and often for prey capture. To determine whether mutations in the FoxP2 gene could be associated with echolocation, we sequenced FoxP2 from echolocating and non-echolocating bats as well as a range of other mammal species. We found that contrary to previous reports, FoxP2 is not highly conserved across all nonhuman mammals but is extremely diverse in echolocating bats. We detected divergent selection (a change in selective pressure) at FoxP2 between bats with contrasting sonar systems, suggesting the intriguing possibility of a role for FoxP2 in the evolution and development of echolocation. We speculate that observed accelerated evolution of FoxP2 in bats supports a previously proposed function in sensorimotor coordination. FOXP2 in communication was first suggested following the discovery that mutations at this locus cause impaired speech-related motor coordination  and comprehension (dysphasia) FOXP2 orthologues show almost no variation across distantly related species of reptile, bird and mammal FOXP2 might be related to the emergence of language FoxP2 knockout mice The role of the forkhead transcription factor Echolocating bats face especially challenging sensorimotor demands, using vocal signals for orientation and prey capture. They can emit echolocation pulses at rates of up to 200 sounds per second, interpret the resulting echoes within time-windows as short as several milliseconds and make motor responses such as changes in flight manoeuvres during these short time intervals FOXP2/FoxP2 expression in the brains of other vertebrate species. Sensory nuclei, including the superior and inferior colliculi, show localised expression in the brains of the adult mouse and human foetus FOXP2/FoxP2 mRNA expression in the brains of mammals and birds FOXP2/FoxP2 functions in sensorimotor coordination, raises the possibility that FoxP2 might be involved in the neural circuits that underpin bat echolocation. Furthermore, bats are also one of only a few groups of vertebrates to exhibit vocal learning FoxP2 expression in the brains (area X) of some songbirds Several of the neural areas implicated in echolocation in bats have also been shown to be associated with Rousettus) has subsequently evolved a simple from echolocation based on tongue clicking Our understanding of the phylogenetic relationships among extant bat species has increased considerably over the past two decades Microchiropteran echolocation calls can be broadly classified as either predominately low duty cycle , frequency modulated (FM) or high duty cycle (>30%) constant frequency (CF) FoxP2 gene could be associated with echolocation in bats, we sequenced FoxP2 from echolocating and non-echolocating bat species, as well as a range of other mammals. We isolated mRNA and used reverse transcriptase-mediated polymerase chain reaction (RT–PCR) to amplify the complete gene in bats from six families, as well as representatives from five other mammalian orders and one reptile. Alignments of new sequences with published FoxP2 sequences from mouse, primates and birds, and those obtained from archived genomic bacterial artificial chromosome (BAC) libraries, revealed high amino acid conservation across most vertebrates FoxP2 exons that showed particularly high levels of variability and therefore surveyed these exons in a much wider range of bat species, as well as 18 cetacean species comprising 15 echolocating toothed whales and dolphins (suborder Odontoceti) and three non-echolocating baleen whales (suborder Mysticeti). This extensive survey confirmed that non-synonymous variation among bats exceeds levels recorded across all other vertebrates, but did not suggest that equivalent accelerated evolution was also a feature of echolocating cetaceans.To assess whether mutations in the FoxP2 gene in 13 bat species, 7 other eutherian mammal species and 1 reptile. After combining with archived sequences, including those obtained from genomic BAC libraries, our analyses of complete FoxP2 gene sequences were based on 13 bats, 22 additional (non-bat) eutherian mammals, 1 non-eutherian mammal (platypus), two birds and one reptile (see We sequenced the complete tile see .P<0.001) and synonymous sites   . The disP<0.001) .FoxP2 sequences between bats and taxa from related orders within the superorder Laurasiatheria. Significant variation in ω was detected with around 16% of sites identified as evolving under divergent selection  (ω for bats over other laurasiatherians (0.23 versus 0.14) reflects the greater number of non-synonymous changes in this group. We repeated the analysis comparing the two major recognised lineages of bats (Yinpterochiroptera and Yangochiroptera) and found evidence for divergent selection at 19% sites  with respective ω values of 0.23 and 0.14  were excluded from the analyses . On the other hand, we found evidence of a change in the strength of selection P<0.001) . The higand 0.14 . Thus moFoxP2 protein structure and found that, on average, the observed changes had less effect than other possible replacements  based on 10,000 randomisations; Z test; P = 0.02). This finding, together with the absence of stop codons, suggests that overall FoxP2 remains constrained in bats despite the comparatively high level of variation. However, at the same time, the distribution of predicted impact scores of observed replacements  was positively skewed  with some changes expected to have a considerable influence on physiochemical properties.We used a Multivariate Analysis of Protein Polymorphism Based on our alignments of complete gene sequences in bats, we identified two highly variable regions (exons 7 and 17). We undertook targeted sequences of these coding regions in a wider range of echolocating and non-echolocating bat species, as well as echolocating and non-echolocating cetaceans and additional eutherian mammals. In total, we obtained exon data for 65 mammal species, including 42 bats (from 10 families) and 18 cetaceans (from two families), two birds and one reptile .Nycteris). Variation at both exons appeared to correspond well to echolocation