Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.Mycobacteriophages are viruses that infect mycobacterial hosts such as  For example, it is not uncommon to find morons, segments of DNA present in one genome but absent from a related genome, which typically contain an open reading frame flanked by a promoter and a terminator Here we describe the sequence determination of eighteen new mycobacteriophage genomes isolated from geographically dispersed locations across the United States. The majority of these phages were isolated, sequenced, and annotated by freshman students in a structured and integrated education and research program supported by the Howard Hughes Medical Institute (HHMI) Science Education Alliance (SEA). Genomic comparison with previously described mycobacteriophage genomes reveals many new insights into mycobacteriophage diversity, evolution, and biological functions. First, we do not see any close correlation between genome type and geographical location or time of isolation. Second, it is evident that the mycobacteriophage population at-large remains under-sampled, because new singleton phages with genomes entirely unrelated to known phages – as well as new relatives of previously classified singleton genomes – can still be isolated. Third, the newly sequenced genomes provide insights into the mechanisms for genome variability including mycobacteriophage mobile elements (MPME), homing endonucleases and inteins. Lastly, we describe new insights into the basis of superinfection immunity among the Cluster A mycobacteriophages, and identify an unusual example of immunity theft.M. smegmatis mc2155 M. smegmatis lawns , and eleven were recovered after enrichment in liquid cultures of M. smegmatis . The isolation of Puhltonio and description of some of its characteristics has been reported recently Thirteen new mycobacteriophages were isolated at 12 different institutions in the autumn of 2008 as part of a freshman research course in phage discovery and genomics administered by the Howard Hughes Medical Institute (HHMI) Science Education Alliance (SEA) Nucleic acids were isolated from each of the 18 phages and all were shown to have dsDNA genomes. Complete genome sequences were determined either by shotgun Sanger sequencing or by 454 pyrosequencing. Sequence ambiguities and genome ends were resolved by direct sequencing of genomic DNA templates using oligonucleotide primers. Eight of the phages were shown to have circularly permuted genomes, and for annotation purposes the left end was defined as either codon one of the large terminase gene, or such as to correspond to the left end of closely related genomes . Ten of −50, the 9,014 predicted genes assembled into 2,345 phams, of which 1,108 (47.2%) are orphams (phams with only a single gene member). The largest pham contains 104 members, and the mean pham size is 3.84 (median is 2.0). The pham assignments are shown in All of the newly sequenced genomes were annotated for open reading frames (orfs), tRNA genes, and other features. All of the predicted protein-coding genes – together with those of the 62 previously annotated genomes – were used to assemble a database in the program Phamerator , which compares each of the predicted amino acid sequences with all others and sorts the genes into phams Each of the 18 newly isolated mycobacteriophages was examined by electron microscopy (data not shown). Sixteen of these have siphoviral morphologies—long flexible non-contractile tails—and two (ET08 and LRRHood) have myoviral morphologies—shorter contractile tails; all have isometric icosahedral heads, with the exception of Island3, which is prolate (data not shown). The length∶width ratio of the Island3 capsid is 2.5∶1, which is similar to those measured for mycobacteriophages of Cluster I, Brujita and Che9c Each of the newly sequenced genomes was compared with known mycobacteriophage genomes using the dotplot program Gepard Phages Eagle, Peaches, SkiPole and RedRock are all members of Cluster A as evident by nucleotide sequence comparison , but theIsland3 has close similarity with Cluster I phages , Fig. 2ATM4 was previously identified as a singleton phage with, by definition, no other closely related genomes. However, two of the new phages described here – Angelica and CrimD –have evident sequence similarity to TM4 , Fig. 2BattP site cannot be readily predicted from bioinformatic analysis. BlastN searches of regions close to the integrase gene identified no segments of nucleotide similarity to tRNA genes within the M. smegmatis chromosome. A number of smaller segments of sequence similarity (<20 bp) were identified, one of which may correspond to an attP common core, presumably reflecting integration into a non-tRNA attB site. LeBron encodes a number of genes of interest including two predicted Holliday-junction enzymes, one related to RusA (gp 76) and one an endonuclease VII-like protein (gp96) (LeBron is a singleton phage and thus presents a new genome type not closely related at the nucleotide level to any previously sequenced mycobacteriophages . It has n (gp96) .Among the LeBron putative virion structure and assembly genes, eight  are most closely related to predicted structural proteins of Wildcat . These include the major capsid subunit (gp8) and major tail subunit (gp13) proteins, which share a common ∼70 aa C-terminal extension, a property described previously for proteins of analogous function in both Bxb1 As the number of sequenced mycobacteriophage genomes has expanded over time, the collection has grown in unpredictable patterns. The majority (39 of 62) of previously described and genomically characterized mycobacteriophages were isolated in Pennsylvania, USA, but the distributed phage discovery education program implemented by the HHMI Science Education Alliance provides a geographically broad basis for isolating new phages, raising the question as to whether there are geographical constraints to the types of phages that are present and can be isolated with the commonly used procedures. The locations of the new phages' isolation are shown in An alternative explanation for non-random recovery of phage types is that there are temporal patterns of phage prevalence. We have therefore also examined the emergence and accumulation of mycobacteriophage isolates for which complete genome sequences have been determined . While aattB sites cannot be predicted readily for Peaches and SkiPole, but BlastN analysis suggests that the attB sites for Eagle and Hope are the host tRNA-gly (Msmeg_4767) and tRNA-Arg (Msmeg_6349) genes respectively, which are the known integration sites for L5 attB site for LeBron, and this is also true for Pumpkin and all of its Cluster E relatives .Nine of the newly sequenced phages encode integrases, of which two are serine-recombinases (Peaches and SkiPole), and seven are tyrosine-recombinases . Because of the small common core sequences used by serine-integrases M. smegmatis tmRNA (Msmeg_2093), strongly suggesting that the tmRNA gene provides the attB site for these phages to integrate. While the use of tmRNA genes for phage integration has been noted previously CrimD and Angelica encode very similar tyrosine integrases  whose closest relatives are those of the F1 subcluster phages (∼44% aa identity). BlastN analysis of the nucleotide sequence immediately upstream of these integrases shows that both phages contain a 23 bp sequence with identity to the 3′ half of the Brujita, Island3, and the previously described Che9c constitute Cluster I, with the closely related Brujita and Island3 forming Subcluster I1. These two genomes are very closely related with only about 100 nucleotide differences across the syntenic parts of the genomes, which extend across the entire genome with two notable exceptions near the right ends , Fig. S165 for Island3 gene 66 .Expansion of the A cluster and the addition of new Subclusters A3 and A4 provides insights into the superinfection immunity systems among these phages. The basis of immunity has been established previously in L5 (Subcluster A2) and Bxb1 (Subcluster A1) We have established the immunity patterns for the Cluster A phages that form stable lysogens . Two of Phylogenetic comparison of Cluster A repressors shows that they have generally evolved in concert with their overall genomes, with clades closely corresponding to cluster assignments . The A1 The repressor-DNA recognition systems of the Cluster A genomes are non-canonical because of the asymmetry of the recognition sites We have examined each of the A cluster phages and identified 23–36 potential binding sites in each genome . AlthougA comparison of the site sequences and the repressor helix-turn-helix motif reveals some interesting correlations, including some very apparent ones involving positions 2, 3, and 4 in the stoperator nucleotide sequences. For example, all of the sites contain a T at position 2 with the exception of the Cluster A2 phages, which have a G in that position . In the 43–46, all of which have homologues located in other mycobacteriophage genomes. The homologues of genes 45 and 46 are in Cluster F1 genomes, and gp43 has a single homologue that is present in the Cluster A1 phage KBG (gp86). Interestingly, LRRHood gp44 is a close relative to the repressor proteins of Cluster A phages discussed above, having only a single amino acid difference with Bxb1 gp69 ; there is strong experimental support for the repressor function of Bxb1 gp69 69 suggesting that it has been acquired recently in evolutionary time. However, none of the Cluster C phages have previously been shown to be temperate, and we have not been