types/phylogenetic boundaries, with almost complete conservation across groups of confamilial species but contrasting signatures between families.Alignments of exons 7 and 17 revealed the extent of accelerated amino acid evolution among bat lineages compared with other vertebrates as well as high conservation among vertebrates in general see  and S5. FOXP2/FoxP2 gene has been of tremendous interest in recent years, with sequence variation and patterns of expression linked to human speech defects FoxP2 sequences undertaken to date and report that when compared with other groups, this gene shows greater variation in bats.The FoxP2 has clearly undergone accelerated evolution in bats compared to other vertebrates, the basis for this change is not clear. Significant divergent selection based on differences in the dN/dS ratios (ω) was detected between laurasiatherians and bats, and also between the two major clades of bats, indicative of a change in the strength of natural selection between these groups. In these tests, higher dN/dS ratios were found in all bats and the clade Yinpterochiroptera, respectively, and values were almost identical, indicating that the detected change in selective pressure has probably occurred since the Yangochiroptera and Yinpterochiroptera diverged. Furthermore, both analyses gave similar results when fruit bats were removed, and thus the divergent selection cannot be attributed to the inclusion of taxa that might have lost the ability (and need) to echolocate.Although dN/dS ratio in the Yinpterochiroptera could arise from either a relaxation in selection pressure or a short burst of Darwinian selection. Although explicit tests for positive selection proved negative, the power of such tests to detect positive selection where sequence variability is low is very limited and prone to false negatives (type 2 error) FoxP2 in echolocating bats is unlikely. First, the fact that FoxP2 is implicated in sensorimotor coordination in mammals and echolocation bats face exceptional challenges in such coordination provide a priori reasons to expect full FoxP2 functionality in all echolocating bats. Second, FoxP2 is highly conserved across divergent lineages of vertebrates including birds, reptiles and mammals, and there is no reason why to suspect that FoxP2 should be any less important in bats than other taxonomic groups. Third, an absence of stop codons, and the non-random distribution of nonsynonymous changes among exons in bat FoxP2 sequences, which are also predicted to have less impact on the protein's physiocochemical properties than random changes, all suggest that the amino acid replacements are not due to relaxed selection. For these reasons we contend that the changes we report are more likely to have some adaptive significance. The observed correlation between substitution rates at neighbouring non-synonymous and synonymous sites could arise by hitchhiking effects, in which selection increases the fixation probability of linked weakly deleterious sites The higher FoxP2 variation in bats might relate to their capacity for vocal learning, which has been described in a number of species FoxP2 might be associated with the evolution of language, a number of studies have screened FoxP2 in vertebrate species that show evidence of vocal learning, which is widely considered to be a critical substrate for the evolution of human language. In addition to bats, vocal learning has also been reported in some lineages of birds FoxP2 expression studies in avian brains have proven inconclusive Rhinolophus) and the fact that both changes do not occur together in any bat, mean that if variation in FoxP2 has a role in vocal learning then it is not straightforward.One possible explanation for higher FoxP2 plays a role in the sensorimotor demands that are peculiar to bat echolocation rather than echolocation in general. Indeed while all cetaceans shared three amino acid substitutions  no differences were observed between echolocating and non-echolocating baleen cetaceans, also supporting an earlier comparison of one example from each suborder We instead speculate that observed variation in bats might be associated with aspects of echolocation. As mentioned, amino acid variation at both exons 7 and 17 in bats corresponds well to echolocation types/phylogenetic boundaries, with almost complete conservation across groups of confamilial species but contrasting signatures between families. Such high sequence diversity at exons 7 and 17 in bats relative to other mammals, including echolocating cetaceans, indicates that FoxP2 variation is linked to echolocation in bats. FoxP2 has previously been implicated in sensorimotor disorders with respect to vocalisations and orofacial coordination The absence of consistent shared amino acids across echolocating bats and cetaceans is not necessarily inconsistent with our hypothesis that FoxP2 does function in echolocation, then the signature at exon 17 might be more suggestive that echolocation has evolved twice in bats. Here, fruit bats that do not have laryngeal echolocation (Yinpterochiroptera) (echolocation status 1 and 2 in Rhinolophus (T641A and D663E) and Hipposideros (V637E and D663E), with further non-synonymous substitutions in the hipposiderid genera Aselliscus (I602M and E657D) and Coelops . In cetaceans, evidence regarding the evolution of echolocation is also equivocal but fossil evidence indicates that early species were toothed rather than possessing baleen and could hear underwater, though there is no evidence to date of anatomical specialisations associated with echolocation A lack of clear differences in nonsynonymous variation at exon 7 between echolocating and non-echolocating lineages in both cetaceans and bats can also be explained if the common ancestor in both groups had echolocation and this ability was subsequently lost in some lineages. This scenario has been previously been suggested for bats FoxP2 sequence variation with respect to other cases of convergence in bat echolocation are also informative. The methionine to isoleucine substitution (M637I) in Hipposideros has also arisen independently in the yangochiropteran species Pteronotus parnellii but not in its congeners P. quadridens or P. macleayii. It is therefore interesting that P. parnellii is the only species of non-rhinolophoid bat to have evolved Doppler-shift compensation in echolocation, a feature that it shares with the genus Hipposideros. A striking example of rapid FoxP2 evolution is that of the yangochiropteran genus Nycteris, which, unlike its closest surveyed relatives, shows independent evolution of nasal emission, multi-harmonic call structure and prey location by passive listening Nycteris also shows the highest rate of non-synonymous change at exon 17 of all species surveyed, differing from both the mammalian consensus and its closest relative by eight amino acids  Tadarida sequence and its close relative Chaerephon plicata (family Molossidae) further highlight the high degree of non-synonymous diversity in this order.Trends in FoxP2 sequence diversity in bats presented in this study, together with evidence of significant divergent selection and a lack of evidence of positive selection at this time, all point to a need for more work to elucidate the role of this protein in bats. Based on current findings, we suggest that echolocation in bats might have involved the recruitment of the FoxP2, and, more generally, that the protein might function in the mobilisation of downstream genes (or genetic cascades) involved in one or more aspects of the development or regulation of complex sensorimotor coordination. To investigate these speculations, data on the expression of FoxP2 in the bat brain, with particular attention to the neural pathways implicated in echolocation, would allow informative comparisons with published results from human, mouse and bird brains. To date, the delineated FoxP2 protein domains thought to be involved in DNA binding and transcription regulation FoxP2 in sensorimotor integration and rapid motor learning.Comparatively high levels of Bat wing membrane biopsies were collected from five localities in China. Some individuals were sacrificed as part of an ongoing surveillance programme for coronaviruses, and their liver and brain tissue stored in liquid nitrogen for RNA preservation. Additional genetic material from bats and other mammals was obtained from tissue banks held at the Institute of Zoology in Beijing and the Institute of Zoology in London.FoxP2 coding sequence from mRNA transcripts. Total RNA was isolated from brain and liver tissue using RNAiso kits  and reverse-transcribed to first-strand cDNA using Invitrogen SuperScript™ II RT and oligo-dT primers. We designed two primers pairs based on conserved sections to amplify two overlapping regions, spanning exons 2 to 10 (5′-GGT ATT AAG TCA TGA TGC AGG A-3′ and 5′-TCG CAT GTG CAA GTG GGT C-3′) and exons 8 to 17 (5′-GGC TGT GAA AGC ATT TGT GAA G-3′ and 5′-ATG GTT GTG GAG TGG TTA TGA AG-3′). Due to technical problems with amplifying the second region in one species (Megaderma spasma), we also designed the degenerate primer 5′-GAG GTY KCA CAA GYC AGT TCT CAT TCC -3′ to work with primer 5′-GGC TGT GAA AGC ATT TGT GAA G-3′.We used RT-PCR to amplify the complete PCR products from cDNA were ligated into a pMD19-T vector (TaKaRa) and cloned. Positive clones were sequenced using Big Dye Terminator on an ABI 3730 DNA sequencer (Applied Biosystems). Complete sequences were reconstructed by overlapping shorter sequences, allowing us to verify that nucleotide changes were not attributable to RT-PCR artefacts.FoxP2 gene sequences, we identified two regions of especially high variability that correspond to exon 7 (188 bp) and exon 17 (145 bp). To survey these exons in a wider range of bat species, DNA was isolated with DNeasy kits (Qiagen) for PCR-based targeted amplification. For exon 7, we designed the degenerate primer 5′-CTG GCT TAA GTC CTG CYG ARA TTC-3′ to use with the published intronic primer 5′-GAA TAA AGC TCA TGA GAT TTA CCT GTC-3′ From alignments of whole FoxP2 gene in one individual each of thirteen species of bat  as well as pig (Sus scrofa), goat (Capra hircus), donkey (Equus asinus), cat (Felis catus), hog-badger (Arctonyx collaris), oriental water shrew , rabbit (Oryctolagus cuniculus) and one reptile species, the red-eared slider terrapin (Trachemys scripta).For whole gene alignments, we sequenced 2100–2200 bp of the FoxP2 sequences of six primate species , chimpanzee , gorilla , orang utan , white-handed gibbon , rhesus macaque  two bird species  and budgerigar ) and mouse . We obtained additional FoxP2 sequences by searching archived genomic BAC libraries  using the BLASTN tool (http://www.ncbi.nlm.nih.gov/BLAST/) for olive baboon , common marmoset , small-eared galago , gray mouse lemur , African elephant , nine-banded armadillo , African hedgehog , common shew , and one non-eutherian mammal species, the platypus .We also obtained from GenBank the published whole FoxP2 gene in 13 bat species, we also sequenced exons 7 (188 bp) and 17 (145 bp) in a wider range of bats . For 12 bat species  DNA was sequenced from multiple individuals (2-5) to confirm the results and in all cases no variation was found between conspecifics.In addition to sequencing the complete FoxP2 evolution is a common feature of vertebrate echolocation, we also sequenced 15 echolocating toothed whales (Atlantic white-sided dolphin (Lagenorhynchus acutus), Blainville's beaked whale (Mesoplodon densirostris), bottlenose dolphin (Tursiops truncatus), common dolphin (Delphinus delphis), Cuvieŕs beaked whale (Ziphius cavirostris), harbour porpoise (Phocoena phocoena), killer whale (Orcinus orca), long-finned pilot whale , northern bottlenose whale (Hyperoodon ampullatus), pygmy sperm whale (Megaptera novaeangliae), Risso's dolphin (Grampus griseus), Sowerby's beaked whale (Mesoplodon bidens), sperm whale (Physeter catodon), striped dolphin  and white-beaked dolphin  as well as three species of non-echolocating baleen whale , humpback whale (Megaptera novaeangliae) and minke whale ). To improve taxonomic coverage further, we also included