successful in isolating stable LRRHood lysogens; we note also that no Cluster C genomes encode either an integrase or other functions associated with prophage maintenance. The presence of a putative repressor gene of the Cluster A type is therefore highly unexpected.Two of the newly sequenced phages, ET08 and LRRHood are clearly members of Subcluster C1. Although LRRHood shares close similarity to other Subcluster C1 phages, it contains a 1.35 kbp segment that is absent from closely related genomes such as Cali . This se43–46 is flanked by 29 bp direct repeats , entered the genome adventitiously very recently and has not yet been removed by random deletion.We note that the data do not allow us to rule out an interesting alternative interpretation of gene http://www.neb.com/neb/inteins.html) showed that ET08 has at least five inteins within gene products gp3, gp79, gp202, gp239 and gp248, the most of any mycobacteriophage sequenced to date. ET08 gp3 and its homologues are predicted to encode nucleotidyltransferases, and the exteins are very closely related (92–96% amino acid identity). Two other Cluster C orthologs  contain a related intein, but it shares only 67% amino acid identity, suggesting that ET08 gp3 has a recent intein insertion. Two of the ET08 intein-containing proteins (gp202 and gp239) are nearly identical across their spans to homologues in other Subcluster C1 phages, although some of the related proteins are intein-less. ET08 gp202 shares 99% amino acid identity with intein-containing homologues in ScottMcG gp208, Spud gp208, Catera gp206 and Rizal gp206, and the exteins are 98–99% identical to the intein-less homologues Bxz1 gp204, LRRHood gp207, and Cali gp207; more distantly related versions of this intein are present in the unrelated genomes Omega gp11 and Kostya gp19 (49% and 39% identity respectively). ET08 gp239 shares 98% identity with Spud gp245, Rizal gp242, Cali gp243, LRRHood gp245, ScottMcG gp245 and Catera gp242, and the exteins are 99–100% identical to Bxz1 gp239.Several mycobacteriophage inteins, sequences able to splice out of a protein precursor, have been described previously, one of which has been shown to have novel biochemical activities Two of the ET08 inteins (in gp79 and gp248) appear to have been acquired recently and are absent from all other homologues in other phages . The extMycobacteriophage Hope is a member of Cluster G and all of the Cluster G phages, BPs, Halo, Angel, and Hope are extremely similar at the nucleotide sequence level 56, near the 5′ end  as well as an upstream ∼380 bp region that is non-coding, AT-rich, and likely contains control sequences. Fang contains an additional gene, 67, which is also of unknown function. The second group contains two genes of similar size to UncleHowie 64 and 65 (genes 65 and 66 in Colbert), as well as an upstream gene, 64; all are of unknown function. Interestingly, the sequence upstream of Colbert 64 also contains an AT-rich region that is consistent with a regulatory function  function . These m40, which is absent from other Cluster E genomes at that syntenic location  – in which freshman undergraduate students at 12 colleges and universities engaged in phage discovery and genomics. The other six phages were isolated within the Phage Hunters Integrating Research and Education (PHIRE) program at the University of Pittsburgh 4, 1 mM CaCl2, 68.5 mM NaCl), and M. smegmatis mc2155. The soil extract was filtered through a 0.22 µm filter and 50 µl of this sample was plated with 0.5 ml late-exponential-phase M. smegmatis mc2155 in 4.5 ml 0.35% mycobacterial top agar (MBTA) with 1 mM CaCl2. Phages isolated through the SEA Alliance were either found through direct plating or by enrichment cultures. Phages isolated by direct plating were found using the same protocol as PBI. Phages found via enrichment were isolated as follows: approximately one gram of a soil sample and 5 ml of late log/early stationary phage M. smegmatis mc2155 was incubated in 50 ml of 7H9 plus 1 mM CaCl2 at 37°C shaking for 24 hours. Any remaining cells were then pelleted by centrifugation, and the supernatant was filter-sterilized through a 0.22 µ filter. The sterilized filtrate was serially diluted with phage buffer and plated as above with MBTA and M. smegmatis mc2155. For both direct plating and enrichment plates, the MBTA/phage/bacterial mixture was distributed evenly on a plate of 7H10 agar (Difco) supplemented with carbenicillin, cycloheximide, 1 mM CaCl2 and 10% albumin dextrose complex (ADC). Plaques were picked into phage buffer and replated with bacteria for several rounds of infection to purify phage isolates. After several rounds of purification, high-titer phage stocks were prepared at PBI using CsCl gradients and ultracentrifugation . DNA from SEA Alliance phages was harvested by treating 10 ml of a filtered phage crude lysate (about 109 plaque forming units (pfu)/ml) with RNaseA and DNaseI for 30 min at 37°C followed by a 60-minute incubation at room temperature. Intact particles were then precipitated with 30% polyethylene glycol (PEG) 8000/3.3 M NaCl overnight at 4°C and then pelleted by centrifugation at 10000 g for 20 min. DNA was extracted from the phage pellet using the Promega Wizard DNA clean-up kit as per manufacturer's instructions.All phages isolated at the Pittsburgh Bacteriophage Institute (PBI) were isolated by co-plating of soil extracts, prepared with Phage Buffer , G Browse −50, are different to those used previously, and were derived by optimizing pham assembly over a range of possible values . GenBank accession numbers are shown in At PBI, double-stranded DNA was phenol extracted from the dialyzed CsCl banded phages, and pyro-sequenced at the University of Pittsburgh Genomics and Proteomics Core Laboratories (UPGPCL) as described previously At the University of Pittsburgh, electron microscopy was performed by placing a suspension of virion particles on a grid with a carbon-coated nitrocellulose film, staining with 2% uranyl acetate, and examining the grid in an FEI Morgagni 268 transmission electron microscope equipped with an AMT digital camera system. Schools participating in the SEA program used locally available electron microscopy resources to obtain micrographs.Figure S1Genome maps of 18 newly sequenced mycobacteriophage genomes. Genome maps for each of the 18 newly sequenced mycobacteriophage genomes were generated using Phamerator. The order of the genomes from top to bottom is according to cluster, numerically[#alphabetically?] from A to singleton. Genes are represented as colored boxes above (rightwards transcribed) or below (leftwards transcribed) each genome, and are colored according to their pham assignation. The pham number is shown above or below each gene with the number of pham members in parentheses. The nucleotide similarity between adjacently displayed genomes is represented by the coloring between genomes, with the strength of the relationship represented according to the color spectrum, with violet being the most similar.(PDF)Click here for additional data file.Figure S2Nucleotide sequence comparison of 17 Cluster A mycobacteriophage genomes. Nucleotide sequences of all 17 Cluster A genomes were concatenated into a single file and compared against themselves using Gepard (PDF)Click here for additional data file.Figure S3Annual changes in types of isolated mycobacteriophages. A. Annual changes in the total accumulated numbers of sequenced mycobacteriophages are shown by cluster. Date reflects date of isolation. The accumulated numbers of subclusters within Cluster A (B) and Cluster B (C) are also shown. Some weak patterns in phage isolation are evident. For example, by 2004 there were seven Subcluster C1 phage genomes, but then no more were isolated until 2008. Likewise, by 2006 there were more A1 subcluster genomes than any other, but no new ones have been isolated since then. In contrast, the number of Subcluster B1 phages more than doubled during this period. Because of the high genetic diversity of the mycobacteriophage population, the sizes of each of the cluster or subcluster groups is small and statistically significant numbers of phages will not be available until there is a 5 to10 –fold increase in the total number of sequenced mycobacteriophage genomes. Subtle changes in isolation methods – including enrichment versus direct plating – could influence the types of phages recovered, as well as changes at the population level.(PDF)Click here for additional data file.Figure S4Immunity patterns of Bxb1, DD5, Jasper, SkiPole, Che12 and L5. Ten-fold serial dilutions of phages were spotted onto either a non-lysogen or a lysogen as indicated, and incubated. Che12 and L5 are homoimmune, and Bxb1, DD5, Jasper, and SkiPole are homoimmune.(PDF)Click here for additional data file.Figure S5Derivation of consensus sequences for Cluster A genome stoperator sites. The 13 bp consensus stoperator sequence for each of 16 Cluster A phages was derived by searching for related sequences in each genome. The base distribution of each of the 13 positions is shown for each genome, with the consensus shown below. A single consensus base was assigned if it occurred in greater than 85% of the sites, and if not then standard abbreviations are used, W: A or T; D: G, T or A; V: G, C or A; M: A or C; N: anything. All sites listed have no more than two deviations from the consensus. For L5, the sites are the same as those described previously (XLSX)Click here for additional data file.Table S1Author contributions.(XLSX)Click here for additional data file.Table S2Pham assignment table.(XLS)Click here for additional data file.