the red and white giant squirrel  and the short-eared elephant-shrew (Macroscelides proboscideus: Macroscelidea).In addition, to determine whether rapid FoxP2 sequences are EU076391-EU076411 inclusive. For exons 7 they EU076412 and EU087927-EU087965 inclusive, and for exon 17 they are EU087966-EU088011 inclusive. Information on the taxonomic groupings of all sequenced species is given in GenBank accession numbers of all new complete Nucleotide sequences (1995 bp) were aligned using the software ClustalX For each pair of complete sequences, we derived the total number of Nei-Gojobori synonymous and nonsynonymous differences dS and dN) for each branch assuming independent dS/dN (ω) ratios per lineage dN values using the software PHYLODRAW We constructed a phylogeny based on published studies et al's modified Branch-Site Model A ω0<1) and neutral selection (ω1 = 1), occur across the tree. Two additional sites evolve under purifying or neutral selection on the background branches but under positive selection (ω2>1) on the branch under consideration (foreground). A likelihood ratio test (LRT) was conducted by comparing the likelihood result of this model to a null model in which all parameters were the same except that ω2 is fixed at 1  on the foreground. We repeated this analysis four times, to test for positive selection along branches ancestral to all bats, to all Yangochiroptera, to all Yinpterochiroptera and to all fruit bats.We tested for evidence of positive selection along selected lineages using Zhang FoxP2 has in some way become recruited into neural pathways involved in bat echolocation, it might be associated with a change in selective pressure between echolocating bats and other mammals, and also between bat lineages that exhibit contrasting forms of echolocation system. Such changes in selection pressure can be small and detectable even when specific tests of positive selection prove negative. To test for evidence of divergent selective pressures among selected pairs of clades, we applied the modified version of Bielawski and Yang's maximum likelihood-based Clade Model C dS/dN (ω) ratio among sites with a proportion of sites evolving under different selective constraints between a pair of clades ω0<1) and neutral evolution (ω1 = 1), but to differ at a third site class under divergent selection (ω2clade 1≠ω2 clade 2). We compared the model results with those obtained from the site model M1a  to conduct a LRT with 3 degrees of freedom.If ω0, and found that estimates of the proportion of sites under divergent selection were consistent.We first test for evidence of divergent selection  between bats and their nearest known relatives in the superorder Laurasiatheria, represented by a clade comprising donkey, pig, goat, shrew, Asian shrew, hedgehog, cat and badger. We then compared the two major recognised lineages of bats (Yinpterochiroptera and Yangochiroptera). Both tests were repeated but with fruit bat sequences excluded, to assess whether any changes in selection pressure are likely to be due to relaxation resulting from a loss of echolocation. For the same reason we directly compared fruit bats and non-fruit bats within the clade Yinpterochiroptera. We tested the robustness of our results and tested for suboptimal likelihood peaks by rerunning the model under a range of initial values of FoxP2 protein than random non-synonymous changes. Amino acid sequences of FoxP2 orthologues of all mammals excluding bats, birds and reptiles were aligned and, to correct for phylogenetic affiliations, were weighted based on PAML-derived branch lengths and known phylogeny topology We also undertook a Multivariate Analysis of Protein Polymorphism (MAPP) Table S1Summary of sequences surveyed in the study. Accession numbers are given for sequences obtained from GenBank.(0.17 MB DOC)Click here for additional data file.Table S2FoxP2 gene  with variable sites shown. For all species represented in FOXP2 orthologue. Amino acids that were linked to the evolution of language are given in red and those only found in bats are given in blue.(0.18 MB DOC)Click here for additional data file.Table S3FoxP2 gene  with variable sites shown. For abbreviations, see (0.17 MB DOC)Click here for additional data file.Table S4FoxP2. For abbreviations, see Exon 7 of (0.13 MB DOC)Click here for additional data file.Table S5FoxP2. For abbreviations, see Exon 17 of (0.10 MB DOC)Click here for additional data file.Figure S1Cladogram showing PAML-derived maximum likelihood estimates of rates of non-synonymous and synonymous substitutions. Bat lineages are coloured to show the two major divergent forms of laryngeal echolocation (blue = high duty constant frequency (CF) calls with at least partial Doppler shift compensation, orange = low duty cycle calls, green = short, broadband multi-harmonic, violet = the absence of laryngeal echolocation in fruit bats. Estimates of the number of non-synonymous substitutions (dN) and synonymous substitutions (dS) under a free ratio model are given on the branches as (dN/dS).(0.05 MB DOC)Click here for additional data file.


Animal studies have demonstrated that paternal obesity increases the risk of metabolic  and reproductive disorders in offspring. Epigenetic changes within sperm are clear mechanistic candidates that are associated with both changes to the father’s environment and offspring phenotype. Specifically there is emerging evidence that a father’s sperm microRNA content both responds to paternal environmental cues and alters the gene expression profile and subsequent development of the early embryo. We used a mouse model of high fat diet (HFD) induced obesity to investigate whether male obesity could modulate sperm microRNA content. We also investigated whether this alteration to a father’s sperm microRNA content lead to a similar change in the sperm of male offspring. Our investigations were initially guided by a Taqman PCR array, which indicated the differential abundance of 28 sperm borne microRNAs in HFD mice. qPCR confirmation in a much larger cohort of founder males demonstrated that 13 of these microRNAs were differentially abundant  due to HFD feeding. Despite metabolic and reproductive phenotypes also being observed in grand-offspring fathered via the male offspring lineage, there was no evidence that any of the 13 microRNAs were also dysregulated in male offspring sperm. This was presumably due to the variation seen within both groups of offspring and suggests other mechanisms might act between offspring and grand-offspring. Thus 13 sperm borne microRNAs are modulated by a father’s HFD and the presumed transfer of this altered microRNA payload to the embryo at fertilisation potentially acts to alter the embryonic molecular makeup post-fertilisation, altering its growth trajectory, ultimately affecting adult offspring phenotype and may contribute to paternal programming.The prevalence of obesity is increasing worldwide and has tripled in men of reproductive age since the 1970s. Concerningly, obesity is not only comorbid with other chronic diseases, but there is mounting evidence that it increases the non-communicable disease load in their children ( Obesity and its consequences for lifelong health and well-being are an increasing burden for health systems worldwide. Globally more than 2.7 billion adults are classified as overweight or obese  and obesOf significant concern, human epidemiological investigations provide evidence that an increased male BMI, or increased food intake, can lead to male transmission of non-communicable diseases to descendants. For example, a presumed excess of grandpaternal food was associated with reduced survivability  and an iAnimal models have clearly demonstrated that programming can be paternally initiated, whereby a father’s pre-conception high fat diet increases the risk of metabolic disturbances in offspring –21. UsinA prime candidate mechanism, by which paternal environmental cues are passed to the next generation, are non-genetic alterations in sperm such as DNA methylation, Histone modifications, DNA damage, and in particular small non-coding RNAs (sncRNA) –25. MatuSperm borne microRNAs have been demonstrated to be important for embryo development, as evidenced by embryonic arrest in embryos deficient of sperm derived microRNA  and thatWe therefore hypothesised that paternal diet-induced obesity during spermatogenesis can also regulate the abundance of microRNA in spermatozoa. Therefore, our aim was to utilise our mouse model of obesity to identify sperm borne microRNAs that are altered by high-fat diet induced obesity. Furthermore given that grand-offspring born to male offspring also have metabolic and reproductive phenotypes  we also aka fathers; C57BL/6NHsd, Envigo, IN, USA) were fed either a control diet , or a high fat diet , which were otherwise matched for nutritional content. All male mice were housed individually post weaning prior to mating , so a dominance structure was not established between males within the same litter that might affect sex hormones profiles and thus potentially skew reproductive and sperm microRNA outcomes. Animals had ad libitum access to feed and water for 10 weeks (5 to 15 weeks of age) and during experiments where fasting was not required. After 10 weeks on the diets (15 weeks of age) a subset of males  were mated with CD fed female C57Bl6 mice to generate male offspring, approximately 1 week prior to sperm collection. Male offspring  were weaned and maintained on the CD until 15 weeks of age and mated with CD fed female C57Bl6 mice to generate the grand-offspring; then offspring sperm samples were collected post mortem.Male C57Bl6 mice , 10 h dark (lights off at 2000) illumination cycle. All procedures were conducted during the light phase of the light cycle and all mice in each group were subjected to all experiments in the order in which they are described.In all cohorts of mice each mouse had total body weight measured weekly and body composition determined by post mortem dissection and weighing each dissected tissue after 10 weeks of diet regimens (for fathers) or at 15 weeks of age for offspring. Tissues (including adipose tissue) and organs of each animal were dissected and weighed post-mortem.The animal ethics committee of the University of Adelaide approved all experiments, and the animals were handled in accordance with the Australian Code of Practices for the Care and Use of Animals for Scientific Purposes.-1 of 25% glucose solution  and then one week later intraperitoneal insulin challenge (IPTT) was performed fed after ip injection of 0.75 IU kg-1 insulin . Tail blood glucose concentration was obtained by a tail vein nick and measured using a glucometer  at timepoints 0 , 15, 30, 60 and 120 minutes. Data is expressed as mean blood glucose concentration per group using area under curve  or area above the curve (AAC) analysis .Following 9–10 weeks of diet treatment in fathers (14–15 week of age), intraperitoneal glucose tolerance test (IPGTT) was performed after 6 h of fasting by intra-peritoneal (ip) injection of 2 g kgFor blood hormone measurements, male mice were fasted overnight (14 h), and a blood sample was obtained by cardiac puncture immediately prior to sacrifice, serum was immediately separated by centrifugation  and frozen at -80°C until further analysis. Fasting serum glucose, cholesterol, triglycerides, non-esterified fatty acids (NEFAC test) and HDL cholesterol (HDLC3) were measured in duplicates on a COBAS INTEGRA 400 . Fasting serum insulin and leptin levels were measured in duplicates by Sensitive Rat Insulin radioimmunoassay  and Mouse Leptin RIA, respectively .Spermatozoa were isolated from the vas deferens and cauda epididymis which had been dissected out of the mouse and gently squeezed into 1 ml GIVF medium  with all tissue remnants removed, immediately after animal sacrifice and overnight fasting. Most of the GIVF/sperm solution was carefully aspirated, pelleted and resuspended/washed in 1 ml of PBS and counted as previously described  and as b2, 6% CO2 and 89% N2. Sperm concentration was determined using a Neubauer haemocytometer and sperm motility was determined by count