Next, we identified NADPH oxidase as the primary source of ROS in atherosclerotic lesions. Treatment of aortic segments from AS mice with apocynin, which interferes with NADPH oxidase activation in part by preventing translocation of the subunit p47phox, significantly reduced superoxide levels. Moreover, addition of apocynin to the drinking water of AS mice produced a decrease in lesion size as compared to untreated AS mice, with the effect most pronounced in the thoracoabdominal aorta but absent from the aortic arch. Granulocyte function in AS+apocynin mice was suppressed, confirming efficacy of apocynin treatment. We conclude that apocynin attenuates the progression of atherosclerosis in hypercholesterolemic mice, potentially by its ability to inhibit generation of superoxide by NADPH oxidase.Of the multiple sources of reactive oxygen species (ROS) in the blood vessel, NADPH oxidases are the primary source. Whereas several studies have implicated NADPH oxidases in the initiation of atherosclerosis, their roles in disease progression are incompletely understood. Our objective was to determine the potential clinical relevance of inhibiting NADPH oxidase in established atherosclerosis. Using a hypercholesteremic murine model of atherosclerosis (ApoE The mechanisms of atherosclerosis are complex, involving initial events of transendothelial migration of inflammatory cells into the vascular wall and formation of fatty streaks and later events that include vascular cell activation and more complex lesion development. The oxidative modification hypothesis implicates reactive oxygen species (ROS) in the pathogenesis of atherosclerosis. As such, lowering of vascular wall ROS should reduce clinical complications associated with atherosclerosis. However, the disappointing results of antioxidant trials in limiting cardiovascular events in humans have highlighted the importance of targeting the enzymatic source of ROS.phox (or its homolog NoxO1), and p67phox (or its homolog NoxA1). The expression of several of these subunits is elevated in vessels from humans with coronary atherosclerosis [Several studies have implicated NADPH oxidase in the development and early progression of atherosclerosis. NADPH oxidase is a primary source of superoxide in the blood vessel, particularly in disease states. NADPH oxidases are multi-subunit complexes that include several Nox homologs that serve as the site of electron transfer from NADPH to molecular oxygen to generate superoxide. Of the seven Nox isoforms, only Nox1, Nox2, Nox4 and Nox5 are expressed in the blood vessel, and they differ in their cell-specific expression, mode of activation, subunit requirements, and function [phox [Studies investigating of the role of NADPH oxidases in atherosclerosis have primarily utilized genetic models with deficiency or overexpression of a particular subunit –14. For phox ,9, Nox1 phox , or Nox2phox  but not phox  attenuatThe objective of our study was to determine whether pharmacologic inhibition of NADPH oxidase activity after the development of atherosclerosis either blocked progression or induced regression. In this study, we identify a time-dependent increase in plaque area and ROS levels in the aorta of hypercholesterolemic mice. Treatment with the NADPH oxidase inhibitor apocynin beginning at 17 weeks of age and continued for an additional 17 weeks reduced vascular ROS and attenuated lesion progression.−/− LDLR−/− atherosclerotic (AS) mice on normal chow, which have total cholesterol levels of 600–800 mg/dL . Moreover, treatment with apocynin significantly decreased superoxide production in the AS aortic segments (phox to the membrane (We next determined the enzymatic source of the increased vascular superoxide in AS by incubating aortic segments from AS mice with inhibitors of mitochondria (rotenone), xanthine oxidase (oxpurinol), arachidonic metabolism (indomethacin), and flavo enzymes (dipheneylene iodonium/DPI). As compared to vehicle control, treatment with polyethylene glycol-superoxide dismutase (PEG-SOD) abolished superoxide production as detected by dihydroethidium (DHE) fluorescence , confirmal layer , consistsegments . In the membrane , consistphox or Nox2 reduces lesion size in the aortic sinus of hypercholesterolemic mice [We next examined whether treatment with apocynin after the development of atherosclerosis in mice either blocked progression or induced regression by treating mice with apocynin beginning at ~17 weeks of age until ~35 weeks of age. Apocynin had no effect body weight or total cholesterol levels . Treatmemic mice ,10,11.in vivo, we examined the respiratory burst activity of blood leukocytes. There was marked reduction in respiratory burst activity of leukocytes collected from apocynin treated mice with only 475 ± 229 leukocytes increasing superoxide generation in response to PMA compared to 2419 ± 420 leukocytes in control mice  of either sex and ranging in age from 15 to 45 weeks of age. C57BL/6 mice (Jackson Laboratories) were used as controls and were studied at 30 weeks of age. All mice were maintained with normal diet  throughout the study. In some studies, apocynin (3 mM) was added to the drinking water of mice starting at ~17 weeks of age and were maintained on apocynin for a duration of ~17 weeks. Age matched AS mice maintained on regular drinking water were used as controls. Mice were sacrificed between the ages of 15 and 45 weeks by injection of pentobarbital . The aortic arch, thoracic and abdominal aortae were removed, placed in Krebs buffer (4 °C) and dissected free of loosely adhering tissue. Arches were then incubated in phenol red free Dulbecco’s Modified Eagle’s Medium (DMEM) for approximately 1 h at 36 °C before performing lucigenin-enhanced chemiluminescence as described below. After chemiluminescence assays, arches were prepared for lesion size assessment by oil red O staining. In other experiments, aortae were processed for membrane fraction as described below.All experiments were performed within the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals, and the protocols approved by the Animal Care and Use Committee at the University of Iowa. The atherosclerotic mice used in this study were apolipoprotein E and low density lipoprotein receptor deficient mice  by enzymatic assay (Boehringer Mannheim).Aortic segments (4 mm) were rinsed in cold PBS and flash-frozen in OCT as previously described . SectionSuperoxide production was measured in aortic arches by lucigenin-enhanced chemiluminescence. Vessel segments were incubated in phenol red free DMEM for approximately 1 h at 36 °C. In some experiments, segments were treated with apocynin . Segments were then placed in cuvettes with 0.5 mL phosphate buffered saline (PBS) and lucigenin (25 μM). After a 1.5 min delay time, measurements were taken every 30 seconds for 5 min on a Monolight luminometer. Measurements were reported as emitted light in relative light units (RLU) and were averaged across the 5-minute reading time. Data were normalized to the surface area for each vascular segment, which was obtained with NIH Image J.g for 1 h at 4 °C. The pellet was resuspended in RIPA buffer supplemented with protease inhibitor cocktail, followed by Western blotting with anti-p47phox . The supernatant was blotted with anti-GAPDH.Aortic segments were incubated in phenol red free DMEM for 1 h at 37 °C, followed by treatment with apocynin (1 mM) for 60 min, then cytokine mix  to activate NADPH oxidase for an additional 10 min. Aortae were flash frozen in liquid nitrogen, homogenized, sonicated, and centrifuged to remove debris. Supernatants were then centrifuged at 100,000× The staining of lipids was performed by oil red O. Following chemiluminescence measurements, the aortic arch, thoracic and abdominal aortae were cut longitudinally and pinned to lay flat. Tissue was then fixed in 7% formaldehyde, rinsed in 60% isopropanol for 30 seconds, and stained in oil red O for 10 min. Tissue was then rinsed in 60% isopropanol briefly and then washed in running water for 2–3 min. Hematoxylin was used as a counter stain. NIH Image J was used to assess the area of the atherosclerotic lesion. Lesion size was calculated as a ratio of oil red O positive aortic tissue to total surface area.phox (1:100) and p22phox (1:100) polyclonal antibodies and appropriate secondary antibodies as previously described [Aortic sections (8 μm thick) were washed with PBS and treated first with normal horse serum, and then with p47escribed . SectionLeukocyte respiratory burst activity was examined as previously described . ApproxiGranulocyte function in mice was assessed in a nitroblue tetrazolium (NBT) slide test in a modified protocol as previously described . In the p value < 0.05 was considered statistically significant.Data are presented as mean ± standard error. Statistical significance was assessed by one-way ANOVA with Tukey’s or Dunnett’s post-test analysis. A In conclusion, our data add to the existing literature, which has primarily relied on genetically modified mice, by demonstrating that a pharmacologic inhibitor of NADPH oxidase can attenuate atherosclerosis. Furthermore, our data extend the role of NADPH oxidases in atherogenesis by revealing that inhibiting NADPH oxidase after disease development is sufficient to limit progression.


LASS2 and GALNT1 were present in cancer patients, while ARHGEF39 and FOXO3 were found only in non-cancer urinary vesicles. Previous studies have pointed to the involvement of those genes in tumour progression and metastasis.Bladder cancer is one of the most common cancers and, together with prostate carcinoma, accounts for the majority of the malignancies of the genitourinary tract. Since prognosis ameliorates with early detection, it will be beneficial to have a repertoire of diagnostic markers that could complement the current diagnosis protocols. Recently, cell-secreted extracellular vesicles have received great interest as a source of low invasive disease biomarkers because they are found in many body fluids, including urine. The current work describes a pilot study to generate an array-based catalogue of mRNA associated to urinary vesicles, and also a comparison with samples obtained from bladder cancer patients. After an analysis of presence/absence of transcripts in bladder cancer EVs, a list of genes was selected for further validation using PCR technique. We found four genes differentially expressed in cancer samples.  Cancer of the urinary bladder is among the five most common malignancies in the Western world ,2 and thin situ. Cytology is a non-invasive technique with high specificity (90%–96%) [The current standard of care for the detection of bladder cancer is based on flexible cystoscopy, usually combined with urine cytology. Cystoscopy is an invasive technique with a high sensitivity 91%) , althoug90%–96%) , but it 1% , alth90%–96%) ,11.The study of extracellular vesicles (EVs) has emerged in the last years as an alternative to identify non invasive biomarkers. Urine contains EVs  and, impg for 10 min, filtered through a 0.22 μm pore membrane, and immediately frozen at −80 °C. The samples were classified as “cancer” when both cystoscopy and subsequent histological analysis were positive. Meanwhile, the control group included samples with negative results for both cystoscopy and cytology analyses.The samples were obtained from patients with different urinary tract conditions, suffering different symptomatology, such as haematuria, dysuria or frequent urination, and were cited for exploratory cystoscopy. The study follows all the ethical regulations and was approved by the Basque Ethical Committee for Clinical Research (identification number CEIC 09-27). Previous to sample collection, patients were informed about the study and requested to sign the informed consent. For each sample, all the urine was collected by spontaneous micturition previous to cystoscopy, centrifuged at 2,000 × g for 10 min, and then ultracentrifuged at 100,000 × g for 75 min. The pellet was re-suspended in 150 uL of PBS and kept at −80 °C until processed. To purify EVs from urine, the stored samples were thawed, centrifuged at 2,000 × Size distribution within EVs preparations was analyzed by measuring the rate of Brownian motion using a NanoSight LM10 system , which is equipped with a fast video capture and Nano-particle Tracking Analysis (NTA) software. NTA post-acquisition settings were kept constant for all samples, and each video was analyzed to give the mean, mode, and median vesicle size, as well as an estimate of the concentration . For cryTotal RNA isolation was achieved by RNeasy columns . Their integrity, size and quantification were evaluated in RNA Pico Chips with a Bioanalyzer . ® System, Epicentre (Cat. Num. TAN07924) and subsequent amplification, labeling and hybridization were performed according to Whole-Genome Gene Expression Direct Hybridization Illumina Inc.’s protocol. Raw data were extracted with GenomeStudio analysis software (Illumina Inc.) in the form of GenomeStudio’s Final Report (sample probe profile).Whole genome expression characterization was conducted using Human HT12 v4 BeadChips . cRNA synthesis was obtained with TargetAmp™ Nano-g™ Biotin-aRNA Labeling Kit for the IlluminaIn the R statistical computing environment, using the lumi Bioconductor package , raw datp-values per probe and sample. We regarded a transcript as significantly present in a sample if its corresponding probe’s p-value was lower than 0.01. For the assessment of differentially detected transcripts, a linear model was fitted to the data and empirical Bayes moderated t-statistics were calculated using the limma package from Bioconductor [For the calling of present transcripts, GenomeStudio provides detection onductor .cDNA was synthesized from 0.1–1 ng of RNA using Superscript III (Invitrogen) following the manufacturer’s recommendations. Primers were designed using Primer3 , and theA total of five urine samples from bladder cancer patients  and six samples from non-cancer patients  were characterized by whole transcriptome arrays. An additional three cancer  and five control  samples were considered for the validation step. We isolated urinary vesicles from urine samples and characterized them by electron microscopy and particle tracking analysis applying NTA techniques. Purified EVs observed under electron microscopy offered a heterogeneous morphology A. AccordWhole genome expression analysis detected 4,102 transcripts across the studied samples, which is about one tenth of the probes tested by the Human HT12 v4 BeadChips (Illumina Inc.). The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE51843) We observed a high variability among samples, with a very low correlation among them , even wip-value of 0.017. The analyzed samples share few common genes, even within groups  than in the cancer samples . The corresponding Mann-Whitney test resulted in a oups see A. Specifoups see B and onloups see . As expeoups see . Out of oups see .In order to validate the array results by PCR, we collected a new set of samples and designed primers for a group of transcripts selected according to different criteria, such as whether they were differentially present between groups, within a wide range or intensities in the array, and also some genes that were previously associated with cancer according to CaGE website  Supplem. We obseLASS2 and GALNT1 were only present in samples from cancer group, meanwhile ARHGEF39 (in the array named Chromosome 9 open reading frame 100 (C9orf100)) and FOXO3 were absent in cancer group and could be detected in control group. The amplification obtained by PCR was visualized in an electrophoresis gel and presented in Among the tested genes, four genes were present in one group and absent in the other. g. Instead of applying intermediate steps to release EVs from the protein complex trap [28S ribosomal gene in all the samples by PCR. The presence of ribosomal fractions and the enrichment in short RNAs both were observed in urinary EVs purified from voided urine [The amount of total RNA we isolated from EVs was very low, ranging from 50 pg to 30 ng, even though we employed a commercial kit that has been reported to maximize the yield of mRNA . We shoulex trap , we sedilex trap ,33. The ed urine . HoweverAccording to Vesiclepedia , the onlWe have observed great variability among samples, and only 4 transcripts were present in all the urine EVs analyzed by array analysis. In addition, there was a lack of correlation both inter and intra-group see , reflectFOXO3, GALNT1, ARHGEF39 and LASS2, whose presence in urine differs between cancer and non cancer patients. We amplified ARHGEF39 and FOXO3 transcripts in non-cancer samples but we could not find them in EVs from cancer patients. ARHGEF39 is a member of the Dbl-family guanine nucleotide exchange factors (GEFs), which are important activators of Rho GTPases [FOXO3, absent in cancer samples, has previously been associated with tumor suppression. Furthermore, it has been described the role of FOXO3 in cancer and cell proliferation [FOXO3 mRNA expression was lower in cancerous versus non cancerous bladder tissues [The results point to four genes, named  GTPases . FOXO3, feration ,37, and  tissues . GALNT1 and LASS2 only in EVs obtained from cancer patients. Both genes have been reported to be involved in bladder cancer progression [GALNT1 has an important role in transforming growth factor signaling [LASS2. Different reports correlate LASS2 expression with the degree of invasion and recurrence in carcinomas [On the contrary, we have found gression ,40. GALNignaling , and it ignaling .A similarcinomas ,44. The reduced number of samples and the high variability in the transcripts detected for each sample reduce the possible biological meaning obtained from this pilot study. Although the causes for this elevated variability remain unclear and further research is needed to unmask them, our results highlight that certifying biomarkers for disease out of the RNA-content in urinary vesicles will require big cohorts of patients. In summary, our work offers a catalogue of transcripts that can be found in urinary EVs. Furthermore, the observed differences between samples from patients with and without bladder cancer suggest that EVs contain valuable information for diagnosis and prognosis. Whether the combination of these four genes may be as useful for diagnosis and prognosis as other genetic signatures obtained from urinary sediment in bladder cancer ,40,45, w