of duplicate measures of 200 sperm [Spermatozoa were extracted into GIVF medium (Vitrolife AB) and allowed to swim out for 10 min at 37°C in 5% O00 sperm  and repo00 sperm .6 spermatozoa for each male mouse investigated (no pooling of samples) with TRI reagent , using Glycogen as a RNA carrier. RT-PCR was performed with multiplexed TaqMan primers, and the RT product pre-amplified with Megaplex PreAmp Primers. miRNA expression profiling was performed on 384-well microfluidic TaqMan Rodent MicroRNA Array cards v. 3.0  amplified on a 7900 HT Real Time PCR system. After quality control in SDS v.2.3 and RQ Manager v.1.2, the raw Ct data were quantile normalised using the normQpcrQuantile function of the R qPCRNorm package [Total RNA was isolated from 4-8x10 package . The degie strict filtering) that were differentially abundant in the sperm of HFD fathers. A core network analysis was then performed using only experimentally validated interactions between molecules (ie strict setting) and to predict the molecular networks that these mRNAs targets are known to function in.The Ingenuity Pathway Analysis® (IPA) tool was used to generate a list of mRNA targets experimentally confirmed to be direct targets of the microRNAs  as well as equal variance by a Levene’s test . Experimental outcomes were analysed using a paired Student’s t-test, a Mann Whitney U test for non-parametric data (Graphpad Prism), or univariate general linear modelling using cohort of animals as a covariate (SPSS), when appropriate. Levels of significance were set at n = 14) were heavier (+24.8%), as a proportion of total body weight they had increased adiposity (+46.6%). Specifically perirenal (+65.3%), gonadal (+57.2%), and omental (+65.3%) adipose depots were greater as a proportion of total body weight (n = 13). Akin to human obesity, HFD feeding also increased fasted blood sera metabolite concentrations, including cholesterol (+47.3%), high density lipoprotein cholesterol low serum 3 , and non-esterified free fatty acids  (We used our mouse model of high fat diet (HFD) induced obesity, where HFD consumption for 10 weeks induces obesity without any overt changes to glucose homeostasis ,22,44,47y weight  compared +32.4%)  compared +32.4%) . Thus th6/mL for CD (n = 13) and 8.41 ± 0.44 x 106/mL for HFD (n = 14)) but sperm motility was significantly reduced (CD (n = 13)– 83.1 ± 2.6%; vs HFD (n = 14)– 65.1 ± 4.4%; p = 0.0023) compared with CD fed fathers. These data confirm our previous findings that diet induced obesity impairs sperm parameters in both mouse models and in humans [Conventional sperm analysis showed that sperm count was unaltered by HFD feeding . The expression of 371 mouse miRNAs could be detected (raw Ct<35) in all 8 CD/HFD samples. LIMMA analysis of quantile normalised Ct values of these 371 miRNAs   .n = 4 per CD/HFD F0 males) and additional males from a total of three separate cohorts of mice . This included a subset of a cohort we had previously published that demonstrated transgenerational transmission of reproductive and metabolic phenotypes into offspring and grand-offspring due a paternal HFD [To build on the preliminary array findings, individual Taqman microRNA qPCR assays for all 28 candidate microRNAs were performed on the sperm from a larger sample that included both the samples used for the array  that we found to be invariable and ubiquitous endogenous in the Taqman PCR array data set and then validated by the individual Taqman qPCR assays in the extended cohort . The smaIn Addition 53 mouse sperm microRNAs were only detected in the sperm from either CD or HFD fed fathers  using a ie diet induced obesity) would have the same impact on sperm microRNA content in the F1 males, who themselves have metabolic disturbances without increased adiposity. We have previously reported that the grand-offspring born through the male offspring lineage also have metabolic and reproductive disturbances , and 16.2 ± 7.13% increase in oocyte ROS) that have some phenotypic overlap with those of the male offspring investigated here  [Many paternal environmental challenges have been demonstrated to initiate transgenerational effects and epigenetic disturbances in offspring, such as endocrine disruptors , stress isation) ,22. Thusisation) , in our An Ingenuity Pathway Analysis® with strict filtering and analysis settings was used to both (i) generate a list of 311 experimentally confirmed mRNA targets  of the 1The 13 sperm borne miRNAs differentially regulated in a mouse model of paternal obesity may act as epigenetic signals transmitted to offspring, by targeting maternally stored mRNA transcripts in the fertilised egg, initiating a cascade of molecular events that reprogram embryonic development that ultimately impairs the long term metabolic and reproductive health of adult offspring. Furthermore, despite grand-offspring mice born to the male offspring displaying some similarities in metabolic and reproductive phenotypes to their offspring fathers, there was limited evidence that any of these microRNAs were also dysregulated in the offspring’s sperm. It must be noted that no dysregulation was observed in the sperm of the offspring presumably due to the variation of microRNA abundance detected within both groups of offspring. This suggests that either a different set of sperm borne microRNAs or other epigenetic/genetic marks are transmitted to grand-offspring that induce these phenotypes and that father to offspring signals may also comprise of more than sperm microRNAs. Taken together this implies that the set of sperm borne microRNAs identified as altered in these fathers’ sperm that were associated with programming in offspring, might be unique to the obese state, as male offspring born to obese fathers using this model of obesity were not themselves obese .ie n = 4), mainly between the HFD father samples. The last microRNA (miR-196a-5p) did not pass the quality test applied to all raw data prior to the LIMMA analysis, but interestingly it was detected in HFD fed fathers sperm but not in CD fed fathers (using a 35 Ct cut-off limit