Quercus montana (CO) and white oak Q. alba (WO) (from North America), and oriental cork oak Q. variabilis (OO) and Mongolian oak Q. mongolica (MO) (from Asia) can escape predation and successfully establish from only taproots. During germination in autumn, cotyledonary petioles of acorns of CO and WO elongate and push the plumule out of the cotyledons, whereas OO and MO extend only the hypocotyls and retain the plumule within the cotyledons. Experiments showed that the pruned taproots (>6 cm) of CO and WO acorns containing the plumule successfully germinated and survived, and the pruned taproots (≥12 cm) of OO and MO acorns without the plumule successfully regenerated along with the detached acorns, thus producing two seedlings. We argue that these two distinct regeneration morphologies reflect alternative strategies for escaping seed predation.Early germination of white oaks is widely viewed as an evolutionary strategy to escape rodent predation; yet, the mechanism by which this is accomplished is poorly understood. We report that chestnut oak  Quercus), in which there is strong evidence for a complex ecological and evolutionary relationship between acorns and rodents that act as both seed predators and keystone dispersal agents of these fruits , in contrast to those of red oaks (subgenus Lobatae), exhibit no dormancy and germinate immediately at or even before seed fall, rapidly producing robust taproots   (S. carolinesis) shows a strong innate tendency to store red oak acorns and excise those of white oak (Tamias sibiricus) repeatedly use their incisors to prune acorns of Quercus mongolica from anchored radicles to arrest or postpone germination, and that other rodents in NA, including the eastern gray squirrel (S. carolinensis), similarly prune Quercus montana acorns from well-developed radicles (Xerospermophilus spilosoma) in central Mexico .Much of this rodent–oak relationship centers on specific acorn characteristics and rodent responses (some of which are heritable) to these acorn traits . For exataproots , 2006. Ttaproots . Althougtaproots , 2007, staproots . Moreove. niger)  in NA anidianus) , 2010 exidianus) . At leashite oak . ElsewheQ. montana[Q. alba, hereafter CO and WO, respectively) elongate and extend well beyond the apical end of the acorn to the point at which the plumule (embryonic stage of the epicotyl) and radicle diverge, resulting in a separation of the two by 1–2 cm , autumn germination of acorns is instead characterized by elongation of the hypocotyls (≍1 cm), which results in the plumule and cotyledonary petioles remaining inside the acorn  in an oak forest in central China , by fastening a plastic tag on each of the taproots of the germinating acorns. All acorns had taproots penetrating >10 cm into the soil. Similarly, in early October 2010, we tagged 100 MO acorns (20 per transect) in an oak forest in northeast China , and in November 2010, we tagged 100 CO acorns (20 per transect) in an oak forest in northeast Pennsylvania  . All three field studies were conducted during years of unusually high acorn mast. In each survey, tagged acorns were checked the following spring and examined for evidence of pruning by rodents and germination success.We collected acorns of WO, OO, and MO from the field and conducted germination experiments on both the acorns and taproots following simulation of taproot pruning. Taproots of each of these three species collected from the field were categorized into six groups according to the length of taproots after pruning: 2, 4, 6, 8, 12, and 16 cm. We imitated rodent pruning by detaching the taproots above or below the plumule from the acorns of WO, and by cutting the taproot at the apical end of the acorn where the radicle emerges for OO and MO acorns. Parallel experiments were conducted on CO as part of a more extensive study on CO pruning . For CO, one individual pruned acorn or taproot of each length was planted into 20 replicate pots (diameter × height = 15 cm × 20 cm) containing organic composite soil. For WO, 18 pruned acorns or taproots of each length were planted randomly into plastic trays (length × width × height: 4 cm × 4 cm × 10 cm). For OO and MO, 30 pruned acorns and 30 taproots of each length were planted into five replicate pots (diameter × height = 40 cm × 20 cm).Q. rubra, hereafter RO) acorns following cold stratification (storage at 4°C for at least 90 days) and initial germination. We separated cotyledons (with the plumule) from the taproots (without the plumule) of RO seedlings of various lengths . One individual taproot of each length and cotyledons containing the plumule were planted per pot (diameter × height = 15 cm × 20 cm) and these six treatments were replicated 10 times. We did not test the viability of cotyledons without the plumule and taproots with the plumule, because RO acorns exhibit different germination morphology from WO acorns; that is, the plumule does not emerge until the taproot reaches 8–10 cm . Therefore, we removed the cotyledons of RO at different stages of seedling development  to determine whether seedlings can establish without cotyledons. Each of these treatments was replicated 12 times. Pots and trays were kept in the laboratory and cultured at room temperature; acorns and taproots were watered regularly and measured at regular intervals after planting. Seedling survival rates were determined 8 weeks after planting.For comparison, we removed the taproots of northern red oak (n= 16) and 48% (n= 13) of pruned taproots of OO and MO regenerated into seedlings, respectively; while 64% (n= 7) of CO pruned taproots with the plumule regenerated into seedlings in the field , implying a threshold response to nutritional reserves in the pruned taproots. Although taproots without the plumule successfully regenerate into seedlings, white oak species in Asia require a higher threshold level of stored reserves in taproots. These results demonstrate the importance of nutritional reserves in the pruned taproots in supporting seedling establishment of both NA and Asian oaks.Q. aliena, Q. aliena var. acuteserrata, Q. acutissima, and Q. liaotungensis) throughout Asia appear to show a similar germination pattern to OO and MO, in which the plumules are retained in the acorns, but hypocotyls (1–2 cm) are extended out of the cotyledons following autumn germination. Likewise, the formation of cotyledonary petioles (1–3 cm) and the extension of taproot containing the plumule are evident in several other white oaks from NA . Cotylethe soil , furtherna in NA ; and posronments , it alsoIndeed, the geographic ranges over which these two germination strategies occur suggest far more than an exclusive response to environmental conditions. We suggest that these two germination morphologies represent independent strategies for dealing with rodent predation, and that the process of rodent pruning and subsequent establishment of oak seedlings from taproots in NA, and from both taproots and acorns in Asia, is a significant mechanism contributing to oak dispersal and regeneration.


The discovery of adrenal incidentalomas due to the widespread use of sophisticated abdominal imaging techniques has resulted in an increasing trend of adrenal gland specimens being received in the pathology laboratory. In this context, we encountered three uncommon adrenal incidentalomas.The aim of this manuscript is to report in detail the three index cases of adrenal incidentalomas in the context of a 13-year retrospective surgical pathology review.The three index cases were investigated and analyzed in detail with relevant review of the English literature as available in PubMed and Medline. A 13-year retrospective computer-based histopathological surgical review was conducted in our laboratory and the results were analyzed in the context of evidence-based literature on adrenal incidentalomas.A total of 94 adrenal specimens from incidentalomas were identified, accounting for 0.025% of all surgical pathology cases. In all 76.6% were benign and 23.4% were malignant. A total of 53 females (56.4%) and 41 males (43.6%) aged 4 to 85 years were identified. The benign lesions included cortical adenoma (43.1%), pheochromocytoma (29.3%) and inflammation/fibrosis/hemorrhage (8.3%). Metastatic neoplasms were the most common malignant lesions (50%) followed by primary adrenocortical carcinomas (31.8%) and neuroblastoma (13.6%). These cases were discovered as adrenal incidentalomas that led to surgical exploration.The three index cases of adrenal incidentalomas with unusual pathologies were encountered that included (a) adrenal ganglioneuroma, (b) periadrenal schwannoma and (c) primary adrenal pleomorphic leiomyosarcoma. These cases are discussed, with a literature and clinicopathological review.Adrenal lesions are uncommon surgical specimens in the pathology laboratory. However, higher detection rates of adrenal incidentalomas aided by the ease of laparoscopic adrenalectomy has resulted in increased adrenal surgical specimens leading to unsuspected diagnostic and management dilemmas. Accurate pathological identification of common and uncommon adrenal incidentalomas is essential for optimal patient management. At autopsy, adrenal masses are among the most common tumors, reported in 3% of the population over 50 years of age ,2. Such The aim of this manuscript was to review and document the trend of adrenal specimens received in our surgical pathology laboratory over a 13-year period with special emphasis on three uncommon pathologies of adrenal ‘incidentalomas’ that were encountered, including ganglioneuroma, periadrenal schwannoma, and primary pleomorphic leiomyosarcoma.Pathological diagnosis of three unusual lesions resulting from the analysis of adrenal incidentaloma specimens prompted a detailed review of all adrenal lesions over the past 13 years (1997 to 2010) available in the Laboratory Information System (LIS). The LIS, operational since 1996, is a robust, reliable, centralized electronic data repository, which is administered by the Saskatoon Health Region for the Department of Pathology and Laboratory Medicine in the city of Saskatoon, Canada. All surgical material was retrieved and analyzed to confirm the histomorphological diagnosis. Histopathological review and clinicopathological correlation with the recorded patient information, including sex, age, laterality, and final diagnosis, was conducted.A complete review using the keywords ‘adrenal’ with ‘ganglioneuroma’, ‘schwannoma’, and ‘leiomyosarcoma’ was completed within the scope of the English language as available in PubMed/Medline databases. Primary references were read and analyzed, and their reference lists were used to retrieve secondary sources. PubMed’s ‘Related Articles’ feature was an additional tool in the identification of relevant articles. Additionally, a literature review on the current status of adrenal incidentalomas was undertaken.This study was conducted with ethics approval from the University of Saskatchewan Biomedical Research Ethics Review Committee.The surgical pathology review of 381,200 cases over the 13-year period (1997 to 2010) retrieved 