of detection), consistent with a large up-regulation as previously reported. This may be due to the multiplex nature of the method used for cDNA generation that might result in inconsistent representation of specific microRNAs in the cDNA versus a single assay in the subsequent qPCRs, or the variance of some microRNAs between the multiple cohorts. Thus highlighting the importance of qPCR confirmation of array data with a larger sample size from multiple cohorts.Of the four sperm microRNAs  that we previously reported as altered in fathers sperm due to this HFD regimen [OCT4, SOX2 and KLF4 [OCT4, SOX2 and KLF4, reducing pluripotency within these embryos, which has important ramifications for the development of the pluripotent epiblast, which will ultimately develop into the fetal pole. A high fat diet has been shown to reduce the formation of the epiblast and also increases the amount of pregnancy loss in this mouse model of obesity [It is possible that the sperm microRNAs that are differentially abundant in obese fathers enact their presumed perturbation to cellular functions by targeting maternally stored mRNAs post-fertilisation, as has recently been demonstrated for transmission of paternal stress cues . Althougand KLF4 ,56. Moreand KLF4 . Since s obesity . Further obesity , and if  obesity –54. Impo obesity ,20,22,58DNMT1 mRNA, reducing its expression and leading to hypomethylation [DNMT3A expression in colon and breast cancer cell lines [DNMT3A and DNMT3B, respectively, for mRNA degradation. Overall the predicted targeting of components of the DNA methylation machinery provides the means for longer term changes to gene expression within the embryo and potentially even subsequent offspring. Furthermore given that offspring sperm does not overtly demonstrate the same compliment of differentially abundant microRNAs, DNA methylation might also be important for the phenotypic transmission from offspring to grand-offspring.The differentially abundant sperm microRNAs may potentially also be acting upon DNA methylation post fertilisation. Indeed there are multiple lines of evidence that microRNAs can alter methylation patterns. Specifically microRNA-126-3p (increased in sperm from HFD fathers) binds the 3′-untranslated region of hylation . Intereshylation . MicroRNll lines ,61, and ll lines . But wheThe tumour suppressor p53 is known to enhance post-transcriptional maturation of multiple miRNAs, including microRNA-143/microRNA-145 (increased in the sperm from HFD fathers), as a response to DNA damage . InteresMicroRNAs-145a-5p, 30a-5p, 126-3p, 184-3p, and 143-3p are the most differentially abundant as a result of diet induced obesity, perhaps indicating their potential to enact aberrant maternal mRNA decay in the embryo. Furthermore 8/13 microRNAs altered by a father’s obesity are amongst the microRNAs whose abundance are most altered in sperm during epididymal transit . Thus ieg microRNA-221/222 [ie the tsRNA fraction [Similar to our findings, the abundance of sperm borne microRNA-30a-5p was increased by a model of paternal stress . This mo-221/222 ; microRN-221/222 ; microRN-221/222 ; microRN-221/222 ). It has-221/222 , entire -221/222 , or specfraction ) are capThe array used to interrogate sperm microRNAs was limited to 641 murine microRNAs of an estimated 1,400 that are present  and did Diet and/or exercise interventions have been used in a similar model of diet induced obesity in fathers to demonstrate that this type of intervention strategy can be effective for ameliorating detrimental effects of paternal diet-induced obesity on metabolic parameters and sperm function , embryonWe have demonstrated that the abundance of 13 sperm borne microRNAs are modulated by a father’s consumption of a high fat diet and speculate that the transmission of this altered microRNA content to the embryo at fertilisation acts to alter the mRNA content of the embryo, as has been previously demonstrated. This altered embryonic mRNA content then acts to alter growth trajectory previously observed for embryos fertilised by obese fathers and ultimately confers sub-optimal adult metabolic and reproductive outcomes described for offspring sired by obese fathers. Although the same microRNAs are most likely not altered in the sperm of male offspring, despite the presence of metabolic/reproductive phenotypes in grand-offspring sired by the male offspring. Thus we have identified a potential mechanism that may, in part, form the basis of obesity initiated paternal programming of the first generation of offspring.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Six stratified freshwater lakes were chosen to study the distribution and diversity of the Calvin-Benson-Bassham (CBB) cycle, the reductive tricarboxylic acid (rTCA) cycle, and the recently discovered archaeal 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) pathway. Eleven primer sets were used to amplify and sequence genes coding for selected key enzymes in the three pathways. Whereas the CBB pathway with different forms of RubisCO  was ubiquitous and related to diverse bacterial taxa, encompassing a wide range of potential physiologies, the rTCA cycle in Epsilonproteobacteria and Chloribi was exclusively detected in anoxic water layers. Nitrifiying Nitrosospira and Thaumarchaeota, using the rTCA and HP/HB cycle respectively, are important residents in the aphotic and (micro-)oxic zone of deep lakes. Both taxa were of minor importance in surface waters and in smaller lakes characterized by an anoxic hypolimnion. Overall, this study provides a first insight on how different CO2 fixation strategies and chemical gradients in lakes are associated to the distribution of chemoautotrophic prokaryotes with different functional traits.While mechanisms of different carbon dioxide (CO Recent 2)  and in b2, whereby the Calvin-Benson-Bassham (CBB) cycle, the reductive tricarboxylic acid (rTCA) cycle, the reductive acetyl-CoA pathway and the recently discovered 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle are most widely distributed in different habitats  is the most prominent enzyme, and the gene coding for the large subunit of RubisCO is known to serve as a marker for the analysis of autotrophic organisms, including bacteria, using the CBB cycle . From thronments . Forms IThaumarchaeota, which assimilate inorganic carbon with an energy efficient variant of the HP/HB cycle originally described for hyperthermophilic Crenarchaeota , the three others were much smaller than 1 km2 and 10.5 to 57 m deep , and Nitrosomonas cryotolerans. Most members of the N. oligotropha lineage are known for their ability to grow effectively at low ammonium concentrations  produced specific PCR amplicons in most depths of the deep lakes ACH, STA and ZUR, with exception of the epilimnion of STA  of the genome sequence. Mechanisms to reduce light-induced damage could also have important implication for the presence of Thaumarchaeota in our study lakes, because several studies have shown that photoinhibition might be an important factor determining the distribution and relative contribution of ammonia oxidizing Thaumarchaeota in aquatic systems , was phylogenetically affiliated with uncultured thaumarchaeal clone sequences recovered from brackish water sediments and several ammonia oxidizing Nitrosopumilaceae isolates  or a very low proportion (ACH) of Thaumarchaeota in the epilimnion of the large lakes, with a strong increase of their abundance in the hypolimnion (Thaumarchaeota in the surface water (3 and 10 m) of ZUR were below 1%  recorded in 100 m depth. However, the number of hcd genes was two magnitudes higher in ZUR with a maximum value of 0.384 x 106 genes ml−1 in 80m depth  in the rTCA cycle were successfully amplified from deep water of HEC (25 and 50 m) and PIB . All of them are capable to extract energy from the oxidation of reduced sulfur compounds. Phylotypes related to green phototrophic Chlorobi were exclusively found in PIB.Genes coding for the key enzyme ATP citrate lyase  and in the hypolimnion of PIB, while in water samples obtained from EGE and HEC marker genes for the rTCA cycle related to Nitrospira were not identified. Sequences of aclA genes retrieved from selected hypolimnion water samples of ACH, PIB, STA and ZUR formed a distinct cluster which was affiliated with different nitrite-oxidizing  and complete ammonia oxidizing (comammox) Nitrospira . Phylogenetic analyses also suggest that the ATP citrate lyase gene is not a suitable phylogenetic marker to differentiate between both metabolisms  niche that was missed by the sampling scheme, it is more likely that the actual environmental conditions determine their existence. Redundancy analysis (RDA) showing correlations between hcd gene abundances and selected environmental variables, indicates the highest relationship between nitrate, the final product of nitrification, conductivity and depth  and HEC (14 m)  . In thesfor NH4+ . Certainep lakes . Consideep lakes  that Tha2. The metabolic lifestyle of the closest relatives using Form IC and II RubisCO for CO2 fixation in the CBB cycle is diverse, but especially RubisCO cbbL IC phylotypes are often associated with facultative chemolithoautotrophs  to identify stratification gradients. Based on these data, eight to ten samples were taken along the water column at the deepest part of the lakes, with an increased sampling resolution along pronounced oxygen gradients . Major anion and cation compositions of the lake water samples were analysed by ion chromatography . Total organic carbon (TOC) and dissolved organic carbon (DOC) were determined using a TOC/TN-Analyzer . In addition to oxygen probe measurements, parallel determination of oxygen concentration at sampling depths were carried out using the Winkler titration method.2 fixation pathways. PCR was performed with the HotStarTaq® Plus Master Mix Kit  following the manufacturer’s instructions. The annealing temperatures used for individual primer sets are listed in aclA genes  in the field, immediately frozen on dry ice and stored in the laboratory at −20°C until use. DNA extraction was performed with the PowerWater® DNA isolation kit  according to the manufacturer’s protocol. lA genes . One μl hcd genes were analysed by quantitative PCR using primers specifically designed for this study  and molecular biology-grade water. The reactions had an initial denaturing step of 10 min at 95°C, followed by 35 cycles including 15 s denaturing at 95°C, 15 s of primer annealing at 56°C and 45 s of primer extension at 60°C. Primer specificity was assessed by melt curve analysis and confirmed by running amplicons on agarose gel electrophoresis. Standard curves for quantification were based on 10-fold dilutions ranging from 101 to 108 copies of DNA of a hcd amplicon of an environmental clone selected from clone libraries produced for the diversity studies (procedure see above). A GeneJET Plasmid Miniprep Kit  was used to extract the plasmids, which were linearized with BcuI restriction enzyme , purified with a Qiaquick PCR Purification Kit  and measured fluorometrically .DNA concentrations in lake water samples were measured using a Quantus fluorometer  and QuantiFluor® dsDNA chemistry  according to the protocols recommended by the manufacturer. Thaumarchaeal is study . The optis study . Samples primers , were us of 56°C . All qPCThaumarchaeota  and the BLAST algorithm within the IMG/M analysis system for metagenomes (https://img.jgi.doe.gov/). Deduced amino acids were aligned using Clustal W or MUSCLE algorithm as implemented in MEGA 6.0 software (Closest relatives to nucleotide sequences and deduced amino acid sequences were obtained using NCBI’s sequence similarity search tools BLASTN and BLASTP (cbbL-Form IA), KY447334 – KY447878 (cbbL-Form IC), KY447879 – KY448163 (cbbM), KY448164 – KY448240 (hcd), KY458233 – KY458281 (hcd), KY458282 – KY458375 (acl-Nitrospira), KY458376 – KY458390 (acl).Sequences data have been submitted to GenBank databases under accession numbers KY418180 – KY418605 (Additional Supporting Information may be found in the online version of this article at the publisher’s website:Supplementary Information