94 adrenal lesions (incidence of 0.025%). Patient ages ranged from 4 to 85 years, with a mean of 51.6 years (median 55 years). The majority (54%) were between the ages 40 to 60. Fine-needle aspiration specimens were rare (4/94). Females (56.4%) had a slightly higher preponderance than males (43.6%). There was a slight predominance for left-sided lesions, with 55 (58.5%) pathologies detected in the left adrenal gland and 39 (41.5%) in the right in our series. Over three-quarters (76.6%) of the pathologies diagnosed were benign  with renal cell carcinoma as the commonest site of origin  followed by lung adenocarcinoma  and solitary cases  of colorectal adenocarcinoma, gastroesophageal adenocarcinoma, B cell lymphoma and urinary bladder carcinoma. Primary malignant lesions included seven adrenocortical carcinomas , three neuroblastomas  and one pleomorphic leiomyosarcoma   scintiscan, which were negative, ruling out pheochromocytoma. Urinary free cortisol and demonstration of a normal diurinal rhythm of cortisol secretion by the administration of the dexamethasone suppression test were also negative ruling out subclinical cortical functional tumors. A laparoscopic left adrenalectomy was carried out. Her recovery was uneventful, and she was discharged 48 h later.The left adrenal tumor was firm, tan, non-encapsulated and measured 3.5 × 2.2 × 2.4 cm in maximum dimension. On cross-sectioning, the mass was well demarcated with a stretched rim of normal-appearing adrenal tissue on either side Figure . On micrA 64-year-old non-smoker with uncontrolled hypertension (average 220/90 mmHg), poorly controlled type II diabetes, atrial fibrillation, depression and bilateral idiopathic blepharospasm underwent abdominal CT scanning for the investigation of abdominal wall swelling. A soft tissue mass on the medial aspect of the left adrenal gland measuring 3.1 × 2.7 × 4.1 cm was visualized incidentally. No additional mesenteric or retroperitoneal lymphadenopathy was identified, and the right adrenal gland, kidneys, spleen, and pancreas appeared normal. A renal Doppler ultrasound confirmed enlargement of the left adrenal gland, and further imaging with CT and MRI showed a solid enhancing mass with imaging features inconsistent with a benign adenoma. Based on his elevated blood pressure, and a 1-month history of biweekly episodes of sweating that lasted 5 minutes without associated chest pain, headache, shortness of breath or palpitations he was clinically suspected to have a pheochromocytoma. At two examinations, urine catecholamines were modestly elevated though metanephrines, vanillyl mandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were normal on repeated testing. Due to the larger size of the tumor (4 to 4.5 cm), he underwent surgical exploration with a left laparoscopic radical adrenalectomy. Postoperatively his blood pressure remained stable and as there were no complications, the patient was discharged in 48 h.The specimen weighed 65 g and consisted of the left adrenal gland wrapped in mature adipose tissue. A large nodule was seen protruding adjacent to the adrenal gland, measuring 3.8 × 2.8 × 3.5 cm. Cut sections of the specimen revealed a well demarcated, fibrous mottled whitish-yellow lesion that grossly appeared to be directly adjacent to the adrenal gland with no direct involvement. On microscopic examination, the nodule was composed of (a) hypercellular areas of spindle cells with abundant eosinophilic cytoplasm, indistinguishable cell borders, and wavy nuclei with focal palisading areas Figure  and (b) A 28-year-old female who smoked half a pack of cigarettes a day with no significant medical history was being investigated for abdominal pain. A CT scan was ordered, which revealed a large left adrenal mass adherent to the kidney. Preoperative investigations including hormonal studies to evaluate catecholamine excess by the assessment of plasma metanephrines, urinary catecholamines and a MIBG scintiscan were negative, ruling out pheochromocytoma. Urinary free cortisol and demonstration of a normal diurinal rhythm of cortisol secretion by the administration of the dexamethasone suppression test were also negative ruling out subclinical cortical functional tumors. The larger size of the mass was highly suspicious for malignancy; therefore an open surgical intervention was planned. At surgery, via a left thoracoabdominal incision an en-bloc resection of the left adrenal mass was carried out including left nephrectomy, partial diaphragmatic resection with reconstruction and placement of a left thoracostomy. Postoperatively, the only significant complication was an asymptomatic left-sided pneumothorax that resolved spontaneously upon removal of the chest tube. She was discharged with an outpatient referral to the Cancer Center in 6 weeks. No further postoperative information is available for review in the current health record system.The gross specimen weighing 1,492 g consisted of the left kidney and adrenal gland, with portions of the diaphragm. A large 16.5 × 13.5 × 12.5 cm ragged, irregular, pale-tan rubbery multinodular mass with hemorrhage and necrosis was seen completely replacing the left adrenal gland with no residual normal tissue being identified. On microscopic examination, the tumor was surrounded by a pseudocapsule distinct from the adjacent renal parenchyma Figure  and compIn situ hybridization for Epstein-Barr virus was negative. A diagnosis of primary pleomorphic leiomyosarcoma was suggested due to the cells being strongly positive to SMA. The final diagnosis of high-grade primary pleomorphic leiomyosarcoma of the adrenal gland was confirmed with corroborative external consultation due to the rarity of this lesion.Immunohistochemical examination showed the neoplastic cells to be strongly positive for vimentin and smooth muscle actin (SMA) Figure  with negImprovements in non-invasive sophisticated abdominal imaging techniques coupled with their growing popularity and acceptance have resulted in the increasing detection of clinically silent adrenal lesions, ‘incidentalomas’ . Such trin vivo functional characterization of the adrenal glands due to the uptake and accumulation of the radio tracer. In this context, MIBG and N-59 scans for functioning adrenal medullary and cortical tissues, respectively, are undertaken to rule out any subclinical medullary/cortical functional tumors.An optimal diagnostic approach for the management of adrenal incidentalomas has yet to be determined; however, certain recommendations have been proposed ,2,10. A et al., preoperative CT and MRI tend to significantly underestimate the actual histological size of adrenal tumors larger than 3 cm by 18.1% and 20%, respectively. In this context, it is recommended that three-dimensional reconstruction of spiral CT or MRI images may overcome this problem of size underestimation [Preoperative radiological imaging including CT scans and MRI are used to determine size of the adrenal tumor, which dictates the need for surgical intervention and influences the surgical approach employed by the surgeon. However, as seen in the study by Kouriefs timation ,13. CT itimation . Similartimation . Additiotimation . The siztimation . Factorstimation . These ptimation . The firtimation . This prtimation .Cystic masses in the adrenal are rare and documented incidence varies between 0.064% to 0.18% in autopsy series ,18. FourThe following section focuses on the discussion of three rare lesions encountered as adrenal incidentalomas in our surgical pathology practice.Ganglioneuromas are benign, slow-growing neoplasms that arise from primordial neural crest tissue containing mature Schwann cells, ganglion cells, and nerve fibers . These tMRI and CT features vary between tumors due to the mixed composition of ganglion and Schwann cells . In thisDefinitive diagnosis of ganglioneuroma of the adrenal gland is only by microscopic examination. Ganglioneuromas are encapsulated on gross examination, with a solid, homogeneous gray-white cut surface as seen in Figure Schwannomas are slow-growing benign nerve sheath tumors that originate from neural crest cells and most commonly arise in the head, neck, upper and lower extremities, and trunk ,25. RareSchwannomas arising specifically within the adrenal gland are extremely rare ,29. It iOn gross examination adrenal/periadrenal schwannomas often appear as a solitary, well circumscribed tumor enveloped in a fibrous capsule with a solid firm white-tan and homogeneous cut surface that may contain cystic changes or hemorrhagic regions ,32. PeriLeiomyosarcoma is a mesenchymal malignancy of the smooth muscle that most commonly arises in the myometrium, retroperitoneum, and the respiratory tract . Primaryin situ hybridization of the tumor.The exact etiology of these neoplasms remains unclear. Immunocompromised patients including transplant recipients, those on chemotherapy and those with HIV/AIDS are predisposed to the development of smooth muscle tumors (SMT). They are also reported to have an increased risk for developing adrenal leiomyosarcoma . In thisOptimal management of this rare lesion recommends complete resection with a minimum of 3 cm margins, though this is often not feasible due to local invasion . Venous Primary adrenal pleomorphic leiomyosarcoma is an extremely rare lesion, with only three cases reported in the English literature as listed in Table Adrenal lesions are rare specimens in the surgical pathology laboratory. With increased and improved use of sophisticated diagnostic imaging techniques it is predicted that the number of adrenal incidentalomas detected will continue to rise. Laparoscopic or open surgical exploration of such lesions will result in increased specimens for pathological analysis. In this context, awareness of common and uncommon benign and malignant lesions of the adrenal medulla and cortex is vital for accurate pathological diagnosis to guide optimal patient management.The authors of this article have no relevant financial relationships with commercial interests to disclose.All authors participated in the creation and revisions of this manuscript. All authors read and approved the final manuscript.


Analysis of gene expression patterns leads to functional understanding of biological processes. Quantitative real-time PCR has become the most commonly used technique for in-depth studies of gene expression. To quantify variation in specific gene expression, accurate and reliable normalization across different samples and tissues is necessary. This can be achieved by selecting one or more suitable reference genes to compare the target mRNA transcript levels. In the present work, we illustrate the first evaluation of potential internal control or reference genes across different developmental stages of eggplant for reliable quantification of transcripts by real-time PCR.18s rRNA, APRT, GAPDH, Cyclophilin, Actin, and RuBP) in a set of tissues representing six developmental stages of eggplant. The candidate genes were cloned from cDNA and analysed by real-time PCR. The expression data analyzed by three statistical methods  identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant. This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant.We have evaluated the stability in expression of six candidate reference genes (18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant. Gene expression analysis is an important tool to understand the functional aspect of genes. Quantification of the steady-state mRNA by real-time PCR is identified as the most reliable and commonly used approach for basic research, molecular medicine and biotechnology ,2. Gene 18s rRNA, 25s rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ubiquitin (UBQ) are some of the commonly used internal control genes as they are likely to be expressed at constant levels regardless of experimental conditions [Some of the house keeping genes involved in basic cellular activities such as nditions . Howevernditions ,13,14. Tnditions ,16. Sevenditions -20.in vitro regeneration leading to high genetic transformation efficiency [Eggplant popularly known as Brinjal in India, is an agronomically important non-tuberous Solanaceous crop primarily grown for its large oval fruit. In addition to being a popular vegetable, it has therapeutic value and used for the treatment of several diseases such as diabetes, arthritis, asthma and bronchitis . It can ficiency . Genomic18s rRNA, APRT, GAPDH, Cyclophilin, Actin, RuBP from the cDNA resulted in 416 bp, 454 bp, 586 bp, 265 bp, 333 bp, and 269 bp amplicons respectively, which were cloned and sequenced , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Cyclophilin, Actin, Ribulose-1,5-bisphosphate carboxylase (RuBP) were selected based on previous studies that relied on them as candidate reference genes [2 software. These Ct mean values were further used for the calculation of expression stability  showed an SD value higher than 1, which disqualified their utility as reference genes. Remaining three genes were selected for further analysis as they showed SD value less than 1. Further processing of the data using pair wise correlation and regression analysis showed the inter gene relations and eliminated APRT, as the gene with the least correlation (r = 0.481) (Table 18s rRNA and Cyclophilin) showed a strong and significant correlation with an ‘r’ value of 0.990 for 18S rRNA and 0.895 for Cyclophilin indicating their stable expression levels. Accordingly, the BestKeeper index (p) was found to be 0.001 and 0.016 respectively for 18s rRNA and Cyclophilin  and CV (%Ct)) were calculated for each of the candidate reference genes in the samples for identifying the overall stability in gene expression. Three candidate reference genes (1) Table . The anaon r = 0.81  allowed sorting of genes ranked on the basis of their expression stability (M) from most stable to least stable in the order of 18s rRNA, APRT and Cyclophilin were identified as appropriate reference genes.Therefore, based on the elaborate statistical analysis The selected genes were analyzed under various situations to assess their utility to serve as reference genes:The utility of the identified genes was further analyzed in four different eggplant varieties using real-time PCR Table . An exprFurther to the validation of the selected genes in different eggplant varieties, their utility was also established in the analysis of transgenics Table B. The reMeloidogyne incognita)  Table C. It wasThese above analyses ultimately confirmed the identification and utility of the three housekeeping genes as the appropriate reference genes for qRT-PCR analysis of gene expression in eggplant.UBQ10 gene shows highly stable expression in Arabidopsis[18s rRNA, APRT, GAPDH, Cyclophilin, Actin and RuBP were selected and their expression examined across six developmental stages of eggplant variety, Pusa Purple Long. In order to minimize bias introduced by the validation approach, the results were analyzed by three different complementary statistical strategies to select best internal controls for normalization of gene expression studies. Best keeper selects the least variable gene using the geometric mean of the raw data [18s rRNA, APRT and Cyclophilin were identified as appropriate genes for normalization. Nevertheless, earlier studies revealed that the expression of both 18s rRNA and Cyclophilin were affected by both biotic and abiotic stress in potato [18s rRNA exhibited most stable expression in plants grown under various environmental conditions in rice [GAPDH was found to be a suitable reference gene for measuring the gene expression in different tissues/organs of sugarcane [Brassica juncea[GAPDH with three other genes as suitable reference genes across different developmental stages.Gene expression profile studies provide an important insight into the biological processes of plant molecular biology research, hence are recognized as crucial steps in identifying gene function. Among the various techniques available to quantify gene expression, qRT-PCR is the most commonly used method -28. It iabidopsis,29. So fraw data . NormFinraw data . Using tn potato . On the  in rice . Interesugarcane . A reporca juncea revealedMajor crux of the investigation was to demonstrate the utility of the identified genes for expression analysis under different experimental conditions. The study successfully provided evidence in establishing stable expression of the identified genes across different eggplant varieties. The expression of the genes was stable in different transgenic plants and also under biotic stress deliberately induced by challenging with nematodes. This provided an unequivocal evidence for the utility of these identified genes for expression analysis by qRT-PCR in eggplant.18s rRNA, APRT and Cyclophilin as reference genes in eggplant, suitable for normalizing real-time PCR data. This is the first study to identify the appropriate reference genes in eggplant. With the growing genomic resources in eggplant, these genes would enable accurate and reliable gene expression data analysis over a wide range of samples/experimental conditions for functional genomics as well as in translational research.We have cloned, sequenced and identified three genes viz., Eggplant seeds (CV. Pusa Purple Long) were procured from National Seeds Corporation Limited, Indian Agricultural Research Institute (IARI), New Delhi. Seeds were germinated and plants were grown under controlled conditions. Young leaves, stems and roots were collected for analysis at 20 days, while flower buds, open flowers and mature leaves were collected after 45–50 days. Three independent biological replicates were collected for each of the samples and immediately frozen in liquid nitrogen.18s rRNA, adenine phosphoribosyl transferase (APRT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Cyclophilin, Actin and Ribulose-1,5-bisphosphate carboxylase (RuBP) were obtained from the GenBank database. Using primer quest tool in Integrated DNA Technology website, primers were designed for these sequences to be used in real-time PCR . Details of the genes and their primers for both cDNA amplification and quantification by real-time PCR are given in Tables Nucleotide sequences of six housekeeping genes; Young leaves of one month old plants were ground in liquid nitrogen and genomic DNA was extracted by using NucleoSpin plant II kit (Macherey-Nagel). The genomic DNA was quantified by using Nanodrop ND-1000 spectrophotometer (Thermo Scientific) and used for PCR.Total RNA was extracted initially from the leaves for cloning the target genes and subsequently from various other plant tissues like young leaf, mature leaf, root, shoot, flower bud and open flower using Spectrum Plant Total RNA Kit (Sigma-Aldrich). The quality and concentration of each of the RNA samples was determined using Nanodrop ND-1000 spectrophotometer (Thermo Scientific), Bioanalyzer (Agilent 2100). Only those RNA samples having a ratio of 1.9 to 2.1 at 260/280; 2.0 to 2.5 at 260/230 and RIN (RNA integrity number) more than 7.0 were used for the analysis. The integrity of RNA was also checked by agarose gel electrophoresis. About 500 ng of each of the RNA was reverse transcribed using cDNA synthesis Kit  according to the manufacturer’s instructions and used for further analysis.18s rRNA, APRT, GAPDH, Cyclophilin, Actin and RuBP were PCR amplified from both the genomic DNA and cDNA of eggplant leaf; PCR amplification reactions were performed in 50 μl reaction volume containing 5 μl 10 x assay buffer, 200 μM each of dATP, dCTP, dGTP and dTTP (Fermentas), 0.5 μM each primer (Table μl genomic DNA (50 ng)/cDNA. The PCR cycles consisted of initial denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 60 s, annealing at 60°C for 30 s and extension at 72°C for 1 min with a final extension at 72°C for 10 min. The amplified products were later resolved on 1.2% agarose gel.er Table , 0.5-2 uEscherichia coli DH5α were transformed with the recombinant plasmids. Positive clones were selected by blue white colony screening. Inserts in the clones were confirmed by restriction digestion with EcoRI and sequenced by ABI solid sequencing platform (Safelab).The amplified cDNA PCR products were cloned into pGEM-T Easy cloning vector (Promega) according to the manufacturer’s instructions. Freshly prepared competent cells of 2 thermal cycler (Eppendorf). A master mix for each sample was prepared with SYBR Green I, blue dye, ROX passive reference and stabilizers, PCR Core Reagents (Eurogentec). Reaction mix of 10 μl was prepared by adding 2.5 ng of cDNA and 750 nM each of the specific primers  was performed using SYBR Green I technology in Realplexrs Table . The ampThree statistical softwares; BestKeeper, NormFinder and geNorm were used for measurement of stability of expression of the six candidate genes.BestKeeper computed the gene expression variation for the six reference genes in all the samples based on crossing points (CP) . PrimaryNormFinder utilizes a model based approach to establish expression stability of candidate reference genes. It uses raw data as an input in the form of expression values generated using the comparative Ct method. It estimates the overall expression variation of the candidate reference genes and the variation between sample subgroups .The geNorm was used to calculate candidate reference gene stability values (M) using raw expression data. Expression stability measure is calculated as the mean of pairwise variation of a gene compared to that of all other genes .Seeds of four popular eggplant varieties, Pusa Shyamla, Pusa Ankur, Pusa Kranti and Pusa Uttam were procured from Division of Vegetable Science, Indian Agricultural Research Institute, New Delhi. Total RNA was extracted from 15 days old seedlings and used for cDNA synthesis, followed by expression analysis.The selected genes were also validated in randomly selected lines of transgenic eggplant using the leaf tissue and compared with the wild type. Total RNA extracted from the leaf samples of transgenic plants was used for qRT-PCR.Meloidogyne incognita) challenged eggplants. The roots of 15 days old plants were inoculated with approximately 300 freshly hatched infective second stage juveniles of M. incognita. The nematode inoculated plants were grown in a growth chamber at 27°C, 70% relative humidity and 16 hr light and 8 hr dark conditions for 30 days for the nematode to complete its lifecycle. Subsequently, the leaves were used for real time PCR to study the expression of the selected genes.The shortlisted reference genes were also validated in nematode (The authors declare that they have no competing interest.UR conceived and designed the experiments and contributed in the manuscript preparation. NPG performed the qRT-PCR, statistical data analysis and preparation of the manuscript. PP carried out the cloning of housekeeping genes and helped in script editing. PKT performed the statistical analysis and wrote the script. DK generated the transgenic and the nematode infected eggplant material. RS improved and carted the script. All authors read and approved the final manuscript.


Thereafter, the two most promising methods were tested by artificial insemination of frozen-thawed semen (3 times a week for 3 weeks using 300 million spermatozoa/hen), followed by candling of incubated eggs, assessment of fertilization, embryonic death, and hatching rate. The survival rate of live, intact spermatozoa was greatest (p≤0.05) in pellet method and the slow programmable protocol (with 10% ethylene glycol) (28.6 and 23.5%). The two best protocols  were subsequently tested in vivo with artificial insemination. The pellet method yielded a 64% fertility rate compared to slow protocol with only 30% fertility. Regardless, both freezing protocols significantly increased embryonic deaths compared to the control group . During the 3-week in vivo trial, fertility increased and early embryonic death decreased over time. According to the results the guinea fowl sperm could tolerate the fast freezing in pellet better than the slower freezing rates and resulted acceptable fertility rate.Conservation of indigenous poultry species is an important part of the new Hungarian agricultural strategy. Semen cryopreservation is the most practical method for the long term storage of poultry genetic material. The objective was to compare four protocols for cryopreservation of guinea fowl sperm  and three cryoprotectants . The efficiency of the methods was examined by  Presently, indigenous poultry genetic materials are stored in vitro in only four national gene banks  in vivo management, in vitro conservation is a strategic tool to secure genetic diversity, considering the risk of epidemic diseases Biodiversity and gene conservation are of broad interest all over the world. Although much of the focus is on endangered species, these issues are also important for domestic livestock. With intense animal breeding and better, more homogenous environmental conditions, genetic diversity of domestic species has decreased rapidly Semen cryopreservation is presently the most practical method for long-term storage of genetic material in poultry species Schramm and HübnerIn the apparently sole study of cryopreservation of guinea fowl spermatozoa, an intermediate freezing rate (15°C/min) and 6% dimethyl-formamide (DMF) was used; 37% sperm survival and 20% fertility were achieved Sperm cryopreservation using slower freezing methods and pellet form using dimethyl-acetamide (DMA) looks also promising. This simple cryoconservation technique does not require complicated equipment, making it appropriate for widespread applications In the present study four freezing protocols with various cooling rates  and cryoprotectants  were tested for cryopreservation of guinea fowl spermatozoa.Keeping of animals and animal welfare prescriptions were according to the Hungarian Animal Protection Law (1998. XXVIII). Institute for Small Animal Research and Co-ordination Centre for Gene Conservation provides a permission for experimental animal research from the National Food Chain Safety Office, Animal Health and Animal Welfare Directorate, Budapest (permission number: 1/1512/49/15/2/2012). All applied experimental methods were permitted the own Institutional Ethical Review Board (No.7/2011) of the Institute for Small Animal Research and Co-ordination Centre for Gene Conservation.ad libitum access to a commercial poultry ration and to water.Thirty rural Hungarian guinea fowl males (1 year old) were kept in individual cages, exposed to a 16L:8D photoperiod  and given in vitro assessment of semen quality was done before freezing and after thawing (fresh and frozen/thawed semen). Following sperm volume measurement, motility was determined by subjective scoring (0–5) by a well-trained person, while sperm concentration was estimated with a spectrophotometer . Sperm morphology normal, abnormal and live/dead cell ratio was examined with aniline blue-eosin staining. This procedure is based on eosin  that is excluded by live, intact cell membranes but dead sperm membranes permit the eosin to stain the sperm. The aniline blue  provides a clear blue background in the smear to enhance the contrast of the white, unstained “live” sperm or the pinkish, stained “dead” sperm. Slides were evaluated microscopically  using an oil immersion objective and 1200× magnification. A total of 200 spermatozoa were assessed per slide.Males were selected firstly according to their tractability and response of sperm collection. Following a training period, assessment of semen quality was done to selecting the best sperm donors. For a two-month interval, semen was collected twice a week by dorso-abdominal massage .In the study, four freezing protocols with various cooling rates  and three cryoprotectants (CPs) were tested: 10% ethylene glycol , 6% dimethyl-formamide  and 6% dimethyl-acetamide  After sperm collection, pooled semen was diluted 1∶3 with Lake’s extender Pretreatment of the semen was similar to the previous protocol, except the equilibration period, which lasted 5 minutes at 5°C for both 10% EG or 6% DMF. Aliquots (200 µL) from the samples were put into the cryo vials and placed in the freezing machine. Freezing started at 5°C with an intermediate freezing rate (−15°C/min) until −30°C, than samples were cooled (−30°C/min freezing rate) to −60°C. Cryo vials were placed into liquid nitrogen (LN). Thawing was done out at 5°C for 20 min in a cooler cabin (as described above).Dilution and precooling of the semen were exactly the same as in fast programmable protocol. However, the cryo vials were put onto a vial rack which was placed in a plastic box at 4 cm above the liquid nitrogen. Thawing was carried out at 38°C in an incubator for 3 min .After sperm collection, pooled semen was diluted 1∶1 with Tselutin’s extender in vitro assessment of frozen-thawed sperm) were tested with artificial insemination . Ten females (controls) were inseminated with fresh, diluted semen, whereas the remaining 20 were inseminated with frozen/thawed semen from slow and pellet method (10 birds per protocol). Artificial insemination was done within 10 minutes after thawing thrice weekly for 3 weeks using 250–300 million spermatozoa/hen/AI. The fertility of 300 incubated eggs was checked by candling at the 10th day of incubation. The rate of the true fertility  was determined. Embryos that had died in the oviduct were detected by propidium iodide staining of the germinal disc. Egg yolks were separated from the albumen and placed into 0.9% NaCl solution. After visual fertility examination germinal discs seeming to be infertile were removed from the vitelline membrane, put into 0.9% NaCl solution and stained on a slide with propidium iodide . In case of fertile egg, propidium iodide (DNA-specific fluorescent dye of red color) stains the nucleus of the dividing embryonic cells which appear in lighting points Thirty female guinea fowls were placed in individual cages . The two best freezing methods .For statistical analysis of the data The quality of spermatozoa after freezing and thawing is shown . The besThe best (p≤0.05) survival rate of live, intact spermatozoa was in the pellet method and slow programmable protocol with 10% EG with 28.6 and 23.5%, respectively. The efficiency of the fast programmable protocol was rather low in both cases (12 and 15.5%), slow protocol with 6% DMF (11.4%) whereas freezing in nitrogen vapor produced the lowest survival rate (7.9 and 8.6%), irrespective of the type of CPs .in vitro assessments). Therefore, spermatozoa cryopreserved by these two methods were used for artificial insemination (in vivo test). To the end of the 3-week insemination period, fertility rates were 91.7, 29.1 and 63.6%, and rate of normal embryos were 83, 12, and 55% in fresh samples (control), slow and pellet method protocols, respectively. In sperm cryopreserved by the slow freezing, only dead embryos were detected in the first two weeks in the eggs. The rate of early embryonic deaths (embryo died in the oviduct) was quite high in the first week of insemination period , but by the third week it had decreased significantly except for the slow protocol’ group . The rate of the embryos that died during incubation decreased in all experimental groups to the end of the examined period . The significantly highest rate of abnormal spermatozoa was detected in slow protocol using EG. Regarding specific defects, damage to the midpiece was the most common abnormality in all protocols (data not shown). Based on the present experiment, cryoprotectant had a lesser impact on sperm morphology than the rate of freezing. To our knowledge, there are no data available regarding sperm abnormalities of frozen thawed guinea fowl semen. However analysed the abnormal sperm ratio in frozen-thawed gander spermatozoa, the rate of midpiece injuries, namely “bent neck”, were the most prevalent Based on slow protocol, only 18–30% of eggs were fertilized; moreover 80–90% of them contained early dead embryos. According to earlier reports frozen–thawed avian spermatozoa are associated with an increased incidence of early embryonic mortality. Although only a single spermatozoon is required to activate an ovum, in the case of avian species, polyspermic fertilization occurs and an optimal number of sperm number are necessary for good fertility. The role of the other spermatozoa penetrated into the ovum is not fully understood yet. Previous studies reported that both too low When semen was preserved by the Furthermore, perhaps morphologically normal spermatozoa contained cryopreservation-induced damage to nuclear structures, including DNA Tselutin et al.,In conclusion, the pellet method was the most effective way for conservation of guinea fowl spermatozoa with a high rate of fertility. There are no data available about the long term duration of sperm in pellet form.